JCTH Associate Editors Dr

JCTH Associate Editors Dr. Timothy Billiar Dr. Kittichai Promrat OWNED BY THE SECOND AFFILIATED HOSPITAL OF CHONGQING MEDICAL UNIVERSITY Presbyterian University Hospital Alpert Medical School of Brown University Pittsburgh, USA Providence, USA Dr. John Birk Prof. Cheng Qian University of Connecticut Third Military Medical University Southwest Farmington, USA Hospital Chongqing, China Editors-in-Chief Prof. Li-Min Chen Dr. Arielle Rosenberg University Paris Descartes Prof. Hong Ren Peking Union Medical College Chengdu, China Paris, France General Editor-in-Chief The Second Affiliated Hospital of Chongqing Prof. Cheng-Wei Chen Prof. Naoya Sakamoto Medical University, China Hokkaido University Nanjing Military Command Sapporo, Japan Dr. Harry Hua-Xiang Xia Shanghai, China Editor-in-Chief Dr. Michael Schilsky Novartis Pharmaceuticals Corporation, USA Prof. Aziz A. Chentoufi Medicine King Fahad Medical College Yale University New Haven, USA Prof. George Y. Wu Riyadh, Saudi Arabia Comprehensive Editor-in-Chief Prof. Xiao-Guang Dou Dr. Tawesak Tanwandee University of Connecticut Heath Center, USA Siriraj Hospital, Mahidol University Shengjing Hospital of China Medical University Bangkok, Thailand Managing Editors Shengyang, China Prof. Fu-Sheng Wang Dr. Jean-François Dufour 302 Miltitary Hospital of China Huai-Dong Hu University of Bern Beijing, China Chongqing, China Bern, Switzerland Zhi Peng Prof. Lai Wei Prof. Marko Duvnjak Peking University People’s Hospital Chongqing, China Clinical Hospital Centre “Sestre milosrdnice” Beijing, China Zagreb, Croatia Executive Editor Prof. Faripour Forouhar Dr. Xue-Feng Xia University of Connecticut The Methodist Hospital Research Institute Houston, USA Hua He Farmington, USA Houston, USA Dr. Johannes Haybaeck Dr. Ke-Cheng Xu Medical University Graz Fuda Cancer Hospital Technical Editor Graz, Austria Guangzhou, China Wan-Chen Hu Prof. Jin-Lin Hou Prof. Man-Fung Yuen Wuhan, China Nanfang Hospital of Southern Medical The University of Hong Kong Queen Mary University Hospital Guangzhou, China Hong Kong, China Prof. Ke-Qin Hu Prof. Da-Zhi Zhang University of California The Second Affiliated Hospital of Chongqing Orange, USA Medical University Chongqing, China Prof. Ji-Dong Jia Capital Medical University Dr. Lan-Jing Zhang Beijing, China University Medical Center of Princeton Contact information Plainsboro, USA Telephone +86 23 63727251 Dr. Joseph Lim Yale University Fax +86 23 63701383 New Haven, USA E-mail [email protected] Prof. Lun-Gen Lu Address 74 Linjiang Road, Shanghai Jiaotong University School of Yuzhong District, Medicine Shanghai, China Chongqing, P. R. China, 400010 Dr. John Luk Johnson & Johnson Medical Ltd. Shanghai, China Publisher Prof. Yen-Hsuan Ni National Taiwan University Xia & He Publishing Inc. Taipei, Taiwan 11415 Ashford Willow, Sugar Land, Texas, 77478, USA Prof. Jun-Qi Niu Jilin University Changchun, China JOURNAL OF CLINICAL AND TRANSLATIONAL HEPATOLOGY

Call for papers JCTH is a new, comprehensive specialist journal focusing on the recent progress in clinical and basic research with direct applications to clinical management of liver diseases. The studies published in JCTH will represent the most current trends in the field of hepatology, highlighting the topically relevant subjects of nations worldwide. Publications in JCTH will be presented in formats that emphasize clarity of the study’s objectives and implications of its findings, using high quality visual aids to enhance the manuscript’s esthetic appeal as well as its impact. For our upcoming issue, we encourage you and your group to submit original articles that showcase your work in hepatology and topically relevant reviews to promote our readers’ understanding of the field.

CONTENTS 2016 4(2):76–168

Original Articles

Accessibility to Oral Antiviral Therapy for Patients with Chronic Hepatitis C in the United States Sammy Saab, Melissa Jimenez, Tiffany Fong, Crystal Wu, Sherona Bau, Zoha Jamal, Jonathan Grotts and David Elashoff ...... 76

Postoperative Risk of Hepatic Decompensation after Orthopedic Surgery in Patients with Cirrhosis Eric M. Nyberg, Michael Batech, T. Craig Cheetham, Jose R. Pio, Susan L. Caparosa, Mary Alice Chocas and Anshuman Singh ...... 83

Effects of Granulocyte Colony-Stimulating Factor on Patients with Liver Failure: a Meta-Analysis Qiao Yang, Ying Yang, Yu Shi, Fangfang Lv, Jiliang He and Zhi Chen ...... 90

Review Articles

Interferon-Free Treatments for Chronic Hepatitis C Genotype 1 Infection Alireza FakhriRavari, Mazyar Malakouti and Rebecca Brady ...... 97

Current Management of Alcoholic Hepatitis and Future Therapies Behnam Saberi, Alia S. Dadabhai, Yoon-Young Jang, Ahmet Gurakar and Esteban Mezey ...... 113

Nonalcoholic Lipid Accumulation and Hepatocyte Malignant Transformation Juanjuan Gu, Min Yao, Dengbing Yao, Li Wang, Xuli Yang and Dengfu Yao ...... 123

Acetaminophen-Induced Hepatotoxicity: a Comprehensive Update Eric Yoon, Arooj Babar, Moaz Choudhary, Matthew Kutner and Nikolaos Pyrsopoulos ...... 131

Hepatitis B Reactivation with Novel Agents in Non-Hodgkin’s Lymphoma and Prevention Strategies Oluwatobi O. Ozoya, Lubomir Sokol and Samir Dalia ...... 143 Immune Activation in the Liver by Nucleic Acids Qian Sun, Qingde Wang, Melanie J. Scott and Timothy R. Billiar ...... 151

The Evolving Nature of Hepatic Abscess: A Review Marianna G. Mavilia, Marco Molina and George Y. Wu ...... 158 Original Article

Accessibility to Oral Antiviral Therapy for Patients with Chronic Hepatitis C in the United States

Sammy Saab*1,2, Melissa Jimenez2, Tiffany Fong2, Crystal Wu2, Sherona Bau2, Zoha Jamal2, Jonathan Grotts3 and David Elashoff3

1Department of Medicine, University of California, Los Angeles, CA, USA; 2Department of Surgery, University of California, Los Angeles, CA, USA; 3Department of Biostatistics, University of California, Los Angeles, CA, USA

Abstract © 2016 The Second Affiliated Hospital of Chongqing Medical University. Published by XIA & HE Publishing Inc. All rights re- Background: Hepatitis C (HCV) direct acting antiviral agents served. (DAAs) are safe, effective, and tolerable. Most contraindications to interferon-based treatment are no long applicable. The aims of this study were to understand the predictors of approval to drug accessibility. Methods: We studied Introduction all consecutive patients with HCV prescribed DAAs between – October 2014 and July 2015. Data on demographic, socio- Up to 5 7 million Americans are believed to be infected with economic status, comorbidities, baseline laboratory values, chronic hepatitis C (HCV) Most of these individuals are unaware of their disease and are in the baby-boomer age and assessment of liver disease severity, insurance, and spe- 1 cialty pharmacy type were collected. Multivariate analyses group. Chronic hepatitis C infection is an important medical were performed to identify predictors of prescription approval. concern that has both hepatic and extrahepatic manifestation.2 The viral infection can lead to cirrhosis, hepatocellular Results: In total, 410 patients were prescribed DAAs between 2–4 October 2014 and July 2015. Of those, 332 (81%) patients carcinoma (HCC), and liver failure. Indeed, HCV is the most common indication for liver transplantation in the were insurance approved for therapy. Of the 332 patients 5 accepted, 251 were accepted after the first prescription United States. Extrahepatic manifestations, such as cryoglobulinemia and kidney disease, are also important causes of attempt, and 38 were accepted after the second and third 6,7 attempts. The number of attempts for the other 43 approved morbidity and mortality. patients was unknown. Older age (p =0.001),employment Achieving a sustained viral response (SVR) is associated (p = 0.001), lack of comorbidities (p = 0.02), liver transplan- with a number of improved clinical outcomes, including liver- related outcomes and overall survival.8,9 Interferon has been tation (p = 0.018), and advanced liver disease (p =0.001) 10 were more likely associated with obtaining approval. House- the cornerstone of antiviral therapy for almost 2 decades. hold income was not associated with insurance approval. However, its utility is limited by a number of contraindications, such as uncontrolled depression and autoimmune In the multivariate analysis, Medicare insurance (odds ratio 11,12 – disorders. The introduction of all-oral direct-acting [OR]) 2.67, 95% confidence interval [CI] 0.96 7.20), lack 13,14 of nonliver comorbidities (OR 2.72, 95% CI 1.35–5.43), and agents (DAAs) has revolutionized the treatment of HCV. the presence of advanced liver disease (OR 1.82, 95% CI Compared to interferon, DAAs have been shown to increase 1.04–3.24) independently predicted drug approval. Conclu- SVR and improve tolerability compared to interferon. Few serious adverse effects are seen with DAAs, and rarely do sion: Despite the availability of DAAs for HCV, barriers 15 from insurance carriers continue to impair widespread use. patients discontinue use of DAAs due to adverse effects. Patients with advanced liver disease, Medicare, and without Antiviral therapy with DAAs has been not only been demonstrated to be safer, more effective, and better tolerated than comorbidities are most likely to be insurance approved 16–20 for DAAs. interferon-based therapy but also more cost effective. Nevertheless, DAAs are expensive and access to these drugs is not guaranteed. Thus, we examined the accessibility of these drugs to patients with HCV. We hypothesized that there Keywords: Hepatitis C; Healthcare access; Antiviral therapy. Abbreviations: 3D, ombitasvir-paritaprevir-ritonavir and dasabuvir; AASLD, are substantial barriers to HCV antiviral therapy for patients, American Association for the Study of Liver Diseases; ALT, alanine transaminase; even after specialty referral. AST, aspartate transaminase; CI, confidence interval; CPD, cardiopulmonary disease; DAAs, direct acting agents; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; HIV, human immunodeficiency virus; HMO, Patient and Methods health maintenance organization; IDSA, Infectious Diseases Society of America; INR, International Normalized Ratio; IQR, interquartile range; OR, odds ratio; PPO, preferred provider organization; SD, standard deviation; SVR, sustained Study populations viral response; ZCTA, zip code tabulation area. Received: 17 March 2016; Revised: 12 April 2016; Accepted: 14 April 2016 q Consecutive HCV patients treated with DAAs at the University DOI: 10.14218/JCTH.2016.00011. of California Los Angeles (UCLA) since the approval of Correspondence to Sammy Saab, Pfleger Liver Institute, UCLA Medical Center, 200 Medical Plaza, Suite 214, Los Angeles, CA 90095, USA. Tel: +1-310-206- sofosbuvir/ledipasvir were identified through an administra- 6705, Fax: +1-310-206-4197, E-mail: [email protected] tive UCLA database. The study period was between October

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2014 and July 2015. We collected demographic information, Results such as gender, age, and ethnicity. Baseline laboratory, radiological, and histological results, as applicable, were During our study period, 410 patients were prescribed all oral also recorded. Laboratory tests were obtained within 30 antiviral therapy. Baseline demographic information is found days of the prescription being sent to pharmacy. Liver in Table 1. The mean age (± SD) of our cohort was 59.6 disease severity, defined through clinical findings, laboratory (± 10.9) years. The majority of patients were male. Almost tests, liver biopsy, and/or radiological evaluation, was classi- 50% of our cohort was unemployed, and most patients were fied as either advanced (stage 3–4) or nonadvanced (stages insured with Medicare. Less than 10% of the patient cohort 0–2). Decompensated manifestations were defined by the had a nonliver related comorbidity. Extrahepatic manifesta- presence of ascites, encephalopathy, variceal bleeding or tions were found in over a quarter of patients, and 22.4% of esophageal varices without bleeding, liver cancer, or jaun- patients had some manifestation of hepatic decompensation. dice. Extrahepatic manifestations were defined by the pres- Almost 20% of the patient cohort was transplant recipients. ence of debilitation/fatigue, type 2 diabetes mellitus, Of the employed patients, most had incomes between porphyria cutanea tarda, cryoglobulinemia, lymphoma, or $50,000 and $100,000. vasculitis. Nonliver comorbidities were defined by the pres- Of the nontransplant recipients, 58.7% of the patients were ence of cardiopulmonary disease, hemodialysis, stroke, non- classified as having advanced liver disease. Baseline labora- HCC malignancy, human immunodeficiency virus (HIV), and tory test values are shown in Table 2. Genotype 1 was the pre- diabetes. dominant viral genotype and was found in 85% of patients. The Generally, prescriptions were sent to specialty pharma- mean HCV viral load was 5,430,135 IU/mL. The mean aspar- cies. These pharmacies were responsible for the completion tate aminotransferase (AST), alanine aminotransferase (ALT), of authorization forms and, if necessary, appeals for drug total bilirubin, and platelet count were 63 IU/mL, 70 IU/mL, approvals. Insurance carrier and specialty pharmacy were 0.88 mg/dL, and 170 3 103/mL respectively. noted. Pharmacies were stratified in our analysis according to The insurance approval rate for our cohort was 81.0% whether they were privately or publically owned (company (332/410), and 36.3% and 63.7% of the prescriptions were traded on a stock exchange). The stratification of pharmacies sent to privately and publically owned pharmacies, respec- was important to exclude effort bias by the two types of tively. Of the 332 patients approved for therapy, 251 were pharmacies. Patient income was estimated by residential approved without the need for appeal to the insurance zip code and zip code tabulation area (ZCTA) through the company. Thirty-four were approved after one appeal, and United States Census Bureau.21 The study was approved by four were approved after the second appeal. Information on the Institutional Review Board. Patient consent was not the time from submission to insurance decision was known in required since this was a retrospective analysis. There was 368 patients, and the mean (± SD) time from submission to no funding for this study. drug approval was 28.1 (± 46.0) days. Patients with Medicaid were less likely to be approved and waited the longest for the final decision (Fig. 2). The likelihood of drug therapy approval Outcomes varied according to insurance plan: Medicaid (80%, 36/45), Medicare (92%, 153/166), non-Medicaid health maintenance Our main outcome was insurance approval for oral antiviral organization (HMO) (78%, 42/54), and preferred provider therapy. We recorded prescription approval or denial for each organization (PPO) (70%, 101/145). Drug therapies are patient. The time between prescription submission and the shown on Table 3. pharmacy’s decision was recorded. The number and the Predictors of approval were age (p = 0.001), work status decision of appeals were also documented. Generally, pre- (p = 0.001), lack of comorbidities (p = 0.02), liver transplan- scription packets containing necessary study results and tation (p = 0.018), and severity of liver disease (p = 0.001). clinical information, including indication for antiviral therapy, The results of multivariate analysis identified Medicare insur- were sent to specialty pharmacies. If the insurance provider ance (OR 2.67, 95% confidence interval [CI]), lack of comor- declined drug approval, it was our practice to appeal the bidities (OR 2.72, 95% CI 1.35–5.43), and the presence of decision up to two times. advanced liver disease (OR 1.82, 95% CI 1.04–3.24) as independent predictors of drug approval (Table 4). Statistical Analysis Discussion Data are presented as mean ± standard deviation (SD), median with interquartile range (IQR), or number in group The results of our study demonstrate the insurance accessi- with percent of group based on the distribution of the data. bility limitations for patients with chronic hepatitis C infection Univariate analysis was performed using a t-test for normally in our practice. The overall insurance approval was 81%. distributed continuous data, Wilcoxon-Rank Sum test for non- Predictors of approval were age, work status, liver trans- normally distributed continuous data, and Fisher’s exact plantation, type of insurance, severity of liver disease, and test or Chi-sqaure test for discrete data. We used a multi- the lack of medical comorbidities. The presence of extra- variate logistic regression model to derive adjusted odd- hepatic manifestations, hepatic decompensation, HCC, and ratios (OR) for predictors of patients that were approved for other co-existent liver diseases were not found to be asso- DAAs by their insurance provider. The multivariate logistic ciated with the likelihood of obtaining DAAs. Initially, the regression model was built using variables with a p value American Association for the Study of Liver Diseases below 0.10 on univariate analysis. All tests were two-sided, (AASLD)/Infectious Diseases Society of America (IDSA) joint and a p value below 0.05 indicated statistical significance. The guidance recommended prioritization of patients with HCV and R Statistical Programming Environment was used to conduct highlighted liver transplant recipients and patients with severe analysis (R Core Team Vienna, Austria). liver damage as a highest prioritization. More recently, the

Journal of Clinical and Translational Hepatology 2016 vol. 4 | 76–82 77 Saab S. et al: Access to hepatitis C antiviral therapy

Table 1. Demographics and baseline results

Overall Approved Denied n/N (%) n/N (%) n/N (%) Characteristics N = 410 N = 332 N = 78 OR 95% CI p-Value

Mean age - years 59.6 (10.9) 60.5 (10.5) 55.6 (11.7) 0.001 Gender 1 Female 172/410 (42%) 139/332 (41.9%) 33/78 (42.3%) Reference Male 238/410 (58%) 193/332 (58.1%) 45/78 (57.7%) 1.02 0.61–1.67 Work status 0.001 Disabled 103 (25.1%) 92 (27.7%) 11 (14.1%) Reference Employed 193 (47.1%) 143 (43.1%) 50 (64.1%) 0.34 (0.16–0.67) Retired 86 (21%) 77 (23.2%) 9 (11.5%) 1.02 (0.4–2.66) Unemployed 28 (6.8%) 20 (6%) 8 (10.3%) 0.3 (0.11–0.86) Non-Liver 59/410 (14.4%) 41/332 (12.3%) 18/78 (23.1%) 0.47 0.26–0.89 0.02 comorbidities Co-existent Liver 20/410 (4.9%) 14/332 (4.2%) 6/78 (7.7%) 0.53 0.2–1.53 0.238 Diseases Extra-hepatic 116/410 (28.3%) 91/332 (27.4%) 25/78 (32.1%) 0.8 0.47–1.38 0.406 manifestations Hepatic 84/410 (20.5%) 74/332 (22.3%) 10/78 (12.8%) 1.95 1.0–4.2 0.063 decompensations HCC 4 (1%) 2 (0.6%) 2(2.6%) 0.23 0.03–1.94 0.165 Liver transplant 83/410 (20.2%) 75/332 (22.6%) 8/78 (10.3%) 2.55 1.24–5.97 0.018 recipient Severity of Liver 0.001 Disease (Non-transplant) Advanced 152/327 (46.5%) 131/257 (51%) 21/70 (30%) Reference Non-advanced 175/327 (53.5%) 126/257 (49%) 49/70 (70%) 0.42 0.24–0.7 Income 0.2 < 50,000 88/399 (22%) 74/322 (23%) 14/77 (18.2%) Reference 50,000–100,000 266/399 (66.7%) 216/322 (67.1%) 50/77 (64.9%) 0.82 0.41–1.53 > 100,000 45/399 (11.3%) 32/322 (9.9%) 13/77 (16.9%) 0.47 0.2–1.11 Percent below 11.1 (8.2–18.2) 11.8 (8.2–18.6) 10.3 (7.9– 1.02 0.99–1.06 0.172 poverty, 15.9) median (IQR) Pharmacy N = 410 N = 332 N = 78 0.602 Privately owned 149/410 (36.3%) 123/332 (37%) 26/78 (33.3%) Reference Publically traded 261/410 (63.7%) 209/332 (63%) 52/78 (66.7%) 0.85 0.5–1.42 Type of insurance < 0.001 Medicaid 45 (11%) 36 (10.8%) 9 (11.5%) Reference Medicare 166 (40.5%) 153 (46.1%) 13 (16.7%) 2.94 (1.14–7.37) Non-Medicaid HMO 54 (13.2%) 42 (12.7%) 12 (15.4%) 0.88 (0.32–2.3) PPO 145 (35.4%) 101 (30.4%) 44 (56.4%) 0.57 (0.24–1.25) Days from Rx 10 (3–27) 8 (2–24) 16 (5–54) 0.99 0.99–1 0.001 request to Insurance Decision Median (IQR) - days

Abbreviations: PPO, Preferred Provider Organization; HMO, Health Maintenance Organization; CPD, Cardiopulmonary Disease; HCC, Hepatocellular Carcinoma; IQR, Interquartile Range.

78 Journal of Clinical and Translational Hepatology 2016 vol. 4 | 76–82 Saab S. et al: Access to hepatitis C antiviral therapy

Fig. 1. Flow chart of approvals and denial for patients with timeline of attempts.

Table 2. Baseline laboratory results

Overall n/N (%) Approved n/N (%) Denied n/N (%) Characteristics N = 410 N = 332 N = 78 OR 95% CI p-Value

Genotype 0.803 1 349/410 (85.1%) 283/332 (85.2%) 66/78 (84.6%) Reference 2 22/410 (5.4%) 19/332 (5.7%) 3/78 (3.8%) 1.48 0.49–6.42 3 30/410 (7.3%) 23/332 (6.9%) 7/78 (9.0%) 0.77 0.33–2 4 9/410 (2.2%) 7/332 (2.1%) 2/78 (2.6%) 0.82 0.19–5.56 Viral load (IU/mL) 2,010,000 2,100,000 1702,612 ––0.579 Median (IOR) (579,500–5,405,000) (57,8750–5,585,000) (662,000–4,310,000) AST (U/L) 46.5 (32–76.2) 48 (31–77) 45 (35.2–72.8) ––0.644 Median (IQR) ALT (U/L) 54 (32–89) 54 (30–89.5) 58.5 (39–87.8) ––0.197 Median (IQR) Bilirubin, 0.5 (0.4–0.8) 0.6 (0.4–0.8) 0.5 (0.4–0.9) ––0.37 Total (mg/dL) Median (IQR) Alkaline 83 (63–111) 86 (65.2–114) 73 (59.2–100) ––0.027 phosphatase (U/L) Median (IQR) Albumin (g/dL) 4.3 (3.8–4.5) 4.2 (3.8–4.4) 4.4 (4–4.6) ––0.002 Median (IQR) Platelet count 169 (109.5–227) 161 (103–223) 181 (141.5–238.5) ––0.019 (x10E3/uL) Median (IQR) Creatinine (mg/dL) 0.9 (0.8–1.1) 0.9 (0.8–1.1) 0.8 (0.7–1) ––0.077 Median (IQR) INR Median (IQR) 1.1 (1.1–1.2) 1.1 (1–1.2) 1.1 (1.1–1.2) ––0.89

Abbreviations: AST, Aspartate Transaminase; ALT, Alanine Transaminase; INR, International Normalized Ratio; IQR, Interquartile Range.

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Fig. 2. Time to decision by insurance type. same joint guidance recommended all patients should be patients with Medicaid insurance. In addition, patients with treated for HCV because of the potential for extrahepatic Medicare insurance generally had higher approval rates. manifestations.22 Patients with Medicare were more likely than any other Several other studies have also highlighted the insurance insurance to be approved for DAAs. Of the 166 patients with barriers to obtaining DAAs. For instance, the study by Do et al. Medicare, 153/166 (92.2%) were approved for treatment. described an overall insurance approval of 77.5%.23 The The ease of Medicare patients to start antiviral therapy major predictors of drug approval were public insurance and may serve as a disincentive for select insurance carriers to advanced liver disease. In contrast, our study stratified deny patients with the expectation they will be eventually patients based on public insurance and found there was a approved. The criteria for drug approval vary across insurance substantive difference between patients with Medicaid and companies. For instance, Medicaid requires a minimal fibrosis Medicare insurances. The results of a separate study by stage of 2 to be approved for treatment, whereas a particular Re et al., which was recently presented at the AASLD national PPO insurance company limits the use of DAAs to patients meeting, revealed an insurance approval of 84%.24 Similar to with at least bridging fibrosis assessed only through a liver our findings, they found a high denial rate for DAAs therapy in biopsy and elastrography.25

Table 3. Treatment regimens

Characteristics Overall Approved n/N (%) Denied n/N (%)

Treatment regimen N = 332 N = 78 Ledipasvir/Sofosbuvir 324 261/332(78.6%) 63/78 (80.8%) Ledipasvir/Sofosbuvir + Ribavarin 39 30/332 (9.0%) 9/78 (11.5%) Sofosbuvir + Ribavarin 37 33/332 (9.9%) 4/78 (5.1%) Sofosbuvir + Simeprevir 2 1/332 (0.3%) 1/78 (1.3%) 3D 12 6/332 (1.8%) 6/78 (7.7%) 3D + Ribavarin 3 1/332 (0.3%) 2/78 (2.6%)

Abbreviation: 3D, Ombitasvir-Paritaprevir-Ritonavir and Dasabuvir.

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Table 4. Results of multivariate analysis

Characteristics OR 95% CI p-value

Age (years) 1.02 0.99–1.04 0.261 Work status 0.354 Disabled Reference Employed 0.56 0.24–1.26 Retired 0.68 0.24–2.01 Unemployed 0.37 0.12–1.17 Insurance 0.023 Medicaid HMO Reference Medicare 2.66 0.96–7.2 Non-Medicaid HMO 1.11 0.39–3.1 PPO 0.78 0.31–1.82 Lack of comorbidities 2.72 1.35–5.43 0.006 Extrahepatic manifestations 0.74 0.41–1.34 0.313 Liver transplant recipient 1.85 0.85–4.46 0.123 Stage of fibrosis 0.035 Non-advanced fibrosis Reference Advanced fibrosis 1.82 1.04–3.24

Abbreviations: PPO, Preferred Provider Organization; HMO, Health Maintenance Organization.

Although studies have shown consistently that antiviral that patients with advanced liver disease, Medicare, and therapy with DAAs for HCV is cost effective, almost 20% of those without comorbidities are most likely to be insurance our patient cohort was declined treatment by the insurance approved for DAAs. carrier.16–20 A potential reason for this lack of approval is the upfront costs associated with curing hepatitis C. The average Conflict of interest wholesale cost for an antiviral therapy course is almost $90,000, and the benefits of curing HCV are not realized for None years after SVR is achieved. For instance, the cost effectiveness of treating HBV has been shown, even though the clinical Author contributions impact is not found for many years, because the treatment costs are amortized.26,27 Study concept and design (SS, SB, TF, CW, DE), data There are a number of limitations to our study. First, our acquisition (MJ, TF, CW, SB), data analysis and interpretation analysis was limited by the start point of our specialty (MJ, JG, DE), drafting of the manuscript (SS, MJ, TF, CW), hepatology clinic. Multiple barriers to therapy exist prior to critical revision of the manuscript (SS, SB, JG, MJ), statistical when the patients walks into our office, and these can be analysis (JG, DE), administrative, technical, or study super- stratified into patient, provider, and system constructs.30–32 vision (SS, DE). Second, the results of our study may not be generalizable to other practices, as the patients in our cohort were prescribed DAAs at an academic center with experienced hepatologists References and a dedicated staff. The exclusive use of a specialist, who may have an inherent interest in drug approval, could have [1] Chak E, Talal AH, Sherman KE, Schiff ER, Saab S. Hepatitis C virus infection in USA: an estimate of true prevalence. Liver Int 2011;31:1090–1101. doi: 10. influenced the likelihood of DAA approval. Another limitation 1111/j.1478-3231.2011.02494.x. is the availability of DAAs from all drug formularies and the [2] Younossi ZM, Kanwal F, Saab S, Brown KA, El-Serag HB, Kim WR, et al. The fluidity of treatment criteria. The large cohort and the long impact of hepatitis C burden: an evidence-based approach. Aliment Pharma- – entry study period should hopefully mitigate this limitation col Ther 2014;39:518 531. doi: 10.1111/apt.12625. [3] Freeman AJ, Dore GJ, Law MG, Thorpe M, Von Overbeck J, Lloyd AR, et al. and the effects on our results. Lastly, our results pertain to Estimating progression to cirrhosis in chronic hepatitis C virus infection. Hep- the United Health care system, and the findings cannot be atology. 2001;34:809–816. doi: 10.1053/jhep.2001.27831. extrapolated to other regions of the World. For instance, in [4] Kanwal F, Hoang T, Kramer JR, Asch SM, Goetz MB, Zeringue A, et al. Increas- many countries, public, not private, medical insurance pre- ing prevalence of HCC and cirrhosis in patients with chronic hepatitis C virus infection. Gastroenterology. 2011;140:1182–1188.e1. doi: 10.1053/j.gastro. dominates. As a result, antiviral therapies are funded by the 2010.12.032. government and accessibility does not depend on insurance [5] Kim WR, Stock PG, Smith JM, Heimbach JK, Skeans MA, Edwards EB, et al. approval. OPTN/SRTR 2011 Annual Data Report: liver. Am J Transplant 2013;1:73–102. Despite the effectiveness, safety, and tolerability of DAAs, doi: 10.1111/ajt.12021. [6] Stefanova-Petrova DV, Tzvetanska AH, Naumova EJ, Mihailova AP, Hadjiev barriers to access these medications continue, even at EA, Dikova RP, et al. Chronic hepatitis C virus infection: prevalence of extra- specialty hepatology clinics. The results of our study highlight hepatic manifestations and association with cryoglobulinemia in Bulgarian

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patients. World J Gastroenterol. 2007;13:6518–6528. doi: 10.3748/wjg.13. chronic hepatitis C virus genotype 1 infection. Aliment Pharmacol Ther. 6518. 2015;41:544–563. doi: 10.1111/apt.13081. [7] Lee MH, Yang HI, Lu SN, Jen CL, You SL, Wang LY, et al. Chronic hepatitis C virus [20] Chahal HS, Marseille EA, Tice JA, Pearson SD, Ollendorf DA, Fox RK, et al. infection increases mortality from hepatic and extrahepatic diseases: a com- Cost-effectiveness of Early Treatment of Hepatitis C Virus Genotype 1 by munity-based long-term prospective study. J Infect Dis. 2012;206:469–477. Stage of Liver Fibrosis in a US Treatment-Naive Population. JAMA Intern doi: 10.1093/infdis/jis385. Med. 2016;176:65–73. doi: 10.1001/jamainternmed.2015.6011. [8] Koh C, Heller T, Haynes-Williams V, Hara K, Zhao X, Feld JJ, et al. Long-term [21] http://factfinder.census.gov/faces/nav/jsf/pages/community_facts.xhtml, outcome of chronic hepatitis C after sustained virological response to interferon- accessed August 2015. based therapy. Aliment Pharmacol Ther 2013;37:887–894. doi: 10.1111/apt. [22] www.HCVguidelines.org. Accessed November, 2015. 12273. [23] Do A, Mittal Y, Liapakis A, Cohen E, Chau H, Bertuccio C, et al. Drug Author- [9] van der Meer AJ, Veldt BJ, Feld JJ, Wedemeyer H, Dufour JF, Lammert F, ization for Sofosbuvir/Ledipasvir (Harvoni) for Chronic HCV Infection in a et al. Association between sustained virological response and all-cause Real-World Cohort: A New Barrier in the HCV Care Cascade. PLoS One. mortality among patients with chronic hepatitis C and advanced hepatic 2015;10:e0135645. doi: 10.1371/journal.pone.0135645. [24] Lo Re V, Gowda C, Urick PN, Halladay J, Binkley A, Carbonari DM, et al. fibrosis. JAMA. 2012;308:2584–2593. doi: 10.1001/jama.2012.144878. Incidence and determinants of denial of DAA therapy by type of insurance [10] Saab S, Jackson C, Nieto J, Francois F. Hepatitis C in African Americans. during the first 6 months of the modern HCV treatment era. AASLD 2015. Am J Gastroenterol. 2014;109:1576–1584. doi: 10.1038/ajg.2014.243. 13–17 Nov, 2015. San Francisco. [11] Russo MW, Fried MW. Side effects of therapy for chronic hepatitis C. Gastro- [25] http://www.dhcs.ca.gov/Pages/HepatitisC.aspx. Accessed. December 2015. enterology. 2003;124:1711–1719. doi: 10.1016/S0016-5085(03)00394-9. [26] Yuan Y, Iloeje UH, Hay J, Saab S. Evaluation of the cost-effectiveness of [12] Slim J, Afridi MS. Managing adverse effects of interferon-alfa and ribavirin in entecavir versus lamivudine in hepatitis BeAg-positive chronic hepatitis B combination therapy for HCV. Infect Dis Clin North Am. 2012;26:917–929. patients. J Manag Care Pharm. 2008;14:21–33. doi: 10.18553/jmcp.2008. doi: 10.1016/j.idc.2012.08.006. 14.1.21. 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Cost-effectiveness and budget 156-4-201202210-00004. impact of hepatitis C virus treatment with sofosbuvir and ledipasvir in the [29] Chen DS, Locarnini S, Wallace J. From the big three to the big four. Lancet – United States. Ann Intern Med. 2015;162:397 406. doi: 10.7326/M14- Infect Dis. 2015;15:626–627. doi: 10.1016/S1473-3099(15)00026-2. 1336. [30] Stepanova M, Younossi ZM. Interferon-free regimens for chronic hepatitis C: [17] Najafzadeh M, Andersson K, Shrank WH, Krumme AA, Matlin OS, Brennan T, barriers due to treatment candidacy and insurance coverage. Dig Dis Sci. et al. Cost-effectiveness of novel regimens for the treatment of hepatitis 2015;60:3248–3251. doi: 10.1007/s10620-015-3709-6. C virus. Ann Intern Med. 2015;162:407–419. doi: 10.7326/M14-1152. [31] McGowan CE, Monis A, Bacon BR, Mallolas J, Goncales FL, Goulis I, et al. [18] Hagan LM, Sulkowski MS, Schinazi RF. Cost analysis of sofosbuvir/ribavirin A global view of hepatitis C: physician knowledge, opinions, and perceived versus sofosbuvir/simeprevir for genotype 1 hepatitis C virus in interferon- barriers to care. Hepatology. 2013;57:1325–1332. doi: 10.1002/hep. ineligible/intolerant individuals. Hepatology. 2014;60:37–45. doi: 10.1002/ 26246. hep.27151. [32] Afdhal NH, Zeuzem S, Schooley RT, Thomas DL, Ward JW, Litwin AH, et al. [19] Younossi ZM, Park H, Saab S, Ahmed A, Dieterich D, Gordon SC. Cost- The new paradigm of hepatitis C therapy: integration of oral therapies into effectiveness of all-oral ledipasvir/sofosbuvir regimens in patients with best practices. J Viral Hepat. 2013;20:745–760. doi: 10.1111/jvh.12173.

82 Journal of Clinical and Translational Hepatology 2016 vol. 4 | 76–82 Original Article

Postoperative Risk of Hepatic Decompensation after Orthopedic Surgery in Patients with Cirrhosis

Eric M. Nyberg1, Michael Batech2, T. Craig Cheetham2, Jose R. Pio2, Susan L. Caparosa2, Mary Alice Chocas1 and Anshuman Singh*1,3

1Department of Orthopaedics, Kaiser Permanente, San Diego, USA; 2Department of Research & Evaluation, Kaiser Permanente, Pasadena, USA; 3Department of Orthopaedics, University of California at San Diego, San Diego, USA

Abstract © 2016 The Second Affiliated Hospital of Chongqing Medical University. Published by XIA & HE Publishing Inc. All rights Background and Aims: Previous studies have shown reserved. increased hepatic decompensation in patients with cirrhosis undergoing surgery. However, there are little data available in cirrhotics undergoing orthopedic surgery compared to cir- Introduction rhotics who did not undergo surgery. The aim of this study was to examine the demographics, comorbid conditions, and clin- Chronic liver disease is a leading cause of morbidity and ical factors associated with hepatic decompensation within mortality worldwide, and in 2011, cirrhosis was the 11th most 90 days in cirrhotics who underwent orthopedic surgery. common cause of death in the United States.1 As the inci- Methods: This is a retrospective matched cohort study. Inclu- dence of chronic liver disease and cirrhosis increases, more $ sion criteria were cirrhosis diagnosis, age > 18 years, 6 patients with cirrhosis are undergoing surgery of all types. months continuous health plan membership, and a procedure Previous studies have demonstrated an elevated risk of peri- code for orthopedic surgery. Up to five cirrhotic controls with- operative morbidity and mortality in patients with cirrhosis out orthopedic surgery were matched on age, gender, and cir- who undergo nonhepatic surgery.2–6 Additional studies have rhosis diagnosis date. Data abstraction was performed for evaluated perioperative risks and complictions in cirrhotics demographics, socioeconomics, clinical, and decompensation undergoing orthopedic surgery and compared them to non- data. Chart review was performed for validation. Multivariable cirrhotic controls.7–14 A recent study by Kim et al. evaluated analysis estimated relative risk of decompensation. Results: 609 patients with chronic liver disease who underwent Eight hundred fifty-three orthopedic surgery cases in cirrhotics surgery, including 246 patients who had cirrhosis and 363 were matched with 4,263 cirrhotic controls. Among the cases patients without cirrhosis.15 The cirrhotic group had markedly and matched controls, the mean age was 60.5 years, and higher postoperative morbidity and mortality than those 52.2% were female. Within 90 days after surgery, cases had without cirrhosis. more decompensation compared to matched controls (12.8% Between 1995 and 2011, Deleuron et al. compared 363 vs 4.9%). Using multivariable analysis, orthopedic surgery, a cirrhosis patients with 109,159 noncirrhosis patients who had 0.5 g/dL decrease in serum albumin, and a 1-unit increase in undergone total hip arthroplasty (THA) or total knee arthro- Charlson Comorbidity Index were associated with a significant plasty (TKA).15 This Danish registry-based historical cohort increase in decompensation within 90 days of surgery. Diabe- study showed that cirrhosis patients who underwent THA tes, chronic obstructive pulmonary disease, and chronic or TKA for primary osteoarthritis had worse outcomes than kidney disease were seen with increased frequency in cases reference patients undergoing the same procedures. Notably, vs. matched controls. Conclusions: Cirrhotics who under- cirrhotic patients had a high risk of readmission for infection, went orthopedic surgery had a significant increase in hepatic renal failure, and liver disease. These results indicated that decompensation within 90 days of surgery compared to the increased risk applied to all cirrhosis patients and was not matched controls. An incremental decrease in serum albumin restricted to severe cases. Orozco et al. compared hepatitis and an incremental increase in the Charlson Comorbidity Index C and a nonhepatitis C groups who underwent orthopedic were significantly associated with hepatic decompensation surgery and found that hepatitis C patients without significant after surgery. fibrosis did not have an increased risk of complications. However, for TKA there was a correlation between greater Keywords: Cirrhosis; Orthopedic surgery. fibrosis and higher infection rates. Liao et al. evaluated Abbreviations: CCI, Charlson Comorbidity Index; CPT, Childs-Pugh-Turcotte; the complication rate after instrumental lumbar surgery EMR, electronic medical record; ICD-9, International Statistical Classification of between noncirrhotic patients and cirrhotic patients.14 The Diseases and Related Health Problems-9; IRB, Institutional Review Board; KPSC, rate of complications after instrumented lumbar surgery Kaiser Permanente Southern California; MELD, Model for End-Stage Liver Disease; THA, total hit arthroplasty; TKA, total knee arthroplasty. was significantly higher in patients with cirrhosis than in Received: 09 December 2015; Revised: 20 January 2016; Accepted: 24 February control patients, especially with subjects who had a Child- 2016 q Turcotte-Pugh (CPT) score of 6 or more. The dominant DOI: 10.14218/JCTH.2015.00049. postoperative complication in this study was deteriorated *Correspondence to: Anshuman Singh, Department of Orthopedics, The Garfield Specialty Center, 5893 Copley Drive, San Diego, CA 92111, USA. Tel: +1-213-359- hepatic encephalopathy, which occurred even in patients 2269, E-mail: [email protected] with stable liver disease.

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The risk of complications in cirrhotic patients after orthopedic surgery requires further evaluation. To date, the focus of research has been to compare individuals with cirrhosis to those without cirrhosis. The authors are not aware of any studies that have specifically evaluated the effect of orthopedic surgery on hepatic decompensation in cirrhotic patients compared to cirrhotic controls not undergoing orthopedic surgery. We feel that a cirrhotic control group is important since cirrhosis in itself is associated with a certain risk of decompensation due to the natural history of the disease. The aim of the present study was, therefore, to evaluate the risk of postoperative hepatic decompensation in patients with cirrhosis after undergoing orthopedic surgery compared to cirrhotic controls who did not undergo orthopedic surgery.

Methods

Study Population

This retrospective matched cohort study was conducted with Kaiser Permanente Southern California (KPSC). KPSC is an integrated health care delivery system comprising 14 hospitals, 214 outpatient clinics, and serving approximately 4 million members. All healthcare encounters were captured by a comprehensive electronic medical record (EMR) and by a thorough claims system. These data include information on patient demographics, socioeconomic status, coded diagnoses and procedures, laboratory test results, and other relevant information. The study was approved by the Fig. 1. Selection criteria for cases and controls. KPSC Institutional Review Board (IRB), and the IRB waived the requirement for patient informed consent as this was a database study without direct patient contact. The KPSC the CCI was calculated as an estimate of health status.17,18 member population is varied and reflects that of the overall The cases undergoing surgery were matched with cirrhotic Southern California population.16 controls who did not undergo surgery using a second index date defined as the date of surgery for the case. Inclusion criteria Hepatic decompensation events that occurred 90 days after the surgery index date were accessed. Hepatic decompen- Participants were included if they received a diagnosis of sation was defined as new onset or worsening of 1) ascites, cirrhosis by International Statistical Classification of Diseases 2) spontaneous bacterial peritonitis, 3) variceal bleeding, and Related Health Problems-9 (ICD-9) from 01 January 2003 and/or 4) hepatic encephalopathy. Decompensation events to 31 December 2013, were $ 18 years old, and had $ 6 were identified electronically using ICD-9 codes. months continuous health plan membership (Fig. 1). Validation of data Cases and Controls To validate the electronic algorithm and to verify decompen- Cirrhotic cases and controls had the same inclusion criteria sation events, chart review was performed on a 10% random with the exception of cirrhotic cases having undergone sample of control subjects and with all surgery subjects orthopedic surgery based on Current Procedural Terminology included in the analysis. To increase algorithm accuracy for procedure codes. Up to five cirrhotic controls not undergoing hepatic encephalopathy, codes for common medications used orthopedic surgery were matched to each case based on to treat this complication, including rifaximin and lactulose, age, gender, cirrhosis diagnosis date, and surgery date. See were included in the electronic algorithm. Additionally, tar- Figure 1 for patient disposition. geted chart review was performed to verify that lactulose and rifaximin were given for hepatic encephalopathy and Data collection not another medical reason. Further, in all cases with abdominal imaging, paracentesis, and/or upper endoscopy within Data on baseline demographics, socioeconomics, comorbid 90 days after surgery, targeted chart review was performed to conditions, lab values, Charlson Comorbidity Index (CCI), and double-check specifically for ascites, spontaneous bacterial Model for End-Stage Liver Disease (MELD) score were col- peritonitis, or variceal bleeding. lected during the pre-index period, which was defined as 6 months prior to the index surgery date (primary index Statistical analyses date). Baseline demographics included age, gender, race/ ethnicity, and neighborhood household income. Household Descriptive statistics were presented as means and range income was estimated on the basis of address using neigh- for continuous variables and frequency (percentages) for borhood income from the US Census. The Quan adaption of categorical variables. Multivariable conditional robust Poisson

84 Journal of Clinical and Translational Hepatology 2016 vol. 4 | 83–89 Nyberg E.M. et al: Hepatic decompensation after orthopedic surgery regression was used to estimate relative risk of decompensa- significant (2-fold) increase in the relative risk of developing tion. SAS Enterprise Guide 4.3 (SAS Institute Inc., Cary, NC, a decompensation event within 90 days after surgery com- USA) was used for all data analyses. pared to matched cirrhotic controls. Multivariable analysis showed that the relative risk of decompensation was signifi- Results cantly increased for each 0.5 g/dL decrease in serum albumin (from 4.0 g/dL) and for each one point increase in the CCI. Patient Characteristics Discussion A total of 853 patients with cirrhosis who underwent orthopedic surgery (cases) were matched with 4,263 cirrhotic In this study of 853 patients with cirrhosis who underwent controls. Among the cases and matched controls, the mean orthopedic surgery, the adjusted relative risk of decompen- age was 60 years, and 52% were female (Table 1). The dis- sation within 90 days after surgery was 2-fold higher than tribution among ethnicities and neighborhood household that of 4,263 patients with cirrhosis (controls) matched by income is shown in Table 1. The baseline CCI was similar age, gender, and date of cirrhosis diagnosis. The rate of overall between cases and controls, as shown in Table 1. Addi- decompensation within 90 days after surgery in cases was tionally, baseline comorbid conditions and selected laboratory 12.8% versus 4.9% in controls (Fig. 2). The baseline CCI was parameters are shown in Table 2. The cases had less similar among cases vs. controls, however, individual labora- advanced liver disease by the selected laboratory parameters tory parameters at baseline suggest that the cases were and had higher percentages of the selected comorbid selected for as having a lower severity of liver disease com- conditions. pared to controls. This is implied by a baseline lower platelet count in cirrhotic controls than in cases. Lower platelet count Rate of Complications is a commonly used surrogate marker for degree of portal hypertension in cirrhotics. Additionally, controls had lower Within 90 days after surgery, patients with cirrhosis who baseline albumin compared to cases, while bilirubin was underwent orthopedic surgery had more decompensation slightly higher among cases compared to controls. Despite events compared to matched controls (12.8% vs 4.9%) this apparent selection for healthier patients, cases had a (Fig. 2). Among the cases that had a decompensation event, 2-fold increased rate of decompensation within 90 days 55% (60/109) underwent nonelective surgery and 78% after surgery. These findings support the suggestion of Rai (85/109) underwent a procedure associated with a higher et al. that the risk versus benefit of the procedure should be volume of blood loss (78% moderate blood loss vs 22% carefully assessed and discussed in patients with concurrent low blood loss) (Table 3). liver disease who are undergoing a surgical procedure.19 Using robust Poisson regression with a decompensation Determining surgical risk in cirrhotic patients is difficult, event as a binary outcome, the crude and multivariable and multiple scoring systems have sought to quantify this adjusted relative risk estimates for decompensation events risk. Kim et al. showed that the type of surgery, CPT score, within 90 days after surgery were compared to that in a and MELD score were independently associated with post- matched cohort (Table 4). Multivariable analysis showed a operative morbidity and mortality in patients with cirrhosis.20

Table 1. Demographic characteristics of patients with cirrhosis who had orthopedic surgery matched with patients with cirrhosis who did not have orthopedic surgery, by age, gender, and date of cirrhosis diagnosis, 2003–2013, n = 5116

Orthopedic Surgery (Cases) No Orthopedic Surgery (Controls) n = 853 (16.7%) n = 4,263 (83.3%) p-value

Age (years, continuous) 0.8527 Mean (Range) 60.5 (18.0–91.0) 60.4 (18.0–93.0) Gender 0.9896 Female 445 (52.2%) 2225 (52.2%) Male 408 (47.8%) 2038 (47.8%) Race/Ethnicity White 485 (56.9%) 1888 (44.3%) Black 68 (8.0%) 421 (9.9%) Hispanic 236 (27.7%) 1444 (33.9%) Asian/Pacific Islander 53 (6.2%) 296 (6.9%) Other 11 (1.3%) 214 (5.0%) Neighborhood Household Income (Category)1 < 45,000 USD 199 (23.6%) 1185 (28.3%) 45,000 – 80,000 USD 429 (50.9%) 2017 (48.1%) > 80,000 USD 215 (25.5%) 990 (23.6%) 1 Neighborhood income is not reported income but is estimated on the basis of members’ addresses using neighborhood income from US Census tract information.

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1 Table 2. Comorbid conditions and laboratory parameters of patients with cirrhosis who had orthopedic surgery matched with patients with cirrhosis who did not have orthopedic surgery, by age, gender, and date of cirrhosis diagnosis, 2003–2013, n = 5116

Orthopedic Surgery (Cases) No Orthopedic Surgery (Controls) p-value

Charlson Comorbidity Index (categorical) 0.0966 # 1 92 (10.8%) 540 (12.8%) > 1 761 (89.2%) 3669 (87.2%) Chronic Heart Failure 0.2222 No 666 (78.1%) 3364 (79.9%) Yes 187 (21.9%) 845 (20.1%) Diabetes Mellitus 0.0083 No 483 (56.6%) 2587 (61.5%) Yes 370 (43.4%) 1622 (38.5%) COPD 0.0013 No 723 (84.8%) 3733 (88.7%) Yes 130 (15.2%) 476 (11.3%) Chronic Kidney Disease 0.0082 No 657 (77%) 3408 (81%) Yes 196 (23%) 801 (19%) MELD Score 0.0002 N 481 1427 Mean (Range) 8.5 (6–31) 9.8 (6–44) INR <0.0001 N 652 1666 Mean (Range) 1.2 (0.9–3.2) 1.3 (0.9–5.2) Serum Creatinine (mg/dL) (categorical) 0.7464 # 1.5 mg/dL 675 (87.3%) 2037 (87.8%) > 1.5 mg/dL 98 (12.7%) 284 (12.2%) Total Bilirubin (mg/dL) (categorical) 0.0249 # 2.0 mg/dL 503 (87.5%) 1603 (83.6%) > 2.0 mg/dL 72 (12.5%) 314 (16.4%) Serum Albumin (g/dL) (categorical) 0.0001 > 3.5 g/dL 266 (56.2%) 683 (46.0%) < 3.5 g/dL 207 (43.8%) 802 (54.0%) Platelet Count (1000/mL) (categorical) 0.0005 $ 75 3 1000/mL 727 (89.2%) 1901 (84.2%) <753 1000/mL 88 (10.8%) 357 (15.8%) 1 The closest lab measured within 180 days of the cases’ surgery date was used.

The present study shows that the MELD score and platelet to analyze outcomes on postoperative morbidity and mortal- count trend toward statistical significance as a predictor of ity using CTP, MELD, and MELD-sodium (MELD-Na) scores.21 the risk of decompensation within 90 days of orthopedic Kim et al. utilized CTP and MELD-based indices to compare surgery. Due to the retrospective design of the present the abilities of liver indices to predict mortality for patients study, one or more parameters included in the MELD score with liver cirrhosis who underwent elective surgery.20 The were frequently missing, therefore, a trend toward signifi- authors concluded that a large-scale study is needed to vali- cance is notable. Further, multivariable analysis showed that date how well liver indices that assess the severity of hepatic the relative risk of decompensation increased significantly decompensation predict perioperative adverse events.20 The with a corresponding incremental change in serum albumin present study strengthens the indication that these scoring and CCI (Table 4). systems are good predictors of perioperative risk. Further, Causey et al. performed a single-center retrospective this applies in cirrhotics undergoing orthopedic surgery com- review of 64 cirrhotic patients who underwent nontransplant pared to cirrhotics not undergoing orthopedic surgery, sug- surgery under general anesthesia over a 6-year period of time gesting that events in the perioperative period precipitate

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Fig. 2. Decompensation within 90 days after surgery among patients with cirrhosis who had orthopedic surgery matched with patients with cirrhosis who did not have orthopedic surgery, by age, gender, and date of cirrhosis diagnosis, 2003–2013. hepatic decompensation at a significantly higher rate than score of 10 or greater had the highest risk of complications. that occurring due to the natural history of cirrhosis alone. Using cirrhotics instead of healthy controls, our study demon- As the population in general is becoming more obese, it is strates that exposure to orthopedic surgery is an independent expected that an increasing number of patients with obesity- risk factor for decompensation in this population. related cirrhosis will undergo elective surgery. A study by Hsieh et al. have demonstrated an association between Tiberi et al. compared 115 patients with cirrhosis and matched volume of blood loss and urgency of surgery and postoper- controls without cirrhosis who underwent THA or TKA from ative morbidity and mortality.7 Similarly, we showed that a 2000 to 2012.11 The authors concluded that patients with higher percentage of cases with a decompensation event cirrhosis undergoing THA or TKA are at increased risk for had urgent surgery and underwent a procedure associated medical and surgical complications. Patients with cirrhosis with higher blood loss (Table 3). had longer hospital stays, more early post-operative hospital To our knowledge, this is the first matched cohort study to readmissions, and more frequent discharges to a skilled evaluate the rate of hepatic decompensation in cirrhotic nursing or short-term rehabilitation facility compared with patients undergoing orthopedic surgery compared to cirrhotic controls. Within 90 days of the procedure, the cirrhotic group patients not undergoing surgery. A strength of this study had more hip dislocations, infections, and revision surgeries. is that it reflects a “real world” population including a large In addition, the study found a correlation between increasing number of patients matched with up to five controls. The MELD scores and increased rates of complications; a MELD ethnic and socioeconomic distribution of the study population

Table 3. Association of surgical urgency and procedural blood loss with decompensation in 90 days (n = 863)

No decompensation in Decompensation in 90 days, n = 744 (87.2%) 90 days, n = 109 (12.8%)

Elective procedure status Non-elective 230 (30.9%) 60 (55.0%) Elective 514 (69.1%) 49 (45.0%) Procedure blood loss Low loss 349 (46.9%) 24 (22.0%) Moderate loss 395 (53.1%) 85 (78.0%)

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Table 4. Crude and multivariable adjusted relative risk estimates for decompensation within 90 days of surgery (using robust Poisson regression with decompensation event as a binary outcome)

Multivariable Crude adjusted

Orthopedic surgery No surgery Reference Reference Had a surgery 2.57 (2.05, 3.21) 2.05 (1.60, 2.62) Age (5-year increase) 1.08 (1.03, 1.13) 0.98 (0.93, 1.04) Gender Female Reference Reference Male 1.30 (1.02, 1.65) 1.02 (0.78, 1.32) Race/Ethnicity White Reference Reference Black 0.98 (0.67, 1.44) 0.76 (0.46, 1.24) Hispanic 0.96 (0.75, 1.23) 1.01 (0.76, 1.34) Asian/Pacific Islander 1.07 (0.72, 1.61) 1.23 (0.82, 1.85) Neighborhood household income < 45,000 USD 1.07 (0.82, 1.38) 0.92 (0.68, 1.25) 45,000 – 80,000 USD Reference Reference $ 80,000 USD 1.03 (0.78, 1.36) 1.08 (0.80, 1.47) MELD score (5-unit increase) 1.28 (1.21, 1.35) 1.06 (0.99, 1.14) Serum albumin albumin (0.5-g/dL decrease) 1.59 (1.49, 1.70) 1.49 (1.37, 1.62) Platelet count (25-unit decrease) 1.14 (1.09, 1.19) 1.03 (0.99, 1.07) Charlson Comorbidity Index (1-unit increase) 1.22 (1.17, 1.27) 1.11 (1.06, 1.17)

is consistent with that of the general population of Southern Conclusions California.16 Further, the study was conducted within a large healthcare delivery system with an EMR allowing comprehen- In summary, this study demonstrates a significant (2-fold) sive evaluation of the medical care provided to patients, increase in the risk of hepatic decompensation in patients minimal ingress and egress, and thorough manual chart with cirrhosis within 90 days of undergoing orthopedic review was performed to validate the electronic algorithm surgery compared to cirrhotics not undergoing orthopedic surgery. This finding reinforces the need for perioperative and to confirm decompensation. optimization of patients with cirrhosis and supports close We matched cases with controls by age, gender, and monitoring of cirrhotic patients in the months following cirrhosis diagnosis date; however, a limitation of the study is orthopedic surgery. that we were not able to match cases and controls by markers of severity of liver disease. This was not possible due to Acknowledgments incomplete clinical data for matching all criteria within an appropriate time period defined as 180 days. Additionally, we This publication was made possible by a Kaiser Permanente were unable to determine baseline CPT score and ASA score Southern California Regional Research Committee grant, due to the nature of the study. Further limitations include which is partially supported by the Southern California Per- drawbacks that are characteristic of retrospective studies, manente Medical Group Research and Evaluation Department and those limitations inherent in using an electronic algorithm and Direct Community Benefit Investment funds. for data abstraction. These limitations include a risk of not identifying certain factors due to deficiencies in coding Conflict of interest although an extensive chart review was performed, as noted above. None

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Author contributions [10] Moon YW, Kim YS, Kwon SY, Kim SY, Lim SJ, Park YS. Perioperative risk of hip arthroplasty in patients with cirrhotic liver disease. J Korean Med Sci 2007; 22:223–226. doi: 10.3346/jkms.2007.22.2.223 Conception and design, interpretation of data, write-up, and [11] Tiberi JV 3rd, Hansen V, El-Abbadi N, Bedair H. Increased complication rates preparation of manuscript (EMN), study design, analysis after hip and knee arthroplasty in patients with cirrhosis of the liver. Clin and interpretation of data, write-up, and critical review of Orthop Relat Res 2014;472:2774–2778. doi: 10.1007/s11999-014-3681-z manuscript (MB), conception and design, interpretation of [12] Deleuran T, Vilstrup H, Overgaard S, Jepsen P. Cirrhosis patients have data, write-up, and critical review of manuscript (TCC), data increased risk of complications after hip or knee arthroplasty. Acta Orthop 2015;86:108–113. doi: 10.3109/17453674.2014.961397 collection, write-up, and critical review of manuscript (JRP), [13] Orozco F, Post ZD, Baxi O, Miller A, Ong A. Fibrosis in hepatitis C patients data collection, write-up, and critical review of manuscript predicts complications after elective total joint arthroplasty. J Arthroplasty (MAC), data collection, write-up, and critical review of manu- 2014;29:7–10. doi: 10.1016/j.arth.2013.03.023 script (SLC), conception and design, interpretation of data, [14] Liao JC, Chen WJ, Chen LH, Niu CC, Fu TS, Lai PL, et al. Complications asso- write-up, and critical review of manuscript (AS). ciated with instrumented lumbar surgery in patients with liver cirrhosis: a matched cohort analysis. Spine J 2013;13:908–913. doi: 10.1016/j.spinee. 2013.02.028 [15] Kim TH, Um SH, Yim SY, Seo YS, Yim HJ, Jeen YT, et al. The risk of perioper- References ative adverse events in patients with chronic liver disease. Liver Int 2015; 35:713–723. doi: 10.1111/liv.12529 – [1] Heron M. Deaths: leading causes for 2011. Natl Vital Stat Rep 2015;64:1 96. [16] Koebnick C, Langer-Gould AM, Gould MK, Chao CR, Iyer RL, Smith N, et al. [2] Pandey CK, Karna ST, Pandey VK, Tandon M, Singhal A, Mangla V. Perioper- Sociodemographic characteristics of members of a large, integrated health ative risk factors in patients with liver disease undergoing non-hepatic care system: comparison with US Census Bureau data. Perm J 2012;16: surgery. World J Gastrointest Surg 2012;4:267–274. doi: 10.4240/wjgs. 37–41. doi: 10.7812/TPP/12-031 v4.i12.267 [17] Romano PS, Roos LL, Jollis JG. Adapting a clinical comorbidity index for [3] Muir AJ. Surgical clearance for the patient with chronic liver disease. Clin use with ICD-9-CM administrative data: differing perspectives. J Clin Epide- Liver Dis 2012;16:421–433. doi: 10.1016/j.cld.2012.03.008 miol 1993;46:1075–1079; discussion 1081-1090 doi: 10.1016/0895-4356 [4] Nicoll A. Surgical risk in patients with cirrhosis. J Gastroenterol Hepatol 2012; (93)90103-8. 27:1569–1575. doi: 10.1111/j.1440-1746.2012.07205.x [18] Quan H, Sundararajan V, Halfon P, Fong A, Burnand B, Luthi JC, et al. Coding [5] Friedman LS. Surgery in the patient with liver disease. Trans Am Clin Climatol algorithms for defining comorbidities in ICD-9-CM and ICD-10 administrative Assoc 2010;121:192–204; discussion 205. – [6] Csikesz NG, Nguyen LN, Tseng JF, Shah SA. Nationwide volume and mortality data. Med Care 2005;43:1130 1139. doi: 10.1097/01.mlr.0000182534. after elective surgery in cirrhotic patients. J Am Coll Surg 2009;208:96–103. 19832.83 doi: 10.1016/j.jamcollsurg.2008.09.006 [19] Rai R, Nagral S, Nagral A. Surgery in a patient with liver disease. J Clin Exp – [7] Hsieh PH, Chen LH, Lee MS, Chen CH, Yang WE, Shih CH. Hip arthroplasty in Hepatol 2012;2:238 246. doi: 10.1016/j.jceh.2012.05.003 patients with cirrhosis of the liver. J Bone Joint Surg Br 2003;85:818–821. [20] Kim DH, Kim SH, Kim KS, Lee WJ, Kim NK, Noh SH, et al. Predictors of [8] Shih LY, Cheng CY, Chang CH, Hsu KY, Hsu RW, Shih HN. Total knee arthro- mortality in cirrhotic patients undergoing extrahepatic surgery: comparison plasty in patients with liver cirrhosis. J Bone Joint Surg Am 2004;86-A: of Child-Turcotte-Pugh and model for end-stage liver disease-based indices. 335–341. ANZ J Surg 2014;84:832–836. doi: 10.1111/ans.12198 [9] Cohen SM, Te HS, Levitsky J. Operative risk of total hip and knee arthroplasty [21] Causey MW, Steele SR, Farris Z, Lyle DS, Beitler AL. An assessment of differ- in cirrhotic patients. J Arthroplasty 2005;20:460–466. doi: 10.1016/j.arth. ent scoring systems in cirrhotic patients undergoing nontransplant surgery. 2004.05.004 Am J Surg 2012;203:589–593. doi: 10.1016/j.amjsurg.2012.01.009

Journal of Clinical and Translational Hepatology 2016 vol. 4 | 83–89 89 Original Article

Effects of Granulocyte Colony-Stimulating Factor on Patients with Liver Failure: a Meta-Analysis

Qiao Yang*1, Ying Yang2, Yu Shi2, Fangfang Lv1, Jiliang He3 and Zhi Chen2

1Department of Infectious Diseases, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, China; 2State Key Lab of Diagnosis and Treatment of Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Disease, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China; 3Institute of Environmental Medicine, Medical College, Zhejiang University, Hangzhou, Zhejiang, China

Abstract Introduction

Background and Aims: It remains controversial whether Liver failure (LF) is a syndrome characterized by severe granulocyte colony-stimulating factor (G-CSF) prolongs sur- hepatic decompensation, resulting in jaundice, infection, vival in liver failure (LF) patients. This meta-analysis was per- hepatic encephalopathy, hepatorenal syndrome, and multi- formed to evaluate the effect of G-CSF on patients with LF. system organ failure.1 The costs associated with intensive Methods: PubMed, EMBASE, and Web of Science databases care and short-term mortality in LF patients have remained were searched to identify English language randomized con- quite high over the past few decades.2 There is, therefore, an trolled trials comparing G-CSF with control therapy published urgent need for more effective adjuvant therapies against LF. before14 February 2015. A meta-analysis was performed to Granulocyte colony-stimulating factor (G-CSF) is a growth examine changes in liver function and patient survival. The factor that stimulates bone marrow to produce granulocytes association was tested using odds ratio (OR) or risk ratio and stem cells and release them into the circulation.3 A sig- (RR) with 95% confidence intervals (CI). Results: Five nificant body of observational and randomized data have randomized controlled trials were eligible for the meta-analy- indicated a potential role of G-CSF for end-stage liver dis- sis. Significant amelioration of prothrombin time and total bi- eases.4–7 Nevertheless, it remains uncertain whether G-CSF lirubin in LF patients was attributed to G-CSF therapy (OR, use can influence the prognosis for established patients with −0.064; 95% CI,−0.481 to 0.353; p< 0.001; and OR, LF. Recently, several studies have shown that G-CSF mobilized −0.803; 95% CI, −1.177 to −0.430; p = 0.000, respectively). CD34+ hematopoietic stem cells and improved liver regener- Treatment with G-CSF resulted in improved Model for End- ation in LF patients.8–12 Although the optimal time, dose, and Stage Liver Disease and Child-Turcotte-Pugh scores (OR, duration of G-CSF treatment and which type of patients with −1.741; 95% CI, −2.234 to −1.250; p = 0.000; and OR, LF are most likely to benefit from G-CSF treatment are unclear, −0.830, 95% CI, −1.194 to −0.465; p = 0.000, respectively). data suggest that G-CSF may be a promising adjuvant therapy A lower incidence of sepsis was found in patients treated with G- for patients with LF. In the present meta-analysis, the benefit CSF (RR, 0.367; 95% CI, 0.158 to 0.854; p = 0.020). G-CSF of G-CSF use for patients with LF was assessed, providing therapy significantly increased survival rate in LF patients (RR, evidence for the therapeutic use of G-CSF in patients with LF. 2.25; 95% CI, 1.517 to 3.338; p = 0.000). Conclusions:The results of this meta-analysis indicate that G-CSF treatment in Methods patients with LF significantly improved liver function, reduced the incidence of sepsis, and prolonged short-term survival. Search strategy and selection of the papers © 2016 The Second Affiliated Hospital of Chongqing Medical University. Published by XIA & HE Publishing Inc. All rights Systematic searches of PubMed, Embase, and Cochrane reserved. databases for articles published before February 2015 were performed for possible publications. Reports cited in the references and relevant reviews were also searched, which included potentially missed studies. The following terms were used in the search procedure: (‘liver failure’ or ‘hepatic failure’ or ‘fulminant hepatitis’ or ‘severe hepatitis’ or ‘end-stage liver disease’) and (‘Granulocyte colony-stimulating factor’ or Keywords: Granulocyte colony-stimulating factor; Liver failure; Liver regeneration; ‘G-CSF’). The retrieved studies were carefully examined to Stem cells. exclude potential duplicates or overlapping data. Titles and Abbreviations: CI, confidence interval; CTP, Child-Turcotte-Pugh; G-CSF, granulo- abstracts selected from the initial search were first scanned, cyte colony stimulating factor; LF, liver failure; MELD, Model for End-Stage Liver Disease; OR, odds ratio: RR, risk ratio. and the full papers of potential eligible studies were reviewed. Received: 28 March 2016; Revised: 23 April 2016; Accepted: 24 April 2016 Eligibility of the studies was assessed independently by q DOI: 10.14218/JCTH.2016.00012. two investigators. The inclusion criteria of studies were the *Correspondence to: Qiao Yang, Department of Infectious Diseases, Sir Run Run following: the outcomes of interest were liver function, clinical Shaw Hospital, School of Medicine, Zhejiang University, Block 2 1st floor, No. 3 Qingchun East Road, Hangzhou, Zhejiang 310016, China. Tel: +86-571- severity indices, the survival of liver or hepatic failure, and 86006229, Fax: +86-571-86044817, E-mail: [email protected] fulminant hepatitis or end-stage liver disease; the study of

90 Journal of Clinical and Translational Hepatology 2016 vol. 4 | 90–96

Fig. 1. Flow diagram of the selection process. Five studies assessed G-CSF use and outcomes in LF. Abbreviations: G-CSF, granulocyte colony-stimulating factor; LF, liver failure.

interest was G-CSF usage, with information available on the and reporting of patient withdrawal and dropout. Studies with odds ratio (OR) or risk ratio (RR) estimates with 95% con- scores more than 4 were defined as high-quality. fidence intervals (CIs). Only studies published in English were included. Statistical methods analysis

Data extraction and quality assessment ORs were calculated to assess the advantage of G-CSF compared to control therapy. Rate ratios were calculated to Data were extracted independently by two authors, and any show how many times higher the probability of death or the discrepancies were resolved by discussion among the incidence of sepsis was in patients receiving G-CSF compared authors. The following information was obtained from each to those receiving controls therapies. RR with 95% CIs was trial: publication details (title, the first author, origin of the calculated using a fixed- or random-effects model. Hetero- study), study design, participants’ details (the numbers of geneity between individual studies was assessed by I2 test, patients enrolled, participant demographics), the intervention where a value greater than 50% was indicative of substantial details, the duration of follow-up, and primary outcomes heterogeneity. A random-effects model was used when the (liver function, clinical severity indices, survival, and side heterogeneity was significant, and a fixed-effects model was effects). applied otherwise. Because of the limitations of test power Quality assessment of the included studies was conducted when less than 10 studies are included, further statistical by two authors using the Jadad quality scale.13 Each study tests were not conducted. A p value < 0.05 was considered was evaluated by examining the allocation sequence gener- statistically significant. STATA 11.0 software was used in all ation, allocation concealment, blinding of outcome assessors, analyses (Stat Corporation, College Station, Texas, USA).

Journal of Clinical and Translational Hepatology 2016 vol. 4 | 90–96 91 2Junlo lncladTasainlHepatology Translational and Clinical of Journal 92

Table 1. Characteristics of included studies

Source and Study Number Age (mean 6 SD Sex Jadad Study years Country design Blinding Duration of patients or median) (M:F) Treatment Survival scores

1 Duan et al. 2013 China RCT Double 3 mon 27 43.5 (29–63) 22:5 G-CSF+SMT 13 2 28 45.9 (22–65) 22:6 SMT 6 2 Garg et al. 2012 India RCT Double 2 mon 23 40 (30–65) 20:3 G-CSF+SMT 16 3 24 40 (19–55) 21:3 Placebo+SMT 7 3 Xing et al. 2013 China Cohort NA 6 mon 21 48.19 6 9.74 16:5 G-CSF 5 1 23 47.86 6 9.74 19:4 6 4 Singh et al. 2014 India RCT Open- 3 mon 23 41.7 6 7.5 23:0 G-CSF+SMT 18 3 label 23 44.3 6 13 23:0 SMT 5 5 Nancy UK Cohort Open- 4 day 6 32.6 (24–54) 3:3 G-CSF – 2 Rolando et al. 2000 label 8 31.3 (25–41) 2:6 – 2016 Abbreviations: G-CSF, granulocyte colony stimulating factor; RCT, randomized controlled trial; SMT, standard medical treatment. ioY. Qiao o.4|90 | 4 vol. tal et – -S hrp o ie failure liver for therapy G-CSF : 96 Qiao Y. et al: G-CSF therapy for liver failure

Fig. 2. Meta-analysis of the neutrophils and CD34+ cell counts in peripheral blood between G-CSF and control therapy. (A) Neutrophil counts in peripheral blood. (B) CD34+ cell counts in peripheral blood. Abbreviation: G-CSF, granulocyte colony-stimulating factor.

Results dosage of 5 mg/kg per day for 6 consecutive days; peripheral neutrophil counts were quantified twice a week for 2 weeks. The data were extracted at the peak level. In the other Description of the included studies study,12 patients were assigned to receive G-CSF at consecutive doses, and data on a safe and effective dose at 96 h Our systematic literature search identified 731 articles for were extracted. The pooled analysis represented an OR of eligibility, of which 21 were potentially relevant reports after 1.619 (95% CI 1.062 to 2.175, p = 0.000) with significant reviewing titles and abstracts. Among the remaining 21 full- heterogeneity (I2 = 76%, p= 0.041) (Fig. 2A). Three text articles, nine articles were excluded because the data studies8,9,11 evaluated CD34+ cells in peripheral blood; did not involve the potential benefit of G-CSF in LF patients, Garg et al.9 was excluded, as the required data were unavail- three articles were excluded because they were not original able. The estimated pooled OR value showed that CD34+ cells articles, three articles were excluded because of an absence were significantly increased in the peripheral blood of of acquired data, and one article was excluded because patients receiving G-CSF therapy (OR = 1.273, 95% CI the same authors published overlapping data on the same 0.836 to 1.710, p = 0.000), with significant heterogeneity patients. For those patients, clinical data was extracted by between the studies (I2 =85.6%,p = 0.008) (Fig. 2B). integrating the overlapping articles. In total, five articles met the inclusion criteria for the final meta-analysis (Fig. 1).8–12 Five clinical trials involving the potential effects of G-CSF in Amelioration in liver function parameters LF patients met the inclusion criteria, and the characteristics of these studies are shown in Table 1. Among these trials, two Data on liver function parameters were available in three were conducted in India,9,11 two were conducted in China,8,10 trials comparing G-CSF with control therapy.10,11 No signifi- and one was conducted in the UK.12 Three of the studies were cant difference was found in prothrombin time (PT) between randomized controlled trials (RCTs),8,9,11 and two were pro- G-CSF and control therapy groups (OR = −0.064, 95% spective cohort studies.10,12 One study only enrolled patients CI −0.481 to 0.353, p = 0.764). No substantial heterogeneity with severe alcoholic hepatitis,11 one study admitted patients was detected (I2 = 61.4%, p = 0.107) (Fig. 3A). For serum – with LF due to acetaminophen overdose,12 two studies levels of total bilirubin (TBIL),9 11 the pooled OR value was included only patients with hepatitis B virus (HBV)-associated significantly reduced in patients receiving G-CSF (OR = LF,8,10 and the remaining study included LF patients with −0.803, 95% CI −1.177 to −0.430, p = 0.000), with signifi- various etiologies.9 cant heterogeneity across studies (I2 = 94.0%, p = 0.000) (Fig. 3B). Mobilization of neutrophils and CD34+ cells in peripheral blood Improvement in clinical severity indices

Two of the studies assessed neutrophil counts after G-CSF The scores of Model for End-Stage Liver Disease (MELD) in administration.8,12 In one study,8 patients in the experimen- LF patients for meta-analysis were available from two pub- tal groups were administered G-CSF subcutaneously at the lications.8,9 MELD scores were significantly improved in G-CSF

Fig. 3. Meta-analysis of the levels of prothrombin time and total bilirubin between G-CSF and control therapy. (A) The levels of prothrombin time. (B) The level of total bilirubin. Abbreviation: G-CSF, granulocyte colony-stimulating factor.

Journal of Clinical and Translational Hepatology 2016 vol. 4 | 90–96 93 Qiao Y. et al: G-CSF therapy for liver failure

Fig. 4. Meta-analysis of MELD and CTP scores between G-CSF and control therapy. Abbreviations: CTP, Child-Turcotte-Pugh; MELD, Model for End-Stage Liver Disease; G-CSF, granulocyte colony-stimulating factor. groups compared to control groups (pooled OR= −1.741, Safety of G-CSF in LF patients 95% CI −2.234 to −1.250, p = 0.000), with significant heterogeneity between studies (I2 = 96.1%, p = 0.000) (Fig. 4A). No significant adverse reactions were reported in these The results of Child-Turcotte-Pugh (CTP) scores for the meta- studies. One patient had a high fever, and that dose of G-CSF analysis were obtained from three trials.8,9,11 The estimated was omitted and did not disturb the therapy.9 pooled OR value showed that CTP scores were significantly decreased in G-CSF groups compared to the control groups Publication bias (pooled OR= −0.830, 95% CI −1.194 to −0.465, p = 0.000) (Fig. 4B). A significant heterogeneity was found among these There was no evidence of publication bias in this study. 2 studies (I = 95.3%, p = 0.000). However, because of the limited number of included studies, it is difficult to confirm whether publication bias existed in the Reduction in the occurrence of sepsis current meta-analysis.

The results of analyses for the association between G-CSF Discussion use and the occurrence of sepsis are shown in Figure 5.8,9 The results demonstrated that the incidence of sepsis was This meta-analysis summarized the results of five studies on lower in patients treated with G-CSF than with standard the clinical benefit of G-CSF in patients with LF. The results medical treatment (SMT) (RR = 0.367, 95% CI 0.158 to indicated that use of G-CSF improves liver function, alleviates 0.854, p = 0.020). No significant heterogeneity was found clinical severity, and increases survival of LF due to a variety among the studies (I2 = 0.0%, p = 0.686). of causes. LF is a life-threatening medical syndrome with high short- Prolongation in survival term mortality. Liver transplantation remains the most effective therapy for the disease, but the shortage of donor organs In four studies, the association between short-term survival limits its availability in most patients with LF;14 hence, it is benefit and G-CSF use in LF patients with various etiology was – imperative to develop novel therapeutic strategies for LF. assessed.8 11 In total, 42 of 94 G-CSF users died from multi- Promising results have been reported with G-CSF therapy organ failure, whereas 74 of 98 G-CSF non-users died. The in several high-quality clinical trials involving patients with pooled analysis of the survival benefit represented an RR various liver diseases.15–22 G-CSF mobilizes bone marrow of 2.25 (95% CI 1.517 to 3.338, p = 0.000), with no substan- hematopoietic stem cells to aid liver regeneration, and it is tial heterogeneity across studies (I2 = 30.1%, p = 0.232). After excluding the study that applied case-control analysis of a cohort study,10 the RR increased even further for patients with G-CSF therapy (RR = 2.682, 95% CI 1.732 to 4.151, p = 0.000), indicating no heterogeneity among studies (I2 = 0.0%, p = 0.666) (Fig.6).

Fig. 5. Meta-analysis of the occurrence of sepsis between G-CSF and Fig. 6. Meta-analysis of the association between G-CSF use and liver control therapy. failure mortality.

94 Journal of Clinical and Translational Hepatology 2016 vol. 4 | 90–96 Qiao Y. et al: G-CSF therapy for liver failure most likely via this mechanism that G-CSF improves survival belonged to UK) and, therefore, the conclusion may not be in acute and chronic LF.23–24 As suggested by previous generalized to other population. studies, bone marrow-derived stems cells, together with hep- In conclusion, the meta-analysis comprehensively analyzed atocytes and intrahepatic stem cells, may represent the third the data on the benefit of G-CSF therapy for patients with LF, proliferative component of the liver regenerative process.5 In and the results suggested that G-CSF use in collaboration with our review, we provided evidence that peripheral CD34+ cells SMT may improve LF survival. These findings have consider- in G-CSF groups were elevated significantly, which may also able importance in clinical practice since the mortality of LF is contribute to liver regeneration. In addition, G-CSF exerts high and the availability of liver transplantation is not widely autocrine and paracrine effects to promote the oval cell reac- attainable. Future studies, especially large size, controlled tion in the liver, cooperatively improving the liver function.25 longitudinal follow-up studies are necessary to strengthen In addition, G-CSF modulates innate and adaptive immune the evidence supporting the use of G-CSF as an adjuvant response in LF patients. Several studies in vitro and in vivo therapy for LF survival. have documented that G-CSF improved neutrophil function and decreased episodes of sepsis and sepsis-related death Acknowledgments in LF patients.8,9,21 Our analysis also showed that peripheral neutrophil counts were higher and the incidence of sepsis was This study was supported by the National Natural Science lower in G-CSF treated groups. Importantly, G-CSF therapy Foundation of China (81400622), the 12–5 State S&T Proj- increased the frequencies of dendritic cells and reduced ects of China (2012ZX10002007) and the Public Technology IFN-g secreting CD8+ T cells, thus reducing immune-mediated Research and social development project of Zhejiang Prov- liver injury.26 ince China (2014C33177). Furthermore, we comprehensively analyzed in this meta- analysis the clinical severity indices of LF, and the results Conflict of interest suggested that G-CSF therapy may result in progressive improvement in MELD and CTP scores at 2 to 3 months. In None addition, patients with G-CSF therapy achieved significantly lower short term mortality compared with standard medical Author contributions treatment only. However, there were some confusing matters left unre- Conceived and designed the meta-analysis (QY, FL, ZC), solved, including the types of LF patients who responded well analyzed the data (QY, YS, YY), wrote the manuscript to G-CSF therapy and the optimal dose and duration of G-CSF (QY, YY), revised the manuscript (QY, JH), checked the manu- adjuvant therapy. Meanwhile, attention should be given to script (FL, ZC). the safety of G-CSF administration to induce bone marrow derived cell mobilization in patients with LF. Although G-CSF therapy was shown to be well tolerated in these studies, it is References important to identify the optimal dose and duration of G-CSF that would achieve desirable hepatoprotective effects and [1] Jalan R, Gines P, Olson JC, Mookerjee RP, Moreau R, Garcia-Tsao G, et al. Acute-on chronic liver failure. J Hepatol 2012;57:1336–1348. doi: 10. the fewest adverse effects. Most of the included studies 1016/j.jhep.2012.06.026. adopted the dosage of 5 mg/kg$d for 5 consecutive days. [2] Olson JC, Wendon JA, Kramer DJ, Arroyo V, Jalan R, Garcia-Tsao G, et al. One of the studies included in this meta-analysis investigated Intensive care of the patient with cirrhosis. Hepatology 2011;54:1864– 1872. doi: 10.1002/hep.24622. the dose-response effect of G-CSF on patients with ALF due to [3] Gianni AM, Siena S, Bregni M, Tarella C, Stern AC, Pileri A, et al. Granulocyte- 12 acetaminophen overdose and found that G-CSF at the dose macrophage colony-stimulating factor to harvest circulating haemopoietic of 50 mg/m2 led to augmented neutrophil counts, which was stem cells for autotransplantation. Lancet 1989;2:580–585. doi: 10.1016/ similar to those achieved at higher doses, and fewer side S0140-6736(89)90711-3. [4] Theocharis SE, Papadimitriou LJ, Retsou ZP, Margeli AP, Ninos SS, Papadimi- effects as compared to higher doses of therapy. Because of triou JD. Granulocyte-colony stimulating factor administration ameliorates the limited data, dose-response analysis for the association liver regeneration in animal model of fulminant hepatic failure and encephal- between G-CSF use and LF survival could not be conducted opathy. Dig Dis Sci 2003;48:1797–1803. doi: 10.1023/A:1025463532521. [5] Gaia S, Smedile A, Omedè P, Olivero A, Sanavio F, Balzola F, et al. Feasibility and the optimal duration of G-CSF in LF is still unclear. and safety of G-CSF administration to induce bone marrow-derived cells The present meta-analysis is restricted by several limita- mobilization in patients with end stage liver disease. J Hepatol 2006;45: tions. First, the number of studies involved in the meta- 13–19. doi: 10.1016/j.jhep.2006.02.018. analysis was small, and thus, the reliability of some of the [6] Spahr L, Lambert JF, Rubbia-Brandt L, Chalandon Y, Frossard JL, Giostra E, et al. Granulocyte-colony stimulating factor induces proliferation of hepatic subgroup analyses might be less than if the number of studies progenitors in alcoholic steatohepatitis: a randomized trial. Hepatology was greater. Second, the etiology of the underlying LF did not 2008;48:221–229. doi: 10.1002/hep.22317. achieve homogeneity. Most of the etiologies in the analysis [7] Lorenzini S, Isidori A, Catani L, Gramenzi A, Talarico S, Bonifazi F, et al. Stem were alcohol, hepatitis B, and acetaminophen overdose. Due cell mobilization and collection in patients with liver cirrhosis. Aliment Phar- macol Ther 2008;27:932–939. doi: 10.1111/j.1365-2036.2008.03670.x. to the inadequate number of available studies, the subgroup [8] Duan XZ, Liu FF, Tong JJ, Yang HZ, Chen J, Liu XY, et al. Granulocyte-colony analyses based on different etiology were not conducted. stimulating factor therapy improves survival in patients with hepatitis B Thirdly, the dose and duration of G-CSF usage differed among virus-associated acute-on-chronic liver failure. World J Gastroenterol 2013; 19:1104–1110. doi: 10.3748/wjg.v19.i7.1104. different studies, and some data were not available from each [9] Garg V, Garg H, Khan A, Trehanpati N, Kumar A, Sharma BC, et al. Granulo- study, which may cause less accurate estimates of the benefit cyte colony-stimulating factor mobilizes CD34(+) cells and improves survival of G-CSF therapy. Another drawback of the meta-analysis is of patients with acute-on-chronic liver failure. Gastroenterology 2012;142: – the regional restriction of the included studies. The studies 505 512.e1. doi: 10.1053/j.gastro.2011.11.027. [10] Xing TJ, Xu HT, Xian JC, Shen ML, Li H, Ye J, et al. Mechanism and efficacy of were conducted in Asia and Europe (two studies belonged to mobilization of granulocyte colony-stimulating factor in the treatment of the mainland of China, two belonged to India, and one chronic hepatic failure. Hepatogastroenterology 2013;60:170–175.

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[11] Singh V, Sharma AK, Narasimhan RL, Bhalla A, Sharma N, Sharma R. Gran- HCV-positive patients with end-stage liver disease. Stem Cell Res Ther 2014; ulocyte colony-stimulating factor in severe alcoholic hepatitis: a randomized 5:70. doi: 10.1186/scrt459. pilot study. Am J Gastroenterol 2014;109:1417–1423. doi: 10.1038/ajg. [20] Wan Z, You S, Rong Y, Zhu B, Zhang A, Zang H, et al. CD34+ hematopoietic 2014.154. stem cells mobilization, paralleled with multiple cytokines elevated in [12] Rolando N, Clapperton M, Wade J, Wendon J. Administering granulocyte patients with HBV-related acute-on-chronic liver failure. Dig Dis Sci 2013; colony-stimulating factor to acute liver failure patients corrects neutrophil 58:448–457. doi: 10.1007/s10620-012-2458-z. – defects. Eur J Gastroenterol Hepatol 2000;12:1323 1328. doi: 10.1097/ [21] Rolando N, Clapperton M, Wade J, Panetsos G, Mufti G, Williams R. Granulo- 00042737-200012120-00010. cyte colony-stimulating factor improves function of neutrophils from patients [13] Jadad AR, Moore RA, Carroll D, Jenkinson C, Reynolds DJ, Gavaghan DJ, et al. with acute liver failure. Eur J Gastroenterol Hepatol 2000;12:1135–1140. Assessing the quality of reports of randomized clinical trials: is blinding doi: 10.1097/00042737-200012100-00011. necessary? Control Clin Trials 1996;17:1–12. doi: 10.1016/0197-2456(95) [22] Han Y, Yan L, Han G, Zhou X, Hong L, Yin Z, et al. Controlled trials in hepatitis B 00134-4. [14] Schiano TD, Kim-Schluger L, Gondolesi G, Miller CM. Adult living donor liver virus-related decompensate liver cirrhosis: peripheral blood monocyte trans- transplantation: the hepatologist’s perspective. Hepatology 2001;33:3–9. plant versus granulocyte-colony-stimulating factor mobilization therapy. doi: 10.1053/jhep.2001.21489. Cytotherapy 2008;10:390–396. doi: 10.1080/14653240802129901. [15] Piscaglia AC, Arena V, Passalacqua S, Gasbarrini A. A case of granulocyte- [23] Yannaki E, Athanasiou E, Xagorari A, Constantinou V, Batsis I, Kaloyannidis P, colony stimulating factor/plasmapheresis-induced activation of granulocyte- et al. G-CSF-primed hematopoietic stem cells or G-CSF per se accelerate colony stimulating factor-positive hepatic progenitors in acute-on-chronic recovery and improve survival after liver injury, predominantly by promoting liver failure. Hepatology 2015;62:649–652. doi: 10.1002/hep.27708. endogenous repair programs. Exp Hematol 2005;33:108–119. doi: 10. [16] Gurakar A, Fagiuoli S, Gavaler JS, Hassanein T, Jabbour N, Wright HI, et al. 1016/j.exphem.2004.09.005. The use of granulocyte-macrophage colony-stimulating factor to enhance [24] Theocharis SE, Papadimitriou LJ, Retsou ZP, Margeli AP, Ninos SS, Papadimi- hematologic parameters of patients with cirrhosis and hypersplenism. J triou JD. Granulocyte-colony stimulating factor administration ameliorates Hepatol 1994;21:582–586. doi: 10.1016/S0168-8278(94)80105-3. liver regeneration in animal model of fulminant hepatic failure and encephal- [17] Maiwal R, Kumar A, Sarin SK. Liver regeneration during acute-on-chronic liver opathy. Dig Dis Sci 2003;48:1797–1803. doi: 10.1023/A:1025463532521. failure using growth factors: in vivo or ex vivo indulgence of bone marrow? [25] Piscaglia AC, Shupe TD, Oh SH, Gasbarrini A, Petersen BE. Granulocyte- Gastroenterology 2013;145:901–904. doi: 10.1053/j.gastro.2013.08.031. colony stimulating factor promotes liver repair and induces oval cell migra- [18] Lyra AC, Soares MB, da Silva LF, Braga EL, Oliveira SA, Fortes MF, et al. – Infusion of autologous bone marrow mononuclear cells through hepatic tion and proliferation in rats. Gastroenterology 2007;133:619 631. doi: 10. artery results in a short-term improvement of liver function in patients with 1053/j.gastro.2007.05.018. chronic liver disease: a pilot randomized controlled study. Eur J Gastroenterol [26] Khanam A, Trehanpati N, Garg V, Kumar C, Garg H, Sharma BC, et al. Altered Hepatol 2010;22:33–42. doi: 10.1097/MEG.0b013e32832eb69a. frequencies of dendritic cells and IFN-gamma-secreting T cells with granulo- [19] Salama H, Zekri AR, Medhat E, Al Alim SA, Ahmed OS, Bahnassy AA, et al. cyte colony-stimulating factor (G-CSF) therapy in acute-on- chronic liver Peripheral vein infusion of autologous mesenchymal stem cells in Egyptian failure. Liver Int 2014;34:505–513. doi: 10.1111/liv.12415.

96 Journal of Clinical and Translational Hepatology 2016 vol. 4 | 90–96 Review Article

Interferon-Free Treatments for Chronic Hepatitis C Genotype 1 Infection

Alireza FakhriRavari*1, Mazyar Malakouti2 and Rebecca Brady1

1Department of Pharmacy Practice, University of the Incarnate Word Feik School of Pharmacy, San Antonio, Texas, USA; 2University of Texas Health Science Center, San Antonio, Texas, USA

Abstract It is estimated that 3–4 million individuals are newly infected with HCV each year.3 As many as 185 million Hepatitis C virus (HCV) infection affects as many as 185 persons worldwide and approximately 3.4–4.4 million individ- million people globally, many of whom are chronically infected uals in the United States are chronically infected with HCV.3,4 and progress over time to cirrhosis, decompensated liver These numbers are perhaps an underestimate, since it is dif- disease, hepatocellular carcinoma, and eventually death ficult to account for infected individuals who are homeless or without a liver transplant. In the United States, HCV genotype incarcerated. Once exposed to HCV, individuals will develop 1 constitutes about 75% of all infections. While interferon and acute infection in as early as 2 weeks and as late as 26 weeks ribavirin therapy was the cornerstone of treatment for many (Fig. 1).5,6 While spontaneous clearance of HCV is relatively years, interferon-free treatments have become the standard rare, it can occur in 18%–34% of acutely infected patients.7 of care with the emergence of new direct-acting agents, The remainder of infected individuals, if left untreated, will resulting in more effective treatment, shorter duration of progress to chronic infection over a period of 6 months. therapy, better tolerability, lower pill burden, and ultimately About 25% of chronically infected persons will develop cir- better adherence. This review will summarize the evidence for rhosis. Progression to cirrhosis was thought to be a slow the currently available combination therapies as well as process, occurring over 25–30 years; but new data suggest emerging therapies in phase 3 trials for treatment of HCV that this progression may occur faster, over 5 to 10 years in genotype 1. some individuals, especially those over 58 years of age.8 © 2016 The Second Affiliated Hospital of Chongqing Medical Once cirrhosis is established, progression seems to slow University. Published by XIA & HE Publishing Inc. All rights down, and 25% of patients will develop hepatocellular carci- reserved. noma (HCC) and/or decompensated liver disease (DLD), and eventually death.8 In fact, more than 350,000 people die annually worldwide from HCV-related complications.9 Introduction Virology Hepatitis C virus (HCV) is the most common cause of chronic hepatitis, which leads to severe complications over time.1 In HCV is an enveloped positive-sense viral ribonucleic acid (RNA) the mid-1970’s, it was shown that most post-transfusion that belongs to the Hepacivirus genus of viruses in the Flavi- cases of hepatitis were neither due to hepatitis A nor B viridae family.2 The viral RNA uses the host’s hepatocyte virus, the only known hepatitis viruses at the time. Hence, it ribosomes for translation into a polyprotein that is processed was referred to as the “non-A non-B hepatitis” until its dis- into 10 polypeptides, each with distinct functions.10 The lack of covery in 1989.2 Hepatitis C has a great disease burden proofreading in HCV replication machinery results in a great and cost in the Western world, as it is the leading cause of number of viral mutations, contributing to a high level of cirrhosis, liver cancer, and a primary indication for liver trans- variation.11 Multiple HCV variants in the same infected individ- plantation.1 Over the 27 years since its discovery, many ual are referred to as “quasispecies”. These variations differ advancements have been made in understanding the virus greatly based on geographic origin and lead to various HCV and developing effective and safe treatments. genotypes. There are seven major HCV genotypes, each with about 30% sequence divergence, whose prevalence varies 3,12 Keywords: Hepatitis C; HCV; Genotype 1; Direct acting antiretroviral agents. geographically (Table 1). Each genotype is grouped into a Abbreviations: AASLD, American Association for the Study of Liver Diseases; number of subtypes, each with about 20% sequence diver- ALT, alanine aminotransferase; ART, anti-retroviral therapy; AUC, area under the gence, denoted by letters a, b, etc.13,14 In the United States, curve; DAAs, direct-acting agents; DLD, decompensated liver disease; ESRD, end-stage renal disease; FDA, Food and Drug Administration; GFR, glomerular HCV genotype 1 constitutes about 75% of all infections fol- filtration rate; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; HIV, lowed by HCV genotypes 2 and 3 constituting the remaining human immunodeficiency virus; IDSA, Infectious Diseases Society of America; 25%.3 Disease association is largely similar across all geno- RAV, resistance associated variant; RNA, ribonucleic acid; SVR, sustained virologic types, but treatment response varies.13 response; ULN, upper limit of normal. Received: 24 February 2016; Revised: 22 March 2016; Accepted: 27 March 2016 q DOI: 10.14218/JCTH.2016.00007. Clinical manifestations *Correspondence to: Alireza FakhriRavari, Department of Pharmacy Practice, University of the Incarnate Word Feik School of Pharmacy, 4301 Broadway CPO – 99, San Antonio, Texas 78209, USA. Tel: +1-210-883-1142, Fax: +1-210-822- Acute hepatitis C is asymptomatic in 70% 85% of infected 15 1516, E-mail: [email protected] individuals. Those infected persons who show signs and

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Fig. 1. Progression of hepatitis C. Abbreviations: DLD, decompensated liver disease; HCC, hepatocellular carcinoma; HCV, hepatitis C virus. symptoms are more likely to clear the virus naturally.7 These inject drugs, have certain medical conditions, including symptoms can include malaise, jaundice, and influenza-like persons who received clotting factor concentrates produced symptoms.15 Elevated aminotransferase levels are often before 1987 or who were on hemodialysis long-term, have present in the setting of acute infection. Chronic hepatitis C HIV, or have persistently abnormal ALT levels. Recipients of is also asymptomatic in most individuals. Alanine aminotrans- transfusions or organ transplants before July 1992 and those ferase (ALT) levels typically fluctuate independent of symp- who were notified that they received blood from a donor who toms in the setting of chronic infection.16 Insulin resistance later tested positive for HCV infection should be screened. often occurs in chronically infected persons.17 Steatosis is Children born to HCV-positive women and healthcare pro- usually present in those chronically infected with HCV geno- viders exposed to needle sticks or HCV positive blood should 18 type 3. In individuals with advanced disease progression, be tested for HCV.21 symptoms of decompensated cirrhosis, such as esophageal varices, ascites, coagulopathy, encephalopathy, or HCC, can be present. Some individuals can also display extrahepatic HCV life cycle symptoms, such as cryoglobulinemia, vasculitis, porphyria 19,20 cutanea tarda, and membranous glomerulonephritis. The HCV virions exist as lipoviroparticles, which are able to enter hepatocytes by endocytosis trapped inside of endosomes. The low pH environment of endosomes results in Screening and diagnosis uncoating of the virion and the release of viral RNA into the cytoplasm. The viral RNA is readily translated in the rough The diagnosis of HCV is based on two broad categories of endoplasmic reticulum into a polypeptide consisting of 10 serological assays to detect antibodies against hepatitis C and structural and nonstructural (NS) proteins. The structural molecular assays to detect or quantify HCV RNA. For acute proteins include the capsid protein C and the envelope hepatitis C, detectable HCV RNA by polymerase chain reaction glycoproteins E1 and E2. The nonstructural proteins include (PCR), even in the setting of negative anti-HCV antibodies, is the porin p7, the autoprotease and assembly factor NS2, the considered definitive proof of the infection. It generally takes serine protease and RNA helicase NS3, the NS3 protease about 12 weeks for the anti-HCV antibody to become detect- cofactor NS4A, the organizer of replication complex and able, whereas HCV RNA can be detected in blood as early as membranous web NS4B, the regulator of replication and 1 week after exposure. Newly detectable HCV RNA and antiviral assembly NS5A, and the RNA-dependent RNA polymer- HCVantibodies with documentation of negative tests within the ase NS5B. The resulting HCV polypeptide is co- and post- prior 6 months are also suggestive of acute HCV infection. It translationally cleaved by cellular proteases and viral NS2/3 sometimes becomes difficult to distinguish between acute and and NS3/4A proteases to release the 10 HCV proteins.13 chronic HCV infection if prior documentation of negative tests The next step in the HCV life cycle is the formation of the are absent. If the antibody is nonreactive, then chronic HCV replication complex consisting of NS3, NS4A, NS4B, NS5A, infection is unlikely. A positive HCV RNA result is evidence of and NS5B proteins. The HCV RNA template binds to NS5A HCV infection. Usually, if both HCV RNA and anti-HCV antibody protein in the replication complex.22 NS5B protein replicates are detectable, the patient has chronic HCV infection.21 the template, and NS3 protein separates the nascent and HCV testing is recommended for all adults born from 1945 through 1965. The testing is also recommended for those who template RNA strands. Cholesterol and fatty acid biosynthesis are important to HCV replication by forming membrane- associated RNA replication complexes. Assembly of HCV 10 Table 1. Prevalence of hepatitis C virus (HCV) genotypes worldwide requires lipid droplets. NS5A protein is also important in the assembly of the replication complex.10,23 NS2 protein HCV Prevalence coordinates virion assembly through interactions with the genotype of HCV Geographic Location glycoproteins, p7, NS3, and NS5A. Lastly, the mature virus 1a 36%–55% United States is released from cells through the Golgi apparatus as lipoviral particles.10 1b 23%–25% Europe, Japan, and China 2 13%–16% Europe, United States, and Central Africa Pathogenesis 3 8%–13% Southeast Asia The HCV replication cycles in the hepatocytes cause cell necrosis 4 1%–2% Middle East and Northern by multiple mechanisms, including immune mediated cytolysis. Africa The virus can also cause hepatic steatosis (more likely with 5 < 1% South Africa HCV genotype 3), oxidative stress, and insulin resistance. This 6 < 1% Southeast Asia continuous necroinflammatory response most likely causes 7 < 1% Unknown progressive fibrosis and scarring of the liver, which leads to cirrhosis over time.18,24

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Treatment response (a decrease in HCV RNA level of $ 2 log IU/mL while on treatment but a detectable level at the end of treat- The combination of peginterferon and ribavirin was consid- ment), virologic breakthrough (a detectable HCV RNA level ered the standard of care for patients with HCV for many while on treatment after previously undetectable), and years.25 This combination did not have a high rate of virologic relapse (an undetectable level of HCV RNA during treatment cure, particularly in patients with HCV genotype 1, and but a detectable level after stopping treatment). Analyses of 48 weeks of treatment was typically required.26–29 Poor studies using interferon-based treatment have shown that response to interferon-based therapy was associated with cir- SVR is associated with lower all-cause mortality in patients rhosis, non-CC interleukin-28B (IL28B) genotype (also with HCV infection and advanced hepatic fibrosis and in known as IFNL3 gene, encoding interferon lambda-3), black patients with HCV-HIV coinfection, including both liver- – race, human immunodeficiency virus (HIV) coinfection, stea- related and nonliver-related mortality.44 47 tosis, and insulin resistance.28,30–34 In addition, severe adverse effects as well as administration via subcutaneous Sofosbuvir and simeprevir injection resulted in poor patient adherence. The introduction of direct-acting agents (DAAs), boceprevir and telaprevir, in Sofosbuvir (SovaldiÒ) is a once-daily, film-coated tablet manu- 2011, led to interferon-sparing combinations, resulting in factured by Gilead Sciences. It was approved by the FDA on shorter duration of therapy with a higher rate of virologic December 6, 2013. Sofosbuvir is currently indicated for the cure (Table 2).35 These first-generation protease inhibitors treatment of HCV genotypes 1 through 4 as a component of a were costly, had a pill burden with a thrice-daily schedule, combination antiviral treatment regimen.48 It is a phosphora- and added more adverse effects, which made it challenging midate prodrug and needs to be converted to its active metab- for the patients to remain adherent and leading to high rates olite (GS-461203) within hepatocytes.49 The phosphoramidate of resistance and clinical failure.36 As more classes of DAAs moiety of the prodrug improves bioavailability and transport were introduced, agents from two or more classes could be into hepatocytes. It can be taken with or without food.50 As a combined to eliminate the need for peginterferon, which was uridine nucleotide analogue, GS-461203 binds to the NS5B previously needed to reduce the emergence of resistance to catalytic site, induces chain termination, and increases the protease inhibitors. These new interferon-free therapies are number of errors in the growing RNA chain.49 It has activity better tolerated by patients and are more effective in achiev- against HCV genotypes 1 through 6 in vitro.50 The pharmacoki- ing a high rate of virologic cure.4 The results of clinical trials netic properties of sofosbuvir are shown in Table 3. Because have confirmed that non-CC IL28B genotype, which is 80% of sofosbuvir is eliminated via urine, it is not recommended associated with poor response to peginterferon-containing for patients with a glomerular filtration rate (GFR) < 30 mL/min/ regimens,37 is not associated with poor response to inter- 1.73 m2 or end-stage renal disease (ESRD). Increases of – feron-free treatments.38 40 However, patients with cirrhosis 5-fold or greater in the area under the curve (AUC) of active and/or HCV genotype 1a remain difficult to treat compared to metabolite have been observed in these patients in a pharma- patients without cirrhosis and those with HCV genotype 1b. cokinetic study.51 Furthermore, patients with creatinine clear- The primary outcome in HCV clinical trials is sustained ance < 60 mL/min were excluded in clinical trials. However, an virologic response 12 weeks after the end of treatment ongoing study is evaluating lower doses of sofosbuvir (such as (SVR12), defined as undetectable HCV RNA serum levels. 100 mg once daily) in patients with severe renal impairment Historically, SVR 24 weeks after the end of treatment (SVR24) or ESRD (NCT01958281 and NCT02563665). Sofosbuvir is gen- was used for the primary endpoint. However, studies have erally well tolerated as part of interferon-free combination shown that SVR12 has a 98% positive predictive value and a therapies. However, post-marketing surveillance has revealed 99% negative predictive value compared to SVR24.41,42 As a a risk of serious and potentially fatal bradycardia when sofosbu- result, the United States Food and Drug Administration vir is taken with amiodarone.52 (FDA) guidance for industry has indicated use of SVR12 as Simeprevir (OlysioÒ) is a second-wave, first-generation the primary efficacy end point in clinical trials.43 Patients NS3/4A serine protease inhibitor manufactured by Janssen. who have never received treatment before are considered It was approved by the FDA on November 22, 2013. Sime- treatment-naïve. Those patients who received treatment previr is currently indicated for the treatment of HCV geno- in the past and failed treatment are considered treatment- type 1 or 4 as part of a combination antiviral treatment experienced, including null response (a decrease in HCV regimen.53 It reversibly inhibits NS3/4A serine protease RNA level of < 2 log IU/mL while on treatment), partial inhibitor by noncovalently binding to the active site, which

Table 2. Efficacy of historic treatments for HCV genotype 1

Year Trials Regimen SVR24 Relapse Treatment

1991 HIT*26 IFN for 24 weeks 2%–3% ;80% Naïve 1991 HIT*,26 IHIT*27 IFN for 48 weeks 7%–11% ;46% Naïve 1998 HIT*,26 IHIT*27 IFN + RBV for 24 weeks 16%–18% ;42% Naïve 1998 HIT*,26 IHIT*27 IFN + RBV for 48 weeks 28%–31% ;24% Naïve 2001 IHIT 2001*28 PegIFN + RBV for 48 weeks 42% ;18% Naïve 2009 IDEAL29 PegIFN + RBV for 48 weeks ;40% 20–31% Naïve

* Included genotypes 1 and other genotypes. Abbreviations: IFN, interferon alpha; PegIFN, pegylated interferon alpha; RBV, ribavirin; SVR, sustained virologic response.

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Table 3. Pharmacokinetics of NS5B polymerase inhibitors

Agent Sofosbuvir49,71 Dasabuvir76 Beclabuvir110

Dosage 400 mg daily 250 mg twice a day with food 75 mg twice a day Half-life 0.5 h (parent drug), 27 h 5.5–6h 8h (active metabolite) Protein-binding 60% > 99% Unknown Elimination Urine (80%), feces (14%) Feces (94%), urine (2%) Feces, urine (< 10%) Pregnancy Category B Category B Unknown Substrate P-glycoprotein, BCRP CYP 2C8, P-glycoprotein, BCRP CYP 3A4 Inhibition — UGT 1A1 OATP 1B1, P-glycoprotein

Abbreviations: BCRP, breast cancer resistance protein; CYP, cytochrome P450; UGT, uridine diphosphate glucuronosyltransferase; OATP, organic anion-transporting polypeptides. interferes with cleavage of the HCV polyprotein and as a result treatment. There was no on-treatment failure. The relapse prevents viral replication.54 It has activity against HCV geno- rate was significantly higher in the 8-week arm with 17% types 1, 2, 4, 5, and 6, with no activity against genotype 3 in (27/155) compared to 3% (4/155) in the 12-week arm. vitro.55 The pharmacokinetic properties of simeprevir are There was no difference in SVR12 based on genotype 1 shown in Table 4. It should be taken with food to improve subtype, presence of the baseline Q80K resistance mutation, absorption.54 It is mainly metabolized by CYP 3A and excreted or whether patients were treatment-naïve or –experienced. primarily in the feces with less than 1% excreted in the A post-hoc analysis showed that an SVR12 rate of 96% is urine.56 Maximum daily doses of 10 mg of rosuvastatin and achieved with 8 weeks of treatment in patients with a baseline 40 mg of atorvastatin are recommended if used concomi- HCV RNA level less than 4 million IU/mL. The most frequent tantly with simeprevir.57 Common adverse effects of simepre- adverse events were nausea, headache, and fatigue. No vir in clinical trials were fatigue, headache, nausea, insomnia, patients discontinued treatment due to an adverse event. pruritus, rash, and photosensitivity. The combination of sime- In OPTIMIST-2,59 a single arm, open-label phase 3 trial in previr and sofosbuvir has been evaluated in phase 3 studies. the United States and Canada, 103 patients with compensated In OPTIMIST-1,58 an open-label phase 3 trial in the United cirrhosis were assigned to receive 12 weeks of simeprevir States and Canada, 310 patients without cirrhosis were 150 mg once daily plus sofosbuvir 400 mg once daily. The randomized to receive either 8 or 12 weeks of simeprevir study included both treatment-naïve and –experienced 150 mg once daily plus sofosbuvir 400 mg once daily. The patients. Overall, SVR12 was achieved in 83% (86/103) of study included both treatment-naïve and –experienced patients, including 88% (44/50) of treatment-naïve and 79% patients. Patients treated previously with a DAA containing (42/53) of treatment-experienced patients. In the absence of regimen were excluded. Overall, SVR12 was achieved in 83% the Q80K mutation, which only occurs in genotype 1a, SVR12 (128/155) of patients who received 8 weeks of treatment rate was 84% (26/31) in patients with genotype 1a and 92% and 97% (150/155) of patients who received 12 weeks of (35/38) in patients with genotype 1b. However, lower rates of

Table 4. Pharmacokinetics of NS3/4A protease inhibitors

Agent Simeprevir54,56 Paritaprevir76 Asunaprevir109 Grazoprevir96

Dosage 150 mg daily with food 150 mg daily with 200 mg twice a day 100 mg daily food Half-life 10–13 h (healthy adults), 5.5 h 17–23 h 31 h 41 h (HCV infected adults) Protein-binding > 99% 97%–99% > 99% 99.8% Elimination Biliary excretion (91%), Feces (88%), urine Feces (84%), Feces (> 90%), CYP 3A (9%) urine (< 1%) urine (1%) Pregnancy risk Category B Category B Unknown Unknown Substrate CYP 3A CYP 3A, P- CYP 3A, P-glycoprotein, CYP 3A, P-glycoprotein, glycoprotein, BCRP, OATP 1B, OATP 2B BCRP, OATP 1B OATP 1B Inhibition CYP 1A2, intestinal CYP BCRP, P-glycoprotein, CYP 2D6 (moderate), Intestinal BCRP 3A4, OATP 1B and OATP 1B P-glycoprotein (weak), P-glycoprotein OATP 1B (weak)

Abbreviations: BCRP, breast cancer resistance protein; OATP, organic anion-transporting polypeptides.

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SVR12 were achieved in the presence of the Q80K mutation with a dual mechanism of action and is only available in (74% (25/34) in patients with genotype 1a). There was no combination with sofosbuvir. It binds to domain 1 of the difference in SVR12 based on genotype 1 subtype. However, NS5A protein and blocks its ability to regulate HCV replication IL28B TT genotype was associated with lower rates of SVR12. within the replication complex, and it inhibits assembly and On-treatment failure occurred in 3% (3/103) of patients. release of viral particles.68 It has activity against HCV geno- One of these patients discontinued treatment due to an type 1, 4, and 5, with limited activity against genotype 2 and 3 adverse event. The remaining two patients with viral break- in vitro.69 The pharmacokinetic properties of ledipasvir are through had multiple reports of missed doses. The relapse shown in Table 5. It can be taken with or without food.67 rate was 13% (13/103), mostly occurring at follow-up week However, the absorption of ledipasvir is pH-dependent, and 4. The most common adverse events were headache, administration of antacids within 4 h should be avoided.70 fatigue, and nausea. Serious adverse events, none of which While both sofosbuvir and ledipasvir are P-glycoprotein sub- were considered related to study treatment, occurred in five strates, neither of them is a CYP substrate, resulting in patients, and three patients discontinued study treatment as a minimal drug interactions. Ledipasvir is also an inhibitor of result. P-glycoprotein.71 Common adverse effects of sofosbuvir- Sofosbuvir has a high barrier to resistance. No resistant ledipasvir in clinical trials were fatigue and headache, and less variants have been detected in clinical trials when used as part commonly nausea, diarrhea, and insomnia.38–40 Bilirubin and 60,61 62 of a dual or triple therapy. In the ELECTRON trial, lipase elevations were also observed. This combination has however, when sofosbuvir was used as monotherapy to treat been evaluated in several phase 3 studies. HCV genotype 2 or 3, one patient developed S282T resistance In ION-1,38 an open-label, phase 3 trial in the United States mutation. This mutation has not been isolated in patients and Europe, 865 treatment-naïve patients with HCV genotype 1 with HCV genotype 1. Simeprevir, on the other hand, seems were randomized to one of the four groups to take sofosbuvir- to have a lower barrier to resistance. Although the presence of ledipasvir 400 mg–90 mg by mouth once daily with or without baseline Q80K polymorphism was originally associated with a ribavirin for either 12 weeks or 24 weeks. A minimum creatinine decreased rate of SVR12 when simeprevir was used in combi- clearance of 60 mL/min was required. SVR12 was achieved in nation with peginterferon and ribavirin,63,64 it was not 99% (211/214) of patients who received sofosbuvir-ledipasvir observed in the COSMOS phase 2 trial when simeprevir was without ribavirin for 12 weeks. Furthermore, SVR12 was used with sofosbuvir.65 Nonetheless, Q80K polymorphism achieved in 97% of patients with cirrhosis, which constituted seemed to result in decreased SVR12 in OPTIMIST-2.59 There- 16% of the patients in the study. Only two patients in the fore, screening for Q80K polymorphism is recommended study had relapse, and they both had cirrhosis. There was no at baseline when using this combination in patients with HCV relapse in groups that received ribavirin. Overall, 10 patients genotype 1a. It is not clear if extending treatment to 24 weeks discontinued treatment prematurely owing to adverse events will increase efficacy in cirrhotic patients with HCV genotype 1a (four received sofosbuvir-ledipasvir for 24 weeks and six in the presence of the Q80K mutation. Thus, it may be reason- received sofosbuvir-ledipasvir plus ribavirin for 24 weeks), able to avoid this combination in patients with the Q80K muta- and all 10 patients achieved SVR. All 10 patients received tion. Although data are lacking, cross-resistance across the at least 8 weeks of therapy before discontinuation. No patient HCV protease inhibitor class is a concern. Hence, simeprevir- containing combinations should be avoided in patients who in the groups receiving 12 weeks of treatment discontinued have failed other first-generation HCV protease inhibitors, therapy prematurely. Of the 33 patients who had a serious such as boceprevir and telaprevir.66 adverse event, only eight were in the 12-week groups, one who received sofosbuvir-ledipasvir and seven who received sofosbuvir-ledipasvir plus ribavirin. The most common Sofosbuvir and ledipasvir adverse events were fatigue, headache, insomnia, and nausea. Groups that received ribavirin had higher rates of TM Sofosbuvir-ledipasvir (Harvoni ) is a once daily, fixed-dose, events associated with ribavirin therapy: fatigue, insomnia, film-coated, one-pill combination manufactured by Gilead asthenia, rash, cough, pruritus, and anemia. Laboratory abnor- Sciences. It was approved by the FDA on October 10, 2014. malities included changes in hemoglobin level (;−2 g/dL) and It is currently indicated for the treatment of HCV genotype 1, hyperbilirubinemia, both of which are associated with ribavirin 4, 5, or 6.67 Ledipasvir is a first-generation NS5A inhibitor therapy.

Table 5. Pharmacokinetics of first-generation NS5A inhibitors

Agent Ledipasvir71 Ombitasvir76 Daclatasvir92

Dosage 90 mg daily 25 mg daily with food 60 mg daily* Half-life 47 h 21–25 h 12–15 h Protein-binding 99.8% 99.9% 99.0% Elimination Feces (86%), urine (1%) Feces (90%), urine (2%) Feces (88%), urine (7%) Pregnancy Category B Category B Unknown Substrate P-glycoprotein, BCRP P-glycoprotein CYP 3A, P-glycoprotein Inhibition P-glycoprotein, BCRP — P-glycoprotein, OATP 1B, BCRP

* 30 mg daily with 3A inhibitors, 90 mg daily with 3A inducers. Abbreviations: BCRP, breast cancer resistance protein; OATP, organic anion transporter peptide.

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Similarly, ION-2 was an open-label, phase 3 trial in the involving 335 patients co-infected with HIV-1 and HCV United States that randomized 440 treatment-experienced genotype 1 or 4.72 All patients were required to receive a patients with HCV genotype 1 to one of four groups.39 The protocol-approved antiretroviral regimen for HIV-1 (emtrici- patients were previously treated with peginterferon and tabine/tenofovir disoproxil fumarate plus efavirenze, ralte- ribavirin, 53% of which had received the treatment with a gravir, or rilpivirine) for at least 8 weeks prior to the first-generation protease inhibitor, mostly telaprevir or boce- screening for the study and to have evidence of HIV-1 viral previr. Nonresponders made up 44% of the patients, and the suppression (HIV-1 RNA < 50 copies/mL) with a CD4+ remainder had either relapse or virologic breakthrough on count >100 cells/mcL. A minimum creatinine clearance of their previous therapy. About 20% of the study patients 60 mL/min was required. Patients received sofosbuvir- had cirrhosis. A minimum creatinine clearance of 60 mL/min ledipasvir 400 mg–90 mg by mouth once daily for 12 weeks. was required. While SVR12 was achieved in 94% (102/109) Only eight patients (2%) were infected with HCV genotype 4. of all patients who received 12 weeks of treatment with sofos- Overall, 20% of patients had cirrhosis, 45% were treatment- buvir-ledipasvir without ribavirin, only 86% of patients with naïve, and 55% were treatment-experienced (of whom 36% cirrhosis achieved SVR12 in this group. Adding ribavirin to had failed previous DAAs). SVR12 was achieved in 96% (322/ the regimen did not improve outcomes in cirrhotic patients. 335) of patients. This rate was 94% among patients with cir- However, this outcome was improved to 100% with 24 weeks rhosis and 97% among treatment-experienced patients. All 13 of treatment. The rate of relapse was 6% with 12 weeks of patients who had relapsed previous treatment with sofosbuvir treatment, but relapse was reduced to 0% with 24 weeks and ribavirin had SVR. One patient died after 4 weeks of treat- of treatment. Overall, 11 patients had a relapse, 10 of which ment. Two patients had a virologic breakthrough during treat- occurred within 4 weeks after the end of treatment. No patient ment that was suspected to be due to poor adherence. Ten had a relapse after 12 weeks post-treatment. None of the patients had a relapse, all of whom were black with seven patients discontinued treatment prematurely due to adverse patients having the TT allele in the IL28B gene. Exploratory events. Patients in the groups that received ribavirin had univariate analysis identified black race and the presence of higher rates of adverse events associated with ribavirin the TT allele to be associated significantly with relapse. None therapy: fatigue, nausea, insomnia, arthralgia, cough, rash, of the patients discontinued treatment prematurely because of irritability, dyspnea, and anemia. The rates of laboratory an adverse event. Most adverse events were mild to moderate, abnormalities were similar in all groups with the exception of most commonly headache, fatigue, diarrhea, and nausea. Rare hyperbilirubinemia, which developed in more patients who grades 3 and 4 serum laboratory abnormalities were reported, received sofosbuvir-ledipasvir plus ribavirin than in those who including elevations in lipase, creatine kinase, and serum received only sofosbuvir-ledipasvir. Changes in hemoglobin glucose. No significant drug interactions were identified in level (;−2.5 g/dL) were associated with ribavirin therapy, as this study. the groups that did not receive ribavirin only experienced non- Unlike sofosbuvir, ledipasvir has a low barrier to resist- significant changes in hemoglobin level (;−0.5 g/dL). ance. NS5B resistance-associated variant (RAV) S285T was 38–40,72 ION-3 was an open-label, phase 3 trial in the United States not detected in ION-1, ION-2, ION-3, or ION-4. One that randomized 647 treatment-naïve noncirrhotic patients patient with relapse in ION-4 had NS5B RAV L159F, but this infected with HCV genotype 1 to one of the three groups to patient also had NS5A RAVs. Among treatment-experienced take sofosbuvir-ledipasvir 400 mg-90 mg by mouth once daily patients in ION-4, three patients had the L159F variant at with or without ribavirin for 8 weeks or without ribavirin for 12 baseline, but all achieved SVR12. Both patients with relapse weeks.40 A minimum creatinine clearance of 60 mL/min was in ION-1 had NS5A-resistant variants at baseline (L31M in 38 required. SVR12 rate was 94% (202/215) in patients who genotype 1a and Y93H in genotype 1b). Of the 11 patients received treatment without ribavirin for 8 weeks. However, who had a relapse in ION-2, seven patients received 12 weeks 39 11 patients (5%) in this group had a relapse, 10 of whom of treatment without ribavirin. NS5A-resistant variants were infected with HCV genotype 1a. Data submitted to the were present at baseline in four of these patients. The remain- FDA showed that nine out of 11 patients with relapse in this ing four patients with a relapse received 12 weeks of treat- group had a viral load of $ 6,000,000 IU/mL at baseline. In ment with ribavirin. NS5A-resistant variants were present at the group that also received ribavirin, nine patients (4%) had baseline in two of these patients. Nevertheless, all 11 patients a relapse, seven of whom were infected with HCV genotype who had a relapse had NS5A-resistant variants at the time of 1a. As a result, the FDA has approved the 8 week regimen the relapse. Of the 23 patients who had a relapse in ION-3, 15 only for treatment-naïve noncirrhotic patients who have a patients had NS5A resistant variants at the time of relapse, 40 baseline viral load < 6,000,000 IU/mL. Three patients discon- nine of whom had the variants at baseline. Of the 10 tinued treatment prematurely due to adverse events, includ- patients who had a relapse in ION-4, four patients had NS5A ing one patient in the group receiving 8 weeks of treatment resistant variants at baseline and eight patients had them at with sofosbuvir-ledipasvir plus ribavirin reported to have a the time of relapse. The two patients who had a virologic road accident. No single serious adverse event occurred in breakthrough, possibly due to poor adherence, did not have more than one patient. The most common adverse events resistance-associated NS5A variants at baseline but did have were fatigue, headache, and nausea. Patients who received such emergent variants at the time of failure. ribavirin experienced greater changes in hemoglobin level (;−1.9 g/dL) than those who did not receive ribavirin Ombitasvir-paritaprevir-ritonavir and dasabuvir (;−0.2 g/dL). Only three patients who received 8 weeks of sofosbuvir-ledipasvir with ribavirin had hyperbilirubinemia. Ombitasvir-paritaprevir-ritonavir and dasabuvir,copackaged as TM Otherwise, the rates of laboratory abnormalities were Viekira Pak by Abbvie, are together known as the “3D” similar among groups. combination since three DAAs are included. The combination ION-4 was a single-group, open-label, phase 3 trial in the was approved by the FDA on December 19, 2014. It is currently United States, Puerto Rico, Canada, and New Zealand, indicated for the treatment of HCV genotype 1, including

102 Journal of Clinical and Translational Hepatology 2016 vol. 4 | 97–112 FakhriRavari A. et al: Hepatitis C genotype 1 infection treatment patients with compensated cirrhosis.73 Ombitasvir-paritapre- Patients with a creatinine clearance < 60 mL/min were vir-ritonavir is a coformulated fixed-dose tablet (12.5 mg/ excluded. SVR12 rate for group 1 was 97% (85/88) and 75 mg/50 mg), and dasabuvir is a separate tablet (250 mg).74 100% (91/91) for group 2. No patients from either group Ombitasvir is a first-generation NS5A inhibitor with pangeno- experienced on-treatment virologic failure or post-treatment typic antiviral activity.75 The pharmacokinetic properties of relapse. Of the three patients who did not achieve SVR12, ombitasvir are shown in Table 5. It is a P-glycoprotein substrate, two patients discontinued the study drug due to an adverse and it is eliminated primarily through the feces with only 2% event, and one patient was lost to follow-up after 4 weeks. eliminated through the urine.76 Paritaprevir is a NS3/4A serine The most commonly reported adverse events were fatigue, protease inhibitor.The pharmacokinetic properties of paritapre- headache, and nausea, which occurred significantly more fre- vir are shown in Table 4. It is a substrate of P-glycoprotein as well quently in group 1 than in group 2. Patients in group 1 also 76 as CYP 3A, and it is an inhibitor of P-glycoprotein. It is primarily experienced significantly more events of insomnia, anemia, eliminated through the feces with only 9% eliminated through rash, and increased blood bilirubin levels, which are known to the urine. Ritonavir,which does not have activity against HCV,is be associated with ribavirin treatment. a potent inhibitor of CYP 3A4 enzyme and is used as a pharma- In PEARL-III,84 a multicenter, double-blind, placebo- 77 cokinetic booster to increase paritaprevir plasma levels. Dasa- controlled phase 3 trial in Europe, Russia, and the United buvir is a non-nucleoside NS5B inhibitor. The pharmacokinetic States, 419 treatment-naïve patients with HCV genotype 1b properties of dasabuvir are shown in Table 3. It is a substrate of without cirrhosis were randomized in a 1:1 ratio to receive P-glycoprotein and CYP 2C8, and it is primarily eliminated either ribavirin twice daily according to body weight or match- 76 through the feces. Since the half-life of dasabuvir is only ing placebo for 12 weeks. All patients received open-label 3D about 6 h, it must be taken twice a day. The 3D combination combination for 12 weeks. Notably, patients with a creatinine should always be taken with a meal since food increases the clearance < 60 mL/min were excluded. SVR was 99.5% 78 absorption of the included agents. In patients with mild, mod- (209/210) in the ribavirin group and 99% (207/209) in erate, or severe renal insufficiency, no dosing adjustment is patients who did not receive ribavirin. Only one patient in the required; although this regimen has not been adequately study, who was in the group receiving ribavirin, had virologic studied in patients with renal dysfunction since patients with a failure during treatment. The two patients in the group not creatinine clearance < 60 mL/min were excluded from most receiving ribavirin who did not achieve SVR12 were lost to studies. RUBY-I study is an ongoing trial investigating the follow-up at week 12. The most common adverse events safety and efficacy of this regimen in patients with severe were headache and fatigue. Adverse events were more fre- renal impairment or ESRD.79 It is contraindicated in patients quently reported in the group receiving antiviral regimens with hepatic decompensation (Child-Pugh B and C), patients that contained ribavirin. receiving medications highly dependent on CYP 3A metabolism Similar to PEARL-III, PEARL-IV was a multicenter, double- for which significant increases in plasma levels is dangerous, blind, placebo-controlled phase 3 trial in Canada, the United patients on moderate or strong inducers of CYP 3A or strong States, and the United Kingdom.84 Instead of patients with inducers of CYP 2C8, patients on strong inhibitors of CYP 2C8 HCV genotype 1b, it randomized 305 treatment-naïve patients (since this may result in increased levels of dasabuvir and QT with HCV genotype 1a without cirrhosis in a 1:2 ratio to receive prolongation), or known hypersensitivity to ritonavir.80 This either ribavirin twice daily according to body weight or match- combination was well tolerated in clinical trials. The most ing placebo for 12 weeks. All patients received open-label 3D common (greater than 10%) adverse effects have been combination for 12 weeks. Again, patients with a creatinine fatigue, nausea, pruritus, other skin reactions, insomnia, and asthenia. Its concomitant use with ethinyl estradiol-containing clearance < 60 mL/min were excluded. The SVR rate was medications can lead to significant elevations in hepatic amino- 97% (97/100) in the ribavirin group and 90% (185/205) in transferase levels.81 patients who did not receive ribavirin. Logistic-regression anal- The addition of ribavirin to the 3D combination is recom- ysis showed that IL28B CC genotype was associated with an mended for all patients except those with HCV genotype 1b increased rate of SVR12. The most common adverse events without cirrhosis.73 Ribavirin can cause significant adverse were headache and fatigue. Adverse events were more fre- effects, including hemolytic anemia. It is also highly terato- quently reported in the group receiving antiviral regimens genic and embryocidal, and extreme care must be given to that contained ribavirin. avoid pregnancy during therapy and for 6 months after finish- SAPPHIRE-I was a multicenter, double-blind, placebo- ing the treatment; this pertains both to treatment of women controlled phase 3 trial in North America, Europe, and Aus- 85 receiving ribavirin and women whose male partners are receiv- tralia. It randomized 631 treatment-naïve patients with HCV ing ribavirin therapy.82 Treatment with ribavirin is contraindi- genotype 1, no cirrhosis, and a plasma HCV RNA level of at cated in pregnant women and men whose female partners are least 10,000 IU/mL in a 3:1 ratio to receive a 12-week treat- pregnant, hemoglobinopathies, and co-administration with ment course with 3D regimen plus ribavirin (group A) or a didanosine. Furthermore, ribavirin must be dosed carefully matching placebo regimen (group B). Patients in group B based on body weight, changes in hemoglobin, and renal were eligible to receive treatment at the end of study. Patients function.82 with a creatinine clearance < 60 mL/min were excluded. About In PEARL-II,83 a multicenter, open-label phase 3 trial in 68% of the patients had HCV genotype 1a, and 31% had IL28B Europe and the United States, 179 patients with HCV genotype CC genotype. The overall SVR12 rate was 96.2% (455/473) in 1b, without cirrhosis, who had previously received and failed group A, including 95.3% (307/322) among patients with HCV treatment with peginterferon and ribavirin were randomized to genotype 1a and 98% (148/151) among patients with receive 3D combination either with ribavirin (group 1) or HCV genotype 1b. The rates were similarly high in all sub- without ribavirin (group 2) for 12 weeks. Ribavirin was dosed groups. Furthermore, logistic-regression analysis showed based on body weight twice daily: 1,000 mg daily if body that patient characteristics, including IL28B genotype, did weight < 75 kg or 1,200 mg daily if body weight $ 75 kg. not significantly affect the rate of SVR12. The most common

Journal of Clinical and Translational Hepatology 2016 vol. 4 | 97–112 103 FakhriRavari A. et al: Hepatitis C genotype 1 infection treatment adverse events were fatigue and headache; the frequency of regimen. The overall SVR12 rate was 97% (33/34), including which did not differ significantly between the two study groups. 97% (28/29) among patients with HCV genotype 1a and 100% Similar to SAPPHIRE-I, SAPPHIRE-II was a multicenter, (5/5) among patients with HCV genotype 1b. The most double-blind, placebo-controlled phase 3 trial in North common adverse events were fatigue, headache, and cough. America, Europe, and Australia.86 Instead of treatment-naïve Only one patient discontinued treatment, which was due to patients, it randomized 394 patients with HCV genotype 1, rash, memory impairment, and anxiety. without cirrhosis, a plasma HCV RNA level of at least The 3D regimen has a low barrier to resistance. Due to 10,000 IU/mL who had previously failed treatment with pegin- a very low rate of virologic failure, resistance-associated terferon and ribavirin in a 3:1 ratio to receive a 12-week treat- variants were not observed in PEARL-II and PEARL-III.83,84 ment course with 3D regimen plus ribavirin (group A) or a In PEARL-IV,84 two of the three patients who received ribavirin matching placebo regimen (group B). Patients in group B were but did not achieve SVR12 had virologic failure. A total of eligible to receive treatment at the end of study. Patients that 18 patients had virologic failure, 16 of whom were not receiv- had received a HCV protease inhibitor, and those with a creati- ing ribavirin. Of these 16 patients, six had a virologic rebound nine clearance < 60 mL/min were excluded. About 58% of the while on treatment, and 10 had a relapse after treatment. All patients had HCV genotype 1a and 11% had IL28B CC genotype. 18 patients who had virologic failure had at least one resist- The overall SVR12 rate was 96% (286/297) in group A, including ance-associated variant. The most frequently detected var- 96% (166/173) among patients with HCV genotype 1a and 97% iants were D168V in NS3, M28T and Q30R in NS5A, and (119/123) among patients with HCV genotype 1b. The rates S556G in NS5B. In SAPPHIRE-I,85 one patient had virologic were similarly high in all subgroups, including IL28B genotype, failure while on treatment, and seven patients had a relapse. prior relapsers, prior partial responders, and prior null respond- Each of these patients had at least one resistance-associated ers. The investigators reported adverse events in 91.2% of variant, most frequently D168V in NS3, M28T and Q30R in patients in the active-regimen group and 82.5% of patients in NS5A, and S556G in NS5B for HCV genotype 1a and Y56H the placebo group; severe adverse events occurred in 2% of and D168V in NS3, L31M and Y93H in NS5A, and S556G in patients in the active treatment group and in 1% of the patients NS5B for HCV genotype 1b. In SAPPHIRE-II,86 seven patients in the placebo group. The two most common adverse events had a relapse, all of whom were adherent to treatment. Five of were headache and fatigue. Pruritus was the only adverse these patients had resistance-associated variants at the time events that occurred in more than 10% of patients in either of relapse, most frequently D168V in NS3, M28V and Q30R in group that had a higher frequency in the active-regimen group NS5A, and S556G in NS5B for HCV genotype 1a and Y56H and than the placebo group. D168A in NS3, Y93H in NS5A, and C316N and S556G in NS5B 87 In TURQUOISE-II,87 a multicenter, open-label, phase 3 for HCV genotype 1b. In TURQUOISE-II, virologic failure controlled trial, 380 patients with HCV genotype 1, a plasma occurred in 13 patients receiving 12 weeks of treatment HCV RNA level of at least 10,000 IU/mL, and compensated (group A), with one patient failing while on treatment, the cirrhosis (Child-Pugh class A) were randomized to receive remaining 12 relapsing, and four patients receiving 24 either 12 (group A) or 24 weeks (group B) of treatment with weeks of treatment (group B). Seven of the 12 patients with 3D regimen plus ribavirin. Patients with prior therapy with a relapse in group A had HCV genotype 1a and a prior null DAAs (e.g., telaprevir and boceprevir) and those with creati- response. Only two of the 17 patients who had virologic nine clearance < 60 mL/min were excluded. About 69% of failure did not have resistance-associated variants at the patients had HCV genotype 1a, 58% were treatment-experi- time of virologic failure. The remaining 15 patients had resist- enced, and 42% were treatment-naïve. For group A, the ance-associated variants in two or more of the drug targets, overall SVR12 rates were 92% (191/208), 89% (124/140) most commonly D168V in NS3 and Q30R in NS5A for HCV among all patients with HCV genotype 1a, 92% (59/64) genotype 1a and D168V in NS3, Y93H in NS5A, and G316Y 88 among treatment-naïve patients with HCV genotype 1a, and and M414I in NS5B for HCV genotype 1b. In CORAL-I study, 86% (65/76) among treatment-experienced patients with the only patient who had a relapse (on day 3 of treatment) HCV genotype 1a. For group B, the overall SVR12 rates had resistance-associated variants R155K in NS3, M28T and were 96% (165/172), 94% (114/121) among all patients Q30R in NS5A, and G554S in NS5B at the time of relapse but with HCV genotype 1a, 93% (52/56) among treatment- not at baseline. naïve patients with HCV genotype 1a, and 95% (62/65) among treatment-experienced patients with HCV genotype Sofosbuvir and daclatasvir 1a. For patients with HCV genotype 1b, SVR12 rates were TM 99% (67/68) in group A and 100% (51/51) in group B. No Daclatasvir (Daklinza ) is a once-daily, oral tablet manufac- specific adverse event led to premature discontinuation by tured by Bristol-Myers Squibb. It is available as 30 mg or more than one patient, and no pattern in the types of 60 mg tablets. It was approved by the FDA on July 24, 2015. adverse events leading to discontinuation was observed. Daclatasvir is currently indicated for the treatment of HCV The CORAL-I Study,88 which was a single-arm, phase 2 trial, genotypes 1 and 3 in combination with sofosbuvir.89 It is a enrolled 34 liver transplant recipients (at least 12 months prior first-generation HCV NS5A inhibitor, which prevents the rep- to screening) with recurrent HCV genotype 1 and a plasma HCV lication of RNA and assembly of the virion by inhibiting con- RNA level of at least 10,000 IU/mL to receive 3D regimen plus formational change of NS5A protein through impairing ribavirin for 24 weeks. Patients had no evidence of cirrhosis, phosphatidylinositol-4-kinase III a activation.90 It has activity and those with a creatinine clearance < 55 mL/min were against HCV genotypes 1 through 6 in vitro.91 The pharmaco- excluded. About 85% of the patients had HCV genotype 1a, kinetic properties of daclatasvir are shown in Table 5. It can be 24% had IL28B CC genotype, and 71% were previously taken with or without food. While co-administration with treated with peginterferon and ribavirin. The median time proton pump inhibitors results in decreased daclatasvir expo- since liver transplantation was 3.3 years, and 85% of patients sure, the interaction is not clinically significant.92 Although were receiving a tacrolimus-based immunosuppressive high-fat meals reduce its absorption, no dosage adjustments

104 Journal of Clinical and Translational Hepatology 2016 vol. 4 | 97–112 FakhriRavari A. et al: Hepatitis C genotype 1 infection treatment are recommended.92 This effect is not observed with low-fat, of patients with advanced cirrhosis and 95% (39/41) of low-caloric meals (approximately 277 total kcal).89 Daclatas- patients post-transplant achieved SVR12. In patients with vir is a substrate of CYP 3A. Therefore, a dose reduction to 30 HCV genotype 1, SVR12 rate was 82% (37/45) in patients mg of daclatasvir is needed when used in combination with a with advanced cirrhosis and 95% (39/41) in patients post- strong CYP 3A inhibitor, such as ketoconazole, atazanavir, transplant. There was no difference based on gender, age, ritonavir, or cobicistat. No dosage adjustments are needed IL28B, or HCV RNA level in the advanced cirrhosis cohort when used in combination with moderate CYP 3A inhibitors. with HCV genotype 1. Overall, the combination was tolerated The use of daclatasvir in combination with strong CYP 3A well. Only one patient in each arm discontinued treatment inducers is contraindicated. A dose increase to 90 mg of due to adverse effects. Most common adverse effects daclatasvir is needed when used in combination with a mod- included headache, fatigue, anemia, diarrhea, nausea, and erate CYP 3A inducer, such as dexamethasone, efavirenz, or arthralgia. nevirapine.92 About 88% of the drug is eliminated in feces, Unlike sofosbuvir, daclatasvir has a low barrier to resist- and 7% is excreted in the urine. Therefore, renal adjustment ance. While monotherapy with daclatasvir selects for resistant of daclatasvir dosage is unnecessary. Daclatasvir is highly variants in both genotype 1a and 1b, most of these variants protein bound and is unlikely to be removed by dialysis. are susceptible to NS3 protease inhibitors and NS5B polymer- When used in combination with sofosbuvir, daclatasvir is gen- ase inhibitors, so daclatasvir as part of a combination therapy erally well tolerated. The most common adverse effects are remains effective.95 In ALLY-2,93 none of the patients in the headache, fatigue, and nausea. Daclatasvir, in combination study had a HCV virologic breakthrough. Overall, 12 patients with sofosbuvir, has been evaluated for treatment of HCV had a relapse, 10 of whom were in the 8-week treatment genotype 1 in phase 3 trials. arm, and nine patients had HCV genotype 1a. Baseline NS5A In ALLY-2,93 an open-label, multi-center phase 3 trial in resistance-associated polymorphisms did not seem to affect the United States, 203 patients co-infected with HIV-1 and significantly response. Only three of the 12 patients with HCV genotypes 1 through 4 were randomly assigned to one relapse had daclatasvir-resistance polymorphism at baseline. of the three arms. The study included both HCV treatment- In ALLY-1,94 nine patients had a relapse in the advanced cir- naïve and –experienced patients. Patients who were HCV rhosis group, whereas only three patients had a relapse in the treatment-naïve were assigned in a 2:1 ratio to receive post-transplant group. One patient with advanced cirrhosis either 12 weeks or 8 weeks of daclatasvir 60 mg once daily, had a breakthrough. All 13 patients who had virologic failure with dose adjustment for concomitant medications, plus had NS5A resistance variants present at the time of failure. sofosbuvir 400 mg once daily. Patients previously treated Only four of these patients had these variants at baseline. No for HCV were assigned to receive 12 weeks of treatment at NS5B-S282 variants were detected at baseline or failure. the same doses. The standard 60 mg dose of daclatasvir was adjusted to 30 mg in patients receiving ritonavir-boosted pro- Elbasvir and grazoprevir tease inhibitors and to 90 mg in those receiving efavirenz or TM nevirapine. Patients with creatinine clearance less than 50 Elbasvir-grazoprevir (Zepatier ) is a once daily, fixed-dose, mL/min were excluded. About 83% of the patients had HCV film-coated one-pill combination manufactured by Merck & Co. genotype 1, and 29 patients (14%) had compensated cirrho- It was approved by the FDA on January 29, 2016 for the sis. Among previously treated patients, 94% had received an treatment of HCV genotypes 1 and 4.96 Grazoprevir is a interferon-containing regimen, and 22% had received a HCV second-generation NS3/4A protease inhibitor, which has protease inhibitor. The rate of SVR12 was 96.4% in treat- shown high potency against HCV genotypes 1, 2, 4, 5, and 6, ment-naïve patients with HCV genotype 1 who received 12 with less potency against genotype 3.97 It also has activity weeks of treatment, 75.6% with 8 weeks of treatment, and against variants that are resistant to earlier first-generation 97.7% in treatment-experienced patients who received 12 NS3/4A protease inhibitors.98 The pharmacokinetic properties weeks of treatment. There was no difference in the rate of of grazoprevir are shown in Table 4. Less than 1% of grazopre- SVR12 based on any subgroups except for baseline viral vir is renally excreted; and, hence, dose adjustments in the load in the 8-week treatment group. Patients with baseline setting of chronic kidney disease is not necessary.99 It is a viral load < 2 million IU/mL had SVR12 rate of 100% substrate of CYP 3A, P-glycoprotein, and the organic anion (18/18) compared to 62% (20/32) in patients with baseline transporter protein 1B and an inhibitor of intestinal breast viral load $ 2 million IU/mL. Although a small number of cancer resistance protein.100 Thus, co-administration with patients had cirrhosis, there was similar rate of SVR12 strong CYP 3A inducers or efavirenz is contraindicated.96 (92%) among these patients. The combination of daclatasvir Elbasvir is a second-generation NS5A inhibitor. It has activity and sofosbuvir was well tolerated. None of the patients dis- against genotype 1, 2a, 3, 4, 5, and 6, including variants that continued treatment due to an adverse event. The most are resistant to first-generation NS5A inhibitors, such as dacla- common adverse events were fatigue, nausea, and head- tasvir and ledipasvir.101,102 However, testing for the presence ache. Two patients died during post-treatment follow-up. of NS5A resistance-associated polymorphisms is recommen- ALLY-1 was an open-label, phase 3 trial that enrolled 113 ded prior to the initiation of treatment in patients with HCV patients infected with HCV (any genotype) who either had genotype 1a.96 The pharmacokinetic properties of elbasvir advanced cirrhosis or had recurrence of HCV infection post- are shown in Table 6. Elbasvir is a substrate of CYP 3A and liver transplant.94 Both treatment-naïve and –experienced P-glycoprotein. No clinically meaningful pharmacokinetic inter- patients were included. The study consisted of 60 patients actions between elbasvir-grazoprevir with tacrolimus, myco- with advanced cirrhosis and 53 patients post-liver transplant. phenolate mofetil, and prednisone have been identified, but All patients received 12 weeks of daclatasvir 60 mg once daily cyclosporine seems to increase exposure to elbasvir-grazopre- plus sofosbuvir 400 mg once daily plus ribavirin initially vir.103 Co-administration with famotidine or pantoprazole does 600 mg per day, adjusted to 1,000 mg per day based on not affect the pharmacokinetics of elbasvir-grazoprevir.104 hemoglobin levels and renal function. Overall, 83% (50/60) There are no drug interactions between elbasvir-grazoprevir

Journal of Clinical and Translational Hepatology 2016 vol. 4 | 97–112 105 FakhriRavari A. et al: Hepatitis C genotype 1 infection treatment

Table 6. Pharmacokinetics of second-generation NS5A inhibitors the Netherlands, Spain, and Sweden.106 The study randomized 224 HCV genotype 1 infected adult patients with chronic Agent Elbasvir96 Velpatasvir96,120 kidney disease stage 4–5, of whom 76% were hemodialysis Dosage 50 mg once daily 100 mg once dependent, to receive either grazoprevir 100 mg/elbasvir daily 50 mg once daily or placebo for 12 weeks. Patients receiving placebo were given active treatment for 4 weeks after the end Half-life 24 h 16–19 h of the treatment period (week 16). About 80% of the patients Protein- 99.9% Unknown were HCV treatment-naïve, and the remaining 20% had pre- binding viously received an interferon regimen. Only 6% of the Elimination Feces (> 90%), urine Feces (99%), patients had cirrhosis. Six of 122 patients in the treatment (1%) urine (1%) group were excluded from the modified full analysis for reasons other than virological failure. SVR12 rate was Pregnancy Unknown Unknown 99.1% (115/116). The frequencies of adverse events were risk similar between the two groups; most commonly headache, Substrate CYP 3A, P-glycoprotein nausea, and fatigue. There were no drug-related serious P-glycoprotein adverse events. Two cases of congestive heart failure Inhibition — Intestinal BCRP, occurred in the treatment group within 14 days of the end of P-glycoprotein treatment, one of which was judged to be drug-related. There were no treatment discontinuations due to an adverse event Abbreviation: BCRP, breast cancer resistance protein. in the treatment group. One patient in the treatment group died from cardiac arrest, but it was not considered related to study drugs. Although first-generation HCV protease inhibi- 105 and either tenofovir or raltegravir. However, boosted HIV-1 tors have been shown to cause a reversible decline in renal protease inhibitors are not recommended for use in combina- function,107 no differences in renal function were noted tion with elbasvir-grazoprevir due to potential drug-drug inter- between the groups in this study. actions. The combination elbasvir-grazoprevir has been C-EDGE CO-INFECTION was an open-label, single-arm, evaluated in phase 3 trials. multicenter, phase 3 trial in Europe, the United States, and C-EDGE Treatment-Naïve was an international, single- Australia.108 The study enrolled 218 treatment-naïve adult blind, phase 3 trial that randomized 421 adult patients patients co-infected with HCV and HIV-1 to receive grazopre- infected with HCV genotypes 1, 4, and 6 in a 3:1 ratio to vir 100 mg/elbasvir 50 mg fixed-dose combination tablet receive treatment with a fixed-dose combination tablet of once daily for 12 weeks without food restriction. About 86% grazoprevir 100 mg/elbasvir 50 mg or a matching placebo of the patients were infected with HCV genotype 1, 13% with 106 once daily, without regards to food, for 12 weeks. The genotype 4, and the remaining 2 patients (1%) with genotype study was conducted in Australia, the Czech Republic, 6. With the exception of seven patients, most patients France, Germany, Israel, Puerto Rico, South Korea, Sweden, received anti-retroviral therapy (ART) with undetectable HIV Taiwan, and the United States. About 91% of the patients had RNA. ART-naïve patients had to have CD3 or CD4 T-cell counts HCV genotype 1, of which 50% were genotype 1a, and 22% of of at least 500 cells/mcL and HIV RNA viral load of less than patients had cirrhosis. The majority of the patients from Aus- 50,000 copies/mL to be included. Otherwise, patients had to tralia, Sweden, and the United States had HCV genotype 1a. have CD3 or CD4 T-cell counts of at least 200 cells/mcL and Patients with creatinine clearance less than 50 mL/min were undetectable HIV RNA levels. Patients with decompensated excluded. SVR12 was achieved in 92% of the patients with liver disease, cirrhosis who were Child-Pugh class B or C, HCV genotype 1a and 99% of patients with genotype 1b. Fur- and hepatitis B virus co-infection were excluded. SVR12 was thermore, SVR12 was achieved in 97% of cirrhotic patients achieved in 96.3% of patients infected with HCV genotype 1 and 94% of non-cirrhotic patients. One patient with HCV gen- and in all 35 patients with cirrhosis. The treatment was genotype 1a had a breakthrough. A relapse occurred in nine erally well tolerated. The most common adverse events were patients with HCV genotype 1a and one patient with genotype fatigue, headache and nausea. Six patients experienced 1b. Elbasvir-grazoprevir was well-tolerated with a similar serious adverse events, but they were not considered to be safety profile between the active and placebo groups. related to treatment. Two patients had a late increase in Serious adverse events were reported in about 2.8% of ALT/AST > 5x upper limit of normal (ULN) after treatment patients in both groups, none of which was considered drug week 4, and this increase normalized without discontinuation related. Although two patients in the treatment group died, of treatment. There was no notable change in CD3 or CD4 neither death was considered drug related. The most common T-cell count at treatment week 12 or follow-up week 12. adverse events were headache (17%), fatigue (15%), and Elbasvir-grazoprevir has a high barrier to resistance.102 In nausea (9%), which were similar in frequency in both C-EDGE Treatment-Naïve,106 virologic analysis of the 10 groups. Three patients discontinued treatment, two patients patients with HCV genotype 1a who had virologic failure with elevated aminotransferase levels and one patient with showed that NS3 RAVs were present in six patients (most palpitations and anxiety on day 4 of treatment. One patient commonly Q80K and D168A), and NS5A RAVs were present in the placebo group discontinued placebo pills because of a in all 10 patients (most commonly M28V/A/G, Q30H/L/R, rash on day 2. Two patients discontinued treatment due to L31M, and Y93H). However, an association of genotype 1a elevations in aminotransferase level. RAVs with virologic failure was present only in patients with C-SURFER was a double-blind, phase 3 trial consisting baseline viral levels greater than 800,000 IU/mL. In of a randomized study of safety and an observational study C-SURFER,106 only one patient relapsed. This noncirrhotic of efficacy in the United States, Argentina, Australia, patient with diabetes had a baseline HCV genotype 1b viral Canada, Estonia, France, Israel, South Korea, Lithuania, load > 800,000 IU/mL and relapsed 12 weeks after the end of

106 Journal of Clinical and Translational Hepatology 2016 vol. 4 | 97–112 FakhriRavari A. et al: Hepatitis C genotype 1 infection treatment treatment. Analysis showed that this patient had an NS5A protease, or nonnucleoside NS5B polymerase inhibitors was L31M mutation at baseline. In C-EDGE CO-INFECTION,108 not permitted. Overall, 91% (379/415) of patients achieved four noncirrhotic patients with genotype 1a relapsed after an SVR12. In the treatment-naïve group, 92% (287/312) of having undetectable HCV RNA levels at the end of treatment. patients achieved SVR12, while in the treatment-experienced Two of these patients had D168A variant in the NS3 region, group, 89% (92/103) achieved SVR12. Fewer patients and three had Q30R/K variants in the NS5A region. In achieved SVR12 with HCV genotype 1a in both the treat- patients with HCV genotype 1, SVR12 was achieved in 87% ment-naïve (90%) and –experienced (85%) group compared of patients who had baseline NS5A resistance-associated to HCV genotype 1b (98% and 100%, respectively). The rates variants compared to 98% in patients without resistance- of SVR12 were similar across all subgroups. The most associated variants. Baseline NS3 resistance-associated var- common side effects, which occurred in more than 10% of iants, as well as Q80K polymorphism in genotype 1a, did not patients, included headache, fatigue, diarrhea, and nausea. seem to affect SVR12. The UNITY-2 trial was a multinational, phase 3 trial in the United States, Canada, France, and Australia, with a two- Daclatasvir, beclabuvir, and asunaprevir cohort, four-arm, design.115 It enrolled 202 compensated- cirrhotic patients with chronic HCV genotype 1 and a plasma Daclatasvir-asunaprevir-beclabusvir, or DCV-TRIO, is a fixed- HCV RNA level of at least 10,000 IU/mL to receive open-label dose, film-coated tablet under development by Bristol Myers DCV-TRIO twice daily for 12 weeks. Additionally, patients Squibb. It contains three direct-acting agents: 30 mg dacla- were randomized in double-blind fashion to receive ribavirin tasvir, 200 mg asunaprevir, and 75 mg beclabuvir. Daclatasvir or matching placebo twice daily. One-hundred twelve patients is currently FDA-approved for the treatment of HCV genotype were treatment-naïve, and 90 patients were treatment- 3 in combination with sofosbuvir.89 Asunaprevir is a second- experienced. Patients previously exposed to NS5A, NS3 pro- wave first-generation protease inhibitor that inhibits NS3 tease, or nonnucleoside NS5B polymerase inhibitors and those serine protease, ultimately preventing the cleavage of the with a creatinine clearance < 50 mL/min were excluded. 109 HCV polypeptide into individual proteins. The pharmacoki- Overall, 93% (188/202) of patients achieved SVR12. In treat- netic properties of asunaprevir are shown in Table 4. It has ment-naïve groups, SVR12 rate was 98% (54/55) in the group in vitro activity against HCV genotypes 1, 4, 5, and 6, with receiving ribavirin and 93% (53/57) in the group receiving 109 less potency against HCV genotypes 2 and 3. Unlike NS5A placebo. The treatment-experienced group on ribavirin inhibitors, NS3 protease inhibitors are more specific to geno- achieved an SVR12 rate of 93% (42/45), and the placebo type as the viral protease active site can vary according to group achieved an SVR12 rate of 87% (39/45). Fewer patients genotype. In addition, asunaprevir has a low barrier to resist- with HCV genotype 1a achieved SVR12, as shown in the UNITY-1 ance, such that it should be used in combination with other trial; however, improved SVR12 rates in the HCV genotype 1a DAA similar to daclatasvir. Beclabuvir is a non-nucleoside population were seen with the addition of ribavirin. Patients NS5B inhibitor and acts by binding allosterically to the NS5B with HCV genotype 1a in the treatment-naïve group that RNA polymerase, ultimately inhibiting the elongation of the received ribavirin had an SVR12 of 97% (38/39) compared nascent viral RNA chain.110 This process differs from nucleo- to 90% (36/40) in those without ribavirin; and in the treat- side NS5B inhibitors that bind directly to the active site of the ment-experienced group, SVR12 was 91% (32/35) and 86% RNA polymerase. This difference in mechanism between the (30/35), respectively. Evaluation of statistical significance non-nucleoside and nucleoside NS5B inhibitors is what deter- was not reported. SVR12 rates were similar across all sub- mines the degree of barrier to resistance. Nucleoside NS5B groups. The most common side effects, which were reported polymerase inhibitors have a high barrier to resistance since in more than 10% of patients, included headache, nausea, the RNA polymerase active site must maintain a particular diarrhea, fatigue, insomnia, and pruritus. Three serious, amino acid sequence for viral replication, whereas non- treatment-related adverse events occurred and included nucleoside NS5B inhibitors like beclabuvir have a low barrier anemia, aminotransferase and bilirubin elevations, and riba- to resistance since they bind elsewhere to RNA polymerase. As such, beclabuvir is best used in combination with virin overdose. 111 DCV-TRIO has a low barrier to resistance. In UNITY-1 other DAAs. The pharmacokinetic properties of beclabuvir 114 are shown in Table 3. Beclabuvir has in vitro activity against trial, virologic failure occurred in 34 patients while data HCV genotypes 1, 3a, 4a, and 6, with reduced potency against were missing for two patients. Eight patients had virologic HCV genotype 2.112 Daclatasvir, asunaprevir, and beclabuvir breakthrough, and 21 patients had relapse (15 treatment- all undergo CYP 3A metabolism and are excreted mainly in the naïve patients and six treatment-experienced patients). The feces. Additionally, all and have increased exposure with renal majority of treatment failures were in patients infected with insufficiency.110,113 Patients enrolled in trials taking the com- HCV genotype 1a. Of the 31 patients with HCV genotype 1a bination tablet were instructed to take it with food. DCV-TRIO infection with available baseline and failure genetic sequenc- fixed-dose combination was evaluated recently in two phase ing, 30 patients had RAVs emerge in NS5A (most frequently 3 trials. Q30), 29 had RAVs emerge in NS3 (most frequently R155), The UNITY-1 trial was a multi-national, phase 3 trial in the and 12 had RAVs emerge in NS5B (most frequently P495). United States, Canada, France, and Australia, with an open- Both patients with HCV genotype 1b who had virologic label, single-group, uncontrolled design.114 It enrolled failure had a different genotype at the time of failure. In 415 non-cirrhotic patients with HCV genotype 1 and a UNITY-2 trial,115 emergent RAVs in those patients with viro- plasma HCV RNA level of at least 10,000 IU/mL to receive logic failure occurred primarily in patients infected with HCV DCV-TRIO twice daily for 12 weeks. Three-hundred twelve genotype 1a. Twelve patients infected with HCV genotype 1a patients were treatment-naïve, and 103 were treatment- with virologic failure had emergent RAVs: 11 NS5A (Q30), 10 experienced. Patients with a creatinine clearance < 50 NS3 (R155K), and two NS5B (P495). Only one patient with mL/min were excluded. Previous exposure to NS5A, NS3 HCV genotype 1b had an emergent NS5A RAV.

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Sofosbuvir and velpatasvir Nine patients died during the study, mostly due to complications of end-stage liver disease, none of which were consid- Velpatasvir (formerly known as GS-5816) is a second- ered to be related to therapy. generation NS5A protease inhibitor and is under development Both sofosbuvir and velpatasvir have a high barrier to in combination with sofosbuvir by Gilead Sciences. It has resistance. In ASTRAL-1,118 two patients had virologic relapse shown pangenotypic antiviral activity in vitro.116 The pharma- by post-treatment week 4, both of whom had NS5A-resistant cokinetic properties of velpatasvir are shown in Table 6. No variants at baseline. However, 99% of patients with baseline 119 clinically important drug interactions between sofosbuvir and NS5A-resistant variants achieved SVR12. In ASTRAL-4, a velpatasvir have been identified in pharmacology studies. total of 22 patients had virologic failure, nine of whom had Velpatasvir is primarily eliminated in the feces with < 1% of HCV genotype 1. In the arm receiving sofosbuvir-velpatasvir the dose excreted in the urine.117 The combination of sofos- for 12 weeks, three patients with genotype 1a and two buvir and velpatasvir was evaluated in phase 3 trials. patients with genotype 1b had relapse, while one patient ASTRAL-1 was a double-blind, multi-center, phase 3 trial in with genotype 1a in the arm receiving 12 weeks of sofosbu- the United States, Canada, Europe, and Hong Kong.118 The vir-velpatasvir plus ribavirin had relapse. In the arm receiving study enrolled 741 patients with HCV genotype 1, 2, 4, 5, or 6, 24 weeks of sofosbuvir-velpatasvir, two patients with HCV 68% of whom were treatment-naïve. The remaining patients genotype 1a and one patient with genotype 1b had relapse. were previously treated with peginterferon or peginterferon Among patients with HCV genotype 1 and baseline NS5A plus ribavirin with or without a protease inhibitor, 51% of resistance-associated variants, the rate of SVR12 was 80% whom had a virologic relapse or breakthrough and 48% who and 90% with 12 and 24 weeks of sofosbuvir-velpatasvir, were non-responders. Patients, with the exception of those respectively, and 100% with the addition of ribavirin. All with HCV genotype 5, were randomized to receive sofosbuvir patients with baseline NS5B resistance-associated variants 400 mg/velpatasvir 100 mg once daily or placebo for achieved SVR12. 12 weeks. Patients with creatinine clearance of less than 60 mL/min and those who had received previous treatment Conclusions with any NS5B inhibitors or NS5A inhibitors were excluded. Overall, 121 patients had cirrhosis, 210 patients had HCV gen- The American Association for the Study of Liver Diseases otype 1a, and 118 patients had HCV genotype 1b. SVR12 was (AASLD) and Infectious Diseases Society of America (IDSA) achieved in 99% of all patients, including 98% of patients with have jointly developed a HCV Guidance, which provides up- HCV genotype 1a and 99% of patients with HCV genotype 1b. to-date recommendations for testing, managing, and treat- The treatment was well tolerated, with only one patient dis- ing hepatitis C (Table 7).21 The recommended regimens for continuing treatment due to an adverse events. Common treatment-naïve noncirrhotic patients with HCV genotype 1 adverse events included headache (29%), fatigue (20%), are 12 weeks of simeprevir plus sofosbuvir, 12 weeks of nasopharyngitis (13%), nausea (12%), insomnia (8%), diar- sofosbuvir-ledipasvir, 12 weeks of ombitasvir-paritaprevir- rhea (8%), asthenia (7%), arthralgia (6%), cough (6%), back ritonavir plus dasabuvir without ribavirin for HCV genotype pain (5%), and myalgia (4%), all of which occurred in the 1b and with ribavirin for HCV genotype 1a, and 12 weeks of placebo arm with similar frequencies. daclatasvir plus sofosbuvir. For treatment-naïve patients In ASTRAL-4,119 an open-label, multicenter, phase 3 trial in with cirrhosis, the recommended regimens are simeprevir the United States, 267 patients with decompensated cirrhosis plus sofosbuvir for 24 weeks, sofosbuvir-ledipasvir for (Child-Pugh-Turcotte class B) were randomly assigned at a 12 weeks, ombitasvir-paritaprevir-ritonavir plus dasabuvir 1:1:1 ratio to one of three arms to receive fixed-dose sofos- without ribavirin for 12 weeks for HCV genotype 1b and buvir 400 mg/velpatasvir 100 mg once daily for 12 weeks, with ribavirin for 24 weeks for HCV genotype 1a, and dacla- sofosbuvir-velpatasvir plus ribavirin for 12 weeks, or sofosbu- tasvir plus sofosbuvir for 24 weeks. vir-velpatasvir for 24 weeks. About 60% of patients had HCV The recommended regimens for treatment-experienced genotype 1a, and 18% had genotype 1b, with 55% of patients noncirrhotic patients with HCV genotype 1 are 12 weeks of having received prior treatment with either peginterferon and simeprevir plus sofosbuvir, 12 weeks of sofosbuvir-ledipasvir ribavirin or a protease inhibitor regimen. Patients with crea- (with ribavirin if prior treatment failure with sofosbuvir plus tinine clearance of less than 50 mL/min, those who had ribavirin), 12 weeks of ombitasvir-paritaprevir-ritonavir plus received previous treatment with any NS5A inhibitor or dasabuvir without ribavirin for HCV genotype 1b and with nucleotide analogue NS5B inhibitor, and patients who had ribavirin for HCV genotype 1a, and 12 weeks of daclatasvir undergone liver transplantation were excluded from this plus sofosbuvir. For treatment-experienced patients with study. Among patients with HCV genotype 1, SVR12 rate cirrhosis, the recommended regimens are simeprevir plus was 88% with 12 weeks of sofosbuvir-velpatasvir, 96% with sofosbuvir for 24 weeks, sofosbuvir-ledipasvir for 24 weeks 12 weeks of sofosbuvir-velpatasvir plus ribavirin, and 92% (with ribavirin if prior treatment failure with sofosbuvir plus with 24 weeks of sofosbuvir-velpatasvir. This regimen was ribavirin), sofosbuvir-ledipasvir with ribavirin for 12 weeks well tolerated. A total of nine patients discontinued treatment (if prior treatment failure with telaprevir, boceprevir, or due to an adverse events, one of whom had received 12 simeprevir), ombitasvir-paritaprevir-ritonavir plus dasabuvir weeks of treatment, four had received 12 weeks of ribavirin- with ribavirin for 12 weeks for HCV genotype 1b and for containing treatment, and four had received 24 weeks of 24 weeks for HCV genotype 1a, and daclatasvir plus sofosbu- treatment. The most common adverse events in all groups vir for 24 weeks. were fatigue (29%), nausea (23%), and headache (22%). Over the past few years, we have seen great advance- Anemia, diarrhea, and insomnia were also common among ments in the treatment of HCV. New treatments have shown patients who received ribavirin. Reductions in hemoglobin, high cure rates in clinical trials. For most treatment-naïve lymphocytes, and platelets were common in all patients patients without cirrhosis, ribavirin-free treatments are effec- but more frequently in patients who were receiving ribavirin. tive. However, treatment-experienced patients with cirrhosis

108 Journal of Clinical and Translational Hepatology 2016 vol. 4 | 97–112 FakhriRavari A. et al: Hepatitis C genotype 1 infection treatment

Table 7. Recommendations by American Association for the Study of Liver Diseases and Infectious Diseases Society of America

Genotype Cirrhosis Treatment-naïve Treatment-experienced

GT1b No Cirrhosis Recommended: Recommended:* GZR/EBR 3 12 weeks (I, A) GZR/EBR 3 12 weeks (I, A) LDV/SOF 3 12 weeks (I, A) LDV/SOF 3 12 weeks (I, A) OBV/PTV/r + DSV 3 12 weeks (I, A) OBV/PTV/r + DSV 3 12 weeks (I, A) SMV + SOF 3 12 weeks (I, A) SMV + SOF 3 12 weeks (I, A) DCV + SOF 3 12 weeks (I, B) DCV + SOF 3 12 weeks (IIa, B) Recommended:** LDV/SOF + RBV 3 12 weeks (IIb, C) Recommended:*** LDV/SOF 3 12 weeks (I, A) DCV+SOF 3 12 weeks (IIa, B) GZR/EBR + RBV 3 12 weeks (IIa, B) Compensated Recommended: Recommended:* Cirrhosis GZR/EBR 3 12 weeks (I, A) GZR/EBR 3 12 weeks (I, A) LDV/SOF 3 12 weeks (I, A) LDV/SOF + RBV 3 12 weeks (I, A) OBV/PTV/r + DSV 3 12 weeks (I, A) LDV/SOF 3 24 weeks (I, A) Alternative: OBV/PTV/r + DSV 3 12 weeks (I, A) SMV + SOF 6 RBV 3 24 weeks (I, A) Alternative:* DCV + SOF 6 RBV 3 24 weeks (IIa, B) DCV + SOF 6 RBV 3 24 weeks (IIa, B) SMV + SOF 6 RBV 3 24 weeks (IIa, B) Recommended:** LDV/SOF + RBV 3 24 weeks (IIb, C) Recommended:*** LDV/SOF + RBV 3 12 weeks (I, A) LDV/SOF 3 24 weeks (I, A) DCV + SOF 6 RBV 3 24 weeks (IIa, B) GZR/EBR + RBV 3 12 weeks (IIa, B) Decompensated Recommended: Prior SOF failure: Cirrhosis LDV/SOF + RBV low-dose 3 LDV/SOF + RBV low-dose 3 12 weeks (I, A) 24 weeks (II, C) DCV + SOF + RBV low-dose 3 12 weeks (I, B) RBV ineligible: DCV + SOF 3 24 weeks (II, C) LDV/SOF 3 24 weeks (II, C) GT1a No Cirrhosis Recommended: Recommended:* GZR/EBR 3 12 weeks (I, A)**** GZR/EBR 3 12 weeks (I, A)**** LDV/SOF 3 12 weeks (I, A) LDV/SOF 3 12 weeks (I, A) OBV/PTV/r + DSV + RBV OBV/PTV/r + DSV + RBV 3 12 weeks (I, A) 3 12 weeks (I, A) SMV + SOF 3 12 weeks (I, A) SMV + SOF 3 12 weeks (I, A) DCV + SOF 3 12 weeks (I, B) DCV + SOF 3 12 weeks (IIa, B) Alternative: Alternative:* GZR/EBR + RBV 3 GZR/EBR + RBV 3 16 weeks (IIa, B)***** 16 weeks (I, B)***** Compensated Recommended: Recommended:* Cirrhosis GZR/EBR 3 12 weeks (I, A)**** GZR/EBR 3 12 weeks (I, A)**** LDV/SOF 3 12 weeks (I, A) LDV/SOF 3 24 weeks (I, A) Alternative: LDV/SOF + RBV 3 12 weeks (I, A) OBV/PTV/r + DSV + RBV 3 Alternative:* 24 weeks (I, A) OBV/PTV/r + DSV + RBV SMV + SOF 6 RBV 3 24 weeks (I, A) 3 24 weeks (I, A) DCV + SOF 6 RBV 3 GZR/EBR + RBV 3 16 weeks (I, B)***** 24 weeks (IIa, B) DCV + SOF 6 RBV 3 GZR/EBR + RBV 3 24 weeks (IIa, B) 16 weeks (IIa, B)***** SMV + SOF 6 RBV 3 24 weeks (IIa, B)

(continued)

Journal of Clinical and Translational Hepatology 2016 vol. 4 | 97–112 109 FakhriRavari A. et al: Hepatitis C genotype 1 infection treatment

Table 7. Recommendations by American Association for the Study of Liver Diseases and Infectious Diseases Society of America (continued ) Genotype Cirrhosis Treatment-naïve Treatment-experienced

Decompensated Recommended: Prior SOF failure: Cirrhosis LDV/SOF + RBV low-dose 3 12 weeks LDV/SOF + RBV low-dose 3 24 weeks (I, A) (II, C) DCV + SOF + RBV low-dose 3 12 weeks (I, B) RBV ineligible: DCV + SOF 3 24 weeks (II, C) LDV/SOF 3 24 weeks (II, C)

(Class of recommendation, level of evidence) *prior treatment failure with PEG-IFN and RBV; **prior treatment failure with SOF + RBV; ***prior treatment failure with (telaprevir, boceprevir, or SMV) + PEG-IFN + RBV or SMV + SOF (no prior NS5A treatment); ****if no baseline high fold-change NS5A RAVs; *****if baseline high fold-change NS5A RAVs. Abbreviations: EBR, elbasvir; DCV, daclatasvir; DSV, dasabuvir; GT, genotype; GZR, grazoprevir; LDV, ledipasvir; OBV, ombitasvir; PEG-IFN, pegylated-interferon; PTV/r, paritaprevir/ritonavir; RBV, ribavirin; SMV, simeprevir; SOF, sofosbuvir. remain difficult to treat and, hence, the need for ribavirin- [8] Butt AA, Yan P, Lo Re V 3rd, Rimland D, Goetz MB, Leaf D, et al. Liver fibrosis containing regimens for these patients. Emerging therapies progression in hepatitis C virus infection after seroconversion. JAMA Intern Med 2015;175:178–185. doi: 10.1001/jamainternmed.2014.6502. could potentially eliminate the need for ribavirin in the coming [9] Mohd Hanafiah K, Groeger J, Flaxman AD, Wiersma ST.Global epidemiology of years. While most DAAs have a low barrier to resistance, hepatitis C virus infection: new estimates of age-specific antibody to HCV using them in combination seems to overcome this limitation. seroprevalence. Hepatology 2013;57:1333–1342. doi: 10.1002/hep.26141. Further evaluation is needed to establish the role of resistance [10] Liang TJ, Ghany MG. Current and future therapies for hepatitis C virus infection. N Engl J Med 2013;368:1907–1917. doi: 10.1056/NEJMra1213651. testing in clinical practice. As more regimens become avail- [11] Moradpour D, Penin F, Rice CM. Replication of hepatitis C virus. 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Safety and antiviral activity of MK-5172, a Sofosbuvir and Velpatasvir for HCV in patients with decompensated cirrho- next generation HCV NS3/4A protease inhibitor with a broad HCV genotypic sis. N Engl J Med 2015;373:2618–2628. doi: 10.1056/NEJMoa1512614. activity spectrum and potent activity against known resistance mutants, in [120] Mogalian E, German P, Kearney BP, Yang CY, Brainard D, McNally J, et al. Use genotype-1 and -3 HCV-infected patients. Presented at HEP DART 2011, of multiple probes to assess transporter- and cytochrome P450-mediated Hawaii, December 4–8, 2011. abstract. drug-drug interaction potential of the pangenotypic HCV NS5A inhibitor [98] McCune JM. The dynamics of CD4+ T-cell depletion in HIV disease. Nature Velpatasvir. Clin Pharmacokinet 2016;55:605–613. doi: 10.1007/s40262- 2001;410:974–979. doi: 10.1038/35073648. 015-0334-7.

112 Journal of Clinical and Translational Hepatology 2016 vol. 4 | 97–112 Review Article

Current Management of Alcoholic Hepatitis and Future Therapies

Behnam Saberi*1, Alia S. Dadabhai1, Yoon-Young Jang2, Ahmet Gurakar1 and Esteban Mezey1

1Division of Gastroenterology and Hepatology, The Johns Hopkins University School of Medicine, Baltimore, MD, USA; 2Department of Oncology, The Johns Hopkins University School of Medicine, Baltimore, MD, USA

Abstract Liver transplantation has been reported with good results in highly selected patients but is controversial due to limited Alcohol is one of the most common etiologies of liver disease, organ supply. and alcoholic liver disease overall is the second most common © 2016 The Second Affiliated Hospital of Chongqing Medical indication for liver transplantation in the United States. It University. Published by XIA & HE Publishing Inc. All rights encompasses a spectrum of disease, including fatty liver reserved. disease, alcoholic hepatitis (AH), and alcoholic cirrhosis. AH can range from mild to severe disease, with severe disease $ being defined as: Discriminant Function (DF) 32, or Model Introduction for End-stage Liver Disease (MELD) $ 21, or presence of hepatic encephalopathy. Management of the mild disease Approximately two-thirds of the US adult population drinks consists mainly of abstinence and supportive care. Severe alcohol each year, and 7.4% report heavy drinking and meet AH is associated with significant mortality. Currently, there is the criteria for alcoholism.1 However, only a minority of heavy no ideal medical treatment for this condition. Besides alcohol drinkers will develop alcoholic liver disease (ALD), likely due cessation, corticosteroids have been used with conflicting to environmental and genetic factors.2 The economic impact results and are associated with an inherent risk of infection. of ALD is significant, with a total cost of around $185 billion. Overall steroids have shown short term benefit when com- Of this cost, 14% (around $27 billion) is related to health pared to placebo, but they have no obvious long term care cost.3 In 2000, the mortality from ALD (reported as benefits. Pentoxifylline does not improve survival in patients age-adjusted death rate) was 25 and 15 per 100,000 for His- with severe AH and is no longer recommended based on the panics and non-Hispanics, respectively. Alcoholic cirrhosis results of the STOPAH (Steroid Or Pentoxifylline for Alcoholic was accounted for about 37% of liver-related deaths, which Hepatitis) trial. Anti-tumor necrosis factor (TNF) agents are is higher than death from cirrhosis due to hepatitis C.4 ALD associated with increased risk of life threatening infections still remains the second most common indication for liver and death. Currently, early stage trials are underway, mainly transplantation after hepatitis C, based on the most recent targeting novel pathways based on disease pathogenesis, annual Organ Procurement and Transplantation Network including modulation of innate immune system, inhibition (OPTN)/Scientific Registry of Transplant Recipients (SRTR) of gut-liver axis and cell death pathways, and activation of report.5 With new direct antiviral agents (DAAs) for treatment transcription factor farnesyl X receptor (FXR). Future treat- of hepatitis C,6 ALD will be one of the leading causes of liver ment may lie in human induced pluripotent stem cell (iPSC) disease in developed countries in the near future. Therefore, technology, which is currently under investigation for the study there is an urgent need for new therapies for the manage- of pathogenesis, drug discovery, and stem cell transplantation. ment of ALD and more importantly alcoholic hepatitis (AH). This review focuses on the current medical and surgical man- Keywords: Alcoholic hepatitis; Corticosteroids; Liver transplantation; Induced agement of patients with AH and future therapies. pluripotent stem cell. Abbreviations: AASLD, American Association for the Study of Liver Disease; AH, Spectrum of liver disease alcoholic hepatitis; ALD, alcoholic liver disease; ALT, alanine aminotransferase; AST, aspartate aminotransferase; AUROC, area under the receiver operating characteristic; CDCA, chenodeoxycholic acid; CI, confidence interval; DF, discriminant The clinical and pathologic spectrum of ALD ranges from function; FXR, farnesyl X receptor; GGT, gamma glutamyl transpeptidase; INR, alcoholic fatty liver disease (alcoholic steatosis) to cirrhosis international normalized ratio; iPSC, induced pluripotent stem cell; LPS, lipopoly- with various survival rates. ALD can be divided into: alcohol saccharide; MELD, model end-stage liver disease; NAC, N-acetylcysteine; NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis; OPTN, Organ induced steatosis (microvesicular, macrovesicular), alcoholic Procurement and Transplantation Network; OR, odds ratio; PBC, primary biliary hepatitis and alcoholic cirrhosis. There is usually overlap of cirrhosis; OCA, obeticholic acid; PT, prothrombin time; PTX, pentoxifylline; RR, different pathological findings in a given patient.2,7 relative risk; SBP, spontaneous bacterial peritonitis; SRTR, Scientific Registry of Transplant Recipients; STOPAH, Steroid Or Pentoxifylline for Alcoholic Hepatitis; TLR4, toll-like receptor 4; TNF tumor necrosis factor; UNOS, United Network for Alcoholic fatty liver Organ Sharing; UTI, urinary tract infection. Received: 21 February 2016; Revised: 27 March 2016; Accepted: 28 March 2016 q Alcohol induced fatty liver is the earliest and most common DOI: 10.14218/JCTH.2016.00006. presentation of ALD which develops in more than 80% of *Correspondence to: Behnam Saberi, Division of Gastroenterology and Hepatol- 8 ogy, The Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA. heavy drinkers. This condition should be differentiated from Tel: +1-410-614-2543, Fax: +1-410-614-7340, E-mail: [email protected] nonalcoholic fatty liver disease (NAFLD), which is associated

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This is an Open Access article distributed under the terms of the Creative Commons Attribution-Noncommercial 4.0 Unported License, permitting all non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Saberi B. et al: Management of alcoholic hepatitis with metabolic syndrome. ALD and NAFLD have a similar patho- the patients.15,23 Laboratory findings are typical for aspartate logic spectrum, and they have been described to coexist in aminotransferase (AST) and alanine aminotransferase (ALT) the clinical setting.9,10 Alcohol induced fatty liver disease is elevation usually less than 300 U/L with a ratio of > 2/1.24,25 usually asymptomatic and may completely resolve after 2–6 However, in patients with foamy fatty degeneration, higher weeks of alcohol abstinence.2,11 Weight loss by low calorie AST levels have been reported.20 Patients with AH usually diet in conjunction with increase physical activity reduces have high bilirubin and an increase in prothrombin time hepatic steatosis in patients with NAFLD.12 (PT)/international normalized ratio (INR). Leukocytosis in the absence of infection can be seen in patients with AH, Alcoholic cirrhosis but the absence of leukocytosis does not rule out the diagnosis.26,27 In those patients who continue to drink, the risk of developing Diagnosis of different forms of ALD based only on clinical cirrhosis will increase to about 30%.13 There is an increased presentation and laboratory findings is difficult.28 Another risk of developing cirrhosis with ingestion of 60–80 g/day challenge is that different types of ALD can develop concom- of alcohol for 10 years or longer in men and 20 g/day for itantly. As mentioned earlier, more than half of the patients women; however, only 6%–41% of those drinking this with AH may have alcoholic cirrhosis as well.2 Liver biopsy amount of alcohol will develop cirrhosis.1 Alcoholic cirrhosis remains the gold standard for diagnosis of AH; however, the has a high mortality rate, with a worldwide reported mortality indication for liver biopsy remains controversial.28 In some rate of 38%–50% of all cirrhosis-related death. The long-term centers, transjugular liver biopsy is performed to confirm the prognosis of alcoholic cirrhosis is dependent on alcohol diagnosis of AH when the diagnosis remains uncertain, abstinence.1,14 despite reviewing the clinical and laboratory data especially, and when corticosteroid with its potential side effects is 29 Alcoholic hepatitis (AH) being considered for treatment. In some studies, hepatocellular damage (hepatocellular ballooning, presence of Superimposed episodes of AH may occur in patients with ALD Mallory bodies), inflammatory infiltrate (mainly polymorpho- that could range from mild to severe and life threatening nuclears), and pericellular fibrosis have been used for diag- 15,30–32 episodes. The true prevalence of AH is not known, but studies nosis of AH. evaluating the histology of patients with ALD suggest that AH might be present in about 10%–35% of alcoholic Treatment patients.15,16 Severe AH is not a benign disease and has a reported 28 day mortality as high as 50% without treat- Alcoholic abstinence and supportive care ment.17,18 More than half of the patients with AH might have cirrhosis at the time of liver biopsy or will develop cir- Alcohol abstinence is the most important predictor for rhosis during the course.2,19 When evaluating survival in determining long-term survival in patients with AH. In the patients with AH, it is important to distinguish the patients study by Potts et al., 5 year survival was significantly higher who have concomitant cirrhosis. Even if the AH component in AH patients who remained abstinent compared to those AH is treated, the cirrhosis might not reverse, and the risk of patients who relapsed or continued to drink (75% vs. 27% and death from complications of portal hypertension and cirrhosis 21%, respectively, p =0.005).33 Nutritional support and man- remains. agement of complications of portal hypertension are other important factors in the care of patients with AH. Other variants of ALD Nutrition Other forms of ALD have been reported in the literature. Foamy fatty degeneration was reported in 21 heavy drinkers, Malnutrition as well as obesity in alcoholic patients are well- presenting mainly with jaundice and hepatomegaly. The recognized phenomena that can critically impact the develop- laboratory findings were significant for marked elevation of ment and progression of liver disease.34–36 The degree of serum aminotransferases, more prolonged elevation of alka- malnutrition is correlated closely with the development of all line phosphatase and bilirubin, and elevated cholesterol the serious complications of liver disease (e.g., ascites, ence- with no leukocytosis when compared to patients with AH. phalopathy, and hepatorenal syndrome) as well as the overall The liver pathology was consistent with mainly foamy fatty mortality.37 The etiology of nutritional deficiencies in alco- change (microvesicular fat) and megamitochondria. Foamy holics is multifactorial and include: decreased caloric intake, fatty degeneration has been reported to have a good prog- inadequate consumption of nutrients, impaired metabolism of nosis with rapid recovery in all patients with alcohol absti- vitamins due to possibly concomitant chronic pancreatitis, nence.20,21 Another variant of ALD is Zieve syndrome, first disruption of the gut microbiome and mucosal integrity, gastri- described in 1957, which was defined as a triad of jaundice, tis, and diarrhea/vomiting.38–41 Overall, patients who are not hyperlipidemia, and transient hemolytic anemia in patients meeting their nutritional needs by oral diet should receive sup- with alcoholic steatohepatitis.22 plementation. An enteral route is preferred whenever possible, as it maintains the gut mucosal integrity and prevents bacterial Diagnosis of AH translocation.42 Despite improvement in nutritional parameters and liver tests in most studies, only a few studies have For diagnosis of AH, a detailed history, including the history shown survival benefit with nutritional supplementation, and of alcohol intake, and physical examination is critical. On the majority of studies have not demonstrated a change in examination, apart from signs of portal hypertension and mortality.39,41 In review of five randomized clinical trials eval- jaundice, patients with AH might have fever in the absence of uating alimentary augmentation, no survival benefit was noted infection. Hepatic bruit may be present in more than half of in the supplemented group (17%–35% mortality) compared to

114 Journal of Clinical and Translational Hepatology 2016 vol. 4 | 113–122 Saberi B. et al: Management of alcoholic hepatitis the controlled group (16%–39% mortality).42 In a randomized a significantly lower 2-month survival than patients with no control trial of vitamin E vs. placebo in patients with AH, infection (46.4 +/− 6.9% vs. 77.5 +/− 3.2%, p < 0.00001). vitamin E improved serum hyaluronic acid but had no benefi- Therefore, it is very important to differentiate infection on cial effect on liver function and survival.43 admission from infection that occurs after initiation of steroids, as survival rates are completely different. Overall, infection was more common in steroid null responders than Medical management responders. In multivariate analysis, only Lille model and MELD score were independently associated with survival, Corticosteroids whereas infection was not. This study suggested that the development of infection after steroid therapy is most likely Corticosteroids are the current main treatment for severe secondary to absence of improvement of liver function; AH (defined as DF $ 32, or MELD $ 21, or presence of however, the study could not rule out the possibility that ste- hepatic encephalopathy) in patients who do not have any roids contributed to infection susceptibility.56 In a recent contraindications for steroid treatment.44,45 Corticosteroids meta-analysis of clinical trials, the group of patients that work by changing the balance of cytokines, reducing pro- received steroids had a higher overall incidence of infection inflammatory cytokines such as tumor necrosis factor 53 (TNF)-a, and increasing anti-inflammatory cytokines such than the control group. as interleukin 10.46 Data from clinical trials and meta- analyses of corticosteroids have been conflicting.47–52 A recent Cochrane meta-analysis concluded that, overall, Response to steroids there is no clear evidence that steroids are effective in the management of AH. However, this meta-analysis concluded Lille score that glucocorticosteroids did significantly reduce mortality in the subgroup of trials with patients with a Maddrey score of Lille score was primarily described to assess the steroid 32 or higher or in patients who had hepatic encephalopathy. response in patients with severe AH after 7 days of treatment. In addition, this study showed that steroids reduced mortal- A total of 295 patients with biopsy proven, severe AH (DF $ 32 ity in low bias-risk studies. The potential for bias was due 53,54 or hepatic encephalopathy) treated with steroids on admission to heterogeneity of data. To overcome this issue, with an overall 6-month survival of 65.4 +/− 2.9 were evaluated Mathurin et al. analyzed individual data from five random- in a cohort of patients. In univariate analysis, the prognostic ized controlled trials and showed that steroids have survival values of clinical and biological variables predicting 6-month advantage for severe AH (defined as DF $ 32 or hepatic survival were studied. Ten variables reached statistical signifi- encephalopathy), with a 28 day survival of 85% among cance. Subsequent multivariate analysis of six objective varia- treated patients compared to 65% for patients receiving bles using logistic regression was calculated as Lille score, placebo (p =0.001).45 Prednisolone 40 mg for a total of ranging from 0 to 1. Lille score = [3.19 – 0.101 * (age in 4 weeks (28 days) or other regimens have been used in the trials. Prednisolone is preferred (but not demonstrated years) + 0.147 * (albumin day 0 in g/L) + 0.0165 * (evolution in m − − to be better) over prednisone, as the latter requires conver- bilirubin level in M) 0.206 * (renal insufficiency) 0.0065 * m − sion within the liver to its active form, prednisolone.54 (bilirubin day 0 in M) 0.0096 * (prothrombin time in seconds)]. The area under the receiver operating characteristic (AUROC) curve of the Lille model was 0.89 +/− 0.02, which was Contraindications for steroids validated and shown to be superior than Child-Pugh score, Maddrey score, MELD score, and Glasgow scores for prediction The main contraindications for steroid treatment in patients of 6-month survival. Patients above the ideal cutoff of 0.45 with AH are renal failure, active gastrointestinal bleeding, showed a marked decrease in 6-month survival compared with uncontrolled hyperglycemia, acute pancreatitis, psychosis, those below the cutoff (25% +/− 3.8% versus 85% +/− 2.5%, and infection.54 Here, we discuss infection as a contraindica- p < 0.0001). This cutoff was able to identify approximately tion for steroid therapy. 75% of the observed deaths, and was suggested for early iden- Infections are common and could be fatal in patients with tification and management of subjects with substantial risk of AH.55 Around 25% of patients with severe AH have some form death who would not respond to steroid therapy according of infection at the time of presentation.56 Infection was con- to the Lille model.58 In a follow-up study with individual sidered an absolute contraindication for steroid therapy, but 57 analysis of five randomized trials, patients were classified into recently the position on this matter has changed. In the study by Louvet et al., in a group of patients with severe AH three groups according to their percentile distribution of Lille the most common infections at the time of presentations score and survival analysis: complete responders (Lille score # # were: spontaneous bacterial peritonitis (SBP) 44%, urinary 0.16; 35th percentile), partial responders (Lille score – $ tract infections (UTI) 37%, respiratory infections 13%, and 0.16 0.56; 35th-70th percentile), and null responders (Lille $ others including skin infections in 11%. The 2-month survival 0.56; 70th percentile). Twenty-eight-day survival was of patients infected and treated before initiation of steroids strongly associated with these groupings (91.1 +/− 2.7% vs. was similar to that of patients not infected before steroids. 79.4 +/− 3.8% vs. 53.3 +/− 5.1%, respectively, p < 0.0001). In the follow-up, infection developed in 24% of patients. Overall, these studies show that steroid therapy is beneficial The pattern of infections after steroid treatment was different mainly in complete responders and partial responders but not for those patients who had infection prior to steroid therapy. in null responders. Therefore, steroids should not be continued The most common infections were pulmonary 40.3%, fol- beyond 7 days in null responders based on their Lille score, as lowed by SBP or bacteremia 28.1%, UTI 17.5%, and other there will be no survival benefit in this population and the use of infections 14.1%. Patients infected after corticosteroids had corticosteroids, in general, are not without risks.45

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Pentoxifylline Combination therapy (PTX plus corticosteroids) vs. corticosteroid monotherapy Pentoxifylline (PTX) is a phosphodiesterase inhibitor and a possible TNF-a inhibitor.54,59 The first pilot study with PTX was conducted in 1991 by McHutchison et al. in patients with In a multicenter randomized control trial of 270 patients with severe AH (DF score $ 32), and they showed that PTX could severe AH, there was no statistical difference in 6-month reduce the development of hepatorenal syndrome and mortal- survival between 4 weeks of dual therapy with prednisolone ity compared to those receiving placebo.60,61 Subsequently, a and PTX vs. prednisolone monotherapy. The study was underpowered to detect a significant difference in hepatore- study was conducted from Rancho Los Amigos California in nal syndrome between the two groups.75 The analysis of 2000 by Akriviadis et al. in 101 patients with severe AH in a pooled data from three clinical trials did not show survival 4 week double-blind randomized trial (PTX 400 mg orally, three benefit from PTX and steroid combination therapy when times daily) compared to placebo. The study confirmed that compared to steroid monotherapy.63 PTX was also studied treatment with PTX improves short-term survival in patients as a salvage option in steroid null responders, but there with AH, and the benefit was attributed to a decrease in the was no benefit.29,76 These findings were confirmed in the 62 risk of developing hepatorenal syndrome. multicenter European STOPAH trial, as discussed previously. The American Association for the Study of Liver Disease Taken together, this data demonstrates that there is no ther- (AASLD) guidelines recommend the use of PTX for severe apeutic benefit of dual therapy in comparison with steroid 2,63 AH if there are contraindications to corticosteroids. monotherapy in the management of patients with severe A few trials have evaluated the efficacy of PTX in the manage- AH.66 ment of AH in comparison to placebo. Recent meta-analysis of four clinical trials, three in abstract and one in paper format, Anti-TNFs showed a reduced incidence of fatal hepatorenal syndrome with PTX when compared to placebo (relative risk (RR): 0.47, Experimental and clinical evidence has shown that TNF-a is a 0.26–0.86, p < 0.01) but no survival benefit at 1 month major contributor to the pathogenesis of AH, and elevated 63–67 (RR: 0.58, 0.31–1.07, p =0.06). In the recent study TNF-a is a major predictor of decreased long-term survival in STOPAH (Steroid Or Pentoxifylline for Alcoholic Hepatitis) trial these patients.77,78 The role of Infliximab and etanercept that will be discussed, PTX had no impact on survival compared have been studied in the treatment of AH and will be dis- to placebo in the treatment of severe AH.68 cussed here.

PTX vs. Corticosteroids Etanercept

In the meta-analysis of three trials that compared the efficacy In a multicenter study by Boetticher et al., 48 patients with of PTX vs. prednisolone in severe AH, two as papers and one severe AH were randomized to receive either etanercept as an interim report, there were no significant differences (TNF-a neutralizing molecule) or placebo for 3 weeks. The between treatment groups.63,69–71 In a recent multicenter results showed similar 1-month mortality (22.7% vs. 36.4%, randomized noninferiority trial comparing PTX vs. predniso- odds ratio (OR) 1.8; 95% confidence interval (CI), 0.5-6.5). lone, the authors concluded that the efficacy of PTX is not The 6-month mortality was significantly higher in the etaner- statistically equivalent to the efficacy of prednisolone, sup- cept group than in the placebo group (57.7% vs 22.7%, porting prednisolone as a preferred option.72 OR, 4.6; 95% CI, 1.3-16.4; p = 0.17). The rate of infectious Although it has been difficult to confirm the superiority of serious adverse events were significantly higher in the PTX or corticosteroids in the treatment of severe AH in a etanercept group than the placebo group (34.6% vs. 9.1%, p = 0.04), suggesting that the increase in late mortality in sufficiently large population,73 this was recently studied in patients receiving etanercept was related to impaired liver the STOPAH trial in more than thousand patients. In this regeneration or suppression of the immune system by multicenter double blinded randomized trial, 2 3 2 factorials etanercept.79 were designed to study the benefit of both prednisolone 40 mg and/or PTX 400 mg TID for 4 weeks. In total, 1,103 patients with a clinical diagnosis of severe AH (DF $ 32) Infliximab were randomized into four arms: A. Placebo/Placebo; B. Pre- In a multicenter study by Naveau et al., 36 patients with dnisolone/Placebo; C. PTX/Placebo; D. PTX/Prednisolone. At severe AH were randomized to receive intravenous infliximab 28 days, the percentage of death in the arms were 17, 14, 19, (10 mg/kg) in weeks 0, 2, and 4 or placebo. All patients and 13, respectively. Prednisolone reduced the risk of 28-day received prednisolone 40 mg/day for 4 weeks. The probability mortality, but this benefit was not sustained at 3 months and of death at 2 months was higher in patients who received 1 year. As expected, infection was more common in the pre- infliximab than in those who received placebo, but the differ- dnisolone group (13%) than in the no prednisolone group ence was not statistically significant. This study had to be (7%) (p = 0.002). This study clearly shows that PTX has no stopped early by the follow-up committee and the sponsor. The impact on disease progression and should no longer be used frequency of severe infection within 2 months was significantly for treatment of severe AH.68,74 It is worth mentioning that higher in the group that received infliximab than that in the transjugular liver biopsy was not a requirement for enrolling placebo group and was thought to be related to significantly patients in the trial.74 lower stimulation capacity of neutrophils.80

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N-acetylcysteine (NAC) transplantation, and only one reported harmful drinking. There was no graft loss due to alcohol in the study.91 A study evaluating combination therapy with prednisolone Short- and long-term outcomes of liver transplant in plus NAC in comparison to prednisolone monotherapy patients with severe AH were reported by Singal et al. showed a significant decrease in mortality with combination using the United Network for Organ Sharing (UNOS) data- therapy at 1 month (8% vs. 24%, p = 0.006) but not at base. Fifty-five patients that were transplanted for AH were 6 months (27% vs. 38%, p = 0.07). Death due to hepatorenal compared with 165 matched patients transplanted for alco- syndrome was less frequent in the combination group than holic cirrhosis, and 1, 3, and 5 year graft survival was 87%, in the steroid monotherapy group at 6 months (9% vs. 82%, and 75%, respectively, and 1, 3, and 5 year patient 22%, p = 0.02). Infections were also less frequent in the pre- survival was 93%, 87%, and 80%, respectively.92 Recidi- dnisolone-NAC group compared to prednisolone only group.31 vism after liver transplant is very variable. Twelve percent At this time, it is not clear if dual therapy with steroid and NAC to 49% of patients with ALD will restart some amount of should become the standard of care in the management of drinking and 10%–15% of patients will resume heavy drink- patients with AH as opposed to steroid alone.81 ing. However, the risk of graft loss due to recidivism is only 0%–5% in AH patients receiving a liver transplant, but is Liver transplantation reported to be higher in some series. Overall, harmful drinking in the post-transplant period can affect the long-term Although liver transplant represents the definite treatment for transplant outcomes, mainly due to poor compliance with patients with decompensated alcoholic cirrhosis, it remains immunosuppressive medications and alcohol induced liver 93–95 an ethically controversial option for the management of injury. patients with ALD, including AH.82,83 The main controversy In summary, early liver transplant is effective in the is based on the public opinion that ALD is self-inflicted and management of patients with severe AH who failed steroid given the high demand for limited organs, the allocation treatment. Patient selection remains extremely controversial should be made to patients who “deserve the liver trans- and needs to be further determined. The patients should plant”.84–86 The other concern is the risk of recidivism.87 be selected based on strict criteria, and it should be empha- Most transplant centers use the 6-month abstinence rule to sized that patients with severe AH who do not respond to achieve two goals: first, this will allow time for the liver to steroids have a very short-term mortality rate without liver 91 recover from the hepatitis due to recent alcohol exposure by transplantation. abstinence and medical treatment; and second, the patient’s commitment to sobriety can be assessed during this time, Future Therapies based on the rationale that duration of abstinence prior to transplant might predict abstinence after liver transplanta- As discussed in this review (summarized in Fig. 1), the tion.83,88,89 Nevertheless, the 6-month rule requirement medical management of AH patients at this time is limited usually is not met by most patients with severe AH that are to steroids. Use of corticosteroids can be associated with sig- null responders to steroids since these patients usually have a nificant risk, such as infection, especially in this sick popula- recent history of alcohol use. Furthermore, pretransplant tion of patients. In addition, the long-term benefit of steroids abstinence does not necessarily predict recidivism after liver is still not established. Recent data suggest that PTX lacks 68 transplant.84,85,90 The other argument against the 6-month efficacy and is no longer recommended (STOPAH trial). rule for liver transplant in patients with severe AH is the Therefore, effective new pharmacologic therapies with high mortality in Lille null responders within this time more favorable side effect profiles are greatly needed for period. Mathurin et al. found that 75% of patients with the treatment of AH patients. One of the challenges of drug severe AH who were not responsive to steroids who did not discovery in the field is the lack of ideal animal models that 96 undergo liver transplant died within the 6-month follow-up entirely mimic human disease. Multiple new clinical trials period.91 are underway with novel therapeutic targets based on Another approach introduced recently is to proceed with essential pathways involved in the pathogenesis of AH. This early liver transplant in patients with severe AH. Liver trans- subject was reviewed in few reviews and at a recent AASLD plant was performed in 26 patients with severe AH who were meeting. Here, we will briefly discuss some of the most null responders to steroid treatment, and outcomes were important therapeutic targets. Alcohol disrupts the intestinal compared to matched controls. These patients were highly epithelial barrier, resulting in increased gut permeability to selected based on a thorough psychosocial evaluation and microbiota. The bacterial endotoxin lipopolysaccharide (LPS) included only 2% of total patients that presented with severe increases in portal circulation and activates toll-like receptor 4 AH. The selection criteria included a lack of response to (TLR4), leading to activation of the innate immune system medical therapy, severe AH as the first liver decompensating and release of various cytokines. Blockage of these pathways event, strong family support, absence of a coexisting severe could have therapeutic implications for the treatment of 16,29,97 psychiatric disorder, and agreement by the patient to adhere AH. to lifelong total alcohol abstinence. The transplant had to be approved by the liver transplant group. The centers used Gut-Liver Axis only 2.9% of available grafts for this indication for liver transplant. The patients with AH who received early liver Disruption of the intestinal epithelial barrier results in transplant had a significantly higher cumulative 6-month increased gut permeability, and has been suggested as an survival than patients who were not transplanted (77.8 +/− 8% essential pathway in the pathophysiology of AH.97,98 Probiot- vs. 23 +/− 8% respectively, p < 0.001), with benefit ics have been used in the management of ALD. Short-term remaining through 2 years of follow-up. Only three patients use of VSL#3 in alcoholic cirrhosis patients was shown to resumed alcohol drinking at 720, 740, and 1,140 days after decrease markers of oxidative stress and plasma cytokines

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Fig. 1. Proposed algorithm for management of patients with alcoholic hepatitis. * Note that pentoxifylline is not included in this algorithm. Based on recent STOPAH trial,68 it was shown that pentoxifylline had no impact on disease progression and is no longer recommended for the treatment of severe alcoholic hepatitis. ** In a separate analysis, patients were classified into three groups based on their Lille score and survival, and cutoff of 0.56 determined nonresponse: complete responders (Lille score # 0.16; # 35th percentile), partial responders (Lille score 0.16–0.56; 35th–70th percentile) and null responders (Lille $ 0.56; $ 70th percentile).45 Abbreviations: AH, alcoholic hepatitis; DF, discriminant function; GI, gastrointestinal; MELD, Model for End-stage Liver Disease; PHTN, portal hypertension; w, week(s).

(TNF-a,IL-6,andIL-8).99 Kirpich et al. conducted a pros- alcoholic patients, those who were treated with probiotics had pective, randomized, clinical trial to study the effects of significantly lower AST, ALT, gamma glutamyl-transpeptidase alcohol and probiotics on the bowel flora and alcohol-induced (GGT), and total bilirubin levels than those treated with stand- liver injury on 66 hospitalized patients. Patients were ran- ard therapy alone.100 Currently there are ongoing clinical trials domized to receive 5 days of Bifidobacterium bifidum and evaluating the effect ciprofloxacin, amoxicillin clavulanate, Lactobacillus plantarum 8PA3 versus standard therapy alone, Lactobacillus rhamnosus, zinc, and rifaximin in the manage- abstinence plus vitamins. After 5 days of probiotic therapy in ment of patients with AH.101

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Innate immune system hepatic progenitor cells, and mature hepatocyte-like cells under defined conditions.112–114,116 The two main uses of Activation of the innate immune system is one of the main human iPSC technology for liver diseases are drug discovery steps in the development of AH. Activation of kupffer cells and cell transplantation.122 Using iPSCs for drug development, causes release of IL-1b, a potent pro-inflammatory cytokine. would require establishing patient iPSC-based disease models IL1b binds to its receptor IL-1R1, which leads to the recruit- for testing potential drugs. The human iPSC-hepatic differen- ment of other inflammatory cells.102,103 It was shown recently tiation technology has allowed us to establish effective large- that an IL1b receptor antagonist was protective against alco- scale drug screening and drug discovery studies using an holic liver injury in an animal model.103 Use of the IL1b recep- iPSC-based liver disease model, which is critical for translating tor antagonist Anakinra in combination with PTX and zinc is the iPSC technology into novel therapies for untreatable liver currently being tested in the management of AH.101 diseases.116 More recently, it was observed that exposure to Another key cytokine, IL-22, was suggested alone or in ethanol at the pathophysiological dosage induced apoptosis combination with steroids to be a potential therapeutic option during differentiation of human iPSC-derived endoderm cells to ameliorate ALD, due to its multiple beneficial properties, into hepatic progenitor cells and significantly reduced prolifer- including its antioxidant, antiapoptotic, antisteatotic, antimi- ative activity of mature stage hepatic cells.123 Importantly, crobial, and proliferative properties. Therefore, IL-22 might increased amounts of lipid droplets were detected in ethanol- be useful in treating patients with AH. Additionally, side effects treated iPSC-derived hepatocytes compared to controls, indi- due to IL22 may be minimal, as IL-22R expression is restricted cating the possibility of modeling ALD using human iPSCs. expression to epithelial cells, such as hepatocytes.104 Pathogenesis research using patient-relevant iPSC models of AH will aid in the discovery of better cellular and molecular Apoptosis Pathways targets for drug development. Regenerative therapy using iPSCs will require in vivo functional testing of these cells. Apoptosis is a prominent feature in the pathophysiology of Perhaps a more immediate use of human iPSC-derived cells AH.105 Excessive hepatocyte apoptosis stimulates inflamma- is the in vitro screening of candidate drugs, as improving and tion and results in the production of proinflammatory cytokines evaluating the in vivo function for most iPSC-derived cell and reactive oxygen species by the innate immune system.106 types is still in the future. Although there are many technical Therefore, inhibition of apoptosis with different caspase inhib- hurdles to overcome before establishing iPSC-based tailored itors can be therapeutic in patients with AH. The caspase inhib- therapy for ALD patients, the potential of pluripotent stem cell itor GS-9450 was shown to be effective in the treatment of therapy is great. Together with continued improvements in patients with nonalcoholic steatohepatitis (NASH).107 The current therapies, iPSC technology provides an additional effectiveness of a pancaspase inhibitor compared to placebo new therapy, but much more work needs to be done to dem- is currently being evaluated in patients with severe AH who onstrate their true value in the clinic. have contraindication to steroids.29 Conclusions Farnesoid X receptor (FXR) ALD remains one of the leading causes of liver disease in the FXR is a nuclear receptor for bile acids, and its activation has United States. The clinical and pathological spectrum of been shown to be essential in the treatment of primary disease includes alcoholic fatty liver, alcoholic hepatitis and biliary cirrhosis (PBC).108,109 Obeticholic acid (OCA) is a semi- alcoholic cirrhosis. In general, diagnosis of AH solely based on synthetic analogue of the primary bile acid chenodeoxycholic clinical and laboratory data might be challenging, as there is acid (CDCA), which selectively activates FXR.110 In the FLINT significant overlap between various forms of ALD. Liver biopsy trial, treatment with OCA improved insulin resistance and remains the gold standard when the diagnosis is unclear, liver histology in patients with NASH. A phase II clinical trial especially when steroids are being considered. The most of OCA in AH is currently underway.108 important predictor for determining long-term survival in AH patients is alcohol abstinence. Steroids have some short term Potential of human induced pluripotent stem cell benefit in treatment of severe AH, but long term effect is (iPSC) based therapy for ALD unclear. Overall, PTX does not improve outcomes in patients with severe AH. Liver transplant has shown good results in the Challenges remain for developing therapeutic approaches management of AH patients who have failed medical therapy, targeting ALD, including AH, partly because of the lack of however patient selection remains a major challenge. There human-relevant model systems to study the effect of alcohol are multiple ongoing clinical trials targeting novel pathways on the liver.111 In recent years, human iPSCs have been involved in the pathogenesis of AH. Human iPSCs are currently generated from diverse human somatic cells,112–117 and being used as a novel technology for studying the patho- these cells can then be differentiated into a spectrum of genesis of ALD with potential for drug discovery. mature human cell types, including functional hepato- 112–114,116,118–123 cytes. This development has enabled us to Conflict of interest access an unlimited supply of hepatocytes, which was one of the major challenges in the past. Moreover, human iPSCs None retain the same genetic information of the donor (i.e., patient) tissues, making iPSCs a promising resource to study human diseases.112,113,116,117,120 Human iPSC lines from Author contributions healthy donors and multiple liver disease patients have been established.112–114,119 Using step-wise hepatic differentiation Designing the manuscript, collecting the data, drafting the protocols, these iPSCs can be induced to definite endoderm, article and reading and approving the final draft of the

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Outcomes versus prednisolone for severe alcoholic hepatitis: a randomized controlled after liver transplantation for alcoholic hepatitis are similar to alcoholic cir- trial. World J Gastroenterol 2009;15:1613–1619. doi: 10.3748/wjg.15. rhosis: exploratory analysis from the UNOS database. Hepatology 2012;55: 1613 1398–1405. doi: 10.1002/hep.25544 [70] Garrido García JR, Sánchez Her- nández G, Melchor López A, Elizalde [93] Cuadrado A, Fabrega E, Casafont F, Pons-Romero F. Alcohol recidivism Barrera CI, Sánchez Vargas L. Pentoxifilina versus esteroide en la sobrevi- impairs long-term patient survival after orthotopic liver transplantation for vencia a corto plazo en hepatitis aguda alcohólica severa. Med Int Mex alcoholic liver disease. Liver Transpl 2005;11:420–426. doi: 10.1002/lt. 2012;28:227–233. 20386

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[94] Pageaux GP, Bismuth M, Perney P, Costes V, Jaber S, Possoz P, et al. Alcohol [109] Hirschfield GM, Mason A, Luketic V, Lindor K, Gordon SC, Mayo M, et al. relapse after liver transplantation for alcoholic liver disease: does it matter? Efficacy of obeticholic Acid in patients with primary biliary cirrhosis and J Hepatol 2003;38:629–634. doi: 10.1016/S0168-8278(03)00088-6 inadequate response to ursodeoxycholic Acid. Gastroenterology 2015; [95] Mackie J, Groves K, Hoyle A, Garcia C, Garcia R, Gunson B, et al. Orthotopic 148:751–761.e758. doi: 10.1053/j.gastro.2014.12.005 liver transplantation for alcoholic liver disease: a retrospective analysis of [110] Neuschwander-Tetri BA. Targeting the FXR nuclear receptor to treat liver survival, recidivism, and risk factors predisposing to recidivism. Liver disease. Gastroenterology 2015;148:704–706. doi: 10.1053/j.gastro. Transpl 2001;7:418–427. doi: 10.1053/jlts.2001.23789 2015.02.037 [96] Bertola A, Mathews S, Ki SH, Wang H, Gao B. Mouse model of chronic and [111] Weber SN, Wasmuth HE. Liver fibrosis: from animal models to mapping of binge ethanol feeding (the NIAAA model). Nat Protoc 2013;8:627–637. doi: human risk variants. Best Pract Res Clin Gastroenterol 2010;24:635–646. 10.1038/nprot.2013.032 doi: 10.1016/j.bpg.2010.07.013 [97] Szabo G. Gut-liver axis in alcoholic liver disease. Gastroenterology 2015; [112] Choi SM, Liu H, Chaudhari P, Kim Y, Cheng L, Feng J, et al. Reprogramming – 148:30 36. doi: 10.1053/j.gastro.2014.10.042 of EBV-immortalized B-lymphocyte cell lines into induced pluripotent stem [98] Yan AW, Schnabl B. Bacterial translocation and changes in the intestinal cells. Blood 2011;118:1801–1805. doi: 10.1182/blood-2011-03-340620 microbiome associated with alcoholic liver disease. World J Hepatol 2012; [113] Liu H, Kim Y, Sharkis S, Marchionni L, Jang YY. In vivo liver regeneration – 4:110 118. doi: 10.4254/wjh.v4.i4.110 potential of human induced pluripotent stem cells from diverse origins. ’ [99] Loguercio C, Federico A, Tuccillo C, Terracciano F, D Auria MV, De Simone C, Sci Transl Med 2011;3:82ra39. doi: 10.1126/scitranslmed.3002376 et al. Beneficial effects of a probiotic VSL#3 on parameters of liver dysfunc- [114] Liu H, Ye Z, Kim Y, Sharkis S, Jang YY. Generation of endoderm-derived – tion in chronic liver diseases. J Clin Gastroenterol 2005;39:540 543. doi: human induced pluripotent stem cells from primary hepatocytes. Hepatology 10.1097/01.mcg.0000165671.25272.0f 2010;51:1810–1819. doi: 10.1002/hep.23626 [100] Kirpich IA, Solovieva NV, Leikhter SN, Shidakova NA, Lebedeva OV, Sidorov [115] Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, PI, et al. Probiotics restore bowel flora and improve liver enzymes in human et al. Induced pluripotent stem cell lines derived from human somatic cells. alcohol-induced liver injury: a pilot study. Alcohol 2008;42:675–682. doi: Science 2007;318:1917–1920. doi: 10.1126/science.1151526 10.1016/j.alcohol.2008.08.006 [116] Choi SM, Kim Y, Shim JS, Park JT, Wang RH, Leach SD, et al. Efficient drug [101] Louvet A, Mathurin P. Alcoholic liver disease: mechanisms of injury and screening and gene correction for treating liver disease using patient- targeted treatment. Nat Rev Gastroenterol Hepatol 2015;12:231–242. specific stem cells. Hepatology 2013;57:2458–2468. doi: 10.1002/hep. doi: 10.1038/nrgastro.2015.35 26237 [102] Dinarello CA, Simon A, van der Meer JW. Treating inflammation by blocking [117] Ye Z, Zhan H, Mali P, Dowey S, Williams DM, Jang YY, et al. Human-induced interleukin-1 in a broad spectrum of diseases. Nat Rev Drug Discov 2012; pluripotent stem cells from blood cells of healthy donors and patients 11:633–652. doi: 10.1038/nrd3800 with acquired blood disorders. Blood 2009;114:5473–5480. doi: 10.1182/ [103] Petrasek J, Bala S, Csak T, Lippai D, Kodys K, Menashy V, et al. IL-1 receptor antagonist ameliorates inflammasome-dependent alcoholic steatohepatitis blood-2009-04-217406 in mice. J Clin Invest 2012;122:3476–3489. doi: 10.1172/JCI60777 [118] Chaudhari P, Prasad N, Tian L, Jang YY. Determination of Functional Activity [104] Ki SH, Park O, Zheng M, Morales-Ibanez O, Kolls JK, Bataller R, et al. of Human iPSC-Derived Hepatocytes by Measurement of CYP Metabolism. – Interleukin-22 treatment ameliorates alcoholic liver injury in a murine Methods Mol Biol 2016;1357:383 394. doi: 10.1007/7651_2014_145 model of chronic-binge ethanol feeding: role of signal transducer and activa- [119] Choi SM, Kim Y, Liu H, Chaudhari P, Ye Z, Jang YY. Liver engraftment poten- tor of transcription 3. Hepatology 2010;52:1291–1300. doi: 10.1002/hep. tial of hepatic cells derived from patient-specific induced pluripotent stem – 23837 cells. Cell Cycle 2011;10:2423 2427. doi: 10.4161/cc.10.15.16869 [105] Natori S, Rust C, Stadheim LM, Srinivasan A, Burgart LJ, Gores GJ. Hepa- [120] Chun YS, Chaudhari P, Jang YY. Applications of patient-specific induced plu- tocyte apoptosis is a pathologic feature of human alcoholic hepatitis. J ripotent stem cells; focused on disease modeling, drug screening and ther- Hepatol 2001;34:248–253. doi: 10.1016/S0168-8278(00)00089-1 apeutic potentials for liver disease. Int J Biol Sci 2010;6:796–805. doi: 10. [106] Chakraborty JB, Oakley F, Walsh MJ. Mechanisms and biomarkers of apop- 7150/ijbs.6.796 tosis in liver disease and fibrosis. Int J Hepatol 2012;2012:648915. doi: 10. [121] Jang YY, Ye Z, Cheng L. Molecular imaging and stem cell research. Mol 1155/2012/648915 Imaging 2011;10:111–122. [107] Ratziu V, Sheikh MY, Sanyal AJ, Lim JK, Conjeevaram H, Chalasani N, et al. [122] Sharkis SJ, Jones RJ, Civin C, Jang YY. Pluripotent stem cell-based cancer A phase 2, randomized, double-blind, placebo-controlled study of GS-9450 therapy: promise and challenges. Sci Transl Med 2012;4:127ps129. doi: in subjects with nonalcoholic steatohepatitis. Hepatology 2012;55:419–428. 10.1126/scitranslmed.3003920 doi: 10.1002/hep.24747 [123] Tian L, Prasad N, Jang YY. In Vitro Modeling of Alcohol-Induced Liver Injury [108] Ali AH, Carey EJ, Lindor KD. Recent advances in the development of farne- Using Human-Induced Pluripotent Stem Cells. Methods Mol Biol 2016; soid X receptor agonists. Ann Transl Med 2015;3:5. 1353:271–283. doi: 10.1007/7651_2014_168

122 Journal of Clinical and Translational Hepatology 2016 vol. 4 | 113–122 Review Article

Nonalcoholic Lipid Accumulation and Hepatocyte Malignant Transformation

Juanjuan Gu#1, Min Yao#2, Dengbing Yao3, Li Wang4, Xuli Yang1 and Dengfu Yao*1

1Research Center of Clinical Medicine and Department of Oncology, Affiliated Hospital of Nantong University, Nantong, Jiangsu, China; 2Department of Immunology, Medical School of Nantong University, Nantong, Jiangsu, China; 3School of Life Sciences, Nantong University, Nantong, Jiangsu, China; 4Department of Medical Informatics, Medical School of Nantong University, Nantong, Jiangsu, China

Abstract among all malignancies, with its mortality rate being almost equal to its morbidity rate; moreover, a large portion of cases Worldwide incidence of hepatocellular carcinoma (HCC) is has been localized to the inshore area of the Yangtze River. steadily increasing, highlighting its status as a public health The development of HCC is a complex multi-step process concern, particularly due to its significant association with involving multiple genes. Major risk factors of HCC include other comorbidities, such as diabetes. However, nonalcoholic hepatitis B virus (HBV) or hepatitis C virus (HCV) infection, fatty liver disease (NAFLD) has emerged as a primary risk alcoholic liver disease, and exposure to nitrosamines, afla- factor, with its own prevalence increasing in recent years, and toxin, and other harmful substances.3 Chronic persistent it has gradually caught up with the historical primary etio- infection with hepatoviruses remains the main pathological logical factors of infection with hepatitis B virus and hepatitis factor of inducing cirrhosis and HCC in nonalcoholic individuals C virus, exposure to aflatoxin, or alcohol liver disease. The in China; however, the recent decades have seen a sweeping deeply worrisome aspects of all of these high risk factors, change in the Chinese dietary structure and lifestyle, and however, are their remarkable presence within populations. this has been accompanied by a drastic increase in incidence Systemic and genetic mechanisms involved in the malignant of fatty liver disease (FLD).4 transformation of liver cells, as well as useful biomarkers of The median prevalences of alcoholic fatty liver disease early stage HCC are being investigated. However, the exact (AFLD) and nonalcoholic fatty liver disease (NAFLD) have mechanisms underlying the interrelation of NAFLD and HCC been reported as 4.5% and 15.0%, respectively.5 Unfortu- remain largely unknown. In this review, some of the recent nately, when left untreated, both nonalcoholic steato-hepatitis advances in our understanding of liver lipid accumulation are (NASH) and alcoholic hepatitis can progress to liver fibrosis, summarized and discussed to provide insights into the relation- cirrhosis and liver cancer; although, the exact underlying ship between NAFLD and hepatocyte malignant transformation. mechanisms that lead to malignant transformation of infected © 2016 The Second Affiliated Hospital of Chongqing Medical liver cells remain largely unknown. Most HCC patients die quickly University. Published by XIA & HE Publishing Inc. All rights because of rapid tumor growth, and surgical operation or liver reserved. transplantation remains the only effective treatment available for HCC.4 This review summarizes the latest advances in our collective understanding of the relationship between NAFLD and hepatocyte malignant transformation. Introduction

th Hepatocellular carcinoma (HCC) is the 5 most common Mitochondria and fatty b-oxidation malignant tumor in the world, ranking as the 3rd most frequent cause of cancer mortality.1,2 In China alone, HCC ranks as 2nd Mitochondria

Keywords: Nonalcoholic fatty liver disease; Hepatocellular carcinoma; Metabolism. The liver is one of the most important organs for maintaining Abbreviations: CPT, carnitine palmitoyltransferase; ER, endoplasmic reticulum; energy supply and carrying out lipid metabolism in the FFA, free fatty acid; ERK, extracellular signal-regulated kinase; GWAS, genome- human body. The peroxisomal compartment in hepatocytes wide association studies; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; host several essential metabolic conversions. Such conver- HCV, hepatitis C virus; HSC, hepatic stellate cell; IL-6, interleukin-6; IR, insulin resistance; miR, microRNA; NAFLD, nonalcoholic fatty liver disease; NASH, non- sions, however, are defective in peroxisomal disorders, as alcoholic fatty hepatitis; NF-kB, nuclear factor kappa B; NKT, natural killer T cells; a result of either failure to properly or adequately import OPN, osteopontin; PNPLA, patatin-like phospholipase domain-containing protein; enzymes into the organelle or presence of mutations in the RNS, reactive nitrogen species; ROS, reactive oxygen species; SNP, single nucleo- enzymes themselves or their cognate transporters that are tide polymorphism; SREBP, sterol regulatory element binding protein; TAGE, toxic advanced glycation end-products. necessary to transfer the substrates across the peroxisomal Received: 17 March 2016; Revised: 13 May 2016; Accepted: 14 May 2016 membrane (Fig. 1). Hepatic pathology is one of the cardinal q DOI: 10.14218/JCTH.2016.00010. features in disorders of peroxisome biogenesis and peroxi- # These authors contributed equally to this work somal b-oxidation, although it only rarely determines clinical *Correspondence to: Professor Dengfu Yao, Research Center of Clinical Medicine, Affiliated Hospital of Nantong University, Nantong 226001, Jiangsu Province, China. fate. Besides these morphological changes, however, the Tel: +86-513-85052297, Fax: +86-513-85052554, E-mail: [email protected] peroxisome malfunctions cause other observable impacts

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Fig. 1. Abnormality of fatty acid oxidation in mitochondria. Carnitine palmitoyltransferase (CPT)-I or CPT-II regulate fatty acid metabolism. Mitochondrial fatty acid b-oxidation requires successive carnitine acyltransferases to translocate acyl-coenzyme A (acyl-CoA) from the cytoplasm into the mitochondrial matrix. CPT-I, as an initial and rate-limiting enzyme, generates acyl-carnitines that traverse mitochondrial membranes via specific transporters to enter the mitochondrial matrix. Then, CPT-II pro- duces acyl-CoA from acyl-carnitine for fatty acid b-oxidation to acetyl-CoA, after which carnitine crosses the mitochondrial inner membrane and binds to the endogenous or exogenous acyl-CoA to prevent accumulation of acyl-CoA, causing cell poisoning. Abbreviations: CoA, Coenzyme A; ACC, Acetyl-CoA carboxylase; TCA, Tricarboxylic acid cycle; UCP, Uncoupling protein; I II III IV, Electron transfer complex.

124 Journal of Clinical and Translational Hepatology 2016 vol. 4 | 123–130 Gu J. et al: NAFLD in HCC on various cellular compartments, such as the mitochondria and endoplasmic reticulum (ER).6

Carnitine level

Carnitine is a physiological substance that is essential for the proper metabolism of fat and energy production; it serves in transport of both long and medium fatty acid chains. Carnitine attracts these fatty acid chains, breaks them down and then carries them to the mitochondria of the cells where they are metabolized (burned). The ultimate result is that the body burns more fats, and in the process, provides the body with more natural energy. Previous studies have revealed that the circulating carnitine analogue 3-(2, 2, 2-trimethyl hydrazinium) propionate dihydrate (THP) affects increased liver weight due to lipid accumulation in wild-type mice (Fig. 2).7,8

Carnitine palmitoyltransferases (CPTs)

Hepatic CPT-II is a mitochondrial protein that is transported to the mitochondrial inner membrane. It, together with CPT-I, oxidizes long-chain fatty acids in mitochondria. Mutation of its encoding gene or defects in the protein product are associated with mitochondrial long-chain fatty acid oxidation disorders. Decreased activity of CPTs is a hallmark of disorders of mitochondrial fatty acid oxidation with autosomal recessive modes of inheritance. The CPT variants exert a dominant- negative effect on the homo-tetrameric protein of the enzyme (Fig. 3), featuring reduced activities, thermal instability, decreased fatty acid b-oxidation (30;59%), decreased intracellular ATP (48;79%), marked reduction of mitochondrial membrane potential at 418C, and shortened half-lives; moreover, these variant proteins are polyubiquitinated and rapidly degraded by a lactacystin-sensitive proteasome 6 pathway. The unstable CPT-II variants, with decreased Fig. 2. Hepatic lipid accumulation with carnitine analogues treatment. enzymatic activities, may cause mitochondrial fuel utiliza- A-D. Oil red O staining of liver lipids. A & B. Liver tissues of control mice. C & D. tion to fall below the phenotypic threshold during conditions Liver tissues of experimental mice. E. Weight changes in different tissues of mice of high fever, and thus might represent potential mecha- treated with carnitine analogues. nisms for NAFLD.

Abnormal liver lipid accumulation can be mediated by hepatic inflammatory cytokines or hepatitis virus infection and reactions with polyunsaturated fatty acids Lipid accumulation derived from hepatocyte membrane phospholipids that result in production of reactive aldehydes as lipid oxidation Lipid accumulation in liver or HCC will cause alterations in (LPO) byproducts. One of such byproducts, 4-hydroxynone- tumor-associated molecular signaling, including pathways nal (4 HNE), can react with DNA to form mutagenic exocyclic involving the common factors of nuclear factor-kappa B etheno-DNA adducts, preferentially forming in codon 249 of (NF-kB), c-Jun N-terminal kinase (JNK) activation protein-1, TP53 and resulting in inactivation of the tumor suppressor as well as overexpression HCC-related genes. For example, p53 and providing a secondary growth advantage and cellular hepatic unsaturated fatty acids inhibit the expression of resistance to apoptosis.11 phosphatase and tensin homologue (PTEN) via activation of an NF-kB/mammalian target of rapamycin (mTOR) complex.9 PTEN is a tumor suppressor gene, which regulates the PKB/ Adipokines serine-threonine kinase protein kinase B (akt) pathway, and its deficiency induces liver cell proliferation, thereby inhibiting cell Accumulating basic and clinical data have demonstrated that apoptosis and promoting tumor formation; these processes leptin plays an important role in hepatic stellate cell (HSC) have been confirmed in mouse models of liver-specific PTEN activation and hepatic fibrosis, in many aspects of both. First, deficiency that show a phenotype resembling NASH features of leptin can stimulate production of the tissue inhibitor of metal- human, with development of liver steatosis, hepatocyte loproteinase-1 (TIMP-1) via the Janus kinase/signal transducer inflammation and/or fibrosis.10 and activator of transcription (JAK/STAT) pathway, thereby DNA damage can affect hepatic lipid metabolism as well. promoting fibrogenesis. Second, it can act on hepatic endo- Reactive oxygen species (ROS) are known to be an important thelial cells, thereby promoting tube formation and migration factor in carcinogenesis. ROS have been shown as increased in and affecting angiogenesis.12 Finally, the level of leptin expres- NAFLD patients with contiguous DNA damage; their induction sion is significantly higher in NAFLD patients, and it has been

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Fig. 3. CPT-II gene mutation and hepatic lipid accumulation. A. CPT-II gene exons 1–5. B. Sequence fragments of the CPT-II gene exon 4 were amplified on the mitochondrial inner membrane. Mutation analysis was carried out using the specific primers to sequence the 1974 nucleotides encoding 658 amino acids. Comparison with the original sequence from GenBank identified two substitution sites: 1618 (G/A) and 1858 (T/C), for V368I and F448L amino acid changes respectively.

confirmed in mouse models that presented with increased Furthermore, tumor necrosis factor (TNF) is a major angiogenesis or progression from NASH to HCC.9,13 adipose-derived cytokine and a potent activator of pro- Adiponectin produced by adipose tissue is an anti- oncogenic pathways involving mTOR, JNK, NF-kB and extrac- inflammatory cytokine and regulates the metabolism of ellular signal-regulated kinases (ERKs). Interleukin (IL)-6 can blood glucose and hepatic fatty acid; furthermore, adiponec- bind to its receptors on liver or non-parenchymal cells to tin is decreased in patients with NAFLD. According to the data promote the binding of signal-transducing receptor (gp130) from animal models xenografted with HCC cell lines, this to the IL-6 receptor (R) complex to activate JAK1; the cytokine is able to increase JNK activation and induce cell activation and phosphorylation of STAT3 factor then promotes apoptosis with AMPK alteration, which could also inhibit mTOR 12,15 phosphorylation or xenograft growth in nude mice.12 Addi- cell proliferation and anti-apoptosis, indicating that the tionally, it has been shown to inhibit tumor growth and meta- coefficient action of adiponectin from adipose tissues, along stasis by suppression of tumor angiogenesis and of levels with related cytokines, affect fatty acid metabolism and hep- of circulating adiponectin in NASH model, which favours atocyte malignant transformation via a multitude of signaling tumourigenesis.14 molecules or pathways.

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Inducing roles of related proteins of fibrogenic biomarkers and the process of cell death.19 In addition, fatty liver represents a pro-metastatic microenviron- Sterol regulatory element binding proteins (SREBPs) ment, with human HCC in the tissue promoting HCC migration and proliferation.20 SREBPs are the main regulators of hepatic lipogenesis and activate hepatic steatosis at the early stage. In HCC, SREBP1 Immune cells can significantly induce lipogenesis and its overexpression is 9 associated with poor prognosis. SREBP1c mRNA has been Immune cells activated by metabolic stress will migrate to the reported as up-regulated in HCC tissues, as compared with liver, interact with cells in that tissue and drive the progres- adjacent non-cancerous or normal liver tissues. SREBP1 has sion from NAFLD to HCC. For example, the metabolic activa- also been shown to activate the fat pathway in human HCC tion of intrahepatic CD8+ T or NKT cells can promote NASH cell lines. Moreover, inhibition of SREBP1 expression results in and HCC via crosstalk with liver cells. The NKT cells primarily cell growth arrest and apoptosis, as well as increased cell 16 cause steatosis in liver tissues by secreting a type II trans- proliferation ability. membrane protein (a TNF ligand super-family member, TNFSF14); moreover, it has been shown that both the Tripartite motif 24 (TRIM24) CD8+ T and NKT cells cooperatively induce liver injury in a study of a new developed C57BL/6 mouse model that reca- a TRIM24 (previously known as TIF1 ) is an epigenetic co- pitulates the key features of metabolic syndrome by feeding a regulator of some gene transcription that directly or indirectly choline-deficient high-fat diet.21 Hepatic immune cells recog- inhibits mouse hepatic lipid accumulation, liver cell inflam- nize cell injury or pathogen invasion via their intracellular or mation, liver fibrosis and hepatocyte damage. Additionally, -/- surface-expressed pattern recognition receptors, subse- global expression analyses of TRIM24 livers unveiled sig- quently initiating signaling cascades that trigger the release naling pathways (unfolded protein response or endoplasmic of factors promoting inflammatory response during NAFLD reticulum nuclear) that are closely associated with some fea- progression;22 this phenomenon supports the theory that tures of patients with NAFLD, inflammatory stress, cell apop- transition from NASH to HCC involves the liver cell lympho- tosis and tissue damage. The loss of TRIM24 expression in toxin-b receptor (LTbR) and NF-kB signaling. liver tissues over time could lead to the progression from NAFLD to NASH or HCC in a time-dependent manner.17 Polyploidization Osteopontin (OPN) Polyploidization represents a huge change in the genome. — — Human hepatic OPN is a multifunctional protein involved in During liver development and even throughout adult life some pathological conditions including immunity, inflamma- physiological polyploidization is likely to occur, but in NAFLD it tion, fibrosis and the development of HCC in different tissues. takes place mainly due to cell cycle suspension in the S/G2 Its deficiency in obese mice (fed with a high-fat diet) was phases. Furthermore, hepatic oxidative stress promotes shown to reduce hepatic steatosis and hepatocyte inflamma- histopathological polyploidization, suggesting that it should tion; in addition, these mice showed attenuated liver cell be an early event in NAFLD that consequently contributes to 23 swelling, portal leukocyte infiltration and macrophage accu- HCC development. mulation. OPN is induced by Hedgehog signaling, and may directly promote pro-fibrogenic responses in steatohepatitis; it also acts as a paracrine factor secreted by the bile duct or Alterations of small molecules natural killer T (NKT) cells and as an autocrine factor promoting fibrosis in HSCs. Silencing of OPN gene transcription by Oxidative stress specific shRNA leads to an increase in Bax expression, a decrease in protein expression of Bcl-2/Bcl-xL and X-linked A serious imbalance was found between limited antioxidant inhibitor of apoptosis and in activation of NF-kBaswellas defenses and excessive formation of reactive species produced induction of mitochondria-mediated apoptosis in the HCC by hepatic oxidative stress, such as the ROS or reactive 18 cell line of LM3. nitrogen species (RNS).14 Obese persons can have increased free fatty acid (FFA) uptake, which in turn would stimulate FA Promoting role of related cells oxidation to compensate for excessive liver fat storage and accelerate b-oxidation, leading to increased production of HSCs ROS that could damage the mitochondrial membrane and DNA. Oxidative stress may also induce ER dysfunction that is Human HSCs in the perisinusoidal space between sinusoids associated with liver malignancy. The ER plays an important and hepatocytes are the predominant fibrogenic cells of liver role in NAFLD pathogenesis; moreover, consecutive increas- tissues and are activated by hepatocyte injury, whereby ing oxidative stress, inflammation and activation of NF-kB and they trans-differentiate from a quiescent state to proliferative JNK signaling pathways lead to the accumulation of intra- matrix-producing myofibroblasts. The subsequent excessive cellular lipids.9 HCC is characterized by high activation of ERK, production of extracellular matrix might result in liver cirrho- which occurs via PIK13 activation. ER stress also activates sis. Human HSCs express amphiregulin and TNF-a converting the nuclear factor erythroid 2-related factor 2 and transcrip- enzyme (TCE) in a time-dependent manner. Human amphir- tion factor-6, both of which regulate the small heterodimer egulin can increase cell proliferation through the EGFR, phos- partner nuclear receptor, leading to its suppression. This phatidylinositol–3 kinase (PI3K) and p38 mitogenic signaling process, in turn, activates cyclin D1 and increases tumorige- pathways, and can also significantly induce up-regulation nicity and hepatocyte proliferation.24

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Iron deposition a contributing factor to hepatocarcinogenesis.11 Hyperinsuli- nemia is characterized by up-regulated expression of the Liver is the body’s main storage site for iron, due to its rich hepatic insulin-like growth factor-1 (IGF1), which subse- reticuloendothelial system.25 Excess liver iron may increase quently stimulates cellular proliferation and inhibits cell apop- NASH risk and its progression to HCC.26,27 Abnormal iron tosis. Insulin also activates the insulin receptor substrate-1 deposition in liver is more frequent in NASH patients, and nec- (IRS-1), which has been shown as up-regulated in HCC.26 roinflammation may be the driving factor. Iron and the Indeed, IRS-1–mediated signals may act as survival factors, coexistence of hyperinsulinemia are risk factors for NASH promoting hepatocyte proliferation via mitogen-activated development and together they may contribute to insulin protein kinase and PI3K, and protecting against transforming resistance, disease progression and HCC. Iron deprivation growth factor b1 (TGF-b1)–induced apoptosis in HCC can suppress HCC growth, as shown in both in vivo and development.24 in vitro experiments.25,28 Genetic factors Alcohol Data from genome-wide association studies (GWAS) have Recent data suggest that chronic alcohol intake results in the shown that patatin-like phospholipase domain-containing induction of hepatic cytochrome P450 2E1 (CYP2E1), which protein 3 (PNPLA3) on chromosome 22 is involved in the leads to generation of ROS with direct and indirect carcino- metabolism of triglycerides and is a genetic factor that pro- genic consequences.29 Genetic factors regulating alcohol motes NASH development. The single nucleotide polymor- metabolism could predispose towards development of alco- phism (SNP) rs738409 is closely related to fatty liver and holic pancreatitis. Studies have also revealed that alcohol involved in fibrosis progression of NAFLD. The CT, K2240X) is also expressed in liver, and the common polymorphism represent a paradigm of rare variant influence on liver fat of its gene rs2228603 has been shown as associated with content and HCC risk. Additionally, mutations in telomerase HCC in alcoholic liver disease.30 reverse transcriptase (TERT) have been associated with hepatic steatosis, and deficiency of TERT can reduce response MicroRNA (miR) to liver damage, thereby inducing the formation of steatosis and fibrosis. In conclusion, the occurrence of NAFLD-HCC miRs are highly conserved, small non-coding RNAs (about seems to be influenced by common genetic variants, such as 18;25 nucleotides in length) that serve to regulate tran- those in PNPLA3, and by rare genetic variants, such as those in scription or translation of target genes and fatty acid metabo- the APOB and TERT genes.39 lism. Both miR-197 and miR-99 have been associated with liver fibrosis in NASH patients. Some studies have also identified Microbiota and toxic substances miR-122 as a key regulator of glucose and lipid metabolism in liver tissues,31 and NAFLD patients have been shown to have Gut microorganisms significantly higher levels of circulating miR-122, miR-34a, and miR-16. During the development of NAFLD, patients with In intestine, there are trillions of microorganisms including simple steatosis to steatohepatitis have shown that the sero- bacteria, archaea, yeasts and viruses that collectively repre- logical levels of miR-122 and miR-34a are closely positively sent the intestinal ecosystem that is involved in energy correlated with disease severity, liver enzyme activities, fibro- harvesting and fat storage. Several studies support the sis stage and active inflammation;32 this finding suggests theory of a relationship existing between intestinal microbial that alteration of circulating miR-122 could be an early changes and obesity and its related complications, including event in the transition of nonalcoholic steatohepatitis to IR and NAFLD. Homoeostasis disorders between bacteria and hepatocarcinogenesis.33 host lead to increased intestinal permeability and production of pro-inflammatory molecules, endotoxemia and cytokines. Insulin resistance The relationship between the gut microbiota and NAFLD is dependent upon altered metabolism of choline and bile acids Hepatic steatosis is defined as lipid accumulation in hepato- and production of endogenous alcohol. It has also been cytes and is very frequently found in adults and obese implicated in the IR development that is involved in NAFLD adolescents. Etiologically, obesity and its associated insulin pathogenesis via various mechanisms. The gut-liver axis is resistance (IR) or excess alcohol intake are the most frequent the way by which bacteria and their potential hepatotoxic causes of liver steatosis. Insulin, as a key hormone, regulates products (lipopolysaccharides, DNA, RNA, etc.) can easily lipogenesis and lipolysis in adipose depots. Under IR condi- reach the liver.40,41 tions, the adipose tissue becomes resistant to the anti-lipolytic Primary bile acids, which are derived from cholesterol, effect of insulin and fatty acid release becomes increased;34 become secondary bile acids via the action of intestinal this process is accompanied by increased lipolysis and/or microbes. Bile acids combing with farnesoid X receptor (FXR) fat intake, possibly promoting hepatic triglyceride synthesis inhibits cholesterol 7 a-hydroxylase (CYP7A1) transcriptions and resulting in liver lipid accumulation.35,36 to affect bile acid synthesis or SREBP-1c transcription, thereby Lipid accumulation in liver tissues causes IR occurrence reducing triglyceride synthesis and enhancing the PPARa through the activation of the NF-kB pathway, leading to signaling to promote fatty acid b-oxidation. If the bile acids hyperinsulinemia and the activation of PI3K/Akt that may be bind to the G-protein-coupled cell surface receptor 5 (TGR5),

128 Journal of Clinical and Translational Hepatology 2016 vol. 4 | 123–130 Gu J. et al: NAFLD in HCC the result could be inhibition of inflammation through sup- are related to increased risk of HCC occurrence, regardless of pression of the NF-kB pathway in macrophages.12 Many sex.48 Hepatic lipid accumulation promotes the release of pro- genetic and environmental factors have been suggested to inflammatory cytokines and progression from steatosis to contribute to the development of obesity and NAFLD, but the NASH. Both diabetes and obesity lead to oxidative stress and exact mechanisms remain unknown and require further lipid peroxidation of phospholipids, resulting in cell injury, investigations. necrosis or hepatocyte malignant transformation.49 Prospec- tive studies with longer follow-up periods should be designed Toxic substances to screen NASH or NAFLD populations for specific biomarkers of malignant transformation of hepatocytes.50,51 NAFLD has been implicated in some medical conditions, including IR, obesity, metabolic syndrome, hyperlipemia, hyperten- Perspectives sion, cardiovascular disease and diabetes. Glyceraldehyde- derived advanced glycation end-products (glycer-AGEs) are the In the past decade, the discussion around the substantial predominant components of toxic AGEs (TAGE). Increasing increase in NAFLD related to hypernutrition and HCC has data have suggested that TAGE, along with its cognate recep- become a cocktail party cliché, but its impact on public health tors, might change intracellular signaling and gene expression cannot be dismissed. With our understanding of the relation- of pro-inflammatory molecules, and may also elicit generation ships of both gradually deepening, we now see that NAFLD of oxidative stress in liver cells such as the human HSCs. might promote the malignant transformation of hepatocytes Circulating TAGE levels were shown to be significantly higher in as a result of liver lipid accumulation, hepatotoxicity, endo- NASH patients than in those with simple steatosis or in healthy plasmic reticulum dysfunction, IR and abnormal fat metabo- subjects. Moreover, in the NASH patients, the TAGE levels lism. Although the exact mechanisms underlying the NAFLD inversely correlated with adiponectin.42 tumor-promoting mechanisms triggered by hypernutrition In addition, dietary or genetic obesity induces alterations remain to be explored,52 it is well recognized that those of gut microbiota, thereby increasing the levels of deoxycholic patients with excessive fat deposition are at greater risk of acid (DCA), a gut bacterial metabolite known to cause DNA tumor-promoting inflammation conditions and should be damage. Tumor cells often show higher rates of fatty acid treated in time to avoid hepatocyte malignant transformation. synthesis and lipid accumulation; moreover, increased lipid availability in liver tissues might provide ATP and structural Acknowledgments support for cancerous cell proliferation. The overexpression of key genes involved in regulation of lipogenesis correlates This work was supported by grants from the Projects of the with tumor growth and poor prognosis of HCC. Hepatic lipid Priority Academic Program Development of Jiangsu Higher accumulation is accompanied by distinct patterns of perilipin 15 Education Institution (Qinglan Plan), Nantong Society Under- expression. These findings suggest that abnormality of taking and Technological Innovation (HS2014078), National hepatic lipid accumulation might promote hepatocyte malig- Natural Science Foundation (81200634 and 81370982) and the nant transformation. International S. & T. Cooperation Program (2013DFA32150) of China. NAFLD in hepatocarcinogenesis Conflict of interest Liver derangements in lipid metabolism, importing FFAs and manufacturing, storing and exporting lipids could lead to None NAFLD development.43 Although the majority of NAFLD patients presented with steatosis only, about 20% of patients present with NASH (as defined by microscopic findings and Author contributions consisting of liver injury, steatosis, parenchymal and portal inflammation, and distinctive fibrosis). Dysregulation of the Contributed equality to this work (JJG, MY, DBY), concept and hormonal axes and cytokines in patients with NAFLD pro- literature search (JJG, LW), drafting the manuscript (JJG, MY, motes a greater impairment of the cycle between metabolic XLY, DBY), and critical revision for intellectual content (YDF). and chronic inflammatory stimulus that might lead to malig- 44 nant transformation of hepatocytes. References In liver tissues, the DCA in the enterohepatic circulation provokes senescence-associated secretory phenotype human [1] Ding J, Wang H. Multiple interactive factors in hepatocarcinogenesis. Cancer HSCs, which in turn secrete tumor-promoting factors, Lett 2014;346:17–23. doi: 10.1016/j.canlet.2013.12.024. and thus facilitating HCC development (as evidenced in [2] Hamid AS, Tesfamariam IG, Zhang Y, Zhang ZG. Aflatoxin B1-induced hep- animal models following exposure to chemical carcinogen).45 atocellular carcinoma in developing countries: Geographical distribution, mechanism of action and prevention. Oncol Lett 2013;5:1087–1092. High leptin level and low adiponectin level are hallmarks of [3] Fan JG, Farrell GC. Epidemiology of non-alcoholic fatty liver disease in China. obesity and involved in NAFLD pathogenesis and carcinogen- J Hepatol 2009;50:204–210. doi: 10.1016/j.jhep.2008.10.010. esis.46 Obesity-promoted HCC has been shown to be depend- [4] Wei Z, Doria C, Liu Y. Targeted therapies in the treatment of advanced hep- – ent on increasing levels of IL-6 and TNF, which result in liver atocellular carcinoma. Clin Med Insights Oncol 2013;7:87 102. doi: 10.4137/ CMO.S7633. inflammation and oncogenic STAT3 activation. The long-term [5] Fan JG. Epidemiology of alcoholic and nonalcoholic fatty liver disease in chronic inflammatory condition of obesity plus the higher China. J Gastroenterol Hepatol 2013;28 Suppl 1:11–17. doi: 10.1111/jgh. levels of IL-6 and TNF might represent a risk factor for HCC.47 12036. A prospective study involving 25337 patients with HCC [6] Yao D, Mizuguchi H, Yamaguchi M, Yamada H, Chida J, Shikata K, et al. Thermal instability of compound variants of carnitine palmitoyltransferase demonstrated that both excess body weight (SRR = 1.48, 95% II and impaired mitochondrial fuel utilization in influenza-associated ence- CI: 1.31–1.67) and obesity (SRR = 1.83, 95% CI: 1.59–2.11) phalopathy. Hum Mutat 2008;29:718–727. doi: 10.1002/humu.20717.

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130 Journal of Clinical and Translational Hepatology 2016 vol. 4 | 123–130 Review Article

Acetaminophen-Induced Hepatotoxicity: a Comprehensive Update

Eric Yoon, Arooj Babar, Moaz Choudhary, Matthew Kutner and Nikolaos Pyrsopoulos*

Rutgers New Jersey Medical School, University Hospital, Newark, New Jersey, USA

Abstract compounds, including herbs and alternative medications, are metabolized in the liver microsomes. The most detrimental Hepatic injury and subsequent hepatic failure due to both clinical presentation is fulminant liver failure, where patients intentional and non-intentional overdose of acetaminophen without a history of liver disease present with hepatic (APAP) has affected patients for decades, and involves the encephalopathy and coagulopathy preceding jaundice. Acet- cornerstone metabolic pathways which take place in the aminophen, also known as APAP (in the United States), microsomes within hepatocytes. APAP hepatotoxicity remains paracetamol (in Europe and other areas of the world) or a global issue; in the United States, in particular, it accounts N-acetyl-p-aminophenol, is one of the most commonly utilized for more than 50% of overdose-related acute liver failure compounds worldwide; its use as an anti-pyretic or analgesic and approximately 20% of the liver transplant cases. The drug has been predominant since 1955, particularly due to pathophysiology, disease course and management of acute the fact that it is easily accessible in various formulations as liver failure secondary to APAP toxicity remain to be precisely an over-the-counter medication.1 Indeed, APAP is reported to elucidated, and adverse patient outcomes with increased be regularly consumed by over 60 million Americans on a morbidity and mortality continue to occur. Although APAP weekly basis, making it the most widely utilized analgesic hepatotoxicity follows a predictable timeline of hepatic and antipyretic in the United States.2 Advertised as safe in failure, its clinical presentation might vary. N-acetylcysteine doses up to 4000 mg every 24 hours by the United States (NAC) therapy is considered as the mainstay therapy, but Food and Drug Administration (FDA), consumption at this liver transplantation might represent a life-saving procedure dose generally does not yield any toxic effects.2,3 Stand- for selected patients. Future research focus in this field may alone APAP is not the only drug formulation that should be benefit from shifting towards obtaining antidotal knowledge suspected in potential APAP toxicity, however. As such, it at the molecular level, with focus on the underlying molecular may be difficult to recognize APAP toxicity, partly due to its signaling pathways. availability in various formulations, such as tablets, liquids, © 2016 The Second Affiliated Hospital of Chongqing Medical rectal suppositories and intravenous liquids, as well as in com- University. Published by XIA & HE Publishing Inc. All rights bination supplements sold as over-the-counter and prescrip- reserved. tion products for analgesia.2 Reported cases of APAP-induced hepatotoxicity first emerged in the United States in the mid-1980s, and since Introduction then all signs point towards a growing incidence. It has been reported that this is one of the most common pharmaceutical products to cause DILI.1–5 Mortality rates have been Drug-induced liver injury (DILI) is not an uncommon adverse approximated at 0.4% in overdose patients, translating to event encountered in clinical practice, since a vast number of 300 deaths annually in the United States.1 Although toxic ingestions causing hepatic failure are usually in excess of Keywords: Acetaminophen toxicity; Hepatotoxicity; APAP; Paracetamol; Acute liver failure (ALF). 150 mg/kg, an increasing number of reports has arisen to Abbreviations: AIF, apoptosis-inducing factor; ALF, acute liver failure; ALT, suggest that lower doses of APAP may confer acute liver – alanine aminotransferase; APACHE II, acute physiology and chronic health eval- injury and liver failure.1 4 A so-called “therapeutic misadven- uation II; APAP, acetaminophen; AST, aspartate transaminase; ATP, adenosine ture” phenomenon, as coined by Zimmerman et al., is becom- triphosphate; DAMP, damage-associated molecular pattern; DILI, drug-induced liver injury; EndoG, endonuclease G; FDA, Food and Drug Administration; ing increasingly acknowledged, as some patients have GDCA, glycodeoxycolic acid; GDH, glutamate dehydrogenase; GSH, glutathione; presented with acute liver failure despite consumption of HMGB1, high mobility group B1; HPLC, high-pressure liquid chromatography; INR, “safe” doses of APAP. Some of these patients may have international normalized ratio; IV, intravenous; JNK, c-Jun-N-terminal kinase; some specific risk factors, such as nuances in APAP metabo- KCH, King’s College Hospital; KIM 1, kidney injury molecule 1; MELD, model for end-stage liver disease; miRNA, microRNA; mtDNA, mitochondrial DNA; lism at the mitochondrial and molecular level, which are NAC, N-acetyl cysteine; NAPQI, N-acetyl-para-benzo-quinone imine; nDNA, currently under investigation in hopes of further elucidating nuclear DNA; NK, natural killer; NKT, natural killer T cells; PO, oral; SFN, sulfor- their contributions to this life-threatening condition. aphane; SOFA, sequential organ failure assessment; SULT, sulfotransferase; UGT, UDP-glucoronosyl transferase. Received: 22 December 2015; Revised: 07 April 2016; Accepted: 18 May 2016 Epidemiology q DOI: 10.14218/JCTH.2015.00052. *Correspondence to: Nikolaos Pyrsopoulos, Division of Gastroenterology & Hep- APAP is one of the most commonly used analgesics in the atology, Rutgers New Jersey Medical School, University Hospital, 185 S. Orange Avenue, Newark, New Jersey 07101, USA. Tel: +1-973-972-5252; Fax: +1-973- United States and reportedly the most common cause of acute 2,3,5 972-3144, E-mail: [email protected] liver failure in the United States. In the United States,

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This is an Open Access article distributed under the terms of the Creative Commons Attribution-Noncommercial 4.0 Unported License, permitting all non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Yoon E. et al: Acetaminophen-induced liver toxicity around 30,000 patients are admitted to hospitals every year and hepatotoxicity. There are three phases of APAP metabo- for treatment of APAP hepatotoxicity.6 While there appears lism. The majority (;90%) of the APAP is funneled into phase II to be a near even ratio of patients who take excessive APAP metabolic pathways, in which APAP conjugation is catalyzed doses knowingly and unknowingly (52% vs. 48%), both by UDP-glucuronosyl transferases (UGT) and sulfotransferase groups are susceptible to liver failure and generate referrals (SULT), with conversion to glucouronidated and sulfated for liver transplantation.2,6 Although the majority of patients metabolites that are eliminated from the body in the urine8 experience mild adverse reactions, such as hepatitis, choles- (Figure 1). A small, measurable amount of APAP (;2%) is tasis or asymptomatic liver enzyme elevation, APAP hepatotox- excreted in the urine without having undergone any metabo- icity is generally estimated to account for approximately 48% lism.8 Another portion of APAP (;10%) is shunted by hepatic – of acute liver failure diagnoses.1 3 Moreover, studies have cytochrome CYP 2E1 (to a lesser extent with CYP 1A2 and 3A4) shown that 29% of patients with acute liver failure secondary to phase I oxidation, in which a highly reactive toxic metabolite, to APAP toxicity undergo liver transplant, and that these cases N-acetyl-para-benzo-quinone imine (NAPQI), is formed.9–13 have a 28% mortality rate.1 Phase III involves metabolite transport in the form of biliary With the advent of APAP/opioid combination analgesics excretion that requires transporters.8 and the ever-growing number of prescription narcotics APAP hepatotoxicity occurs through formation of the noxious being prescribed by medical practitioners, a larger number NAPQI metabolite, which is present in excessive quantities, as of patients have been placed in harm’s way as they are at augmented by features of glutathione (GSH) depletion, oxida- significant risk of APAP hepatotoxicity. Less than a decade tive stress and mitochondrial dysfunction leading to depletion ago, in 2010, over 130 million prescriptions for APAP/hydro- in adenosine triphosphate (ATP) stores.3,9,13 There is evidence codone were filled in the United States.3 Reportedly, 63% of to support the theory that the metabolic activation of APAP unintentional overdoses of APAP occur with use of the opioid/ generates NAPQI that binds to a number of cellular proteins, APAP combination, with an additional 17% of adults suffering especially mitochondrial proteins. Adherence to mitochondrial liver injury.5,6 Fortunately, the United States FDA has recog- proteins, especially in the setting of GSH depletion, is important nized the dangers of the prescription combination of APAP because mitochondrial protein binding depletes native antiox- and narcotic analgesics and, in January 14, 2014, implemented idant functions and also alters the mitochondrial ATP-synthase a fixed amount of APAP approved per dosage unit of combina- a-subunit, leading to ineffective ATP production.9,13 tion analgesics tablets in effective practice. Epidemiologic Other mechanisms of hepatotoxicity include the formation studies have demonstrated that at least 6% of filled prescrip- of toxic free radicals, such as peroxynitrite, from the reaction tions for either acetaminophen alone or acetaminophen in of superoxide and nitric oxide, subsequently forming nitro- combination with opioids exceeded 4000 mg per day of acet- tyrosine adducts inside the mitochondria.9,13 GSH repletion 3,5,6 aminophen, which is a cause for real concern. Further not only provides surplus cysteine as an energy substrate for epidemiologic studies have demonstrated that there is a true the Krebs cycle, it also serves the important role of scavenging lack of knowledge regarding the harmful potential of APAP. for free radicals and peroxynitrite.9,10 Mitochondria, which are It is not uncommon for patients to confuse non-steroidal anti- critical for cellular respiration and metabolism, suffer damage inflammatory (NSAIDs) medications, such as naproxen or to their own mitochondrial DNA by the actions of reactive ibuprofen, with APAP. While providing health literacy education oxygen species and peroxynitrite compounds, and they have to teach distinguishment of APAP and NSAIDs is important, been directly implicated in the process leading to cessation of some have thought it possibly more beneficial to mitigate the ATP synthesis.9,11 chance of inappropriate APAP over-ingestion by reducing the Many biochemical studies have been performed in murine pack size of APAP preparations sold, which they believe may models. However, when HepaRG stem-cell derived human 7 reduce the incidence and severity of APAP hepatotoxicity. hepatocytes were subjected to APAP, the same mechanisms What is clear is that APAP overdose and hepatotoxicity is a of hepatotoxicity were demonstrated—starting with GSH preventable public health concern that deserves awareness depletion and moving through protein adduct formation, by both clinicians and the patient population alike. superoxide and peroxynitrite formation and lysosomal iron uptake into mitochondria.9–11,13,14 Oxidative stress com- Pharmacology and Hepatotoxicity pounded with lysosomal iron uptake within the mitochondria leads to mitochondrial membrane dysfunction via disruption APAP absorption occurs rapidly in the duodenum, owing to its of the mitochondrial membrane permeability transition pore, – property as a weak acid.8 If a patient consumes food around triggering cell necrosis.9 11,13,14 Organelle swelling leads to the same time of APAP ingestion, there may be a delay in the cellular necrosis and the release of mitochondrial contents, time of, but not the extent of, drug absorption.8 Much like con- such as apoptosis-inducing factor (AIF) and endonuclease current food consumption causing time-delay in APAP absorp- G (EndoG), which in turn migrate to the nuclei and cause tion, a patient with chronic liver disease is at risk of prolonged DNA fragmentation.12 Cellular swelling, karyolysis, karyor- drug serum half-life (by an average of 2.0 to 2.5 hours, and up rhexis, vacuolization, inflammation and release of cellular to more than 4 hours), especially if extended-release APAP contents (alanine aminotransferase, ALT) are key processes formulations are consumed. While an overdose of APAP yields of hepatocyte necrosis and related death in humans, as peak serum concentrations (10 – 20 mg/mL) within 4 hours, a shown by the biochemical evidence of severe elevations in – patient taking the medication safely will achieve peak concen- aminotransferases, especially ALT.9 11 trations within 1.5 hours, with a half-life of 1.5 – 3hours.8 In non-toxic ingestion of APAP, the processing of NAPQI APAP metabolism occurs within liver microsomes at the occurs with rapid conjugation by hepatic GSH to form non- microscopic level. While the pathways are well elucidated toxic mercaptate and cysteine compounds that are excreted and the fine details of their molecular mechanisms are beyond in urine.8 The body requires a delicate balance between pro- the scope of this manuscript, it is worth noting that not all duction and detoxification/transport of harmful reactive patients suffer the same fate when it comes to APAP ingestion species and protein adducts, and when these defenses are

132 Journal of Clinical and Translational Hepatology 2016 vol. 4 | 131–142 Yoon E. et al: Acetaminophen-induced liver toxicity

Fig. 1. Acetaminophen (APAP) metabolic pathway. overwhelmed at hepatotoxic doses of APAP, the glucoronida- adducts, lead to oxidative injury and hepatocellular necrosis. tion and sulfonation pathways become saturated, pushing the An approximately 70% reduction of GSH has been proposed majority of APAP to be metabolized to NAPQI by the CYP as the threshold for protein adduct binding to occur; however, 2E1 pathway and resulting in GSH depletion and build-up of this theory has been called into question as APAP-protein toxic NAPQI.9,10 Myeloperoxidase and cyclooxygenase-1 are adducts have been detected at 1 hour after APAP treatment, enzymes that also function in the processing of NAPQI into a time that that would precede GSH depletion.8 It is important non-reactive metabolites. In addition, hepatocytes have the to note, however, that a clear inverse relationship has been ability to induce innate and adaptive immune defenses to demonstrated between GSH concentration and APAP meta- 11 dampen the inflammatory response during necrosis. It has bolic activation activity.8 Findings from current studies of been suggested that perhaps the role of the innate immune APAP hepatotoxicity seem to favor the mechanism of toxic system, as mediated by the natural killer (NK) and natural injury being related to mitochondrial and endoplasmic reticula killer T cells (NKT) which are abundant in hepatocytes, is to leading to hepatocyte injury and necrosis. cause the release of pro-inflammatory cytokines and chemo- kines that enhance hepatocellular cytotoxicity.15 Damage- associated molecular pattern (DAMP) species released into Factors influencing APAP-related hepatotoxicity the circulation, such as nuclear fragments and mitochondrial DNA (mtDNA), appear to recruit inflammatory cells via the The most essential determining factor in both the develop- innate immune system as the liver’s attempt to remove ment and severity of APAP hepatotoxicity is the drug’s ingested necrotic cell debris and promote the liver’s recovery phase.12,16 dose, but some argue that the length of time from APAP APAP-induced cell death of hepatocytes takes on the ingestion to N-acetylcysteine (NAC) therapy (“time to NAC”)is 15,17–20 characteristic changes of necrosis. The hepatotoxicity of equally if not more important. Many other factors are APAP rests predominantly with the highly toxic and reactive confluent in the development and severity of liver injury after compound NAPQI, which forms covalent bonds with sulf- APAP over-ingestion (Table 1). Liver metabolism during glu- hydryl groups on cysteine and lysine molecules within the couronidation or sulfation, CYP activity and maintenance of mitochondria of hepatocytes and which spontaneously reacts hepatic GSH supply depends on patient factors such as age, with GSH and binds to hepatic proteins.8,11 The protein nutritional status, pre-existing liver disease, concurrent use adducts formed, specifically those of mitochondrial protein of alcohol and other liver-metabolized medications, genetic

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Table 1. Factors influencing APAP-related hepatotoxicity returned to normal.19 Yet, while asymptomatic, chronically elevated aminotransferases may be of concern to healthcare Factors Clinical implications providers, leading to further costly diagnostic studies or Dose and C APAP toxicity with acute, high doses changes/restrictions in necessary medications. pattern of use EtOH B APAP toxicity with acute EtOH Alcohol ingestion C APAP toxicity with chronic EtOH The pairing of alcohol and APAP is a reality that deserves ingestion attention, as both substances are ubiquitous in society. Furthermore, individuals with the intent of self-harm may Herbs and C APAP toxicity with impedance of over-ingest both substances. Intuitively, the co-ingestion of medications hepatic glucouronidation alcohol and APAP is expected to cause acute hepatotoxicity. Age and genetic C APAP toxicity with advancing age and As it turns out, the manner in which alcohol is consumed plays factors impaired glucouronidation a significant role in who may or may not suffer APAP-related 8,11,20–24 Nutritional C APAP toxicity in malnourished patients hepatotoxicity. The interplay between alcohol and status APAP, which is a competitive substrate for CYP 2E1 metabolism, is complex.20–24 According to a prospective observatio- Chronic liver C APAP toxicity, especially in chronic nal study of approximately 360 patients who presented with disease EtOH abuse acute APAP overdose, the concurrent acute ingestion of

Abbreviations: APAP, acetaminophen; EtOH, ethyl alcohol. alcohol and APAP resulted in the prevalence of hepatotoxicity, as defined by ALT > 1000 U/L or an international normalized ratio (INR) of > 1.3, in 5.1% of patients versus 15.2% of predispositions, and most importantly, the acuity or chronicity patients who did not consume alcohol among all of the APAP of APAP overuse.3,15,17,20–30 consumers who presented as above or below the “200-line” on the Rumack-Matthew nomogram; moreover, this finding was repeated regardless of the time between ingestion and Dose and pattern of use assessment.21 Thus, acute ethanol ingestion may serve as a protective mechanism against APAP hepatotoxicity, but Zimmerman’s “therapeutic misadventure” describes acute whether this phenomena takes place only in alcoholic patients liver injury following APAP consumption and failure of APAP (versus nonalcoholic patients) is unclear.8,20–22 The mecha- therapy, when used at or below the daily maximum recom- nism behind this interesting finding appears to be ethanol’s mended dose of 4000 mg. However, there are many other competitive utilization of the CYP 2E1 substrate, which dimin- patients who intentionally over-ingest APAP (with clear evi- ishes the NAPQI byproduct of phase I APAP metabolism.21,22 dence from careful history taking and measured APAP levels), or the more indolent and accidental over-ingestion of doses Another suggested hypothesis is that ethanol may directly over 4000 mg per day (not as obvious when eliciting the enhance the activity of NAD(P)H:quinone reductase, serving “ to reconvert quinone metabolites back to native APAP and patient history), which we would like to term therapeutic 21 misfortune.” The United States FDA clearly states “severe thereby limiting the accumulation of toxic metabolites. liver damage may occur if you take more than 4000 mg of Chronic alcohol ingestion, however, augments APAP hep- acetaminophen in 24 hours.”31 In these extreme circumstan- atotoxicity by up-regulating, enhancing and increasing the ces, prompt medical assessment and therapeutic intervention synthesis and activity of CYP 2E1 and decreasing GSH stores is necessary to administer therapy that will allow for salvage and synthesis, thus enhancing liver necrosis and worsening 5,8,21 of a damaged and/or failing liver. prognosis. Chronic alcoholism and APAP overdose may There is no question that the upper threshold on the potentiate liver failure, but there is no indication that the com- maximum daily allowed dose of APAP has been the subject bination of alcoholism and taking therapeutic amounts of 21 of controversy. An example of such is the voluntary adjust- APAP will necessarily cause hepatotoxicity. Therefore, one ments made by McNeil, the pharmaceutical producer of the has to be wary of characterizing all alcoholics as being at Tylenol® brand of APAP, wherein the maximum daily APAP higher risk of APAP hepatotoxicity. Regular ethanol consump- dose was reset from 4000 mg to either 3000 mg per day or tion of greater than 250 mg/dL appears to be the threshold for 3250 mg per day for consumers taking the 500 mg or 325 mg enhancement of CYP 2E1 enzymatic activity; this condition tablet preparations, respectively.32 Many of the other manu- represents individuals who drink 18 or more standard alco- 21 facturers have not followed suit, however. For increased holic drinks. A more reasonable explanation of why chronic patient safety in APAP consumption, the FDA has worked to alcohol abusers have higher APAP-related hepatotoxicity is a mitigate APAP hepatotoxicity by legislating regulations to limit tendency among these individuals to delay seeking medical prescription combination drug products to have no more than attention after APAP ingestion, which would prolong their 325 mg of APAP per dosage unit and have urged healthcare “time to NAC”—the only demonstrated treatment available providers to cease prescribing combination drug products for salvage of a hepatotoxic liver. with more than 325 mg of APAP per tablet.31 The data suggests that chronic alcohol consumers who One study reported that ingestion of the recommended experience “therapeutic misadventure” are at increased risk – maximum amount of 4000 mg of APAP for 2 weeks resulted in of APAP hepatotoxicity.20 24 It appears that a single dose the asymptomatic elevation of alanine transaminase, up to of APAP, at 325 mg to 500 mg for as-needed analgesic or three times normal, in 40% of patients.19 These transaminase anti-pyretic purposes, will not lead to acute APAP hepatotox- elevations did not amount to any clinical significance, and icity in an alcoholic patient, especially if APAP is taken less than after the APAP was discontinued the transaminase levels 8 hours from alcohol consumption. APAP is often prescribed,

134 Journal of Clinical and Translational Hepatology 2016 vol. 4 | 131–142 Yoon E. et al: Acetaminophen-induced liver toxicity and can be prescribed, to patients who require an anti-pyretic Even though chronic malnutrition is common among liver or analgesic despite recent history of alcohol ingestion. disease patients, there is a subset of malnourished patients, namely those with the psychiatric illness anorexia nervosa, which merits special attention. This subpopulation may Herbals and medications other than APAP undertake intentional APAP overdoses, representing intent of self-injury. Patients with anorexia nervosa have low GSH There are many prescribed and over-the-counter substances stores already, as is expected in the malnourished state, but that interact with liver metabolism; these interactions can their rate of CYP 2E1 activity is also diminished; thus, the risk result in harmful or protective tendencies, and have been of APAP toxicity appears not to be exacerbated in these reported for a plethora of particular herbs and medications. nutritionally-depleted patients in particular, due to this Medications that stimulate the CYP system can predispose unique circumstance.29 patients to APAP hepatotoxicity by causing enhanced production of NAPQI via the oxidative pathway.25 Case series reports have concluded that in 50% of adult patients with APAP- Phosphate in APAP hepatotoxicity induced acute liver failure, the life-threatening condition was Early murine models demonstrated that mice with adequate preceded by ingestion of potentially hepatotoxic herbs within ATP supply underwent hepatocyte apoptosis when exposed to 8 weeks of hospital presentation.4 The prescription agents excessive APAP. In fasting animals, the opposite was true, with known for their hepatotoxic tendencies include anti-epileptic necrosis being the more prominent response to excessive drugs, such as phenobarbital, phenytoin and carbamazepine, APAP. Phosphate has historically been associated with poor as well as anti-tuberculosis drugs, such as isoniazid and rifam- outcomes in patients with APAP hepatotoxicity, with early pin. Over-the-counter herbs and dietary supplements, such studies showing an association between low phosphate levels as St. John’s wort, garlic and germander, may mechanistically and increased morbidity and mortality.35 However, findings enhance the CYP system.1 An alternative mechanism by which from more recent studies have led to the proposal that patients medications may enhance APAP hepatotoxicity involves com- with APAP hepatotoxicity and high levels of serum phosphate petitive utilization of the glucouronidation pathway, resulting have worse outcomes. Levels greater than 3.7 mg/dL at 48–72 in increased metabolism towards the CYP system and thus hours post-ingestion have been reported as associated with enhanced formation of hepatotoxic substrates.25 Importantly, higher mortality from acute liver failure.36,37 Findings from opioid therapies can reduce the level of hepatic GSH and other research studies have contradicted this proposition, thereby potentiate APAP-induced liver injury.8 however, showing that the association between hyperphos- It is important to note that some natural products and phatemia and liver failure is only evident in patients with remedies have been demonstrated to have protective acute renal failure (with 50–80% of patients with acute liver potential against APAP hepatotoxicity.8 One proposed mech- failure having concomitant acute renal failure38). It is well- anism underlying this phenomenon is the competitive known that with acute kidney injury, serum phosphate levels utilization of CYP 2E1 in herbal drug metabolism that would rise; disruption of pathways necessary for adequate cell regen- decrease APAP’s conversion into its toxic metabolites, such as eration and ATP production facilitates serum phosphate levels NAPQI.25,33,34 The Schisandra plant species, ubiquitous in its remaining high, with some researchers postulating an associ- use as a traditional East Asian herb, has been used in Korea, ation of this with increased mortality. The association between China and Japan as a remedy for hepatitis; in addition, the hypophosphatemia and better clinical outcomes seems to be Wuzhi Tablet, a recently introduced alcohol extract prepara- secondary to enhanced cell regeneration with hepatocyte tion of S. sphenanthera, has shown significant hepatoprotec- phosphate uptake from the serum to generate ATP.39,40 tive effects against APAP-induced liver injury.34 Treatment Given the importance of serum phosphate levels, members with one particular herbal agent, Schisandrol B, the active of the Acute Liver Failure Study Group (commonly referred extractant from S. sphenanthera, has been shown to dec- to as ASFLG) have included serum phosphate levels in their rease APAP hepatotoxicity as well as to lead to features of prognostic scoring system for mortality related to APAP liver regeneration by inhibiting NAPQI-initiated injury via the hepatotoxicity.41 blockade of CYP activity, the down-regulation of p53/p21 and the up-regulation of other signaling pathways to facilitate Age and genetic factors hepatic tissue repair.34 APAP, an analgesic utilized by patients from infancy into Nutritional status the geriatric years, shows age-related hepatotoxic tendencies. In general, younger patients are better able to overcome The collective evidence shows a clear association between acute liver failure as a result of APAP hepatotoxicity, probably malnourishment and APAP hepatotoxicity. Malnutrition is due to the larger hepatic cell mass that is present in this associated with conditions consistent with GSH depletion, population before the cell damage occurs, as well as the which may support its role of placing patients at further risk better capacity of those cells for non-toxic metabolism and for hepatotoxicity. In a poor nutritional state, GSH reserves their improved capacity for regeneration.20 Patients over become easily exhausted and hepatic glucouronidation is 40yearsoldwhooverdoseonAPAPportendahigherriskof stunted, leading to decreased levels of available GSH that acute liver failure, liver transplantation and death.5 In would otherwise be scavenged for formation of reactive general, APAP metabolism appears age-dependent, with metabolites in phase I of APAP metabolism. Poor nutritional elderly patients being at higher risk of hepatotoxicity after status is often associated with chronic alcoholism, and thus acute overdose of APAP than the pediatric population.42 Con- alcoholic patients with APAP-induced hepatotoxicity may fall sidering the current widespread use of APAP alone and in opiate victim to both decreased body GSH stores as well as enhance- combinations among adults of advanced age (to treat cancer ment of the CYP enzymatic activity.30 pain or chronic musculoskeletal pains), one must not forget

Journal of Clinical and Translational Hepatology 2016 vol. 4 | 131–142 135 Yoon E. et al: Acetaminophen-induced liver toxicity to monitor patients for complications of long-term and high- NAC should be of the utmost priority since the benefit of dose APAP use. therapy may outweigh the risks of untreated APAP toxicity.1,47 Various genetic polymorphisms in CYP enzymes and their Although more studies retrospectively investigating pregnant levels of activity may explain why APAP is metabolized women would be helpful, for now the judicious use of APAP with excessive or diminished oxidative capabilities.26,43,44 should be advised as APAP remains the most common drug The enzymes UGT (glucouronidation), SULT, CYP 450, GST, overdose in pregnancy.46 N-deacetylase (deacetylation), NAT2 (deacetylation), and fatty acid amide hydrolase are involved in APAP metabolism Clinical manifestations and have been shown to be related to both hepatic and neph- 45 rotoxic effects of the analgesic medication. It appears that Identification of APAP overdose is critical, as significant mor- genotypic changes of these enzymes leads to potentially bidity and mortality may be prevented with early therapy. different risk/benefit ratios when APAP is ingested. Compara- Many patients have only minimal and non-specific symptoms tive studies of Caucasian and African populations showed that that are comparable to viral prodrome; these symptoms more mercapturic acid and cysteine conjugate metabolites include malaise, nausea with or without vomiting, and abdo- were recovered in the former (at nearly a 2:1 ratio), indicating minal pains. There are four established sequential stages of 43 that increased hepatic metabolism occurs in that group. APAP hepatotoxicity, which should be considered upon pre- Another study, which compared Caucasian and Chinese pop- sentation to clinic. It is vital, however, to keep in mind that ulations indicated the increased susceptibility of the former to while each stage is designated by a general time range APAP hepatotoxicity reflects the lower CYP 2E1 and 2D6 activ- following the APAP over-ingestion, clinical symptoms and 45 ity in the latter. Thus, although no specific limitations on laboratory results are dependent on the formulation (com- APAP intake have been proposed based on ethnic background, bined opiate-APAP preparations, extended-release, etc.) or further studies on this topic may yield useful clinical informa- doses of APAP ingested, including co-ingestions (i.e. chronic or tion about the safety profile amongst a diverse patient acute alcohol ingestion, herbal supplements, or prescription population. drugs, as previously discussed), and the existence of preceding liver disease. Chronic liver disease Stage I occurs within the first 24 hours of ingestion and is characterized by the non-specific symptoms of nausea, vom- APAP metabolism is reduced in patients with cirrhotic livers, iting, malaise, lethargy and diaphoresis. Aspartate transami- as compared to those with normal (non-diseased) livers. nase (AST) and ALT values are usually normal, although in Chronic liver disease patients who use alcohol infrequently immense overdose, elevated values can be detected in as do not appear to be at an elevated risk of developing APAP little as 8–12 hours. 21,22,24 hepatotoxicity. Although ideally a different choice of Stage II occurs within 24 to 72 hours and is characterized anti-pyretic or analgesic may be used, a less than 4000 mg by improvement or resolution of stage I symptoms (also per day use of APAP in the cirrhotic patient may be safe in the known as the latent period). However, elevations of AST and short term. More conservative dose limits, such as 2000 mg ALT typically begin to occur. Severe cases of APAP overdose per day, have been recommended, especially for patients in can present with tender hepatomegaly (with right-upper quad- whom the liver disease is marked with hepatic decompensa- rant pain), jaundice and coagulopathy. Approximately 1;2% 21 tion or active alcohol abuse. of patients may also experience renal failure in the setting of acute tubular necrosis with or without hepatic necrosis.48 Pregnancy Stage III occurs within 72 to 96 hours after the initial APAP overdose, and is noted by the return of stage I symptoms APAP is considered the most frequently prescribed analgesic in along with marked AST and ALT elevations (possibly > 3000 pregnancy.46 Pregnancy is a high-risk state for many medica- IU/L) in conjunction with jaundice, encephalopathy, coagul- tions, and APAP is no exception. Unfortunately, APAP toxicity opathy and lactic acidosis. Maximal liver injury occurs in this in pregnancy can result in significant morbidity and mortality stage. Renal failure, and on rare occasions pancreatitis, can for both the mother and the fetus.46 APAP can freely cross the occur as a complication.49 This stage has the highest risk of selective maternal-fetal barrier of the placenta, after which it mortality, which is most frequently due to multi-organ failure. can then be metabolized by fetal hepatocytes, causing fetal Lactic acidosis portends a poor prognosis (by King’s College hepatic necrosis if appropriate therapy with NAC is not admin- Hospital (KCH) criteria), and the mechanism of lactic acide- istered in a timely manner.47 Even though there is heightened mia is two-fold: NAPQI being present in excess and causing activity of glucouronidation and oxidative pathways, causing mitochondrial dysregulation, followed in subsequent hours the increased clearance of APAP in pregnancy, no evidence by tissue hypoxia and decreased hepatic metabolism and exists to suggest pregnancy as a predisposing risk factor for clearance of lactate, leading to a “two-hit” effect on worsening APAP toxicity.1 Moreover, despite that fact that APAP can cross of the lactic acidosis condition.12,13 Certain objective data, the placental barrier, the only instances when the fetus has such as a prothrombin time peaking at > 180 seconds, been shown to be at risk is when the mother suffers from along with prothrombin time that continues to rise beyond severe APAP hepatotoxicity, which is usually apparent based 4 seconds after the APAP overdose, confers about a 90% mor- on the history of presenting illness. Some case reports have tality without liver transplantation.50 If a patient becomes described loss of pregnancy following high-dose ingestion of obtunded and comatose as a result of the APAP overdose, APAP (up to 29 g) within a day of hospital presentation and intubation with mechanical ventilation becomes necessary. others have described successful fetus delivery following Stage IV occurs after 96 hours following the recovery from ingestion of 15 g and aggressive NAC rescue therapy.48 stage III. Normally, stage IV lasts between 1–2 weeks, but Should either APAP “therapeutic misadventure” or intentional its duration can be prolonged depending upon the severity of overdose be suspected, aggressive antidote intervention with the ingestion as well as the preparation of APAP ingested.

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If liver biopsies had been performed in previous stages, the rates of 60% for severe hepatotoxicity and 5% for mortality.57 histologic recovery period may take several months longer A “high toxicity line” also exists parallel to the “probable tox- than the clinical recovery. Chronic hepatitis has not been icity line”, and this begins at 300 mg/mL at 4 hours, equating reported as a complication of APAP overdose-associated to a 90% incidence of severe hepatotoxicity and 24% mortal- acute liver failure.1 ity.57 In the United States, Australia and New Zealand, a more Poor prognostic signs include multi-organ failure, which conservative measurement has been established, starting may involve cerebral edema, renal failure, profound hypo- at a 4-hour APAP concentration of 150 mg/mL and known as glycemia and lactic acidosis, any signs of which should the “treatment line,” which is intentionally set lower to prompt an immediate liver transplant evaluation.4,6,8,50,52,53 account for inaccuracies in the history of APAP ingestion and In clinical practice, signs of unintentional toxic ingestion typ- inherent laboratory error in APAP measurement.54,59 This line ically present later, when the above-mentioned complications sits 25% below the “probable toxicity line.” Studies have are already apparent and have progressed. Indeed, further already shown that the “treatment line” (also known as the studies to elucidate the prevalence of APAP-related hepato- “150 line”) serves well to identify and help protect high-risk toxic patients may be beneficial to identify the clinical stage of patients who may already have diminishing APAP levels.54,59 APAP hepatotoxicity in order to better triage patient treat- Further conservative measures to decrease the APAP meas- ment, improve disposition, and design the best follow-up. urement threshold to a “100 line” have not presented dem- APAP hepatotoxicity is one of few bona fide causes of liver onstrated increases in patient health success rate; rather, use injury that can raise serum aminotransferases > 10,000 IU/L. of such a strategy would subject more patients to overtreat- The most worrisome time for maximal organ damage occurs ment without much added benefit, leading to increasing between 3 and 5 days following the acute ingestion, strad- healthcare costs as patients are often treated for acute APAP dling stage III and stage IV hepatotoxicity; thus, prompt hepatotoxicity in medical intensive care units to ensure close recognition of APAP toxic ingestion with expedient initiation of monitoring of vital signs and the ability for frequent neuro- therapeutic intervention becomes important to prevent acute logic assessments.54,59 liver failure. In general, due to the benign nature of NAC therapy, if there is any doubt about the concentration of serum APAP or General approach and diagnostic tools the timing of ingestion, treatment with NAC is safe and indicated. The initial purpose of the Rumack-Matthew nomo- Patient history and physical examination are paramount, gram was for acute ingestions of APAP and its use has not especially in delineating the time course and formulation of been validated for chronic ingestions, extended-release prep- APAP ingested. APAP level measurements are commonly arations or co-ingestions of APAP with other substances that performed when a patient initially presents in the acute care may be hepatically cleared. This limitation proves clinically setting with a toxic ingestion of unknown substance, altered significant as many individuals with APAP over-ingestion 21 mental status and/or suspicion of intent of self-harm. It is encompasses this broad-spectrum population. critical to obtain a 4-hour APAP level, or as close to a 4-hour level as possible, as this value guides therapy and impacts Evaluations after repeated overdoses patient outcome. Upon presentation, additional laboratory studies are necessary to obtain other important clinical Patients with unintentional APAP overdoses have usually parameters, including arterial blood gas (to investigate acid/ ingested APAP often over many days as an analgesic or anti- base status), coagulation profile, basic metabolic panel, pyretic therapy. Symptoms of hepatotoxicity may have hepatic function tests, and urine drug screen (to determine already begun by presentation. Jaundice, right-upper quad- possible co-ingestions).54 A negative APAP reading should rant pain, nausea, vomiting, hepatomegaly and encephalop- not be interpreted as APAP-related hepatic injury having not athy indicate high levels of APAP ingestion, and thus when taken place, as levels may be undetectable while liver these symptoms are observed the patient’s APAP level should damage has already occurred. However, clinicians should be be checked.27 The Rumack-Matthew nomogram that was pre- cautious in interpreting APAP levels in patients with direct viously described is not applicable in this case, and instead hyperbilirubinemia > 10 mg/dL, as a falsely elevated APAP treatment with NAC would be appropriate for a finding of APAP concentration can be the result of bilirubin’s interaction with levels > 20 mg/mL, with or without ALT elevation.56,60 NAC the enzymatic measurement of APAP.55 This clinical context should also be administered when a patient has excessive becomes especially important because DILI can manifest APAP intake with elevated ALT levels, even though serum significant hyperbilirubinemia. APAP levels are undetectable. NAC does not appear to be of benefit when APAP levels are undetectable, patients are Evaluations after acute single overdose asymptomatic, or ALT level is normal.

Use of the Rumack-Matthew nomogram in management of Evaluations after established hepatotoxicity and liver failure APAP overdose is acceptable when the acuity of ingestion is known to be within 24 hours.54,56–59 The nomogram plots Because the overwhelming majority (;90%) of patients with the independent time in hours versus APAP concentration. APAP hepatotoxicity will recover spontaneously from their The important values from the nomogram plotting are acute ingestion and instead require appropriate therapy, points on the “probable toxicity line,” which include an APAP with intensive care monitoring using cardiac telemetry and level of 200 mg/mL at 4 hours and 25 mg/mL at 16 hours after frequent laboratory checks. Admission at a liver transplant acute ingestion. Patients who fall above these levels at the facility is important for patients who develop signs of acute appointed times are at risk of severe hepatotoxicity (defined liver failure, which include encephalopathy, coagulopathy and as AST > 1000 IU/L).56–62 NAC therapy is recommended in metabolic acidosis. The workup and management of fulmi- such clinical scenarios, to overcome the reported incidence nant hepatic failure does not differ under conditions of APAP

Journal of Clinical and Translational Hepatology 2016 vol. 4 | 131–142 137 Yoon E. et al: Acetaminophen-induced liver toxicity hepatotoxicity, except for the use of NAC as an antidote. dysfunction and to promote oxidant stress.17 Indeed, high Although the APAP toxicity-associated acute liver failure has levels of protein adducts have been detected in the serum of better outcome than other causes of liver failure, mortality is patients with APAP overdose.17 Detection of elevated levels still significant in ;30% of cases without a liver transplant (for of these APAP-protein adducts by high-pressure liquid chro- those who are deemed in need of a transplant).51,52 matography (HPLC) can be used for clinical indication of APAP The development of a prognostic model for survivability of hepatotoxicity. However, such testing remains a specialized APAP hepatotoxicity, with or without liver transplant, was an method, available to certain institutions, and thus has not important achievement to delineate need for emergency spread to all health centers that handle cases of APAP hepa- transplantation. The KCH in London, United Kingdom devel- totoxicity. For HPLC interpretation of suspected APAP hepato- oped a prognostic model based on two groups of patients, toxicity cases, liver centers will have to look to hire expert those with APAP-induced acute liver failure and those with technicians who can provide these services in an emergency non-APAP-induced acute liver failure. The first check is to setting. obtain a blood gas measurement of arterial pH; if arterial pH is Serum markers of mitochondrial damage and death, includ- < 7.3 or blood lactate is > 3.0 mg/dL after adequate volume ing glutamate dehydrogenase (GDH), nuclear DNA (nDNA) and resuscitation, irrespective of level of encephalopathy, the mtDNA have been investigated as clinically useful surrogate patient meets the KCH Criteria. If acidosis or lactate does markers capable of indicating mitochondrial lysis following not meet the KCH criteria, another check is performed to hepatocyte necrosis in APAP hepatotoxicity.12,64–68 There is determine whether the patient has a lactate > 3.5 mg/dL after evidence that patients who were non-survivors of APAP- early volume resuscitation. If this criteria is not met, the final induced acute liver failure had a statistically significant set of criteria will be considered, which includes grade III or VI increase in GDH, nDNA, and mtDNA fragment levels, as com- encephalopathy with prothrombin time > 100 seconds or INR pared to patients that recovered and survived their acute liver > 6.5, with serum creatinine > 3.4 mg/dL. In the absence of a failure, inferring that more significant mitochondrial damage liver transplant, the APAP-associated acute liver failure portends a higher mortality rate.65 The highest activity of patients who met the KCH criteria reportedly have 80–90% GDH in plasma has been found in patients with markedly ele- – mortality.51 53 vated ALT levels emanating from zone 3 of the liver, where Various other scoring systems have been evaluated as well. GDH is most highly expressed and where APAP is known to The model for end-stage liver disease (MELD) has not proven case the greatest extent of liver injury.12 Moreover, the time 53 superior to the KCH criteria or INR alone. One study com- course of the release of GDH and mtDNA has been shown to pared various scoring systems, including the KCH criteria, the correspond well to the release of ALT from hepatocytes under- sequential organ failure assessment (SOFA), the MELD and the going necrosis. acute physiology and chronic health evaluation II (APACHE II), In addition to GDH, nDNA and mtDNA, the intranuclear as prognostic indicators for their ability to reduce mortality product high mobility group B1 (HMGB1), a chromatin protein rates due to APAP hepatotoxicity. While the KCH criteria involved in nuclear DNA organization and transcription regu- had excellent specificity, it was at the expense of sensitivity, lation, is also detectable in serum in the setting of hepatocyte yielding a limited ability to distinguish patients who would necrosis.64 The detection of these serum biomarkers, also survive with conservative medical management versus those referred to as DAMPs, may help in the future decision-making 52 that would die without a liver transplant. The SOFA score, of therapeutic designs and transplant evaluations, as well as to owing to its measures of physiologic parameters of multiple identify future potential targets of medical therapy.66 organ systems, was superior in discriminating patients who The association between a rise in levels of bile acids and 52 would benefit most from a liver transplant evaluation. those of ALT (indicating hepatocyte injury) was investigated Similar to any solid tumor transplant candidate, candidates as another biomarker that might be potentially useful for for transplant treatment for APAP hepatotoxicity should predicting prognosis of APAP hepatotoxicity. The process of undergo a thorough psychiatric evaluation and assessment bile acid transport from hepatocytes into the biliary tract via to determine the level of social support needed. This type of canalicular transporters is sensitive to disturbances, as evi- evaluation becomes especially important in cases for which denced by elevated bilirubin levels that occur upon minor liver the APAP-associated liver failure was a consequence of inten- injury. A research group found that one specific bile acid, tional ingestion, as a repeat episode of over-ingestion will glycodeoxycholic acid (GDCA), rose significantly higher in the inevitably cause harm to the transplanted organ and squan- group of non-surviving APAP-associated liver failure patients dering of a limited resource. than in the group of patients who survived their liver failure despite having similar ALT levels as the first group.64 Due to Serum biomarkers of APAP-induced liver injury the increased efficiency in medical diagnostics afforded by HPLC and mass spectroscopy techniques, prognostic-related The detection of one or more biomarkers in the setting of findings of bile acid level may add to the armamentarium of APAP hepatotoxicity is desirable, especially in clinical scenarios findings from studies on how to better predict patient morbid- where the diagnosis of APAP hepatotoxicity is unclear. Multiple ity and mortality, and perhaps can even be used to shift treat- serum biomarkers have been described as potential indi- ment decision-making on surgical transplantation versus cators, not only to identify hepatocyte injury and necrosis medical therapy for APAP overdose. but also to help predict patient outcomes based on presence On the molecular level, human microRNAs (miRNA) have or absence of certain intracellular or intramitochondrial also been identified as potential early biomarkers for indicat- markers.12,17,63–69 ing APAP-induced hepatotoxicity at a stage before alanine APAP protein adducts that form upon NAPQI conjugation aminotransferases become elevated. One miRNA in particular, with protein sulfhydryl groups of cysteine in GSH or cellular the miRNA-122, was shown in a cohort who had taken proteins readily interact with mitochondria. These mitochon- APAP to significantly increase before ALT levels rose.67 This drial protein adducts are thought to cause mitochondrial finding was reaffirmed in another study that demonstrated a

138 Journal of Clinical and Translational Hepatology 2016 vol. 4 | 131–142 Yoon E. et al: Acetaminophen-induced liver toxicity corresponding increase of serum miRNA-122 and miRNA-375, with established APAP hepatotoxicity or those with high risk along with an increase in APAP protein adducts, all of which of developing this condition. NAC replenishes and maintains were also seen prior to ALT elevation.68 Urinary miRNA-375 hepatic GSH stores by providing cysteine, the substrate was also detected at high levels in that study.68 Furthermore, which detoxifies reactive metabolites of APAP.23,24,56,75 In that study had also enrolled children who had presented to addition, it may reduce NAPQI back to APAP by enhancing the hospital with accidental APAP overdose, and thus applic- the sulfonation pathway of APAP metabolism.56,75 Adminis- ability to the adult population may be better reserved for a tering NAC to patients with APAP overdose may reduce mor- time after other studies are completed with a greater adult tality from 5% to 0.7% of patients, and thus timely population. administration is critical.71 Table 2 lists commonly accepted One research group has sought to capture one aspect of indications for treatment with NAC in the setting of acute the wide systemic effects of APAP hepatotoxicity by focusing over-ingestion. in on kidney failure following acute APAP ingestion. Specifi- Delaying therapy to APAP hepatotoxic patients is danger- cally, expression of the kidney injury molecule-1 (KIM-1) was ous. While the goal is to treat patients before the develop- quantified, and it was found that patients who died from ment of acute liver failure, treatment should still be given to APAP-associated acute liver failure or who required liver patients who are found to be in active hepatic failure since transplant had significantly higher KIM-1 values on day 1 of NAC appears to improve hepatic perfusion and oxygen presentation, as compared to those who spontaneously delivery, to refine mitochondrial energy metabolism, and survived.69 Elevations of a kidney injury marker should not to facilitate scavenging of reactive oxygen and nitrogen come as a surprise, however, as acute kidney injury evalua- species.56,60–62 NAC can be delivered as either intravenous tion in the form of serum creatinine is included in scoring (IV) or oral (PO) preparations, with dosing based on the systems such as the APACHE II and KCH criteria. KIM-1 meas- patient’s body weight (maximum dose of 110 kg for oral and urements appear to outperform traditional serum creatinine 100 kg for IV).54,56,71 as a measure of acute organ damage, and this possibility The standard formulations for NAC are as follows. For the deserves further research attention. IV regimen, a loading dose of 150 mg/kg in 200 cc is given for Detection of biomarkers is certain to gain more clinical 15 minutes, followed by 50 mg/kg in 500 cc over 4 hours significance in the future management of APAP hepatotoxicity and then 100 mg/kg in 1000 cc over 16 hours.4,54,56,71 For in the modern era of outcome-based medical care. Collective the PO regimen, a loading dose of 140 mg/kg is given, fol- qualitative or quantitative results of these biomarkers may lowed by 70 mg/kg every 4 hours for a total of 18 total serve to predict potential morbidity and/or mortality of liver doses.54,56,71 Renal or hepatic dose adjustment is unneces- disease and failure, leading to appropriate triage of patients sary. Patients at high risk of hepatotoxicity should have serial into those requiring emergency liver transplantation versus APAP level and hepatic function panels measured before the patients who are likely to have good clinical improvement completion of NAC. NAC should be continued if APAP levels with NAC therapy alone. More validated studies need to be remain > 10 mg/mL or ALT is persistently elevated, with re- done to make this correlation apparent. evaluation of the status after 12 hours.54,56 NAC therapy can be considered complete if APAP levels are undetectable and In the hospital: acute management of APAP overdose ALT level has returned to normal.56 However, if the patient continues to present evidence of liver dysfunction beyond Physically blocking APAP from the GI tract: gastrointestinal persistent ALT elevation (satisfying KCH criteria of acidosis, decontamination elevated creatinine, hyperbilirubiniemia, coagulopathy and encephalopathy), then NAC therapy should be continued as One proposed method of initial therapy is to physically inhibit drug absorption or remove the offending agent from the Table 2. Clinical indications for use of NAC gastrointestinal tract. If a patient is fortunate enough to seek out and obtain early medical attention (typically within 1 Severe APAP toxicity with ALT:AST > 1000 4 hours of the acute APAP ingestion), activated charcoal can 2 Initiation of NAC within 24 hours of ingestion be effective in limiting drug absorption, unless there are obvious contraindications, such as an unprotected airway or 3 Serum APAP levels from 140 mg/L at 4 hours to compromise of the gastrointestinal tract.70 Gastric lavage, 50 mg/L at 10 hours activated charcoal ingestion, and vomiting induced by ipece- 4 Acute poisoning (ingested in 1 hour) with no other cauanha can all reduce drug absorption, but weak evidence products containing acetaminophen in the past 24 suggests activated charcoal as the preferred method of gas- hours trointestinal decontamination.71 The only patients who may 5 Acute poisoning with no ingestion of sustained release benefit from activated charcoal beyond 4 hours of ingestion formulations are those who ingested extended-release APAP formulations or co-ingestion of drugs that delay gastric emptying time.70 6 Baseline normal ALT, AST and INR Use of activated charcoal is not without risks, however, and 7 Used ideally within the first 8–10 hours with risk of one must be wary of aspiration pneumonia, vomiting, diar- hepatotoxicity being < 5%, especially if APAP level is rhea, constipation, ileus, and interference with the patient’s above the treatment line on the Rumack-Matthew regular medications.72 nomogram 8 Empirical use when APAP levels cannot be obtained NAC within 8 hours of ingestion

NAC is a cysteine prodrug and hepatic GSH precursor, and Abbreviations: APAP, acetaminophen; ALT, alanine aminotransferase; AST, aspar- should be administered immediately as an antidote in patients tate aminotransferase; INR, international normalized ratio; NAC, N-acetylcysteine.

Journal of Clinical and Translational Hepatology 2016 vol. 4 | 131–142 139 Yoon E. et al: Acetaminophen-induced liver toxicity these indicators portend a poor prognosis regardless of trans- systems but also necessitates a multidisciplinary approach aminase levels.62 Patients with severe hepatotoxicity or acute that includes psychiatric evaluation, which may be challeng- liver failure should continue on NAC at 6.25 mg/kg/h until a ing for patients who have intentionally overdosed on APAP. liver transplant takes place or liver dysfunction is reversed In one study, 20% of the center’s liver transplantation cases (evidenced by regression of AST or ALT, resolved encephalo- were due to APAP overdose, which was the most common pathy, and/or INR > 1.5) with an undetectable APAP cause of acute liver failure prompting transplantation; level.54,56 Table 3 describes how NAC therapy functions to mit- however, only 12% of those patients presenting to this same igate hepatocyte necrosis and to promote liver regeneration. center with acute liver failure from APAP overdose actually While there are two treatment options, the clinical scenario received a transplant.51 In a large multicenter study of liver should determine the route of NAC administration (PO versus failure cases from the United States’ Acute Liver Failure Study IV). The efficacy between the two preparations appears Group, 54% of patients had APAP-related liver failure and the same, although no head-to-head trial currently exists. were compared to non-APAP related liver failure patients; IV NAC is preferentially given to patients with acute liver the APAP-associated liver failure cases fared better in 2-year failure and to those who refuse or have a contraindication for survivorship if they were not transplanted, as compared to the PO formulation; contraindications include coma, pancrea- transplanted patients.73 Conservative medical therapy has a titis, ileus or gastrointestinal tract insufficiency. IV NAC has a good outcome when prognostic scoring systems point against 10–20% risk of anaphylactic reaction, and transfusion should proceeding with transplant, and it appears that physicians are not be halted for minor reactions of flushing.60,72 Other iden- making better-informed decisions in regards to transplanta- tified side-effects of NAC include nausea, vomiting, diarrhea tion of patients who present with acute liver failure related to or constipation, and rarely fever, headache, drowsiness or APAP hepatotoxicity. hypotension. Steroids, diphenhydramine and bronchodilators can help resolve the anaphylactoid reactions and should be Alternative therapeutic thoughts in APAP hepatotoxicity given so that NAC therapy can be continued under close monitoring.72 The oral formulation has a sulfur component, and Targeting APAP hepatotoxicity at the molecular level as an thus has an unpleasant taste and smell that can cause nausea alternative to NAC therapy has been the goal of many and vomiting; switching PO to IV therapy is an option. This research groups. One particular group has been investigating switch can be mitigated, however, by using a nasogastric tube metformin as a potential therapeutic intervention to reduce for direct gastrointestinal access or by taking the medication hepatotoxicity after APAP exposure. The proposed therapeutic with sodas or juice to make the medication more tolerable. mechanism involves the down-regulation of c-Jun-N-terminal kinase (JNK) by inducing Gadd45-beta activity, which ulti- 74 Liver transplantation mately disrupts a mechanism of mitochondrial damage. One investigative group in South Korea has reported on Liver transplantation can be a lifesaving procedure when APAP the use of sulforaphane (SFN) as a potential protector against 75 ingestion has progressed to irreversible liver failure. Prog- oxidant-induced tissue injury. SFN is a dietary isothiocyna- nostic factors including MELD, KCH criteria and APACHE II nate synthesized from a precursor found in vegetables such scores have all been investigated as objective indicators as cauliflower, broccoli, kale, cabbage, collards and Brussels 75 to select liver transplantation candidates.51,52,62 Qualifying sprouts as well as other genera, such as the radish. SFN has for liver transplantation not only requires a projected poor been previously shown to have protective effects against outcome and high risk of mortality based on these scoring oxidative stress-damaged tissues, such as the brain, heart, pancreas and kidney.75 In primary hepatocytes, cells pre- treated with SFN were shown to be protected against APAP- induced liver injuries via antioxidant actions by blocking Table 3. Role of NAC generation of reactive oxygen species, depletion of GSH and 1 Protection against reactive oxygen species by peroxidation of lipids.75 increasing Nrf2 and HO-1 mRNA levels Another group in China has suggested an association between adequate hepatocellular serotonin levels in murine 2 Protection against mitochondrial dysfunction, which models and protection against hepatocyte inflammation, causes release of acylcarnitines in peripheral oxidative stress, GSH depletion, peroxynitrite formation, vasculature elevated hepatocyte proliferation and reduction in several 3 Elimination of JNK activation and GDH release enzymes described in the process of hepatocyte necrosis.76 4 The mechanism of protection during the early Human hepatocyte models are a next logical step, and thus metabolism phase primarily involves improved more investigations need to be carried out before either sero- scavenging of the reactive metabolite NAPQI due to tonin or SFN can be realistically considered on the therapeutic accelerated GSH synthesis level. 5 Scavenging of the reactive metabolite NAPQI and decreasing of protein binding during the early phase Conclusion occurs through increasing levels of GSH APAP ingestion and subsequent hepatotoxicity is a critical 6 Provision of increased amounts of cysteine to allow problem that continues to plague individuals across the world, regeneration of GSH well beyond clearance of APAP, due to the cheap cost of APAP contributing to its being a allowing for hepatocyte regeneration ubiquitous analgesic and anti-pyretic drug available through consumer pharmacies and as prescription-only medication Abbreviations: JNK, c-Jun-N-terminal kinase; GD, glutamate dehydrogenase; GSH, glutathione; NAC, N-acetylcysteine; HO-1, heme oxygenase-1; NAPQI, formulations. Since APAP is responsible for nearly half of the N-acetyl-para-benzo-quinone imine; Nrf2, nuclear factor erythroid 2-related factor 2. cases of acute liver failure in the United States and remains

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Toxicol Appl Pharmacol 2015;284:180–187. doi: 10.1016/j.taap. doi: 10.1007/BF03160941. 2015.02.013. [49] Caldarola V, Hassett JM, Hall AH, Bronstein AB, Kulig KW, Rumack BH. Hem- [69] Antoine DJ, Sabbisetti VS, Francis B, Jorgensen AL, Craig DG, Simpson KJ, orrhagic pancreatitis associated with acetaminophen overdose. Am J Gastro- et al. Circulating kidney injury molecule 1 predicts prognosis and poor enterol 1986;81:579–582. outcome in patients with acetaminophen-induced liver injury. Hepatology [50] Harrison PM, O’Grady JG, Keays RT, Alexander GJ, Williams R. Serial pro- 2015;62:591–599. doi: 10.1002/hep.27857. thrombin time as prognostic indicator in paracetamol induced fulminant [70] Underhill TJ, Greene MK, Dove AF. A comparison of the efficacy of gastric hepatic failure. BMJ 1990;301:964–966. doi: 10.1136/bmj.301.6758.964. lavage, ipecacuanha and activated charcoal in the emergency management [51] Bailey B, Amre DK, Gaudreault P. Fulminant hepatic failure secondary to of paracetamol overdose. Arch Emerg Med 1990;7:148–154. doi: 10.1136/ acetaminophen poisoning: a systematic review and meta-analysis of prog- emj.7.3.148. nostic criteria determining the need for liver transplantation. Crit Care Med [71] Chun LJ, Tong MJ, Busuttil RW, Hiatt JR. Acetaminophen hepatotoxicity and – 2003;31:299 305. doi: 10.1097/00003246-200301000-00048. acute liver failure. J Clin Gastroenterol 2009;43:342–349. doi: 10.1097/ [52] Cholongitas E, Theocharidou E, Vasianopoulou P, Betrosian A, Shaw S, Patch MCG.0b013e31818a3854. D, et al. Comparison of the sequential organ failure assessment score with [72] Buckley N, Eddleston M. Paracetamol (acetaminophen) poisoning. BMJ Clin the King’s College Hospital criteria and the model for end-stage liver disease Evid 2007;2007. score for the prognosis of acetaminophen-induced acute liver failure. Liver [73] Reddy KR, Schilsky ML, Stravitz R, Ellerbe C, Durkalski V, Fontana RJ, et al. Transpl 2012;18:405–412. doi: 10.1002/lt.23370. Liver transplantation for acute liver failure: results from the NIH Acute Liver [53] Craig DG, Ford AC, Hayes PC, Simpson KJ. Systematic review: prognostic Failure Study Group. Hepatology 2012;56:246A–247A. tests of paracetamol-induced acute liver failure. Aliment Pharmacol Ther [74] Kaplowitz N, Win S, Than TA, Liu ZX, Dara L. Targeting signal transduction 2010;31:1064–1076. doi: 10.1111/j.1365-2036.2010.04279.x. [54] Rumack BH. Acetaminiphen hepatotoxicity: the first 35 years. J Toxicol Clin pathways which regulate necrosis in acetaminophen hepatotoxicity. J – Toxicol 2002;40:3–20. doi: 10.1081/CLT-120002882. Hepatol 2015;63:5 7. doi: 10.1016/j.jhep.2015.02.050. [55] Buehler MC, Curry SC. False positive acetaminophen levels associated with [75] Noh JR, Kim YH, Hwang JH, Choi DH, Kim KS, Oh WK, et al. Sulforaphane hyperbilirubinemia. Clin Toxicol (Phila) 2005;43:167–170. doi: 10.1081/ protects against acetaminophen-induced hepatotoxicity. Food Chem Toxicol CLT-200057853. 2015;80:193–200. doi: 10.1016/j.fct.2015.03.020. [56] Heard KJ. Acetylcysteine for acetaminophen poisoning. N Engl J Med 2008; [76] Zhang J, Song S, Pang Q, Zhang R, Zhou L, Liu S, et al. Serotonin deficiency 359:285–292. doi: 10.1056/NEJMct0708278. exacerbates acetaminophen-induced liver toxicity in mice. Sci Rep 2015;5: [57] Prescott LF, Illingworth RN, Critchley JA, Stewart MJ, Adam RD, Proudfoot AT. 8098. doi: 10.1038/srep08098. Intravenous N-acetylcysteine: the treatment of choice for paracetamol poi- [77] Bliebel W, Kim S, D’Silva K, Lemmer ER. Drug-induced liver injury: review soning. Br Med J 1979;2:1097–1100. doi: 10.1136/bmj.2.6198.1097. article. Dig Dis Sci 2007;52:2463–2471. doi: 10.1007/s10620-006-9472-y.

142 Journal of Clinical and Translational Hepatology 2016 vol. 4 | 131–142 Review Article

Hepatitis B Reactivation with Novel Agents in Non-Hodgkin’s Lymphoma and Prevention Strategies

Oluwatobi O. Ozoya1, Lubomir Sokol2 and Samir Dalia*3

1Emergency and Trauma Center, Tampa General Hospital, Tampa, FL, USA; 2Department of Hematological Malignancies, H. Lee Moffitt Cancer Center, Tampa, FL, USA; 3Oncology and Hematology, Mercy Clinic Joplin, Joplin, MO, USA

Abstract Introduction

Hepatitis B virus (HBV) infection remains an endemic disease Hepatitis B virus (HBV) is a DNA virus that resides exclusively in most parts of the world despite available prophylactic in human reservoirs and is transmitted through body fluids. vaccines. Non-Hodgkin’s lymphoma is the most common The infective form remains prevalent globally despite avail- hematological malignancy, and certain patients undergoing able prophylactic immunization. Endemicity is present in Asia, therapy are at increased risk of HBV reactivation. Rituximab, Africa, the Middle East, parts of Eastern Europe, and South a monoclonal antibody, is well studied in HBV reactivation, but America, and up to 400 million people are chronically newer agents have been implicated as well. Here, we review infected.1 Although the United States is non-endemic for novel agents suspected in HBV reactivation and effective HBV, up to 1 million people have chronic HBV, and the risk strategies to prevent HBV reactivation. Fifteen years of liter- of acute infection persists. Estimated age-adjusted preva- ature were reviewed in order to better understand the reac- lence in the US is 4.7% for core antibody (anti-HBc) and tivation rates of hepatitis B in patients with non-Hodgkin’s 0.27% for surface antigen (HBsAg).2 In addition, HBsAg prev- lymphoma. Anti-CD20 antibodies continue to be the main alence rate is thought to be 0.75% in those who receive medications that can lead to HBV reactivation, and HBV immunosuppressive therapy and is highest among those with reactivation rates have decreased with increased awareness. co-infection of the human immunodeficiency virus (HIV).3 HBV reactivation is uncommon when using other novel agents. Vaccination efforts have contributed to the decrease in anti- Entecavir and lamivudine remain the agents of choice to HBc prevalence rate from 1.9% in 1999 to 0.6% in 2006 for prevent HBV reactivation in high risk patients. In conclusion, those aged 6 to 19 years. Among adults older than 50 years the immunosuppressive effect of NHL and its therapy provide a of age, the prevalence rate decreased marginally from pathway for HBV reactivation, especially in patients treated 7.7% in 1999 to 7.2% in 2006.2 Moreover, HBV has been impli- with anti-CD20 antibody. Since many HBV positive patients cated in the incidence of non-Hodgkin’s lymphoma (NHL),4–7 are often excluded from clinical trials of novel agents in NHL, which increases with advancing age for all race and gender more aggressive post-market surveillance of new agents, well- subgroups.8,9 designed best practice advisories, and timely case reports are NHL is one of the most prevalent hematological malignan- needed to reduce the incidence of HBV reactivation. Lastly, cies globally and contributed to over 190,000 deaths in large prospective investigations coupled with well-utilized best 2008.10 NHL is also common in the US, where almost practice advisories need to be conducted to understand the 20,000 patients will die from this disease each year, despite impact of more potent novel NHL therapy on HBV reactivation. available chemotherapy.11 Cytotoxic chemotherapy, which is © 2016 The Second Affiliated Hospital of Chongqing Medical highly immunosuppressive, has been linked to reactivation University. Published by XIA & HE Publishing Inc. All rights of HBV infection in seropositive and seronegative cases reserved. (undetectable anti-HBc and anti-HB surface antigens).12–18 The mechanisms underlying HBV reactivation in NHL therapy include the active roles of acquired viral genomic mutations, Keywords: Hepatitis B virus; HBV reactivation; Non-Hodgkin’s lymphoma; chronic viral infection, immunosuppression, and exaggerated Rituximab; Anti-CD20 antibody; Best practice advisories. immune response following cessation of chemotherapy.7,19–21 Abbreviations: AASLD, American Association for the Study of Liver Diseases; ACIP, Advisory Committee on Immunization Practices; AGA, American Gastroen- The definition of HBV reactivation can vary, but it must include terological Association; ALT, alanine aminotransferase; anti-HBc, HBV core anti- a 10-fold rise in HBV DNA in a patient with past or latent body; APASL, Asian-Pacific Association for the Study of the Liver; ASCO, American HBV infection and/or levels > 10,000 copies/mL (4 log Society of Clinical Oncology; BTK, Bruton’s kinase; CDC, Centers for Disease 7,22,23 Control and Prevention; CI, confidence interval; CLL, chronic lymphocytic leuke- copies/mL). A serum transaminase level three times mia; CPOE, computerized physician order entry; DLBCL, diffuse large B-cell lym- the upper limit of normal or alanine aminotransferase (ALT) phoma; EASL, European Association for the Study of Liver Disease; FDA, Food and over 100 IU/L7,19,24 in a patient with known HBV seropositivity Drug Administration; HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus; may also be considered HBV reactivation.22 HBV reactivation HIV, human immunodeficiency virus; HR, hazard ratio; NCCN, National Compre- hensive Cancer Network; NHL, non-Hodgkin’s lymphoma; OR, odds ratio; PI3Kd, is important in patients treated for NHL, as it increases the phosphatidylinositol 3-kinase delta inhibitors; RR, risk ratio. likelihood of stopping therapy, may lead to liver failure, and Received: 13 February 2016; Revised: 25 April 2016; Accepted: 26 April 2016 24–26 q can increase the risk of mortality. DOI: 10.14218/JCTH.2016.00005. Chemo-immunotherapy induced HBV reactivation can be *Correspondence to: Samir Dalia, Oncology and Hematology, Mercy Clinic Joplin, 100 Mercy Way, Joplin, MO 64804, USA. Tel: +1-417-782-7722, preventable if detected early. A vast majority of the NHL Fax: +1-417-556-3063, E-mail: [email protected] or [email protected] literature on HBV reactivation has focused on rituximab. With

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This is an Open Access article distributed under the terms of the Creative Commons Attribution-Noncommercial 4.0 Unported License, permitting all non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Ozoya O.O. et al: Hepatitis B reactivation with novel agents the advent of newer immunotherapy agents, less is under- In addition to rituximab, ofatumumab was included in stood about the incidence and prevention of HBV reactivation. the Food and Drug Administration (FDA) reactivation warning This review aims to summarize recent data on the reactiva- 4 years after its approval in 2009.24,37 A search of the FDA tion of HBV with novel agents in NHL therapy and effective Adverse Event Reporting System database yielded 32 cases methods to prevent HBV reactivation in these patients. of rituximab-associated HBV reactivation and one case associated with ofatumumab (http://www.fda.gov/Drugs/DrugSaf- ety/ucm366406.htm). Data in support of ofatumumab in HBV Review Criteria reactivation is still sparse, and a recent European Phase IV trial in advanced chronic lymphocytic leukemia (CLL) categorically Relevant articles for this review were identified by searching reported no case of HBV reactivation in patients treated with PubMed, Embase, Ovid Medline, and Scopus using the following ofatumumab.38 Obinutuzumab, recently approved by the FDA “ ” terms, alone and in combination: Hepatitis B reactivation , for CLL in 2013, has a black box warning for HBV reactivation. “ ” “ ’ ” “ novel agents , Non-Hodgkin s lymphoma , immunosup- However, no published data exist to support this report.39,40 A ” “ ” “ pression , immunocompromised host , immunosuppressive search from the FDA Adverse Event Reporting System data- ” “ ” “ ” “ ” “ ” agents , hepatitis B , hepatitis B virus , HBV , reactivation , base did not yield any data to support this report (http:// “ ” “ ” management and prevention . Full text articles of all www.fda.gov/Drugs/DrugSafety/PostmarketDrugSafety selected studies were retrieved, and if a paper was selected InformationforPatientsandProviders/ucm111085.htm#O; for inclusion, the bibliographic references were scrutinized to http://www.fda.gov/drugs/informationondrugs/approved identify additional relevant studies. The period of the search drugs/ucm373263.htm). A Japanese Phase I study of obinutu- was restricted to a 15 year period to limit the search to novel zumab in refractory B-cell NHL excluded patients with seropos- – agents in the treatment of NHL (2000 2015). itive HBV status,41 thereby precluding the usefulness of identifying adverse events through clinical trials.42 Hepatitis B reactivation in NHL therapy Although there is no FDAwarning yet, few case reports of HBV reactivation have been reported with other monoclonal agents Hepatitis B reactivation in susceptible individuals has been used to treat NHL. Alemtuzumab (anti-CD52) therapy, mainly linked with chemotherapy through amplification of viral repli- used in CLL, increased HBV DNA level to 7.3 log copies/mL in 43 cation and immune function recovery following chemotherapy one patient, while mogamulizumab in adult T-cell leukemia- induced-immunosuppression.20,21,26,27 Reactivation is also lymphoma increased HBV DNA to a range of 2.1 to 9.1 log 44,45 attributable to profound immunosuppression induced by lym- copies/mL during therapy for four different patients. Other ’ phocytotoxic monoclonal antibodies27,28. In the largest study novel agents, such as the small molecule inhibitors [Bruton s (10,729 patients) on HBV reactivation in patients receiving kinase (BTK) inhibitors and phosphatidylinositol 3-kinase d chemotherapy, predictors included male gender, race (Asian, delta inhibitors (PI3K )], have been linked to the occurrence Black), rituximab use, and presence of hepatitis B risk factors. of autoimmune hepatitis, but it is unclear if HBV reactivation 20,46,47 The risk of reactivation was high in individuals with anti-HBc can occur. Idelalisib, a potent, small-molecule inhibitor d or anti-HBs and even higher in those with seropositive of PI3K has demonstrated favorable treatment response in HBsAg.28 patients with indolent NHL who are refractory to rituximab 48,49 The severity of chemotherapy-induced HBV reactivation in and other previous chemotherapy. Asymptomatic elevated – NHL has been categorized as low risk with azathioprine and transaminase levels was reported in 47% 48% of such – methotrexate moderate risk with commonly used cytotoxic patients, and 13% 25% had grade 3 elevations, although most 48,49 chemotherapy, such as CHOP (cyclophosphamide, doxorubicin, cases resolved following dose reduction. It is unclear if vincristine, and prednisone), high-dose corticosteroids (predni- these are negligible laboratory abnormalities or an indication d solone > 20 mg), and fludarabine. Patients who received anti- that patients with HBV risk factors treated with PI3K inhib- 50,51 CD20 monoclonal antibodies, such as rituximab, were at the itors may develop overt HBV reactivation. Clinical trials highest risk for HBV reactivation.29,30 of these agents in combination with rituximab are underway, Among the monoclonal anti-CD20 antibodies, rituximab- and the outlook in regard to HBV reactivation is guarded until 42,52 associated HBV reactivation has been the most commonly more post-market surveillance data emerge. Table 1 reported event.7,20,26 Two meta-analyses have demonstrated summarizes confirmed and suspected novel agents with HBV more than a five-fold increased risk of HBV reactivation with reactivation sequela. rituximab chemotherapy based on HBcAb serum level (risk ratio (RR) of 5.52, 95% confidence interval (CI) 2.05–14.85, Prevention Strategies p < 0.001)12 and odds ratio (OR) of 5.73, 95% CI 2.01–16.33; Z=3.33,p < 0.001.13 The first published meta-analysis HBV screening, best practice advisories, and HBV reported a 55% liver failure rate,13 while another reported that vaccination 43% of participants developed adverse hepatic-related events.7 In addition, early studies on HBV reactivation rates from ritux- Screening for both HBV reactivation and HBV risk factors is imab combined chemotherapy reported rates up to 56%, espe- the first step in preventing reactivation in patients undergoing cially in HBV endemic regions.22,31,32 However, more recent therapy for NHL.28,30,53 Organizational policies on screening studies have reported lower reactivation rates (< 2.7%) and have significantly increased screening rates over the last lower mortality rates,14,15,33,34 even in high prevalent regions. 6 years from 20% to 90% at large oncology centers in the This discrepancy may be explained by improved defined criteria US.24,28 Policies on screening modalities are also gradually and awareness ofHBV reactivation.14,35 Inaddition, reactivation shifting from a targeted approach to a universal approach. rates may be reduced due to early diagnosis and increased A targeted screening approach for patients undergoing knowledge of the management of chronic hepatitis B and the chemo-immunotherapy on the basis of HBV risk status associated HBV reactivation in oncologic therapy.30,36 is limited since chronic hepatitis may be asymptomatic, and

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Table 1. Novel agents and HBV reactivation status

HBV reactivation Data a Agent Target Indication Status source References

Monoclonal antibodies Rituximab* CD20 Relapsed or refractory indolent FDA boxed FDA 13–15,30,31,37 lymphoma maintenance therapy in warning AERS B-cell NHL Ofatumumab* CD20 Relapsed/refractory CLL FDA boxed FDA 37,38 warning AERS Obinutumumab* CD20 Rituximab-refractory patients FDA boxed FDA 39–41 warning AERS Alemtuzumabx CD52 Refractory B-CLL +HBVr but no Case 27,43 FDA warning reports yet Mogamulizumabx CC Aggressive adult T-cell leukemia- +HBVr but no Case 44,45 chemokine lymphoma (ATL) and peripheral T-cell FDA warning reports receptor lymphoma yet Small molecule inhibitors Ibrutinibx BTK Low-grade NHL Immune Clinical 46,47 inhibitors hepatitis trials Idelalisibx PI3Kd Relapsed/Refractory low-grade NHL Immune Clinical 46,47 inhibitors hepatitis/ trials Transaminitis a Agent – Confirmed* and suspectedx agents with HBV reactivation sequela Abbreviations: B-CLL, B-cell chronic lymphocytic leukemia; BTK, Bruton’s kinase; FDA, Food and Drug Administration; FDA AERS, FDA Adverse Event Reporting System; HBV, hepatitis B virus; NHL, non-Hodgkin lymphoma; PI3Kd, phosphatidylinositol 3-kinase delta inhibitor. sufficient information may not be elicited during initial patient patients receiving chemotherapy or immunotherapy should encounters to adequately assess the risk of HBV infec- be considered for testing and prophylaxis. On the other tion.54,55 On the other hand, universal HBV screening for all hand, the NCCN57 and the American Association for the patients newly diagnosed with cancer who require chemo- Study of Liver Diseases (AASLD)61 suggested excluding immunosuppressive therapy results in a more direct reduc- from screening, patients with low risk of HBV and candidates tion in reactivation rates.24 In the US, hospital-enforced uni- for cancer therapy associated with low risk of reactivation, versal screening policies in some large cancer treatment because supporting evidence was insufficient. A tenable centers provide evidence that increasing pre-therapy screen- explanation for the disparity in screening approach may be ing and prophylaxis for HBV can result in zero-to-minimal consideration given to patients from regions with high HBV cases of HBV reactivation.28,53 Currently, the National Com- prevalence. At this time, clinicians should determine if prehensive Cancer Network (NCCN) recommends that all screening for HBV is needed in each individual patient receiv- patients receiving an anti-CD20 antibody should be screened ing chemotherapy. In the future, we hope that better data are for HBV prior to exposure to the agent.56 Screening should be available to help allocate resources for patients in need of for both HBsAg and anti-HBc since HBV DNA undetected in HBV screening prior to chemotherapy.53 serum can persist in tissues11,56,57 and the antigen may be Improved coordination of primary care and specialist absent in indolent cases of HBV.58 Patients who are found to healthcare professional services is required to screen be positive for either should be considered for HBV prophy- promptly all patients at risk of HBV infection irrespective laxis prior to chemo-immunosuppressive therapy. of negative cancer status. Screening prescription by health- According to clinical opinion in the oncologic literature, care professionals to patients, prior to immunosuppressive there is clear consensus on adopting screening to identify therapy still needs to be enhanced.54,55,62 Improved screen- patients who are at high risk of HBV and also candidates for ing rates to prevent HBV reactivation can be achieved with anti-CD20 monoclonal antibody. There are differences in improved data management and alert systems integrated opinion regarding the necessity to screen all patients who into existing electronic medical records.55,63 Between 2012 will receive chemotherapy irrespective of HBV risk status. and 2013, a cancer center study showed the role of a best Guidelines from the American Gastroenterological Associa- practice advisory (alert system) incorporated in the compu- tion (AGA),26 the European Association for the Study of Liver terized physician order entry (CPOE) application in increasing Disease (EASL),59 the American Society of Clinical Oncology HBV screening rates prior to therapy. At the end of the (ASCO),53 and the Asian-Pacific Association for the Study program, the study reported an improved screening rate for of the Liver (APASL)60 recommend that low and high risk patients treated with biologic agents from < 50% to > 85%.63

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Since prior HBV infection prior HBV infection is a baseline risk [95% CI, 6.4% to 27.0%]; p = .002) with entecavir com- factor for HBV reactivation, prevention efforts need to main- pared to lamivudine.72 Importantly, despite the lower risk of tain a clear distinction between identifying risk factors for HBV viral resistance with tenofovir, adefovir, and entecavir com- reactivation and for HBV infection.28,36 Notable risk factors for pared to lamivudine,77 these newer agents on rare occasions HBV infection include groups such as healthcare workers, were associated with mild renal dysfunction with long-term injecting drug users, recipients of blood products or dialysis, use.20,78,79 Therefore, dosage of these agents needs to be household contacts of people with chronic HBV infection, adjusted in renal impairment, and renal function should be incarcerated persons, people with multiple sex partners, assessed at least every 3 months.57,79 men who have sex with men, and unvaccinated or partially Another study that explored steroid-free chemotherapy in vaccinated travelers to high endemic regions.36 Low and the prevention of HBV reactivation in NHL reported a lower intermediate endemic countries require catch-up immuniza- incidence and severity of reactivation when compared to tion targeted at these high risk groups. In addition, programs steroid-containing chemotherapy.80 Despite the overall sur- that mandate HBV vaccination for entry into schools, colleges, vival rate, which was higher in the steroid-containing group, and work-places remain an effective strategy to reduce HBV a limitation of this approach may be deciding between survival infection in low-intermediate endemic regions.36 following rituximab-containing therapy and the risk of HBV The Advisory Committee on Immunization Practices reactivation. Finally, more studies are needed to understand (ACIP) of the Centers for Disease Control (CDC) recommends the efficacy of antivirals when both rituximab and steroids are universal vaccination of individuals, especially male adults, used.81 who test positive, since the risk of hepatitis B remains highest Therapeutic intervention for HBV reactivation in aggressive in this group.36 This recommendation is important because lymphomas/NHL is indicated in instances such as missed HBV adult males have a higher incidence of NHL compared to status and failed or interrupted prophylactic therapy.27,82–84 women of the same age group.8,9,64 For children, universal Failure of prophylactic lamivudine may result in withdrawal vaccination at childbirth with follow-up remains appropriate hepatitis, viral breakthrough, or mortality from liver failure.85,86 for high endemic (>8%) regions. On a global scale, newborns Predictors of lamivudine failure in a study include: elevated who received a birth dose of HBV vaccine increased from 27% baseline HDV DNA titer ($ 2,000 IU/mL) (hazard ratio [HR], to 38% between 2006 and 2014. Birth cohorts who received 9.94; p = 0.0063) and the use of rituximab (HR, 3.19; three doses in 2008 and 2014 increased from 69% to 82%. p = 0.027) for viral breakthrough, while for withdrawal hepatitis, Although these data indicate that HBV vaccination rates are a high baseline HBV DNA titer (HR, 5.90; p= 0.007), liver cirrhosis rising, there is still room for improvement in high endemic (HR, 10.4; p = 0.002), and distant metastasis (HR, 5.14; 1,65 areas. Since the last two decades, the CDC has recom- P = 0.008) were independent risk factors.86 Therapeutic inter- mended routine post-vaccination tests for anti-HBs and ventions also depend on the presence of secondary liver failure annual booster doses for sustained immunity among high and prognosis of the lymphoma. Newer antivirals may be used 66 risk groups and immunocompromised persons. Therefore, where liver failure is absent.27,75 For patients with HBV reactiva- targeted and universal vaccination efforts should not wane, tion resulting in liver failure, the AGA recommends liver trans- because the risk of exposure to HBV continues to persist. plantation.26,75 Favorableoutcomeswererecordedinpatients who underwent liver transplantation and antiviral therapy, Reactivation prophylaxis and therapy having established prior complete remission of disease and a favorable prognosis of the lymphoma type.75,87 HBV seropositive individuals who require chemotherapy or Based on better outcomes from recent data, recent ASCO anti-CD20 antibody therapy should be started on antivirals in and NCCN guidelines on HBV reactivation recommend, when a timely manner. Globally, lamivudine has been the most possible, that patients who are HBV positive (HBsAg-positive utilized nucleos(t)ide analogue for the prevention of HBV and/or anti-HBc positive) and require chemo-immunotherapy reactivation during chemo-immunotherapy.33,67–70 Prognosis should be placed on prophylaxis with entecavir for the of patients with lamivudine following HBV reactivation duration of treatment.53,61 Other agents, including tenofovir remains favorable, despite few cases of viral resistance.71 and lamivuidine, can also be used, but there may be more Newer nucleos(t)ide are now favored in reactivation manage- resistance to lamivudine therapy. Newer antiviral medications ment.71–75 Newer nucleos(t)ide analogues, such as entecavir, are readily favored considering the relatively high barrier to adefovir, and tenofovir, have been used because of their lower viral resistance compared to lamivudine for both prophylactic viral resistance.27,57 In a notable study, Li and others com- and therapeutic purposes. The literature suggests that pro- pared entecavir with lamivudine as prophylaxis for hepatitis B phylaxis against rituximab associated HBV-reactivation reactivation in lymphoma patients undergoing chemotherapy. should be extended up to 12 months following oncologic The entecavir group exhibited a significantly lower rate of HBV therapy and should be combined with HBV viral load surveil- reactivation (0% vs 12.4%, p = 0.024) and a lower incidence lance every 3 months.30,53,57 Although NCCN warns against of aborted chemotherapy compared to the lamivudine group lamivudine as a routine prophylactic agent due to risk of viral (5.9 vs 20.2%, p = 0.042).74 resistance, AASLD, EASL, ASIF, and APASL recommend lam- The stage of NHL disease is a useful criterion when ivudine as a viable prophylactic agent based on its pharmaco- – considering use of nucleos(t)ide analogues. Studies have economic and safety profiles.59 61,88 Specifically, the AASLD shown that the efficacy of entecavir is higher than that of endorses lamivudine when the planned duration of chemo- lamivudine, especially in advanced disease.72,76 In advanced therapy is less than 12 months and entecavir or tenofovir diffuse large B-cell lymphoma (DLBCL), Huang and colleagues for chemotherapy lasting beyond 12 months.20,61,89 In addi- compared entecavir and lamivudine and demonstrated that tion, the EASL advocates for use of baseline HBV DNA < 2,000 there was a lower reactivation rate (6.6% vs 30%; difference, IU/mL for initiating lamivudine while values > 2,000 IU/mL 23.4% [95% CI, 10.2% to 36.6%]; p = .001) and premature are indications for prescribing entecavir or tenofovir.59 chemotherapy cessation (1.6% vs 18.3%; difference, 16.7% In addition, the APASL and ASIF recommend lamivudine for

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Table 2. Modalities for preventing HBV reactivation

Reactivation Prevention Key Characteristics Modality References Indication/Advantages Drawbacks

Pre-NHL therapy screening Targeted screening Screen NHL patients receiving Missed cases 53,61,62 anti-CD20 antibody with (asymptomatic HBV infection) + HBV risk status Universal screening Screen all patients receiving False HBV positive cases 26,59,60,63 anti-CD20 antibody irrespective Added cost to therapy of HBV risk status Best Practice Advisory Increase HBV screening rates Implementation cost 56,64 (EHR alert system) HBV Vaccination Catch-up vaccination Low NHL endemic regions Missed cases 36,54,66 Reduced HBV reactivation rates Universal vaccination High endemic regions Surveillance cost 1,36,54,66 Reduced HBV reactivation rates Reactivation Prophylaxis Entecavir Agent of choice in advanced Expensive Renal 61,72,73,74,78 disease. Least viral resistance insufficiency (rare) Lamivudine Most used and studied Withdrawal hepatitis 31,32,33,34,55, agent. Cheapest Lamivudine failure (Viral 68,71,85,89 resistance, viral breakthrough) Adefovir Less viral resistance Expensive Renal 27,76,80,81 dysfunction (rare) Hypophosphatemia (rare) Tenofovir Least viral resistance Expensive Renal 19,27,70,71 dysfunction (rare) Hypophosphatemia (rare) Low dose/steroid free Reduced incidence Potentially adverse 83,84 chemo-immunotherapy of reactivation survival outcomes from undertreated NHL disease Reactivation Treatment Lamivudine Most used agent globally Risk of acquired 33,59,60,69,70,91 viral resistance Newer antivirals Lamivudine failure. Liver Depends on prognosis 27,53,61,68,77 (Entecavir, Adefovir, failure must be absent of lymphoma type Tenofovir) Liver transplantation If liver failure is present Depends on cost, 26,75 with HBV reactivation disease prognosis and post-transplant sequelae

BPA- Best Practice Advisory is synonymous with Electronic Health Records (EHR) alert system. treatment-naïve patients with evident or impending hepatic There are groups of patients in whom routine antiviral derangement.60,88 We urge clinicians to follow HBV seropos- therapy may not be indicated. These groups include patients itive patients closely while using novel agents for NHL, to who do not meet the criteria for reactivation (the rise in HBV ensure that reactivation does not occur. Clinicians should DNA < 10-fold in patients with past or latent infection, ALT level < three times the upper limit of normal), younger also remember to screen patients for HBV who are receiving patients in the immune-tolerant phase (normal ALT levels chemotherapy without rituximab, if they are at high risk for despite positive HBsAg and HBV DNA and minimal hepatic reactivation. If a patient is positive for HBV, then prophylaxis inflammation), and patients in the inactive carrier phase. In should be initiated. The literature remains unclear at this time addition, patients are considered low risk with agents such as whether universal screening for HBV should be done in all low dose corticosteroids or immunosuppressive agents (e.g., patients receiving chemotherapy. azathioprine and 6-mercaptopurine), and may be excluded

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150 Journal of Clinical and Translational Hepatology 2016 vol. 4 | 143–150 Review Article

Immune Activation in the Liver by Nucleic Acids

Qian Sun, Qingde Wang, Melanie J. Scott and Timothy R. Billiar*

Department of Surgery, University of Pittsburgh, Pittsburgh, PA, USA

Abstract (PRRs) expressed by both immune and nonimmune cells.1,2 This pathway leads to the eradication of pathogens through Viral infection in the liver, including hepatitis B virus (HBV) numerous clearance mechanisms that involve the production and hepatitis C virus (HCV) infection, is a major health of inflammatory mediators, cell recruitment, and activation. problem worldwide, especially in developing countries. The Similarly, the same PRRs can be activated in response to infection triggers a pro-inflammatory response in patients sterile injuries by endogenous activators known as damage- that is crucial for host defense. Recent studies have identified associated molecular patterns (DAMPs). For example, nucleic multiple transmembrane and cytosolic receptors that recog- acids of microbial or host origin represent a major category of nize pathogen-derived nucleic acids, and these receptors are both PAMP and DAMP. These “non-self” or “self-originated” essential for driving immune activation in the liver. In addition molecules are detected by PRRs, including toll-like receptors to sensing DNA/RNA from pathogens, these intracellular (TLRs), nucleotide-oligomerization domain (NOD)-like recep- receptors can be activated by nucleic acids of host origin in tors (NLRs), and retinoic acid-inducible gene (RIG)-I like response to sterile injuries. In this review, we discuss the receptors (RLRs), which initiate cell signaling and gene expanding roles of these receptors in both immune and expression. nonimmune cells in the liver. The liver, by virtue of its anatomical position “downstream” © 2016 The Second Affiliated Hospital of Chongqing Medical from the gastrointestinal tract and as the target of many University. Published by XIA & HE Publishing Inc. All rights heterotrophic viruses, is adapted to recognize invading reserved. pathogens as well as toxic insults that lead to liver injury. These innate immune recognition pathways are present in essentially all of the resident cells in the liver, which include Introduction not only resident cells of the immune system such as Kupffer cells but also the parenchymal cells or hepatocytes. The liver The activation of innate immune pathways is crucial for host integrates its important and unique roles in metabolism with defense in response to invasive pathogens and during sterile its functions in local and systemic immune responses. If inflammation, as observed after tissue trauma or ischemia. In damaged, the liver can co-opt these metabolic and immune the setting of microbial infection, pathogen-associated responses to regenerate its mass. All of these responses molecular patterns (PAMPs), which are conserved microbial involve the robust synthesis and turnover of nucleic acids that motifs, can be recognized by pattern recognition receptors must be compartmentalized or sequestered to regulate appropriate nucleic acid sensing. Here, we review recent advances in our understanding of nucleic acid sensing by Keywords: Nucleic acid; Innate immunity; Intracellular receptor; Hepatocytes. Abbreviations: ADAR1, adenosine deaminase acting on RNA1; AIM2, absent in PRRs in liver cells. melanoma 2; ASC, apoptosis-associated speck-like protein containing a caspase recruitment domain; CARD, caspase activation and recruitment domain; cGAS, cyclic GMP-AMP synthase; CMV, cytomegalovirus; CpG, cytosine-phosphate- Intracellular nucleic acid sensors in the liver guanine; DAI, DNA-dependent activator of IRF; DAMP, damage-associated molecular pattern; dsRNA, double-stranded RNA; HMGB1, high-mobility group box 1; IFI16, gamma-interferon-inducible protein 16; IFN, interferon; IL, interleukin; Innate immune sensors involved in the recognition of nucleic I/R, ischemia/reperfusion; iNOS, inducible nitric oxide synthase; IRF, interferon acids can be divided into two groups based on their sub- regulatory factor; ISGs, interferon-stimulated genes; LGP2, laboratory of genetics cellular localization. TLRs, including TLR3, TLR7, TLR8, and and physiology 2; LRR, leucine-rich repeat; MDP, muramyldipeptide; MDA5, mel- TLR9, are membrane-bound receptors that monitor endoly- anoma differentiation-associated protein 5; mtDNA, mitochondrial DNA; MyD88, 3,4 myeloid differentiation primary-response protein 88; NF-kB, nuclear factor kB; sosomes for pathogen or host derived nucleic acids. NLRs, NOD-like receptors; NOD2, nucleotide-binding oligomerization domain A second group of receptors recognize nucleic acids in the protein 2; PAMP, pathogen-associated molecular patterns; pgRNA, pregenomic cytoplasm and is further classified here into those that recog- RNA; PKR, protein kinase R; Pol-III, polymerase III; poly[I:C], polyinosinic- nize cytosolic DNA and those that detect pathogen- or host- polycytidylic acid; PRRs, pattern recognition receptors; RANTES, regulated upon activation, normal T cell expressed and secreted; RIG-I, retinoic acid-inducible derived RNA. gene I; RLRs, RIG-I like receptors; ROS, reactive oxygen species; ssRNA, single- stranded RNA; STAT1/2, signal transducer and activator of transcription 1/2; Nucleic acid sensing by TLRs STING, stimulator of IFN genes; TLRs, Toll-like receptors; TNFa, tumor necrosis factor alpha; TRAF6, tumor necrosis factor receptor-associated factor 6; TRIF, TIR domain-containing adaptor inducing IFN-b; UTRs, untranslated regions. TLRs are transmembrane receptors composed of ligand Received: 15 January 2016; Revised: 24 February 2016; Accepted: 7 March 2016 q binding ectodomain and transmembrane domains as well as DOI: 10.14218/JCTH.2016.00003. a cytosolic domain that is crucial for the recruitment of down- *Correspondence to: Timothy R. Billiar, Department of Surgery, University of Pittsburgh, Suite F1281, 200 Lothrop Street, Pittsburgh, PA 15213, USA. Tel: stream adaptors, including myeloid differentiation primary- +1-412-647-1749, Fax: +1-412-647-3247, Email: [email protected] response protein 88 (MyD88) and TIR domain-containing

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Cytosolic RNA sensors Cytosolic DNA sensors

RLRs are a group of well-characterized cytosolic RNA sensors Compared to cytosolic RNA sensors, receptors for cytosolic responsible for intracellular dsRNA-induced type-I IFN pro- DNA are controversial and elusive. Two major signaling path- duction.12 All three members of the RLR family, RIG-I, mela- ways have been described for cytosolic DNA-sensing: one is noma differentiation-associated protein 5 (MDA5), and mediated by the adaptor protein STING to induce IFN pro- laboratory of genetics and physiology 2 (LGP2), are broadly duction, and the other is mediated by activation of the expressed by both immune and nonimmune cells such as inflammasome to trigger production of IL-1b and IL-18. hepatocytes, making them essential in the host response DNA-dependent activator of IRF (DAI, also known as ZBP1), against viral infection in the liver.9,13 gamma-interferon-inducible protein 16 (IFI16), and the RNA Both RIG-I and MDA5 recognize dsRNA, including the helicase DDX41 have all been reported to recognize dsDNA in synthetic analog poly (I:C). However, they have been shown the cytosol and trigger type I IFN production through the to respond preferentially to RNA ligands of variable lengths. STING/TBK1/IRF3 pathway.4,9 In addition, a recently discov- MDA5 generally recognizes long dsRNA molecules (> 2 kb), ered cytosolic receptor cyclic GMP-AMP synthase (cGAS) was whereas RIG-I preferentially binds to shorter fragments of shown to detect cyclic dinucleotides derived from bacterial dsRNA with an uncapped 5′ triphosphate end.9,12 A few cells and to induce type I IFN in a STING-dependent studies have shown that RIG-I may also be involved in host manner.4,21 immune response to cytosolic viral and bacterial dsDNA, but it Most of the intracellular nucleic acid-sensing pathways was suggested that RNA polymerase III (Pol-III) was required discussed thus far lead to the production of IFNs vital to for this process as Pol-III converts pathogen dsDNA to combat viral infection. However, there is yet another group of dsRNA.3,9,14 Very little is known about the RNA ligands that nucleic acid-sensing receptors that have been shown to cause can activate LGP2, but recent studies suggest that its ATPase the maturation of pro-inflammatory cytokines IL-1b and activity may regulate RIG-I- and MDA5-dependent type I IFN IL-18, cytokines important in host response to bacterial production in response to viral infection.12,15 infection as well as sterile inflammation. A typical inflamma- Upon recognition of cytosolic dsRNA by RIG-I and MDA5, some contains a NLR or an absent in melanoma 2 (AIM2), the N-terminal caspase activation and recruitment domains apoptosis-associated speck-like protein containing a caspase- (CARDs) associate with the adaptor protein mitochondrial recruitment domain (ASC), and procaspase-1. Inflamma- antiviral signaling (MAVS, also known as IPS1), and the some activation leads to autocleavage of caspase-1, forming interaction subsequently leads to the activation of transcrip- the active caspase-1 p10/p20 tetramer that cleaves pro-IL-1b tion factors IRF3, IRF7, and NF-kB.12 As discussed above, and pro-IL-18 into their active forms.22 In addition, inflamma- activation of these transcription factors results in the produc- somes play unique roles in hepatocytes, which do not produce tion of IFNs and pro-inflammatory cytokines, which are IL-1b or IL-18. Others have shown that caspase-1 activation important in host defense against viruses. The transmem- regulates enzymes involved in glycolysis,23 while we have brane protein stimulator of IFN genes (STING) was also shown that inflammasome activation upregulates mitochon- shown to be important for RIG-I-dependent IFN production, drial autophagy in hypoxic hepatocytes.24 Recent research although the details of the mechanism remain unclear.9 has identified AIM2 as the receptor for cytosolic dsDNA In addition to RLRs, a few other cytosolic RNA sensors have derived from bacteria and viruses.25 However, little is known recently been described. Protein kinase R (PKR) is a serine/ about the ligand specificity of AIM2, and it appears that AIM2 threonine kinase that can be activated by dsRNA.12 Upon can be activated by dsDNA from various sources, whether

152 Journal of Clinical and Translational Hepatology 2016 vol. 4 | 151–157 Sun Q. et al: Nucleic acid sensing by immune system from bacteria, virus, or host.4,26 It was also recently shown A few RLRs have been identified that sense cytosolic HCV that cytosolic mitochondrial DNA (mtDNA) and more specifi- RNA. RIG-I binds to HCV RNA and signals IRF3 activation to cally, oxidized mtDNA, contributes to the secretion of IL-1b induce the expression of IFNs and ISGs that are essential for and IL-18 by activating the NLRP3 inflammasome.27 However, viral elimination.37,39–41 RIG-I signaling has been proposed to whether NLRP3 can directly sense mtDNA that is released into be negatively regulated by formation of a multimeric complex the cytosol is still unclear. that is controlled by the internal repressor domain at the C terminus of RIG-I.42 Another RLR LGP2 was shown to bind to Recognition of HBV by intracellular nucleic acid HCV RNA, but it functions as a negative regulator of host sensors in the liver defense mediated by RIG-I.42 Interestingly, a recent study suggested that MDA5 also plays a critical role in IFN response Human HBV is a small DNA virus that causes hepatic inflam- during HCV infection, and the function of RIG-I maybe limited 35 mation and is associated with substantial morbidity world- only to early sensing of the virus infection. wide.28 Around 400 million people worldwide are chronically HCV RNA replication was also shown to trigger dsRNA- infected with HBV, substantially increasing their risk of devel- induced antiviral pathways mediated by PKR in human hep- 43 oping cirrhosis and hepatocellular carcinoma.28 Although the atoma cells. Interestingly, the viral nonstructural 5A protein underlying mechanisms leading to host defense against HBV was found to bind PKR and block the PKR-dependent IFN infection remain to be clearly defined, there is emerging evi- response, suggesting the potential role of PKR as a cytosolic dence that innate immunity and especially IFN response are HCV RNA sensor. NALP3, a NLR that triggers activation of the essential for clearance of intrahepatic HBV.13,29,30 Moreover, inflammasome, has also been indicated to sense HCV IFN and nucleoside analogs are currently available for HBV and induce the secretion of IL-1b through assembly of the 44,45 treatment, suggesting the important role of host innate inflammasome complex. However, no studies have dem- immune response in viral elimination.30,31 onstrated that NLRP3 directly binds nucleic acids (DNA The HBV genome comprises a relaxed partially dsDNA of or RNA), and thus it is possible that NLRP3 in this case is acti- approximately 3.2 kb.30,32 Upon entry and uncoating of HBV vated by increased reactive oxygen species (ROS) production 45 in hepatocytes, the relaxed circular DNA (rcDNA) is converted instead. Another study by Li et al. suggests that TLR3 senses into covalently closed circular DNA, which is then transcribed HCV dsRNA that is longer than 80-100 base pairs, resulting in to generate four RNA species: the 0.7 kb, 2.1 kb, 2.4 kb, and NF-kB activation and the production of regulated upon activa- 3.5 kb viral RNA transcripts.32 The RNA species are exported tion, normal T cell expressed and secreted (RANTES), macro- to the cytoplasm and then translated into HBV proteins. The phage inflammatory protein-1 alpha (MIP-1a), MIP-1b, IP-10, 46 longest 3.5 kb viral RNA is also known as the pregenomic RNA and IL-6 in hepatocytes. (pgRNA) that serves as the template for reverse transcription and synthesis of new rcDNA. Recognition of adenovirus and cytomegalovirus (CMV) Given the life cycle of HBV, both DNA and RNA can by intracellular nucleic acid sensors in the liver potentially serve as PAMPs to activate cytosolic nucleic acid sensors. Indeed, two recent studies have identified the roles Adenovirus and CMV are dsDNA viruses that typically target of two RNA sensors, RIG-I and MDA5, in the recognition of tissues other than the liver.47 However, in immunocompro- 30,32 HBV virus and host defense response (table 1). In the mised and liver transplant patients, adenovirus and CMV first study, the authors showed that the RNA sensor RIG-I infections are associated with the development of hepatitis was essential for the recognition of HBV genotypes A, B, and possibly fulminant liver failure.47,48 CMV, for example, and C and for induction of type III IFN expression in human accounts for symptomatic infection in 40% of pediatric liver 30 hepatocytes. Moreover, their study suggests that the 5′-ε transplant recipients, and it is associated with substantial region of HBV pgRNA interacts with RIG-I and is a key morbidity and mortality in these patients.49 element for RIG-I dependent IFN-l1 induction. However, for An IRF3-dependent type I IFN response was reported in genotype D HBV, the second study showed that MDA5, but not macrophages in response to adenovirus infection.14,50,51 RIG-I, is critical for the expression of IFN-stimulated genes A variety of DNA sensors, including DDX41, Pol-III, and 32 (ISGs) following infection. The discrepancy between the cGAS, have been identified to sense cytosolic dsDNA derived two studies could be due to distinct genomic sequence of from adenovirus.14,50,51 Infection of a macrophage-like cell 33 HBV genotype D. Interestingly, AIM2-dependent inflamma- line was shown to trigger upregulation of IRF3 primary some formation and the production of IL-1b/IL-18 have been response genes, such as ISG56 and IFN-b, within 2-6 hours 34 found during HBV infection. and to induce later the phosphorylation of signal transducer and activator of transcription 1/2 (STAT1/2), a type I IFN sec- Recognition of HCV by intracellular nucleic acid ondary signaling molecule, to amplify further the antiviral sensors in the liver response.50,51 Interestingly, as an enzyme responsible for transcribing DNA template into RNA, Pol-III was reported to HCV infects nearly 200 million people worldwide, and 80% of sense adenoviral dsDNA in the cytosol and trigger RIG-I- infected individuals develop chronic inflammation that may dependent IFN production.14 eventually progress to cirrhosis or liver cancer.35,36 The HCV Mouse CMV infection was shown to activate two nucleic genome contains a positive-sense RNA comprised of highly acid receptors on the endosomal membrane, TLR9 and TLR3, structured 5′ and 3′ untranslated regions (UTRs) and a and trigger two nonredundant pathways mediated by MyD88 large open reading frame that encodes HCV structural and and TRIF, respectively.52 Both of the receptors are required nonstructural proteins.37 Similar to the treatment of HBV, for type I IFN production in response to mouse CMV infection, HCV infection is treated with an IFN-a-based therapy,38 sug- whereas neither pathway is enough to provide full protection gesting the important role of innate immune response in host against the virus.52 Two cytosolic DNA sensors, DAI and defense against HCV. IFI16, were also shown to be important for the induction of

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Table 1. Nucleic acid sensing by pattern recognition receptors (PRRs) in liver infection and diseases

Nucleic Experimental Signaling Cytokine Virus PRR Acid System Pathway production References

Adenovirus DDX41 dsDNA Macrophage TBK1/STING- IFNb 51 IRF3 Pol-III/RIG-I dsDNA Macrophage RIG-I-IRF3 IFNb 14 cGAS dsDNA Macrophage STING-IRF3 IFNb 50 CMV IFI16 dsDNA Macrophage STING-IRF3 IFNb 54 DAI dsDNA Fibroblast IRF3 IFNb 53 AIM2 dsDNA In vivo Inflammasome IL-1b/IL-18 25 TLR9/TLR3 dsDNA In vivo MyD88/TRIF IFNa/b/g 52 HBV AIM2 dsDNA Glomerular Inflammasome IL-1b/IL-18 34 mesangial cell MDA5 RNA Hepatocyte IRF3 IFNb 32 RIG-I pgRNA Hepatocyte IRF3/NFkB IFNl 30 HCV NLRP3 RNA Hepatocyte/ Inflammasome IL-1b 44,45 Macrophage TLR3 DsRNA Hepatocyte NF-kB RANTES, MIP-1a, 46 MIP-1b, IP-10 and IL-6 PKR RNA Hepatocyte IRF1 – 43 RIG-I RNA Hepatocyte IRF3 IFNa/b/ISG 37,39,42 MDA5 dsRNA Hepatocyte IRF3 IFNb/l 35 mtDNA NLRP3 Oxidized Macrophage Inflammasome IL-1b 27 dsDNA TLR9 dsDNA In vivo MyD88 IL-1b/IL-6/IFNa 8,67 Apoptotic or TLR9 dsDNA In vivo MyD88 IL-1b/IL-18/IL-6/TNFa 56,57 necrotic DNA Histone/DNA TLR9 – Kupffer cells MyD88 IL-1b/IL-18/IL-6/TNFa 59,68 Plasmodium MDA5 RNA Hepatocyte IRF3/IRF7 IFNa/b/ISG 66

Abbreviations: AIM2, absent in melanoma 2; cGAS, cyclic GMP-AMP synthase; CMV, cytomegalovirus; DAI, DNA-dependent activator of IRF; dsRNA, double-stranded RNA; HBV, hepatitis B virus; HCV, hepatitis C virus; IFI16, gamma-interferon-inducible protein 16; IFN, interferon; IL, interleukin; IRF, interferon regulatory factor; ISG, interferon-stimulated gene; MDA5, melanoma differentiation-associated protein 5; mtDNA, mitochondrial DNA; MyD88, myeloid differentiation primary-response protein 88; pgRNA, pregenomic RNA; PKR, protein kinase R; Pol-III, polymerase III; PRRs, pattern recognition receptors; RANTES, regulated upon activation, normal T cell expressed and secreted; RIG-I, retinoic acid-inducible gene I; STING, stimulator of IFN genes; TLRs, Toll-like receptors; TNFa, tumor necrosis factor alpha; TRIF, TIR domain-containing adaptor inducing IFN-b.

IFN-b following human CMV infection in macrophages.53,54 In other hand, has been shown to induce IL-1b secretion upon addition, AIM2 was suggested to sense dsDNA derived from binding to NLRP3.27 However, although mtDNA can be pulled mouse CMV and induce the release of IL-1b and IL-18 down with NLRP3 by immunoprecipitation, whether NLRP3 in vivo.25 IL-18, in turn, was shown to induce further IFN-g directly interacts with mtDNA remains to be determined. production by natural killer cells in the spleens of CMV-infected In models that induce sterile injury in the liver, such as mice and results in enhanced control of virus replication.25 liver ischemia/reperfusion (I/R) and acetaminophen overdose, DNA released from host cells serves as a major source Recognition of host nucleic acids in the liver of DAMP to trigger inflammation and liver injury. In an acetaminophen-induced hepatotoxicity model, for example, Cytosolic nucleic acids derived from the host itself represent a activation of TLR9 by DNA released from apoptotic and group of DAMPs that cause sterile inflammation in the context necrotic hepatocytes was shown to trigger the upregulation 56,57 of cell stress and tissue damage. DNA derived from mitochon- of pro-IL-1b, pro-IL-18, and TNFa. Interestingly, in the dria is of particular interest given its similarities to bacterial model of liver I/R, disruption of TLR9 activation by injecting DNA, which contains immune activating unmethylated CpG inhibitory CpG immediately before I/R was shown to reduce motifs.8,55 Mitochondria damaged by cellular stress are significantly the release of proinflammatory cytokines IL-6 degraded by autophagy in immune cells as well as nonim- and TNF by immune cells.58 Moreover, it has been shown mune cells, including hepatocytes.8,24 It was reported that that circulating levels of histones, proteins associated with mitochondrial DNA that escapes from autophagy causes DNA in the nucleus, are significantly increased after liver IL-1b and IL-6 production through activation of TLR9 on the I/R. These extracellular histones are able to enhance DNA- lysosomal membrane.8 Cytosolic oxidized mtDNA, on the mediated TLR9 activation during this process.59 Our own

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Fig. 1. Nucleic acid sensing pathways via intracellular DNA/RNA receptors. Under normal biological conditions, hepatocytes exhibit abundant nucleic acid synthesis. Under pathological conditions, mitochondria DNA and cellular RNA transcripts serve as damage-associated molecular patterns (DAMPs) to trigger inflammatory response by activating cytosolic receptors and downstream signaling pathways. In addition, RNA/DNA can be released to the extracellular space, leading to activation of endosomal receptors, such as toll-like receptor (TLR)3, TLR7, and TLR9. While stimulation of TLRs lead to inflammatory cytokine production, primarily via myeloid differentiation primary-response protein 88 (MyD88), tumor necrosis factor receptor-associated factor 6 (TRAF6), nuclear factor kappa B (NF-kB), and mitogen activated protein kinase (MAPK) pathways, dsRNA predominantly causes activation of interferon (IFN) pathways by phosphorylation of interferon regulatory factors (IRFs). data also suggest that AIM2, an inflammasome protein that damage in liver under sterile conditions. Moreover, a recent triggers activation of caspase-1, is essential for sensing cyto- study by Pestal et al. showed that ADAR1 negatively regulates solic dsDNA in hepatocytes after redox stress induced by the RLR-MAVS-IFN pathway and plays an essential role in hemorrhagic shock with resuscitation (manuscript submit- organ development.63 We have found that hepatocytes ted), which represents another mechanism of the recognition utilize TLR3 and PKR to detect the dsRNA mimic Poly (I:C) of nucleic acids derived from host itself (Fig. 1). Rather than leading to IRF3 and 7 upregulation and type 1 IFN production leading to cytokine release, activation of AIM2 and caspase-1 (manuscript submitted). Taken together, the recent observa- in hepatocytes is responsible for upregulation of cellular tions on the role of self-sensing nucleic acids in the liver could adaptive responses, such as mitochondrial autophagy and explain, in part, the pathways leading to type I IFN-induced 64 subsequent hepatoprotection in response to redox stress. liver damage in I/R. The role of self-derived nucleic acids in Cellular RNA is a potent and important signal of danger driving both immune responses and hepatic immunopathol- that activates innate immune response in hepatocytes under ogy is a fertile area for further investigation. pathologic conditions. We have provided evidence that adenosine deaminase acting on RNA1 (ADAR1), an RNA Cell-specific nucleic acid sensing in the liver editing enzyme, is important to prevent detection of cytosolic 60,61 self RNA in hepatocytes (Fig. 1). We showed in a cell Many of the nucleic acid sensing pathways described above model that suppression of ADAR1 expression leads to lead to an antiviral response that is dependent on cytokine enhanced RIG-I activation and IFN production following I/R production. Given that hepatocytes are the major targets of that results in increased organ damage.62 Deletion of ADAR1 liver viruses, such as HBV and HCV, and they do not produce gene in mice also leads to type I IFN production and tissue high levels of cytokines,24 it is likely that the nucleic acid

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J Virol 2005;79:2689– injury. Hepatology 2010;51:621–632. doi: 10.1002/hep.23365. 2699. doi: 10.1128/JVI.79.5.2689-2699.2005. [59] Huang H, Evankovich J, Yan W, Nace G, Zhang L, Ross M, et al. Endogenous [38] Fried MW, Shiffman ML, Reddy KR, Smith C, Marinos G, Goncales FL Jr, et al. histones function as alarmins in sterile inflammatory liver injury through Toll- Peginterferon alfa-2a plus ribavirin for chronic hepatitis C virus infection. like receptor 9 in mice. Hepatology 2011;54:999–1008. doi: 10.1002/hep. N Engl J Med 2002;347:975–982. doi: 10.1056/NEJMoa020047. 24501. [39] Saito T, Owen DM, Jiang FG, Marcotrigiano J, Gale M. Innate immunity [60] Wang G, Wang H, Singh S, Zhou P, Yang S, Wang Y, et al. ADAR1 prevents induced by composition-dependent rig-i recognition of hepatitis c virus rna. liver injury from inflammation and suppresses interferon production in hep- Nature 2008;454:523–527. doi: 10.1038/nature07106. atocytes. Am J Pathol 2015;185:3224–3237. doi: 10.1016/j.ajpath.2015. [40] Lee HC, Narayanan S, Park SJ, Seong SY,Hahn YS. Transcriptional regulation of 08.002. IFN-l genes in hepatitis C virus-infected hepatocytes via IRF-3.IRF-7.NF-kB [61] Yang S, Deng P, Zhu Z, Zhu J, Wang G, Zhang L, et al. Adenosine deaminase complex. J Biol Chem 2014;289:5310–5319. doi: 10.1074/jbc.M113.536102. acting on RNA 1 limits RIG-I RNA detection and suppresses IFN production [41] Li K, Chen Z, Kato N, Gale M Jr, Lemon SM. Distinct poly(I-C) and virus-acti- responding to viral and endogenous RNAs. J Immunol 2014;193:3436– vated signaling pathways leading to interferon-beta production in hepato- 3445. doi: 10.4049/jimmunol.1401136. – cytes. J Biol Chem 2005;280:16739 16747. doi: 10.1074/jbc.M414139200. [62] Wang H, Wang G, Zhang L, Zhang J, Zhang J, Wang Q, et al. ADAR1 sup- [42] Saito T, Hirai R, Loo YM, Owen D, Johnson CL, Sinha SC, et al. 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Journal of Clinical and Translational Hepatology 2016 vol. 4 | 151–157 157 Review Article

The Evolving Nature of Hepatic Abscess: A Review

Marianna G. Mavilia*1, Marco Molina2 and George Y. Wu3

1University of New England College of Osteopathic Medicine, Biddeford, ME, USA; 2Department of Radiology, UCONN Health, Farmington, CT, USA; 3Department of Medicine, Division of Gastroenterology-Hepatology, UCONN Health, Farmington, CT, USA

Abstract in Taiwan.16 In the early 1900s, mortality was as high as 75%–80%,4 while today, mortality is markedly decreased, Hepatic abscess (HA) remains a serious and often difficult to ranging from 10%–40%.8 This is due to improvements in anti- diagnose problem. HAs can be divided into three main biotic therapy and interventional procedures for the treatment categories based on the underlying conditions: infectious, of HA.3,4,6 malignant, and iatrogenic. Infectious abscesses include those Although mortality is improved, it is still high, making early secondary to direct extension from local infection, systemic diagnosis of HA exceedingly important to the clinical outcome. bacteremia, and intra-abdominal infections that seed the HA can be difficult to diagnose, and the symptomatology is portal system. However, over the years, the etiologies and variable. Often, objective findings are nonspecific,16,17 and risks factors for HA have continued to evolve. Prompt recog- therefore, diagnosis relies largely on imaging.2 nition is important for instituting effective management and The aim of this review is to describe some of the changes in obtaining good outcomes. important risk factors, mechanisms, and patterns of develop- © 2016 The Second Affiliated Hospital of Chongqing Medical ment of HA and to review the current recommendations for University. Published by XIA & HE Publishing Inc. All rights the diagnosis and treatment of this condition. reserved. Risk Factors

There are many risk factors associated with the development Introduction of HA and increased mortality from HA. These factors are detailed in Table 1. Risk factors predisposing patients to HA Hepatic abscess (HA) can be defined as an encapsulated range from diabetes mellitus (DM), cirrhosis, general collection of suppurative material within the liver paren- immune-compromised state, use of proton pump inhibitor 1,2 chyma, which may be infected by bacterial, fungal, and/ (PPI) medications, gender, and age. 2 or parasitic micro-organisms. Since the majority of HAs in DM is a predisposing factor for HA that is well documented 2,3 the Western world are infected with bacteria, pyogenic in the literature. 5,18,19 Studies have found DM as a concom- liver abscess will be the focus of this review. itant disease in 29.3%–44.3% of patients with HA.5,19 Diabetic In the early 1900s, the most common cause of HA was patients are also more likely to present with multiple 4 pylephlebitis secondary to appendicitis. In the late 1900s, abscesses.19 There are several pathophysiologic features of 4,5 biliary tract disease emerged as the most frequent culprit, DM that contribute to higher infection risk.18,20 For instance, 2,6,7,8 and it remains the most common cause of HA today. More hyperglycemia is known to alter neutrophil metabolism.21 Dia- recently, there has been an increase in the incidence of HA betics also have been shown to have impaired polymorphonu- arising in association with malignancies and their treatment, clear leukocyte (PMN) chemotaxis and phagocytosis,18,20 4,9 including HA from liver metastasis and as complications of which weakens their immune defense against infections and transarterial chemoembolization (TACE) or radiofrequency leaves them more susceptible to abscess formation. 10–14 ablation (RFA). Like diabetics, patients with liver cirrhosis have an 15 Although the frequency of HA varies by region, the overall increased risk of HA due to their immune-compromised incidence is fairly low, ranging from 2.3 cases per 100,000 state.7 Cirrhotics are 15.4 times more likely to develop HA 15 hospital admissions in North America to 275.4 per 100,000 than the general population.7,22 Other conditions and treatments may compromise the Keywords: Liver abscess; Liver neoplasms; Iatrogenic disease; Risk factors. immune system and render it inadequate to counteract Abbreviations: ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, pathogens. These include various immunodeficiencies, che- aspartate aminotransferase; CEUS, contrast-enhanced ultrasound; CT, computed tomography; DM, diabetes mellitus; EBA, enterobiliary anastomosis; FDA, Food motherapy, solid malignancies, immunosuppression therapy 20 23 and Drug Administration; GGT, gamma glutamyl transpeptidase; HA, hepatic after organ transplant, as well as splenectomy, all of abscess; HCC, hepatocellular carcinoma; INR, international normalized ratio; IV, which have been associated with an increased risk of HA. intravenous; MRI, magnetic resonance imaging; PD, percutaneous drainage; PMN, The use of PPI medications has also been found to polymorphonuclear leukocyte; PO, oral, PPI, proton pump inhibitor; RFA, radio- 24 frequency ablation; SD, surgical drainage; TACE, transarterial chemoemboliza- increase the risk of HA formation. This is presumably tion; US, ultrasound; WBC, white blood cell count. because PPI medications increase the gastric pH, which Received: 18 January 2016; Revised: 25 February 2016; Accepted: 09 March 2016 24 q decreases the natural gastric defense against bacteria. In DOI: 10.14218/JCTH.2016.00004. a large case-control study, Wang et al. demonstrated a dose- *Correspondence to: Marianna G. Mavilia, University of New England College of Osteopathic Medicine, 11 Hills Beach Road, Biddeford, ME 04005, USA. Tel: response relationship between HA formation and dose of PPI 24 +1-617-435-1185, Fax: +1-860-679-6582, Email: [email protected] over a 90 day period. Although this was a large study,

158 Journal of Clinical and Translational Hepatology 2016 vol. 4 | 158–168

Table 1. Risk factors for development of hepatic abscess (HA) and were conducted in the same region and used very similar increased mortality from HA sample sizes, the difference in findings could be explained Increased risk of Increased mortality by the inclusion criteria. Chen et al. included only primary developing HA from HA HA, whereas Lee et al. did not specify the diagnosis as primary or secondary. Diabetes mellitus*5,8,19 Malignancy16 The theme of immune compromise is also seen in the Liver cirrhosis*22 Diabetes mellitus*5,16 mortality rates for HA. Mortality rates for HA in patients with *22 associated malignancy was reported to be double that of Immune-compromised Liver cirrhosis 26 20 cancer-free patients. As reported by Lin et al., patients with state hepatocellular carcinoma (HCC) and HA had a higher 60-day Use of PPI24 Male gender*16 mortality rate compared to those with HCC only.15 Addition- Advanced age19 Multiorgan failure16 ally, cirrhosis was associated with a 4-fold increase in the risk 22 *16 5 of death from HA. Mortality was also reported to increase Male gender Sepsis 4 when HA contained mixed organisms or fungal infection. Infection with mixed organisms4 Categorization HA rupture16 Abscess size > 5 cm5 For the purposes of this review, we have divided HA into three Respiratory distress16 subgroups based on category: infectious, malignant, and iatrogenic. There are some areas of overlap between catego- Hypotension16 ries, as depicted in Figure 1. Jaundice16 16 Extrahepatic involvement Infectious Abscess

*Diabetes mellitus, liver cirrhosis and male gender are risk factors for both development and increased mortality of HA. Pathogens may gain access to the liver through contiguous spread from infection of neighboring tissue, from blunt or penetrating trauma to the abdomen,9,27 and through hema- encompassing 10 years of data, it was conducted exclusively togenous spread (Fig. 2).2 The latter most commonly occurs in Taiwan, where the incidence of HA in general is high. Addi- due to systemic bacteremia or in intra-abdominal infections. tionally, control subjects were not matched for comorbidities However, bacteremia is only detectable in 43% of HAs,9 or indication for PPI use. Both of these factors may have had making diagnosis of HA in this instance increasingly difficult. some influence on their findings. In cases of intra-abdominal infection, including appendicitis Most cases of HA present at advanced age. One study 19 and diverticulitis, bacteria can seed the portal vessels, reported a mean age > 57 years. This finding suggests that causing pylephlebitis and portal pyemia, ultimately leading to older individuals are more susceptible to bacterial infection 6,7 21 HA formation. A recent cohort study of 54,147 patients and thus abscess formation. However, more research is with diverticular disease and 216,588 matched controls needed to clarify the mechanism of this association. found the incidence of HA to be 2.44-fold higher in those In a 10-year audit of experience with HA from 1989 to with diverticular disease compared to controls.28 This study 1999, Lee et al. found the ratio of males to females presenting included patients with both diverticulosis and diverticulitis.28 5 17 with HA was about 2 to 1. This was confirmed by Pang et al. Of them, 10%–25% had diverticulitis or related complications 25 and Lin et al. during the 4–8 year follow-up period.28 Since diverticulitis seems more likely to cause mucosal defect or perforation Morbidity and Mortality than diverticulosis, the inclusion of diverticulosis patients is interesting and suggests that the incidence of HA in this As previously mentioned, mortality remains high, although it group may have been even higher if the study had examined has decreased over time. Several associated factors and only diverticulitis patients. With the development of diagnostic comorbid conditions have been implicated in the risk of methods to facilitate early detection and surgery to resolve the death from HA.8 A study by Chen et al. divided a group of infection before spread can occur, appendicitis and diverticuli- 134 patients with primary HA into a mortality and a survivor tis have become much less frequent causes of HA. group and identified several risk factors for increased mortal- Nonmetastatic colorectal cancer has also been associated ity, including male gender, malignancy, multi-organ failure, with HA formation. This link is thought to be due to local and HA rupture.16 Other signs and symptoms found to be destruction of the mucosa by the tumor, which allows invasion associated with the mortality group in this study included of bacteria and entry into the bloodstream.18,29–31 Similar respiratory distress, hypotension, jaundice, and extrahepatic cases have also been reported postpolypectomy, where involvement, such as endopthalmitis.16 The latter mentioned polyp removal caused a microperforation in the colon.32 signs were indicators of higher comorbidity, which not Under these circumstances, bacteria gain access to the surprisingly were associated with higher mortality in those portal venous system and then to the liver bed.8,20 In some patients.16 A similar study by Lee et al. found that diabetes, of these cases, the HA was the presenting sign30 and has been sepsis, and the presence of larger abscesses (> 5 cm) were considered to be a herald of silent colon cancer.5 additional factors associated with higher mortality5 that were Currently, the most common route of infection is the biliary not identified as significant in the study by Chen et al.16 In tree, responsible for 30%–50% of cases of HA.7,33 Biliary contrast to that study, Lee et al. did not find a significant infections occur mainly in the setting of obstruction from difference in mortality by gender.5 Although these studies gallstone disease, malignancy, or stricture,4,6 leading to

Journal of Clinical and Translational Hepatology 2016 vol. 4 | 158–168 159 Mavilia M.G. et al: Evolving nature of hepatic abscess

Fig. 1. The gray areas. Fig. 1 depicts a comparison of the sub-groups of HA and also delineates the areas of overlap between them.

proliferation of bacteria in the biliary tract, ascending cholan- Secondary infection of primary liver tumor gitis, and invasion of the liver parenchyma. Although many types of bacteria have been identified in Primary HCC can spontaneously develop in an area of central HAs, there have been several microbiological trends. Prior to necrosis,1 which can become infected with bacteria.7,15 It is 1980, Escherichia coli was the most common pathogen in HA also possible for HCC to cause biliary obstruction,7,15 poten- worldwide.3,15 Currently, this trend has shifted to Klebsiella tially leading to ascending cholangitis and HA, as described pneumonia, particularly in Asia, where it accounts for 50%– above. 15 73% of cases.3,16,29 This may be related to the association HA can be the initial manifestation of HCC. These indi- of HA with DM. Diabetic patients are more susceptible to viduals tend to have a worse prognosis because the diagnosis 15 K. pneumonia infections.19,21,34 Clostridium perfringens has of HA often delays the diagnosis of underlying HCC. Some also been reported in the literature more frequently, but it is signs that were reported to be helpful in identifying superin- still a rare cause of HA.20 Depending on the primary source fected malignancy included a thickened wall, the presence of of infection, multiple organisms can be involved in HA.9 septations, aerobilia, portal thrombosis, and gas within the 2,8 Eltawansy et al. reported the presence of mixed organisms abscess. in 14%–55% of cases.20 Secondary infection of liver metastases

Malignant Abscess HA formation from liver metastasis is quite rare. A compilation of 1,262 cases of HA over 32 different studies found that only Malignant abscesses can be divided into three subcategories: 3% were associated with hepatic metastasis.9 However, in secondary infection of a primary liver tumor, secondary compiling these cases, it is possible that inclusion criteria infection of a metastatic liver lesion, and superinfection of may have differed between studies and skewed the results. spontaneous necrosis. There are a number of case reports of metastatic liver lesions

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Fig. 2. Routes of infection. arising from metastatic melanoma,27 rectal cancer,35 colon four patients with RFA and EBA performed at the same time, no cancer,31,36,37 esophageal carcinoma,9,37 and pancreatic patients developed HA, presumably because peri-operative cancer37,38 among others. antibiotic coverage prevented biliary contamination and HA formation.13 Superinfection of spontaneous necrosis Although the overall incidence of HA following TACE and RFA is low, the impact is increasing because of the increased TACE and RFA are frequently used in the treatment of use of ablative techniques for HCC palliation39,40 and the inoperable HCC10,11,39 as well as liver metastasis. Both pro- increase in incidence of HCC.41 cedures induce necrosis of the tumor and some surrounding Given the known risk of HA in this situation, the effect of liver tissue. This area of necrosis may serve as a nidus for antibiotic prophylaxis for both TACE and RFA has been infection. In addition, some mechanisms specific to the studied, but its role remains controversial.10,13 Currently, TACE procedure predispose individuals to abscess formation. there are no guidelines for prophylaxis before or during For example, TACE-induced necrosis suppresses reticuloen- TACE.10 Hoffman et al. found that only one out of the 10 dothelial cell activity, leading to decreased immunity.11 There patients with prior EBA undergoing RFA treatment developed is also an immunosuppressant effect from the chemothera- HA after antibacterial prophylaxis.14 These authors recom- peutic agents used.39 mended antibiotic prophylaxis before RFA intervention plus The incidence of HA has been reported to range from 0%– a prolonged course for at least 10 days postprocedure.14 1.4% following TACE,10,11 and 0.1%–0.7% following RFA.12,13 However, given the small sample size, more convincing evi- The incidence increases in patients who have a pre-existing dence is required to support the use of prophylaxis. enterobiliary anastomosis (EBA).12,13 In these patients, a dys- Shin et al. revealed that the time from TACE to diagnosis of functional sphincter of Oddi allows retrograde contamination of HA ranged from 2–90 days, and the median survival time the biliary tract,14 which occurs in 90% of patients with EBA.14 after this diagnosis was 6 months.10 This finding illustrates For patients with previous EBA treated specifically with RFA, the importance of monitoring patients for signs of infection the incidence of HA increases up to 86%.12 For these post procedure and early detection and treatment of HA. reasons, EBA may be a contraindication for TACE and RFA.2 Given the significantly increased risk, less than 2% of patients who underwent RFA had pre-existing EBA in a large study by Iatrogenic Abscess Elias et al.13 Interestingly, this same study demonstrated that when EBA and RFA were done at the same time, there was no In addition to TACE and RFA, several other procedures have increased risk for HA formation.13 However, this was a very been associated with an increased risk of HA formation. The small subgroup of the study with only four patients. In these incidence of HA following biliary procedures has been

Journal of Clinical and Translational Hepatology 2016 vol. 4 | 158–168 161 Mavilia M.G. et al: Evolving nature of hepatic abscess reported to be up to 26%.2 Biliary stenting, sphincterotomy, Table 2. Clinical findings and EBA are known to contaminate the biliary tract with bac- Signs and symptoms teria,38 allowing for ascending infection.42 Matthews et al. reported that HA after a biliary procedure tended to be more Malaise16 89% indolent compared to those from biliary obstruction.33 47 Surgical procedures on the hepatobiliary system can disturb Chills 69% the liver’s blood supply,33 leading to ischemic necrosis. Simi- Fever17,46,47 59%–90% larly, arterial embolization used in the treatment of abdominal Tachycardia17 52% trauma can also cause ischemic necrosis of the liver and sub- 46,47 – sequent abscess formation.2 In addition, complications of sur- Nausea 43% 68% gical procedures, such as biliary stricture, can result in Abdominal pain16,17,46 39%–84% 33 secondary infection of susceptible tissues. Vomiting45,47 30%–32% “ Choledochoduodenostomy can be complicated by sump 45 syndrome,”43 in which bile flow through a segment of the sur- Right pleural effusion 28% gically altered common bile duct become stagnant.43 This Weight loss47 26% complication contributes to bacterial proliferation and predis- Jaundice17,47 19%–21% poses patients to cholangitis and HA.43 16,46 – Blunt trauma and some surgical procedures can produce Ascites 18% 21% hematomas in the liver. Although hematoma is a rare compli- Murphy’s sign16 16% cation of laparoscopic cholecystectomy, Brown et al. described Hepatomegaly16,46 16%–52% a case in which a large hematoma on computed tomography Guarding17 14% (CT) was found on postoperative day 6 with early signs of infection.44 The development of hematoma during laparo- Respiratory distress16 13% scopic cholecystectomy may have been associated with prior Hypotension16,17,46 13%–30% use of nonsteroidal anti-inflammatory medications.44 Diarrhea47 11% 16 Diagnosis Anorexia 11%

Because most of the symptoms of HA are due to infection and are nonspecific,2,17 it can be quite difficult to diagnose in a (IV) iodinated contrast, CT can reveal rim enhancement and timely manner. enhancing internal septations, if present, due to increased vas- The most commonly reported signs and symptoms cularity of the abscess wall and septa.47 On MRI, HA will be (Table 2) include fever in most but not all cases, abdominal hyperintense on T2-weighted images and hypointense on non- – pain, and hypotension.2,5,16,17,45 47 The percentage of patients contrast T1-weighed images. Depending on the proteinaceous affected by each symptom encompasses a fairly wide range, content of the HA, some may demonstrate hyperintense signal which reflects the high degree variability in clinical findings. on noncontrast T1-weighted images. After the administration The difficulty in making the diagnosis is also reflected in of gadolinium, HAs demonstrate similar enhancement charac- the reported delay in time of onset of symptoms to time of teristics seen with CT. 17 diagnosis, which on average is one week. Although CEUS is not Food and Drug Administration (FDA)- Laboratory findings in patients with HA are also relatively approved for liver imaging in the United States, it can be useful nonspecific. The most common abnormalities (Table 3) are elevated white blood cell count (WBC), elevated C-reactive protein, hypoalbuminemia, elevated aspartate aminotrans- Table 3. Laboratory findings ferase (AST), elevated alanine aminotransferase (ALT), ele- Abnormal laboratory finding* vated alkaline phosphatase (ALP), elevated gamma glutamyl transpeptidase (GGT), elevated bilirubin, and elevated inter- C-reactive protein 100% national normalized ratio (INR).17 While laboratory testing alone is not diagnostic,47 labora- Hemoglobin 82% tory abnormalities usually prompt imaging studies that do Bilirubin 75% lead to the diagnosis. GGT 75% Diagnosis of HA is made by imaging in 90% of cases.2 Imaging can also help identify the underlying cause in some WBC 74% cases.2 The primary methods of diagnostic imaging are con- ALT 73% ventional ultrasound (US) and CT. Both methods carry a sen- Albumin 73% sitivity of 96%–100% for detection of HA.47 However, Lin ALP 71% et al. found that 25% of patients had equivocal results in the emergency department, and 14% had a false negative AST 67% 25 result on US. HA are typically hypo-echoic on US and may INR 13% have varying degrees of internal echogenicity depending on the presence of septations or gas (Fig. 3).47 *Adapted from Pang TC, Fung T, Samra J, Hugh TJ, Smith RC. Pyogenic liver ’ When US is nondiagnostic, CT, magnetic resonance imaging abscess: an audit of 10 years experience. World J Gastroenterol 2011;17:1622- 1630. doi:10.3748/wjg.v17.i12.1622. (MRI), or contrast enhanced US (CEUS) should be used to 25 Abbreviations: ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, make the diagnosis. HAs have lower attenuation compared alanine aminotransferase; GGT, gamma glutamyl transpeptidase; INR, interna- 47 to normal liver tissue on noncontrast CT. With intravenous tional normalized ratio; WBC, white blood cell count.

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Fig. 3. Ultrasound (US). A. US demonstrates a hypoechoic abscess with heterogeneous echogenicity centrally consistent with septations and internal debris (blue arrow). B. Color Doppler US demonstrates peripheral hypervascularity surrounding the abscess cavity. in the diagnosis of HA. CEUS is performed by injection of ing 67%–95% and 18%–32% of cases, respectively.16,17,52 contrast medium through a peripheral IV line and subsequent With regard to consistency, abscesses were solid in 58% of views are taken through the arterial phase (10-30s post- cases and cystic in 42% of cases.52 Furthermore, gas was injection), the portal phase (30-120s postinjection), and the present in about 17% of cases.52 These radiological features late phase (> 120s postinjection).48 Many studies have can help both with classification of HA and selection of the most identified characteristic features of HA on CEUS, including appropriate treatment approach. Improvements in imaging rim enhancement in the arterial phase,48,49 enhancement techniques have led to more efficient diagnosis and decreased of septa creating a honeycomb appearance in loculated mortality.53,54 HAs,48,49 and washout of the lesion during the late phase.48 A study by Popescu et al. evaluated 41 patients with confirmed Treatment HA for these three findings using CEUS.48 They concluded that CEUS yielded a “conclusive diagnosis” in 93% of cases and Prior to 1980, treatment of HA primarily consisted of open suggested that this method may be superior to conventional surgical drainage.54 However, percutaneous drainage (PD) 48 US. However, by using only patients with a confirmed diag- has gained much popularity since its advent in 195355 and nosis of HA, this study was not well controlled. In contrast, Liu has emerged as the first line treatment for HA.2,56 Smaller et al. examined patients with various focal liver lesions, includ- abscesses have even been managed medically with antibac- ing HA, infected granulomas, and inflammatory pseudotu- terial therapy alone.2,54 The development of effective antibi- 50 mors. Like Popescu et al.,Liuet al. demonstrated the same otics and advances in minimally invasive procedures by three characteristic findings in HA, with 93.8% having rim interventional radiology have largely been responsible for enhancement, 68.8% showing septal enhancement, and the decrease in mortality rate of HA.17,52 80.6% demonstrating late phase washout.50 However, this study also found rim enhancement in 20% of infected granu- Medical management lomas and 50% of the pseudotumor cases.50 Additionally, all of the infected granulomas and pseudotumors demonstrated Management with antibiotics alone has been shown to be 50 late phase washout. These latter findings make it difficult effective for small abscesses, < 3–5 cm in diameter.2 Hope to use these three characteristics findings as criteria for a et al. reported a 100% success rate for eight patients with “definitive diagnosis” as suggested by Popescu et al. Although unilocular abscess measuring < 3 cm in diameter with anti- the diagnostic value of CEUS may be slightly overstated by biotics alone.54 In a larger study of 176 patients, an 81.2% some authors, it is well described in the literature as providing success rate after antibacterial treatment was reported.57 51 clearer images than conventional US. CEUS allows for better However, abscess characteristics, such as size and loculation, visualization of the septations and consistency within the were not clearly stated in this group. abscess,48,49.,51 which may better inform selection of a treat- Biksup et al. proposed that smaller abscesses in difficult ment approach. anatomical positions, such as in the caudate lobe, may be These imaging techniques also play a role in diagnostic US best treated conservatively due to the increased risks associ- or CT-guided needle aspiration, which can confirm the diag- ated with invasive management.58 nosis of HA as the material sampled during this procedure can Antibacterial therapy should begin as soon as blood has been be analyzed to determine the etiological agent. This proce- obtained for identification of organisms.2 Patients are typically dure also plays a therapeutic role in percutaneous drainage, treated initially with IV antibiotics, followed by a course of oral which will be discussed later. (PO) antibiotics. Recommendations for duration range from Other important features that can be observed from 3 weeks IV plus 1–2 months PO to 2–3 weeks IV plus 1–2 imaging studies include location, size, number of abscesses, weeks PO.47 Treatment duration depends on both response to consistency, and presence of gas within the abscess. The right treatment, as determined by repeat US imaging, and resolution lobe of the liver is the most common location,16,47 accounting of fever and leukocytosis.47 for 68.7% of cases in one study.16 Solitary abscesses (Fig. 4) Empiric coverage for gram-negative bacilli, gram-positive are more common than multiple abscesses (Fig. 5), represent- cocci, as well as anaerobic bacteria has been recommended.2

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Fig. 4. Dynamic contrast-enhanced computed tomography (CT). A. Late arterial phase CT demonstrates hypervascular, peripheral enhancement of the abscess seen in Figure 4 (blue arrow). B. Portal venous phase CT demonstrates conspicuity of internally enhancing septations (blue star), likely representing intervening hepatic parenchyma. Note the multilocular nature of the abscess, which has implications for potential treatments (blue arrows).

This is usually accomplished with eithera third-generation ceph- contamination, and a relatively lower cost compared to surgi- alosporin plus metronidazole or piperacillin/tazobactam.47,53 cal drainage (SD).60 However, some common pathogens associated with HA are It has been reported that PD fails in 15%–36% of 17,45 resistant to both ampicillin and the fluoroquinolones.47 Treat- cases. There are a few possible reasons for these failures. ment is becoming complicated as the incidence of a hyper- HAs that are multiloculated can pose a challenge to drain all 53,54 resistant K. pneumonia increases in some parts of the world.47 compartments. In addition, HAs containing viscous fluid and necrotic tissue may be difficult to drain completely45,53,54 due to blockage of the catheter.45,53 Pang et al. found that Interventional treatment hypoalbuminemia was a significant risk factor for PD failure.17 The reason for this increase in risk is somewhat PD is the most commonly selected option for first-line treatment unclear. However, a low albumin level may indicate severity of HA. HA can be drained either with needle aspiration or by of any underlying sepsis.17 In spite of its minimal invasive- 2 insertion of a pigtail catheter drain under US or CT guidance. ness, PD has been reported to be complicated by hemorrhage With percutaneous needle aspiration, a 16-18 Ga needle is and biliary fistulae.17 inserted into the abscess cavity, and contents are aspirated 56 until it is evacuated completely. Similarly, during percutane- Surgical treatment ous catheter drainage, an 8-14 F pigtail catheter is inserted into 56 the lesion and left in place. It is then drained by gravity until Surgery is indicated as the initial management for HA 56 empty. Several studies have found percutaneous catheter rupture,53,54 peritonitis,17 anatomically difficult access,43,54 drainage to be more effective than percutaneous needle aspira- and co-existing pathology requiring surgery.53,54 Initial surgi- tion, as it has higher success rates.56,59 cal management may also be indicated for larger abscesses PD carries many benefits, including being a minimally- measuring > 3–5 cm in diameter.53,54 However, some contro- invasive procedure,53,54 obviating the need for general anes- versy remains in the literature regarding the best approach thesia.53,54,60 It also has a lower risk of adhesion formation, for treatment of large abscesses.

Fig. 5. Magnetic resonance imaging (MRI). A. T2-weighted image demonstrates multiple (at least six) small hyperintense abscess cavities in the right hepatic lobe (blue arrows). Note the hyperintense, edematous hepatic parenchyma (blue star). B. Noncontrast T1-weighted fat-sat image demonstrates varying degrees of T1 hyperintensity in the abscess cavities consistent with proteinaceous debris. C. Postcontrast T1-weighted fat-sat image demonstrates peripheral or rim enhancement around each of the abscesses.

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Interventional versus surgical treatment morbidity (9.1%) compared to the PD group (0%).46 In contrast, Tan et al. found no significant difference in morbidity Since 2001, the number of interventional drainage procedures between these two groups.53 It is likely that the difference has more than doubled, while the number of surgical drainage in sample size and relative distribution between treatment procedures has decreased by about 20%.60 Selection of a groups accounted for the different findings of these two treatment method depends largely on the size and consistency studies. of the abscess. One study of 48 patients with large (> 3 cm), unilocular abscesses treated with PD plus antibiotics reported an 83% success rate.54 Large, multiloculated HAs treated with Other considerations in selecting the treatment PD and antibiotics had only a 33% success rate, whereas a approach large multi-loculated group treated by SD was 100% successful.54 Comparison of these findings with findings from similar In addition to abscess size and consistency, the type of studies is shown in Table 4. This study, among others, demon- abscess should be considered when choosing a treatment strated better clinical outcomes with SD for patients with larger strategy (Fig. 6). Several reports recommended that iatro- abscesses.53,54 However, Ferraioli et al. demonstrated similar genic abscesses due to surgical procedures should be success rates of 95.4% and 93.4% for PD and SD, respec- treated surgically.2 In addition, because malignant HAs con- tively.46 Data for the SD group in Ferraioli’s study was collected taining necrotic tissue are at higher risk for PD failure,45 during an earlier time period (beginning in 1981) than the PD malignant abscesses may be less likely to respond to PD. group (1998), and changes in medical imaging and manage- Nevertheless, because PD is minimally invasive, it might be ment during this time may have impacted the results. In addi- reasonable to attempt PD if accessible, and only if unsuccess- tion, both Ferraioli et al. and Tan et al. allocated patients to ful, resort to SD.46,47 either a PD or SD group based on clinical judgment of the treating physician.46,53 Hope et al. defined the study groups Complications by abscess size alone.54 This difference may account for some of the discrepancy in success rates between these It has been reported that 15.7% of patients develop compli- three studies. cations from HA.16 This includes septic metastasis leading to When selecting a treatment approach, it is important to extrahepatic complications, such as endopthalmitis,16,61 consider the morbidity and mortality of these procedures. In septic pulmonary embolism,52 infection of the lungs, central the past, SD has been associated with a high mortality rate, nervous system, and the eyes.47 Abscess rupture is another ranging from 10%–47%.56 One might expect this finding to reported complication,16 with spontaneous rupture occurring be true as patients selected for surgery are typically those in 6.1% of cases.52 There was a higher reported incidence of with larger abscesses, difficult anatomic access, multiple HA rupture in abscesses infected with Klebsiella compared to comorbidities, or other complicating factors. In comparing other bacteria.47 HAs can also erode the diaphragm, causing the modalities, many studies have found no significant differ- pleural effusion, empyema, pneumonia, pericarditis, bron- ence in mortality between PD and SD.46,53,54 However, Fer- chopleural fistulas, or duodenobronchofistulas.36 Multiorgan raioli et al. found that the SD group had a significantly higher failure can also occur as a consequence of HA.16

Table 4. Treatment success rates

Treatment

Study Medical Radiological Surgical

Alkofer et al.45 2011 70% 75.4% 91.6% n=17 n=57 n=12 Mean diameter 3.7 cm Mean diameter 8.7 cm Mean diameter 9.14 cm Ferraioli et al.46 2008 – 95.4% 93.2% n = 108 n=44 Mean diameter 5.1 cm Mean diameter 5.3 cm Hope et al.54 2008 100% 83% 100% n=8 n=48 n=27 Diameter < 3 cm Diameter > 3 cm Diameter > 3 cm Uni-locular Multi-locular 33% n=24 Diameter > 3 cm Multilocular Tan et al.53 2005 – 72.2% 93.8% n=36 n=44 Diameter > 5 cm Diameter > 5 cm Bamberger et al.57 1996 81.2% –– n = 176

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Fig. 6. Treatment strategies of HA*. *Adapted from Hope WW, Vrochides DV, Newcomb WL, Mayo-Smith WW, Iannitti DA. Optimal treatment of hepatic abscess. Am Surg 2008;74:178-182.

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168 Journal of Clinical and Translational Hepatology 2016 vol. 4 | 158–168 Editorial Board

Dr.Subrat Kumar Acharya Dr. Tatsuo Kanda Dr. Qing-Feng Sun New Delhi, India Chiba, Japan Wenzhou, China Dr. Piero Luigi Almasio Dr. Anastasios Koulaouzidis Dr. Jian Sun Palermo, Italy Edinburgh, United Kingdom Guangzhou, China Prof. Gianfranco D. Alpini Prof. Ming Kuang Prof. Gloria Taliani Bryan, USA Guangzhou, China Roma, Italy Dr. Masahiro Arai Dr. Ashish Kumar Prof. Hong Tang Tokyo, Japan New Delhi, India Chengdu, China Dr. Moustafa Awady Prof. Manoj Kumar Prof. Rolf Teschke Cairo, Egypt New Delhi, India Hanau, Germany Prof. Sina Aziz Prof. Laurentius A. Lesmana Prof. Vladimir Maximovich Tsyrkunov Karachi, Pakistan Jakarta, Indonesia Grodno, Republic Belarus Dr. Gyorgy Baffy Prof. Jun Li Dr. Claudio Tiribelli Boston, USA Nanjing, China Trieste, Italy Prof. Mahmoud Mohamed Bahgat Dr. Bing Liu Prof. Zheng-Kun Tu Dokki, Egypt Guangzhou, China Changchun, China Dr. Chalermrat Bunchorntavakul Dr. Man-Qing Liu Dr. Adriana Vince Bangkok, Thailand Wuhan, China Zagreb, Croatia Dr. Peter Buch Prof. Feng-Min Lu Prof. Gen-Shu Wang Miami, USA Beijing, China Guangzhou, China Dr. Wendy Cao Dr. Rohit Loomba Dr. Le-Yi Wang New York, USA San Diego, USA Columbus, USA Prof. Flair José Carrilho Prof. Xiong Ma Prof. Benjamin Wong São Paulo, Brazil Shanghai, China Hong Kong, China Dr. Jayanta Roy Chowdhury Dr. Mazyar Malakouti Prof. Catherine Y. Wu New York, USA San Antonio, USA Farmington, USA Dr. Phunchai Charatcharoenwitthaya Prof. Qing Mao Prof. Qing Xie Bangkok, Thailand Chongqing, China Shanghai, China Prof. Mark J. Czaja Prof. Yi-Min Mao Dr. Yong-Ning Xin New York, USA Shanghai, China Qingdao, China Dr. Anna Mae Diehl Prof. Eduardo Martinez Prof. Hui-Ping Yan Durham, USA Hidalgo, Mexico Beijing, China Prof. Gülsüm Özlem Elpek Dr. Dominik Andre Megger Prof. Ming Yan Antalya, Turkey Bochum, Germany Shangdong, China Prof. Jian-Gao Fan Prof. Xiao-Hui Miao Prof. Dong-Liang Yang Shanghai, China Shanghai, China Wuhan, China Dr. Yu-Chen Fan Prof. Albert D. Min Prof. Eric M. Yoshida Jinan, China San Diego, USA Vancouver, Canada Prof. Eduardo Fernández-Martínez Dr. Paul Naylor Dr. Hong You Hidalgo, Mexico Detroit, USA Beijing, China Dr. Heather L. Francis Dr. Mohamed Othman Prof. Yu-Feng Yuan Bryan, USA El Paso, USA Wuhan, China Prof. María Teresa Pérez-Gracia Prof. Calvin Q. Pan Prof. Cihan Yurdaydin Valencia, Spain New York, USA Ankara, Turkey Prof. Ahmet Gurakar Prof. Jin-Jiang Pang Dr. Maysaa El Sayed Zaki Baltimore, USA Shanghai, China Mansoura, Egypt Prof. Steven-Huy Bui Han Dr. Giovanni Perricone Prof. Ji-Ming Zhang Los Angeles, USA Milan, Italy Shanghai, China Prof. Ying Han Dr. Nikolaos T. Pyrsopoulos Prof. Xin-Xin Zhang Xi'an, China Newark, USA Shanghai, China Dr. Kazuhiko Hayashi Dr. Farzin Roohvand Dr. Yuan-Yuan Zhang Nagoya, Japan Tehran, Iran Chengdu, China Prof. Peng Hu Prof. Sammy Saab Prof. Yue-Xin Zhang Chongqing, China Los Angeles, USA Xinjiang, China Prof. Yan Huang Dr. Behnam Saberi Prof. Ying-Ren Zhao Changsha, China Baltimore, USA Xi'an, China Prof. Yuan Huang Dr. Regina Santella Dr. Ming-Hua Zheng Nanchang, China New York, USA Wenzhou, China Prof. Hartmut Jaeschke Prof. Jia Shang Dr. Sen-Lin Zhu Kansas City, USA Zhengzhou, China Guangzhou, China Dr. Wasim Jafri Dr. Ke-Qing Shi Aga Khan, Parkistan Wenzhou, China Prof. Lenox Jeffers Prof. Gamal Shiha Miami, USA Mansoura, Egypt Dr. Di Jia Dr. Ashwani Singal Boston, USA Birmingham, USA Dr. Wei Jia Dr. Robert Smolic Kunming, China Osijek, Croatia Dr. Jia-Ji Jiang Dr. Martina Smolic Fuzhou, China Osijek, Croatia Prof. Xiang-Jun Jiang Dr. Jonathan G. Stine Qingdao, China Charlottesville, USA