Easl Postgraduate Course •

THE INTERNATIONAL LIVER CONGRESS™ 2018 CONGRESS™ LIVER INTERNATIONAL THE

EASL POSTGRADUATE COURSE •

11-12 APRIL 2018 POSTGRADUATE COURSE VIRAL HEPATITIS VIRAL COURSE POSTGRADUATE

• e y 11-12 2018 APRIL ou 10-14 n ext Ap y ri ear in l VIENNA www.ilc-congress.eu ILC2018-CongressApp-A4.pdf 1 07.03.2018 10:59:16

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Specialties

Cirrhosis and Liver tumours complications

General hepatology Cholestasis and autoimmune

Metabolism, alcohol Viral and toxicity hepatitis

CY The 2018 Postgraduate course

CMY K focuses on viral hepatitis.

Fields Liver transplant and surgery Public health Basic and translational science Imaging and interventional What’s your colour? Follow the colour codes and pictogrammes throughout the programme book to find the sessions of interest to you! 11-12 APRIL 2018

PARIS, FRANCE

TABLE OF CONTENTS

GENERAL INFORMATION 5 EASL GOVERNING BOARD 6 WELCOME MESSAGE FROM THE COURSE ORGANISERS 8 PROGRAMME 9 ACRONYMS 12

SESSION 1 – HBV 15 WHERE DO WE STAND WITH DIAGNOSIS OF HBV INFECTION PHASE AND HBV-INDUCED LIVER DAMAGE IN HBeAg-POSITIVE CARRIERS? 16 WHEN AND HOW TO TREAT A HIGHLY VIRAEMIC HBeAg-POSITIVE PATIENT, AND HOW TO MONITOR AND GUIDE THE TREATMENT 22 HOW TO MANAGE PREGNANT WOMEN TO MINIMISE THE RISK OF HBV TRANSMISSION 31

SESSION 2 – HCV 35 DIFFICULT-TO-CURE PATIENTS IN 2018 36 RETREATMENT STRATEGIES IN 2018 43 HCC RISK POST-DAA TREATMENT: A BALANCED PERSPECTIVE 48

SESSION 3 – HDV 69 ASSESSING LIVER FIBROSIS IN HEPATITIS DELTA VIRUS: INVASIVE VS. NON-INVASIVE TECHNIQUES. IS THE LIVER BIOPSY STILL NECESSARY AS A DIAGNOSTIC TOOL AND IN LONG-TERM SURVEILLANCE? 70 FACTS AND CONTROVERSIES IN TREATMENT OF HEPATITIS DELTA WITH PegIFNα 74 THE RISK OF DECOMPENSATION AND HCC DEVELOPMENT IN PATIENTS WITH HDV-RELATED CIRRHOSIS 84 THE ROLE OF LIVER TRANSPLANTATION IN HEPATITIS DELTA 92

SESSION 4 – HEV 105 HOW TO BECOME INFECTED WITH HEV IN EUROPE 106 DIAGNOSTICS IN HEV INFECTION 110 HEPATITIS E VIRUS: NATURAL HISTORY 115 TREATMENT OF HEPATITIS E 122

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SESSION 5 – HBV 125

WHERE DO WE STAND ON DIAGNOSING HBV INFECTION PHASE, AND HBV-INDUCED LIVER DAMAGE IN HBeAg-NEGATIVE CARRIERS? 126 PegIFN VS. ORAL THERAPY IN HBeAg-NEGATIVE CHB PATIENTS 132 CAN WE STOP NUCLEOS(T)IDE ANALOGUE THERAPY IN HBeAg-NEGATIVE CHRONIC HEPATITIS B PATIENTS? 141 MANAGEMENT OF HBeAg-NEGATIVE PATIENTS WHO STOPPED LONG-TERM TREATMENT WITH NUCLEOS(T)IDE ANALOGUES 149

SESSION 6 – HCV 159

PRACTICAL HCV TREATMENT IN PATIENTS WITH DECOMPENSATED CIRRHOSIS 160 REMOVING PATIENTS FROM THE TRANSPLANT LIST WITH ANTI-HCV TREATMENT 169 HCV TREATMENT IN THE IMMUNOSUPPRESSED PATIENT 175 PROS AND CONS OF TRANSPLANTING HCV-INFECTED ORGANS 184

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GENERAL INFORMATION

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GENERAL INFORMATION EASL EASL POSTGRADUATE COURSE POSTGRADUATE EASL 2018 CONGRESS™ LIVER INTERNATIONAL THE Prof. Analisa Berzigotti Analisa Prof. Hemming Hemming Bern, SwitzerlandBern, SECRETARY SECRETARY THE HOME OF HEPATOLOGY HOME THE GENERAL Oslo, Norway P rof. Tom Tom rof. P Karlsen

Prof. Marco Marzioni SCIENTIFIC COMMITTEE MEMBERS COMMITTEE SCIENTIFIC EASL GOVERNINGEASL BOARD Ancona, Italy Ancona, VICE-SECRETARY Prof. Markus Cornberg Markus Prof. Hannover, Germany Prof. Frank Tacke Frank Prof. Aachen, Germany Aachen, Birmingham, United Kingdom United Birmingham, Prof. Philip N. Newsome Philip Prof. Prof. Mauro Bernardi Mauro Prof. Dr Alejandro Forner Alejandro Dr TREASURER Barcelona, Spain Bologna, Italy 11-12 APRIL 2018

PARIS, FRANCE

EU POLICY EDUCATIONAL COUNCILLORS COUNCILLOR GENERAL INFORMATION

Prof. Francesco Negro Prof. Massimo Pinzani Prof. Helena Cortez-Pinto Geneva, Switzerland London, United Kingdom Lisbon, Portugal

ILC 2018 HONORARY PRESIDENT

Prof. Tilman Sauerbruch Bonn, Germany

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WELCOME MESSAGE FROM THE COURSE ORGANISERS

On behalf of the European Association for the Study of the Liver (EASL), we are delighted to welcome you to Paris for the ILC 2018 EASL postgraduate course on “Viral hepatitis”.

Although potent antiviral medicines are available to combat hepatitis B and C, the achievement of the ambitious goals set by the WHO in 2016 regarding the elimination of viral hepatitis

GENERAL INFORMATION as a global health threat poses substantial challenges. From the public health standpoint, the identification of undiagnosed populations, a proper linkage to specialized care, and the unfettered access to potent antivirals are among the issues to be tackled. From the clinical point of view, proper management requires careful assessment of patients’ features, including past treatment history, characterization of specific viral strains, liver disease severity, comorbidities and concomitant medications. In addition, the full control of viral replication (eventually leading to viral clearance, like in most hepatitis C patients) may not always lead to complete control of the associated disease (both hepatic and extrahepatic) and its progression: many open questions remain that warrant further research. Finally, newly emerging (hepatitis E) and re-emerging (hepatitis Delta) types of viral hepatitis pose serious problems in terms of screening and therapy, respectively.

We have structured the 2018 postgraduate course in the form of 6 distinct clinical case discussions, each animated by a leading hepatologist, with the contribution of relevant experts. The aim of this course is to foster the team discussion and management of complex viral hepatitis cases, in the wake of the arrival of new potent antivirals and the WHO strategy of viral hepatitis elimination by the year 2030.

The organisers and the faculty wish you an enjoyable time in Paris and they hope you find the course stimulating and informative.

Maurizia BRUNETTO Francesco NEGRO Italy Switzerland

Jean-Michel PAWLOTSKY Heiner WEDEMEYER France Germany

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PROGRAMME

WEDNESDAY 11 APRIL 2018

HBV

Case presentation, Anna LOK, United States GENERAL INFORMATION Where do we stand with diagnosis of HBV infection phase and HBV-induced liver damage in HBeAg-positive carriers? Maria BUTI, Spain

11:30 -12:30 Why and how to treat a highly viraemic HBeAg-positive patient? How to monitor and guide the treatment? Patrick KENNEDY, United Kingdom

How to manage pregnant women to minimize the risk of HBV transmission Henry CHAN, Hong Kong

HCV

Case presentation, Alessandra MANGIA, Italy

Difficult-to-cure patients in 2018 Christophe HEZODE, France 12:30-13:30 Retreatment strategies in 2018 Jordan FELD, Canada

HCC risk post-DAA treatment: a balanced perspective Geoffrey DUSHEIKO, United Kingdom

13:30-14:00 Lunch

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HDV

Case presentation, Mario RIZZETTO, Italy

Assessing liver fibrosis in hepatitis delta virus: invasive vs. non-invasive techniques. Is the liver biopsy still necessary as a diagnostic tool and in long-term surveillance? Benjamin HEIDRICH, Germany

GENERAL INFORMATION Facts and controversies in treatment of hepatitis Delta with 14:00-15:30 PegIFNα Cihan YURDAYDIN, Turkey

The risk of decompensation and HCC development in patients with HDV-related cirrhosis Grazia NIRO, Italy

The role of liver transplantation in Hepatitis Delta Didier SAMUEL, France

15:30-16:00 Coffee break

HEV

Case presentation, Sven PISCHKE, Germany

How to become infected with HEV in Europe Robert A. DE MAN, Netherlands

Diagnostics in HEV infection 16:00-17:30 Darius MORADPOUR, Switzerland

Hepatitis E virus: natural history Harry DALTON, United Kingdom

Treatment of hepatitis E Nassim KAMAR, France

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THURSDAY 12 APRIL 2018

HBV

Case presentation, Rafael ESTEBAN, Spain

Where do we stand on diagnosising HBV infection phase, and HBV-induced liver damage in HBeAg-negative carriers? George PAPATHEODORIDIS, Greece GENERAL INFORMATION PegIFN vs. oral therapy in HBeAg-negative CHB patients Pietro LAMPERTICO, Italy 08:30-10:00 Can we stop nucleos(t)ide analogue therapy in HBeAg-negative chronic hepatitis B patients? Fabien ZOULIM, France

Management of HBeAg-negative patients who stopped long-term treatment with nucleos(t)ide analogues Thomas BERG, Germany

10:00-10:30 Coffee break

HCV

Case presentation, Kosh AGARWAL, United Kingdom

Practical HCV treatment in patients with decompensated cirrhosis Xavier FORNS, Spain

Removing patients from the transplant list with anti-HCV treatment 10:30-12:00 Norah TERRAULT, United States

HCV treatment in the immunosuppressed patient Alessio AGHEMO, Italy

Pros and cons of transplanting HCV-infected organs Marina BERENGUER, Spain

12:00-13:30 Lunch

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ABBREVIATIONS AND ACRONYMS

AASLD American Association for EASL European Association for the Study of Liver Diseases the Study of the Liver

ACLF acute-on-chronic liver failure EBL endoscopic band ligation

ADV adefovir EIA enzyme immunoassays

AE adverse events ETV entecavir GENERAL INFORMATION AFP alpha-fetoprotein GBS Guillain-Barré syndrome

ALF acute liver failure GP glecaprevir/pibrentasvir

ALT alanine aminotransferase HAART highly active antiretroviral therapy ANRS Agence Nationale de Recherches sur le Sida et HAV hepatitis A virus les hépatites virales HBIG hepatitis B immune globulin APASL Asian Pacific Association for the Study of Liver Diseases HBRN Hepatitis B Research Network

ARFI acoustic radiation force HBV hepatitis B virus impulse imaging HBeAg hepatitis B e antigen

AST aspartate aminotransferase HBsAg hepatitis B surface antigen

AUROC area under the receiver HCV hepatitis C virus operating characteristic curve HCC hepatocellular carcinoma BCLC Barcelona Clinic Liver Cancer HDIN Hepatitis Delta International BEA baseline-event-anticipation Network score HDV hepatitis delta virus BMD bone mineral density HEV hepatitis E virus BMI body mass index HIV human immunodeficiency CHD chronic hepatitis delta virus

CI confidence interval HR hazard ratio

CLD chronic liver disease HVPG hepatic venous pressure CKD chronic kidney disease gradient

CSPH clinical significant portal IFN interferon hypertension INR international normalized ratio

DAA direct-acting antiviral agents IT immune tolerant

DCV declatasvir IU international units

DDI drug-to-drug interactions IgM immuno globulin M

DLBCL diffuse large B cell lymphoma IgG immuno globulin G

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IPAS individualised pathway ROS reactive oxygen species aberrance scoring SNP single nucleotide IVDU intravenous drug users polymorphism

KT kidney transplantation SOF sofosbuvir

LAM lamivudine SRTR Scientific Registry of Transplant Recipients LDV ledipasvir SVR sustained virological response LFT liver function test GENERAL INFORMATION TAF tenofovir alfenamide LT liver transplantation TDF tenofovir disoproxil fumarate LuT lung transplantation TDV tenofovir MELD Model for End-stage Liver Disease TE transient elastography

MC mixed cryoglobulinemia ULN upper limit of normal

MR magnetic resonance UNOS united network for organ sharing NA nucleos(t)ide analogues VA veterans administration NASH non-alcoholic steatohepatitis VEL velpatasvir NAT nucleic acid amplification techniques VOX voxilaprevir

NHL non-Hodgkin lymphoma VR viral response

NK natural killer cells WHO World Health Organization

NPV negative predictive value

NSBB non-selective beta-blockers

NTCP sodium taurocholate co-transporting polypeptide

OLT orthotopic liver transplantation

ORF open reading frames

PegIFNα pegylated interferon alpha

PCR polymerase chain reaction

PI protein inhibitors pSWE point shear wave elastrography

RAS resistance-associated substitutions

RBV ribavirin

ROC receiver operating characteristic curve

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WEDNESDAY 11 APRIL 2018 11:30-12:30 BIOTECH SESSION 1 – HBV SHARE VILLAGE ZONE

MY CY EASL CMY K COMMUNITY CAREER HUB CENTRE Hall 7.2

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WHERE DO WE STAND WITH THE DIAGNOSIS OF HBV INFECTION PHASE AND HBV-INDUCED LIVER DAMAGE IN HBeAg-POSITIVE CARRIERS?

Maria Buti Hospital Universitari VAll d’ Hebron Barcelona, Barcelona, Spain. E-mail address: [email protected] SESSION 1 – HBV TAKE-HOME MESSAGES • The immune tolerant (IT) phase of chronic hepatitis B is established based on hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg)-positive testing with persistently normal alanine aminotransferase levels, and high HBV DNA levels.

• Most IT patients are children or young adults who acquired HBV infection perinatally. These individuals usually remain HBeAg-positive and have high viral replication levels for prolonged periods, although they have minimal liver disease.

• Liver fibrosis can be reliably assessed using non-invasive markers.

• Chronic HBV IT patients should undergo liver function monitoring every 6 to 12 months, using biochemical tests to seek evidence of immune activation and non-invasive markers to evaluate fibrosis progression. HBV therapy is usually not recommended during the IT phase.

WHAT PHASE OF CHRONIC HBV INFECTION WAS THIS PATIENT IN AT THE TIME OF PRESENTATION AND WHAT PHASE WAS SHE IN WHEN THE DECISION WAS MADE TO INITIATE ANTIVIRAL TREATMENT? The outcome of hepatitis B virus (HBV) infection depends on the patient’s age at the time it was acquired. HBV infection acquired prenatally or in early childhood is associated with a high rate of chronicity and is believed to progress through various phases. According to the AASLD and EASL guidelines, these phases are named as follows: immune tolerant (IT) or chronic hepatitis B e antigen (HBeAg)-positive infection, HBeAg-positive immune active or HBeAg- positive chronic hepatitis B, inactive carrier chronic hepatitis B (CHB) or chronic HBeAg- negative infection, and HBeAg-negative immune reactivation or HBeAg-negative chronic hepatitis [1-2]. Table 1 shows the various phases of chronic HBV infection. The IT phase is characterised by high serum HBV DNA concentrations, normal alanine aminotransferase (ALT) levels, and minimal or no liver inflammation on biopsy.

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Table 1. Phases of chronic HBV as proposed by the EASL Guidelines [2].

*Persistently or °°intermittently HBV DNA levels can be between 2,000 and 20,000 IU/ml in some patients without signs of chronic hepatitis.

Given the dynamic nature of CHB infection, serial monitoring of HBV DNA and ALT levels is important to characterise the phase of infection. A single ALT and HBV DNA determination SESSION 1 – HBV are insufficient to assign the infection phase; at least two determinations separated by an interval of 6 months are needed before a patient can be staged as an IT patient or as an HBeAg- positive patient with chronic hepatitis B infection. This phase is more common and prolonged in persons who acquired the infection perinatally, and the rate of spontaneous HBeAg loss is very low in this phase. These patients are highly contagious due to the high HBV DNA levels. It is important to differentiate between the IT phase and the HBeAg-positive chronic hepatitis B phase, the second phase of CHB, which is characterised by the presence of serum HBeAg, high HBV DNA levels, and elevated ALT, moderate or severe necroinflammation in the liver and accelerated progression of fibrosis [1]. This phase may occur after several years of the first phase and may be reached faster and more often in patients infected in adulthood. The outcome of this phase is variable. Of note, some individuals will be in the “grey-zone,” which means that their HBV DNA and ALT levels do not fall into the same phase. One study aimed to validate the categorisation of chronic hepatitis B viral infection into phases based on measures of disease activity, and viral load found that 38% of patients did not fit clearly into any of the groups. The authors stressed that monitoring is essential to categorise these patients [3].

The ALT values are a critical point. The value considered the upper limit of normal (ULN) differs between some international guidelines. The EASL and APASL guidelines establish the normal range at <40 U/L, while the AASLD recommendations place normal ALT levels at <19 U/L for females and <30 U/L for males [1-2,4].

At the time of presentation, this patient would be classified into phase-1 HBV infection. She was asymptomatic, and her laboratory tests showed persistently positive HBeAg, normal ALT concentrations, and high HBV DNA and hepatitis B surface antigen (HBsAg) levels. She was not co-infected with hepatitis C virus (HCV), hepatitis delta virus (HDV), or human immunodeficiency virus (HIV). In addition, liver stiffness determined by vibration-controlled transient elastography (TE) was 4.7 kPa, which is compatible with no or minimal fibrosis. If the AASLD criteria for normal ALT were used (<19 U/L for females and <30 U/L for males), this woman would be considered to have elevated ALT levels, more than 2 times the ULN. Several points should be taken into consideration. First, the patient’s age; usually the median age of onset of the immune active phase is 30 years in those infected at a young age. It is unclear when she acquired HBV infection. HBsAg serology was negative previously, and she was vaccinated against HBV infection, but despite this, she had chronic HBV infection. Her BMI had increased to 23.5 in the last few years, indicating being overweight, a factor associated with elevated ALT levels. In my opinion, her chronic hepatitis B was likely transitioning from the IT to the HBeAg-positive immune active phase. At the time of starting therapy, she was transitioning to an immune active phase.

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The phase of the infection has an impact on the treatment decision. Children and young adults in the IT phase are believed to lack an HBV-specific adaptive immune response and therefore, are not considered candidates for treatment [5]. Classically, patients in the IT phase do not receive therapy, particularly interferon-containing regimens, because of the low likelihood of achieving HBeAg and HBsAg clearance when ALT levels are normal. Studies performed in the seventies showed a relationship between ALT levels and HBeAg seroconversion: the higher the ALT level, the greater the likelihood of achieving HBeAg seroconversion [6]. The long- term clinical impact of suppressing viral replication with nucleos(t)ide analogues (NA) in the IT phase is unknown.

SESSION 1 – HBV HOW SHOULD ACTIVITY AND STAGE OF LIVER DISEASE BE DETERMINED IN PATIENTS WITH CHRONIC HBV INFECTION? The initial evaluation of an HBeAg-positive patient should include a physical examination and a thorough clinical history, with special emphasis on risk factors for co-infection, alcohol use, and the family history of HBV infection and liver cancer. Laboratory tests should assess liver disease activity including complete blood count, platelet count, aspartate aminotransferase (AST), ALT, total bilirubin, alkaline phosphatase, albumin, the international normalized ratio, HBV markers (HBsAg, HBeAg/anti-HBe, HBV DNA quantitation), and tests for patients at risk of co-infection with HCV, HDV, or HIV.

Additionally, an abdominal ultrasound and vibration-controlled TE or serum fibrosis panel (AST-to-platelet ratio index [APRI], FIB-4, or FibroTest) should be done in the first evaluation. How reliable are non-invasive tests for liver fibrosis in staging patients with chronic HBV infection?

The EASL guidelines for treatment-naïve patients with CHB infection and normal ALT have established a TE cut-off of <6 kPa to define non-significant fibrosis and >9 kPa for severe fibrosis/cirrhosis. Patients with a TE score between 6 and 9 kPa fall into the grey area, and liver biopsy is recommended if the results have an influence on therapy, regardless of HBV DNA levels or HBeAg status [7] (Fig. 1).

Similar recommendations are suggested in the APASL guidelines. HBeAg-positive patients with high HBV DNA levels (>20,000 IU/ml) and ALT <2 ULN should undergo non- invasive fibrosis assessment and monitored every 3 months. Biopsy should be considered if non-invasive tests suggest evidence of significant fibrosis, ALT becomes persistently elevated, age is >35 years, or there is a family history of hepatocellular carcinoma (HCC) or cirrhosis. Patients should be considered for treatment if biopsy shows moderate to severe inflammation or significant fibrosis [4].

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Fig. 1. Proposed algorithm for the use of transient elastography (TE) in treatment- naïve patients as proposed by EASL guidelines [7]. HCC, hepatocellular carcinoma; ALT, alanine aminotransferase; ULN, upper limit of normal.

Serum markers of fibrosis, such as the APRI, FIB-4, and FibroTest, are only moderately accurate for identifying persons with significant fibrosis (fibrosis stage 2 or greater on the METAVIR scale). However, they have a good diagnostic accuracy for excluding advanced fibrosis [7] and may be useful aids in decision-making (Table 1). TE has shown good performance for assessing significant HBV-related fibrosis (stage F2) and cirrhosis (stage F4), with areas under the receiver operating curve (AUCs) of 0.65-0.97 and 0.8-0.97, respectively [7]. Nonetheless, TE has some limitations, for example, it is less accurate in predicting the existence of significant fibrosis, and it has been unsuccessful in individuals with high ALT/ bilirubin levels, obesity, and ascites. In addition, TE is more expensive than the non-invasive models.

Assessment of liver fibrosis is more important for establishing antiviral therapy in patients with ALT <2 ULN than in those with ALT ≥2 ULN. Non-invasive models for estimating liver fibrosis are recommended as a means to select patients for liver biopsy. In two studies on HBeAg-positive patients with normal ALT and HBV DNA >2x106 IU/ml, liver biopsy showed only a mild disease state in all cases, and none of the patients had a histological fibrosis score of >1. However, in a study performed in Korea, 28% of HBeAg-positive patients with normal ALT and HBV DNA >2x104 IU/ml had significant histology [8], and in another, conducted in India, 40% of 73 HBeAg-positive patients with persistently normal ALT had significant fibrosis [9]. In these studies, HBV DNA level was a poor surrogate for fibrosis on liver biopsy [7]. Thus, liver fibrosis cannot be predicted based on HBV DNA levels and ALT alone, and for this reason, reliable non-invasive markers are important.

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Is fibrosis the main determinant for treatment decisions? Is there any role for liver inflammation in treatment decisions?

Liver fibrosis is one of the main prognostic factors in viral hepatitis B and other chronic liver diseases, as the degree of fibrosis correlates with the risk of developing cirrhosis and liver- related complications. Before treating a patient, it is important to determine whether there is advanced fibrosis to guide treatment choices, the duration of therapy, and the therapeutic endpoints. Currently, fibrosis is the main determinant for treatment decisions. In chronic hepatitis B, there is a varying degree of predominantly lymphocytic portal inflammation with interface hepatitis and spotty lobular inflammation. Inflammation is minimal in the IT phase, but it is prominent in the immune-reactive phase [5]. Histological studies based on paired biopsies obtained before and after HBeAg seroconversion have consistently demonstrated that SESSION 1 – HBV seroconversion to anti-HBe is followed by a significant improvement or even disappearance of disease activity. Active inflammation is the driving force leading to fibrosis, and increasing grades of inflammation are associated with increased fibrosis. Because the goal of treatment is to stop ongoing necroinflammation and prevent progression to cirrhosis and HCC, follow-up biopsies may be helpful in protocol settings. In HBeAg-positive patients, necroinflammation is usually associated with elevated ALT levels and transitioning to an immune-reactive phase. Currently, treatment is indicated when necroinflammatory activity persists for more than 6 weeks and is associated with elevated ALT levels and HBeAg-positive status. Are there reliable non-invasive tests for determining liver inflammation?

Currently, there are no reliable non-invasive tests for liver inflammation. Most available tests are designed to evaluate fibrosis, and some of them analyse fibrosis and inflammation. Several studies have shown that after suppressing viral replication in patients with chronic hepatitis B, ALT levels normalise and there is an improvement in necroinflammation and fibrosis in CHB. Still today, elevated ALT levels are the best method to evaluate inflammation. What are the indications for liver biopsy in patients with chronic HBV infection?

Traditionally, liver biopsy has been considered the reference method for evaluating tissue damage, such as hepatic fibrosis in patients with chronic liver disease [10]. Currently, liver biopsy is not required to establish treatment decisions. However, it remains within the armamentarium of hepatologists for assessing the aetiology of complex diseases or for clarifying discordance between clinical symptoms, and to assess the extent of fibrosis by non-invasive approaches particularly in patients with ALT values >0.5 times the ULN but >1.0 of ULN, and HBV DNA levels between 2,000 and 20,000 IU/ml.

In this case, liver biopsy can help to identify necroinflammation and steatosis, but ultimately, the results will not greatly change the clinical management. These characteristics, if treated, respond to antiviral therapy.

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References

[1] Terrault NA, Bzowej NH, Chang KM, Hwang JP, Jonas MM, Murad MH. American Association for the Study of Liver Diseases. AASLD guidelines for treatment of chronic hepatitis B. Hepatology 2016;63(1):261-83.

[2] European Association for the Study of the Liver. EASL 2017 Clinical Practice Guidelines on the management of hepatitis B virus infection. J Hepatol. 2017;67(2):370-398.

[3] Sarin SK, Kumar M, Lau GK, Abbas Z, Chan HL, Chen CJ, et al. Asian-Pacific clinical practice guidelines on the management of hepatitis B: a 2015 update. Hepatol Int. 2016;10(1):1-98.

[4] Di Bisceglie AM, Lombardero M, Teckman J, Roberts L, Janssen HL, Belle SH, SESSION 1 – HBV Hoofnagle JH, Hepatitis B Research Network (HBRN). Determination of hepatitis B phenotype using biochemical and serological markers. J Viral Hepat. 2017;24(4):320-329.

[5] Kennedy PTF, Sandalova E, Jo J, Gill U, Ushiro-Lumb I, Tan AT, et al. Preserved T-cell function in children and young adults with immune-tolerant chronic hepatitis B. Gastroenterology. 2012;143(3):637-645.

[6] Perrillo RP, Lai CL, Liaw YF, Dienstag JL, Schiff ER, Schalm SW, et al. Predictors of HbeAg loss after lamivudine treatment for chronic hepatitis B. Hepatology. 2002;36(1):186-94.

[7] European Association for Study of Liver; Asociacion Latinoamericana para el Estudio del Higado. EASL-ALEH Clinical Practice Guidelines: Non-invasive tests for evaluation of liver disease severity and prognosis. J Hepatol. 2015;63(1):237-64.

[8] Park JY, Park YN, Kim DY, Paik YH, Lee KS, Moon BS et al. High prevalence of significant histology in asymptomatic chronic hepatitis B patients with genotype C and high serum HBV DNA levels. J Viral Hepat 2008;15:615–621.

[9] Kumar M, Sarin SK. Hepatitis B virus immunotolerant patients: need to differentiate patients with or without liver disease. Gastroenterology 2009;137(2):742–743.

[10] Tapper EB, Lok ASF. Use of Liver Imaging and Biopsy in Clinical Practice. N Engl J Med. 2017 7;377(23):2296-2297.

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WHEN AND HOW TO TREAT A HIGHLY VIRAEMIC HBeAg-POSITIVE PATIENT, AND HOW TO MONITOR AND GUIDE THE TREATMENT

Patrick T.F. Kennedy*, Upkar S. Gill Hepatology, Centre for Immunobiology, Barts and The London School of Medicine & Dentistry, Queen Mary, University of London, London, United Kingdom. E-mail address: [email protected] SESSION 1 – HBV TAKE-HOME MESSAGES • Patients previously considered ‘immune tolerant’ have hepatitis B virus (HBV)-specific immune responses, which are ostensibly no different to their peers labelled as ‘immune active’.

• This observation has led to a change in the nomenclature of disease phase and patients previously labelled immune tolerant are now referred to as hepatitis B e antigen (HBeAg)- positive chronic infection.

• There is emerging data that this early disease phase is not benign and devoid of disease progression.

• A recent study demonstrated an increased risk of hepatocellular carcinoma development in untreated HBeAg-positive chronic infection.

• EASL guidelines recommend treatment should now be considered in HBeAg-positive chronic infection above the age of 30.

• Oral antivirals can be stopped in HBeAg-positive chronic hepatitis B patients following a period of consolidation therapy after HBeAg to anti-HBe seroconversion, but hepatitis B surface antigen (HBsAg) loss remains the ideal treatment endpoint.

SHOULD TREATMENT BE RECOMMENDED IN HIGHLY VIRAEMIC HBeAg-POSITIVE PATIENTS WITH MINIMAL INFLAMMATION AND MINIMAL FIBROSIS? IS HIGH VIRAEMIA PER SE AN INDICATION FOR TREATMENT? Historically highly viraemic hepatitis B e antigen (HBeAg)-positive patients with minimal inflammation and fibrosis were considered ‘immune tolerant’ and excluded from treatment. Immune tolerance, characterised by the presence of HBeAg, high serum HBV DNA and normal alanine aminotransferase (ALT), was considered to be a benign disease phase with minimal or no disease progression. More recently, this overly simplistic interpretation of the early phase of chronic hepatitis B (CHB) has been challenged. CHB is a dynamic immune mediated liver disease, thus characterising patients based on clinical parameters alone is not without flaw. Moreover, other factors apart from disease phase should be considered when

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PARIS, FRANCE making treatment decisions. These include patient age, ethnicity, infecting genotype, family history of hepatocellular carcinoma (HCC) and comorbidities.

Recent work has highlighted the limitations of serum ALT as a surrogate of immune activity, thus challenging the notion of withholding treatment until there is evidence of significant ALT elevation. HBV-specific immune responses were detected in patients considered immune tolerant and were no different from those found in age-matched peers categorised as immune active [1, 2]. New nomenclature is now used to describe this disease phase, acknowledging that ‘immune tolerance’ may be misleading; the latest EASL guidelines for the management of CHB now refer to this phase as HBeAg-positive chronic infection (Fig. 1) [3]. There is emerging recognition that patients in this disease phase may have a greater risk of disease progression than previously thought, so the critical question now is around the timing of treatment in these patients [3]. SESSION 1 – HBV

Fig. 1. Disease phases of chronic hepatitis B infection reflecting the updated nomenclature. Representation of the changes/fluctuations in serum HBV DNA (solid blue line) and ALT (solid red line) over the typical course of chronic infection in each disease phase. Proposed levels of HBsAg are shown (dashed green line) as predicted throughout each disease phase.

In keeping with this, there is evidence to suggest that high viral load, regardless of inflammation correlates with the development of HCC [4], underlining the importance of HBV DNA level in treatment decisions. Blocking viral replication and reducing the potential for oncogenic progression could be a strategy to employ in the earlier stages of CHB, especially as symptoms of advanced disease often appear late and sometimes at a stage where little can be done to alter the disease course. When patients transition from HBeAg chronic infection to HBeAg chronic hepatitis, it is accepted that there is more immune mediated liver damage, and should this be prolonged, there is increased risk of disease progression and the development of HCC. The early phase of infection may be mistaken as being mechanistically ‘tolerogenic’, but even small fluctuations in ALT and HBV DNA levels may be warning signs of disease progression (Fig. 2). Our recent data challenges the concept of immune tolerant disease [1, 2], and thus adds weight to the argument for early treatment in young patients with CHB.

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Fig. 2. An alternative representation of the natural progression of CHB through disease phases. T cell priming may occur during the early phase of infection, with a distinction between presence of HBV-specific antiviral T cells (dashed green line) and control of viral replication (solid green line), which may be more exaggerated in this phase in younger patients. Potential mechanisms of tolerance include functional exhaustion, innate immune activation, and presence of chemokines (solid pink block). Immune activation associated with fluctuations of ALT and HBV DNA may be associated with further T cell activation. Low levels of viral replication and ALT levels, defined as HBeAg-negative chronic infection may be associated with the T cell control, but with the potential for greater T cell exhaustion with advancing age (solid blue block). Adapted from [5] and [6].

The recent EASL guidelines recommend that treatment should be considered in highly viraemic patients with normal ALT levels above the age of 30. The optimal timing for starting antiviral therapy to reduce complications and the development of HCC remains a subject of debate, but we have demonstrated that events associated with hepatocarcinogenesis may already be present well before this. As noted there is evidence of HBV-specific T cell responses in the early phase of infection. These may, however, not cause recruitment of inflammatory cells until there is evidence of liver inflammation. Even if liver inflammation is considered mild, there may be ensuing liver damage and thus the potential risk for the development of HCC in untreated patients (Fig. 3). It is likely that even with high serum HBV DNA, patients with lower ALT levels, and/or of younger age may respond well to therapy. As younger patients are usually excluded from treatment consideration, there is a paucity of data on treatment outcome in this cohort. However, even with the few studies performed to date, data show that such patients may respond more favourably to treatment. A study by Carey et al., showed that immune tolerant children treated with combination nucleos(t)ide analogue (NA) and pegylated interferon alpha

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(PegIFNα) demonstrated evidence of hepatitis B surface antigen (HBsAg) loss associated with increased HBV-specific T cell proliferation during therapy [7]. In addition, a recent study demonstrated an increased risk of HCC development in untreated HBeAg-positive patients, adding further weight to the argument for earlier therapeutic intervention [8]. Therefore, high viral load alone may be an indication for treatment, with the potential to reduce the risk for HCC development over the longer term. SESSION 1 – HBV

Fig. 3. Representations of conventional and immunological phases of disease. (A) Conventional portrayal of HBeAg-positive chronic infection and chronic hepatitis showing HBV-specific T cell only present in the latter. (B) Immunological display of disease phase indicating the presence of HBV-specific T cells in the early phase of infection but potentially without the recruitment of inflammatory cells, which are thought to only occur at a later stage (with fluctuations of ALT). Increased inflammatory responses, with small ALT fluctuations lead to liver damage and the potential for HCC development over time. Solid red line represents ALT, solid blue line HBV DNA and the dashed purple line represents the potential risk of developing HCC, which is notable even prior to the age of 30. Adapted from [9].

Other than age, factors such as ethnicity, infecting HBV genotype and fibrosis stage should also be considered in these patients when considering treatment. Patients of Afro-Caribbean descent are at risk of developing HCC at an earlier age, and thus these patients may benefit from early therapeutic intervention. Importantly, patients with a family history of HCC should also be considered for early treatment regardless of the evidence of inflammation. HBV genotype is also important, for example genotype C is associated with delayed eAg seroconversion and a higher risk of cirrhosis and HCC than other genotypes. For this reason, these patients may also benefit from earlier treatment [10]. Patients with established fibrosis at an early age should also be treated early to prevent disease progression.

In the case presented here of a young female patient, there was evidence of biochemical activity and increasing median liver stiffness on transient elastography (along with high viral load), which form the basis of the decision to treat. In line with the Prati criteria, the upper limit of normal (ULN) of ALT for a young female is 19 U/L (and 30 U/L for a male) [11], the serum ALT in this case (42 U/L) is more than twice the ULN and likely reflects significant immune activity against the virus, underlining the importance of using these revised “normal” serum ALT levels in treatment decisions.

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WHAT IS THE PREFERRED INITIAL TREATMENT FOR HBeAg-POSITIVE PATIENT WITH HIGH VIRAEMIA, MINIMAL INFLAMMATION AND MINIMAL FIBROSIS? WHAT IS THE RATIONALE FOR THE CHOICE OF TREATMENT? WHAT IS THE LIKELIHOOD OF RESPONSE? The currently available options for the treatment of CHB infection include NAs or PegIFNα, but these therapies have lower efficacy in patients considered immune tolerant. Previously, patients with HBeAg-positive CHB were treated on the development of significant hepatitis interpreted as “immune activity”. In this regard, PegIFNα was offered as a favourable SESSION 1 – HBV treatment option with its ability to induce long-term immunological control with a finite treatment duration. As in the case presented here, PegIFNα was offered as a first-line therapy for these reasons. As noted, data are limited on the treatment of patients with HBeAg-positive chronic infection and thus the ideal treatment strategy remains unknown. Factors associated with improved response to PegIFNα include an elevated ALT and lower HBV DNA, and although there was ALT elevation in the case presented, HBV DNA levels remained high, reducing response rates for PegIFNα. Nonetheless, PegIFNα was offered as first-line therapy and the application of early stopping rules, meant treatment could be individualised for this patient (see below).

The recommended first-line NAs; entecavir (ETV) and tenofovir disoproxil fumarate (TDF) and the more recently licensed tenofovir alfenamide (TAF) are well-tolerated, have a high genetic barrier to resistance, and are highly effective in delivering viral suppression. The rationale for using a NA as a first-line therapy is to lower the viral load, which in turn reduces the risk of complications of CHB and potentially the development of HCC. These drugs have been shown to halt the progression of liver disease and can also result in a significant improvement of histological necroinflammation and fibrosis [12]. Moreover, NAs are safe with little or no reported resistance and are widely used in clinical practice. Notably, newer pipeline therapies are likely to require viral suppression in the first instance prior to their employment, therefore, initial treatment with NAs may be advantageous from this perspective. The significance of this cannot be underestimated given the treatment landscape for CHB is on the cusp of a major change with a multitude of novel antiviral therapies in the pipeline [13]. Thus, it is unlikely that treatment with NAs in young patients will be lifelong as we move closer towards achieving functional cure.

Response to NA therapy is defined as an undetectable HBV DNA by PCR. The likelihood of response is good in compliant patients even with a high viral load; approximately 70-80% will

become aviraemic after 12 months of therapy. Primary non-response is defined as <1 log10 decrease in HBV DNA after 3 months. Although partial virological response is defined as a

decrease of HBV DNA >1 log10 IU/ml, but detectable virus at 12 months, patients with very high viral loads, such as those in HBeAg-positive CHB may require more than 12 months to become aviraemic. Although these patients often have a low ALT, further decreases (in accordance with Prati criteria) may be seen, leading to favourable outcomes. Markers used to determine response include HBV DNA, HBeAg (to check for evidence of seroconversion) and quantitative HBsAg levels can also be measured, although declines in HBsAg tend to be slow.

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HOW SHOULD RESPONSE TO INTERFERON THERAPY BE MONITORED? WHEN SHOULD TREATMENT BE STOPPED BASED ON FUTILITY? PegIFNα therapy offers a finite treatment course and was one of the reasons for considering it as a first-line therapy in the case presented. It is more effective in those of younger age, with moderate viral load and has the advantage of the absence of antiviral resistance. However, its overall success is limited to a small proportion of patients; approximately 10% of those treated achieve functional cure, defined as sustained serum aviraemia and loss of HBsAg. Response to PegIFNα is also more durable and HBeAg (and HBsAg) loss may occur following treatment cessation. Approximately 30% of HBeAg-positive patients have a favourable response to SESSION 1 – HBV PegIFNα with sustained HBeAg seroconversion, and a proportion of these patients go on to achieve HBsAg loss [14]. In HBeAg-positive CHB patients, pretreatment predictors of response include; age, sex, serum ALT ratio, HBV DNA, and HBsAg levels in addition to high necroinflammatory score on liver biopsy. HBV genotypes A and B have been shown to be associated with higher rates of HBeAg seroconversion and HBsAg loss than genotypes C and D. Despite our female patient being relatively young and having evidence of necroinflammation on liver biopsy, the high viral load and less favourable genotype (D), reduces her chances of a favourable treatment response.

The response to interferon therapy should be monitored using the decline of HBV DNA and quantitative HBsAg as indicators. The use of ‘early stopping rules’ based primarily on HBsAg decline at week 12 or 24 of therapy can guide physicians in determining whether treatment can be stopped early owing to suboptimal response; thus avoiding the potential undesirable systemic effects of a full treatment course (Fig. 4). This strategy would allow an early switch to NA therapy, providing an individualised approach to CHB management [14]. In HBeAg- positive disease, a decline of HBsAg levels below 1,500 IU/ml at 12 weeks is a good predictor of HBeAg seroconversion. Conversely, HBsAg levels >20,000 IU/ml for genotype B and C and no decline for genotypes A and D at 12 weeks are associated with a poor response. At 24 weeks, HBsAg levels of >20,000 IU/ml (as in the case presented here), regardless of genotype are associated with a low probability of HBeAg seroconversion, and thus, PegIFNα should be stopped based on futility [14]. Although a decrease in HBV DNA, reduction in HBeAg levels and ALT flares on-treatment are associated with good responses to PegIFNα, these have not been validated in large studies. Furthermore, it remains to be seen whether novel biomarkers such as hepatitis B core-related antigen (HBcrAg) and HBV RNA will provide improved predictors of treatment outcome to enhance response-guided therapy. As in this case, although there was a poor response to PegIFNα, the early switch to NA therapy following PegIFNα may provide greater reductions in HBsAg (and HBsAg loss) due to ongoing immune modulation after PegIFNα cessation [15].

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Fig. 4. Stopping rules for PegIFNα in HBeAg-positive CHB patients using HBsAg levels. (*indicates that in genotype B and C patients if HBsAg level is >20,000 IU/ml then PegIFNα should be stopped).

HOW SHOULD RESPONSE TO ORAL ANTIVIRAL THERAPY BE MONITORED? HOW WOULD YOU MANAGE PATIENTS WHO REMAIN VIRAEMIC AFTER 1 YEAR OF THERAPY? HOW WOULD YOU MANAGE PATIENTS WHO EXPERIENCE VIROLOGIC BREAKTHROUGH? The response to oral antiviral therapy with NAs is measured using levels of HBV DNA and biochemical activity with ALT levels. As noted, virological response is defined as an undetectable HBV DNA by PCR. Patients with elevated serum ALT levels should demonstrate biochemical normalisation within 12 months of therapy initiation, which is associated with better outcomes and the potential to reverse fibrosis if present [12]. On the initiation of NAs, renal and bone profiling should be undertaken to monitor any potential adverse events associated with treatment [16], although these adverse effects are reported to be less common with the newly licensed TAF [17]. The efficacy of NAs in HBeAg-positive disease has been demonstrated in randomised controlled trials showing that 5 years of NA therapy leads to a ~99% cumulative probability of virological response and approximately 50% probability of HBeAg loss. HBsAg loss occurs in up to 10% of patients at 5 years, but this is based on data in patients with an elevated ALT and lower HBV DNA at the initiation of therapy [3].

In patients with a very high viral load, as in the case presented here, some may remain viraemic after 12 months of therapy and more time may be required to achieve undetectable HBV DNA. A consistent, albeit slower, decline in HBV DNA should be observed and if this is not the case, there is cause for concern. The prevention of resistance should rely on the exclusive utility of NAs with a high genetic barrier to resistance (ETV, TDF, TAF). Compliance with therapy should be checked in all cases before concluding that suboptimal response is related to the emergence of resistance or treatment failure. Poor compliance will be the main cause of primary non-response in the majority of cases. Resistance testing and genotyping of HBV strains should be undertaken to exclude resistance mutations. It is noteworthy that treatment with ETV, TDF and TAF, partial virological response is associated with a very high

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PARIS, FRANCE pretreatment viral load rather than the lack of efficacy of these agents. In HBeAg-positive patients who remain viraemic at 1 year of therapy, the rate of decline of HBV DNA over 48 weeks should be assessed, those showing evidence of a continuous decline in viral load should remain on the same agent with an appropriate level of supervision and monitoring. Conversely, there is no place for the utility of older NAs such as lamivudine, adefovir or telbivudine owing to their lower genetic barrier to resistance and patients on these agents should be switched to more potent agents following resistance testing.

Virological breakthrough in compliant patients on potent NAs is rare. A full history should be taken to ensure complete adherence. Should adherence be confirmed, then resistance genotyping should be undertaken and treatment altered accordingly. In some cases, a change of NA may be mandated. Treatment should be modified as soon as resistance is proven to prevent further increases in viral load and the potential for flares in serum ALT and disease progression [3]. SESSION 1 – HBV

IS HBeAg TO ANTI-HBe SEROCONVERSION AN INDICATION TO STOP ORAL ANTIVIRAL THERAPY? HOW SHOULD PATIENTS BE MONITORED AFTER TREATMENT DISCONTINUATION? HBeAg to anti-HBe seroconversion is an indication to stop oral antiviral therapy provided there is a period of consolidation therapy. However, a number of factors should be considered prior to stopping therapy; these include patient age, the degree of fibrosis (treatment should not be stopped in patients with cirrhosis), and a family history of HCC. The ideal treatment endpoint in HBeAg-positive patients is also HBsAg loss and functional cure at an earlier stage of disease might have a greater impact in preventing HCC development.

In HBeAg-positive CHB patients treated with NAs who achieve on-treatment HBeAg seroconversion, associated with undetectable HBV DNA, NA therapy can be discontinued following a period of consolidation therapy. The optimum period of consolidation therapy is debatable, but it is accepted that this should be a minimum of 6 months (but preferably 12 months or more). It is expected that in the majority of these patients there will be durable HBeAg seroconversion along with virological remission (HBV DNA between 2,000-20,000 IU/ml) 3 years following NA cessation. These patients, however, still require close monitoring of HBV DNA and serum ALT following treatment discontinuation. Should there be persistent elevation in HBV DNA (>20,000 IU/ml) and/or evidence of ongoing biochemical activity, these patients would be treated as HBeAg-negative chronic hepatitis and NA therapy should be re-introduced. Biomarkers to better define disease progression and HCC risk in those patients with sustained virological and biochemical responses following consolidation therapy are needed to improve patient care and should be a focus of future research. References

References in bold are required reading

[1] Kennedy PTF, Sandalova E, Jo J, Gill U, Ushiro-Lumb I, Tan AT, et al. Preserved T-cell function in children and young adults with immune-tolerant chronic hepatitis B. Gastroenterology. 2012,143(3):637-645.

[2] Mason WS, Gill US, Litwin S, Zhou Y, Peri S, Pop O, et al. HBV DNA Integration and Clonal Hepatocyte Expansion in Chronic Hepatitis B Patients Considered Immune Tolerant. Gastroenterology. 2016,151(5):986-998 e984.

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[3] European Association for the Study of the Liver. European Association for the Study of the L: EASL 2017 Clinical Practice Guidelines on the management of hepatitis B virus infection. J Hepatol. 2017,67(2):370-398.

[4] Chen CJ, Yang HI, Su J, Jen CL, You SL, Lu SN, et al. Risk of hepatocellular carcinoma across a biological gradient of serum hepatitis B virus DNA level. JAMA 2006,295(1):65- 73.

[5] Gill US, Kennedy PT. Chronic hepatitis B virus in young adults: the need for new approaches to management. Expert Rev Anti Infect Ther. 2014,12(9):1045-1053.

[6] Velazquez VM, Grakoui A. Immune quiescence and hepatitis B virus: tolerance has its limits. Gastroenterology. 2012,143(3):529-532. SESSION 1 – HBV [7] Carey I, D’Antiga L, Bansal S, Longhi MS, Ma Y, Mesa IR, et al. Immune and viral profile from tolerance to hepatitis B surface antigen clearance: a longitudinal study of vertically hepatitis B virus-infected children on combined therapy. J Virol. 2011,85(5):2416-2428.

[8] Kim GA, Lim YS, Han S, Choi J, Shim JH, Kim KM, et al. High risk of hepatocellular carcinoma and death in patients with immune-tolerant-phase chronic hepatitis B. Gut. 2017. [Epub ahead of print].

[9] Bertoletti A, Kennedy PT. The immune tolerant phase of chronic HBV infection: new perspectives on an old concept. Cell Mol Immunol. 2015,12(3):258-263.

[10] Tong S, Revill P. Overview of hepatitis B viral replication and genetic variability. J Hepatol. 2016,64(1 Suppl):S4-S16.

[11] Prati D, Taioli E, Zanella A, Della Torre E, Butelli S, Del Vecchio E, et al. Updated definitions of healthy ranges for serum alanine aminotransferase levels. Ann Intern Med. 2002,137(1):1-10.

[12] Marcellin P, Gane E, Buti M, Afdhal N, Sievert W, Jacobson IM, Washington MK, et al. Regression of cirrhosis during treatment with tenofovir disoproxil fumarate for chronic hepatitis B: a 5-year open-label follow-up study. Lancet. 2013,381(9865):468-475.

[13] Gill US, Kennedy PTF. Current therapeutic approaches for HBV infected patients. J Hepatol. 2017,67(2):412-414.

[14] Sonneveld MJ, Hansen BE, Piratvisuth T, Jia JD, Zeuzem S, Gane E, et al. Response- guided peginterferon therapy in hepatitis B e antigen-positive chronic hepatitis B using serum hepatitis B surface antigen levels. Hepatology. 2013,58(3):872-880.

[15] Gill US, Peppa D, Micco L, Singh HD, Carey I, Foster GR, et al. Interferon Alpha Induces Sustained Changes in NK Cell Responsiveness to Hepatitis B Viral Load Suppression In Vivo. PLoS Pathog. 2016,12(8):e1005788.

[16] Gill US, Zissimopoulos A, Al-Shamma S, Burke K, McPhail MJ, Barr DA, et al. Assessment of bone mineral density in tenofovir-treated patients with chronic hepatitis B: can the fracture risk assessment tool identify those at greatest risk? J Infect Dis. 2015,211(3):374-382.

[17] Agarwal K, Fung SK, Nguyen TT, Cheng W, Sicard E, Ryder SD, et al. Twenty-eight day safety, antiviral activity, and pharmacokinetics of tenofovir alafenamide for treatment of chronic hepatitis B infection. J Hepatol. 2015,62(3):533-540.

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HOW TO MANAGE PREGNANT WOMEN TO MINIMISE THE RISK OF HBV TRANSMISSION

Henry Lik Yuen Chan Department of Medicine and Therapeutics and Institute of Digestive Disease, The Chinese University of Hong Kong, Hong Kong, China. E-mail address: [email protected]

TAKE-HOME MESSAGES SESSION 1 – HBV • Risk of maternal-to-infant hepatitis B virus (HBV) transmission persists despite vaccination to newborns when maternal viral load exceeds 6 log IU/ml.

• Use of tenofovir disoproxil fumarate (TDF) during the third trimester of gestation for mothers with high viral load can greatly reduce the risk of HBV transmission to infants.

• Close monitoring of HBV DNA and alanine aminotransferase for 6 months postpartum is recommended after cessation of TDF.

• TDF is generally safe for both mothers and infants, and breastfeeding is not contraindicated for mothers on TDF.

UNIVERSAL HBV VACCINATION FOR NEWBORNS The World Health Organization recommends a standard 3-dose universal hepatitis B virus (HBV) vaccination with the first dose given within 24 hours after birth. For HBV-infected mothers, co-administration of hepatitis B immunoglobulin with the birth dose vaccine can offer additional protection to newborns against perinatal transmission. Universal vaccination was first introduced in Taiwan in 1984. At the end of 2016, the HBV vaccine had been introduced into 186 countries listed in the World Health Organization, and global coverage of the 3-dose HBV vaccine was estimated to be at 84%. The United Kingdom is one of the last European countries to introduce universal vaccination for newborns; all babies born after August 2017 are eligible for HBV vaccine. Five European countries (Denmark, Finland, Iceland, Norway and Sweden) have yet to introduce the universal HBV vaccination policy due to the low local prevalence of HBV infection.

The introduction of universal vaccination has led to a dramatic reduction in the global prevalence of HBV infection. In Taiwan, where maternal HBV infection is prevalent, among adolescents aged 15 years who received the vaccination at birth, 48.3% were found to have protective anti-HBs levels (≥10 IU/ml), and 72% of the remaining subjects had anamnestic response [1]. Approximately 0.5% of Taiwanese children aged <15 years still have positive hepatitis B surface antigen (HBsAg), and a few subjects with occult HBV infection (negative HBsAg, positive anti-HBc and detectable HBV DNA) were also identified 25 years after the launch of the universal vaccination program [2]. Various reports from China and Taiwan have shown positive maternal hepatitis B e antigen (HBeAg) status, and high maternal viral load are the key factors associated with risk of HBV transmission despite vaccination to newborns [3]. In general, the risk of HBV transmission to a newborn starts to increase when maternal HBV DNA is higher than 6 log IU/ml, and the risk increases exponentially with each logarithmic

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increase in maternal viral load. As serum HBsAg has a good correlation with HBV DNA level in HBeAg-positive mothers, serum HBsAg >4 log IU/ml is also found to associate with increased risk of maternal-to-infant HBV transmission.

ANTIVIRAL THERAPY IN HBV CARRIER MOTHERS As recommended by the Food and Drug Administration in the United States, telbivudine and tenofovir disoproxil fumarate (TDF) are category B anti-HBV drugs suitable for pregnant women. Based on data from antiretroviral registries, lamivudine appears to have a similar safety profile as TDF during pregnancy [4]. To further reduce the risk of HBV transmission to

SESSION 1 – HBV newborns by HBV carrier mothers with a high viral load, many centres have studied the use of antiviral agents in the third trimester at week 28 to 32 of gestation, in addition to vaccination of the newborn [5]. In general, antiviral treatment reduced the relative risk of HBV transmission by 70%. A definitive randomised control trial was conducted in China comparing TDF starting at 30-32 weeks of gestation vs. no antiviral treatment for mothers with HBV DNA >200,000 IU/ml [6]. All infants received a combination of HBV vaccine and hepatitis B immunoglobulin at birth. On per-protocol analysis, 7% of babies born from 88 deliveries without TDF vs. 0% babies from 97 deliveries with TDF treatment had HBV infection (p = 0.01). However, isolated cases of HBV infection have been reported among infants born from HBV-infected mothers who received TDF during the third trimester, and the maternal viral load at delivery was about 4-5 logs IU/ml [7,8].

As the use of lamivudine or telbivudine may induce drug resistance particularly among high viral load carrier mothers, TDF should be the preferred antiviral drug to use during pregnancy. EASL guidelines recommend TDF treatment for all pregnant women with high HBV DNA load (>200,000 IU/ml) or HBsAg level >4 log IU/ml at week 24-28 of gestation [9]. There is insufficient data to recommend the commencement of TDF at an earlier phase of pregnancy, although some experts suggest that TDF could be started in the second trimester for mothers with HBV DNA >9 log IU/ml. Nonetheless, the safety data from antiretroviral registries of the use of TDF during pregnancy, suggest that pregnant women should be offered TDF treatment, and those women on other antiviral drugs should switch to TDF during the pregnancy [9].

STOPPING ANTIVIRAL THERAPY AFTER DELIVERY Pregnancy is a special stage of life when cell-mediated immunity is suppressed to tolerate paternally derived foetal antigens, while the immunology changes are reversed back to normal postpartum. As a result, elevation of alanine aminotransferase (ALT) may develop in the postpartum period in HBV carrier mothers. Approximately 10% to 25% of HBV-infected women develop increased levels of ALT after delivery; most ALT flares are <5 times the upper limit of normal and self-limiting, and they rarely lead to hepatic decompensation [10-12]. Mothers with positive HBeAg and higher HBV DNA seem to have a higher risk of postpartum ALT f lares.

For mothers who are in the phase of HBeAg-positive chronic infection (or immune tolerant phase), which is characterised by a very high viral load but absent of significant hepatic necroinflammation or fibrosis, long-term antiviral treatment may not be desirable or indicated [9]. EASL guidelines recommend that stopping TDF can be considered within 12 weeks after delivery [9]. As antiviral drugs can only suppress HBV DNA replication but cannot eliminate HBV in the liver, viral relapse after stopping antiviral drug after pregnancy should be expected.

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Based on the randomised controlled trial conducted in China, the risk of any ALT elevation postpartum was slightly higher among HBV carrier mothers who stopped TDF (46%) vs. those who were untreated (30%) [6]. Only 1% of mothers developed ALT level >10 times the upper limit of normal after stopping TDF, and ALT was normalised after re-initiation of antiviral therapy. However, among mothers who have indication for antiviral treatment, for example, those who have evidence of significant liver fibrosis, antiviral treatment should not be stopped after delivery, and long-term antiviral treatment should be offered.

Regardless of the use of antiviral therapy, ALT elevation may occur postpartum in HBV carrier mothers. Most ALT elevation develops within the first 6 months postpartum in both untreated mothers and those who have stopped antiviral treatment. A large-scale follow-up study in China revealed two bursts of hepatic flare at 1 month (19.8%) and 3 months (17.1%) after delivery, and the time pattern was similar among mothers with and without antiviral therapy SESSION 1 – HBV [13]. Hence, close monitoring for HBV DNA and ALT at 4 weekly intervals is recommended in the initial 6 months postpartum. Mild ALT elevation can be observed without antiviral therapy as it is usually self-limiting. Re-initiation of antiviral therapy should be considered if ALT is elevated >10 times the upper limit of normal.

SAFETY OF ANTIVIRAL DRUGS The use of antiviral therapy (lamivudine, telbivudine and TDF) is safe for both the mother and the foetus [5]. Based on reports from observational studies, there was no increase in congenital malformation and prematurity of infants or increase in postpartum haemorrhage, caesarean section rate or elevated creatine kinase rate for mothers who received antiviral therapy. According to the label of TDF, breastfeeding is not recommended. However, available data from animal and human studies suggests that the concentration of TDF in breast milk is low and should not be harmful to the infant [14]. It is essential to balance the risk and benefit of TDF treatment for HBV-infected mothers and infants, and the EASL guidelines propose there is no contraindication to breastfeeding in HBsAg-positive women treated with TDF [9]. References

References in bold are required reading

[1] Wu TW, Lin HH, Wang LY. Chronic hepatitis B infection in adolescents who received primary infantile vaccination. Hepatology 2013;57:38-45.

[2] Ni YH, Chang MH, Wu JF, Hsu HY, Chen HL, Chan DS. Minimization of hepatitis B infection by 25-year universal vaccination program. J Hepatol 2012;57:730-5.

[3] Wen WH, Huang CW, Chie WC, Yeung CY, Zhao LL, Lin WT, et al. Quantitative maternal hepatitis B surface antigen predicts maternally transmitted hepatitis B virus infection. Hepatology 2016;64:1451-61.

[4] Bzowej NH. Curr Hepatitis Rep 2010;9:197-204.

[5] Brown Jr RS, McMahon BJ, Lok AS, Wong JB, Ahmed AT, Mouchli MA, et al. Antiviral therapy in chronic hepatitis B viral infection during pregnancy: a systemic review and meta-analysis. Hepatology 2016;63:319-33.

[6] Pan CQ, Duan Z, Dai E, Zhan S, Han G, Wang Y, et al. Tenofovir to prevent hepatitis B transmission in mothers with high viral load. N Engl J Med 2016;374:2324-34.

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[7] Chen HL, Lee CN, Chang CH, Ni YH, Shyu MK, Chen SM. Efficacy of maternal tenofovir disoproxil fumarate in interrupting mother-to-infant transmission of hepatitis B virus. Hepatology 2015;62:375-86.

[8] Greenup AJ, Tan PK, Nguyen V, Glass A, Davison S, Chatterjee U, et al. Efficacy and safety of tenofovir disoproxil fumarate in pregnancy to prevent perinatal transmission of hepatitis B virus. J Hepatol 2014;61:502-7.

[9] European Association for the Study of the Liver. EASL 2017 clinical practice guideline on the management of hepatitis B virus infection. J Hepatol 2017;67:370-98.

[10] Giles M, Visvanathan K, Lewin S, Bowden S, Locarnini S, Spelman T, et al. Clinical and SESSION 1 – HBV virological predictors of hepatic flares in pregnant women with chronic hepatitis B. Gut 2015;64:1810-5.

[11] Chang CY, Aziz N, Poongkunran M, Javaid A, Trinh HN, Lau D, et al. Serum alanine aminotransferase and hepatitis B DNA flares in pregnant and postpartum women with chronic hepatitis B. Am J Gastroenterol 2016;111:1410-5.

[12] Kushner T, Shaw PA, Kalra A, Magaldi L, Monpara P, Bedi G, et al. Incidence, determinants and outcomes of pregnancy-associated hepatitis B flares: a regional hospital-based cohort study. Liver Int 2017;00:1-8.

[13] Liu J, Wang J, Qi C, Cao F, Tian Z, Guo D, et al. Baseline hepatitis B virus titer predicts initial postpartum hepatic flare. J Clin Gastroenterol 2017 Jun 26. doi: 10.1097/ MCG.0000000000000877. [Epub ahead of print].

[14] Ehrhardt S, Xie C, Guo N, Nelson K, Thio CL. Breastfeeding while taking lamivudine or tenofovir disoproxil fumarate: a review of the evidence. Clin Infect Dis 2015;60:275-8.

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WEDNESDAY 11 APRIL 2018 12:30-13:30 SESSION 2 – HCV

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EASL POSTGRADUATE COURSE

DIFFICULT-TO-CURE PATIENTS IN 2018

Christophe Hézode Department of Hepatology, Henri Mondor Hospital, Paris-Est University, INSERM U955, Créteil, Paris, France. E-mail address: [email protected]

TAKE-HOME MESSAGES • Direct-acting antiviral agent (DAA)-naïve, treatment-experienced patients infected with genotype 1a are difficult-to-cure patients with sofosbuvir and first-generation NS5A inhibitor. They should be treated for 12 weeks with ribavirin or 24 weeks without ribavirin, regardless of the fibrosis stage. Options including next-generation NS5A are optimal and

SESSION 2 – HCV – SESSION 2 preferable if available in these patients.

• DAA-naïve, genotype 1a with a hepatitis C virus (HCV) RNA level >800,000 IU/ml at baseline are difficult-to-cure patients with grazoprevir and elbasvir. They should be treated for 16 weeks with ribavirin regardless of the fibrosis stage. Options including next-generation NS5A are optimal and preferable if available in these patients.

• DAA-naïve patients with decompensated cirrhosis infected with genotype 1, 4, 5, or 6 are difficult-to-cure patients with sofosbuvir and first-generation NS5A inhibitor. They should be treated with for 12 weeks with ribavirin. Options including next-generation NS5A are optimal and preferable if available in these patients.

• DAA-naïve, genotype 3 patients with compensated cirrhosis are difficult-to-cure patients with sofosbuvir and first-generation NS5A inhibitor (daclatasvir). They should be treated for 24 weeks with ribavirin. Options including next-generation NS5A are optimal and preferable if available in these patients.

• DAA-naïve, genotype 3 patients with decompensated Child-Pugh B and C cirrhosis, are difficult-to-cure patients with sofosbuvir and first-generation NS5A inhibitor (daclatasvir) for 24 weeks. They remain difficult-to-cure patients with sofosbuvir, velpatasvir and ribavirin for 12 weeks.

INTRODUCTION The development of direct-acting antiviral agents (DAAs) for the treatment of hepatitis C virus (HCV) chronic infection has resulted in high rates of sustained virologic response (SVR) in most DAA-naïve patient populations. There remain some DAA-naïve difficult-to-cure patients defined by an SVR rate around or lower than 90% reported in this population under a well-conducted DAA-based regimen. However, the characteristics of these patients could be different across countries and depend on the availability of DAAs in different regions of the world. Efficacy and predictors of SVR are different between combinations including or excluding sofosbuvir and those with first or next-generation DAAs.

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DAA-NAÏVE, TREATMENT-EXPERIENCED PATIENTS INFECTED WITH GENOTYPE 1A The most currently available DAA treatment regimens for genotype 1 chronic HCV infection include first-generation NS5A inhibitors (e.g., daclatasvir, ledipasvir) in combination with sofosbuvir and are highly effective in DAA-naïve patients. Despite having a limited effect on plasma HCV RNA levels when given as a monotherapy, ribavirin was shown to accelerate the second-slope of viral decline when used in combination with interferon alpha. Ribavirin continues to contribute to the efficacy of DAA regimens by increasing overall SVR rates and allowing for shorter treatment durations, particularly in the most difficult-to-cure patients (e.g., those with cirrhosis or prior treatment experience) [1, 2]. An integrated analysis of 513 genotype 1 patients with compensated cirrhosis treated with sofosbuvir and ledipasvir, with or without ribavirin for 12 or 24 weeks has shown that optimal SVR rates, above 95%, were observed in treatment-experienced patients treated for 12 weeks with ribavirin and those treated for 24 weeks regardless of ribavirin intake. By contrast, SVR was achieved in 90% of those treated for 12 weeks without ribavirin (Fig. 1) [1]. Other large pooled analysis from phase SESSION 2 – HCV – SESSION 2 II and III clinical trials have demonstrated that the presence of a high-level NS5A resistance- associated substitution (RAS) at baseline had a negative impact on SVR rates (85% to 90%) in treatment-experienced patients with or without compensated cirrhosis infected with HCV genotype 1a treated with sofosbuvir and ledipasvir for 12 weeks. The addition of ribavirin and/ or the extension of the treatment duration to 24 weeks were associated with higher SVR rates above 95% [3]. In practice, to optimise efficacy and prevent failure, it is recommended that all treatment-experienced, DAA-naïve patients, infected with genotype 1a, with compensated disease should be treated with sofosbuvir and first-generation NS5A inhibitor for 12 weeks with ribavirin or 24 weeks without ribavirin. If baseline resistance testing is available in these patients, the addition of ribavirin or the extension of therapy is recommended in those with only NS5A RAS detected.

Fig. 1. Efficacy of sofosbuvir and ledipasvir-based regimens in patients with cirrhosis [1]. SVR, sustained virological response; LDV, Ledipasvir; SOF, sofosbuvir; RBV, ribavirin; wk, week.

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When compared with first-generation NS5A inhibitors, velpatasvir demonstrates not only a broad genotypic coverage of HCV isolates but also improved potency against many known NS5A RASs that confer reduced susceptibility to first-generation NS5A inhibitors. Thus, there is a need to understand the potential impact of NS5A RASs on treatment outcome in patients treated with regimens containing next-generation NS5A inhibitors. A pooled analysis, including 1,778 DAA-naïve patients with compensated disease from clinical trials who completed treatment with sofosbuvir and velpatasvir for 12 weeks showed that 99% of genotype 1 patients achieved SVR [4]. Subtype had no impact on the efficacy reported with this regimen. Using a 15% sequencing assay cut-off, the presence of NS5A RASs at baseline did not impact SVR rate in patients infected with genotype 1a or 1b (Fig. 2). Overall, the SVR rate was 97% to 100% independent of the presence of NS5A RASs at baseline in DAA- naïve patients with compensated disease, and genotype 1, 2, 4, 5 or 6 infection treated with sofosbuvir and velpatasvir for 12 weeks [4]. Thus, genotypes 1a difficult-to-cure patients with sofosbuvir in combination with first-generation NS5A inhibitor become easy-to-treat patients with sofosbuvir and velpatasvir. SESSION 2 – HCV – SESSION 2

Fig. 2. Efficacy of sofosbuvir and velpatasvir for 12 weeks in genotype 1 and 3 patients according to the presence of NS5A RAS [4]. SVR, sustained virological response; RAS, resistance-associated substitution.

A recent post hoc analysis of 11 pooled phase II and III clinical trials, including 957 DAA-naïve patients infected with genotype 1a and treated with grazoprevir and elbasvir for 12 weeks, demonstrated that the SVR rate was not optimal, below 95%, in patients with an HCV RNA level >800,000 IU/ml at baseline. This result was observed in treatment-naïve and treatment- experienced patients and those with or without compensated cirrhosis. This suggests that all DAA-naïve genotype 1a patients with an HCV RNA level >800,000 IU/ml are not easy-to- treat patients with this treatment option and should benefit from the extension of therapy to 16 weeks and the addition of ribavirin [5].

The pan-genotypic and next-generation strategy, glecaprevir and pibrentasvir for 12 weeks, yielded an SVR rate of 99% in 90 patients with genotype 1 infection and compensated cirrhosis, suggesting that all genotype 1a patients are no more difficult-to-treat with this option (Fig. 3) [6].

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Fig. 3. Efficacy of glecaprevir and pibrentasvir for 12 or 16 weeks in patients with compensated cirrhosis [6, 9]. TN, treatment-naïve; TE, treatment-experienced; GT, genotype.

DAA-NAÏVE PATIENTS INFECTED WITH GENOTYPE 3 HCV genotype 3 is the second most prevalent genotype worldwide and associated with several clinical features, such as accelerated progression of fibrosis and a greater risk of steatosis and hepatocellular carcinoma. Thus, there is an urgent need for safe and effective treatment of genotype 3 infection. Sofosbuvir and first-generation NS5A inhibitor (daclatasvir) for 12 weeks is a recommended option for non-cirrhotic genotype 3 infected individuals in several clinical guidelines and relates to high efficacy. However, using this option genotype 3 is more difficult-to-treat in patients with cirrhosis. An SVR rate of 86% was observed among genotype 3-infected patients with compensated cirrhosis following 12 or 16 weeks of treatment with sofosbuvir, daclatasvir and ribavirin [7]. A large real-world cohort of patients with genotype 3 infection plus advanced liver disease provides data on the clinical effectiveness of sofosbuvir and daclatasvir with or without ribavirin in a challenging subset of patients with very limited options. For patients who received sofosbuvir and daclatasvir for 24 weeks, an overall SVR rate was 89% (86% with cirrhosis, 98% without) and was similar with and without prior HCV treatment, 90% and 88%, respectively [8]. Among treatment-experienced patients with cirrhosis, SVR was 87% without ribavirin and 80% with ribavirin. Among patients with cirrhosis, SVR was numerically higher in the 24-week groups (86% without ribavirin and 82% with ribavirin), additionally, it was higher in compensated (88%) than in decompensated disease (74%) (Fig. 4) [8]. No additional benefit of ribavirin was observed in compensated or decompensated cirrhosis, although unrandomised treatment allocation and potential selection bias for ribavirin use makes it difficult to assess the significance of this. These data are consistent with other real-world findings in which patients with cirrhosis treated with sofosbuvir, daclatasvir and ribavirin for 24 weeks received no SVR benefit over those treated without ribavirin. In summary, genotype 3 patients with cirrhosis remain difficult-to-treat patients with a sofosbuvir and daclatasvir-based regimen and the addition of ribavirin is recommended.

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EASL POSTGRADUATE COURSE SESSION 2 – HCV – SESSION 2 Fig. 4. Efficacy of sofosbuvir and daclatasvir in genotype 3 patients with cirrhosis in real-life [8]. SVR, sustained virological response; RBV, ribavirin.

Regarding the use of next-generation NS5A inhibitor, velpatasvir, in combination with sofosbuvir, the situation is different for genotype 3 patients. A pooled analysis from clinical trials, including 478 DAA-naïve genotype 3 patients with compensated disease who completed treatment with sofosbuvir and velpatasvir for 12 weeks, showed an SVR rate of 97.5% [4]. All six genotype 3 patients infected with a non-genotype 3a subtype achieved SVR. A lower SVR rate was observed for patients with NS5A RASs at baseline. The SVR rate was 93% and 98% in patients with and without baseline NS5A RASs. However, the negative impact of the presence of NS5A RAS was observed in the 22 patients who harboured only Y93H (86% of SVR) (Fig. 2). The SVR rates in the presence of Y93H in genotype 3 patients with cirrhosis were evaluated but only six genotype 3 patients had cirrhosis and harboured Y93H at baseline. Four of six of these patients achieved SVR after sofosbuvir and velpastavir treatment [4]. In summary, DAA-naïve genotype 3 patients with compensated cirrhosis are not difficult-to-treat patients with sofosbuvir and velpatasvir for 12 weeks.

The other pan-genotypic option, glecaprevir and pibrentasvir, has been evaluated in DAA- naïve genotype 3 patients with compensated cirrhosis. Forty treatment-naïve and 47 treatment- experienced patients were treated for 12 or 16 weeks, respectively [9]. SVR was achieved by 98% and 96% of treatment-naïve and treatment-experienced, respectively (Fig. 3). Thus, glecaprevir and pibrentasvir provide an efficacious treatment option for what was previously one of the most difficult-to-cure HCV patients groups, those with genotype 3 infection and compensated cirrhosis.

DAA-NAÏVE PATIENTS WITH DECOMPENSATED CIRRHOSIS Treatment options are limited for patients chronically infected by HCV with decompensated (Child-Pugh B or C) cirrhosis. These patients have an absolute contraindication to the use of interferon-based regimens, and protease inhibitors should not be administrated in this setting. Thus, treatment of DAA-naïve patients with decompensated cirrhosis should be based on the combination of sofosbuvir and a first or next-generation NS5A inhibitor, namely ledipasvir, daclatasvir or velpatasvir.

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The efficacy and safety of sofosbuvir, ledipasvir and ribavirin have been assessed in DAA- naïve patients infected with genotype 1 or 4 with decompensated cirrhosis in two cohorts. One cohort included patients who had not undergone liver transplantation, and the other cohort enrolled post-transplantation patients. Patients were randomised to receive 12 or 24 weeks of therapy [2]. Among 89 genotype 1 patients without liver transplantation, the SVR rates were 87% and 96% after 12 and 24 weeks of therapy, respectively, in Child-Pugh B patients. The SVR rates were 85% and 78% after 12 and 24 weeks of therapy, respectively, in Child-Pugh C patients. In the post-transplant patients, 95% and 100% of the 40 Child-Pugh B patients achieved an SVR for 12 and 24 weeks of therapy, respectively. For the seven Child-Pugh C patients, SVR was observed in 50% and 80% for 12 and 24 weeks of therapy, respectively. Finally, among all genotype 4 patients SVR12 was achieved by 78% and 94% for 12 and 24 weeks of therapy, respectively [2]. These results indicate that DAA-naïve patients with decompensated cirrhosis before or after liver transplantation, particularly those with Child- Pugh C disease, remain difficult-to-cure patients with sofosbuvir in combination with a first- generation NS5A inhibitor and ribavirin for 12 or 24 weeks.

The efficacy and safety of sofosbuvir with a next-generation NS5A inhibitor, velpatasvir, HCV – SESSION 2 were assessed in DAA-naïve Child-Pugh B patients who were randomised to receive 12 weeks with or without ribavirin or 24 weeks [10]. The highest SVR rates were observed in patients treated for 12 weeks with ribavirin. In genotype 1 patients, 94% and 100% of genotype 1a and 1b patients achieved an SVR, respectively. In the 13 patients with genotype 3 infection treated for 12 weeks and with ribavirin, the SVR rate was high (85%) but not optimal. For the other genotypes, the number of patients was small, but the SVR rate was 100% (Fig. 5). Based on these results, sofosbuvir, and the combination of sofosbuvir, velpatasvir and ribavirin for 12 weeks becomes the standard of care for patients with decompensated Child-Pugh B patients. Unfortunately, especially for genotype 3 patients, no arm of sofosbuvir, velpatasvir and ribavirin for 24 weeks was included in this study. Moreover, no data was available for Child-Pugh C patients. In summary, despite the availability of this option with ribavirin given for 12 weeks, Child-Pugh B patients infected with genotype 3, and Child-Pugh C patients remain difficult-to-cure patients.

Fig. 5. Efficacy of sofosbuvir and velpatasvir-based regimens in patients with decompensated cirrhosis [10]. SOF, sofosbuvir; VEL, velpatasvir; GT, genotype; SVR, sustained virological response; wk, week.

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CONCLUSION Therapeutic options including a first-generation NS5A inhibitor are highly effective. However, some DAA-naïve patients are difficult-to-cure and need therapy optimisation by adding ribavirin and/or extending treatment duration. However, when next-generation NS5A inhibitors are available, the group of difficult-to-treat patients is very limited, mainly genotype 3 patients with Child-Pugh B decompensated cirrhosis and all patients with Child-Pugh C cirrhosis. References

[1] Reddy KR, Bourliere M, Sulkowski M, Omata M, Zeuzem S, Feld JJ, et al. Ledipasvir and sofosbuvir in patients with genotype 1 hepatitis C virus infection and compensated cirrhosis: An integrated safety and efficacy analysis. Hepatology 2015;62:79-86.

[2] Manns M, Samuel D, Gane EJ, Mutimer D, McCaughan G, Buti M, et al. Ledipasvir and sofosbuvir plus ribavirin in patients with genotype 1 or 4 hepatitis C virus infection SESSION 2 – HCV – SESSION 2 and advanced liver disease: a multicentre, open-label, randomised, phase 2 trial. Lancet Infect Dis 2016;16:685-97.

[3] Sarrazin C. The importance of resistance to direct antiviral drugs in HCV infection in clinical practice. J Hepatol 2016;64:486-504.

[4] Hezode C, Reau N, Svarovskaia ES, Doehle BP, Shanmugam R, Dvory-Sobol H, et al. Resistance Analysis in Patients with Genotype 1-6 HCV Infection Treated with Sofosbuvir/Velpatasvir in the Phase 3 Studies. J Hepatol 2017;Dec 5 [Epub ahead of print].

[5] Serfaty L, Jacobson I, Rockstroh JK, et al. A Pragmatic Approach to Optimizing the Efficacy of Elbasvir/Grazoprevir Using Baseline Viral Load in Participants With Hepatitis C Virus (HCV) Genotype (GT)1a Infection: A Post Hoc Analysis of 11 Clinical Trials. Hepatology 2017;66(1 Supp):604A.

[6] Forns X, Lee SS, Valdes J, Lens S, Ghalib R, Aquilar H, et al. Glecaprevir plus pibrentasvir for chronic hepatitis C virus genotype 1, 2, 4, 5, or 6 infection in adults with compensated cirrhosis (EXPEDITION-1): a single-arm, open-label, multicentre phase 3 trial. Lancet Infect Dis 2017;17:1062-8.

[7] Leroy V, Angus P, Bronowicki JP, Dore GJ, Hezode C, Pianko S, et al. Daclatasvir, sofosbuvir, and ribavirin for hepatitis C virus genotype 3 and advanced liver disease: a randomized phase III study (ALLY-3+). Hepatology 2016;63:1430-16.

[8] Hezode C, Lebray P, de Ledinghen V, Zoulim F, Di Martino V, Boyer N, et al. Daclatasvir plus sofosbuvir, with or without ribavirin, for hepatitis C virus genotype 3 in a French early access programme. Liver Int 2017;37:1314-24.

[9] Wyles D, Poordad F, Wang S, Alric L, Felizarta F, Kwo, PY, et al. Glecaprevir/ Pibrentasvir for Hepatitis C Virus Genotype 3 Patients with Cirrhosis and/or Prior Treatment Experience: A Partially Randomized Phase III clinical trial. Hepatology 2017; Sep 19 [Epub ahead of print].

[10] Curry MP, O’Leary JG, Bzowej N, Muir AJ, Korenblat KM, Fenkel JM, et al. Sofosbuvir and Velpatasvir for HCV in Patients with Decompensated Cirrhosis. N Engl J Med 2015;373:2618-28.

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RETREATMENT STRATEGIES IN 2018

Jordan J Feld Toronto Centre for Liver Disease, Toronto General Hospital, University Health Network Sandra Rotman Centre for Global Health, University of Toronto, Toronto, Canada. E-mail address: [email protected]

TAKE-HOME MESSAGES • Retreatment is rarely an emergency.

• Assess for and correct modifiable factors that may have led to treatment failure before retreatment.

• Sofosbuvir/velpastasvir/voxilaprevir is highly effective for retreatment and is the first-line HCV – SESSION 2 salvage regimen for patients who initially did not respond to an NS5A-inhibitor-containing regimen.

• Sofosbuvir/velpatasvir can be used for patients with genotype 1b or 2 who failed with an initial regimen that did not include NS5A inhibitors, whereas those with other genotypes should receive sofosbuvir/velpatasvir/voxilaprevir.

• Glecaprevir/pibrentasvir can be used as a salvage therapy only for patients with genotype 1 or 2 infection exposed to a prior protease and/or polymerase inhibitor but should not be used in those who have received an NS5A inhibitor previously.

• There is likely a very small population of patients not served by current salvage regimens for whom therapy will have to be individualised.

Fortunately, with the revolution in hepatitis C virus (HCV) therapy, the vast majority of patients respond to any of the many first-line direct-acting antivirals (DAA) regimens. However, while only a small percentage of patients do not respond to a course of DAAs, because of the huge number of patients with HCV globally, even a small failure rate amounts to a large absolute number of patients who require retreatment. As first-line therapy moves out of speciality clinics, it is likely that referrals will be limited to harder-to-cure populations and as such, patients requiring retreatment may actually make up a large percentage of those seen in hepatology and infectious disease clinics focused on HCV therapy. Fortunately, highly effective options for retreatment now exist with cure rates comparable to those achieved with first-line therapy.

RELAPSE OCCURS: WHAT NOW? With the extremely high success rates of current DAA regimens, patients and providers ‘expect’ a cure when they embark on a course of HCV therapy. No one expects to be the 2-5% who do not achieve sustained virological response (SVR). As a result, the first reaction to a relapse (or breakthrough) is often surprise and some degree of panic or alarm. Although failure to achieve SVR is obviously disappointing, immediate cure of HCV is rarely an emergency. As such, with few exceptions, retreatment is also rarely an emergency. Reminding patients (and ourselves)

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of this fact is an important first step after the initial shock of a positive HCV RNA at the SVR12 time point. Reassuring patients that effective retreatment options exist is important and should be mentioned before the course of initial therapy. Before rushing into retreatment, it is important to consider why treatment was not effective the first time around.

IDENTIFYING THE CAUSE OF NON-RESPONSE For most patients, there are no easily modifiable factors to explain why treatment did not lead to SVR. However, it is worth reviewing possible causes for treatment failure to ensure that mistakes are not repeated, particularly given the limited options for retreatment after an unsuccessful salvage regimen. Important points to review include:

• Adherence – How many pills were missed and how frequently? If adherence was an issue, what were the reasons? Tolerability?

SESSION 2 – HCV – SESSION 2 • Regimen – Was the regimen used, appropriate for the patient? Ensure that the regimen and duration were appropriate given the genotype and other baseline characteristics (prior treatment history, cirrhosis).

• Duration – Was the appropriate course of therapy given? For example, if a patient was given only 8 weeks of sofosbuvir/ledipasvir did he/she meet eligibility criteria for shortening this regimen (HCV RNA level, no cirrhosis).

• Drug interactions – Were any medications, including over-the-counter or herbal preparations, started or continued during HCV therapy that may have interacted with one or more of the DAAs used? Particular attention should be paid to medications that may reduce DAA absorption or levels, e.g. proton pump inhibitors with ledipasvir or velpatasvir, St. John’s Wort with most DAAs etc.

• Substance use – Did patients continue or relapse to use recreational drugs and/or alcohol during HCV therapy? Could substance use have affected DAA adherence?

• Resistance – Was baseline drug resistance assessed and if so was it managed appropriately (e.g. testing for NS5A resistance in patients with genotype 1a infection treated with elbasvir/ grazoprevir)?

• Other factors – Were there other medical or non-medical factors that may have impacted therapy, e.g. prior gastrointestinal surgery affecting drug absorption. If generics were used, were they from a reliable company licensed for DAA production?

• Re-infection – Is it possible that recurrent viremia reflects a new infection rather than relapse? Risk factors for re-infection should always be explored.

SALVAGE REGIMENS In 2017, two new DAA regimens were approved, both with an indication for retreatment of prior DAA failures. The first was the pan-genotypic combination of sofosbuvir/velpatasvir/ voxilaprevir (SOF/VEL/VOX), which is a fixed-dose single-tablet regimen that combines DAAs with three independent modes of action (nucleotide polymerase inhibitor, NS5A inhibitor and protease inhibitor). The second regimen approved was the pan-genotypic combination of glecaprevir/pibrentasvir (GLE/PIB), a second-generation protease and NS5A inhibitor,

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PARIS, FRANCE which is taken as 3 tablets daily. Notably, it is possible and perhaps likely, that these recent approvals will be the last approvals of new HCV therapies. Although other agents may be developed, they are unlikely to improve upon the results with the current regimens, and as such, SOF/VEL/VOX and GLE/PIB may be the only available salvage regimens available in the foreseeable future. This fact has important implications: firstly, there is no value in waiting for future therapy for retreatment, and secondly, it is critical to get it right with these regimens, as another course of retreatment may not be available.

SOF/VEL/VOX SOF/VEL/VOX was studied as a retreatment option in the POLARIS 1 and 4 trials [1]. In POLARIS 1, patients who had previously failed a regimen containing an NS5A inhibitor were randomised to receive SOF/VEL/VOX or placebo for 12 weeks. Placebo-treated patients were subsequently treated with SOF/VEL/VOX for 12 weeks as well. SOF/VEL/VOX was highly effective, leading to an SVR12 rate of 99% (140/142) in patients without cirrhosis and 93% SESSION 2 – HCV – SESSION 2 (113/121) in those with cirrhosis at baseline. In total, there were seven virological failures (6 relapses and 1 breakthrough) in the study, all of whom had cirrhosis and 6 of 7 of whom had genotype 1a or 3 infection. However, even in those with genotypes 1a and 3, treatment was highly effective with SVR rates of 96% for genotype 1a and 95% for genotype 3. Treatment was similarly effective in the small number of patients with genotypes 2, 4, 5 and 6. Outcomes were almost identical in the delayed treatment arm. Notably, treatment was equally effective in patients with and without baseline resistance-associated substitutions (RAS), and no new RAS were selected in those who did not achieve SVR. Treatment was generally safe and well- tolerated, but the addition of VOX resulted in an increased incidence of non-treatment-limiting diarrhoea. These impressive results led to the approval of SOF/VEL/VOX for retreatment of patients who did not achieve SVR after receiving an NS5A-inhibitor-containing DAA regimen. Because almost all first-line DAA regimens include an NS5A-inhibitor, the results from the POLARIS 1 trial are the most relevant for retreatment.

SOF/VEL/VOX was also studied in patients who had failed a non-NS5A-inhibitor-containing regimen in the POLARIS 4 trial. In this study, patients were randomised to receive SOF/ VEL/VOX or SOF/VEL alone for 12 weeks. Given that these patients had never been exposed to an NS5A inhibitor, it was expected that the SOF/VEL without the addition of VOX might be adequately effective. However, somewhat surprisingly, the triple regimen proved more effective. Of 182 patients who received SOF/VEL/VOX, there was only 1 relapse (and 3 non- virological failures – SVR12 98%). In contrast, of 151 patients who received SOF/VEL alone, 14 relapsed and 1 broke through during therapy (SVR12 90%). Notably, all of the SOF/VEL failures occurred in patients with genotype 1a or 3. This led the US Federal Food and Drug Administration to recommend SOF/VEL/VOX only for patients with genotypes 1a or 3 and SOF/VEL alone for other genotypes with the argument that VOX did not add efficacy for the other genotypes and may increase toxicity. The AASLD/IDSA guidelines took a slightly more conservative approach and recommended SOF/VEL/VOX for patients with genotypes 4, 5 or 6 as well as 1a or 3 with the argument that few patients with these rarer genotypes were included in the POLARIS 4 study.

Collectively these data confirm that SOF/VEL/VOX is an excellent option for all patients who have failed a previous DAA regimen and even SOF/VEL alone can be used in easier-to-cure genotypes (1b and 2) in patients who received a non-NS5A-inhibitor-containing regimen as first-line therapy.

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GLE/PIB Although GLE/PIB is highly effective in non-DAA experienced patients, the data supporting its use as a retreatment option after DAAs is less compelling. In the MAGELLIN-1 study, 91 patients with prior NS3 and/or NS5A-inhibitor experience were randomised to 12 or 16 weeks of GLE/PIB [2]. For patients with no RAS or only NS3 RAS before retreatment, either 12 or 16 weeks was highly effective (SVR 100%), but for those with NS5A RAS and particularly for those with NS5A and NS3 RAS, efficacy dropped off considerably. Although there were no relapses with 16 weeks of therapy, the numbers of patients were too few to draw strong conclusions and data were reported in aggregate rather than by genotype subtype making it hard to interpret given the much greater effect of NS5A RAS in a genotype 1a than genotype 1b background. Based on these results, the AASLD/IDSA guidelines recommend GLE/PIB as a salvage option only for patients with genotype 1 exposed to a previous NS5B (polymerase) and/or NS3 (protease) inhibitor but not to those who have been treated with an NS5A inhibitor (the vast majority). GLE/PIB can also be used for retreatment of patients with genotype 2 infection previously treated with SOF. SESSION 2 – HCV – SESSION 2

OTHER OPTIONS Combining therapies may also be effective. A pilot trial of SOF combined with elbasvir/ grazoprevir led to SVR in 25 of 26 patients who had failed an NS5A or NS3 inhibitor with RAS at the time of retreatment. Other combinations such as SOF with GLE-PIB may be very attractive for certain patients and should be studied.

REMAINING CHALLENGES Although SOF/VEL/VOX, SOF/VEL and/or GLE/PIB are excellent salvage options, there remain some gaps for retreatment.

• Decompensated cirrhosis – Both SOF/VEL/VOX and GLE/PIB contain a protease inhibitor and are thus contraindicated in patients with decompensated cirrhosis, leaving SOF/VEL + ribavirin as the only option for retreatment in these relatively desperate patients.

• Chronic Kidney Disease (CKD) – SOF is not recommended for patients with glomerular filtration rate <30 cc/min, limiting options for patients with advanced CKD who did not respond to an initial course of an NS5A-containing regimen. GLE/PIB can be used in patients with CKD.

• Drug interactions – For patients who need retreatment but require agents that interact with one or all salvage regimens (e.g. amiodarone), treatment options are limited if the ‘offending agent’ cannot be changed.

• New DAA failures – Almost no patients in the retreatment trials had been exposed to VEL, elbasvir/grazoprevir or other more recently approved DAAs. Although the results are expected to be comparable to those seen with similar regimens, it will be important to document such efficacy.

• Salvage regimen failures – Currently there are no clear options for retreatment of the small number of patients who fail a first-line DAA option and then fail SOF/VEL/VOX or GLE/PIB. Retreatment with the same regimen for longer possibly with the addition of

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ribavirin or another DAA may be reasonable, however, there are currently no data to guide therapy.

• Resistance testing – Guidelines do not recommend RAS testing prior to retreatment with SOF/VEL/VOX given the excellent results with or without baseline RAS [3]. I would favour collecting the data now if possible to ensure that there are not clear signature RAS that may require intensified therapy.

CONCLUSION DAAs are remarkably effective meaning that most patients will respond to the first course of treatment. For the small number of patients who do not achieve SVR, it is important to correct modifiable factors, but fortunately, SOF/VEL/VOX, SOF/VEL and to a lesser extent GLE/PIB offer a high chance of cure with retreatment. There is no immediate prospect of a new therapy to fill the small number of remaining treatment gaps, but fortunately, it will be a tiny fraction of patients who require other options. More than retreatment, our biggest challenge continues HCV – SESSION 2 to be identifying patients and getting them into care. References

[1] Bourliere M, Gordon SC, Flamm SL, Cooper CL, Ramji A, Tong M, et al. Sofosbuvir, Velpatasvir, and Voxilaprevir for Previously Treated HCV Infection. The New England journal of medicine 2017, 376(22):2134-2146.

[2] Poordad F, Pol S, Asatryan A, Buti M, Shaw D, Hezode C, et al. Glecaprevir/Pibrentasvir in Patients with HCV Genotype 1 or 4 and Prior Direct-acting Antiviral Treatment Failure. Hepatology 2017; Nov 20 [Epub ahead of print].

[3] Zuckerman A, Chastain CA, Naggie S: Retreatment Options Following HCV Direct Acting Antiviral Failure. Current treatment options in infectious diseases 2017, 9(4):389- 402.

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HCC RISK POST-DAA TREATMENT: A BALANCED PERSPECTIVE

Geoffrey Dusheiko University College London and Kings College Hospital, London, United Kingdom. E-mail address: [email protected]

TAKE-HOME MESSAGES • There is clear evidence from large datasets that direct-acting antiviral agent (DAA)- induced sustained virological response is associated with a reduced risk of de novo incident hepatocellular carcinoma (HCC).

SESSION 2 – HCV – SESSION 2 • Nonetheless, there is a persistent risk of HCC in hepatitis C patients with cirrhosis, despite a sustained virological response.

• All patients with cirrhosis should be closely monitored and followed after successful antiviral therapy.

• It has been suggested that the risk of recurrent HCC is higher after DAA treatment than after interferon treatment. However, accurate comparison requires statistical adjustment for independent risk factors that may explain any observed differences. Selection bias may play a role in evaluating the incidence of recurrent tumour in cohorts treated with DAAs compared to interferon.

• Considerable loss of life expectancy can be affected by DAA treatment of hepatitis C in those with advanced disease, and in those with treated HCC.

INTRODUCTION Chronic hepatitis C virus (HCV) is a hepatotropic virus which can lead to progressive inflammation and fibrosis, resulting in cirrhosis, decompensated cirrhosis and hepatocellular carcinoma (HCC). Current direct-acting antiviral agents (DAAs) are effective and safe treatment options for patients with hepatitis C, across all genotypes, and in patients with cirrhosis or even decompensated cirrhosis. Current therapy effectively achieves a sustained virological response (SVR) in the majority of treated patients, including those with cirrhosis – unlike previous regimens of pegylated interferon alpha (PegIFNα) and ribavirin. The latter was often contraindicated or unsafe in patients with advanced or decompensated cirrhosis. Thus, DAAs have transformed treatment and enhanced treatment rates; treatment is currently limited only by clinical capacity, the availability of funds, and the ability to diagnose and treat new cases [1].

It should be noted that the treatment goals of DAA therapy extend beyond the attainment of an SVR, and importantly include the ability to reduce the incidence of HCC [2]. Several large databases indicate a beneficial effect of DAA treatment [3]. Large cohort studies have suggested a reduction in both HCC incidence and all-cause mortality by eradicating hepatitis C [4].

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IFN-based treatment has not amplified the goal of reducing hepatitis C global morbidity. Although some patients at risk of HCC can attain an SVR, the incidence of HCC has increased over time among patients with hepatitis C and cirrhosis, for example in Denmark [5]. The age-standardised HCC incident rates have remained stable, in British Columbia and Scotland, and have increased in New South Wales [6]. Thus, although an (IFN-induced) SVR has been advantageous, widespread IFN use has had a limited overall impact on HCC prevention. There has also been an adverse impact of delayed notification to increase the risk of advanced liver including decompensated cirrhosis and HCC [7].

The ability to treat cirrhosis has meant large numbers of patients with advanced liver disease have now been treated with DAAs. The success of DAA treatment in patients with cirrhosis has resulted in significant numbers of these ageing patients requiring follow-up and possible management after an SVR. [8, 9].

If DAAs are considered as having a negative effect in patients with cirrhosis, then any increased incidence of HCC would nullify the survival benefits [10]. This syllabus reviews the current literature and balances concerns over a possible increase in HCC after DAA therapy, with the SESSION 2 – HCV – SESSION 2 potential benefit of treating patients at risk HCC development [11].

HCC IN HCV-INDUCED CIRRHOSIS HCV is undisputedly an important worldwide cause of HCC. Age and hepatic fibrosis are important time-dependent risks [12]. A retrospective analysis of the United network for organ sharing (UNOS) organ procurement transplant network database from 2003-2014 has compared HCC-related liver transplant surgical trends between baby boomer and non- baby boomer generations. These trends show that the number of liver transplants required for HCC have increased significantly in the non-alcoholic steatoheptatits (NASH) and hepatitis C groups. The proportion of baby boomers who underwent liver transplant for HCC was highest for HCV (84%) followed by NASH (73%) [13]. Hence, the recommendation for birth cohort HCV screening stems from a greater understanding of a high prevalence of chronic hepatitis C and HCV-related HCC within the baby boomer age group.

ACHIEVEMENT OF AN SVR: BENEFITS WITH IFN, OR DAA-INDUCED SVR HCV viraemic patients with a prior SVR and compensated cirrhosis at the time of diagnosis have improved prolonged overall survival compared to viraemic patients. It is apparent that given the lack of cirrhosis progression, patients with SVR are less likely to ultimately die of end-stage liver disease if treated before hepatic decompensation has occurred [14].

A cohort from Denmark achieving SVR reduced all-cause mortality and liver-related mortality. However, increased incidence rates of HCC and decompensation remained elevated in patients with alcohol abuse after a SVR [15]. Data from 1,323 patients are included in the prospective Agence Nationale de Recherches sur le Sida et les hépatites virales (ANRS) cirrhosis (CirVir) cohort. This cohort of patients are recruited from 35 clinical centres in France from 2006 to 2012 – all of whom had biopsy proven Child-Pugh class A cirrhosis. All patients received treatment with either IFN or DAAs. The median follow-up was 58 months. In total, 668 patients (50%) achieved an SVR. The SVR in this cohort was associated with a decreased incidence of HCC compared with patients without an SVR, and a decreased incidence of

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hepatic decompensation. Therefore, this and other studies have confirmed a reduction in critical events after DAA treatment [16].

To evaluate HCC risk in patients with chronic hepatitis C treated (or not) with PegIFNα and ribavirin, Lee et al. dichotomised the outcomes based on patients achieving SVR. The median follow-up was 31 months. The three-year cumulative HCC development rate in the SVR group was 1.1% but 8.6% in the SVR-minus group. The presence of cirrhosis (HR 9.92, p <0.01), age (HR 1.03, p <0.01), the lack of an SVR (HR 7.02, p <0.01), and no treatment (HR 6.76, p <0.01) were independent predictors for HCC development [17].

A Canadian study evaluated the effect of SVR on the risk of HCC and its incidence post SVR. Individuals were tested for HCV between 1990-2013 and linked with data detailing their medical visits, hospitalisations, cancer prescription drugs and mortality. Patients receiving IFN-based HCV treatments were followed from the end of treatment to HCC diagnosis, death or until December 2012. HCC risk among those who did or did not achieve SVR were analysed using a multivariate proportional hazard model with the Fine and Gray modification for competing risks. Of 8,147 individuals who received HCV treatment, 57% achieved an SVR. SESSION 2 – HCV – SESSION 2 Each group was followed for a median of 5.6 years. HCC incidence rates of 1.1/1,000 person- years among the SVR group and 7.2/1,000 person-years among the non-SVR group occurred. HCC incidence was higher among those with cirrhosis (SVR: 6.4 vs. no SVR: 21/1,000 person- years). In the multivariable model, SVR was associated with a lower HCC risk while cirrhosis, age 50 years, male, genotype 3 were associated with a higher HCC risk [18].

Large-scale Markov models have been developed to simulate the natural history, morbidity and mortality before and after DAA therapy. For example, in Spain in a model of 50,000 patients, a post-DAA strategy predicts the prevention of 8,667 cases of decompensated cirrhosis, 5,471 cases of HCC, as well as 1,137 liver transplants and 9,608 liver-related death. Although the investment required is substantial, the prevention of long-term morbidity and mortality and increased quality of life demonstrate good use of public money [19].

THE RISK OF HCC IS NOT ERADICATED BY AN SVR Evidence suggests that the risk of HCC is not entirely eradicated by the achievement of SVR. We need to consider that particular patients with cirrhosis remain at risk of HCC after a cure of their hepatitis C [20]. Recent studies have evaluated the risk of cirrhosis-related complications in Western cohorts of patients with chronic hepatitis C and bridging fibrosis or cirrhosis. Van der Meer et al., pooled data from 1,000 (IFN) treated patients who achieved an SVR. The median age of the cohort was 52 years and the median follow-up 5.7 years. The cumulative eight-year HCC incidence was 1.8% among patients with bridging fibrosis but 8.7% (95% CI 6-11.4) among patients with cirrhosis (p = 0.058). In those with cirrhosis, the eight-year HCC incidence was 2.6% among patients <45 years, 9.7% among patients aged 45-60 years, and 12.2% in patients older than 60 years of age at the time of treatment. Higher age, lower platelet count and diabetes mellitus were independently associated with the development of HCC [3]. Huang et al. compared 313 patients with either spontaneous HCV clearance or 564 age and sex matched patients who achieved a treatment- induced SVR: 2.2% of the 877 patients developed HCC. Cox regression analysis of factors predicting HCC included SVR, diabetes age and more advanced fibrosis [21]. Other authors have suggested that among patients with an SVR, advanced age, male gender, cirrhosis, decreased platelet counts, increased aspartate aminotransferase (AST), and alpha-fetoprotein (AFP) levels appear to be associated with a higher risk of HCC development [22].

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THE NEED TO TREAT PATIENTS WITH ADVANCED FIBROSIS It is imperative to treat patients with advanced fibrosis because of the risk of disease progression. In an Alaskan cohort, 1.7% of patients with no or mild fibrosis developed end- stage liver disease, compared to 7.9%, 16.4% and 49% with moderate fibrosis, severe fibrosis, and cirrhosis respectively within five years of biopsy. The 5-year outcome of HCC was 1%, 1.1% and 13.4% among patients with mild fibrosis, moderate fibrosis, severe fibrosis and cirrhosis, respectively (p <0.01) [23].

RISK OF HCC AFTER DAA TREATMENT: STUDIES SHOWING AN INCREASED RISK A note of caution was recently sounded by Reig et al. who reported an unexpected increase in the incidence of both de novo (i.e. incident) HCC, recurrent HCC and more aggressive and

faster progression of HCC in patients treated with DAAs [24, 25]. It is important to know HCV – SESSION 2 whether the effects of an SVR are nullified via a pro-tumour activity. Their initial report included patients from four hospitals who had HCV infection and a prior history of treated HCC, which was considered a “complete response” and lacking “non-characterised nodules” at the time they underwent HCV treatment with oral DAAs (patients receiving IFN were excluded). Between 2014 and 2015, 103 patients with prior HCC received DAA treatment. Fifty-eight met the inclusion criteria of the study. The median follow-up was 5.7 months. Three patients died and 16 developed radiological tumour recurrence (27.6%). The authors claimed that these data showed an unexpected high rate and unusual pattern of tumour recurrence, compared with an IFN-induced SVR. Given the hospital unit’s experience and reputation, these data raised significant concerns; however, is it premature, and possibly wrong, to raise a red flag on DAA treatment of those at risk of de novo HCC or recurrent HCC. The question is important given the large increase in treatment numbers of patients in the baby boomer age group.

The statistical analysis of this report has been examined and the data criticised. Camma et al. considered that the wide confidence intervals and small size of the sample were problematic. Definite estimate of the likelihood of HCC recurrence is difficult, due to the high clinical, biological and epidemiological heterogeneity of HCC, and the rates vary among the different HCC treatment groups. A wide range of time had elapsed since HCC treatment and the initiation of DAAs (the median was 11.2 months but the reported range was 1.2-87.7 months). After extracting the individual data, the Kaplan-Meier actuarial probability of HCC recurrence, using HCC treatment as a starting point (rather than DAA initiation) at 6 and 12 months was 7% and 13%, at variance with the reported crude rate of 27% [26].

OTHER STUDIES SUGGESTING A POSSIBLE INCREASE OF INCIDENT OR RECURRENT HCC AFTER DAA THERAPY There is a need to separate incident HCC vs. recurrent HCC, but not all reports have clearly delineated these differences. Ravi et al. reported a high rate of de novo HCC incidence after treatment with DAA therapy for HCV-related cirrhosis in a retrospective analysis. The authors examined patients with cirrhosis treated with DAA therapy between January 2015 and March 2016 at the University of Alabama. Patients with a prior history of HCC were excluded. They analysed the occurrence of de novo HCC within 6 months. In total, 123 patients with HCV cirrhosis received treatment between January 2015 and March 2016. Fifty-seven patients

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were excluded from the analysis leaving 66 patients in the final analysis. Of these, 92% were treated with a combination of ledipasvir and sofosbuvir for 12 weeks and the remainder for 24 weeks. An SVR was observed in 92%. 9% developed HCC either during or within 6 months of treatment. The authors argued that patients with HCV cirrhosis have an HCC incidence of 3% to 5% per year, whereas in this study they observed occurrence of de novo HCC in 9% within 6 months [27].

Conti et al. consecutively analysed 344 patients with cirrhosis treated with DAAs. Fifty-nine patients had previous HCC. DAA therapy induced an SVR in 91% of patients. During 24 weeks of follow-up, HCC was detected in 26 patients (7.6%, 95% CI: 4.99-10.84). Seventeen of 59 patients (28.8%, 95% CI: 17.6-42.07) with a previous HCC, and nine of 285 patients (3.16%, 95% CI: 1.45-5.90) without previous HCC were diagnosed with a tumour. Child-Pugh class B, more severe hepatic fibrosis, a low platelet count and previous HCC were significantly associated with HCC development. By multivariate analysis, Child-Pugh class and a history of HCC independently associated with HCC development [28].

Large numbers of patients have been treated for hepatitis C in Egypt. The incidence rates of SESSION 2 – HCV – SESSION 2 HCC recurrence between DAA exposed and non-exposed patients were compared starting from the date of HCC complete radiological response, and censoring after 2 years. In total, 116 patients were included: 53 treated with DAAs and 63 not treated with DAAs. A recurrence rate of 37% and 25% was observed in each group after a median of 16 and 23 months follow- up, respectively. Poison regression using inverse probability of treatment demonstrated an association between DAAs and HCC recurrence with an incidence rate ratio of 3.83 (95% CI: 2.02-7.25). The authors stressed the need for further mechanistic studies and clinical trials to accurately confirm this role and to identify patient characteristics that may be associated with this event [29].

A Spanish real-world cohort of almost 4,000 genotype 1 patients, treated with combination regimens of DAA, in 35 centres across Spain between April 2015 and February 2016 has been analysed. An SVR of 96% was achieved in this large national database. HCC recurred in 30% of patients with a complete response to therapy for previous HCC. Incident HCC was reported in 0.93% [30].

Data derived from a retrospective multicentre cohort trial in 15 hospitals across Belgium analysed the HCC occurrence and recurrence rates within 6 months after treatment with DAA, with or without PegIFNα. The populations were matched based on fibrosis score. Of the 567 patients included: 77 were treated with PegIFNα plus DAA from 2008-2013, and 490 with DAAs alone between 2013 and 2015. The patients treated with PegIFNα plus DAA were younger than patients treated with DAA without PegIFNα (53 vs. 59 years respectively). 47% of patients treated with PegIFNα plus DAA were on the fibrosis stage METAVIR F4 vs. 67% of patients treated with DAA without PegIFNα (p = 0.001). No difference was observed in early occurrence of new HCC. The authors did observe a high early recurrence rate of HCC in patients treated with DAA without PegIFNα, but these patients were at a higher risk of HCC [31].

The incidence of HCC after SVR in an IFN-free treated group was found to be more than twofold higher than in a IFN-based therapy group in 1,533 patients treated in five institutions across Japan (7.2 vs. 3.0% and 6.2 vs. 3.0%) when excluding patients who underwent a curative resection for HCC. It is important to note the background characteristics: age, and serum AFP levels were higher, and platelet count was lower whereas liver fibrosis was more advanced in patients who achieved an SVR with IFN-free therapy compared with IFN-based therapy [32].

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STUDIES THAT ARE NOT SUGGESTING AN INCREASED INCIDENCE OF HCC Innes et al. examined the frequency of HCC occurrence in patients with advanced liver disease after receipt of IFN-containing or free therapy for hepatitis C infection. They identified HCC naïve individuals with cirrhosis receiving antiviral therapy in Scotland from 1997-2016 who achieved an SVR. Using a Cox regression they compared the risk of HCC occurrence according to treatment regimen. Of the 857 patients that met the study criteria, 31% received a DAA, IFN-free regimen. Individuals receiving an IFN-free treatment were more likely to be older, white, Child-Pugh B or C vs. Child-Pugh A, to be thrombocytopenic, non-genotype 3, and treatment-experienced. DAA therapy was associated with a significant increased risk of HCC: HR 2.48, p = 0.021. However, after multivariate adjustment for baseline factors, no significant risk attributable to IFN-free therapy persisted (HR 1.15, p = 0.74). Therefore, the authors concluded that the higher incidence of HCC following SVR with IFN-free therapy is related to baseline risk factors and patient selection and not the use of IFN-free therapy per se [33].

Large databases have also examined the risk and determinants of HCC patients cured with HCV – SESSION 2 DAA. In a retrospective cohort study, the outcome in HCV-positive patients treated with DAAs in 129 Veterans Administration (VA) hospitals between January 1, 2015 and December 31, 2015 was examined. The authors calculated the annual incidence rates of HCC by SVR to compare the risk of HCC patients with vs. those without SVR, and to identify factors associated with incident HCC among patients with SVR. In total, 22,500 patients were treated with DAAs (19,518 with SVR; 2,982 without SVR: the mean age was 61 years; 39% had cirrhosis), 271 new cases of HCC including 183 in patients with a SVR were observed. Compared with patients without SVR, those with SVR had a significantly reduced de novo HCC risk (0.90 vs. 3.45 HCC/100 person-years, adjusted HR 0.28, 95% CI 0.22-0.36). Patients with cirrhosis had the highest annual incidence of HCC after SVR: 1.82 vs. 0.34/100 person-years in patients without cirrhosis; the adjusted HR was 4.73 (95% CI, 3.34-6.68). Thus, among patients treated with DAA, a SVR was associated with considerable reduction in the risk of HCC; this database therefore provides no evidence that DAAs promote HCC, but in patients with SVR, the absolute risk of HCC remains high in patients with established cirrhosis, who should be considered for ongoing HCC surveillance [34].

An indirect comparison of time to recurrence in patients with successfully treated early HCC and hepatitis C, with those of patients with SVR by IFN-induced or IFN-free methods has been assessed in a Veterans Health Administration retrospective cohort study using patients who terminated treatment by July 1, 2015. A total of 11,464 patients achieved an SVR: 92% in sofosbuvir-based treatment, 86% in ombitasvir-based treatment, and 83% using simeprevir and sofosbuvir therapy [35].

A Japanese study analysed a total of 177 patients receiving DAA after curative treatment of HCC: 89 underwent DAA therapy after initial HCC treatment and the other 88 patients after repeated therapy of 2-10 times. HCC recurrence rates at the end of the 1st and 2nd year were 18 and 22% in patients with single HCC therapy, 28% and 41% in those with 2-3 HCC therapy courses, and 60% and 74% with 3 to 4 times or more therapies. Recurrence rates at the first and second year were 18% and 25% with DAA therapy, and 21% and 46.6% in those without DAA therapy. Multivariate analysis showed DAA therapy significantly decreased occurrence rates with a HR 0.353 (CI 0.91-0.65) after adjustments with covariates of tumour multiplicity, AFP and prothrombin time [36].

An ANRS analysis from three French prospective multicentre ANRS cohorts, including more than 6,000 patients treated with DAA, focused on HCC patients who underwent curative

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procedures before DAA treatment, to assess rates of HCC recurrence in these patients according to antiviral regimen. Of the 267 patients with chronic hepatitis C who were previously treated for HCC that were analysed, 189 received DAA and 78 did not. Rates of recurrence were 1.11/100 and 1.73/100 person-months, respectively. Thus, there was not an increased risk of HCC recurrence after DAA treatment in patients who underwent curative HCC treatment including liver transplantation [37].

A large prospective cohort of HCV patients with cirrhosis and previously successfully treated for HCC has been reported from Italy. 143 consecutive patients with complete response after treatment of HCC were subsequently treated with DAAs. The primary endpoint was the probability of HCC early recurrence. 86% had Child-Pugh class A cirrhosis and 76% Barcelona Clinic Liver cancer (BCLC) classification A 96% SVR was observed. HCC recurrence occurred with a nodular pattern in 83% and was infiltrative in 17% of patients. Six, 12, and 18-month HCC recurrence rates were 12%, 26% and 29%, respectively. The main tumour size and history of prior HCC recurrence were independent risk factors for HCC recurrence by Cox multivariate model. The authors concluded that the probability of HCC early recurrence

SESSION 2 – HCV – SESSION 2 remains high despite HCV eradication by DAAs. The risk was considered comparable but not higher to that reported in the literature, in DAA untreated patients. Previous HCC recurrence and tumour size could be used to stratify the risk of HCC early recurrence [38].

Petta et al. evaluated 443 patients with HCV-related cirrhosis and BCLC stage A/0 who had complete radiologic response after curative resection or ablation. Fifty-eight patients had SVR achieved by IFN-free regimens after HCC cure and 57 patients had SVR achieved by IFN- based regimens after HCC cure. The time to recurrence was similar in patients with SVR by IFN-free or IFN-based (p = 0.49). However, time to recurrence was significantly lower in patients with active HCV infection compared with those with an SVR achieved both by an IFN-free and by IFN-based treatments. The authors concluded that SVR obtained by either IFN-based or IFN-free regimens significantly reduces tumour recurrence without differences related to the antiviral strategy used [39].

In the UK program for the treatment of decompensated cirrhosis and advanced liver disease, outcomes after a year following treatment showed that treatment was beneficial if a timely SVR could be achieved. There was no evidence of increasing liver malignancy [40].

Resolving the question of whether DAA-induced SVR reduces the risk of de novo HCC in HCV infection has been heavily influenced by the large Veterans Affairs national healthcare system cohort. This cohort collected data from 62,354 patients who started antiviral therapy in January 1999 to December 2015. The cohort included 35,871 (58%) patients treated with IFN only regimens, 4,535 (7.2%) with DAA plus IFN regimens, and 21,948 (35%) with DAA only regimens. Patients were retrospectively followed until June 15, 2017 to identify incident cases of HCC. Cox proportional hazards regression was used to determine the association between SVR and HCC risk or between type of antiviral regimen and HCC risk. 3,271 incident cases of HCC were diagnosed during a mean follow-up of 6.1 years. The incidence of HCC was highest in patients with cirrhosis and treatment failure, followed by cirrhosis and SVR, no cirrhosis and treatment failure and no cirrhosis and SVR. SVR was associated with a significantly decreased risk of HCC in multivariable models irrespective of whether the antiviral treatment was DAA only, DAA plus IFN, or IFN only. Treatment with DAAs only or a DAA IFN regimen was not associated with increased HCC risk compared with treatment with an IFN only regimen. A DAA-induced SVR was associated with a 71% reduction in HCC risk. Treatment with DAAs is not associated with increased HCC risk compared with treatment with IFN [41]. Similarly, in Japan, the HCC risk after SVR was similar regardless of whether the latter was achieved by DAAs or IFN-based regimens [42].

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The ERCHIVE study electronically retrieved data from a cohort of HCV-infected veterans in the VA database to investigate whether DAA use was associated with higher rates of HCC incidence compared to treatment with IFN-based regimens. The authors performed a retrospective population-based cohort study. In a cohort of 17,836 patients, SVR was achieved by 66% and 96% of IFN and DAA treated groups, respectively. Among all treated patients the risk of HCC was not higher in the DAA group compared to the IFN group (HR 1.07, 95% CI 0.55-2.08). Among patients with cirrhosis who achieved SVR, neither the HCC incidence rate nor HCC-free survival was significantly different in the DAA group compared to the IFN group (21.2 vs. 22.8 per 1000 person-years; p = 0.78). Untreated persons with cirrhosis had a significantly higher HCC incidence rate (45.3 per 1000 person-years compared to those treated with either IFN or DAAs; p = 0.03); both groups of treated patients had a significantly lower probability of HCC development compared to untreated patients; the authors thus concluded that DAA treatment is not associated with a higher risk of HCC in patients with cirrhosis with chronic hepatitis C in the short-term [43].

Metke et al. prospectively monitored 373 patients with chronic hepatitis C who received IFN- free therapies. The data compared the results to a retrospective control cohort of untreated HCV – SESSION 2 patients with cirrhosis. 158 DAA treated and 184 control patients with cirrhosis were included. The groups did not differ in severity of liver disease nor prevalence of diabetes mellitus. Patients were followed for a median of 440 and 592 days. HCC developed in 6 and 14 patients resulting in an incidence of 2.90 vs. 4.48/ 100 person-years. In the DAA antiviral treatment group, there were no new cases of HCC later than 450 days after treatment initiation. The authors concluded that IFN-free DAA treatment does not alter the short-term risk for HCC in patients with liver cirrhosis. A reduced HCC incidence may become evident after more than 1.5 years of follow-up [44].

Other small reports have suggested that the risk of HCC is similar between PegIFNα and ribavirin treated groups and asunaprevir and daclatasvir treated groups [45], and that DAA treatment could reduce the incidence of HCC [46].

Virlogeux et al. reported 68 consecutive HCV patients with first HCC diagnosis and under remission, who were subsequently treated (or not) with a DAA combination. All patients had cirrhosis. The median age was 62 years, 76 male, and 34% were treated with DAAs with 96% achieving SVR. The median time between HCC remission and DAA initiation was 7.2 months. The median time between DAA start and HCC recurrence was 13 months. The recurrence rate was 1.7/100 person-months among treated patients vs. 4.2/1000 person-months among untreated patients. By multivariate survival analysis the HR for HCC recurrence after DAAs was 0.24 (95% CI 0.1-0.55; p <0.01). Thus, the HCC recurrence rate was significantly lower among patients treated with DAAs compared with untreated patients [47].

There is of course a risk of HCC recurring after resection. However, in addition to secondary prevention through treatment, a tertiary prevention is possible. It has been suggested that one- and two-year cumulative recurrence rates indicate that there is no significant difference between those attaining SVR and non-SVR groups, but later phase 3-5-year and 7-year cumulative recurrence rates are reduced (after IFN therapy) compared to those without SVR [48].

Post-hepatectomy recurrence has been similarly assessed in other studies. Outcomes in a retrospective study of 349 HCC patients who underwent initial radical hepatectomy at a single institution in Japan from January 2005 to December 2014 were analysed. Sixty-eight patients had achieved SVR. Post-hepatectomy overall and recurrence-free survival rates were significantly higher in the SVR group than the non-SVR group. Univariate analysis of post-hepatectomy recurrence-free survival in the SVR group revealed multiple significant factors: AST, indocyanine green retention rate at 15 minutes, and hepatic vascular invasion.

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Multivariate analysis confirmed an interval of less than 30 months between achieving SVR and hepatectomy as an independent prognostic factor of recurrence-free survival. This data suggests that the interval between achieving SVR and hepatectomy is an important predictor of recurrence in HCV-related HCC in patients who achieve an SVR [49].

REVIEWS AND SYSTEMATIC REVIEWS OF THE RISK OF HCC IN HCV PATIENTS Several published studies have assessed the relationship between DAA therapy and HCC in different clinical settings. It is crucially important to separate HCC recurrence in patients with a history of cured HCC from that of de novo HCC occurrence in patients without previously known HCC. It is also apparent that rates of HCC recurrence after DAA therapy are extremely variable, reflecting great heterogeneity in this setting.

The data on de novo HCC incidence are more homogeneous. Several reviewers have stressed SESSION 2 – HCV – SESSION 2 the need for prospective studies [50]. Bang and Song published a systematic review evaluating antiviral efficacy in patients with chronic hepatitis C to assess differences in the development of HCC or mortality, between SVR and non-SVR patients. The methodological quality was assessed using risk of bias tables or the Newcastle Ottawa scale. Publication bias was assessed. In total, 59 studies (4 randomised controlled trials, 15 prospective and 40 retrospective cohort studies) were included. Antiviral treatment was associated with reduced development of HCC vs. no treatment (OR 0.392, 95% CI 0.275-0.557) and this effect intensified when SVR was achieved (vs. no SVR OR: 0.203, 95% CI 0.164-0.251). Antiviral treatment was associated with low all-cause mortality vs. no treatment (OR 0.380 95% CI 0.295-0.489) and liver specific mortality (OR 0.363, 95% CI 0.260-0.508). The authors concluded that antiviral therapy can reduce the development of HCC and mortality particularly when SVR is achieved [51].

A meta-analysis estimates the recurrence and survival probabilities of HCV-related early HCC following complete response after potentially curative treatment and identifies predictors of recurrence and survival. Studies that reported time-dependent outcomes (HCC recurrence or death) after potentially curative treatment of HCV-related early HCC were identified in May 2016. Data on patient populations and outcomes were extracted from each study by three independent observers and combined using a distribution free summary survival curve. The primary outcomes were actuarial probabilities of recurrence and survival. Eleven studies met the inclusion criteria. The pooled estimate of actuarial recurrence rate was 7.4% at 6 months and 47% at 2 years. Pooled estimates of actuarial survival rates were 79% at 3 years and 58% at 5 years. Heterogeneity among studies was highly significant for all outcomes. The univariate meta-regression analysis showed that lower serum albumin, randomised control study design and follow-up were independently associated with a high recurrence risk. Tumour size and AFP levels were associated with higher mortality. This meta-analysis concludes that recurrence risk and survival are extremely variable in patients who were successfully treated with HCV-related HCC; the analysis provides a benchmark for indirect comparisons of the benefits of DAAs for the correct design of randomised controlled trials [52].

Other reviewers have concluded that restriction of treatment is not warranted [53]. Waziry et al. conducted a systematic review comparing the rate of HCC occurrence in patients with HCV-related cirrhosis following DAA vs. IFN treatment and compared the rate of HCC recurrence in patients who received curative treatment following DAA vs. IFN treatment. The review included reports from January 2000 to 2017. Studies were included if they assessed HCC outcomes by type and response to HCV therapy. Random effects meta-analysis were undertaken to determine the combined estimate of HCC incidence. Forty-one studies included

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13,875 patients; of which 26 reported HCC occurrence (IFN 17, DAA 9, prospective 19, retrospective 5, and retrospective-prospective 2) and 17 HCC recurrence (IFN 7, DAA 10, prospective 11, retrospective 5 and retrospective-prospective 1). In studies assessing HCC occurrence, the average follow-up was shorter (1 vs. 5.5 years) and average age older (60 vs. 52 years) in DAA studies. In studies assessing HCC recurrence the average follow-up was shorter (1.3 vs. 5 years) but the average age was similar (64 vs. 66 years) in DAA studies. HCC occurrence was 1.14/100 person-years (95% CI 0.86-1.52) and 2.96/100 person-years (95% CI 1.76-4.96) in IFN and DAA studies, respectively. HCV recurrence was 9.2/100 person-years (95% CI 7.18-11.81) and 12.16/100 person-years (95% CI 5-29.5) in IFN and DAA studies, respectively. By meta-regression, adjusting for study follow-up and age in DAA therapy, was not associated with a higher rate of HCC occurrence (relative risk 0.68; 95% CI 0.18-2.55; p = 0.55) or recurrence (relative risk 0.62, 95% CI 0.11-3.45, p = 0.56) [54, 55].

MECHANISMS OF HCV HEPATOCARCINOGENESIS SESSION 2 – HCV – SESSION 2 Possible theories as to the mechanisms behind tumour relapse after DAA therapy have been enunciated. These explanations include: alterations of immunosurveillance and gene expression, a protective and antineoplastic effect from inflammation secondary to chronic HCV infection that is then abolished with DAA therapy, and delay in radiographic identification of previously undetectable tumours.

The mechanisms of hepatic carcinogenesis are unclear. Hepatocarcinogenesis may occur directly via HCV proteins or transcripts or indirectly through chronic liver inflammation. Hepatitis C infection is clearly the associated driver; there is, as described above, a known high incidence of primary malignancy of the liver in patients with persistent hepatitis C. The tumour promoting processes are not fully understood but chronic inflammation is implicated. Several pathways are of interest, including IFN regulatory factor 5 (IRF5), which may play a critical role in virus Toll-like receptor and IFN-induced signalling pathways. IRF5 is also a tumour suppressor and is modulated and dysregulated during HCV infection, perhaps suggesting a pathological role in HCV-induced HCC [56].

Also, several gene modifications have been associated with the development and progression of HCV to HCC [57]. A strong association between single nucleotide polymorphism (SNP) rs17047200, located within the intron of the tolloid-like 1 gene (TLL1) on chromosome 4, and development of HCC have been reported. The SNP rs17047200 AT/TT was an independent risk factor for HCC along with male sex, older age, lower level of albumin, advanced stage hepatic fibrosis, presence of diabetes, and higher post-treatment level of AFP. It is proposed that this SNP might affect splicing of TLL1 mRNA short variants with high catalytic activity and accelerate hepatic fibrogenesis and carcinogenesis. Such information may provide tests to identify patients at risk for HCC after an SVR to identify pro-oncogenic signatures persisting after an SVR [58]. To date, however, there is little if any evidence to suggest that molecular tumorigenic processes are enhanced by DAA treatment [59].

The immune response to HCV-associated HCC is not fully understood. Natural killer (NK) cell number and function may be associated with cancer progression. However, although it is proposed that NK cell function may be altered by a SVR achieved with IFN treatment is of interest, details of the phenotypic and functional characteristics NK cells in HCC are lacking. NK cell function is regulated by activating and inhibitory receptors, determined by genetic factors and engagement with cognate ligands on transformed or infected cells. The phenotypic and functional characteristics of NK cells in HCC patients undergoing curative treatment in relation to clinical outcome has been evaluated in 70 HCC patients undergoing resection

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or ablation with controls. NK cell cytokine production and cytotoxicity were compared by multivariate analysis between cohorts with different overall survival and time to disease recurrence. Age, Child-Pugh class and NK cell phenotype clustering could independently identify patients with different overall survival. NK cells from patients with better outcome expressed high levels of cytotoxic granules and CD3zeta, and lower levels of natural cytotoxic receptors: whereas cytotoxic function and IFN-gamma production were significantly lower in the cohort of patients with worse outcome compared to controls. These results suggest a role for NK cells in the control of HCC progression and survival, providing a basis for immunotherapeutic strategies to target potentially 8 NK cell responses [60]. However, is not proven that DAA treatment and rapid viral suppression alters NK cell function.

HCV specific CD8 T cells suffer progressive exhaustion during persistent infection although the processes are not fully understood. Peripheral blood HCV specific CD8+ T cells from patients with chronic infection and resolved infection after treatment have been studied. These are functionally impaired, perhaps as a result of co-stimulation failure; dysfunction could also involve negative checkpoint PD-1/PD-L1 regulation [61]. Other gene level statistical aberration

SESSION 2 – HCV – SESSION 2 will require systematic assessment by individualised pathway aberrance scoring (IPAS) to identify differential pathways in HCV patients with cirrhosis and HCC. This will allow us to examine the expression of hub cancer genes to potentially provide novel predictive and prognostic markers for HCC, and to study alterations induced by treatment [62]. At this point, it is only a hypothesis that a rapid suppression of HCV could negatively affect immunologic tumour surveillance or immune restitution [63].

UNDETECTED HCC It could be proposed that individuals found to have HCC after DAA therapy may have been harbouring the tumour before DAA treatment initiation, which was too small for detection by available imaging studies. Although this is biological plausibility, it is difficult to prove unless more sensitive imaging technologies can be utilised.

RISK FACTORS, HETEROGENEITY AND THE NEED FOR STRATIFICATION Prediction of HCC risk: Many different scores have been developed to predict HCC that allow better identification of those at risk. Specific models for assessing risk in the general population are known. It is difficult to know how these risk scores are generalised for predicting risk of recurrence on DAA treatment [64]. Other mathematical models have been produced [65]. Ultimately a genetic-based risk assessment may prove critical [66]. As noted above, a number of possible crucial genes and mRNAs involved in the initiation and progression of HCV-infected cells to HCC have been identified from mRNA hub gene networks [67]. Other markers including wisteria floribunda positive Mac-2 binding protein have been proposed but require validation [68].

An accurate means of determining risk for recurrence or survival after curative resection or ablation in patients with HCV-related HCC is important for stratifying patients according to expected outcomes in an era of DAA therapy. More precise stratification is required to assess the true incidence of de novo HCC and particularly recurrent HCC after treatment of HCV. More data is needed to assess this absolute risk [69] and data to assist risk of occurrence post- treatment [70]. It is possible that post-treatment AFP and fibrosis stage are useful markers to

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The ANRS have built a nomogram based on data of 1,323 patients with HCV cirrhosis enrolled on the French prospective CO13 CirVir cohort. A Cox multivariate model was used to predict HCC. During follow-up of a median of 51 months, 103 and 39 patients develop HCC in the training and validation sets, respectively. Five variables were independently associated with the occurrence of HCC: age >50 years, past excessive alcohol intake, low platelet counts, gamma- glutamyltransferase above the upper limit of normal and absence of an SVR during follow-up. Based on this parameter, an 11 point score was derived and the population stratified into three groups in with a gradated increase from 0 to 30% at 5 years for the patients with lowest and highest scores [72].

AFP concentrations may assist. The AFP score is based on the addition of AFP values at listing to tumour staging may increase the accuracy for predicting recurrence. The French Organisation for Organ Sharing accurately estimated probabilities of recurrence and survival for HCC patients using the AFP score beyond and within Milan criteria: overall five-year SESSION 2 – HCV – SESSION 2 probability of recurrence and survival rates varied according to an AFP score = 2 vs. >2 in the whole population [73]. Others have also suggested that AFP measurements may have additive power for predicting the development of HCC. Tada et al. examined a total of 571 patients with hepatitis C who achieved SVR with IFN-based therapy. Univariate and multivariate Cox proportional hazards models and time-dependent ROC curves were used to analyse the clinical factors associated with the development of HCC. The 5, 10, 15 and 20-year cumulative incidence rates for HCC were 1.7%, 4.8%, 5.8%, and 6.6%. A multivariate Cox proportional hazard model showed that older age, male gender, lower platelet counts, 24 weeks at the end of treatment, as well as a higher AFP (HR 3.6) were independently associated with HCC development [74]. An initial diagnosis of compensated cirrhosis combined with the post SVR albumin levels of less than or equal to 36 g/L, predicted the occurrence of HCC after SVR in patients with chronic hepatitis C [75].

Nagata et al. evaluated the occurrence and recurrence of HCC in chronic hepatitis C patients treated with DAAs with the aim of identifying biomarkers of HCC development after antiviral treatment. The study was a retrospective review of a prospective database of 1,897 chronic hepatitis C patients who were treated with IFN-based (1,145) or IFN-free therapies (752). Cumulative HCC occurrence and recurrence were compared using propensity scored matched analysis. Predictors of HCC were identified. Propensity score matched analysis showed no significant difference in HCC occurrence and recurrence rates between groups treated with IFN-based or IFN-free therapy. A multivariate analysis showed that high levels of post-treatment AFP or wisteria floribunda agglutinin positive Mac-2 binding protein were significantly associated with HCC occurrence and recurrence in patients without severe fibrosis. Post-treatment biomarkers may prove to be helpful in screening those whom it is necessary to follow-up for HCC development [76].

A multicentre cohort study of 1,675 Japanese patients who achieved SVR were divided into two groups; with (152) or without previous HCC (1,523) to evaluate the short-term risk of HCC among patients with SVR by DAAs including those with cirrhosis or previous HCC. Kaplan-Meier method Cox proportional hazard analysis was used to calculate the cumulative HCC incidence and related factors. After a follow-up period (median 17 months), 46 (2.7%) patients developed HCC. One-year cumulative rates of de novo HCC were 0.4% and 4.9% for the non-cirrhosis and cirrhosis groups, respectively (p <0.001). For patients with cirrhosis, serum AFP levels at the end of treatment was the strongest predictor of de novo HCC. The one-year cumulative de novo HCC rates were 1.4% and 13.1% at the end of treatment, AFP

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<9 and >9 ng/ml, respectively. One-year cumulative rates of HCC recurrence were 6.5% and 23% for the non-cirrhosis and cirrhosis groups. For patients with cirrhosis, previous HCC characteristics were significantly associated with HCC recurrence. Sex, age and metabolic factors did not influence the de novo HCC recurrence. The authors concluded that elimination of HCC, serum end of treatment AFP level and previous HCC characteristics would be useful markers for predicting de novo HCC recurrence in patients with cirrhosis [77].

INFLUENCE OF HEPATIC DECOMPENSATION ON HCC OUTCOME Progression of the liver disease influences the ultimate outcome of HCC. The impact of early events (HCC recurrence or hepatic decompensation) within 12 months of complete radiologic response on 5-year overall survival was assessed in a large cohort of patients with HCV and cirrhosis, and successfully treated HCC. In total, 328 consecutive Caucasian patients with HCV-related cirrhosis and BCLC stage 0/A HCC, who had a complete radiological response after curative resection or thermal ablation were prospectively recruited. The primary endpoint SESSION 2 – HCV – SESSION 2 was 5-year overall survival. The observed 5-year survival rate was 44%. The observed HCC early recurrence and early hepatic decompensation rate were 21% and 10%, respectively. Early hepatic decompensation, the presence of oesophageal varices at baseline and age were significantly associated with a 5-year overall survival. Thus, survival in HCV-infected patients with cirrhosis and successfully treated HCC is influenced by early hepatic decompensation, pointing to a role for DAA treatment and ideally, an SVR to arrest progression of the liver disease. These data provide a rationale for treatment to preserve long-term liver function, with the aim of lowering cirrhosis-related mortality [78].

The extent of hepatectomy and portal hypertension influences death from liver failure and can identify a clinical circumstance where the oncological benefit would be borderline, and raise the question whether surgery would be too harmful [79].

RESPONSE TO TREATMENT IF HCC PRESENT It has been suggested that significantly lower SVR rates occur with oral DAAs in hepatitis positive patients with cirrhosis who have or had HCC compared to those without HCC. Failure to achieve SVR was observed in 21% of patients with HCC compared to 12% of patients without HCC (p = 0.009). DAA therapy in the presence of an inactive tumour or after removal of the tumour resulted in excellent SVR rates similar to those without HCC. The primary predictor of DAA treatment failure was the presence of active HCC the time HCV treatment was initiated [80].

CONCLUSIONS There is a persistent risk of HCC in hepatitis C patients after an SVR. This risk for de novo HCC has been observed after both IFN and DAA treatment. Heterogenous cohorts have been studied for clinical outcome, but accurate assessment of the risks is required. Resolving chronic hepatitis C by DAA treatment, despite the higher SVR rates, does not eliminate the occurrence of HCC. Subsets of patients previously treated for HCC still have a risk of tumour recurrence in the short- to medium-term. SVRs are durable in patients with cirrhosis: many patients will have a good long-term clinical prognosis, but the risk of HCC remains. Therefore, all patients with cirrhosis should be closely monitored and followed after successful antiviral therapy.

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However, there is clear evidence from large datasets that a DAA-induced SVR is associated with a reduced risk of cirrhosis, hepatic decompensation, the need for liver transplantation and both liver-related and all-cause mortality. Moreover, SVR will reduce the risk of incidence of de novo HCC.

It has been suggested that the risk of de novo and particularly recurrent HCC is higher than expected after DAA treatment, in comparison to historical patient cohorts treated with IFN-based therapy. However, the comparison is inappropriate without statistical adjustment for independent risk factors that may explain any observed differences. It is not correct to compare the first-generation of the treated DAA population to those who received IFN: most individuals with cirrhosis who received IFN were unsuccessful in achieving SVR. Importantly, meta-analysis comparing both groups for the difference in HCC occurrence and recurrence require correcting for the length of follow-up and patient heterogeneity [81].

Improved risk stratifications are required for comparisons, and the reporting of results demands an accurate propensity score analysis. Thus, better definition of risk predictive host factors is required, for example advanced liver disease, age, accompanying metabolic disease SESSION 2 – HCV – SESSION 2 including diabetes, and persisting hepatic inflammation and elevated AFP as well as viral factors, for example genotype 3. Patients treated with DAAs are often older and in a more advanced stage of cirrhosis than those who had received IFN-based therapy; these factors may have contributed to the observed higher incidence of HCC. IFN-induced SVR was more likely in patients who had a lower risk of HCC development and therefore a lower annual incidence. As DAA treated patients are older and may have parameters that signal a higher risk of HCC, selection bias may play a role in assessing the incidence of tumour in a cohort treated with DAAs compared to IFN [33].

The hypothesis is unproven whether a “re-programming” of tumour surveillance, and a dysregulation of the immune response favouring tumour progression occurs after rapid reduction in viraemia. There may however be clinical circumstances where the oncological benefit would be borderline or even whether surgery would be harmful, so that a separation of optimal from non-optimal DAA treated HCC candidates is required. Also, as the risk persists after SVR is achieved a clinical management strategy is required. Regular surveillance is obligatory even after clinical stabilisation of patients with advanced fibrosis.

Morphological and pathological changes in resected tumours provide observations that may be of value when assisting in predicting risk of recurrence after DAA treatment and SVR. There is limited evidence available to guide clinicians as to which post SVR patient should be monitored or discharged, so that in our present state of knowledge patients with cirrhosis should be followed. Clinical practice update guidelines are necessary to generate best practice recommendations for patients who have achieved SVR [82]. Before DAA therapy is initiated in a patient who has cirrhosis or has undergone treatment for HCC with curative intent, a de novo or recurrent HCC should be meticulously excluded. Even after SVR, these patients still need intensive and assiduous follow-up and surveillance [83, 84].

Thus, among patients treated with DAA, SVR is associated with considerable reduction in the incidence of HCC. There is no firm evidence that DAAs promote HCC but in patients with SVR the relative risk of HCC remains high in patients with established cirrhosis, who require ongoing HCC surveillance. Considerable loss of life expectancy can be countered by treatment of hepatitis C in those with advanced disease. In the future, it may be possible to derive molecular signature of cancer risk even after viral cure [85] and to improve imaging for any residual tumour.

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Reports of unexpected high rates of HCC recurrence after DAA treatment should be understood as an unproven supposition, which may be unfounded. There are important implications for withholding treatment of HCV in patients with cirrhosis as well as in patients with treated HCC. As more patients are treated and additional follow-up time accrues, the benefits of treatment of HCV in patients with treated HCC are likely to accrue. If there are lingering doubts, prospective controlled trials in patients with treated HCC would be required. Finally, it is important to treat in order to prevent cirrhosis and treat before the development of cirrhosis. References

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[44] Mettke F, Schlevogt B, Deterding K, Wranke A, Smith A, Port K, et al. Interferon-free therapy of chronic hepatitis C with direct-acting antivirals does not change the short- term risk for de novo hepatocellular carcinoma in patients with liver cirrhosis. Alimentary pharmacology & therapeutics. 2017.

[45] Nagaoki Y, Imamura M, Aikata H, Daijo K, Teraoka Y, Honda F, et al. The risks of hepatocellular carcinoma development after HCV eradication are similar between patients treated with peg-interferon plus ribavirin and direct-acting antiviral therapy. PloS one. 2017;12(8):e0182710.

[46] Ogata F, Kobayashi M, Akuta N, Osawa M, Fujiyama S, Kawamura Y, et al. Outcome of All-Oral Direct-Acting Antiviral Regimens on the Rate of Development of Hepatocellular Carcinoma in Patients with Hepatitis C Virus Genotype 1-Related Chronic Liver Disease. Oncology. 2017;93(2):92-8.

[47] Virlogeux V, Pradat P, Hartig-Lavie K, Bailly F, Maynard M, Ouziel G, et al. Direct- acting antiviral therapy decreases hepatocellular carcinoma recurrence rate in cirrhotic patients with chronic hepatitis C. Liver international : official journal of the International Association for the Study of the Liver. 2017;37(8):1122-7.

[48] Shinkawa H, Hasegawa K, Arita J, Akamatsu N, Kaneko J, Sakamoto Y, et al. Impact of Sustained Virological Response to Interferon Therapy on Recurrence of Hepatitis C Virus-Related Hepatocellular Carcinoma. Annals of surgical oncology. 2017;24(11):3196- 202.

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[49] Okimoto S, Kobayashi T, Kuroda S, Ishiyama K, Ide K, Ohira M, et al. Prediction of recurrence following hepatectomy in patients with hepatitis C virus infection-related hepatocellular carcinoma who achieved a sustained virological response. Hepatology research : the official journal of the Japan Society of Hepatology. 2017.

[50] Alberti A, Piovesan S. Increased incidence of liver cancer after successful DAA treatment of chronic hepatitis C: Fact or fiction? Liver international : official journal of the International Association for the Study of the Liver. 2017;37(6):802-8.

[51] Bang CS, Song IH. Impact of antiviral therapy on hepatocellular carcinoma and mortality in patients with chronic hepatitis C: systematic review and meta-analysis. BMC gastroenterology. 2017;17(1):46.

[52] Cabibbo G, Petta S, Barbara M, Missale G, Virdone R, Caturelli E, et al. A meta-analysis of single HCV-untreated arm of studies evaluating outcomes after curative treatments of HCV-related hepatocellular carcinoma. Liver international : official journal of the International Association for the Study of the Liver. 2017. SESSION 2 – HCV – SESSION 2 [53] Sparchez Z, Mocan T. Hepatocellular Carcinoma Occurrence and Recurrence after Antiviral Treatment in HCV-Related Cirrhosis. Are Outcomes Different after Direct Antiviral Agents? A Review. Journal of gastrointestinal and liver diseases : JGLD. 2017;26(4):403-10.

[54] Waziry R, Hajarizadeh B, Grebely J, Amin J, Law M, Danta M, et al. Hepatocellular carcinoma risk following direct-acting antiviral HCV therapy: A systematic review, meta- analyses, and meta-regression. Journal of hepatology. 2017;67(6):1204-12.

[55] Wirth TC, Manns MP. The impact of the revolution in hepatitis C treatment on hepatocellular carcinoma. Annals of oncology : official journal of the European Society for Medical Oncology. 2016;27(8):1467-74.

[56] Cevik O, Li D, Baljinnyam E, Manvar D, Pimenta EM, Waris G, et al. Interferon Regulatory Factor 5 (IRF5) suppresses Hepatitis C Virus (HCV) Replication and HCV- Associated Hepatocellular Carcinoma. The Journal of biological chemistry. 2017.

[57] Ezzat WM, Amr KS. Insights for hepatitis C virus related hepatocellular carcinoma genetic biomarkers: Early diagnosis and therapeutic intervention. World journal of hepatology. 2016;8(30):1251-61.

[58] Matsuura K, Sawai H, Ikeo K, Ogawa S, Iio E, Isogawa M, et al. Genome-Wide Association Study Identifies TLL1 Variant Associated With Development of Hepatocellular Carcinoma After Eradication of Hepatitis C Virus Infection. Gastroenterology. 2017.

[59] Vescovo T, Refolo G, Vitagliano G, Fimia GM, Piacentini M. Molecular mechanisms of hepatitis C virus-induced hepatocellular carcinoma. Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases. 2016;22(10):853-61.

[60] Cariani E, Pilli M, Barili V, Porro E, Biasini E, Olivani A, et al. Natural killer cells phenotypic characterization as an outcome predictor of HCV-linked HCC after curative treatments. Oncoimmunology. 2016;5(8):e1154249.

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[61] Moreno-Cubero E, Subira D, Sanz-de-Villalobos E, Parra-Cid T, Madejon A, Miquel J, et al. According to HCV infection stage, IL-7 plus 4-1BB triggering alone or combined with PD-1 blockade increases TRAF1(low) HCV-specific CD8(+) cell reactivity. J Virol. 2017.

[62] Li YL, Zheng MX, Wang G. A personalized approach identifies disturbed pathways and key genes in hepatitis C virus-cirrhosis with hepatocellular carcinoma. European review for medical and pharmacological sciences. 2016;20(20):4266-73.

[63] Werner JM, Adenugba A, Protzer U. Immune reconstitution following HCV clearance with direct antiviral agents: potential consequences for patients with HCC? Transplantation. 2016.

[64] Sherman M. HCC Risk Scores: Useful or Not? Seminars in liver disease. 2017;37(4):287- 95.

[65] Zekri AR, Youssef AS, Bakr YM, Gabr RM, Ahmed OS, Elberry MH, et al. Early

detection of hepatocellular carcinoma co-occurring with hepatitis C virus infection: A HCV – SESSION 2 mathematical model. World journal of gastroenterology : WJG. 2016;22(16):4168-82.

[66] Choi J, Lim YS. Genetic-based Risk Assessment for Hepatocellular Carcinoma in Patients with Hepatitis C: Where Do We Stand? EBioMedicine. 2017;15:6-7.

[67] Poortahmasebi V, Poorebrahim M, Najafi S, Jazayeri SM, Alavian SM, Arab SS, et al. How Hepatitis C Virus Leads to Hepatocellular Carcinoma: A Network-Based Study. Hepatitis monthly. 2016;16(2):e36005.

[68] Sato S, Genda T, Ichida T, Amano N, Sato S, Murata A, et al. Prediction of Hepatocellular Carcinoma Development after Hepatitis C Virus Eradication Using Serum Wisteria floribunda Agglutinin-Positive Mac-2-Binding Protein. International journal of molecular sciences. 2016;17(12).

[69] Bandiera S, Billie Bian C, Hoshida Y, Baumert TF, Zeisel MB. Chronic hepatitis C virus infection and pathogenesis of hepatocellular carcinoma. Current opinion in virology. 2016;20:99-105.

[70] Huang CM, Hu TH, Chang KC, Tseng PL, Lu SN, Chen CH, et al. Dynamic noninvasive markers predict hepatocellular carcinoma in chronic hepatitis C patients without sustained virological response after interferon-based therapy: Prioritize who needs urgent direct-acting antiviral agents. Medicine. 2017;96(46):e8696.

[71] Ji F, Zhou R, Wang W, Bai D, He C, Cai Z, et al. High Post-treatment alpha-Fetoprotein Levels and Aspartate Aminotransferase-to-Platelet Ratio Index Predict Hepatocellular Carcinoma in Hepatitis C Virus Decompensated Cirrhotic Patients with Sustained Virological Response After Antiviral Therapy. Journal of interferon & cytokine research: the official journal of the International Society for Interferon and Cytokine Research. 2017;37(8):362-8.

[72] Ganne-Carrie N, Layese R, Bourcier V, Cagnot C, Marcellin P, Guyader D, et al. Nomogram for individualized prediction of hepatocellular carcinoma occurrence in hepatitis C virus cirrhosis (ANRS CO12 CirVir). Hepatology (Baltimore, Md). 2016;64(4):1136-47.

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[73] Notarpaolo A, Layese R, Magistri P, Gambato M, Colledan M, Magini G, et al. Validation of the AFP model as a predictor of HCC recurrence in patients with viral hepatitis- related cirrhosis who had received a liver transplant for HCC. Journal of hepatology. 2017;66(3):552-9.

74] Tada T, Kumada T, Toyoda H, Kiriyama S, Tanikawa M, Hisanaga Y, et al. Post- treatment levels of alpha-fetoprotein predict long-term hepatocellular carcinoma development after sustained virological response in patients with hepatitis C. Hepatology research : the official journal of the Japan Society of Hepatology. 2017;47(10):1021-31.

[75] Zeng QL, Li B, Zhang XX, Chen Y, Fu YL, Lv J, et al. Clinical Model for Predicting Hepatocellular Carcinomas in Patients with Post-Sustained Virologic Responses of Chronic Hepatitis C: A Case Control Study. Gut and liver. 2016;10(6):955-61.

[76] Nagata H, Nakagawa M, Asahina Y, Sato A, Asano Y, Tsunoda T, et al. Effect of interferon-based and -free therapy on early occurrence and recurrence of hepatocellular carcinoma in chronic hepatitis C. Journal of hepatology. 2017;67(5):933-9. SESSION 2 – HCV – SESSION 2 [77] Ogawa E, Furusyo N, Nomura H, Dohmen K, Higashi N, Takahashi K, et al. Short- term risk of hepatocellular carcinoma after hepatitis C virus eradication following direct- acting anti-viral treatment. Alimentary pharmacology & therapeutics. 2018;47(1):104-13.

[78] Cabibbo G, Petta S, Barbara M, Attardo S, Bucci L, Farinati F, et al. Hepatic decompensation is the major driver of death in HCV-infected cirrhotic patients with successfully treated early hepatocellular carcinoma. Journal of hepatology. 2017.

[79] Cucchetti A, Sposito C, Pinna AD, Citterio D, Cescon M, Bongini M, et al. Competing risk analysis on outcome after hepatic resection of hepatocellular carcinoma in cirrhotic patients. World journal of gastroenterology : WJG. 2017;23(8):1469-76.

[80] Prenner SB, VanWagner LB, Flamm SL, Salem R, Lewandowski RJ, Kulik L. Hepatocellular carcinoma decreases the chance of successful hepatitis C virus therapy with direct-acting antivirals. Journal of hepatology. 2017.

[81] Fangazio S, Camatta D, Tran Minh M, Ceriani E, Minisini R, Pirisi M. Treatment failure after interferon-free treatment of hepatitis C as a clue of a yet undetected hepatocellular carcinoma. Journal of hepatology. 2017.

[82] Jacobson IM, Lim JK, Fried MW. American Gastroenterological Association Institute Clinical Practice Update-Expert Review: Care of Patients Who Have Achieved a Sustained Virologic Response After Antiviral Therapy for Chronic Hepatitis C Infection. Gastroenterology. 2017.

[83] Worns MA, Galle PR, Zeuzem S, Schirmacher P, Manns M, Vogel A. Drug Treatment for Chronic Hepatitis C Infection and Cancer Risk. Deutsches Arzteblatt international. 2017;114(35-36):597-602.

[84] Blanco JR, Rivero-Juarez A. The risk of hepatocellular carcinoma after sustained virological response in patients treated with the new direct-acting antiviral drugs: should we be worry about it? Expert Rev Anti Infect Ther. 2016;14(11):993-6.

[85] Baumert TF, Juhling F, Ono A, Hoshida Y. Hepatitis C-related hepatocellular carcinoma in the era of new generation antivirals. BMC medicine. 2017;15(1):52.

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WEDNESDAY 11 APRIL 2018 14:00-15:30 SESSION 3 – HDV

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ASSESSING LIVER FIBROSIS IN HEPATITIS DELTA VIRUS: INVASIVE VS. NON-INVASIVE TECHNIQUES. IS THE LIVER BIOPSY STILL NECESSARY AS A DIAGNOSTIC TOOL AND IN LONG-TERM SURVEILLANCE?

Benjamin Heidrich Dept. of Gastroenterology, Hepatology & Endocrinology, Hannover Medical School, Hannover, Germany. E-mail address: [email protected]

TAKE-HOME MESSAGES • Liver biopsy is invasive but still the gold standard for assessing the stage of disease in chronic

SESSION 3 – HDV hepatitis delta.

• None of the existing methods for the measurement of liver stiffness have been validated for chronic hepatitis delta.

• Existing non-invasive fibrosis scores need to be validated in patients with chronic hepatitis delta since the existing scores performed are not satisfactory.

• Delta Fibrosis Score and cholinesterase levels could be promising for predicting fibrosis in chronic hepatitis delta.

STAGING OF LIVER FIBROSIS IN CHRONIC HEPATITIS DELTA The main goal of treating patients with chronic viral hepatitis like chronic hepatitis delta is to improve the long-term survival and the quality of life. Both can be achieved by preventing disease progression and consequently the development of hepatocellular carcinoma (HCC). As part of the initial evaluation of all patients being chronically infected with hepatitis B (HBV) and hepatitis D virus (HDV) staging of liver fibrosis is recommended by the EASL clinical practice guideline. However, based on the EASL recommendations the assessment can be done either by liver biopsy or by non-invasive methods like liver stiffness measurement and serum biomarkers. Assessing the stage of liver disease is important to identify patients in need of treatment and for HCC surveillance [1]. Based on EASL recommendations administrating pegylated interferon alpha (PegIFNα) for at least 48 weeks is the current treatment of choice in chronic hepatitis delta with compensated liver disease (Evidence level I, grade of recommendation 1). However, the treatment is associated with only moderate to poor success rates in combination with severe side effects. In the coming years, new and more targeted therapy approaches for HDV might be approved. In the first clinical trials, side effects, as well as success rates of these drugs, are favourable compared to PegIFNα. Therefore, the identification of patients with advanced fibrosis is important. In these patients, treatment with PegIFNα should be initiated immediately, whereas patients without advanced fibrosis could wait until the approval of new drugs [1].

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LIVER BIOPSY FOR ASSESSING STAGE OF FIBROSIS Liver biopsy was, for many years, the reference method for assessing the stage of liver fibrosis in patients with liver diseases including chronic viral hepatitis. Being the reference method implies validation for all liver diseases. Trained pathologists can provide the stage of fibrosis accurately with robust scoring systems like the semi-quantitative METAVIR score if the liver biopsy is of sufficient size. Furthermore, computer-aided morphometric measurement of collagen proportionate area, a partly automated technique, provides an accurate and linear evaluation of the amount of fibrosis [2]. Importantly, liver biopsy is not only used to assess the stage of fibrosis but can be helpful for grading or to identify the aetiology of the underlying liver disease, such as in patients with chronic hepatitis delta and hepatitis delta-associated autoimmune hepatitis or patients with overlapping syndromes. However, taking a liver biopsy is associated with certain disadvantages. First of all, it is an invasive method carrying a risk of rare but potentially life- threatening complications [2]. Furthermore, biopsies represent only small parts of the liver and might not be a representation of the whole liver. Therefore, the length and the caliber are important for sufficient liver biopsies. Interobserver variability is another potential limitation.

To summarise, taking liver biopsies for assessing the stage of liver fibrosis is validated, reliable and available in almost all regions worldwide. However, due to its disadvantages, it cannot be used for close monitoring of patients with chronic hepatitis delta with regular biopsies. SESSION 3 – HDV

NON-INVASIVE METHODS FOR ASSESSING THE STAGE OF FIBROSIS In recent years, non-invasive methods have been developed to overcome certain disadvantages of liver biopsies. In general, non-invasive methods can be divided into two different groups based on the underlying principle for the assessment of liver fibrosis: (1) methods measuring the liver stiffness, and (2) approaches relying on biomarkers [2]. Several of these methods have been established and validated in different liver diseases. In chronic hepatitis B and C, they have been implemented into clinical practice guidelines as alternatives to liver biopsies in certain cases [1–3].

ASSESSMENT OF LIVER STIFFNESS To date, several methods have been developed to measure the liver stiffness as an indicator of liver fibrosis. One of them is transient elastography (TE), which measures the velocity of a low-frequency (50 Hz) elastic shear wave propagating through the liver [2]. Others are point shear wave elastography (pSWE) also known as acoustic radiation force impulse imaging (ARFI), and 2D-shear wave elastography. These techniques are ultrasound-based. Finally, 3-D magnetic resonance (MR) elastography is a further alternative for the non-invasive assessment of liver stiffness [2]. All these methods are non-invasive and could regularly be applied every 6 to 12 months for monitoring the disease progression. However, these methods have certain disadvantages, which may limit their use in clinical routine. Detailed advantages and disadvantages can be reviewed in the EASL-ALEH clinical practice guideline on the use of non-invasive tests for disease staging [2]. Based on the recommendations in the clinical practice guideline, TE is a fast, simple, safe and easy to learn procedure that is widely available. However, it is associated with certain limitations mainly the impossibility of obtaining results in cases of ascites or morbid obesity, and its limited applicability in cases of obesity, and limited operator experience (A1). The alternative techniques (pSWE/ARFI and 2D-SWE) seem to be

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applicable even in obese patients and patients with ascites. However, their quality criteria for correct interpretation are not yet well-defined (A1). MR elastography for monitoring disease progression in the clinical routine is currently not recommended due to its high costs and time-consuming protocols (A1) [2].

None of the mentioned non-invasive methods have been validated in patients with chronic hepatitis delta. When comparing cut-off values for detecting METAVIR F3 or F4 with TE in patients with chronic hepatitis C or B, huge differences can be observed especially for detecting METAVIR F4. Therefore, the use of these methods seems to be limited in chronic hepatitis delta since cut-off values differ between different forms of chronic viral hepatitis. However, TE might be reliable in patients with very high or very low results.

THE USE OF SERUM BIOMARKERS FOR THE ASSESSMENT OF LIVER FIBROSIS IN CHRONIC HEPATITIS DELTA Serum biomarkers can be an alternative approach for regular monitoring of disease stage in patients with chronic liver diseases. Many biomarkers and scores have been proposed for different diseases. Most of them have been established and validated for hepatitis C patients followed by HBV and fatty liver disease. Details can be reviewed in the EASL-ALEH clinical SESSION 3 – HDV practice guideline on the use of non-invasive tests for disease staging [2]. Serum biomarkers have a high availability worldwide, and their costs are low. Therefore, they could easily be used for regular monitoring. However, they are unable to discriminate between intermediate stages of fibrosis, and they are not specific for the liver. Therefore, other conditions influencing the results need to be taken into consideration. EASL recommends the potential use of biomarkers for disease monitoring due to the high applicability (>95%) and good interlaboratory reproducibility. However, clinicians should be aware of factors influencing the test results and the limitations of each biomarker [2]. Importantly, in line with measuring liver stiffness, none of these biomarkers have been validated in patients with chronic hepatitis delta.

VALIDATION OF SERUM BIOMARKERS FOR ASSESSING STAGE OF LIVER DISEASE IN CHRONIC HEPATITIS DELTA In 2016, Lutterkort et al. published the first study on validating existing serum biomarkers in patients with chronic hepatitis delta [4]. They included 100 patients with available liver biopsies from the former HIDIT2 trial. Histological staging was performed by two central blinded pathologists according to ISHAK score. The aim of the study was the identification of patients with advanced fibrosis defined as ISHAK F3-4. Therefore, they aimed for high sensitivity (>80%), positive predictive value (>90%) and area under the receiver operating characteristic curve (AUROC) (0.8). In the first stage, they tested published scores with published cut-off values for the use in chronic hepatitis delta. However, none of the tested scores achieved the predefined assumptions for a reliable use. In a second stage, they tried to adapt the scores for the use in chronic hepatitis delta. However, none of the tested scores could be improved sufficiently [4]. Finally, they established a score for the use in chronic hepatitis delta based on albumin, gamma-glutamyl transpeptidase, cholinesterase and age. Patients with two or more points could be identified as having advanced fibrosis with a sensitivity of 85%, a positive predictive value of 93% and an AUROC of 0.87. They tried to validate the new score in an independent cohort but failed to achieve similar results. Therefore, the score can be a promising serum biomarker in chronic hepatitis delta, but validation in independent cohorts is

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PARIS, FRANCE still missing. At the end of 2016, a second study was published by Takyar et al. on validating existing serum biomarkers for assessing the stage of liver fibrosis in chronic hepatitis delta [5]. In line with the study by Lutterkort et al., they observed a lower performance accuracy of serum fibrosis markers in chronic hepatitis delta compared to patients either being chronically infected with HBV or HCV. Even the adaptation of pre-existing cut-off values for the use in chronic hepatitis delta did not result in a better performance to identify patients with advanced fibrosis reliably. Finally, in 2017 a third study on the use of serum biomarkers in chronic hepatitis delta was published by Abbas et al. [6]. The study identified cholinesterase as a promising serum biomarker for the assessment of liver fibrosis in chronic hepatitis delta, which was also part of the fibrosis score published by Lutterkort et al.

CONCLUSION To summarise, to date non-invasive methods for the assessment of liver fibrosis have not been validated for their use in chronic hepatitis delta. Liver biopsies should be an important diagnostic tool in the care of patients chronically infected with HDV and should not be substituted by non-invasive methods. Patients with intermediate stages of fibrosis cannot be reliably monitored with the available methods. Future studies are needed to validate non- invasive methods especially for the measurement of liver stiffness to offer new diagnostic SESSION 3 – HDV tools in the care of patients with chronic hepatitis delta. Serum biomarkers can be a further alternative as shown by Lutterkort et al. and Abbas et al. However, so far none of the published biomarkers are able to substitute liver biopsies completely. They can only be seen as an additional diagnostic tool. References

References in bold are required reading

[1] European Association for the Study of the Liver. EASL 2017 Clinical Practice Guidelines on the management of hepatitis B virus infection. J Hepatol 2017;67:370–98. doi:10.1016/j. jhep.2017.03.021.

[2] European Association for Study of the Liver, Asociacion Latinoamericana para el Estudio del Higado. EASL-ALEH Clinical Practice Guidelines: Non- invasive tests for evaluation of liver disease severity and prognosis. J Hepatol 2015;63:237–64. doi:10.1016/j.jhep.2015.04.006.

[3] European Association for Study of Liver. EASL Clinical Practice Guidelines: management of hepatitis C virus infection. J Hepatol 2014;60:392–420. doi:10.1016/j.jhep.2013.11.003.

[4] Lutterkort GL, Wranke A, Yurdaydin C, Budde E, Westphal M, Lichtinghagen R, et al. Non-invasive fibrosis score for hepatitis delta. Liver Int Off J Int Assoc Study Liver 2016;37(2):196-204. doi:10.1111/liv.13205.

[5] Takyar V, Surana P, Kleiner DE, Wilkins K, Hoofnagle JH, Liang TJ, et al. Noninvasive markers for staging fibrosis in chronic delta hepatitis. Aliment Pharmacol Ther 2017;45:127–38. doi:10.1111/apt.13834.

[6] Abbas M, Abbas Z. Serum cholinesterase: A predictive biomarker of hepatic reserves in chronic hepatitis D. World J Hepatol 2017;9:967–72. doi:10.4254/wjh.v9.i22.967.

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FACTS AND CONTROVERSIES IN TREATMENT OF HEPATITIS DELTA WITH PegIFNα

Cihan Yurdaydin Department of Gastroenterology, University of Ankara medical school, Cebeci Tip Fakultesi Hastanesi, Dikimevi, Ankara, Turkey. E-mail address: [email protected]

TAKE-HOME MESSAGES • Current treatment with proven efficacy in chronic hepatitis delta (CHD) consists of the use of pegylated interferon (PegIFN), with viral response rates of around 25%.

• Some patients may benefit from one year of treatment with PegIFN, others respond to prolonged courses of treatment, and optimal treatment duration is decided on an individual basis.

SESSION 3 – HDV • The best surrogate marker of treatment efficacy is HBsAg clearance, but it is rarely achieved. Undetectable hepatitis delta virus (HDV) RNA or a more than 2 log decrease of HDV RNA compared to baseline at end of treatment may be considered as a treatment response.

• Nucleos(t)ide analogues (NAs) used for the treatment of hepatitis B are ineffective in CHD when used for only one year. Long-term use of NAs may, however, be effective in CHD.

• New treatments are currently being tested in phase II trials with promising results, and they may contribute to the management of CHD in the near future.

Chronic hepatitis delta (CHD) continues to be a dilemma for patients and physicians for two reasons. First, it was and is the most severe form of viral hepatitis [1]. Second, when CHD treatment is compared to treatments of other forms of chronic viral hepatitis there is a detrimental picture in CHD: the long-term outcome of chronic hepatitis B (CHB) has drastically improved with the use of nucleos(t)ide analogues (NAs) [2]. In chronic hepatitis C (CHC), the availability of combination treatments with direct-acting antivirals has enabled very high cure rates despite shortened treatment durations [3]. Treatment of CHD, on the other hand, has basically not changed in the last 30 years. However, this may soon change as long-term NA use has been reported to be beneficial in patients with HIV-HDV [4, 5, 6]. At this meeting, we will probably see data confirming studies in patients with HIV-HDV co- infection in at least a proportion of patients with CHD. New approaches to treatment are being explored in recent years, and these approaches may change the way we treat patients with CHD [7]. In contrast to developments in CHB and CHC treatment, however, new treatment strategies appear to benefit from approaches using combination treatment with pegylated interferon (PegIFN) [8-10]. Hence, the IFN era may continue in CHD even if we witness approval of new treatment modalities. It is therefore timely to recapitulate our knowledge in treating CHD with conventional or PegIFN.

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TREATMENT OF CHD WITH PegIFN WHEN TO START TREATMENT: In recent years, the two biggest clinical studies ever performed in CHD compared treatment with PegIFN-alpha 2a as monotherapy, with combination therapy of PegIFN with adefovir or tenofovir, respectively [11, 12]. In the first of these studies, the HIDIT-1 Study, patients with advanced liver disease responded similarly to patients with less advanced disease, and patients with compensated cirrhosis appeared to tolerate treatment well, although some serious adverse events were more often seen in patients with advanced liver disease [13]. In the second study, PegIFN therapy appeared to be more effective in patients with compensated cirrhosis compared to patients without cirrhosis [12]. However, two long-term cohort studies, one from Italy and one from Turkey, are contradictory to the data mentioned above. Specifically, in the study from Italy, Romeo et al. reported that in patients with baseline cirrhosis (n = 24) 12.5% developed a lasting viral response (VR) [14]. Patients who developed cirrhosis on follow- up (n = 41) had a similar VR rate (17.1%), whereas patients with baseline chronic hepatitis (n = 25) had a VR rate of 60% [14], indicating a strikingly better VR in patients with less advanced disease (Fig. 1). Similarly, in the cohort study from Turkey, high platelet count was an independent predictor of treatment response [15], suggesting again that early treatment with IFNs is better than delaying treatment in patients with CHD. Both of these studies compared to the two HIDIT studies mentioned above have the drawback of being retrospective, however SESSION 3 – HDV contrary to the HIDIT studies in these two cohort studies patients were treatment-naïve at baseline.

Fig. 1. Viral response to interferon treatment in patients with baseline or follow-up cirrhosis compared to patients with baseline chronic hepatitis [14]. F-up, follow-up; SVR, sustained virological response.

The Hannover Group developed the baseline-event anticipation (BEA) score as a facilitator for treatment-commencement decision. The BEA score is based on variables such as age and sex of the patient, region of origin of the patient, and bilirubin, platelet and INR levels. Patients with BEA-A B and C Scores were reported as mild, moderate and high risk patients, respectively. Patients with BEA B and C scores displayed a hazard ratio of 9.01 and 25.3, respectively compared to patients with BEA-A for developing a liver-related clinical endpoint (Fig. 2) [16]. As the authors pointed out, the BEA score is a reliable marker for deciding which patient is in urgent need of treatment. Overall, based on these data, we suggest starting treatment at an early stage of CHD infection whenever this is possible.

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Fig. 2. BEA scores and their effects on survival [16].

ASSESSMENT OF TREATMENT EFFICACY It is the norm of clinical practice to use surrogate markers for treatment efficacy. These surrogate markers are used to basically predict those patients who will not develop complications of the disease or die from the disease. In CHD, the optimal surrogate marker is similar to CHB, HBsAg clearance. However, this is rarely achieved, and therefore is not practical [17]. Sustained virologic response extrapolated from the hepatitis C literature does not appear to be an optimal surrogate marker since it was shown that a substantial proportion of patients negative for HDV RNA at post-treatment month 6 might have detectable HDV RNA thereafter [18]. However, it needs to be said that in the vast majority of publications, HDV RNA negativity at 6 months treatment-free follow-up was used to define treatment response. The accumulated knowledge from many clinical studies that IFNs are effective in 25 to 30% of patients is based mainly on post-treatment month 6 HDV RNA negativity data. Further, in the analysis by Heidrich et al. [18], none of the patients who became HDV RNA positive after month 6 of post-treatment follow-up developed a clinical event during 5 years of follow-up. Interestingly, in the landmark study by Farci et al. patients who had been treated with IFN for one year, a 2 log decrease at the end of treatment was associated with improved survival [19]. End of treatment HDV RNA negativity or a 2 log decrease of HDV RNA compared to baseline may be reasonable surrogate markers of treatment efficacy. Since new therapies are on the horizon, an expert advisory manuscript is in preparation on the topic of what a reasonable surrogate for treatment efficacy in CDH should be.

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EFFECT OF IFN TREATMENT ON THE NATURAL COURSE OF THE DISEASE Several studies have clearly shown the beneficial impact of IFN treatment on the natural course of CHD (Fig. 3) [20, 21]. IFN treatment was less often associated with clinical endpoints such as hepatic decompensation, development of hepatocellular carcinoma (HCC) and liver-related mortality [18]. In a further study on patients treated with IFN and comparing patients who had a maintained VR to those without a VR, patients with a VR displayed a significant decrease in the occurrence of hepatic decompensation, liver-related mortality and overall clinical endpoints compared to those without a VR (Fig. 4) [15]. HCC also tended to occur less frequently although this was not statistically significant. It should also be underlined that in this study, the two independent risk factors for developing a clinical event was non- response to IFN treatment and baseline cirrhosis [15]. Finally, the study has also shown that in patients having a maintained virologic response, HBsAg clearance is not a rare phenomenon on long-term follow-up, which developed in 37% at a median of 46 months after treatment discontinuation (Fig. 5).

HCC Survival SESSION 3 – HDV

Hepatic decompensation

Fig. 3. Cumulative probability of developing clinical endpoints: IFNa, NAs, no therapy.

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Fig. 4. Improved survival with virologic response to interferon [15]. SESSION 3 – HDV MVR, maintained virologic response.

Fig. 5. HBsAg clearance is frequent in patients with maintained virologic response (MVR) [15].

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TREATMENT DURATION In most studies on the use of conventional or pegylated IFN for the management of CHD, treatment duration is for one year: some studies have used treatment durations of up to 2 years. In general, prolonging treatment beyond one year did not appear to provide additional benefit [17]. This was fortified by the results of the HIDIT-2 Study in which patients had received PegIFN for 2 years and where post-treatment month 6 HDV RNA negativity did not appear to be different from the HIDIT-1 Study, where PegIFN treatment was given for one year. However, results of two recent studies suggest that prolonged IFN treatment may be necessary for higher VR rates [22, 23]. The cohort analysis from Turkey also suggests that prolonged treatment is necessary for a subset of patients. Cumulative VR rates increased with increased treatment durations [15]. It appears that in CHD there is no fixed optimal treatment duration and that treatment duration needs to be decided on an individual basis.

RESPONSE AND NON-RESPONSE TO PegIFN Patients who have not responded to PegIFN therapy represent an important problem. Part of the reason of non-response may be that they had missed the opportunity of responding to treatment because of long-treatment interruptions. It is therefore important to define what SESSION 3 – HDV non-response actually means in a condition where today the only treatment with proven efficacy is treatment with PegIFN. Currently, there is not the luxury for patients who do not achieve undetectable HDV RNA levels at the end of treatment or post-treatment to be labelled as non-responders. Patients who display HDV RNA reductions of 2 logs or more compared to baseline may benefit from prolonging treatment and patients who relapse after treatment may benefit from re-institution of treatment without delay [15, 19]. Patients who at the end of treatment have less than a 1 log decrease compared to baseline, however, may be considered as true non-responders [24], and in those patients, an alternative treatment needs to be considered. One such alternative is to enter them into a clinical trial with new drugs currently testing their efficacy and safety in CHD patients. Another more realistic possibility is to put such patients on long-term treatment with NAs. Long-term NAs were found to be effective in patients with HIV-HDV co-infection when these compounds had been given as part of antiretroviral management [4, 5]. It appears that such a strategy may also work in a subset of patients with CHD without HIV co-infection. NA treatment for one year was, in general, ineffective shown in several studies using different NAs [17]. The difference with long-term use is that it may indirectly decrease HBV cccDNA and HBsAg production, which would decrease HDV propagation to uninfected hepatocytes.

HIV-HDV INFECTION HDV infection is a major cause of hepatic decompensation, HCC and survival. With the availability of very effective treatment in HIV, hepatitis B and very high rates of cure with direct-acting antivirals in hepatitis C, HDV appears to be the main determinant of survival in HIV-infected patients [25]. The effectiveness of PegIFN treatment in patients with HIV-HDV is largely unknown.

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NEW TREATMENTS For the first time, new treatment approaches are seriously being explored for the management of CHD. These drugs tailor various steps in the life cycle of HDV. However, it is clear to say that any approach leading to a functional or complete cure of CHB would be beneficial in CHD as well, as HBsAg is the only product of HBV needed by HDV to propagate infection.

New drugs explored for the treatment in CHD are shown in Table 1. However, three classes of drugs have reached phase II studies. These are hepatocyte entry inhibitors, prenylation inhibitors and nucleic acid polymers (NAPs) [7]. The hepatocyte inhibitor myrcludex B has been assessed both as a monotherapy and in combination with PegIFNα. Recently, three different doses of myrcludex B have been explored, and the final results of the latter study will be presented at EASL ILC 2018. Overall, a combination of myrcludex B with PegIFNα- 2a appears to be superior to monotherapy in inducing HDV RNA decreases [9], and for monotherapy, efficacy appears to be dose-dependent. Myrcludex B needs to be given daily by subcutaneous injection but appears to be well-tolerated.

Table 1. New treatments in CHD [7]. Hepatocyte entry inhibitors Farnesyl transferase inhibitors SESSION 3 – HDV Nucleic acid polymers Small interfering RNAs Immunological approaches: Interferon lambda, TLR agonists, check point inhibitors, HBV vaccines

The prenylation inhibitor lonafarnib has been tested at various doses, in various combinations and for durations up to one year at three sites in separate studies in Hannover, Bethesda and Ankara. Gastrointestinal adverse events represent dose-limiting side effects of lonafarnib. Low-dose lonafarnib in combination with ritonavir or PegIFNα may be the best combination for tolerability and efficacy [10]. Interestingly, lonafarnib treatment for 3 to 6 months may lead to a beneficial post-treatment flare leading to clearance or very low levels of HDV RNA in a subset of patients.

NAPs are hydrophobic oligonucleotides which bind with high affinity to amphipathic protein structures in a sequence-independent fashion. Their mechanism of action is not entirely clear although they most likely act by preventing the extrusion of hepatitis B or D from hepatocytes. They need to be given as an intravenous infusion, and three different formulas have so far been tested in CHB or CHD patients. NAPs have been used both in CHB and CHD as a monotherapy to be followed by sequential combination treatment with PegIFN. They lead to a robust decline in serum HDV RNA as well as HBsAg, and in some patients also clearance of HBsAg with the development of anti-HBs [8]. Characteristics and side effects of these three treatment approaches are displayed in Tables 2 and 3.

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Table 2. Characteristics of novel drug treatment for chronic hepatitis D [7].

Drug Mode of action Administration Phase of route study Myrcludex B Interferes with HDV Subcutaneous, daily for Ib, II entry into hepatocyte 6 months, ± PegIFN through NTCP inhibition Lonafarnib Farnesyl transferase Oral, 2 to 12 months, ± II inhibitor, inhibits ritonavir ± PegIFN virion assembly Rep-2139-Ca Nucleic acid polymer, Intravenous infusion, II binds with high affinity once weekly for 4-6 to amphipathic proteins months ± PegIFN which are required at various stages of the viral life cycle

NTCP, sodium taurocholate co-transporting polypeptide. SESSION 3 – HDV

Table 3. Side effects of the hepatocyte entry inhibitor myrcludex B, the farnesyl transferase inhibitor lonafarnib and nucleic acid polymers [7].

Myrcludex B: • Lipase, amylase elevation in phase I but not in phase II study • Elevation of taurine- and glycine-conjugated bile acids- without apparent clinical consequences • Thrombocytopenia, neutropenia, lymphopenia and eosinophilia: generally mild, transient Lonafarnib: • Gastrointestinal toxicity: anorexia, nausea ± vomiting, diarrhoea, weight loss: dose- dependent and in lower dose cohorts generally mild and well-tolerated Nucleic acid polymers: • Hair loss, dysphagia, anorexia, dysgeusia in HBV Study: related to heavy metal exposure at the trial site? • Administration route related side effects: peripheral grade 1 hyperemia, fever, chills, headache

CONCLUSIONS In conclusion, CHD continues to be treated with IFNs. An individual approach to treatment is recommended when using IFNs especially regarding treatment duration. In clear non- responders to IFN treatment, long-term NA administration appears to be rational. New treatments are on the horizon, which will hopefully change the way we treat CHD although even with new treatment modalities IFN may still be needed.

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References

References in bold are required reading

[1] Yurdaydin C, Idilman R, Bozkaya H, Bozdayi AM. Natural history and treatment of chronic delta hepatitis. J Viral Hepat. 2010;17:749-756.

[2] European Association for the Study of Liver. EASL 2017 Clinical Practice Guidelines on the management of hepatitis B virus infection. J Hepatol. 2017;67:370-98.

[3] European Association for the Study of Liver. EASL Recommendations on Treatment of Hepatitis C 2016. J Hepatol. 2017;66:153-94.

[4] Sheldon J, Ramos B, Toro C, Ríos P, Martínez-Alarcón J, Bottecchia M, et al. Does treatment of hepatitis B virus infection reduce hepatitis delta virus replication in HIV- HBV-HDV coinfected patients? Antiviral Ther. 2008;13:97-102.

[5] Soriano V, Vispo E, Sierra-Enguita R, Mendoza Cd, Fernández-Montero JV, Labarga P, et al. Efficacy of prolonged tenofovir therapy on hepatitis delta in HIV-infected patients. AIDS. 2014;28:2389-94.

[6] Béguelin C, Friolet N, Moradpour D, Sahil R, Suter-Riniker F, Lüthi A, et al. Swiss HIV

SESSION 3 – HDV Cohort Study. Impact of tenofovir on HDV replication in the Swiss HIV Cohort study. Clin Infect Dis. 2017;64:1275-8.

[7] Yurdaydin C. Recent advances in managing hepatitis D. F1000Res. 2017 Aug 30;6:1596.

[8] Bazinet M, Pantea V, Cebotarescu V, Cojuhari L, Jimbei P, Albrecht J, et al. Safety and efficacy of REP 2139 and pegylated interferon alfa-2a for treatment-naive patients with chronic hepatitis B virus and hepatitis D virus co-infection (REP 301 and REP 301-LTF): a non-randomised, open-label, phase 2 trial. Lancet Gastroenterol Hepatol. 2017;2:877-89.

[9] Bogomolov P, Alexandrov A, Voronkova N, Macievich M, Kokina K, Petrachenkova M, et al. Treatment of chronic hepatitis D with the entry inhibitor myrcludex B: First results of a phase Ib/IIa study. J Hepatol. 2016;65:490-8.

[10] Yurdaydin C, Keskin O, Kalkan C, Karakaya F, Çali kan A, Karatayil E, et al. Optimizing lonafarnib treatment for the management of chronic delta hepatitis: The LOWR HDV-1 study. Hepatology 2018. [Epub ahead of print].

[11] Wedemeyer H, Yurdaydin C, Dalekos GN, Erhardt A, Çakalo lu Y, De ertekin H, et al. Peginterferon plus adefovir versus either drug alone for hepatitis delta. N Engl J Med. 2011;364: 322–31.

[12] Wedemeyer H, Yurdaydin C, Ernst S, Caruntu FA, Curescu MG, Yalcin K, et al. 96 weeks of pegylated-Interferon-alpha-2a plus tenofovir or placebo for the treatment of hepatitis delta: the HIDIT-2 study (abstr.) Hepatology. 2013;58:222-3.

[13] Kabaçam G, Dalekos G, Çakalo lu Y, Zachou K, Bock T, Erhardt A, et al. Pegylated interferon based treatment of patients with advanced liver disease due to chronic delta hepatitis. Turk J Gastroenterol. 2012;23:560-8.

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[14] Romeo R, Del Ninno ED, Rumi M, Russo A, Sangiovanni A, de Franchis R, et al. A 28- year study of the course of hepatitis Δ infection: risk factor for cirrhosis and hepatocellular carcinoma. Gastroenterology. 2009;136:1629-38.

[15] Yurdaydin C, Keskin O, Kalkan C, et al. Interferon treatment duration in patients with chronic delta hepatitis and its effect on the natural course of the disease. J Infect Dis. 2018 (in press).

[16] Calle Serrano B, Großhennig A, Homs M, Heidrich B, Erhandt A, Deterding K, et al. Development and evaluation of a baseline-event-anticipation score for hepatitis delta. J Viral Hepat. 2014;21:e154-63.

[17] Yurdaydin C. Treatment of chronic delta hepatitis. Sem Liver Dis. 2012:32:237- 44.

[18] Heidrich B, Yurdaydin C, Kabaçam G, Ratsch BA, Zachou K, Bremer B, et al. Late HDV RNA relapse after peginterferon alpha-based therapy of chronic hepatitis delta. Hepatology. 2014;60:87-97.

[19] Farci P, Roskams T, Chessa L, Peddis G, Mazzoleni AP, Scioscia R, Serra G, et al. Long- term benefit of interferon alpha therapy of chronic hepatitis D: regression of advanced hepatic fibrosis. Gastroenterology. 2004;126:1740-9. SESSION 3 – HDV [20] Manesis E, Vourli G, Dalekos G, Vasiliadis T, Manolaki N, Hounta A, et al. Prevalence and clinical course of hepatitis delta infection in Greece: a 13-year prospective study. J Hepatol. 2013;59:949-56.

[21] Wranke A, Calle Serrano B, Heidrich B, Kischner J, Bremer B, Lehmann P, et al. Antiviral treatment and liver-related complications in hepatitis delta. Hepatology. 2017;65:414-25.

[22] Guedj J, Rotman Y, Cotler SJ, Koh C, Schmid P, Albrecht J, et al. Understanding early serum hepatitis D virus and hepatitis B surface antigen kinetics during pegylated Interferon-alpha therapy via mathematical modeling. Hepatology. 2014;60:1902-10.

[23] Soyer OM, Baran B, Ormeci AC, Gokturk S, Aydin E, Onel D, et al. Comparison of the efficacy of 12 months and longer courses of interferon therapy for the treatment of chronic delta hepatitis: a retrospective cohort study. Postgraduate Med. 2016;128:432-8.

[24] Keskin O, Wedemeyer H, Tuzun A, Zachou K, Deda X, Dalekos GN, et al. Association between level of hepatitis D virus RNA at week 24 of pegylated interferon therapy and outcome. Clin Gastroenterol Hepatol. 2015;13:2342-9.

[25] Soriano V, Sherman KE, Barreiro P. Hepatitis delta and HIV infection. AIDS 2017; 31:875-84.

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THE RISK OF DECOMPENSATION AND HCC DEVELOPMENT IN PATIENTS WITH HDV-RELATED CIRRHOSIS

Grazia Anna Niro*, Rosanna Fontana, Angelo Andriulli IRCCS “Casa Sollievo Sofferenza” Hospital, Gastroenterology and Endoscopy Unit, San Giovanni Rotondo (FG), Italy. E-mail address: [email protected]

TAKE-HOME MESSAGES • Chronic hepatitis D (CHD) is usually a progressive disease with a high risk of liver decompensation and hepatocellular carcinoma (HCC); liver failure is the primary complication. Whether patients with CHD are at a higher risk of HCC than the hepatitis B virus (HBV) mono-infected patient remains controversial.

SESSION 3 – HDV • The clinical outcome is worse in CHD patients with high levels of both hepatitis delta virus (HDV) and HBV replication. HDV genotype 1, the most common worldwide, is associated with a wide spectrum of disease severity. HDV genotype 3 has been implicated in severe and fulminant cases of hepatitis D in South America.

• The conventional Model for End-stage Liver Disease and Child-Pugh scores are commonly used to define the clinical state and predict the risk of disease evolution.

• A specific baseline-event-anticipation score has been proposed to assess HDV liver disease and predict the development of complications.

• It is unclear whether HDV could play by itself an oncogenetic role. Ongoing studies are evaluating the impact of oxidative stress, DNA methylation and histone modification linked to severe HDV necroinflammation.

INTRODUCTION The hepatitis delta virus (HDV) is a hepatotropic virus which induces hepatitis only in individuals who are also infected with hepatitis B virus (HBV). The patients with chronic hepatitis delta (CHD) are mostly anti-HBe positive, have low levels HBV DNA in serum and lack distinctive histological features. Chronic disease develops in 90% of the carriers of the hepatitis B surface antigen (HBsAg) superinfected with HDV. HDV disease is heterogeneous in clinical manifestations, and its progression appears to be dependent on different co-factors in different areas of the world [1]. Is the risk of hepatocellular carcinoma (HCC) increased or not in HDV?

The course of the HDV disease may vary from mild to fulminant but is usually severe and progresses to cirrhosis and liver failure; with 10-15% of cases developing cirrhosis within 2 years and 70-80% within 5 to 10 years (Fig. 1). CHD runs an asymptomatic clinically non- progressive course only in a minority of infected individuals [2]. Though progression to cirrhosis is rapid, when this condition is established, the subsequent course is often stable, and

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PARIS, FRANCE cirrhosis may remain compensated for over a decade (average 13 years) before complications manifest [3]. In 39 subjects chronically infected with HDV, recruited in a prospective study of 200 European patients with compensated HBV cirrhosis, the risk for HCC development, decompensation events, and mortality was increased by a factor of 3.2, 2.2 and 2.0, respectively, compared to HDV-negative patients with cirrhosis. SESSION 3 – HDV

Fig. 1. Progression of fibrosis and cirrhosis in HDV chronic patients.

In Sweden, the risk of HCC was 6-fold higher in patients with HBV and HDV, compared to reference patients with HBV mono-infection. In the survey of Hospital Discharges and Outpatient Registries on which the study was based, half of the patients who developed CHD presented with acute hepatitis; the incidence of HCC was higher in this group than in the patients presenting with a chronic HDV infection; no information on concomitant risk factors was provided by the study [4]. In Mongolia, Oyunsuren et al. investigated the association between the viral pattern and development of HCC: co-infection of HBV with HDV was strongly associated with the development of HCC at a younger age [5]. In a retrospective study, using a national data repository in the USA, the incidence rate of HCC was 2.9-fold higher in HDV-positive vs. HDV-negative individuals [6]. The American cohort included more than 25,000 patients with HBV infection; 8.5% were tested for HDV, and most of the HDV-positive were hepatitis C virus (HCV) co-infected or addicted to substances (Fig. 2). Altogether these studies concluded that HDV patients are at a higher risk of HCC than the HBV mono-infected.

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Fig. 2. Incidence rates of hepatocellular carcinoma (HCC) and decompensation stratified by HDV status. Incidence rates reported as cases per 1,000 person-years. Data taken from Kushner 2015 [6].

In England, data were different. In a retrospective survey in London, which considered 962 HBV-positive patients and 82 of them positive for HDV antibody (8.5%), the risk of HCC was similar in the HBV/HDV co-infected patients and the HBV mono-infected [7].

Reported cross-sectional studies may have a selection bias because of interfering factors; many patients included were drug and alcohol abusers or had poor hygienic conditions. Therefore the increased risk for HCC could not be attributed to HDV alone.

When patients were evaluated over the long-term, complications related to portal hypertension were observed more frequently than HCC development (Table 1). In a homogeneous population of native Europeans with chronic hepatitis and cirrhosis, clinical decompensation (ascites, encephalopathy, jaundice and bleeding) was the dominant complication occurring in a fifth of the patients, whereas HCC developed in a minority (9%). In a study from Milan [9], 46 of 146 patients with cirrhosis developed HCC with an annual rate of 2.8%; HCC was observed after a mean follow-up of 83 months from the diagnosis of cirrhosis. The patients who developed HCC were predominantly males with a mean age of 55 years; markers of HBV and HDV replication were present in 50% and 74% of patients, respectively. More than half of these patients (n = 25) were still alive after 12-324 months from HCC diagnosis: 6 underwent liver transplantation, and 19 were treated with percutaneous ablation techniques [9]. In another Italian cohort, 17 of 188 patients followed for a mean time of 7.8 years, developed HCC [10]. Also, in this study the patients were mostly males (70%) with a mean age of 53 ± 13 years, were anti-HBe positive, and 53% had persistent HDV viremia. Forty-two patients with cirrhosis (22.3% of enrolled patients) experienced one or several episodes of liver decompensation, which included ascites (36 pts), jaundice (25 pts), encephalopathy (12 pts) and bleeding from esophageal varices (9 pts) (Fig. 3) [10].

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Table 1. Rates of hepatocellular carcinoma (HCC) development and events of decompensation in long-term follow-up studies of HDV-infected patients.

Study Patient Observation Decompensation HCC number period number (%) number (%) (years) Gheorghe, 2005 [8] 166 3.5 134 (80) 20 (12) Romeo, 2009 [9] 299 19 54 (18) 46 (15) Niro, 2010 [10] 188 7.8 42 (22) 17 (9) Buti, 2011 [11] 158 13 29 (18) 4 (3) Manesis, 2013 [12] 65 4.2 9 (14) 2 (3) SESSION 3 – HDV

Fig. 3. Outcome of patients with HDV chronic hepatitis and cirrhosis, in a cohort of Italian patients. Taken from Niro et al. [10].

In conclusion, CHD patients stand a high risk of decompensation and HCC. HCC occurrence is influenced by different factors in different areas of the world; cirrhosis certainly plays an important role.

HCC DIFFERENCE IN HBV AND HBV/HDV INFECTION Abbas et al. compared the clinical aspects of HCC in 92 cases of patients with CHD (HDV group) and 92 HBsAg-positive HDV-negative patients (HBV group). In the HDV group, the liver was smaller in size, thrombocytopenia more pronounced, and varices larger than in the HBV group; of interest, in the former group HCC was often multifocal at presentation, and alpha-fetoprotein levels were >1,000 IU/ml [13]. What are the factors of liver disease progression and HCC development in HDV?

The severity of the chronic HDV hepatitis may be dependent on viral and host factors.

HDV genotypes. Eight genotypes of HDV have been identified throughout the world. Genotype 1 is the most common; it is associated with a wide spectrum of disease severity. Patients infected by HDV genotype 2 in Taiwan had fewer adverse outcomes and a longer

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survival compared with genotype 1, either in the acute and the chronic form of the infection [14]. At odds, HDV genotype 3 is characterised by a higher pathogenicity, which is responsible for outbreaks of severe and fulminant hepatitis in northern South America. Genotypes 5 to 8 were described in West and Central Africa, where HDV plays a prominent role in liver cancers.

HBV genotypes. In HDV patients with a poor prognosis (cirrhosis, HCC or mortality due to liver failure), HBV genotype C was prevalent compared to genotype B. HDV genotype 3 patients with CHD of HDV genotype 3 were more frequently co-infected by the genotype F of HBV. This association is due to variations in the antigen-coding region of the HDV, which could interact with HBsAg to create new virions and modulate in this way the infectivity. The combination of HDV genotype 3 and HBV genotype F seems responsible for an aggressive disease pattern, at variance with the milder forms of the disease reported for HDV genotype 2 and HBV genotype B.

HDV and HBV replication. The replicative activity of HDV and HBV has a major influence on the course of liver disease; HDV replication persisting at high titers is associated with a worse prognosis. Due to the dynamic interplays between HDV and HBV, viremia may fluctuate during the follow-up of patients with CHD, so that viral dominance may vary and a serial determination of the replication activity of the two viruses is needed to identify the dominant strain. Early inhibition of HDV replication remarks a favourable outcome [3]. At the stage of cirrhosis, HDV replication tends to decline, and HBV DNA levels may increase, but this SESSION 3 – HDV profile has only a marginal impact on the occurrence of HCC and decompensation. Recent work by Romeo et al. investigated the correlation between HBsAg, HBV DNA, HDV RNA levels and the progression to cirrhosis, HCC and liver decompensation [15]. In non-cirrhotic patients, the amount of circulating HDV RNA impacted the progression to cirrhosis and HCC development; by the receiver operating characteristic curve (ROC) analysis, the predictive cut- off value for cirrhosis was set at 600,000 HDV RNA copies/ml. Once cirrhosis is established, the role of HDV replication as a predictor of a negative outcome was not a determinant.

Low levels of HBV DNA are commonly detected in HDV patients, due to the inhibition of HBV replication by the HDV proteins p24 and p27. Nevertheless, in specific epidemiological settings, such as drug addicts and the human immunodeficiency virus (HIV)-infected patients, 15-30% of patients are HBeAg and/or HBV DNA positive.

Patients, who responded to interferon (IFN) therapy clearing both HDV RNA and HBV DNA had stable disease with occasionally a dramatic improvement in liver histology and also with the regression of liver fibrosis [16]. The clearance of the HBsAg remains the ultimate endpoint of the cure: it provides the only reliable proof that HDV has been eliminated.

HIV-infected patients. The interaction of HDV with HIV leads to a more rapid progression of fibrosis: cirrhosis was diagnosed in 30% of HIV/HDV co-infected patients at presentation, and HDV was a major driver for decompensation, HCC development and mortality. Should we tailor surveillance according to a patients’ profile?

The management of CHD is similar to other forms of chronic viral hepatitis. Patients with HBV/HDV co-infections are assessed for liver disease activity, and stage; based on disease severity and fibrosis stage, treatment and HCC surveillance are enforced.

Different clinical scores are available to classify HDV patients with liver pathology. The commonly used Model for End-stage Liver Disease (MELD) and Child-Pugh score, are independent from the aetiology of the disease. A new score has been designed specifically for HDV liver disease. The baseline-event-anticipation score (BEA score) includes age, sex, region of origin, bilirubin, and platelets counts [17]. Using the hazard ratio (HR) and ROC

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PARIS, FRANCE curves, three groups (BEA-A-B-C) were identified with a mild, moderate and high risk of liver complications. This score needs further validation in a large cohort and in patients with different genotypes. What is known about the mechanisms of oncogenicity in HDV?

The mechanisms of carcinogenesis are not well understood, yet HDV could play by itself an oncogenetic role [18, 19]. The virus exerts epigenetic control over HBV transcription and replication. The two isoforms of the HDV protein, the small hepatitis D antigens or p24 and the large hepatitis D antigens or p27, could inhibit HBV replication by repressing HBV enhancers. The p27 isoform inhibits IFN-α signalling by a complex interference with the Janus kinase, tyrosine kinase 2, signal transducer and activator of transcription, resulting in HBV replication inhibition.

Oxidative stress. The oxidative stress linked to the severe necroinflammation induced by HDV may lead to HCC development. The large HDV antigen regulates several cellular functions, e.g. activation of the STAT3 (signal transducer and activator of transcription 3) and NF-kB (nuclear factor kappa B) pathways; it elicits an increase in reactive oxygen species (ROS) that are implicated in cell transformation and tumorigenesis.

DNA methylation. HDV could exert an oncogenic property by altering DNA methylation events. HDV infection induced the expression of DNA methyltransferases 3b (DNMT3b) and SESSION 3 – HDV was associated with E2F1 transcription factor hypermethylation (Fig. 4) [20].

Histone modification. HDV enhances clusterin expression via histone acetylation. Clusterin is involved in oncogenic pathways and is overexpressed in HCC tissue. Although associated with cell cycle death and inhibition of proliferation, this protein could also increase cell survival and allow tumorigenesis.

Fig. 4. DNA methylation patterns are established and maintained by DNMTs (DNA methyltransferases) enzymes that are essential for proper gene expression patterns. In humans, overexpression of these enzymes has been linked to a variety of cancers. Taken from Rodeniser et al. [20].

What is the experience of the new drugs against HCC related to HDV?

No data on the use of sorafenib or regorafenib specific for HDV-infected patients are available. In the pivotal clinical trials evaluating these drugs, HDV co-infection was an exclusion criterion. This same situation was followed in trial protocols exploring the benefit of immune checkpoint inhibitors, such as tremelimubab and nivolumab [21].

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References

References in bold are required reading

[1] Wranke A, Pinheiro Borzacov LM, Parana R, Lobato C, Hamid S, Ceausu E, et al. Clinical and virological heterogeneity of hepatitis delta in different regions world-wide: The Hepatitis Delta International Network (HDIN). Liver Int 2017;00:1-9.

[2] Rizzetto M, Verme G, Recchia S, Bonino F, Farci P, Aricò S, et al. Chronic hepatitis in carriers of hepatitis B surface antigen, with intrahepatic expression of the delta antigen. An active and progressive disease unresponsive to immunosuppressive treatment. Ann Intern Med. 1983;98:437-41.

[3] Rosina F, Conoscitore P, Cuppone R, Rocca G, Giuliani A, Cozzolongo R, et al. Changing pattern of chronic hepatitis D in Southern Europe. Gastroenterology 1999;117:161-6.

[4] Ji J, Sundquist K, Sundquist J. A population-based study of Hepatitis D Virus as potential risk factor for hepatocellular carcinoma. J Natl Cancer Inst 2012; 104: 790-792

[5] Oyunsuren T, Kurbanov F, Tanaka Y, Elkady A, Sanduijav R, Khajidsuren O, et al. High frequency of hepatocellular carcinoma in Mongolia; association with mono-, or co- infection with hepatitis C, B, and Delta Viruses. J Med Virol. 2006; 78 (12): 1688-95.

[6] Kushner T, Serper M, Kaplan DE. Delta Hepatitis within the Veterans Affairs Medical System in the United States: Prevalence, Risk Factors, and Outcomes. J Hepatol.2015; 63 (3): 586-592.

[7] Cross TJ, Rizzi P, Horner M, Jolly A, Hussain MJ, Smith HM, et al. The increasing prevalence of hepatitis delta virus (HDV) infection in South London. J Med Virol. 2008; 80 (2): 277-82.

[8] Gheorghe L, Iacob S, Simionov I, V dan R, Gheorghe C, Iacob R, et al. Natural History of compensated viral B and D cirrhosis. Rom J Gastroenterol. 2005; 14 (4): 329-35.

[9] Romeo R, Del Ninno E, Rumi MG, Russo A, Sangiovanni A, De Franchis R, et al. A 28-Year Study of the course of Hepatitis Delta Infection: a risk factor for Cirrhosis and Hepatocellular Carcinoma. Gastroenterology 2009;136:1629-38.

[10] Niro GA, Smedile A, Ippolito AM, Ciancio A, Fontana R, Olivero A, et al. Outcome of chronic delta hepatitis in Italy: a long-term cohort study. J Hepatol. 2010; 53 (5): 834-40.

[11] Buti M, Homs M, Rodríguez-Frias F, Funalleras G, Jardì R, Sauleda S, et al. Clinical outcome of acute and chronic hepatitis delta over time: a long-term follow-up study. J Viral Hepat. 2011; 18 (6): 434-42.

[12] Manesis EK, Vourli G, Dalekos G, Vasiliadis T, Manolaki N, Hounta A, et al. Prevalence and clinical course of hepatitis delta infection in Greece: A 13-year prospective study. J Hepatol. 2013; 59 (5): 949-56.

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[13] Abbas Z, Qureshi M, Hamid S, Jafri W. Hepatocellular Carcinoma in Hepatitis D: does it differ from Hepatitis B monoinfection? Saudi J Gastroenterol. 2012; 18 (1): 18-22.

[14] Su CW, Huang YH, Huo TI, Shih HH, Sheen IJ, Chen SW, et al. Genotypes and Viremia of Hepatitis B and D viruses are associated with outcomes of Chronic Hepatitis D patients. Gastroenterology 2006; 130:1625-35.

[15] Romeo R, Foglieni B, Casazza G, Spreafico M, Colombo M, Prati D. High serum levels of HDV RNA are predictors of cirrhosis and liver cancer in patients with Chronic Hepatitis Delta. PLoS One. 2014 21; 9 (3): e92062.

[16] Farci P, Roskams T, Chessa L, Peddis G, Mazzoleni AP, Scioscia R, et al. Long-term benefit of interferon alpha therapy of chronic hepatitis D: regression of advanced hepatic fibrosis. Gastroenterology 2004;126:1740-9.

[17] Calle Serrano B, Groβhenning A, Homs M, Heidrich B, Erhardt A, Deterding K et al. Development and evaluation of a baseline-event-anticipation score for hepatitis delta. J Viral Hep 2014; 21(11):e154-e163.

[18] Abbas Z, Abbas M, Abbas S, Shazi L. Hepatitis D and hepatocellular carcinoma. World J Hepatol 2015;7(5):777-86.

[19] Alfaiate D, Dény P, Durantel D. Hepatitis delta virus: From biological and medical aspects to current and investigational therapeutic options. Antiviral Research 2015; 122:112-29.

[20] Rodenhiser D, Mann M. Epigenetics and human disease: translating basic biology into clinical applications. CMAJ 2006 31; 174 (3): 341-8.

[21] Kudo M. Immune Checkpoint Inhibition in Hepatocellular Carcinoma: Basics and Ongoing Clinical Trials. Oncology. 2017;92(Suppl 1):50-62.

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THE ROLE OF LIVER TRANSPLANTATION IN HEPATITIS DELTA

Bruno Roche2,3,4, Didier Samuel1,2,3,4* 1Assistance Publique-Hopitaux de Paris, Hôpital Paul Brousse, Centre Hépato-Biliaire, Villejuif, France; 2Univ. Paris-Sud, UMR-S 1193, Villejuif, France; 3Inserm, Unité 1193, Villejuif, France; 4Hepatinov, Villejuif, France. E-mail address: [email protected]

TAKE-HOME MESSAGES • Previous control of hepatitis B virus (HBV) infection reduced the incidence of hepatitis D in developed countries. However, the decreasing trend has come to a stop due to an increased proportion of hepatitis delta virus (HDV)-infected patients migrating from endemic HDV areas.

• Liver transplantation is the only treatment option for patients with end-stage liver disease,

SESSION 3 – HDV hepatocellular carcinoma or fulminant hepatitis due to co-infection or super-infection with HDV and HBV.

• Using combination prophylaxis with hepatitis B immune globulins (HBIG) and antivirals, patients infected with HBV and HDV are less at risk of HBV recurrence post-transplantation than patients infected with HBV alone, and have a better survival rate.

• HDV patients are at risk of re-infection by both HBV and HDV. As no antiviral treatment is available for HDV infection, most experts recommend not to discontinue HBIG.

INTRODUCTION Around 5% of the chronic carriers of hepatitis B virus (HBV) worldwide have serological evidence of exposure to hepatitis delta virus (HDV). HDV co-infection is associated with more severe liver disease and a higher incidence of cirrhosis than HBV mono-infection [1, 2]. Liver transplantation (LT) is the only treatment option for patients with end-stage liver disease, hepatocellular carcinoma (HCC) or fulminant hepatitis due to co-infection or super-infection with HDV and HBV. Patients chronically infected with HBV and HDV are less at risk of HBV recurrence post- LT than patients infected with HBV alone, related to a low level of HBV replication at the time of LT, which is a major risk factor of HBV recurrence, and have a better survival rate [3]. The use of potent antivirals against HBV in combination with hepatitis B immune globulins (HBIG) has further reduced the risk of HBV-HDV re-infection. However, HDV patients are at risk of re-infection by both HBV and HDV, and currently, there is no effective treatment against HDV. In this review, we will discuss the epidemiology of hepatitis delta, indications and results of LT, mechanisms of HDV re-infection, and prophylaxis of recurrence.

EPIDEMIOLOGY OF HEPATITIS DELTA The distribution pattern of HDV is worldwide but not uniform (Fig. 1). For example, 90% of HBV carriers are infected with both viruses in the Pacific Islands, whereas the rates decline

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PARIS, FRANCE to 8% in Italy and 6% in Japan. Current estimates suggest that 15-20 million people are infected with HDV. However, these estimates could be inaccurate since systematic screening of HBV-infected individuals is not performed. HDV is divided into 8 genotypes. Genotype 1 is prevalent worldwide, whereas genotype 2 and 4 are found in Japan and Taiwan. Genotype 3 is common in the Amazonian basin whereas genotypes 5-8 are found in Africa. Since the 1990s, the circulation of HDV has declined significantly in Europe, mainly due to the implementation of prophylaxis against HBV (i.e., vaccination, screening of blood products and pregnant women, implementation of safety procedures against blood-borne infections among healthcare workers, improved socioeconomic conditions, and the increased awareness of the general public on sexually transmissible agents), but HDV remains a major health problem in many underdeveloped areas of the world were HBV is uncontrolled [1]. Sequential surveys in Italy documented an anti-HDV antibody rate of 24.6% in 1983, of 14% in 1992 and of 8.3% in 1997. During the period 1987-2014, the proportion of cases in the age group of 0-29 and 30-49 years strongly decreased from 39.8% to 3.3% and from 50.6% to 16.4%, respectively. In contrast, the proportion of cases in patients aged 50 years or older increased from 9.6% to 80.3% [1]. However, recent studies in the UK and Germany have revealed that the decline was not sustained [2]. The main source of HDV maintenance in Europe appears to be the massive immigration rates, but also the small pools of intravenous drug users and the survivors of the HDV epidemic in the 1970-80s [1]. SESSION 3 – HDV

Fig. 1. Worldwide prevalence of HDV and the geographic distribution of its genotypes (adapted from [2]).

INDICATIONS AND RESULTS OF LIVER TRANSPLANTATION FOR DELTA VIRUS INFECTION LT is the only treatment option for patients with end-stage liver disease, HCC or fulminant liver failure related to co-infection or super-infection with HDV and HBV. In Europe, in 2009, 7% and 2% of patients undergoing LT have liver disease that is associated with HBV mono- infection and HBV-HDV co-infection, respectively (Fig. 2) [4].

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3244 Fig. 1. Worldwide prevalence of HDV and the geographic distribution of its genotypes 3245 (adapted from [2]).

3246

3247 Indications and results of liver transplantation for delta virus infection

3248 LT is the only treatment option for patients with end-stage liver disease, HCC or 3249 fulminant liver failure related to co-infection or super-infection with HDV and HBV. In 3250 Europe, in 2009, 7% and 2% of patients undergoing LT have liver disease that is THE INTERNATIONAL LIVER CONGRESS™ 2018 3251 associated with HBV mono-infection and HBV-HDV co-infection, respectively (Fig. 2) 3252 EASL[4]. POSTGRADUATE COURSE

Without HCC Without HCC With HCC With HCC

3253

3254 Fig.Fig. 2. EvolutionEvolution of of liver liver transplantation transplantation for viral for cirrhosisviral cirrhosis in Europe in [8].Europe [8].

In 1993, a large European multicentre study including 372 patients showed that the overall three-year actuarial risk of HBV recurrence was 67% in the patients with HBV-related cirrhosis and 32% in those with HDV-related cirrhosis [3]. For patients with HDV cirrhosis, the three-year actuarial risk of HBV recurrence was 70% and 17% among patients not given

SESSION 3 – HDV immunoprophylaxis and patients given long-term HBIG prophylaxis, respectively (p <0.001). In multivariate analysis, the independent predictors of a lower risk of HBV recurrence after transplantation were HDV co-infection (relative risk 3.92; 95% confidence interval, 2.48 to 6.21; p <0.001), acute liver disease (relative risk 3.76; 95% confidence interval, 1.70 to 8.30; p <0.001), and long-term administration of HBIG (relative risk 3.28; 95% confidence interval, 1.92 to 5.60; p <0.001). The lower risk of recurrence among the patients with HDV cirrhosis could be explained by the fact that 70-90% of patients with HDV co-infection are hepatitis B e antigen (HBeAg)-negative, and most have low serum HBV DNA level due to the inhibitory effect of HDV on HBV replication. The actuarial survival rate of patients undergoing transplantation for HBV cirrhosis and HDV cirrhosis was 48% and 85%, respectively. In multivariate analysis, HDV co-infection (relative risk 6.25; 95% percent confidence interval, 3.13 to 12.42; p <0.001) and long-term administration of HBIG (relative risk 2.22; 95% percent confidence interval, 1.13 to 4.33; p <0.001) were independently predictive of a better survival rate.

A major advance came with the availability of highly effective and well-tolerated antiviral agents against HBV, in combination with HBIG, which reduced the risk of re-infection to less than 5-10% during the first 2 years following LT. As a result, the outcomes of patients with HBV and HDV liver disease undergoing LT are now similar to or better than those of patients undergoing LT for other indications. The European liver transplant registry data show that the five- and ten-year survival rates were 89 and 86% in patients transplanted for HDV cirrhosis without HCC and 78 and 73% in patients transplanted for HDV cirrhosis and HCC, respectively (Fig. 3) [4].

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Without HCC With HCC

Fig. 3. Survival after liver transplantation for viral cirrhosis in Europe [8].

PHYSIOPATHOLOGY OF HDV RECURRENCE HDV is a defective RNA agent requiring the presence of HBV for its life cycle. The transplant model provides new insights into the pathology of HDV and its interactions with HBV. The course of HDV re-infection varies depending on whether or not HBsAg is present. In the few SESSION 3 – HDV cases where HBsAg reappeared, it was associated with a combined HBV-HDV replication; HBV DNA and HDV RNA were present in the serum or HDAg was present in the liver, or both. Acute hepatitis, followed by chronic hepatitis, developed. HBV-HDV recurrence is, in general, less severe than HBV recurrence alone [5].

Ottobrelli et al., speculates that HDV can cause in the grafted liver subclinical infections independently of an overt HBV infection [6]. In these patients, re-infection with HDV was shown by the intermittent finding of HDV RNA in sera and HDAg in the graft, and the absence of HBV markers in sera or liver. However, in most transplant recipients HBV suppression was only transitory, and the recurrence of HBV led to the intrahepatic spread of HDV and the relapse of the disease with typical clinical, virologic and histological patterns. In patients receiving long-term HBIG prophylaxis without HBV recurrence, we observed in the first post-transplant year, serum HDV RNA or liver HDAg in 88% of patients with a histologically normal graft [5]. In the long-term, these markers were detectable in only 5% of the patients. These data suggest that in the transplant setting, HDV replication can be independent from that of HBV but that HDV hepatotoxicity is HBV dependent.

The unexpected finding of HDV viremia in the absence of detectable HBV has raised questions about the biology of HDV in the transplantation setting. The hypotheses for explaining the presence of HDV replication in HBsAg-negative patients are: 1) HBV markers could be present but not detectable; 2) HDV is present in the hepatocytes in the absence of HBsAg but either cannot replicate or has a low replication level; 3) the level of HDV RNA in the liver is much lower in patients without HBsAg than those with HBsAg, and this low level of delta virus may explain the absence of liver graft lesions. Smedile et al., using more sensitive PCR-based assays for HDV and HBV detection, showed that HBV and HDV were always detected coincidentally in serum following the post-LT incubation period [7]. This temporal association confirmed the dependence of HDV on its helper HBV. HDV viremia reached maximum levels only after full expression of HBV had occurred. The failure to observe HBV antigen staining in the graft is likely caused by the fact that pre-LT levels of HBV are 10,000-fold less than the level of HDV, and a proportionately smaller number of hepatocytes would be infected with HBV. Mederacke et al., in a recent study described early HDV RNA kinetics during the first days after LT [8].

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They showed that HDV RNA becomes rapidly negative in serum within 1 to 10 days after LT and that the HDV RNA decline parallels almost exactly with HBsAg decline. However, HDV antigen was detectable in the liver graft in six out of 26 patients up to 19 months post- LT in the absence of liver HBV DNA, cccDNA, serum HBsAg and HDV RNA. This latency of HDV in the graft could be a potential concern, as subsequent HBV co-infection may result in productive HDV infection. The authors suggested that shortening the duration of HBIG administration in HDV/HBV patients could have detrimental consequences as re-infection in the case of HDV latency may lead to chronic hepatitis delta.

PROPHYLAXIS OF HBV/HDV RECURRENCE Risk factors predicting HBV/HDV recurrence

With or without prophylaxis, high HBV DNA level (>105 copies/ml) at transplant, an uncommon feature in HDV co-infected patients, is the strongest predictive factor of HBV recurrence (Table 1) [3, 9, 10]. Surrogate markers for low levels of HBV replication including HBeAg-negative status at transplant, HDV co-infection and fulminant hepatitis are predictive of a low risk of HBV recurrence [3, 9]. SESSION 3 – HDV PRE-TRANSPLANT ANTIVIRAL TREATMENT Patients co-infected with HBV and HDV have low levels of HBV DNA and generally do not require pre-transplant antiviral B therapy. Treatment with nucleos(t)ide analogues is not effective at reducing HDV replication but might be useful in patients with high HBV replication and be of potential benefit when used long-term by gradually reducing HBsAg concentration. There is currently no approved therapy for HDV infection. Pegylated interferon (PegIFN) has demonstrated better response to treatment in comparison with classical IFN. Sustained virological response was observed in 20 to 36% and 31 to 43% using standard IFN and PegIFN, respectively [2]. The use of IFN therapy is limited to patients with compensated cirrhosis and contraindicated in patients with decompensated cirrhosis. Even in the absence of HBV or HDV replication, patients remain at risk of HCC and continued surveillance is needed until the time of LT. Post-transplant prevention of HBV/HDV recurrence a) Combination prophylaxis

In patients with HBV/HDV co-infection, recurrence of HBV infection after LT can lead to HBV/HDV re-infection of the graft. The latter could have deleterious consequences, as no effective antiviral drug is available for the treatment of HDV infection. Thus, many experts recommend long-term administration of combination prophylaxis after LT. In contrast, the risk of HBV recurrence is lower in patients transplanted for HBV/HDV co-infection as compared with patients transplanted for HBV mono-infection.

The use of antivirals pre-transplantation followed by a combination of antivirals and HBIG post-LT provides improved control of HBV replication pre-transplantation and complementary forms of prophylaxis post-transplantation to minimise the risk of re-infection. This regimen is considered as the standard of care against HBV recurrence after LT. Combination prophylaxis allowed a reduction of the HBIG dose required for long-term use. With this combination approach, the HBV recurrence rate at 1 to 2 years post-transplantation has been reduced to 0-10% (Table 1) [8-16].

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PARIS, FRANCE NA 100% 100% 1 years 92% 1 years 88% 5 years 95% 1 years 91% 1 years 81% 5 years Survival copies/ml 5 NA NA NA HCC pre-LT Pre-LT HBV DNA >10 Pre-LT HBV DNA HBIG monoprophylaxis Risk factors for HBV recurrence 15% 0% 0% 8% 6.6% 1 years 1% 4% 5 years HBV recurrence N (%) SESSION 3 – HDV 54 40 15 NA 43 17 Follow-up (months) LAM + HBIG = 18) (n LAM + HBIG IM titrated maintain to anti-HBs IU/L >200 LAM + HBIG IV IU/month 10,000 LAM or famciclovir + HBIG IV titrated to maintain anti-HBs >100 IU/ L LAM ± ADV or TDV + HBIG IV 10,000 IU/ month LAM + HBIG IM 400-800 IU/month Prevention of HBV recurrence 100% 100% 8.5% 15.5% 26% 5% HDV co- infection 10 (38%) 0 16 (27%) NA 21 (41%) (85%) 125 HBV DNA positive (%) LT at 26 25 59 206 51 147 No. of patients Mederacke [8] Caccamo [15] Han [12] Authors (references) Indefinite high-dose IV HBIG Steinmuller [13] Faria [9] Indefinite low-dose IM HBIG Gane [10] Table 1. Prevention 1. Table recurrence HBV of after liver transplantation with antiviral and anti-HBs (HBIG). Ig

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EASL POSTGRADUATE COURSE 97% 90% 83 months 100% Survival Shorter HBIG duration NA Risk factors for HBV recurrence ) ) 5 5 0% (if HBV DNA <10 2 (5.8%) a patient received an HBsAg- positive graft 11% 23% (if23% HBV DNA >10 HBV recurrence N (%) SESSION 3 – HDV 14 28 83 Follow-up (months) LAM + HBIG 6 months then LAM + ADV Antiviral + HBIG (median months) 18 then antiviral LAM + HBIG 1 month then LAM Prevention of HBV recurrence 9.8% 100% 20% HDV co- infection 41 (44.5%) 1 (3%) 0 HBV DNA positive (%) LT at 92 34 20 No. of patients Neff [14] Cholongitas [16] Authors (references) Short-term HBIG and long-term antivirals Buti [11] NA, not available; LT, Liver transplantation;NA, available; not LAM, LT, lamivudine; entecavir; tenofovir; ETV, adefovir; HCC, hepatocellular TDV, ADV, carcinoma; intravenous;IV, IM, intramuscular.

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Combination protocols are heterogeneous with regards to the dosing, duration and routes of HBIG administration (Table 1). In these studies, the proportion of patients co-infected with HBV and HDV was low (between 5 and 26%). Two studies assessed the efficacy of combination prophylaxis in patients transplanted for HBV/HDV co-infection [8, 15]. Caccamo et al. evaluated 46 patients with HBV/HDV co-infection [15]. Twenty-one received HBIG monoprophylaxis and 25 received combination prophylaxis with HBIG + lamivudine (LAM). No HBV/HDV recurrence was observed during the follow-up period. In this study, the combination prophylaxis is cost-efficient as compared with HBIG monoprophylaxis in the first two post-transplant years. Mederacke et al. reported 26 patients transplanted for HBV/HDV co-infection receiving combination prophylaxis [8]. No HBV/HDV recurrence was observed during the study period. The most cost-effective regimen reported to date, is a very low intramuscular (IM) HBIG plus LAM regimen decreasing costs by more than 90% as compared with intravenous (IV) regimen with a recurrence rate as low as 4% at 4 years [10]. More recently, subcutaneous regimens of HBIG have also proven effective, with some advantage in tolerability and possibility of self-administration by the patient at home [17].

Several meta-analyses have compared the use of HBIG, antivirals or both [18, 19]. Despite methodological limitations of studies included in these meta-analyses, combination prophylaxis was significantly superior to antivirals or HBIG alone in preventing HBV recurrence, irrespective of the HBV DNA level at transplantation and reducing overall and HBV-related mortality in some studies. SESSION 3 – HDV

The optimal HBIG protocol is yet to be defined. Further research is needed to determine the dose and duration of HBIG after LT and whether HBIG can be stopped. b) Alternatives to long-term combination HBIG and antiviral prophylaxis

Indefinite combination therapy with HBIG plus a nucleos(t)ide analogue may not be required in all liver transplant recipients. The replication status of the patient prior to the initiation of antiviral therapy should guide prophylaxis. Alternative strategies to consider, especially in patients without detectable HBV DNA prior to transplantation, are the discontinuation of HBIG after some defined interval and continuing treatment with antivirals alone. The high cost of long-term HBIG and the inconvenience of parenteral administration prompt consideration of these other treatment approaches.

In a study of 29 patients (4 were HDV co-infected), high-dose HBIG and LAM were used in the first month, and then patients were randomised to receive either LAM monotherapy or LAM plus HBIG at 2,000 IU IM monthly [11]. None of the patients developed HBV recurrence during the first 18 months but later recurrences developed in four patients on LAM after 5 years of follow-up. Cholongitas et al. reported 34 patients transplanted for HBV/HDV co-infection receiving combination prophylaxis [16]. After a median time of 18 months (range, 6-144 months) HBIG was discontinued. Two patients (5.8%) had HBV/HDV recurrence after HBIG withdrawal (median follow-up: 28 months; range, 12-58). One of them had received an HBsAg-positive liver graft. The authors found that patients with HBV/HDV recurrence have received HBIG for a shorter period than those without recurrence (median: 9 vs. 28 months, p = 0.008). The small number of patients included in this study prevents reliable suggestions, and further larger prospective studies are needed for firm conclusions. An alternative approach is to switch from HBIG/LAM to a combination of antiviral agents that present a greater barrier to the development of resistance than LAM. In a randomised prospective study, 16 of 34 patients (2 were HDV co-infected) receiving low-dose IM HBIG/LAM prophylaxis who were at least 12 months post-LT were switched to adefovir/LAM combination therapy, whereas the remaining patients continued HBIG/LAM [20]. At a median follow-up of 21 months post-

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switch, no patient had disease recurrence, although one patient in the adefovir/LAM group had a low titer of HBsAg in serum but was repeatedly HBV DNA negative. Using the same protocol, Saab et al. switched 61 liver transplant recipients (3 out of 22 with available HDV data were positive) to a combination of a nucleoside (LAM or entecavir) and nucleotide analogue (adefovir or tenofovir) [21]. At a median follow-up of 15 months post-switch, two patients were HBsAg-positive in serum but repeatedly HBV DNA negative. Drug compliance during long- term antiviral therapy may be a very important issue for transplant patients who feel healthy but have a lifelong risk of HBV recurrence.

An ultimate approach was to evaluate the safety of complete and sustained prophylaxis withdrawal in liver transplant recipients at low risk of HBV recurrence. Lenci et al., evaluated a cohort of 30 patients at low risk of recurrence (HBeAg and HBV DNA negative at LT, 23% HDV co-infected) and treated them with a combination of HBIG and LAM (+/- adefovir) for at least 3 years [22]. Sequential liver biopsies were performed and evaluated for the presence of intrahepatic total HBV DNA and cccDNA. Using the absence of intrahepatic total HBV DNA and cccDNA as a guide, HBIG and then antiviral therapy was withdrawn in a stepwise fashion. Six years after completion of prophylactic withdrawal, HBV recurrence (HBsAg seroreversion +/- detectable HBV DNA) occurred in six patients: in one patient after HBIG interruption and in 5 after both HBIG and antiviral cessation. Only three patients required re-starting HBV prophylaxis because of persistent HBV replication, and all achieved optimal control of HBV infection and did not

SESSION 3 – HDV experience clinical events. The other patient who recurred showed only short-lasting HBsAg positivity, with undetectable HBV DNA, followed by spontaneous anti-HBs seroconversion.

The studies to date highlight several key issues to consider with the discontinuation of HBIG post-transplantation: First, the risk of HBV recurrence after cessation of HBIG may increase with the time off HBIG; second, HBsAg positivity in some patients might have deleterious consequences in the setting of HBV/HDV co-infection; third, HBV DNA persists in serum, liver or peripheral blood mononuclear cells even 10 years after LT, in a proportion of HBV transplanted patients who are HBsAg-negative, these reservoirs may serve as a source of HBV and HDV re-infection in the future [23]; fourth, currently we lack the ability to identify patients who may have cleared HBV post-transplantation.

Based on recent data, antiviral monoprophylaxis after HBIG discontinuation may be considered but only in the setting of clinical trials. c) Guidelines and the future prospects for preventing HBV/HDV re-infection

The principles in strategies to prevent HBV/HDV recurrence should be to maximise antiviral potency while minimising the risk for viral resistance, costs, side effects, and inconvenience to patients. HBIG and antiviral combination prophylaxis have been the gold standard for preventing recurrent disease. Today, low-dose IM or subcutaneous HBIG in combination with a potent nucleos(t)ide analogue is the most cost-effective prophylaxis. Preoperative antiviral B therapy could be discussed in patients with low HBV replication and decompensated cirrhosis. Entecavir, tenofovir or a nucleoside/nucleotide combination should be used in preference to LAM. Few studies have evaluated the possibility to use short-term HBIG (12-24 months) then switched to antiviral therapy. Although potent HBV-polymerase inhibitors can control HBV replication, HBsAg reappearance and/or HBV DNA persistence in serum, liver or peripheral blood mononuclear cells might have deleterious consequences in the setting of HBV/HDV co- infection. Patients co-infected with HDV are at risk of re-infection by both HBV and HDV. As no antiviral treatment is available for HDV infection, most experts recommend not to discontinue HBIG (Fig. 4).

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Status HBsAg (+) HBV DNA <104-5 copies/ml HDV RNA positive

Pre-LT - No preoperative antiviral B or Delta therapy - Discuss antiviral B therapy if HBV DNA positive and decompensated cirrhosis

Anhepatic HBIG IV phase at LT

First post- Low dose IV or IM HBIG operative week

Post-LT - IM-SC HBIG to maintain anti-HBs level

at >100 IU/L + second generation anti- SESSION 3 – HDV viral agent - Monitor for HBV/HDV recurrence

Fig. 4. Prophylaxis for prevention of HBV graft recurrence following transplantation for HBV/HDV co-infection. Proposal for guideline. LT, liver transplantation; HBIG, hepatitis B immune globulin; IM, intramuscular; IV, intravenous, SC, subcutaneous.

Transplant recipients should be monitored for HBV/HDV recurrence using HBsAg and HBV DNA every 3 months, HDV RNA every 6 months to detect re-infection and/or the emergence of viral resistance.

CONCLUSIONS Liver transplantation is the only treatment option for patients with end-stage liver disease, HCC or fulminant hepatitis due to co-infection or super-infection with HDV and HBV. Due to low pre-transplant levels of HBV replication, patients chronically infected with HBV and HDV are less at risk of HBsAg reappearance post-transplantation than patients infected with HBV alone and have better survival. HBIG and antiviral combination prophylaxis is the gold standard for preventing recurrent disease. Today, low-dose IM or subcutaneous HBIG in combination with a potent nucleos(t)ide analogue is the most cost-effective prophylaxis. The latency of HBV in the graft could be a potential concern, as subsequent HDV super- infection may result in productive HDV infection. As no antiviral treatment is available for HDV infection, most experts recommend not to discontinue HBIG.

ACKNOWLEDGEMENT The authors thank Vincent Karam (Data Manager of the European Liver Transplant Registry) for his help for collecting data for the present manuscript.

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[8] Mederacke I, Filmann N, Yurdaydin C, Bremer B, Puls F, Zacher BJ, et al. Rapid early HDV RNA decline in the peripheral blood but prolonged intrahepatic hepatitis delta antigen persistence after liver transplantation. J Hepatol 2012;56:115-122.

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[10] Gane EJ, Angus PW, Strasser S, Crawford DH, Ring J, Jeffrey GP, et al. Lamivudine plus low-dose hepatitis B immunoglobulin to prevent recurrent hepatitis B following liver transplantation. Gastroenterology 2007;132:931-937.

[11] Buti M, Mas A, Prieto M, Casafont F, Gonzalez A, Miras M, et al. A randomized study comparing lamivudine monotherapy after a short course of hepatitis B immune globulin (HBIg) and lamivudine with long-term lamivudine plus HBIg in the prevention of hepatitis B virus recurrence after liver transplantation. J Hepatol 2003;38:811-817.

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[13] Steinmuller T, Seehofer D, Rayes N, Muller AR, Settmacher U, Jonas S, et al. Increasing applicability of liver transplantation for patients with hepatitis B-related liver disease. Hepatology 2002;35:1528-1535.

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[15] Caccamo L, Agnelli F, Reggiani P, Maggi U, Donato MF, Gatti S, et al. Role of lamivudine in the posttransplant prophylaxis of chronic hepatitis B virus and hepatitis delta virus coinfection. Transplantation 2007;83:1341-1344.

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[17] De Simone P, Romagnoli R, Tandoi F, Carrai P, Ercolani G, Peri E, et al. Early Introduction of Subcutaneous Hepatitis B Immunoglobulin Following Liver Transplantation for Hepatitis B Virus Infection: A Prospective, Multicenter Study. Transplantation 2016;100:1507-1512.

[18] Loomba R, Rowley AK, Wesley R, Smith KG, Liang TJ, Pucino F, et al. Hepatitis B immunoglobulin and Lamivudine improve hepatitis B-related outcomes after liver transplantation: meta-analysis. Clin Gastroenterol Hepatol 2008;6:696-700.

[19] Cholongitas E, Goulis J, Akriviadis E, Papatheodoridis GV. Hepatitis B immunoglobulin and/or nucleos(t)ide analogues for prophylaxis against hepatitis b virus recurrence after liver transplantation: a systematic review. Liver Transpl 2011;17:1176-1190. SESSION 3 – HDV [20] Angus PW, Patterson SJ, Strasser SI, McCaughan GW, Gane E. A randomized study of adefovir dipivoxil in place of HBIG in combination with lamivudine as post-liver transplantation hepatitis B prophylaxis. Hepatology 2008;48:1460-1466.

[21] Saab S, Desai S, Tsaoi D, Durazo F, Han S, McClune A, et al. Posttransplantation hepatitis B prophylaxis with combination oral nucleoside and nucleotide analog therapy. Am J Transplant 2011;11:511-517.

[22] Lenci I, Baiocchi L, Tariciotti L, Di Paolo D, Milana M, Santopaolo F, et al. Complete hepatitis B virus prophylaxis withdrawal in hepatitis B surface antigen-positive liver transplant recipients after longterm minimal immunosuppression. Liver Transpl 2016;22:1205-1213.

[23] Roche B, Feray C, Gigou M, Roque-Afonso AM, Arulnaden JL, Delvart V, et al. HBV DNA persistence 10 years after liver transplantation despite successful anti-HBS passive immunoprophylaxis. Hepatology 2003;38:86-95.

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WEDNESDAY 11 APRIL 2018 16:00-17:30 SESSION 4 – HEV

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EASL POSTGRADUATE COURSE

HOW TO BECOME INFECTED WITH HEPATITIS E VIRUS IN EUROPE

Robert A de Man Department of Gastroenterology and Hepatology, Erasmus Medical Center, Rotterdam, Netherlands. E-mail address: [email protected]

TAKE-HOME MESSAGES • Hepatitis E virus (HEV) genotype 3 infects humans in Europe and is a zoonosis restricted to pigs, wild boar and deer.

• Transmission has been documented by food containing insufficiently heated pork liver. Food-related outbreaks have also been documented in relation to consumption of infected shellfish.

• Different infection model systems show that inactivation of HEV needs heating to at least 70°C core temperature for at least 2-5 minutes.

• The risk of HEV transfusion from dietary and environmental exposure far exceeds the risk of transfusion to recipients from unscreened donors. All susceptible recipients using SESSION 4 – HEV immunosuppressive drugs should receive dietary advice to decrease the food-related infection risk.

• Person to person transmission of genotype 3 in Europe is too inefficient to cause clinical disease. When normal hygienic measures are followed, there is no rationale for specific isolation measures when a patient is admitted to hospital.

HEPATITIS E VIRUS IN EUROPE HAS AN ANIMAL RESERVOIR Hepatitis E virus (HEV) genotype 1-4 infects pigs, deer and wild boar. Genomic sequence comparison data suggests that in Europe HEV carried by pigs and humans are genetically closely related and that cross-species transmission is occurring. Transmission from the animal to humans after eating undercooked meat has been described in several papers linking the infected animal liver to sporadic cases of hepatitis in patients [1,2,3]. The pig is considered the primary host for HEV. Infected pigs have no symptoms. HEV RNA is present in serum, faeces and pig liver of animals going to the slaughter. A survey of raw uncooked human pig liver in retail services has shown HEV PCR positivity in up to 6.5% in the Netherlands and up to 11% in a United States survey. In the UK, up to 10% of pork sausages showed evidence of the presence of HEV. Virus extracted from positive livers could successfully infect pigs although only when the inoculum contained a high infectious dose. During infection, the virus is shed mostly via faeces. As piglet faeces are almost liquid, environmental contamination occurs. To support this concept HEV RNA has been found in the pig farm environment, in pig sewage, slaughterhouse sewage, ditch manure and a pig water trough. The presence of immunoglobulin G (IgG) HEV antibodies proves previous contact with the virus. The seroprevalence of IgG HEV antibodies in persons working with pigs has increased in some European countries, but

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FOOD AND FOOD-RELATED OUTBREAKS Food-related outbreaks have been documented in relation to pork pies, liver pate, wild boar, undercooked or raw pork, homemade sausages, eating offal, meat (in general), unpasteurized milk, uncooked shellfish (mussels, oysters or both) and figatelli pig liver sauce, which is often consumed without cooking. A French slaughterhouse study showed again the presence of high HEV RNA titers in 1,134 pig livers in 2.8% of those tested HEV RNA was shown. However this was not shown in the paired pork ham muscles [4]. A pivotal study from rural China stresses the environmental factors that have to be taken into account. HEV was studied in the setting of mixed farming of domestic animals (cows and pigs). HEV genotype 4 was shown to be excreted in cow milk. Gavage contaminated with raw milk or even pasteurised milk resulted in active infection in rhesus macaques. A short period of boiling but not pasteurisation could completely inactivate HEV [5].

THERMOSTABILITY OF HEV IN FOOD PRODUCTS Can we inactivate HEV below the infectious dose in the kitchen? Several experiments investigating the relationship between the thermostability and infectivity of HEV have

been done using different experimental set-ups. Faecal suspensions of three different HEV SESSION 4 – HEV virus strains were heated to temperatures between 45°C and 70°C, and residual infectivity was determined in a cell culture system permissive for HEV. Two strains were completely inactivated at 60°C; one strain was moderately more resistant with 80% inactivation at 60°C [6].

In a hepatocarcinoma cell line system infected with HEV, HEV did not grow at 70°C while HEV incubated at 56°C for 30 minutes was still infectious [7].

Using a swine bioassay, Feagins et al. showed that incubation of homogenates of HEV contaminated pig livers in a 56°C water bath for 60 minutes did cause infection in 4/5 inoculated pigs. Pig livers that were boiled for 5 minutes at a minimum internal temperature of 71°C or stir-fried at 191°C for 5 minutes were not able to induce infection in this model. As input in this model, square liver pieces between 0.5 and 1 cm were used, caution should be taken when extrapolating these data to the preparation of pig livers in a household setting [8].

Johne et al. used a titration method using the cell line A549/D3 infected with HEV genotype 3, and subsequently counted the focus forming units by immunofluorescence. Heat treatment for 1 minute resulted in a moderate decrease of infectivity up to 60°C, 2-3.5 log decrease between 65°C and 70°C and no remaining virus was detected at temperatures >80°C. Two minutes heating for 70°C resulted in the absence of detectable virus [9].

TRANSFUSION-ASSOCIATED HEV The risk of a transfusion-associated HEV infection is linked to the frequency of HEV viraemia in blood donors, the donor viral load and the volume of plasma in the final transfused blood components. In countries three levels of endemic HEV can be identified: low with IgG anti-

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HEV <10%, intermediate with IgG anti-HEV 10-20% and high with IgG anti-HEV >20%. Countries with high IgG anti-HEV seroprevalence among donors in Europe are the Netherlands (27%; HEV PCR(+) 1:600 donors), Germany (29.5%, HEV PCR(+) 1:1,200 donors) and France (22.4%, HEV PCR(+) 1:744 donors) [11]. HEV has been transmitted in samples of red blood cells, platelets, pooled granulocytes and fresh frozen plasma. No transmission has been described in plasma-derived products possibly because the European Pharmacopoeia requires HEV screening of plasma pools for the production of SD plasma. In a retrospective study, 225,000 English blood donors were screened with HEV PCR, 79 were viraemic, which is a prevalence of 1:2,848. Follow-up of 43 recipients who had received infected blood components indicated that 18 (42%) had evidence of HEV infection. Ten recipients (23%) developed chronic infection. A similar study in Japan showed evidence of infection in 50% of recipients [12].

The risk of infection was related to the volume of plasma transfused, the viral load with an estimated lowest infectious dose in the UK study of 2.10E4 IU HEV.

The protective role of antibodies is unclear. The proportion of donors who transmitted infection and had antibodies were similar to those with antibodies which did not transmit infection (4/18 vs. 8/25). Immune compromised patients with low antibody titers can become re-infected.

Should all blood products be screened for HEV RNA? In some countries there is a high prevalence of HEV viraemia among asymptomatic donors, a high rate of transmission by infected blood or blood products, there is non-negligible morbidity and mortality related to HEV infection with a lack of EMEA approved efficient antiviral therapy. This risk of transfusion-associated HEV should be confronted with the dietary and environmental risk. SESSION 4 – HEV Based on a UK risk analysis with an annual seroconversion rate of 0.2%, approximately 13 donor exposures equal one year of environmental exposure.

Therefore, from a public health perspective eliminating the dietary risk of HEV transmission is the most effective intervention, which would also improve blood safety. However, until the dietary risk is eliminated transfusion-associated HEV infection in immunosuppressed patients represents a preventable cause of serious morbidity and mortality and indicates a need to improve transfusion safety. The group in need is relatively small in number but may receive a substantial number of transfusions. The final decision on blood donor screening varies by country and is the result of a discussion comprising political, medical, ethical economic and legal aspects [13].

WHAT IS THE RISK OF PATIENT TO PATIENT TRANSMISSION OF HEV? In one series of 2,233 tested individual patients, a family size larger than 4 persons was associated with for anti-HEV positivity with an odds ratio of 3.8 (95% confidence interval 1.8-13.2), which may support the possibility of person to person transmission as has been suggested in genotype 1 outbreaks [10]. However, efficient person to person transmission is unlikely since very few reports mention outbreaks or family clusters. Due to the wide range in the incubation period and the large number of asymptomatic cases, distinction between person to person transmission or infection through a common food source is impossible. Sero- epidemiological studies have found no association with sexual contact or pregnancy.

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References

References in bold are required reading

[1] Tkahashi K, Kitajima N, Abe N, Mishiro S. Complete or near complete nucleotide sequences of hepatitis E virus genome recovered from wild boar, deer and four patients who ate the deer. Virology. 2004;330:501-5.

[2] Tei S, Kitajima N, Takahashi T, Mishiro S. Zoonotic transmission of hepatitis E virus from deer to human beings. Lancet. 2003;362:371-3.

[3] Lewis HC, Wichmann O, Duizer E. Transmission routes and risk factors for autochtonous hepatitis E virus infection in Europe: a systematic review Epidemiol Infect. 2010;18:145-66.

[4] Feurer C, Le roux A, Rossel R, Barnaud E.Dumarest M, Garry P, et al. High load of hepatitis E viral RNA in pork livers but absence in pork muscle at French slaughterhouses. Int J Food Micobiol. 2018;264:25-30.

[5] Huang F, Li Y, Jing S, Wang J, Long F, He Z, et al. Excretion of hepatitis E virus into milk in cows imposes high risks of zoonosis. Hepatology. 2016;64:350-9.

[6] Emerson SU, Arankalle VA, Purcell RH. Thermal stability of hepatitis E virus. J Infect Dis. 2005;192:930-3.

[7] Tanaka T, Takahashi M, Kusano E, Okamoto H. Development and evaluation of an

efficient cell-culture system for hepatitis E virus. J Gen Virol. 2007;88:903-11. SESSION 4 – HEV

[8] Feagins AR, Opriessnig T, Guenette DK, Halbur PG Meng XJ. Inactivation of infectious hepatitis E virus present in commercial pig livers sold in local grocery stores in the United States. Int J Food Microbiol. 2008;123:32-37.

[9] Johne R, Trjnar E, Filter M, Hofmann J. Thermal stability of hepatitis E virus as estimated by a cell culture method. Appl Environ Microbiol. 2016;30(82):4225-31.

[10] Rapicetta M, Kondili LA, Pretolani S, Stroffolini T, Chionne P, Villano U, et al. Seroprevalence and anti HEV persistence in the general population of the republic of San Marino. J Med Virol. 1999;58:49-53.

[11] Izopet J, Lhomme S, Chapuy-Regaud S, Mansuy JM, Kamar N, Abravanel F. HEV and transfusion-recipient risk. Transfusion Clinique et Biologique. 2017;24:176-181.

[12] Hewitt PE, Ijaz S, Brailsford SR, Brett R, Dicks S, Haywood B, et al. Hepatitis E virus in blood components: a prevalence and transmission study in southeast England. Lancet. 2014;384:1766-73.

[13] Bomonovic D, Tedder R, Blumel J, Zaaijer H, Gallian P, Niederhauser C, et al. Hepatitis E and blood donation safety in selected Eurpean countries: a shift to screening? Euro Surveill. 2017;22(16):pli=30514.

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DIAGNOSTICS IN HEV INFECTION

Darius Moradpour Division of Gastroenterology and Hepatology, Centre Hospitalier Universitaire Vaudois, University of Lausanne, Rue du Bugnon 46, CH-1011 Lausanne, Switzerland. E-mail address: [email protected]

TAKE-HOME MESSAGES • Hepatitis E virus (HEV) infection should be included early in the differential diagnosis of acute hepatitis, neuralgic amyotrophy and other neurological manifestations, drug-induced liver injury, as well as chronic hepatitis in immunocompromised patients.

• A combination of serological and molecular testing (anti-HEV IgM and PCR for HEV RNA) is best used to diagnose acute hepatitis E.

• PCR for HEV RNA should be used to diagnose chronic hepatitis E in immunocompromised patients.

INTRODUCTION

SESSION 4 – HEV Hepatitis E virus (HEV) is one of the most common causes of acute hepatitis and jaundice in the world [1, 2]. It is a positive-strand RNA virus classified in the Hepeviridae family [3, 4]. Most human pathogenic strains can be assigned to genotypes 1-4 within the species Orthohepevirus A. The 7.2 kb positive-strand RNA genome encodes three open reading frames (ORF) which are translated into (i) the ORF1 polyprotein, representing the viral replicase, (ii) the ORF2 protein, corresponding to the viral capsid, and (iii) the ORF3 protein, a small protein involved in viral particle secretion.

Two patterns of hepatitis E have been described: On the one hand, HEV genotypes 1 and 2 are transmitted from humans to humans by the faecal-oral route and can cause large, primarily waterborne outbreaks in low-income countries with poor sanitation, infecting about 20 million people and claiming 70,000 lives every year [5]. On the other hand, HEV genotypes 3 and 4 have been recognised as primarily porcine zoonosis in high-income countries, with much higher than anticipated seroprevalence rates, reaching 86.4% in the South of France [6]. Here, we shall focus on HEV genotype 3 infection acquired in Europe.

Hepatitis E represents a growing health concern and has been referred to as “Europe’s new hepatitis problem” [7]. Recent estimates suggest 300,000 new infections per year in Germany and 100,000 in the UK ([2] and refs. therein).

CLINICAL MANIFESTATIONS Most HEV infections are asymptomatic but, in some instances, the infection can be severe. For unknown reasons, middle-aged and elderly men are at risk of developing symptomatic and sometimes severe hepatitis ([8, 9] and refs. therein). Acute-on-chronic liver failure has been observed in patients with underlying liver cirrhosis.

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HEV genotype 3 (and, to a lesser extent, genotypes 4 as well as 7) may persist in immunocompromised patients and cause chronic hepatitis with potential for rapid progression to cirrhosis [2, 10]. In addition, HEV genotype 3 is a cause of diverse neurological manifestations, especially neuralgic amyotrophy (also known as Parsonage-Turner syndrome) [11, 12]. Transaminases are often only moderately elevated in patients with neurological manifestation and may be even normal in some. HEV infection also accounts for 3-13% of suspected drug-induced liver injury [13, 14].

The clinical and laboratory course of acute hepatitis E is schematically illustrated in Fig. 1. HEV infection can be diagnosed either directly by the detection of HEV RNA in blood or stool or indirectly by the detection of anti-HEV antibodies [15]. The incubation period is 2-8 weeks. HEV RNA can be detected in blood and stool 2-3 weeks post-infection. Viraemia lasts 3-6 weeks and excretion in stool possibly longer. If symptoms appear, they usually coincide with transaminase elevation and the appearance of anti-HEV IgM. Anti-HEV IgM persist for 3-6, sometimes up to 12 months. Anti-HEV IgG appears soon after IgM and persist for years. SESSION 4 – HEV

Fig. 1. Clinical and laboratory course of acute hepatitis E. Dashed arrows denote the window of PCR positivity in blood after the appearance of symptoms. ALT, alanine aminotransferase; HEV, hepatitis E virus, IgM, immunoglobulin M; IgG, immunoglobulin G.

Chronic hepatitis E has been observed in immunosuppressed individuals. As for acute hepatitis E, most patients with chronic hepatitis E are asymptomatic. However, chronic hepatitis E may rapidly progress to cirrhosis in some patients. Anti-HEV IgM and IgG are often negative in immunocompromised patients. Hence, PCR for HEV RNA should be performed to diagnose chronic hepatitis E and to monitor the course of viral replication under modification of immunosuppressive treatment and/or antiviral therapy.

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SEROLOGICAL ASSAYS Acute hepatitis E can be diagnosed by the detection of anti-HEV IgM antibodies or by rising titers of anti-HEV IgG by enzyme immunoassays (EIA). However, serological assays for anti- HEV have varying sensitivity and specificity [15-19]. Hence, serological testing is best used in combination with molecular testing (see below). Anti-HEV IgG testing is used primarily for epidemiological purposes. However, differences in sensitivity and specificity of serological assays need to be taken into account in interpreting reported seroprevalence rates [18, 19].

MOLECULAR ASSAYS Nucleic acid amplification techniques (NAT) for the detection of acute or chronic HEV infection. The most frequently used assays for the detection of HEV RNA in blood or stool target conserved regions of the genome, in particular the region of ORF2 overlapping with ORF3, and detect the four major human pathogenic HEV genotypes [20]. These can be determined by sequence analysis. A World Health Organization (WHO) International Standard for HEV RNA and a WHO International Reference Panel for HEV genotypes 1-4 have been developed, allowing to report quantitative results in International Units (IU) per ml and facilitating the evaluation and standardisation of NAT [21].

HEV RNA should be determined in an early plasma or serum sample, as the window of PCR positivity in blood is usually limited to 2-3 weeks after the appearance of symptoms (Fig. 1).

SESSION 4 – HEV In chronic hepatitis E, the persistence of HEV RNA in stools at the end of ribavirin therapy in patients with negative PCR in blood is associated with increased risk of relapse after treatment cessation [22]. The duration of ribavirin treatment should be extended in these cases.

ANTIGEN ASSAYS Detection of HEV capsid antigen by EIA may also be used to diagnose acute and chronic hepatitis E. Interestingly, a recent study suggested that HEV antigen levels may be able to distinguish acute from chronic infection [23]. While HEV antigen testing may represent an interesting alternative in laboratories which do not have access to NAT, its role in clinical practice remains to be determined.

IMMUNOHISTOCHEMISTRY Immunohistochemistry of ORF2 protein can be used to establish a histopathologic diagnosis of acute or chronic hepatitis E in liver biopsies [24].

A suggested diagnostic algorithm for HEV infection is illustrated in Fig. 2.

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Fig. 2. Diagnostic algorithm for hepatitis E. In immunocompetent patients, serological and molecular testing are best used in combination. In the case of high clinical suspicion and negative serology, PCR for HEV RNA should be performed. Molecular testing is also recommended in cases of doubtful anti-HEV IgM positivity. In immunocompromised patients, HEV RNA testing should be used to diagnose HEV infection.

ACKNOWLEDGEMENT The author gratefully acknowledges discussions with Vincent Aubert, Sally A. Baylis, Harry R. Dalton, Nassim Kamar, Sven Pischke, Roland Sahli and Heiner Wedemeyer. References

[1] Hoofnagle JH, Nelson KE, Purcell RH. Hepatitis E. N Engl J Med 2012;367:1237-1244. SESSION 4 – HEV [2] Kamar N, Izopet J, Pavio N, Aggarwal R, Labrique A, Wedemeyer H, Dalton HR. Hepatitis E virus infection. Nat Rev Dis Primers 2017;3:17086.

[3] Debing Y, Moradpour D, Neyts J, Gouttenoire J. Update on hepatitis E virology: implications for clinical practice. J Hepatol 2016;65:200-212.

[4] Nimgaonkar I, Ding Q, Schwartz RE, Ploss A. Hepatitis E virus: advances and challenges. Nat Rev Gastroenterol Hepatol 2018;15:96-110.

[5] Rein DB, Stevens GA, Theaker J, Wittenborn JS, Wiersma ST. The global burden of hepatitis E virus genotypes 1 and 2 in 2005. Hepatology 2012;55:988-997.

[6] Mansuy JM, Gallian P, Dimeglio C, Saune K, Arnaud C, Pelletier B, et al. A nationwide survey of hepatitis E viral infection in French blood donors. Hepatology 2016;63:1145- 1154.

[7] Kupferschmidt K. Europe’s new hepatitis problem. Science 2016;353:862-863.

[8] Fraga M, Doerig C, Moulin H, Bihl F, Brunner F, Müllhaupt B, et al. Hepatitis E virus as a cause of acute hepatitis acquired in Switzerland. Liver Int, 2017 [In press].

[9] European Centre for Disease Prevention and Control. Hepatitis E in the EU/EEA, 2005- 2015. https://ecdc.europa.eu/en/publications-data/hepatitis-e-eueea-2005-2015

[10] Kamar N, Selves J, Mansuy JM, Ouezzani L, Peron JM, Guitard J, et al. Hepatitis E virus and chronic hepatitis in organ-transplant recipients. N Engl J Med 2008;358:811-817.

[11] Dalton HR, Kamar N, van Eijk JJ, McLean BN, Cintas P, Bendall RP, Jacobs BC. Hepatitis E virus and neurological injury. Nat Rev Neurol 2016;12:77-85.

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[12] van Eijk JJJ, Dalton HR, Ripellino P, Madden RG, Jones C, Fritz M, et al. Clinical phenotype and outcome of hepatitis E virus-associated neuralgic amyotrophy. Neurology 2017;89:909-917.

[13] Dalton HR, Fellows HJ, Stableforth W, Joseph M, Thurairajah PH, Warshow U, et al. The role of hepatitis E virus testing in drug-induced liver injury. Aliment Pharmacol Ther 2007;26:1429-1435.

[14] Davern TJ, Chalasani N, Fontana RJ, Hayashi PH, Protiva P, Kleiner DE, et al. Acute hepatitis E infection accounts for some cases of suspected drug-induced liver injury. Gastroenterology 2011;141:1665-1672.

[15] Aggarwal R. Diagnosis of hepatitis E. Nat Rev Gastroenterol Hepatol 2013;10:24-33.

[16] Avellon A, Morago L, Garcia-Galera del Carmen M, Munoz M, Echevarria JM. Comparative sensitivity of commercial tests for hepatitis E genotype 3 virus antibody detection. J Med Virol 2015;87:1934-1939.

[17] Norder H, Karlsson M, Mellgren A, Konar J, Sandberg E, Lasson A, et al. Diagnostic performance of five assays for anti-hepatitis E virus IgG and IgM in a large cohort study. J Clin Microbiol 2016;54:549-555.

[18] Bendall R, Ellis V, Ijaz S, Ali R, Dalton HR. A comparison of two commercially available anti-HEV IgG kits and a re-evaluation of anti-HEV IgG seroprevalence data in developed countries. J Med Virol 2010;82:799-805.

SESSION 4 – HEV [19] Hartl J, Otto B, Madden RG, Webb G, Woolson KL, Kriston L, et al. Hepatitis E seroprevalence in Europe: a meta-analysis. Viruses 2016;(8):8.

[20] Jothikumar N, Cromeans TL, Robertson BH, Meng XJ, Hill VR. A broadly reactive one- step real-time RT-PCR assay for rapid and sensitive detection of hepatitis E virus. J Virol Methods 2006;131:65-71.

[21] Baylis SA, Blumel J, Mizusawa S, Matsubayashi K, Sakata H, Okada Y, et al. World Health Organization International Standard to harmonize assays for detection of hepatitis E virus RNA. Emerg Infect Dis 2013;19:729-735.

[22] Abravanel F, Lhomme S, Rostaing L, Kamar N, Izopet J. Protracted fecal shedding of HEV during ribavirin therapy predicts treatment relapse. Clin Infect Dis 2015;60:96-99.

[23] Behrendt P, Bremer B, Todt D, Brown RJ, Heim A, Manns MP, et al. Hepatitis E virus (HEV) ORF2 antigen levels differentiate between acute and chronic HEV infection. J Infect Dis 2016;214:361-368.

[24] Lenggenhager D, Gouttenoire J, Malehmir M, Bawohl M, Honcharova-Biletska H, Kreutzer S, et al. Visualization of hepatitis E virus RNA and proteins in the human liver. J Hepatol 2017;67:471-479.

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HEPATITIS E VIRUS: NATURAL HISTORY

Harry R Dalton Royal Cornwall Hospital and European Centre for Environment and Human Health, University of Exeter, Truro, United Kingdom. E-mail address: [email protected]

TAKE-HOME MESSAGES • Acute liver failure (ALF) occurs in pregnant females in developing countries with hepatitis E virus (HEV) genotype 1 infection and has a mortality rate of 25%.

• ALF is uncommon in acute HEV genotype 3 infection in developed countries, and excess mortality in pregnant women has not been observed.

• Acute-on-chronic liver failure (ACLF) occurs in patients with underlying chronic liver disease when infected with HEV genotype 1, with mortality rates of up to 70%.

• ACLF is less common with HEV genotype 3, and the mortality rate may be no different from other causes of hepatic decompensation.

• In immunosuppressed transplant recipients, the risk of developing a chronic infection after exposure to HEV genotype 3 is approximately 60%. SESSION 4 – HEV • The risk of developing a chronic infection in such circumstances is increased by the use of potent immunosuppression (e.g. tacrolimus) and a low serum lymphocyte count.

• Transplant patients with untreated chronic HEV infection have rapidly progressive chronic liver disease; 10% develop cirrhosis within 2 years.

• A range of extrahepatic manifestations are associated with HEV infection, the most important of which are neurological.

• 5.5% of patients with acute or chronic HEV infection present with primarily a neurological illness: such patients are not usually jaundiced, and the liver function tests may be normal.

INTRODUCTION Our understanding of HEV has changed completely over the past ten years. Previously, it was considered that HEV was limited to certain developing countries and was only ever seen in high-income countries in travellers returning from hyperendemic areas in South Asia or Africa. We now know that HEV is endemic in most high-income countries and is largely a zoonotic infection, with pigs as the primary host. In such settings, HEV causes both acute and chronic infection, and in many European countries is now the most common cause of acute viral hepatitis, but the incidence varies significantly between and within countries and over time.

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NATURAL HISTORY OF ACUTE INFECTION: HEV GENOTYPES 1 AND 2 The key clinical observations from developing countries with respect to hepatitis E was in the 1950s and 1970s in India. In the mid-1950s in Delhi, there was a major outbreak of unexplained hepatitis and in 1978-9 in Kashmir, with high mortality in pregnant women. These outbreaks were confirmed as being caused by HEV in retrospect and were the first well-documented observations of excess maternal mortality associated with HEV.

HEV genotype 1 and 2 are obligate human pathogens spread oro-faecally via contaminated water. They cause human disease in areas with fragile sanitary infrastructure in Asia (genotype 1), Africa (genotype 1 and 2) and Mexico (genotype 2). Sporadic cases are common but are sometimes interspersed by large outbreaks involving thousands or tens of thousands of cases. More recently, there have been ongoing, stuttering outbreaks in African refugee camps, including a current one in South Sudan and a very recent one in Nigeria.

HEV genotype 1 and 2 usually cause a brief, self-limiting hepatitis in young adults that is indistinguishable clinically from other causes of acute viral hepatitis. The clinical attack rate on exposure is approximately 1 in 5. Chronic infection with HEV genotype 1 and 2 has not been reported so far. The mortality rate in pregnant women is approximately 25%. Deaths are due to acute liver failure (ALF) with associated obstetric complications such as eclampsia and haemorrhage and are associated with a high perinatal infant mortality rate. The cause of the excess maternal mortality is not known. Despite its possible teratogenicity, there has been interest in the use of ribavirin in pregnant women with HEV infection. However, there are

SESSION 4 – HEV currently no data to support the use of ribavirin in such patients.

Some studies also show high mortality in patients with underlying chronic liver disease who develop HEV infection. This includes a study from India which shows that the 12-month mortality rate in such patients with acute-on-chronic liver failure (ACLF) approaches 70%. However, the mortality of HEV-related ACLF varies widely in different studies. Studies from Asia, which mainly involved HEV genotype 1 infections, reported mortality rates of between 0-67% in patients with chronic liver disease when experiencing an HEV super-infection. A recent analysis of 368 ACLF patients described that HEV-associated cases had a more benign course than alcohol-associated ACLF.

NATURAL HISTORY OF ACUTE INFECTION: HEV GENOTYPES 3 AND 4 Acute HEV genotype 3 infection is clinically silent in the vast majority of patients. Only a minority (probably less than 5%) develop symptoms of acute hepatitis with elevated liver enzymes, jaundice and non-specific symptoms such as fatigue, itching and nausea (Fig. 1). However, HEV infection is the major cause of acute viral hepatitis in many European countries and in Germany, UK and France there have been more reported cases of acute hepatitis E than of hepatitis A virus (HAV) or acute hepatitis B virus (HBV) infections in 2015-16.

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Fig. 1. Symptomatic and asymptomatic locally-acquired HEV in developed countries. >95% of infections with HEV genotype 3 and genotype 4 are asymptomatic (A). Only a small minority of patients (mainly older males) develop symptomatic hepatitis (C). Other patients are symptomatic, but currently not diagnosed (B). These include patients who present with primarily a neurological illness. The numerical relationship between A, B and C is uncertain.

Immunocompetent patients with acute hepatitis E clear the infection spontaneously, but there have been a few reports of cases with more prolonged viraemia. Progression to ALF is rare in SESSION 4 – HEV patients with HEV genotype 3 infection. However, single cases of ALF due to HEV infection have been reported in several European countries. In a German single centre study of 80 cases with ALF, HEV RNA was found in 10% of patients and HEV considered as the probable cause. Patients with confirmed acute hepatitis E should be monitored for transaminases (alanine aminotransferase [ALT], aspartate aminotransferase [AST]), bilirubin and INR. Once HEV infection is cleared, patients develop immunity against HEV which is not sterilising. Thus, re- infection with HEV is possible even though the likelihood of developing symptomatic hepatitis is reduced compared to non-immune individuals.

In contrast to HEV in developing countries, HEV genotype 3 and 4 tend to affect older males. In one study from England, the male to female ratio was 3:1, and the median age 63 years. The finding that older males are most likely to develop clinically apparent acute hepatitis on exposure to HEV genotype 3 and 4 is a consistent observation, but unexplained. It seems likely that this relates to host factors, rather than differential exposure, as individuals of all ages appear to be exposed to HEV. One possible explanation is that clinically apparent hepatitis is more likely to be evident in patients with subclinical hepatic steatosis/fibrosis. In a study from England, some patients with hepatitis E were heavy alcohol consumers, and an excess number were diabetic, both of which are risk factors for hepatic steatosis and fibrosis.

Acute hepatitis E is a concern in patients with underlying chronic liver disease. Some cases of ACLF are caused by HEV infection. This is a particular problem in elderly patients where acute hepatitis may take a more severe course. HEV infection may be less relevant in European patients with decompensated cirrhosis. Only 11/343 patients with decompensated chronic liver disease followed in France or the UK had acute hepatitis E and three of those died. Of note, HEV did not alter the mortality in this study, compared to other causes of hepatic decompensation. These findings are in line with data from France showing a low prevalence of HEV infection in patients with severe acute alcoholic hepatitis. There are only a few case reports of HEV genotype 3 and 4 in pregnancy. Excess maternal mortality has not been observed.

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NATURAL HISTORY OF CHRONIC INFECTION: GENOTYPES 3 AND 4 In immunosuppressed patients, HEV infection can fail to clear. Such patients develop chronic hepatitis, but so far this has only been seen in patients infected with HEV genotype 3 or 4. Chronic HEV infection has been defined as a persistence of HEV replication for 6 months. However, in an observational study performed in solid organ transplant patients, it was observed that no spontaneous HEV clearance occurred between 3 and 6 months after infection, and spontaneous HEV clearance occurred only within the first 3 months after infection. These data suggest that in solid organ transplant recipients, patients who are viraemic for more than 3 months after HEV infection can be considered as chronically infected and considered for treatment. However, in a small number of cases, spontaneous clearance has been observed between 3-6 months.

The clinical presentation of chronic HEV infection has mainly been described in the setting of organ transplantation but is similar in other immunosuppressed groups including haematological patients, individuals living with HIV, and rheumatology patients receiving heavy immunosuppression. In a series of 85 solid organ transplant recipients, only one-third of patients were symptomatic, with fatigue as the main symptom. The majority of patients are asymptomatic and present with mild and persistent liver function test (LFT) abnormalities: in one study of chronically infected transplant recipients at diagnosis the ALT was 260 ± 38 IU/L, AST was 155 ± 25 IU/L, and γGT was 308 ± 56 IU/L.

One-third of solid organ transplant recipients infected with HEV have a resolving hepatitis,

SESSION 4 – HEV and the remaining patients develop chronic hepatitis. Other smaller studies show progression to chronic infection occurs in less than 50%. In solid organ transplant recipients infected with HEV genotype 3, rapid progression of liver fibrosis has been observed leading to cirrhosis and, in some cases, decompensation and death. There appears to be no difference in HEV RNA concentration between patients with or without progressive liver fibrosis. Interestingly, liver fibrosis can regress after HEV clearance. Extrahepatic HEV-associated manifestations, i.e. neurological and renal injury, have been observed both during acute and chronic HEV infection (see below). In solid organ transplant patients, a low lymphocyte count at diagnosis and the use of tacrolimus (rather than cyclosporine A) are associated with the development of chronic infection after exposure to HEV. In HIV patients, chronic HEV infection has mostly been described in patients with a CD4+ T cell count <200/mm3. No predictive factor(s) for the development of chronic HEV infection in other immunosuppressed groups have been identified.

EXTRAHEPATIC MANIFESTATIONS Extrahepatic manifestations of HEV infection are increasingly recognised (Table 1), the most important are neurological.

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Table 1. Extrahepatic manifestations of acute and chronic hepatitis E.

Organ system Clinical syndrome Notes Neurological • *Neuralgic amyotrophy See main text • *Guillain-Barré syndrome • *Meningoencephalitis • Mononeuritis multiplex • Myositis • Bell’s palsy, vestibular neuritis and peripheral neuropathy Renal* • Membranoproliferative and See main text membranous glomerulonephritis • IgA nephropathy Haematological • Thrombocytopenia • Mild thrombocytopenia is • Monoclonal immunoglobulin common. Occasionally severe • Cryoglobulinemia • Reported in 25% of cases of acute HEV in UK study. Significance • Aplastic anaemia uncertain • Haemolytic anaemia • Occurs mainly in association with renal disease • Case reports only SESSION 4 – HEV • Case reports only Other • Acute pancreatitis • 55 cases worldwide. HEV • Arthritis genotype 1 only. The pancreatitis is usually mild • Myocarditis • Case reports only • Autoimmune thyroiditis • Case reports only • Myaesthenia gravis • Case reports only • Case reports only

*There is good evidence to support a causal role for HEV and these associated conditions. For the other extrahepatic manifestations, causality remains to be established. Neurological injury

HEV infection has been described in association with a range of neurological injuries. In one study of 126 patients with acute and chronic HEV infection, seven (5.5%) patients presented with neurological injury. To date, approximately 150 cases have been described, mainly from Europe in the context of HEV genotype 3 infection. HEV-associated neurological injury has also been described in Asia in the context of HEV genotype 1 infection. Most (>90%) cases have been documented in the immunocompetent patients, but neurological injury also occurs in the context of chronic infection with HEV genotype 3. Neurological pathology that has been described in association with HEV infection includes neuralgic amyotrophy (NA), Guillain-Barré syndrome (GBS), encephalitis/myelitis, mononeuritis multiplex, Bell’s palsy, vestibular neuritis, myositis and peripheral neuropathy. The best documented are NA, GBS, and encephalitis/myelitis.

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There have been several cohort and case studies of HEV infection in patients with NA. These are almost universally from Europe in the context of HEV genotype 3 infection. In an Anglo/ Dutch cohort study, 5/47 (10.6%) of patients with NA had evidence of HEV infection at the start of their illness. Very recent data from a multicentre study in Europe of 118 patients with NA shows that patients with HEV-associated disease have a distinct clinical phenotype, compared to NA patients without evidence of HEV infection. Patients with HEV-associated NA were significantly more likely to have bilateral involvement of and more extensive damage to the brachial plexus. They were also more likely to have neurological damage outside the brachial plexus, particularly phrenic nerve involvement. Another recent European multicentre study systematically prospectively tested over 450 consecutive patients with acute onset of non- traumatic neurological injury. Evidence of HEV infection was found in 2.4% of cases, three of whom had NA with bilateral involvement, which we now know is the clinical phenotype associated with HEV infection. Finally, it is worth noting that in one centre’s experience (Dalton et al.), the triad of bilateral shoulder pain in a middle-aged male with abnormal LFTs is highly predictive of HEV infection.

There have been three case-control studies on HEV infection and GBS from Holland, Bangladesh and Japan. Collectively these studies confirm the relationship between HEV infection and GBS, as evidence of HEV infection at the start of the neurological illness was found in 5-11% of patients, significantly higher than in controls. In addition, in a very recent cohort study from Belgium, 6/73 (8%) of GBS patients had evidence of HEV infection.

There have been 13 case reports/small case series on HEV infection and encephalitis/myelitis, from Europe, Asia and the USA. Some cases had features of additional involvement of the

SESSION 4 – HEV peripheral nervous system. Five of the cases were in immunocompromised transplant recipients in the context of chronic HEV genotype 3 infection. Several of these patients had a prominent ataxic component to their neurological symptomatology. These patients had a poor outcome with long-term neurological sequelae and two deaths. In one of these patients, ‘quasispecies compartmentalization’ was noted, i.e. there was a significant difference in sequence homology in HEV RNA from the serum and cerebrospinal fluid. This raises the question of whether certain strains of HEV might be neurotropic.

In all of the above studies, the patients with HEV-associated neurological injury generally had only modest abnormalities of liver function and were mostly anicteric. In some patients, the LFTs were normal. Thus, the neurological symptoms and signs dominated the clinical picture. Pathogenic mechanisms are uncertain but could be due to molecular mimicry, which would be congruent with current notions in NA and GBS, or due to direct neurotropism. It seems likely that, at least in the case of NA, GBS and encephalitis/myelitis, the relationship between HEV infection and neurological damage is causal. The evidence to support causality includes the number and homogeneity of cases over time and geographical location; case-control data in GBS; documentation of HEV RNA in the serum and cerebrospinal fluid in some cases with quasispecies compartmentalization; intrathecal anti-HEV IgM synthesis; resolution of neurological symptoms with viral clearance; in vitro data that show HEV can grow on a range of neurological cell lines; and in vivo animal studies that show HEV can cross the blood-brain barrier.

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Renal injury

HEV can cause glomerulonephritis in both immunocompetent and immunosuppressed patients. Renal impairment has been documented in solid organ transplant recipients during acute HEV infection. Cases of membranoproliferative glomerulonephritis with and without cryoglobulinaemia, as well as cases of membranous glomerulonephritis have been reported, mainly in immunosuppressed patients infected with HEV genotype 3. Cases of membranoproliferative and membranous glomerulonephritis have been documented with HEV genotype 1 and 3 in the immunocompetent patient. Renal function improves, and proteinuria levels decrease following HEV clearance, either spontaneously or following therapy. These data suggest the relationship between HEV infection and the associated renal injury is likely to be causal. Of note, in one case, HEV RNA was isolated from the cryoprecipitate obtained from a patient who developed HEV-associated cryoglobulinaemic glomerulonephritis. References

References in bold are required reading

[1] Pischke S, Hartl J, Pas SD, Lohse AW, Jacobs BC, Van der Eijk AA. Hepatitis E virus: Infection beyond the liver? J Hepatol. 2017;66:1082-1095.

[2] Dalton HR, Kamar N, van Eijk JJ, McLean BN, Cintas P, Bendall RP, et al. Hepatitis E virus and neurological injury. Nat Rev Neurol. 2016;12: 77-85.

[3] Dalton HR, van Eijk JJ, Cintas P, Madded R, Jones C, Webb G, et al. Hepatitis E infection and acute non-traumatic neurological injury: A prospective pilot SESSION 4 – HEV multicenter study J Hepatol. 2017;67: 925-932.

[4] Kamar N, Bendall R, Legrand-Abravanel F, Xia NS, Ijaz S, Izopet J, et al. Hepatitis E. Lancet. 2012;379:2477-2488.

[5] Kamar N, Dalton HR, Abravanel F, Izopet J. Hepatitis E virus infection. Clin Microbiol Rev. 2014;27:116-138.

[6] Adlhoch C, Avellon A, Baylis SA, Ciccaglione AR, Couturier E, de Sousa R, et al. Hepatitis E virus: Assessment of the epidemiological situation in humans in Europe, 2014/15. J Clin Virol. 2016;82:9-16.

[7] Kumar Acharya S, Kumar Sharma P, Singh R, Kumar Mohanty S, Madan K, Kumar Jha J, et al. Hepatitis E virus (HEV) infection in patients with cirrhosis is associated with rapid decompensation and death. J Hepatol. 2007;46:387-394.

[8] Kamar N, Garrouste C, Haagsma EB, Garrigue V, Pischke S, Chauvet C, et al. Factors associated with chronic hepatitis in patients with hepatitis E virus infection who have received solid organ transplants. Gastroenterology. 2011;140:1481-1489.

[9] Woolson KL, Forbes A, Vine L, Beynon L, McElhinney L, Panayi V, et al. Extra- hepatic manifestations of autochthonous hepatitis E infection. Aliment Pharmacol Ther. 2014;40:1282-1291.

[10] van Eijk JJ, Dalton H, Ripellino P, Madden R, Jones C, Fritz M, est al. Clinical phenotype and outcome of hepatitis E virus associated neuralgic amyotrophy; an international multicentre retrospective comparative study. Neurology. 2017 Aug 29;89(9):909-91.

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TREATMENT OF HEPATITIS E

Nassim Kamar Department of Nephrology and Organ Transplantation, CHU Rangueil, Toulouse, France. E-mail address: [email protected]

TAKE-HOME MESSAGES • The reduction of immunosuppression is the first-line therapeutic option in transplant patients with chronic hepatitis E virus infection.

• Ribavirin monotherapy is highly effective for treating hepatitis E virus (HEV) infection.

• The mechanism of action of ribavirin is unknown.

• Pegylated interferon is an alternative therapy to ribavirin.

• The role of HEV RNA polymerase mutations on virological response is unknown.

SUMMARY Chronic hepatitis E virus (HEV) infection is described in immunosuppressed patients, mainly in SESSION 4 – HEV solid organ transplant patients. In vitro, it has been shown that several immunosuppressants, i.e. calcineurin inhibitors and mTOR inhibitors, increase HEV replication, while the mycophenolic acid does not. Retrospective studies have shown that decreasing immunosuppression allows HEV clearance in one-third of patients chronically infected with HEV. Hence, it is considered as a first-line therapeutic option. Ribavirin was found to be very effective for treating HEV and was associated with a sustained virological rate (SVR) around 85%. However, the optimal ribavirin dose and treatment duration, as well as the mechanism of action of ribavirin are still undetermined. A rapid decrease in viral load within the first week was associated with SVR, suggesting that it can guide the duration of therapy. Similarly, a persistence of detectable HEV RNA at the end of therapy was associated with an increased risk of relapse after treatment cessation, suggesting that this parameter should be considered before stopping therapy. The mechanisms for treatment failure in ribavirin treated patients are unknown. Several HEV RNA polymerase mutations were described in patients with treatment failure or in patients who relapse. However, some other patients having the same mutations achieved SVR. Hence, their role and impact on virological response are unknown. In non-responders to ribavirin, pegylated interferon can be successfully used. However, since it stimulates the immune system, it is contraindicated in organ transplant patients, except in liver transplant patients. Actually, there is no other alternative therapy to ribavirin.

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Fig. 1. Treatment algorithm for immunocompromised patients who received solid organ transplantation [1]. SESSION 4 – HEV

A few case reports showed a rapid clearance of HEV RNA at the acute phase of HEV infection when patients were given ribavirin. However, these data are uncontrolled, and spontaneous improvement cannot be ruled out.

Ribavirin was used in patients experiencing HEV-associated extrahepatic manifestations. In patients with neurological symptoms, the role of ribavirin is uncertain. However, it has been shown to be effective for treating HEV-associated glomerulonephritis. References

References in bold are required reading

[1] Kamar N, Izopet J, Pavio N, Aggarwal R, Labrique A, Wedemeyer H, Dalton HR. Hepatitis E virus infection. Nat Rev Dis Primers. 2017;3:17086

[2] Kamar N, Izopet J, Tripon S, Bismuth M, Hillaire S, Dumortier J, et al. Ribavirin for chronic hepatitis E virus infection in transplant recipients. N Engl J Med. 2014;370(12):1111-20.

[3] Kamar N, Lhomme S, Abravanel F, Marion O, Peron JM, Alric L, Izopet J. Treatment of HEV Infection in Patients with a Solid-Organ Transplant and Chronic Hepatitis. Viruses. 2016;8(8)222.

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THURSDAY 12 APRIL 2018 08:30-10:00 SESSION 5 – HBV

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WHERE DO WE STAND ON DIAGNOSING HBV INFECTION PHASE, AND HBV-INDUCED LIVER DAMAGE IN HBeAg-NEGATIVE CARRIERS?

George Papatheodoridis Department of Gastroenterology, Medical School of National and Kapodistrian University of Athens University, Laiko General Hospital, Athens, Greece. E-mail address: [email protected]

TAKE-HOME MESSAGES • Patients with HBeAg-negative chronic hepatitis B virus (HBV) infection (previously called “inactive HBV carriers”) typically have normal alanine aminotransferase (ALT) according to traditional values of the upper limit of normal (ULN ~40 IU/L) and undetectable or low (≤2,000 IU/ml) HBV DNA levels.

• Patients with HBeAg-negative chronic HBV infection and persistently normal ALT may sometimes have HBV DNA levels >2,000 and usually ≤20,000 IU/ml.

• Close follow-up for a minimum of 3-12 months and sometimes liver biopsy are mostly required in grey-zone HBeAg-negative cases who present with normal ALT but HBV DNA >2,000 IU/ml, or mildly elevated ALT (1-2 x ULN), or with ALT >2 x ULN but HBV DNA ≤20,000 IU/ml.

• Patients with typical HBeAg-negative chronic hepatitis B presenting with ALT >2 x ULN SESSION 5 – HBV and HBV DNA >20,000 IU/ml should start treatment immediately without necessarily a liver biopsy.

Patients with chronic infection with hepatitis B virus (HBV) and negative HBV e antigen (HBeAg) have been traditionally classified into inactive carriers and those with HBeAg- negative chronic hepatitis B (CHB). In the recent EASL HBV guidelines [1], the term “HBeAg- negative chronic HBV infection” has been suggested as a replacement for the term “inactive carrier state”, while HBeAg-negative CHB continues to include all HBeAg-negative cases with HBV-induced liver damage.

Given the identical serological profile, the differential diagnosis between HBeAg-negative chronic infection and CHB is usually based on alanine aminotransferase (ALT), HBV DNA levels and liver lesions [1]. The differential diagnosis between these two groups is mandatory, as the first needs only to be followed for the risk of future HBV exacerbation, while the latter needs to receive anti-HBV treatment. Typically, patients with HBeAg-negative chronic HBV infection have normal ALT according to traditional values of upper limit of normal (ULN ~40 IU/L) and undetectable or low (≤2,000 IU/ml) HBV DNA levels. In such cases, there is no need for liver biopsy, which would show no or minimal necroinflammation and no or minimal to mild fibrosis probably produced during the preceding HBeAg seroconversion phase [2]. On the other hand, the diagnosis of HBeAg-negative CHB can be made in HBeAg-negative patients with established chronic HBV infection when the following criteria are fulfilled: a) elevated ALT, to establish hepatocellular damage; b) HBV DNA >2,000 IU/ml, to establish active HBV replication; and c) exclusion of other concomitant or superimposed causes of liver disease (e.g. superinfections

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PARIS, FRANCE with other hepatitis viruses [D or C], alcohol abuse, non-alcoholic fatty liver disease, hepatotoxic drug use, or other more rare causes of liver injury [autoimmune, metabolic etc.]). Liver biopsy is considered to be helpful or even critical to confirm the diagnosis of HBeAg-negative CHB in cases in which underlying hepatocellular damage may be missed if only ALT and HBV DNA values are taken into consideration (usually patients with normal ALT), as well as in cases in which the relation between HBV and hepatocellular damage is not straightforward (patients with mild ALT elevations and/or low to moderate HBV DNA levels) [2].

INITIAL FOLLOW-UP OF HBeAg-NEGATIVE CHRONIC HBV PATIENTS Since ALT is the most convenient and cheap surrogate marker of liver injury usually reflecting the degree of HBV-related injuries of hepatocytes, and HBV DNA levels reflect HBV replication, these two parameters represent the basis for the diagnostic approach of all chronic HBV patients in clinical practice. However, a major limitation of ALT as a reliable marker of liver injury is that its levels often fluctuate over time during the variable course of chronic HBV infection. Thus, a single ALT value at baseline is definitely not a reliable marker of the presence and particularly of the absence of liver injury in HBeAg-negative patients. Therefore, a minimum follow-up of 3-6 months with at least 3-4 ALT determinations is required in HBeAg-negative patients with initially normal or mildly elevated (≤2 x ULN) ALT [2]. In contrast, more prompt management is advised in HBeAg-negative patients presenting with ALT >2 x ULN who may need only one additional confirmation of ALT levels if they have high HBV DNA levels (>20,000 IU/ml) as well [1]. In addition, all patients with HBeAg- negative chronic HBV infection should undergo careful physical examination, abdominal ultrasonography and non-invasive assessment of liver fibrosis, if available [1].

Based on their initial follow-up of ALT and HBV DNA values, HBeAg-negative patients can be classified into: a) cases with definite chronic HBV infection (ALT ≤ULN and HBV DNA SESSION 5 – HBV ≤2,000 IU/ml); b) grey-zone cases with normal ALT (ALT ≤ULN and HBV DNA >2,000 IU/ ml); c) grey-zone cases with elevated ALT (ALT 1-2 x ULN regardless of HBV DNA or ALT >2 x ULN and HBV DNA ≤20,000 IU/ml); and d) cases with obvious HBeAg-negative CHB (ALT >2 x ULN and HBV DNA >20,000 IU/ml) [3].

CASES WITH DEFINITE HBeAg-NEGATIVE CHRONIC HBV INFECTION

Patients with initially normal ALT and HBV DNA ≤2,000 IU/ml can be a more safely considered case, with definite HBeAg-negative chronic HBV infection after additional ALT follow-up for a total 12-month period. Liver biopsy is not justified, except perhaps in exceptionally rare cases with clinical or ultrasonographic signs of advanced fibrosis or cirrhosis or liver stiffness >9 kPa at a reliable initial liver elastography [1]. In the vast majority of such cases without the latter signs of advanced liver disease, patients with HBeAg-negative chronic HBV infection should be only monitored for the risk of HBV exacerbation for life. Their follow-up is based on ALT measurements every 6–12 months and periodic HBV DNA tests every 2–3 years [1, 2]. To minimise the risk of missing transient short-lasting ALT flares and worsening liver histology, periodic assessment of liver stiffness with elastography is often advised as well [2].

Recent data suggest that quantitative HBsAg testing can be helpful in deciding the frequency of follow-up in such patients. Patients with HBsAg levels <1,000 IU/ml can undergo ALT

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measurements every 12 months and have HBV DNA and liver fibrosis tests every 3 years, while those with HBsAg levels ≥1,000 IU/ml should better undergo ALT measurements every 6 months and HBV DNA and liver fibrosis tests every 2 years (Fig. 1) [4].

GREY-ZONE HBeAg-NEGATIVE CASES WITH NORMAL ALT It is now well accepted that some patients with HBeAg-negative chronic HBV infection may have HBV DNA levels >2,000 IU/ml (usually ≤20,000 IU/ml) accompanied by persistently normal ALT. These patients have no or minimal hepatic necroinflammatory activity with usually no or mild and rarely moderate liver fibrosis [5]. At the same time, such cases are at a higher risk of having, and most importantly of progressing, to HBeAg-negative CHB and therefore closer follow-up is recommended [6]. Similarly to cases with definite HBeAg-negative chronic HBV infection, liver biopsy is recommended only in the rare such cases with clinical or ultrasonographic signs of advanced fibrosis or cirrhosis or liver stiffness >9 kPa at a reliable initial liver elastography [1]. It should be noted that liver biopsy may reveal moderate fibrosis in up to 50% of HBeAg-negative patients with persistently normal ALT, HBV DNA >2,000 IU/ ml and liver stiffness >6.5 kPa, but whether treatment is required for moderate fibrosis without necroinflammation is rather questionable [7]. SESSION 5 – HBV

Fig. 1. Management of HBeAg-negative chronic HBV patients with normal baseline ALT levels. These patients should be followed with ALT every 3-4 months for one year. Patients with ALT elevations or HBV DNA >20,000 IU/ml or any sign of advanced disease should be managed according to the algorithm in Fig. 1. Patients with ALT persistently 2,000 (usually ≤20,000) IU/ml should undergo liver elastography and may be treated if liver stiffness exceeds 9 kPa. In contrast, such cases with liver stiffness ≤9 kPa may better remain under follow-up with *ALT every 3-4 months and #HBV DNA every 12 months for two additional years and with ¶ALT every 6 months and #HBV DNA every 12 months thereafter, if they remain in the same status. Patients with ALT persistently

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Patients with HBeAg-negative chronic HBV infection, normal ALT and HBV DNA >2,000 IU/ ml, who do not have any of the previously described signs of advanced liver disease, should be closely followed with ALT measurements every 3 months for the first year and every 6 months at least thereafter. Moreover, evaluation of HBV DNA levels and a non-invasive liver fibrosis test should be performed annually for at least 3 years (Fig. 1). If they do not fulfil any indications for treatment during the first 3 years of follow-up, these patients should be monitored for life, like all other cases with definite HBeAg-negative chronic HBV infection [1, 2].

GREY-ZONE HBeAg-NEGATIVE CASES WITH ELEVATED ALT HBeAg-negative patients with mild ALT elevations (1-2 x ULN) regardless of HBV DNA levels, who often have HBV DNA ≤20,000 IU/ml or even ≤2,000 IU/ml, as well as HBeAg- negative patients with HBV DNA ≤20,000 IU/ml regardless of ALT values [5] are rather difficult to differentiated from cases with ΗΒeAg-negative chronic HBV infection who have another cause of ALT elevations (such as non-alcoholic fatty liver disease). A close follow-up for 3-6 months with ideally monthly ALT determinations and a second HBV DNA determination can be quite helpful for clinical decisions. In patients remaining in this subgroup, a liver biopsy should be recommended before any therapeutic decision (Fig. 2) [3]. SESSION 5 – HBV

Fig. 2. Management of HBeAg-negative chronic HBV patients with increased baseline ALT levels. Patients should be initially followed with monthly ALT determinations for 2-3 months, unless they have signs of cirrhosis for which immediate treatment is recommended.

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Patients with ALT >2 x ULN and HBV DNA >20,000 IU/ml should start therapy after the assessment of liver fibrosis severity perhaps with liver elastography. Patients with ALT 1-2 x ULN and/or HBV DNA ≤20,000 IU/ml should undergo liver elastography or biopsy for therapeutic decisions.

Regardless of the cause of liver injury, these patients are at risk of progressive liver injury as well as progressing to HBeAg-negative CHB, particularly those with HBV DNA >2,000 IU/ml. Therefore, a close follow-up for at least 3 years, if they remain untreated, is also recommended, similarly to the follow-up of grey-zone HBeAg-negative cases with normal ALT and HBV DNA >2,000 IU/ml.

CASES WITH OBVIOUS HBeAg-NEGATIVE CHB All HBeAg-negative patients with ALT >2 x ULN and HBV DNA >20,000 IU/ml, which are present either at baseline or develop during follow-up, should be treated immediately. A liver biopsy may offer useful information, but it is not mandatory and should not delay the onset of therapy [1]. As in all HBeAg-negative patients, careful physical examination, abdominal ultrasonography and non-invasive assessment of liver fibrosis are recommended, as these patients are at a higher risk of having developed cirrhosis, which does not affect the decision for treatment but potentially the need for surveillance for hepatocellular carcinoma (HCC) [1]. It should be noted that the cut-off for the diagnosis of at least severe fibrosis is considered to be 12 kPa for chronic HBV patients with elevated ALT, while the elastographic measurements are considered to reliably represent the severity of fibrosis only in patients with ALT elevations up to 5 x ULN and to be confounded by severe inﬂammation in patients with ALT >5 x ULN [1, 8].

SESSION 5 – HBV Patients who start anti-HBV therapy should have the recommended follow-up for the efficacy and safety of their therapeutic agent. In addition, patients with signs of cirrhosis at baseline as well as those with moderate to high HCC risk scores should remain under HCC surveillance [1].

CONCLUSIONS Follow-up remains the cornerstone for the correct diagnosis of the phase of HBeAg-negative chronic HBV patients. Although the differential diagnosis is straightforward in some HBeAg- negative patients with chronic HBV infection (previously called inactive carriers) as well as in some patients with obvious HBeAg-negative CHB, close follow-up with repeated ALT determinations, HBV DNA measurements, non-invasive assessments of fibrosis, abdominal ultrasonography and perhaps liver biopsy may be also required in other cases. In any case, all HBeAg-negative chronic HBV patients should be followed for life or at least until HBsAg loss, which will occur in a minority of cases. Cases who do not currently fulfil treatment indications and thus remain untreated should be followed for the risk of HBV exacerbation, while cases of patients who start therapy should be followed for the efficacy and safety of therapy and possibly for the risk of HCC. In addition, all patients should be strongly advised to avoid or manage co- factors which can worsen the progression of liver disease and usually increase the risk of HCC, such as alcohol abuse, co-infections with other hepatitis viruses or human immunodeficiency virus, smoking, diabetes, metabolic syndrome and obesity [1].

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References

References in bold are required reading

[1] European Association for the Study of the Liver. EASL 2017 Clinical Practice Guidelines on the management of hepatitis B virus infection. J Hepatol 2017;67:370-98.

[2] Papatheodoridis GV, Manolakopoulos S, Liaw Y-F, Lok A. Follow-up and indications for liver biopsy in HBeAg-negative chronic hepatitis B virus infection with persistently normal ALT: a systematic review. J Hepatol 2012;57:196-202.

[3] Papatheodoridis GV. Hepatitis B virus treatment: Which patients can have treatment deferred? Clinical Liver Disease 2013;2:15-7.

[4] Brunetto MR, Oliveri F, Colombatto P, Moriconi F, Ciccorossi P, Coco B, et al. Hepatitis B surface antigen serum levels help to distinguish active from inactive hepatitis B virus genotype D carriers. Gastroenterology 2010;139:483-90.

[5] Papatheodoridis GV, Manesis EK, Manolakopoulos S, Elefsiniotis IS, Goulis J, Giannousis J, et al. Is there a meaningful serum HBV DNA cut-off level for therapeutic decisions in HBeAg-negative chronic hepatitis B virus infection? Hepatology 2008;48:1451-9.

[6] Papatheodoridis GV, Chrysanthos N, Hadziyannis E, Cholongitas E, Manesis EK. Longitudinal changes in serum HBV DNA levels and predictors of progression during the natural course of HBeAg-negative chronic hepatitis B virus infection. J Viral Hepat 2008;15:434-41.

[7] Papatheodoridis GV, Manolakopoulos S, Margariti A, Papageorgiou M-V, Kranidioti H,

Katoglou A, et al. The usefulness of transient elastography in the assessment of patients SESSION 5 – HBV with HBeAg-negative chronic hepatitis B virus infection. J Viral Hepat 2014;21:517-24.

[8] European Association for the Study of the Liver, Asociacion Latinoamericana para el Estudio del Higado. EASL-ALEH Clinical Practice Guidelines: Non-invasive tests for evaluation of liver disease severity and prognosis. J Hepatol 2015;63:237-64.

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PegIFN VS. ORAL THERAPY IN HBeAg-NEGATIVE CHB PATIENTS

Mauro Viganò1 and Pietro Lampertico2* 1Hepatology Division, Ospedale San Giuseppe, Università degli Studi di Milano, Milan, Italy; 2CRC “A. M. and A. Migliavacca” Center for Liver Disease, Division of Gastroenterology and Hepatology, Fondazione IRCCS Cà Granda Ospedale Maggiore Policlinico, Università degli Studi di Milano, Milan, Italy. E-mail address: [email protected]

TAKE-HOME MESSAGES • Currently, there are two different treatment strategies for HBeAg-negative chronic hepatitis B patients: a finite course of pegylated interferon alpha (PegIFNα) and a long-term administration of the more potent and less resistance-prone nucleot(s)ide analogues (NAs), such as entecavir, tenofovir disoproxil fumarate and tenofovir alafenamide.

• One year of PegIFNα achieves a persistent suppression of viral replication with alanine aminotransferase (ALT) normalisation in nearly 20% of HBeAg-negative patients, ultimately leading to HBsAg loss in approximately 30% of them during long-term off-treatment follow-up.

• PegIFN maintains considerable but acceptable side effects which occur in approximately 30% of patients. The cost-effectiveness of PegIFNα can be significantly increased by careful patient selection based upon baseline virus and host features and by applying early on- treatment stopping rules based upon HBsAg kinetics. SESSION 5 – HBV • Long-term administration of NAs has become the most popular treatment strategy worldwide because of the easier management compared to PegIFN and the almost universal virologic and biochemical remission combining with histological improvement and prevention of liver decompensation.

• However, lifelong NAs treatment is associated with high cost, unknown safety, adherence issues, unknown risk of drug-resistance in the long-term, and negligible rates of HBsAg seroclearance.

INTRODUCTION Chronic hepatitis B virus (HBV) infection remains one of the most common infectious diseases, and world’s leading causes of death as an estimated 240 million individuals are chronically infected with HBV worldwide [1]. Hepatitis B envelope antigen (HBeAg)-negative, which is currently the predominant type of chronic hepatitis B (CHB) in many countries, is a difficult-to-cure, progressive disease leading to end-stage liver disease and hepatocellular carcinoma (HCC) [1]. The goal of therapy in such patients is to persistently suppress viral replication with the aim to halt the progression of liver damage and prevent the development of cirrhosis and further liver-related complications [1]. As recommended by the recent EASL Clinical Practice Guidelines on the management of HBV infection [1], there are two main treatment options for HBeAg-negative CHB patients: treatment with pegylated interferon alpha (PegIFNa) or with a nucleos(t)ide analogues (NAs) with high barrier to resistance,

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PARIS, FRANCE i.e. entecavir (ETV), tenofovir disoproxil fumarate (TDF), and the recently approved tenofovir alafenamide (TAF) (Table 1). A 48-week course of PegIFN is the only strategy able to offer a chance for sustained off-treatment response after a finite course of therapy compared with NAs which require long-term administration (Table 1). However, the major limitations to the wide use of PegIFNα are the need for parenteral therapy and the clinical and laboratory monitoring, the side effects profile and contraindication for some patient categories, and the lack of effectiveness in a large proportion of patients (Table 1). Notwithstanding, the cost-effectiveness of PegIFNα can be significantly increased by careful patient selection based upon baseline alanine aminotransferase (ALT), HBV DNA levels, viral genotype, host genetic variants and, above all, by applying early on-treatment stopping rules based upon hepatitis B surface antigen (HBsAg) kinetics. The main advantage of treatment with potent NAs is its high long-term antiviral efficacy leading to undetectable HBV DNA levels in the vast majority of compliant patients with minimal risk of drug-resistance. However, with the hypothetical hindrances of costs and the low rates of HBsAg seroclearance (Table 1). NAs are the treatment of choice in patients with compensated cirrhosis, because of the risk of hepatic decompensation associated with PegIFNα-related flares of hepatitis, in those with decompensated cirrhosis, in patients of advanced age, and in those contraindicated or unwilling to use IFN. Whereas all other patients willing to receive IFN-based treatment can be considered either for PegIFNα or NAs treatment (Table 1).

Table 1. Main concepts and features of current treatment strategies of CHB patients [1]. SESSION 5 – HBV

PegIFNα, pegylated interferon alfa; ETV, entecavir; TDF, tenofovir disoproxil fumarate; TAF, tenofovir alafenamide; NA, nucleoside/nucleotide analogues; eGFR, estimated glomerular filtration rate. 1 See section on ‘PegIFN treatment and NAs treatment’. 2 Dose adjustments in patients with eGFR <50 ml/min are required for all NA, except for TAF (no dose recommendation for TAF in patients with CrCl <15 ml/min who are not receiving haemodialysis). 3 A plateau in serologic responses has been observed beyond treatment year 4. 4 So far no TDF or TAF resistance development has been detected. In this article, we review the efficacy and safety of PegIFNα and NAs in HBeAg-negative CHB patients.

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PegIFN TREATMENT Efficacy and safety of PegIFNα In HBeAg-negative patients, six months after the end of a 48-week course of PegIFNα-2a, the rates of HBV DNA <400 copies/ml and HBsAg loss were 19% and 4%, respectively (Table 2). The 3-year follow-up showed that off-treatment response, defined as HBV DNA <400 copies/ml, was sustained in 18% of patients, whereas at year 5 of follow-up 12% of the patients achieved HBsAg clearance, a percentage that increased to 28% in those with HBV DNA ≤10,000 copies/ml one year after the end of treatment (EOT) [1, 2]. However, treatment with PegIFNα maintains considerable but acceptable side effects, which occur in approximately 30% of patients, even if in rare cases they can be serious. The most frequently reported side effects are: flu-like syndrome, headache, myalgia, fatigue, weight loss, depression, hair loss and local reactions at the injection site, neutropenia and thrombocytopenia [1,2]. PegIFNα is contraindicated in patients with advanced liver disease such as portal hypertension, leukopenia and/or severe thrombocytopenia, severe depression or a history of major psychiatric illness, ongoing pregnancy, active autoimmune diseases, and severe concomitant disease. For this reason, only patients with mild to moderate chronic hepatitis should be considered for PegIFN therapy as hepatitis flares may result in decompensation of liver disease [1]. The ability to predict which patients are most likely to respond or not respond would be useful in guiding treatment decisions, both at baseline, upon host and viral features, and during treatment by applying early on-treatment stopping rules.

Table 2. Results of main studies for the treatment of HBeAg-negative CHB patients at 6 months following 48 weeks of PegIFNα and at 48 or 52 weeks of NA therapy [1]. SESSION 5 – HBV

References: EASL CPG 20121 for all drugs except for TAF.7 4 PegIFNα, pegylated interferon alfa; ETV, entecavir; TDF, tenofovir disoproxil fumarate; TAF, tenofovir alafenamide; LAM, lamivudine; TBV, telbivudine; ADV, adefovir; ALT, alanine aminotransferase. * PegIFNα was given as percutaneous injections once weekly and nucleos(t)ide analogues as oral tablets once daily. # The definition of ALT normalisation varied among different trials (i.e., decrease of ALT to ≤1.25-times the upper limit of normal [ULN] in the ETV or ≤1.3-times the ULN in the TBV trial). The lower quantification limit of HBV DNA assays was different across studies: for TAF studies it was <29 IU/ml. Baseline predictors of PegIFN response

In HBeAg-negative patients, high baseline ALT, low serum HBV DNA, younger age, female gender and HBV genotype were independent predictors of response. Genotypes B or C patients have a better chance of response than genotype D patients. A baseline score system (from 0 to 7) that combined five variables: HBV genotype, HBV DNA, ALT, qHBsAg and age identified patients with a high and low likelihood of response. HBV DNA <2,000 IU/ml and HBV DNA <2,000 IU/ml plus normal ALT 48 weeks after EOT were achieved in 61% and 45% of patients

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PARIS, FRANCE with baseline scores ≥4, respectively, but only in 11% and 8% of patients with scores 0–1 with a 89% and 92% negative predictive value (NPV), respectively [1,2].

Unfortunately, the fluctuating pattern of serum HBV DNA and ALT tends to give a cumbersome prediction of a response by these variables at an individual level. These flaws fostered the search for other pretreatment predictors of a response to IFN-based treatment. The search led to a genome-wide association analysis of SNPs rs12979860 and rs8099917 mapping in the genomic region 3 kb upstream of the gene codifying for IL28B on chromosome 19, now renamed IFN lambda 4 (IFNλ4). However, the preliminary observations of an association between a favourable IFNλ4 polymorphisms and higher chances of sustained virologic and serological responses in HBeAg-negative patients were not confirmed by subsequent studies [3]. Recently, IFNλ4 rs368234815 and rs117648444 variants were reported to strongly predict HBsAg clearance in 126 HBeAg-negative treated with IFN and followed-up for a median of 11 (1-23) years [4]. However, until further studies in large cohorts of homogeneous ethnic and virologic groups of patients confirm these results, a baseline host genetic testing to prioritise CHB patients for PegIFN therapy is not recommended in clinical practice [1]. On-treatment predictors of PegIFN response

Reports of HBsAg decline in HBeAg-negative patients undergoing IFN-based therapy suggested the potential role of this marker for predicting treatment response. In HBeAg- negative patients, a combination of a lack of decrease in HBsAg levels and <2 log10 IU/ml decline in HBV DNA at 12 weeks of PegIFNα treatment predicts a no response in genotype D patients (NPV 100%), allowing to discontinue therapy in approximately 20% of treated patients (Fig. 1). No robust on-treatment stopping rules have been developed for HBeAg- negative patients with genotype B or C, and very few data are available for those with genotype A and E [1, 2]. SESSION 5 – HBV

Fig. 1. Week 12 stopping rules for HBeAg-negative patients treated with PegIFNα. These rules are based upon viral genotype, HBsAg and HBV levels [1].

DE NOVO PegIFN PLUS NA TREATMENT COMBINATION IN NAÏVE PATIENTS Several studies in HBeAg-negative patients testing the combination of PegIFN with first or second-generation NAs did not provide evidence for a superior efficacy of the combination regimen vs. PegIFN monotherapy both in terms of sustained virologic and serological response [1, 2]. Recently, the combination of PegIFN with TDF was evaluated in a multicentre international study enrolling 740 naïve patients (42% HBeAg-positive, 21% genotype D)

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randomised to receive a 48-week PegIFN + TDF (arm A), TDF + PegIFN for 16 weeks followed by TDF for 32 weeks (arm B), TDF alone (arm C), and 48 weeks of PegIFN alone (arm D) [6]. Among the 312 HBeAg-negative patients, the combination arm was superior to all the others. Six months after EOT the rates of HBsAg seroclearance were 5%, 1.3%, 0% and 1.3% in arm A, B, C and D, respectively [5].

ADD-ON PegIFN TO NA Two recent European studies prospectively investigated the “add-on” PegIFN strategy in HBeAg- negative patients with HBV replication fully suppressed by long-term NA treatment [2]. A French randomised controlled, open-label trial, enrolled 185 patients with virologic suppression while on stable NA therapy for at least 1 year. Patients were randomised 1:1, according to qHBsAg, to receive a 48-week course of PegIFNα-2a 180 μg per week in addition to the NA regimen or to continue to receive NA only. The rates of HBsAg loss were significantly higher in the combination group than in the NA group at week 48 (8% vs. 0%, p = 0.0057) but not at week 96 (8% vs. 3%, p = 0.15). Low baseline HBsAg levels predicted HBsAg loss and anti-HBs seroconversion. Twenty percent of the patients discontinued PegIFN due to adverse events (AEs). In the open- label, single arm, multicentre Italian cohort HERMES study, 70 HBeAg-negative genotype D patients who following ETV or TDF monotherapy had serum HBV DNA persistently <20 IU/ml for at least 12 months (mean: 3.2, range: 1.1-8.4 years), HBsAg >100 IU/ml and stable

HBsAg levels in the 3 months preceding enrolment (<0.5 log10 IU/ml decline), received a 48- week course of add-on PegIFNα-2a. At week 48, median HBsAg levels significantly decreased from baseline value of 1,160 IU/ml to 308 IU/ml (p = 0.0001); serum HBsAg decreased ≥50% from baseline in 30 patients (43%), and the proportion of patients with HBsAg <1,000, <500 or <100 IU/ml increased from 47% to 72%, 23% to 56% and 0 to 22%, respectively. One patient (1.4%) seroconverted to anti-HBs at week 24 and successfully withdrew from both NA and IFN treatment, and two additional patients (2.8%) reached HBsAg <10 IU/ml. Sixty-two patients SESSION 5 – HBV (89%) experienced AEs, 8 (11%) and 7 (10%) patients discontinued or temporarily interrupted PegIFN due to AEs respectively, while in 8 (11%) patients PegIFN was down dosed following AEs. Overall, 24 patients (34%) discontinued PegIFN: 11 (16%) for HBsAg non-response, 8 (11%) for AEs, 5 (7%) for compliance or consent withdrawal.

SWITCH FROM NA TO PegIFN No studies on switching to PegIFN are available for NA-treated HBeAg-negative patients.

NAs TREATMENT Long-term efficacy and safety of ETV

The 48-week efficacy data are reported in Table 2. In the VIRGIL Study, including 243 NA- naïve CHB patients (64% HBeAg-negative) treated with ETV monotherapy, the cumulative probability of achieving a virologic response (<80 IU/ml) in HBeAg-negative patients at week 48, 96 and 144 was 89%, 98% and 99%, respectively, with two patients (1%) achieving HBsAg loss and none developing ETV-resistance (ETV-R). A US study of 153 patients treated with ETV showed high response rates in naïve HBeAg-negative patients reaching 100% at year 3. In the Hong Kong cohort, including 132 NA-naïve HBeAg-negative patients on ETV, the rates of HBV DNA undetectability (<12 IU/ml) and ALT normalisation progressively increased to

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PARIS, FRANCE reach 98% and 86% at year 3, respectively. One patient seroconverted to anti-HBs at year 3 while none developed ETV-R. In a Japanese study, 252 NA-naïve HBeAg-negative patients were treated with continuous ETV for 4 years, 100% of the patients achieved undetectable HBV

DNA levels (<2.6 log10 copies/ml), and 91% normalised ALT levels, with no cases of ETV-R. In a field practice study in Italy including 418 NA-naïve patients (83% HBeAg-negative) treated with ETV for 5 years, the rate of viral suppression and ALT normalisation was 100% and 93% of HBeAg-negative patients, respectively. One patient (0.7%) who developed ETV-R at year 3, was successfully rescued by TDF [1]. The US ENUMERATE study of 421 HBeAg- negative patients, reported 96% rate of undetectable serum HBV DNA and 56% rate of ALT normalisation after 5 years of ETV therapy [6], whereas a seven-year study with ETV in 132 Chinese HBeAg-negative patients reported 100% rate of undetectable serum HBV DNA and ALT normalisation and 1% ETV-R rate [7].

Long-term administration of ETV was associated with low rates of serious AEs and discontinuations. After a median of 184 weeks of ETV exposure, the commonly (≥10%) reported AEs were upper respiratory tract infection, headache and nasopharyngitis. Most AEs were mild to moderate, 19% of patients reporting grade 3-4 AEs which were considered related to ETV in 4% of cases ultimately leading to treatment discontinuations in 1% of patients [1]. The favourable safety profile of ETV was also confirmed in field practice studies. Among 3,823 patients exposed to ETV in different countries for 12 to 66 months, no major safety issues were reported. Few cases of lactic acidosis were reported in patients with decompensated cirrhosis under ETV treatment, all of whom had a baseline MELD score >22 points, which resolved in most patients after drug withdrawal. This warning suggests that particular caution should be exercised when administering ETV to any patient with severe liver damage and high baseline MELD scores, and that ETV treatment should be suspended in any patient who develops clinical or laboratory findings suggestive of lactic acidosis [8]. Long-term efficacy and safety of TDF SESSION 5 – HBV The 48-week efficacy data are reported in Table 2. After 8 years, 99% of the 264 HBeAg- negative TDF-treated patients achieved virological response (HBV DNA <400 copies/ml), without evidence for TDF-R, whereas HBsAg seroclearance occurred in 1% of the HBeAg- negative patients. The efficacy of TDF treatment was evaluated in some field practice studies [1]. A German multicentre observational study (GEMINIS) that enrolled 400 CHB patients (69% HBeAg-negative) reported a 92% virologic response (HBV DNA <69 IU/ml) among HBeAg-negative naïve patients with neither virologic breakthrough nor TDF-R after 3 years of treatment. During the same follow-up, the 440 (74% HBeAg-negative) French CHB patients showed 96% rate of virologic response (HBV DNA <69 IU/ml) with HBsAg loss in 8 HBeAg- negative patients. In the multicentre European clinical practice study, 97% of the 374 NA- naïve CHB patients (80% HBeAg-negative) treated with TDF for a median of 39 months, achieved a virologic response at year 4. Five patients (1.7%) cleared HBsAg, and no cases of TDF-R were observed.

While TDF registration studies showed minimum renal toxicity events (~2%) and no significant change in bone mineral density (BMD) over long-term treatment, real-life long-term studies have demonstrated that some patients may experience a decline in estimated glomerular filtration rate (eGFR), hypophosphatemia, chronic tubular disease, Fanconi syndrome and loss of BMD [9]. For this reason, the international guidelines on HBV management recommend in patients at risk of development and/or with underlying renal or bone disease to be administered ETV or TAF instead of TDF [1] (Table 3). TAF (Vemlidy®), a new anti-HBV drug with a better safety profile compared to TDF, has been recently approved for the treatment of CHB adults with compensated liver disease.

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Table 3. Indications for selecting ETV or TAF over TDF according to the recent EASL HBV CPG [1].

* TAF should be preferred to ETV in patients with previous exposure to nucleoside analogues. ** ETV dose needs to be adjusted if eGFR <50 ml/min; no dose adjustment of TAF is required in adults or adolescents (aged at least 12 years and of at least 35 kg body weight) with estimated creatinine clearance (CrCl) ≥15 ml/min or in patients with CrCl <15 ml/min who are receiving haemodialysis. Efficacy and safety of TAF

The 48-week efficacy data are reported in Table 2. The long-term efficacy and safety of SESSION 5 – HBV TAF in treatment-naïve HBeAg-negative CHB adult patients were evaluated in an ongoing, randomised, double-blind, multinational, non-inferiority trials (GS-US-320-0108) [10]. Patients were randomised 2:1 to once daily TAF 25 mg or TDF 300 mg for up to 144 weeks, and then switched to open-label TAF 25 mg/day until week 384. Enrolled were CHB patients with serum HBV DNA levels ≥20,000 IU/ml, serum ALT level ≥60 U/L for men, ≥38 U/L for women, i.e. ≥2 times the upper limit of normal (ULN) of the AASLD range and ≤10 times the ULN by central laboratory range, and an estimated glomerular filtration (eGFR) by Cockcroft-Gault ≥50 ml/min. Excluded were patients with HCC, evidence of clinical hepatic decompensation, co-infection with hepatitis C or D virus or with HIV. HBeAg-negative patients treated with TAF 25 mg (n = 285) and TDF 300 mg (n = 140) for 96 weeks showed similar rates of viral suppression (90% vs. 91%) with only one TAF-treated HBeAg-negative CHB patient (<1%) clearing HBsAg by week 96 [10, 11]. TAF-treated patients showed higher rates of ALT normalisation by both AASLD and central laboratory normal range: 50% vs. 40% (p = 0.035) and 81% vs. 71% ( p = 0.038), respectively. TAF was associated with smaller changes in renal safety parameters than TDF, suggesting that TAF has less impact on renal function. After 96 weeks of treatment of HBeAg-negative patients, median changes in eGFR and the percentage of patients with a ≥25% eGFR decline were significantly different between TAF and TDF-treated patients (-0.6 vs. -3.6 ml/minute, p = 0.011 and 11% vs. 18%, p = 0.046). Overall, through week 96, TAF treatment had significantly less impact on eGFR than TDF at all- time points [12]. After 96 weeks, TAF recipients had significantly smaller median percentage changes in markers of kidney tubular dysfunction such as the urine retinol-binding protein to creatinine (RBP:CR) ratio and the urine beta2-microglobulin to creatinine (B2M:CR) ratio compared to TDF-treated patients (18.5% vs. 53.2%, p <0.001 and 10.8% vs. 59.2%, p <0.001)

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[12]. TAF treatment was associated with significantly smaller mean percentage changes in BMD at the hip (-0.33 vs. -2.51%, p <0.001) and spine (-0.75 vs. -2.57%, p <0.001) than TDF. A higher proportion of subjects treated with TDF also experienced >3% decline in hip and spine BMD compared to TAF-treated patients (spine: 45% vs. 25%, p <0.001 and hip: 39% vs. 14%, p <0.001) [13]. Greater declines in BMD were seen in patients with >1 risk factor for bone loss at baseline including female gender, advanced age, Asian ethnicity and baseline renal impairment. However, in comparison to TDF, the proportion of patients with >3% decline in hip and spine BMD were lower in the TAF group and were less impacted by the presence of multiple risk factors compared to TDF group. By multivariate analysis, independent predictors (odds ratio [OR], 95% CI) for >3% BMD decline in hip or spine at week 96 included study drug treatment (TAF vs. TDF, OR 0.232, 95% CI 0.170-0.317, p <0.0001) age (<50 vs. ≥50 years, OR 0.619, 95% CI 0.430-0.889, p = 0.0095), gender (female vs. male, OR 1.471, 95% CI 1.065-2.032, p = 0.0192), and baseline renal function (eGFR as a continuous variable, OR 0.991, 95% CI 0.984-0.998, p = 0.0086).

CONCLUSIONS HBeAg-negative CHB is a frequent, progressive and difficult-to-cure phase of chronic B infection. The endpoint of therapy is to persistently suppress viral replication to halt progression of liver disease. Two different treatment strategies are currently available: short-term course of PegIFN or long-term therapy with NAs. Young patients with mild to moderate stages of liver disease can benefit from a 48-week course of PegIFNα, while NAs may be preferred in patients with more severe liver disease, in older patients, and in those who do not respond, are unwilling or contraindicated to IFN. Advantages of NAs include excellent tolerability, effective inhibition of HBV replication without the emergence of drug-resistance, high rates of biochemical remission, histological improvement, prevention of clinical decompensation but limited effect on HCC rates. NAs are the only treatment for patients with severe liver disease, SESSION 5 – HBV old patients, or those contraindicated or unwilling to PegIFN and with significant concomitant diseases. However, long-term administration of NAs cannot eradicate HBV, making long- term therapy necessary in most patients, with increasing cost, compliance issues and unproven safety profiles. On the other hand, a course of PegIFN may be the most appropriate first-line treatment strategy when the purpose of treatment is to achieve a sustained immune control after a short treatment course, although its benefits are restricted to a subgroup of patients. To increase the rates of patients who may benefit from this treatment minimising the AEs, a careful patient selection and individualised treatment decisions to achieve a treatment optimisation is required. Young age, high ALT levels, low HBV DNA and virus genotype have been identified as pretreatment predictors of a response, whereas a response-guided therapy approach based on HBsAg levels allow for the identification of non-responders who can stop PegIFN treatment early. Combination of NA and PegIFN is not currently recommended, but many new studies are underway. References

References in bold are required reading

[1] European Association for the Study of the Liver. EASL 2017 Clinical Practice Guidelines on the management of hepatitis B virus infection. J Hepatol 2017;67: 70-398.

[2]. Viganò M, Grossi G, Loglio A, Lampertico P. Hepatitis B: is there still a role for interferon? Liv Int 2018. [Epub ahead of print].

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[3] Galmozzi E, Viganò M, Lampertico P. Systematic review with meta-analysis: do interferon lambda 3 polymorphisms predict the outcome of interferon-therapy in hepatitis B infection? Aliment Pharmacol Ther 2014;39:569-78.

[4] Galmozzi E, Facchetti F, Grossi G, et al. IFNL4 rs368234815 and rs117648444 variants predict off-treatment HBsAg seroclearance in IFN-treated HBeAg-negative chronic hepatitis B patients. Liver Int 2017 Jul 21. doi: 10.1111/liv.13526. [Epub ahead of print]

[5] Marcellin P, Ahn SH, Ma X, Caruntu FA, Tak WY, Elkashab M, et al. Combination of tenofovir disoproxil fumarate and Peginterferon α-2a increases loss of hepatitis B surface antigen in patients with chronic hepatitis B. Gastroenterology 2016;150:134-44.

[6] Ahn J, Lee HM, Lim JK, Pan CQ, Nguyen MH, Ray Kim W, et al. Entecavir safety and effectiveness in a national cohort of treatment-naïve chronic hepatitis B patients in the US – the ENUMERATE study. Aliment Pharmacol Ther 2016;43:134-44.

[7] Lam YF, Seto WK, Wong D, Cheung KS, Fung J, Mak Ly, et al. Seven-Year Treatment Outcome of Entecavir in a Real-World Cohort: Effects on Clinical Parameters, HBsAg and HBcrAg Levels. Clin Transl Gastroenterol 2017;8:e125. doi: 10.1038/ctg.2017.51.

[8] Baraclude https://packageinserts.bms.com/pi/pi_baraclude.pdf.

[9] Lampertico P, Chan HLY, Janssen HLA, Strasser SI, Schindler R, Berg T. Review article: long-term safety of nucleoside and nucleotide analogues in HBV-monoinfected patients. Aliment Pharmacol Ther 2016;44:16–34.

[10] Buti M, Gane E, Seto WK, Chan HL, Chuang WL, Stepanova T, et al. Tenofovir alafenamide versus tenofovir disoproxil fumarate for the treatment of patients with HBeAg-negative chronic hepatitis B virus infection: a randomised, double- blind, phase 3, non-inferiority trial. Lancet Gastroenterol Hepatol 2016;1:196-

SESSION 5 – HBV 206.

[11] Brunetto M, Lim YS, Gane E, Seto WK, Osipenko M, Ahn SH, et al. A phase 3 study comparing tenofovir alafenamide (TAF) to tenofovir disoproxil fumarate (TDF) in patients with HBeAg-negative, chronic hepatitis B (CHB): efficacy and safety results at week 96. J Hepatol 2017;66:S25–S26.

[12] Chuang WL, Agarwal K, Hwang JS, Caruntu F, Wong F, Hann HW, et al. Continued improvement in renal laboratory parameters in CHB patients treated with tenofovir alafenamide (TAF) compared with tenofovir disoproxil fumarate (TDF) over 96 weeks. J Hepatol 2017;66:S695.

[13] Fung S, Chuang WL, Nishiguchi S, Seto WK, Tak WY, Erne EM, et al. Smaller decreases in bone mineral density in chronic hepatitis B patients receiving tenofovir alafenamide compared with tenofovir disoproxil fumarate over 96 weeks. J Hepatol 2017;66:S691–2.

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CAN WE STOP NUCLEOS(T)IDE ANALOGUE THERAPY IN HBeAg-NEGATIVE CHRONIC HEPATITIS B PATIENTS?

François Villeret, Fabien Zoulim* 1INSERM U1052- Cancer Research Center of Lyon (CRCL), 69008 Lyon, France; 2University of Lyon, UMR_S1052, CRCL, 69008 Lyon, France; 3Department of Hepatology, Croix Rousse Hospital, Hospices Civils de Lyon, France. E-mail address: [email protected]

TAKE-HOME MESSAGES • Nucleos(t)ide analogue (NA) therapy may be stopped in non-cirrhotic, virally suppressed, HBeAg-negative chronic hepatitis B patients provided that post-treatment monitoring can be guaranteed.

• Virological remission and HBsAg loss can be observed in a good proportion of patients following NA discontinuation.

• Flares with liver decompensation may be observed in patients with cirrhosis.

• Low HBsAg levels at the end of treatment seem to be predictive of the off-treatment response and subsequent HBsAg loss.

• However, the predictive threshold for HBsAg is not yet established. SESSION 5 – HBV • Novel viral and immunological biomarkers are under evaluation to refine the decision algorithm.

BACKGROUND HBeAg-negative chronic hepatitis B (CHB) is becoming the most frequent form of CHB. Currently, its treatment relies on the use of either pegylated interferon or nucleos(t)ide analogues (NA). The use of pegylated interferon is discussed in a previous chapter by Prof. Lampertico.

In HBeAg-negative CHB patients treated with NAs with a high barrier to resistance, i.e. entecavir (ETV), tenofovir disoproxil fumarate (TDF), tenofovir alafenamide fumarate, viral suppression and alanine aminotransferase (ALT) normalisation can be achieved in >90% and >70% of patients, respectively, after one year of therapy. However, the rate of HBsAg loss, the only endpoint defined for HBeAg-negative patients that would allow treatment cessation, is extremely low (<1%). Since NA therapy does not usually achieve hepatitis B virus (HBV) eradication and rarely results in HBsAg loss, the EASL guidelines recommend lifelong NA therapy in HBeAg-negative CHB patients. Currently, stopping NAs is only recommended if patients achieve HBsAg loss [1].

As long-term NA therapy is thought not to be convenient for patients and associated with cumulative cost, investigators considered stopping NA after achieving viral suppression. Based

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on the pathobiology of CHB, the expectation was that some patients might remain in virological remission, others may experience a virological rebound associated with an immune-induced hepatitis flare that may lead to HBsAg loss, and others may experience a viral relapse with the re-initiation of chronic inflammation. This may depend on the status of the intrahepatic pool of cccDNA, the template for viral gene expression and for viral genome replication, and the level of immunological control of HBV in the liver microenvironment at the time of treatment cessation.

GENERAL CONSIDERATIONS FOR STOPPING NA IN HBeAg- NEGATIVE CHB Recent evidence, accumulating mainly from Asian countries, in which NAs can be discontinued in HBeAg-negative CHB patients who achieve serum HBV DNA undetectability on three separate 6 months occasions, suggests that the discontinuation of NAs might be feasible in these patients [2].

The prediction of sustained virological remission after treatment cessation remains an important issue that needs to be addressed. Indeed, the analysis of the literature does not provide a clear message since studies are heterogeneous, do not always consider the same factors, and include both HBeAg-positive and negative patients. However, an important factor affecting the probability of off-NA virological remission appears to be the duration of on- therapy HBV DNA undetectability [3]. According to the existing data, virological remission defined as HBV DNA 2,000–20,000 IU/ml is maintained in approximately 50% of patients 3 years after NAs cessation if they have remained for more than two years on virological remission during therapy [3]. Since such findings are based on studies with durations of on- therapy virological remission of 2 to 5 years [3], the optimal duration of on-NAs remission before discontinuation remains unclear. SESSION 5 – HBV Since overt hepatitis flares and life-threatening episodes (jaundice, liver decompensation, liver failure) have been rarely reported in patients with pre-existing cirrhosis who discontinue NAs [4], treatment discontinuation is currently discouraged in patients with cirrhosis. Moreover, NAs may be discontinued only in patients who can be followed closely with ALT and HBV DNA determinations at least during the first year following NAs cessation, to allow for rapid retreatment if needed. Unfortunately, no reliable predictor of post-NAs remission has been identified to date.

Retreatment criteria are also important but have yet to be determined [3]. Based on reasonable clinical judgment, treatment indications for naïve CHB patients may also apply NA discontinuation. This is covered in the following chapter by Prof Berg.

CLINICAL AND VIROLOGIC PARAMETERS TO GUIDE NA DISCONTINUATION IN HBeAg-NEGATIVE CHB PATIENTS An interesting study of 188 naïve CHB patients (83 HBeAg-positive, 105 HBeAg-negative patients), who were previously treated with lamivudine (LAM) (treatment duration: 89.3 weeks, range: 52-243 weeks) but stopped the treatment for at least 12 months, looked at predictors of off-treatment responses. The cumulative incidence of HBsAg loss and HBV relapse at year 6 after stopping LAM treatment was 24% and 65.9%, respectively. Cox regression analysis revealed that lower ALT levels at baseline, lower HBsAg levels at the end of treatment (EOT),

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PARIS, FRANCE and longer treatment duration were independent predictors for HBsAg loss; old age, male sex and higher HBsAg levels at the EOT were independent predictors for post-treatment HBV relapse. At the EOT, in HBeAg-negative patients, the HBsAg cut-off values of 120 and 200 IU/ml could predict 79.2% (19/24) of HBsAg loss and 93.3% (28/30) post-treatment sustained response, respectively. Further HBsAg reduction (>0.22 log IU/ml) at month 6 after stopping treatment was an independent predictor for HBsAg loss after adjusting for HBsAg level at EOT. The authors concluded that serum HBsAg levels at EOT could be useful to guide the timing of stopping LAM treatment in CHB patients [5].

A key finding was made from an observational cohort study, following 33 HBeAg-negative patients with CHB, undetectable serum HBV DNA, and normal levels of aminotransferases after long-term (4 or 5 years) treatment with adefovir dipivoxil (ADV). All patients were followed for 5.5 years. During the first few months of treatment discontinuation, all patients experienced virological relapse, and 25 (76%) had a biochemical relapse. During the follow- up period, 18 patients (55%) who had discontinued antiviral therapy achieved sustained response (HBV DNA level <2,000 IU/L and persistently normal ALT levels). Among these, 13 (39%) cleared HBsAg. Fifteen patients (45%) with virological and/or biochemical relapse were retreated with oral antiviral agents (11 during the first 18 months and 4 after the third year), without evidence of liver decompensation; only 1 showed HBsAg loss (6%). Higher pretreatment and EOT levels of ALT, no previous treatment with interferon, and lower level of HBsAg at EOT were significantly associated with HBsAg clearance based on multivariate analysis. HBsAg titers were lower at EOT in patients with sustained response who cleared HBsAg (median 161 IU/ml) compared with relapsers (median 4,837 IU/ml) and sustained responders who did not clear HBsAg (median 5,242 IU/ml) [6].

Another study aimed to test the stopping rule of the Asian Pacific Association for the Study of the Liver (APASL) guidelines in HBeAg-negative CHB patients treated with ETV, i.e. undetectable HBV DNA documented on three occasions ≥6 months apart. Ninety-five

patients (39 with cirrhosis) were treated with ETV for a median of 721 (395-1,762) days before SESSION 5 – HBV stopping therapy and were then monitored for serum HBV DNA and ALT levels at least every 3 months. Within 1 year after stopping ETV therapy, “clinical relapse” (an episode of ALT elevation >2 times the upper limit of normal [ULN] plus HBV DNA >2,000 IU/ml) occurred in 43 (45.3%) of the 95 patients. Of the 39 patients with cirrhosis, 17 (43.6%) relapsed and one (2.6%) developed decompensation. The median duration until relapse was 230 days (74.4% >6 months). Logistic regression analysis showed that baseline HBV DNA ≤2x105 IU/ml was the only significant independent factor for sustained response. The 1-year relapse rate was 29% in patients with a baseline HBV DNA ≤2x105 IU/ml vs. 53% in those with HBV DNA >2x105 IU/ml (p = 0.027). For the latter, consolidation therapy >64 weeks reduced the relapse rate to 33.3% in patients without cirrhosis. HBsAg levels at the EOT was similar in relapsers (mean 828 IU/ml) compared with non-relapsers (mean 565 IU/ml). There was no difference between relapsers and non-relapsers in the magnitude of the decline in the levels of HBsAg and HBV DNA from baseline to 6 months of ETV therapy [4].

In a recent controlled study, HBeAg-negative patients without cirrhosis who had received TDF for ≥4 years, with suppressed HBV DNA for ≥3.5 years, were randomly assigned to either stop (n = 21) or continue (n = 21) TDF monotherapy. Of the patients who stopped TDF therapy, 62% (n = 13) remained off-therapy to week 144. Median HBsAg change in this group was -0.59 log10 IU/ml (range -4.49 to 0.02 log10 IU/ml) vs. 0.21 log10 IU/ml in patients who continued TDF therapy. Four patients (19%) achieved HBsAg loss. Patients stopping therapy had initial fluctuations in viral load and ALT; however, at week 144, 43% (n = 9) had either achieved HBsAg loss or had HBV DNA <2,000 IU/ml. There were no unexpected safety issues identified with stopping TDF therapy in this study. Furthermore, only patients with HBsAg

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levels <25,000 IU/ml at the time of treatment cessation showed a >0.5 log10 decrease in HBsAg levels after stopping TDF. No predictors of HBsAg loss were reported in this randomised study [7].

Since most studies included a relatively small number of patients and had various designs, a recent large real-world prospective study was conducted to investigate the issue of stopping NA therapy in Asian HBeAg-negative CHB patients. Of the 1,075 HBeAg-negative patients treated with NA for 156 (61-430) weeks, 6 showed HBsAg seroclearance during treatment at an estimated annual incidence of 0.15%. Of the patients who remained HBsAg seropositive, 691 (52.3 years old, 86% males, 44.6% cirrhosis) had stopped NA therapy using the APASL stopping rule and then were prospectively followed-up. Baseline and on-treatment clinical and viral features, treatment duration, consolidation duration, time to undetectable HBV DNA, time to normal ALT, EOT HBsAg and HBsAg log reduction were compared between patients with and without HBsAg seroclearance after EOT. During a median off-therapy follow-up period of 155 (2-614) weeks, HBsAg seroclearance was confirmed in 42 patients. The 6-year cumulative incidence was 13% with an estimated annual incidence of 1.78%. Cox regression analysis showed that shorter time to undetectable HBV DNA (<12 weeks), greater

HBsAg reduction during therapy (>1 log10), lower EOT HBsAg level (<100 IU/ml), patients with sustained response and relapsers not retreated were factors for off-therapy HBsAg seroclearance. During follow-up after cessation of therapy, 7 of the 308 patients with cirrhosis suffered from hepatic decompensation, and three died despite retreatment [8].

Overall, the incidence of HBsAg seroclearance after stopping NA seems significantly higher than that during therapy (see Table 1 summarising the main studies). Patients with a more pronounced and rapid response to NA have a better off-treatment response. Patients with clinical relapse who remain untreated have a higher incidence of HBsAg clearance than those who received retreatment, suggesting that transient untreated clinical relapse may drive sufficient immune control to achieve a functional cure of infection. SESSION 5 – HBV

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PARIS, FRANCE ; Tx, Ref. 9 6 5 10 11 12 7 8 HBsAg EOT* IU/ml;2 log reduction >1 log10 161 IU/ml 120 IU/ml 300 IU/ml 50 IU/ml IU/ <25,000 ml <100 IU/ml ; TLV, telbivudine ; TLV, Incidence – 5 yrs23% 39% – 3 yrs 30% – 6 yrs 46% – 6 yrs – 6 yrs55% wks 144 19% 6 yrs13% HBsAg loss 11/53 13/33 ? 20/73 44/119 4/21 42/691 ; TDF, tenofovir disoproxil; TDF, fumarate FU duration 47 mo 5.5 yrs 49 mo 3 yrs 66.8 mo 6 yrs 144 wks 155 wks Tx duration 27 mo 4-5 yrs 93 wks 5.3 yrs 30 mo 151 wks >4 yrs 156 wks SESSION 5 – HBV ; ETV, entecavir; ETV, adefovir Age (yr) 56 52 49 42 52 52 45 52 Male (%) 81 82 78 73 78 79 79 86 Non- LC 35 33 73 33 0 91 21 383 LC 18 0 32 0 73 28 0 308 N 53 33 105 33 73 119 21 (42) 691 Type of NA of Type LAM ADV LAM L A M, A DV, ET V, TDF ET V, L A M, TLV LAM, ETV TDF ET V, TDF, L A M, A DV, TLV Table 1. Summary 1. Table studies of on off-therapy HBsAg seroclearance (modified from Jeng et al., Hepatology In press 2018, [8]). * values HBsAg of EOT at associated with subsequent HBsAg loss. analogue;NA, LAM, nucleos(t)ide lamivudine; ADV, treatment;treatment; of end reference; months; mo, Ref, EOT, FU, wks, follow-up; weeks; yrs, years.

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NOVEL BIOMARKERS TO PREDICT OFF-TREATMENT RESPONSES From all these studies, it is apparent that biomarkers predicting off-treatment responses are missing. Thresholds of HBsAg levels have not yet been defined. It is unknown whether the pool of intrahepatic cccDNA at the time of treatment cessation would predict the outcome when treatment is stopped, as none of the studies have included an EOT liver biopsy analysis. To circumvent this problem, surrogate markers of the cccDNA pool size or transcriptional activity could be interesting to study in the context of stopping NA strategies [13]. Among them, the quantification of serum hepatitis B core-related antigen (HBcrAg) and/or of circulating viral RNAs appears promising. HBcrAg still suffers from its lower limit of detection, especially in NA virally suppressed HBeAg-negative CHB, and circulating viral RNAs are still lacking an approved standardised quantification test.

Other investigators have looked at immune parameters that could predict the outcome after cessation of NA. In a small prospective study, viral and host markers were studied after stopping long-term therapy with NA in patients without cirrhosis with HBeAg-negative CHB. After stopping therapy, 13 of 15 patients experienced a virological relapse. Rebound of HBV DNA and HBcrAg was associated with induction of plasma tumour necrosis factor, interleukin (IL)- 10, IL-12p70, CXCL10 and subsequent decline in HBsAg, with 3 patients (20%) experiencing HBsAg loss after long-term follow-up. However, due to the small size of the study it was not possible to identify a specific type of cytokine or a specific profile predicting HBsAg decline [14].

Another interesting study aimed at characterising the immune profile in CHB with CyTOF, to identify biomarkers of immune control following NA therapy discontinuation. This was a cohort of virally suppressed patients on NA therapy (>24 months) before and after treatment discontinuation. The investigators could distinguish two groups of patients: i) non-flare with maintained normal ALT (<80 IU/ml); and ii) flare with post-treatment elevated ALT (>150 IU/ml) and viral rebound. Their detailed immunological analysis showed that at NA SESSION 5 – HBV discontinuation, patients with detectable frequencies of circulating HBV-specific T cells controlled HBV replication and had no hepatic flare. In patients without viral control who developed hepatic flares, a significant increase was observed in serum CXCL10 and IL10 levels correlating with ALT elevation [15].

PRAGMATIC TREATMENT DECISION ALGORITHM Based on the results of published studies, NA discontinuation can be considered in patients with prolonged viral suppression (see Fig. 1). Patients with cirrhosis should be excluded from this strategy because of the risk of post-treatment flare and hepatic decompensation, observed only in patients with cirrhosis. Stopping NA can only be proposed to patients who can follow a precise post-treatment monitoring. When patients meet all these criteria, the quantification of HBsAg and, when available, the kinetics of response to NA for HBsAg and HBV DNA can help identify those patients with a higher likelihood of post-treatment response and HBsAg loss.

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Fig. 1. Algorithm for stopping NA in HBeAg-negative patients.

References

References in bold are requierd reading SESSION 5 – HBV

[1] European Association for the Study of the Liver. EASL 2017 Clinical Practice Guidelines on the management of hepatitis B virus infection. J Hepatol 2017;67:370-398.

[2] Liaw YF, Leung N, Kao JH, Piratvisuth T, Gane E, Han KH, et al. Asian-Pacific consensus statement on the management of chronic hepatitis B: a 2008 update. Hepatol Int 2008;2:263-83.

[3] Papatheodoridis G, Vlachogiannakos I, Cholongitas E, Wursthorn K, Thomadakis C, Touloumi G, Petersen J. Discontinuation of oral antivirals in chronic hepatitis B: A systematic review. Hepatology 2016;63:1481-92.

[4] Jeng WJ, Sheen IS, Chen YC, Hsu CW, Chien RN, Chu CM, Liaw YF. Off-therapy durability of response to entecavir therapy in hepatitis B e antigen-negative chronic hepatitis B patients. Hepatology 2013;58:1888-96.

[5] Chen CH, Lu SN, Hung CH, Wang JH, Hu TH, Changchien CS, Lee CM. The role of hepatitis B surface antigen quantification in predicting HBsAg loss and HBV relapse after discontinuation of lamivudine treatment. J Hepatol 2014;61:515-22.

[6] Hadziyannis SJ, Sevastianos V, Rapti I, Vassilopoulos D, Hadziyannis E. Sustained responses and loss of HBsAg in HBeAg-negative patients with chronic hepatitis B who stop long-term treatment with adefovir. Gastroenterology 2012;143:629-36 e1.

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[7] Berg T, Simon KG, Mauss S, Schott E, Heyne R, Klass DM, et al. Long-term response after stopping tenofovir disoproxil fumarate in non-cirrhotic HBeAg- negative patients – FINITE study. J Hepatol 2017;67:918-924.

[8] Jeng WJ, Chen YC, Chien RN, Sheen IS, Liaw YF. Incidence and predictors of HBsAg seroclearance after cessation of nucleos(t)ide analogue therapy in HBeAg negative chronic hepatitis B. Hepatology 2017. [Epub ahead of print].

[9] Chan HL, Wong GL, Chim AM, Chan HY, Chu SH, Wong VW. Prediction of off- treatment response to lamivudine by serum hepatitis B surface antigen quantification in hepatitis B e antigen-negative patients. Antivir Ther. 2011;16(8):1249-57

[10] Patwardhan VR, Sengupta N, Bonder A, Lau D, Afdhal NH. Treatment cessation in noncirrhotic, e-antigen negative chronic hepatitis B is safe and effective following prolonged anti-viral suppression with nucleosides/nucleotides. Aliment Pharmacol Ther. 2014;40(7):804-10.

[11] Hung CH, Wang JH, Lu SN, Hu TH, Lee CM, Chen CH. Hepatitis B surface antigen loss and clinical outcomes between HBeAg-negative cirrhosis patients who discontinued or continued nucleoside analogue therapy. J Viral Hepat. 2017;24(7):599-607.

[12] Yao CC, Hung CH, Hu TH, Lu SN, Wang JH, Lee CM, Chen CH. Incidence and predictors of HBV relapse after cessation of nucleoside analogues in HBeAg-negative patients with HBsAg ≤ 200 IU/mL. Sci Rep. 2017;12;7(1):1839.

[13] Testoni B, Levrero M, Zoulim F. Challenges to a Cure for HBV Infection. Semin Liver Dis 2017;37:231-242.

[14] Honer Zu Siederdissen C, Rinker F, Maasoumy B, Wiegand SB, Filmann N, Falk CS, et al. Viral and Host Responses After Stopping Long-term Nucleos(t)ide Analogue Therapy

SESSION 5 – HBV in HBeAg-Negative Chronic Hepatitis B. J Infect Dis 2016;214:1492-1497.

[15] Gill US, Rivino L, Le Bert N, Kunasega-ran K, Tan D, Koh S, et al. Characterisation of the immune profile in Chronic Hepatitis B, with CyTOF, to identify biomarkers of immune control following NUC therapy discontinuation. Hepatology 2015;62(Supplement: 1):294A.

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MANAGEMENT OF HBeAg-NEGATIVE PATIENTS WHO STOPPED LONG-TERM TREATMENT WITH NUCLEOS(T)IDE ANALOGUES

Florian van Bömmel, Thomas Berg* Section of Hepatology, Clinic for Gastroenterology and Rheumatology, University Hospital Leipzig, Leipzig, Germany. E-mail address: [email protected]

TAKE-HOME MESSAGES • Discontinuation of nucleos(t)ide analogues (NA) frequently results in a virologic and biochemical flare that runs through different phases: the lag phase, reactivation phase, and consolidation phase.

• The flares observed during the reactivation phase are often transient and most likely represent a trigger for inducing a long-term HBV-specific immune control, and therefore do not need immediate interventions but close follow-up evaluations.

• In order to guarantee a safe and effective outcome of NA treatment discontinuation, the NA retreatment should be initiated timely enough to prevent harm for the patient, but virologic and biochemical flares should be tolerated to some extent to allow the establishment of an HBV-specific immune control.

• Recommendations are provided on how to follow and monitor patients during the different phases after NA cessation and when starting NA retreatment seems indicated. SESSION 5 – HBV

NA DISCONTINUATION AS A NOVEL TREATMENT APPROACH Discontinuing a long-term treatment with oral nucleos(t)ide analogues (NA) in HBeAg- negative patients has been shown to lead to a stable immune control over the HBV infection in many patients, and even to HBsAg loss in some of those patients in whom a functional cure is otherwise very rare. Because of the encouraging reports from many small studies and the relatively low risk for the patients undergoing NA discontinuation it has been included as an option for HBeAg-negative patients in the recently published European treatment guidelines [1]. However, to date there are no prospective randomised studies that give detailed evidence for the management of patients undergoing NA discontinuation. In this module we aim to characterise the time period following NA discontinuation and give recommendations on how to follow-up patients undergoing NA discontinuation.

HBV DNA RELAPSES AFTER NA DISCONTINUATION MAY TRIGGER IMMUNE RESPONSE OR LIVER DAMAGE Virological relapses are observed in most HBeAg-negative chronic hepatitis B patients who discontinue NAs, and in many of these patients the virological relapses are followed by a

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biochemical relapse [2-8]. Formerly, the relapse after NA discontinuation has mostly been associated with the progression of liver necroinflammation, and hepatic failure has been described in some patients that stopped NAs [4, 5]. A relapse was therefore taken as an indication for prompt retreatment. However, recent studies have shown that the occurrence and the phenotype of the relapse after NA discontinuation may not only be a predictor for the occurrence of significant liver damage but also a trigger for the achievement of immune control [9]. Defining the right time point for retreatment is thus crucial in order to grant safety for the patient undergoing NA discontinuation on the one hand and to allow HBV-specific immune amplification during the relapse on the other hand.

From the present studies, it is suggested that patients run through different phases during the period after NA discontinuation, all of which deserve the special attention and measures by the treating physician in order to provide optimal care for the patient. We suggest here a classification of three phases of the viral relapse and describe their individual characteristics and measures that should be taken to maintain the patient’s safety (Fig. 1). SESSION 5 – HBV

Fig. 1. Suggested phases after discontinuation of long-term NA treatment in patients with HBeAg-negative chronic hepatitis B according to courses of HBV DNA, HBsAg and ALT and possible long-term outcomes (modified according to Lampertico P and Berg T; Hepatology 2018, In press).

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THE LAG PHASE The lag phase describes the period that lies between the time point of NA discontinuation and the reappearance of HBV DNA. During the lag phase, ALT levels and liver function are normal, and the patient should be free of complaints associated to NA discontinuation. Most viral relapses will likely occur shortly after NA discontinuation (i.e., within the first 1-6 months). However, the duration until the occurrence of the relapse may vary and relapses may even occur after more than 12 months [3, 4, 7, 8]. Thus, in a recent study of 130 HBeAg- negative patients from Greece and Taiwan that showed at least 24 months of undetectable HBV DNA during treatment with different NAs, it was found that HBV DNA reappeared in 73% of patients within the first 6 month after NA discontinuation [3]. Until month 12, another 8% of patients had relapsed. Intriguingly, between months 12 and 24, 9% of patients additionally showed a viral relapse. Accordingly, in a prospective study in 184 HBeAg-negative Asian patients the relapse rate was 74.3% at month 6 after NA discontinuation with a peak of relapses between months 3 and 6, and relapses increased to 91.4% at month 12 after NA discontinuation (Fig. 2) [2].

Factors influencing the duration of the lag phase are not well-defined; however, it seems that HBsAg levels either at NA baseline or at the time point of NA cessation may play a role as some studies have shown that the lower the HBsAg levels the lower the risk for virologic relapse [3, 5, 7]. Moreover, from a recent study from Taiwan there is evidence that the time point of viral relapse may be associated with the type of NA that was used [10]. Accordingly, in this prospective analysis the median time to virologic relapse was significantly shorter in patients who had received tenofovir (n = 34), as compared to those who had received entecavir (n = 66; 2.4 vs. 6.2 months, p = 0.035). Other factors that potentially influence the duration of the lag phase could be the duration of the consolidation treatment after HBV DNA has become undetectable, the immune status (including host genetic variants) or the HBV genotype [3, 5, 6, 7, 10]. SESSION 5 – HBV

Fig. 2. Increase of HBV DNA relapses >2,000 IU/ml in HBeAg-negative patients after discontinuation of treatment with entecavir. Most virologic relapses occurred between weeks 12 and 24. At 48 weeks after NA discontinuation, a virologic relapse was seen in almost all patients (modified from Seto et al. [2]).

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Although the present knowledge on the lag phase needs confirmation in large and prospective studies, the possibility of late relapses should be taken into account. As the total rate of patients achieving a viral relapse during the long-term follow-up after NA discontinuation is high (up to 100 percent, Table 1), it seems appropriate to monitor alanine aminotransferase (ALT) and HBV DNA on a monthly basis during this phase.

THE REACTIVATION PHASE The reactivation phase starts with the reappearance of HBV DNA in patients undergoing NA discontinuation, in many cases including an ALT flare, and it ends when HBV DNA and ALT levels have returned to a plateau (Fig. 1). The reactivation of HBV replication is likely the pivotal event that determines the degree of immune control that can be achieved by NA discontinuation. In a recent study, several plasma cytokines, chemokines and growth factors indicating T cell activity were analysed in the serum of HBeAg-negative patients in whom NA treatment was stopped. It was found that they showed increased expression after the reappearance of HBV DNA [9]. It can therefore be hypothesised that the sudden exposure of HBV DNA to the immune system represents a trigger for immune activation, and it seems obvious that this trigger should not be turned down immediately by an early retreatment approach when aiming for achieving a functional cure.

Flares in ALT levels follow many virologic reactivations. Most ALT flares after NA discontinuation were described to be self-limiting, however, some cases with fatal reactivations have been described, all of them having liver cirrhosis at baseline (Table 1). Therefore, this careful selection and surveillance of this phase deserves the special attention of the physician who is monitoring the NA discontinuation.

SESSION 5 – HBV THE CONSOLIDATION PHASE The consolidation phase starts when HBV DNA and ALT levels have reached a plateau and shifts into a long-term outcome (Fig. 1). The duration of this phase is undefined. It is thus possible that HBsAg has already become undetectable during the reactivation phase and the endpoint of NA discontinuation has already been reached. On the other hand, HBsAg losses can still occur after 24 months (or possibly later) after NA discontinuation. The consolidation phase should therefore be considered important for immune control development (Fig. 3) [9]. The monitoring of the patient can be extended to a three-monthly follow-up during the consolidation phase.

THE LONG-TERM OUTCOME The ideal outcome of NA discontinuation is the sustained loss of HBsAg, the functional cure (Fig. 1). Another endpoint is the absence of indication for retreatment. However, as disease reactivation may occur while HBsAg is positive, long-term follow-up of these patients is necessary. In contrast, about 40-60% of patients are on retreatment at this phase according to most series evaluating long-term outcome after NA cessation [3, 5, 7, 8].

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PARIS, FRANCE 0 0 0 0 1 (patient with cirrhosis) 3 (all patients with cirrhosis) Death (n) 0 0 0 0 n.a. 9 (all patients had cirrhosis at baseline) Hepatic decompensation (n)

7 [33%]** at week week at 7 [33%]** 12 55 [42%]** 93 [64%]¥ 42 [26%]** 274 [68.4%] 274 419 [60.6%]** ALT relapse (n [%]) (n any increase in HBV DNA; increase. ¥ any ALT ∑ ∑ SESSION 5 – HBV 21 [100%] 108 [83%]* 108 95 [65.5%]* 163 [91.4%]* 163 n.a. 547 [79.2%]* 547 HBV DNA relapse [%]) (n

144 weeks 24 months 16 months [1–88] 12 months 48 weeks [48-350] 155 weeks [2-614] Follow-up after NA disc. [range] 21 (0) 130 (0) 145 (n.a.) 145 184 (34) 401 691 (308) n (cirrhosis) . [2] et al. [8] et al et al. [4] et al. [5] et al. [6] Berg Seto Papatheodoridis et al. [3] Ha Kuo Jeng Study (first author) Table 1. Rates 1. virologic of Table and biochemical relapses as well as the occurrence hepatic of decompensation and deaths associated with NA discontinuation in selected studies. * Increase HBV of >2,000 DNA IU/ml; ** ULN; Increase >2x ALT of

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Fig. 3. The course of HBsAg, ALT and HBV DNA levels in patients who achieved HBsAg loss after discontinuation of long-term treatment with adefovir (modified from Hadziyannis et al. [7]). In most patients, the decrease in HBsAg levels started during the

SESSION 5 – HBV reactivation phase and HBsAg loss appeared in the consolidation phase.

A loss of HBsAg was shown to occur in rates between 0% and more than 30% of patients [2, 7, 8]. Interestingly, it can appear a long-time after NA cessation, also beyond the second and third year of follow-up. Quantitative HBsAg levels represent a marker that is helpful to identify patients who are about to develop HBsAg loss already during the reactivation phase (Fig. 1). Thus, it has been shown in different studies that HBsAg levels start decreasing during the reactivation phase in those patients who lose HBsAg during the subsequent period (Fig. 3) [5, 7, 8, 9]. As a reverse conclusion, it can be deduced that in patients who do not show a decrease in HBsAg levels during the reactivation phase a HBsAg loss is unlikely to be established. Hence, further studies need to prove whether in those patients without any decrease in HBsAg levels during the reactivation phase, a retreatment can be started in those who have an indication according to current treatment guidelines, irrespective of our proposed retreatment rules [1, 8].

SAFETY MEASURES FOR PATIENTS UNDERGOING NA DISCONTINUATION A retreatment with the same potent NA that was given before treatment cessation has been shown to be effective in rapidly suppressing viral replication but also leads to cessation of ALT flares [7]. Although data from large prospective and randomised studies are missing, a retreatment with tenofovir was shown to lead to a normalisation of ALT and a decrease

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PARIS, FRANCE in HBV DNA levels in the few patients with severe disease reactivation after discontinuation of tenofovir (Fig. 4) [8]. We recommend using a NA with a strong antiviral activity such as tenofovir or entecavir for retreatment. SESSION 5 – HBV

Fig. 4. Individual courses of virologic and biochemical relapse of HBV infections after discontinuation of treatment with tenofovir and response to retreatment. Two patients with early flares and one with persistent high viremia showed prompt response to retreatment with tenofovir (modified from Berg et al. [8]).

One needs to be aware that some fatal courses after NA discontinuation have been reported, even after immediate re-start of NAs. Although it seems that all of the patients suffering a fatal reactivation had liver cirrhosis, the risk needs to be taken seriously. To grant safety for the patients, NA should not be stopped in patients with liver cirrhosis or significant concomitant diseases including extrahepatic HBV manifestations [8]. In those patients in whom NA treatment is being discontinued, we propose early retreatment criteria according to the protocol from the FINITE study (Table 2). In all other patients, not fulfilling immediate retreatment criteria, an individualised retreatment approach has to be applied. Moderate HBV DNA rebounds with and without mild ALT flares following NA discontinuation in patients without cirrhosis should be obviously tolerated for a certain time in order not to terminate the potential HBV-specific immune response being induced by the virologic relapse (Table 2).

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Table 2. Proposed retreatment criteria for non-cirrhotic HBeAg-negative patients after NA discontinuation (modified from Berg T et al. [8]).

1. Confirmed (i.e., two consecutive central laboratory results) increase in direct bilirubin from baseline, and ALT ULN at the confirmatory test 2. Confirmed sustained increase in prothrombin time ≥2.0 s from baseline with appropriate vitamin K levels and elevated ALT 3. Confirmed elevated ALT 10 x ULN with or without associated symptoms 4. ALT 2 x ULN and ≤5x ULN persisting for ≥84 days (12 weeks) as well as a HBV DNA relapse ≥ 20,000 copies/ml 5. ALT 5 x ULN and ≤10 x ULN persisting for ≥28 days (4 weeks)

CONCLUSION Currently, discontinuation of an effective long-term NA treatment represents a promising approach to achieve functional cure or treatment-free survival for selected HBeAg- negative patients. It is helpful to distinguish different phases of the HBV infection after NA discontinuation to provide adequate measures with respect to follow-up monitoring and retreatment approaches because these will differ according to the different phases. The role and type of HBV reactivation after NA cessation for the long-term outcome and the induction of a functional HBV cure needs further investigations to develop new management approaches for those patients. Finally, large-scale studies are needed to confirm the best approach to monitor patients after stopping NAs, and to establish sensitive and specific indicators for starting retreatment.

SESSION 5 – HBV References

[1] European Association for the Study of the Liver. EASL 2017 Clinical Practice Guidelines on the management of hepatitis B virus infection. J Hepatol. 2017 Aug;67(2):370-398.

[2] Seto WK, Hui AJ, Wong VW, Wong GL, Liu KS, Lai CL, et al. Treatment cessation of entecavir in Asian patients with hepatitis B e antigen negative chronic hepatitis B: a multicentre prospective study. Gut 2014;64:667-672.

[3] Papatheodoridis GV, Manolakopoulos S, Su TH, Siakavellas S, Liu CJ, Kourikou A, Yang HC, Kao JH. Significance of definitions of relapse after discontinuation of oral antivirals in HBeAg-negative chronic hepatitis B. Hepatology. 2017 Aug 31.

[4] Kuo YH, Chen CH, Wang JH, Hung CH, Tseng PL, Lu SN, et al. Extended lamivudine consolidation therapy in hepatitis B e antigen–positive chronic hepatitis B patients improves sustained hepatitis B e antigen seroconversion. Scand J Gastroenterol 2010;45:75-81.

[5] Jeng WJ, Chen YC, Chien RN, Sheen IS, Liaw YF. Incidence and predictors of HBsAg seroclearance after cessation of nucleos(t)ide analogue therapy in HBeAg negative chronic hepatitis B. Hepatology. 2017 Nov 6.

[6] Ha M, Zhang G, Diao S, Lin M, Sun L, She H, et al. A prospective clinical study in hepatitis B e antigen–negative chronic hepatitis B patients with stringent cessation criteria for adefovir. Arch Virol 2012;157:285-290

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[7] Hadziyannis SJ, Sevastianos V, Rapti I, Vassilopoulos D, Hadziyannis E. Sustained responses and loss of HBsAg in HBeAg-negative patients with chronic hepatitis B who stop long-term treatment with adefovir. Gastroenterology. 2012;143:629-636.

[8] Berg T, Simon KG, Mauss S, Schott E, Heyne R, Klass DM, et al.; FINITE CHB study investigators [First investigation in stopping TDF treatment after long-term virological suppression in HBeAg-negative chronic hepatitis B]. Long-term response after stopping tenofovir disoproxil fumarate in non-cirrhotic HBeAg-negative patients – FINITE study. J Hepatol. 2017pii:S0168-8278(17)32155-4.

[9] Höner Zu Siederdissen C, Rinker F, Maasoumy B, Wiegand SB, Filmann N, Falk CS, et al. Viral and host responses after stopping long-term nucleos(t)ide analogue therapy in HBeAg-negative chronic hepatitis B. J Infect Dis. 2016;214:1492-1497.

[10] Su TH, Yang HC, Tseng TC, Liou JM, Liu CH, Chen CL, et al. Distinct relapse rates and risk predictors after discontinuing tenofovir and entecavir therapy. J Infect Dis. 2018. [Epub ahead of print]. SESSION 5 – HBV

EASL THE HOME OF HEPATOLOGY 157 62.2018EventsCalendar-A4_CMYK.pdf 1 09.03.2018 18:28:20

MEETINGS

2018 GUT-LIVER AXIS

C 07 - 09 June 2018 Leuven, Belgium M

CMY

ABDOMINAL SONOGRAPHY, NAFLD SUMMIT 2018 A CLINICAL COURSE

07 - 08 September 2018 20 - 22 September 2018 Rotterdam, Netherlands Geneva, Switzerland

DIAGNOSIS AND MANAGEMENT OF VASCULAR AASLD- EASL LIVER DISEASES, MASTERCLASS 2018 A CLINICAL COURSE

23 - 24 November 2018 29 November - 01 December 2018 Bern, Switzerland Washington, United States www.easl.eu/_events THE INTERNATIONAL LIVER CONGRESS™ 2018

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THURSDAY 12 APRIL 2018 10:30-12:00 SESSION 6 – HCV

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PRACTICAL HCV TREATMENT IN PATIENTS WITH DECOMPENSATED CIRRHOSIS

Sabela Lens, Zoe Mariño, Sergio Rodríguez, Juan Carlos García-Pagán, Xavier Forns* Liver Unit, Hospital Clínic, IDIBAPS and CIBEREHD. The University of Barcelona, Barcelona, Spain. E-mail address: [email protected]

TAKE-HOME MESSAGES • Direct-acting antivirals achieve high efficacy rates even in patients with advanced liver disease.

• Protease inhibitors should be avoided in Child-Pugh B/C patients for safety reasons.

• Hepatitis C virus (HCV) clearance may be associated with liver function improvements and a reduction in the number and severity of decompensation events. Patients with Model for End-Stage Liver Disease score >20 are less likely to improve; if they are liver transplant candidates, antiviral treatment should be preferably given after transplantation.

• Hemodynamic studies show a significant decrease of hepatic venous pressure gradient (HVPG) values soon after successful antiviral treatment. However, a significant number of patients with cirrhosis remain at risk of liver decompensation (HVPG ≥10 mmHg) at least in the short-term. Studies with longer follow-up periods are necessary to know if liver architectural changes are irreversible in some patients (“point of no return”) and if these patients can be identified.

• Transient elastography after HCV cure is not an accurate tool to discard the presence of clinically significant portal hypertension. SESSION 6 – HCV – SESSION 6 • Lifelong screening for hepatocellular carcinoma in patients with cirrhosis must be performed, as risk persists after sustained virological response.

BACKGROUND Chronic hepatitis C virus (HCV) infection is one of the main causes of liver failure and liver transplantation in many parts of the world. Survival of patients with cirrhosis and portal hypertension, particularly after the first episode of clinical decompensation, is reduced as compared to patients with compensated disease (Fig. 1).

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Months after decompensation

Fig. 1. Survival curves after liver decompensation according to Child-Pugh state [1].

HCV-infected patients with decompensated cirrhosis have been and remain the most difficult-to-treat population. In the interferon era, the presence of portal hypertension was a contraindication for therapy, since the risk of severe adverse events (including sepsis and clinical decompensation) was very high and the response rates very low. With the appearance of the first-generation protease inhibitors (PI) telaprevir and boceprevir, which were administered in combination with interferon and ribavirin, the proportion of HCV-infected patients who achieved sustained virological response (SVR) increased significantly. Nevertheless, individuals with advanced liver disease were still at high risk of severe adverse events. Indeed, the presence of portal hypertension, as indicated by low platelet count (≤100,000/mm3) and/or liver failure (low albumin levels <35 g/L) identified individuals at high risk of sepsis, worsening of liver function and death [2].

The first direct-acting antiviral (DAA) that could be safely used in patients with decompensated HCV – SESSION 6 cirrhosis was the NS5B polymerase inhibitor sofosbuvir. The combination of sofosbuvir with ribavirin administered for 24 or 48 weeks was assessed in a compassionate use program in patients with advanced liver disease after liver transplantation, including individuals with decompensated cirrhosis [3]. Sustained virological rates in this difficult-to-treat population were achieved in nearly half of treated patients, with an excellent safety profile. Moreover, and despite the study not being a prospective one, clinical improvements (defined by liver function tests and/or liver-related events) occurred in a significant proportion of patients. Overall, the study indicated that HCV clearance was able to impact the liver function even in cases of advanced liver disease.

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WHAT TYPES OF HCV DAAS CAN AND CANNOT BE USED IN DECOMPENSATED PATIENTS? Following the approval of sofosbuvir, a number of different DAA have reached the market in the last few years, including PI (simeprevir, paritaprevir, asunaprevir, grazoprevir, glecaprevir, voxilaprevir), NS5A inhibitors (daclatasvir, ledipasvir, ombitasvir, elbasvir, velpatasvir) and NS5B polymerase inhibitors (sofosbuvir, dasabuvir).

Overall, very few studies have addressed the safety and efficacy of these new compounds in individuals with decompensated cirrhosis. In general, pharmacokinetic studies have shown that, compared with healthy individuals, the steady-state area under the plasma concentration- time curve is significantly increased when using PI in Child-Pugh B and particularly Child- Pugh C patients. In addition, several studies have shown grade 2 and grade 3 abnormalities in liver tests (bilirubin and aminotransferases) when PI were part of the treatment regimen. Due to these reasons, regulatory agencies have either contraindicated (in Child-Pugh C patients) or not recommended (in Child-Pugh B patients) PI as part of treatment regimens. However, it is worth noting that real-life studies have shown reasonable efficacy data (SVR rates 70-80%) using treatment combinations that include a PI (i.e. sofosbuvir and simeprevir) in individuals with past or present clinical decompensation episodes.

The first studies that specifically addressed the efficacy and safety of a treatment combination in patients with decompensated cirrhosis were the SOLAR 1 and 2 studies [4, 5]. In both studies, genotype 1 or 4 infected patients with Child-Pugh B or C cirrhosis (among other treatment groups) were randomised to receive sofosbuvir/ledipasvir with ribavirin, for 12 or 24 weeks. Overall, response rates ranged between 83 and 92%. Although SVR was slightly higher in Child-Pugh B as compared to C, the length of therapy (12 or 24 weeks) did not appear to influence the response rate. Results combining patients with decompensated cirrhosis from both studies are depicted below in Fig. 2. SESSION 6 – HCV – SESSION 6

Fig. 2. Merged SVR rates in patients included in the SOLAR 1 and 2 studies assessing the efficacy of sofosbuvir + ledipasvir + ribavirin during 12 or 24 weeks in patients with Child-Pugh B or C cirrhosis (data taken from [4, 5]).

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Safety using this combination was good, and the most severe adverse events were not related to the study medication. Besides HCV clearance, one of the main goals of treating patients with decompensated cirrhosis is the impact on liver function. This was also captured in the SOLAR studies. Indeed, improvements in Model for End-Stage Liver Disease (MELD) scores occurred in around two-thirds of non-transplanted patients who achieved SVR12 and had measurements for MELD recorded for both baseline and week 12 of follow-up. Although changes in MELD scores were generally small (1-3 points) it is also true that follow-up in these studies was not long enough to provide solid information on the real impact on liver function of viral eradication [4, 5].

Real-life studies, such as the NHS England expanded access programme (EAP) [6] that included more than 400 patients with decompensated cirrhosis also showed that the combination of sofosbuvir plus an NS5A inhibitor (ledipasvir or daclatasvir) was able to clear HCV in more than 80% of treated patients. This viral cure was also associated with significant improvements in liver function. Interestingly, individuals older than 65 years and with more advanced liver disease (as measured by an albumin <35 g/L) were less likely to benefit from therapy.

In the ASTRAL-4 study [7], the pan-genotypic combination of sofosbuvir and velpatasvir was assessed in 267 patients with Child-Pugh B cirrhosis. Individuals were randomised 1:1:1 to receive a 12 or 24 weeks regimen of sofosbuvir-velpatasvir, or the same combination during 12 weeks but with ribavirin. Genotype distribution was: 78% genotype 1, 4% genotype 2, 15% genotype 3, 3% genotype 4, and less than 1% genotype 6. Overall SVR rates were 83% among patients who received 12 weeks of sofosbuvir-velpatasvir, 94% among those who received 12 weeks of sofosbuvir-velpatasvir plus ribavirin, and 86% among those who received 24 weeks of sofosbuvir-velpatasvir. Post hoc analyses did not detect any significant differences in rates of SVR among the three treatment groups (Fig. 3). SESSION 6 – HCV – SESSION 6

Fig. 3. SVR12 rates in the ASTRAL-4 study according to the treatment arm in genotype 1 and 3 (data taken from [7]).

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Despite the small number of individuals with decompensated cirrhosis and infected with genotype 3, data from the study suggests that the contribution of ribavirin may be particularly important in this group of patients to clear HCV. In addition, and despite the short follow-up, it is worth mentioning that of the 250 patients who had liver function scores available at baseline and at post-treatment week 12, 47% had an improvement, 42% had stability, and 11% had a worsening in the Child-Pugh score. Changes in liver function scores were mainly explained by decreases in bilirubin and increases in albumin levels. Safety was good using this combination, and most drug-related side effects were ribavirin related. Nevertheless, as the number of treated individuals is limited, close follow-up of this particular group of decompensated patients with cirrhosis is very relevant.

WHICH PATIENTS WITH DECOMPENSATED CIRRHOSIS SHOULD BE TREATED AND WHICH REGIMEN SHOULD BE USED? From a theoretical point of view, all patients with decompensated cirrhosis should undergo antiviral therapy, unless there are underlying comorbidities that preclude a lower survival than that defined by the liver disease. The goal of antiviral treatment in these patients is not only to clear HCV but to impact on liver function. However, it is important to note that these improvements may be insufficient to avoid liver-related death or the need for liver transplantation, and therefore, not everyone benefits from DAA therapy. Indeed, most deaths among patients with decompensated cirrhosis receiving DAA therapy are related to the severity of the underlying liver disease.

The most relevant data assessing the impact of antiviral therapy on liver function comes from studies including patients with decompensated cirrhosis awaiting liver transplantation. In both a European and a Spanish multicentric study [8, 9] including 103 and 122 patients, respectively, DAA treatment was associated with liver function improvements that allowed “delisting” of around 20-25% of patients with decompensated cirrhosis (Fig. 4).

The main predictors of delisting in both studies were the baseline MELD score and the

SESSION 6 – HCV – SESSION 6 improvement of liver function tests (MELD, albumin) during DAA therapy [8, 9]. The possibility of delisting was very low for patients with a baseline MELD score above 18-20 and for this reason, clinical guidelines suggest that patients with clinical decompensation and a MELD score >20 are less likely to improve.

In the setting of patients awaiting liver transplantation, the MELD cut-off is a useful recommendation to decide which patients are candidates to undergo antiviral therapy. Indeed, individuals with a high MELD score might be better served by transplantation than treatment (which can be indicated after transplantation). However, for those patients who do not have an indication for transplantation and are still candidates for therapy, larger studies with a longer follow-up are clearly needed to better define the impact of HCV eradication on liver function (see below).

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Fig. 4. The comparable cumulative incidence of delisting after antiviral therapy while on the waiting list in two multicentric studies [8, 9]. (A) European multicentric study Belli et al. J. Hepatology 2016 and (B) Spanish multicentric study by Pascasio et al. J Hepatology 2017.

Regarding the treatment regimen to be used in patients with decompensated liver disease, and SESSION 6 – HCV – SESSION 6 based on the studies mentioned above, there are only a few regimens that can be used, mainly sofosbuvir with an NS5A inhibitor (ledipasvir for genotypes 1 and 4; daclatasvir or velpatasvir for all genotypes) with or without ribavirin. The recently updated AASLD guidelines (accessed online, https://www.hcvguidelines.org/) recommend that sofosbuvir plus ledipasvir and low-dose ribavirin (starting at 600 mg/day) or sofosbuvir plus velpatasvir and weight- based ribavirin for 12 weeks can be administered in individuals infected with genotypes 1, 4, 5 or 6. For patients infected with genotype 3, the combination of sofosbuvir and velpatasvir (or daclatasvir) with ribavirin for 12 weeks is the recommended regimen. In patients who are ribavirin ineligible, treatment should be extended to 24 weeks.

For those individuals with decompensated cirrhosis who failed a previous sofosbuvir or NS5A containing regimen, the only choice is to combine sofosbuvir with ledipasvir (genotypes 1, 4, 5, 6) or velpatasvir (all genotypes) with ribavirin for 24 weeks. As stated above, PI (including voxilaprevir) are not approved for use in patients with decompensated cirrhosis.

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FOLLOW-UP OF PATIENTS WITH DECOMPENSATED CIRRHOSIS Most studies assessing the impact of viral eradication on decompensated cirrhosis provide data on MELD scores improvements. Indeed, the proportion of patients with decompensated cirrhosis who were part of the SOLAR and ASTRAL studies and had an improvement of the MELD score after SVR12 ranged from 52 to 74% [4–7]. The limitation of these studies, however, is their short follow-up and the fact that they do not provide detailed information on the impact of viral clearance on portal pressure and specific decompensation episodes over time.

Recently, a prospective study including patients with cirrhosis and clinically significant portal hypertension (CSPH, hepatic venous pressure gradient [HVPG] ≥10 mmHg) undergoing DAA therapy assessed the effect of HCV clearance on portal hypertension [10]. A large number of patients (n = 226) underwent a baseline HVPG measurement, that was repeated 24 weeks after treatment finalisation. Most patients (75%) had oesophageal varices, around 20% were Child-Pugh B, and 29% had a previous episode of clinical decompensation. The overall results showed that HVPG decreased from 15 mmHg to 13 mmHg (p <0.01) (Fig. 5A); the reduction in portal pressure was greater than 10% in 140 (62%) of patients. Nevertheless, 176 (78%) of the patients remained with CSPH at 24 weeks after treatment, and thus, at risk of clinical decompensation (Fig. 5B). SESSION 6 – HCV – SESSION 6

Fig. 5. Changes in HVPG values 24 weeks after the end of therapy. Overall reduction in mmHg (A) and proportion of patients with CSPH at baseline and after SVR (B).

Patients who remained with CSPH in the previous study [11] will undergo a new hemodynamic assessment 96 weeks after end of treatment. Very preliminary data from 56 patients show that HVPG continues to decrease over time (-2 ± 3.2 mmHg at SVR24 and -4.3 ± 3.6 mmHg). Nevertheless, 43 (76%) of these patients still had CSPH 96 weeks after viral clearance (unpublished data).

Liver fibrosis is characterised by the accumulation of extracellular matrix, initially in the perisinusoidal space of Disse and portal tracts. This is then followed by the formation of regeneration nodules, vascular occlusion and angiogenesis that further disrupts the liver architecture [11]. The data mentioned above indicate that fibrosis regression occurs in some patients with very advanced liver disease (clinical decompensation) since a significant decrease in portal pressure is the best indicator of an improvement in liver architecture. However, this is

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PARIS, FRANCE not the case for other patients, highlighting the possibility of a “point of no return” where liver damage is not reversible (or requires an even longer follow-up). Finally, we should be aware that in some individuals, co-factors (such as active alcohol intake, non-alcoholic steatohepatitis) might explain a lack of improvement or even worsening of liver function even after HCV cure.

From a practical point of view, all patients with decompensated cirrhosis should have lifelong screening for hepatocellular carcinoma (HCC), since it is well known that the risk of HCC persists even after HCV eradication [12]. Regarding clinical decompensation, we know that the risk disappears in patients with an HVPG below 10 mmHg [13]. Nevertheless, only a few academic centres have the capability to determine HVPG. Moreover, non-invasive assessment of liver stiffness does not seem to be accurate at excluding the presence of CSPH after antiviral therapy [10]. Thus, patients with a previous episode of clinical decompensation must be followed for a long period, before assuming that the risk of decompensation is removed. Indeed, prospective studies should assess the impact of SVR on specific variables. For instance, the doses of diuretics (or the frequency of large volume paracentesis) should be recorded during and after HCV cure in order to demonstrate significant changes. Similarly, episodes of hepatic encephalopathy and the need for therapy or hospital admissions should also be prospectively documented.

Whether management of gastroesophageal varices in patients achieving HCV clearance is the same than in patients with active HCV infection or not must be evaluated [14]. It is reasonable to think that this is the case in those patients with SVR but who show no clear improvement in liver function or that remain with clinical significant portal hypertension (HVPG >10 mmHg). Indeed, some studies have shown the appearance of EV after SVR [15]. In patients with previous variceal bleeding receiving secondary prophylaxis with endoscopic band ligation (EBL) plus non-selective beta-blockers (NSBB), the recommendation would be to follow NSBB treatment and to continue endoscopy surveillance to confirm variceal eradication (or to reinitiate EBL if there is variceal recurrence). In patients with oesophageal varices on primary prophylaxis, NSBB need to be continued unless there is a marked improvement in liver function (ideally if HVPG can be measured and shown to be below 10 mmHg). The presence of oesophageal varices may be reassessed and if they are no longer present NSBB could be discontinued. If primary prophylaxis is based on EBL, recurrence of varices needs to be periodically checked

for potential additional EBL sessions. Again, if marked improvement after SVR is observed HCV – SESSION 6 and after several endoscopies (perhaps for 2 years) discarding variceal recurrence, follow-up screening endoscopies may be abandoned. Finally, in patients without or with small varices not requiring primary prophylaxis before SVR, after achieving HCV clearance it seems reasonable to follow the same endoscopic follow-up strategy than before SVR [14]. Screening would not be necessary in those individuals in whom HVPG was already lower than 10 mmHg before therapy or in whom HVPG decreased below 10 mmHg after DAA therapy. In the next few years, we will certainly have more solid data to refine the current recommendations. References

[1] Planas R, Montoliu S, Ballesté B, Rivera M, Miquel M, Masnou H, et al. Natural History of Patients Hospitalized for Management of Cirrhotic Ascites. Clin Gastroenterol Hepatol. 2006;4(11):1385–1394.e1.

[2] Hezode C, Fontaine H, Dorival C, Larrey D, Zoulim F, Canva V, et al. Triple therapy in treatment-experienced patients with HCV-cirrhosis in a multicentre cohort of the French Early Access Programme (ANRS CO20-CUPIC) – NCT01514890. J Hepatol. 2013;59(3):434–41.

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[3] Forns X, Charlton M, Denning J, Mchutchison JG, Symonds WT, Brainard D, et al. Sofosbuvir compassionate use program for patients with severe recurrent hepatitis C after liver transplantation. Hepatology. 2015;61(5):1485–94.

[4] Charlton M, Everson GT, Flamm SL, Kumar P, Landis C, Brown RS, et al. Ledipasvir and Sofosbuvir Plus Ribavirin for Treatment of HCV Infection in Patients with Advanced Liver Disease. Gastroenterology. 2015;149(3):649–59.

[5] Manns M, Samuel D, Gane EJ, Mutimer D, McCaughan G, Buti M, et al. Ledipasvir and sofosbuvir plus ribavirin in patients with genotype 1 or 4 hepatitis C virus infection and advanced liver disease: a multicentre, open-label, randomised, phase 2 trial. Lancet Infect Dis. 2016;16(6):685–97.

[6] Foster GR, Irving WL, Cheung MCM, Walker AJ, Hudson BE, Verma S, et al. Impact of direct acting antiviral therapy in patients with chronic hepatitis C and decompensated cirrhosis. J Hepatol. 2016;64(6):1224–31.

[7] Curry MP, O’Leary JG, Bzowej N, Muir AJ, Korenblat KM, Fenkel JM, et al. Sofosbuvir and Velpatasvir for HCV in Patients with Decompensated Cirrhosis. N Engl J Med. 2015;31;373(27):2618–28.

[8] Belli LS, Berenguer M, Cortesi PA, Strazzabosco M, Rockenschaub S-R, Martini S, et al. Delisting of liver transplant candidates with chronic hepatitis C after viral eradication: A European study. J Hepatol. 2016;Sep;65(3):524–31.

[9] Pascasio JM, Vinaixa C, Ferrer MT, Colmenero J, Rubin A, Castells L, et al. Clinical outcomes of patients undergoing antiviral therapy while awaiting liver transplantation. J Hepatol. 2017;67(6):1168–76.

[10] Lens S, Alvarado-Tapias E, Mariño Z, Londoño M-C, LLop E, Martinez J, et al. Effects of All-Oral Anti-Viral Therapy on HVPG and Systemic Hemodynamics in Patients With Hepatitis C Virus-Associated Cirrhosis. Gastroenterology. 2017;153(5):1273–1283.e1.

[11] Marrone G, Shah VH, Gracia-Sancho J. Sinusoidal communication in liver fibrosis and regeneration. J Hepatol. 2016;65(3):608–17. SESSION 6 – HCV – SESSION 6 [12] Meer AJ Van Der, Heathcote EJ, Janssen HLA. Association Between Sustained Virological and Advanced Hepatic Fibrosis. 2013;

[13] Ripoll C, Groszmann R, Garcia-Tsao G, Grace N, Burroughs A, Planas R, et al. Hepatic venous pressure gradient predicts clinical decompensation in patients with compensated cirrhosis. Gastroenterology. 2007;133(2):481–8.

[14] de Franchis R, Baveno VI Faculty R de, Pascal JP, Ancona E, Burroughs AK, Henderson JM, et al. Expanding consensus in portal hypertension: Report of the Baveno VI Consensus Workshop: Stratifying risk and individualizing care for portal hypertension. J Hepatol. 2015;63(3):743–52.

[15] Di Marco V, Calvaruso V, Ferraro D, Bavetta MG, Cabibbo G, Conte E, et al. Effects of Eradicating Hepatitis C Virus Infection in Patients With Cirrhosis Differ With Stage of Portal Hypertension. Gastroenterology. 2016;151(1):130–139.e2.

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REMOVING PATIENTS FROM THE TRANSPLANT LIST WITH ANTI-HCV TREATMENT

Norah Terrault University of California San Francisco, San Francisco, United States. E-mail address: [email protected]

TAKE-HOME MESSAGES • Safe and highly effective hepatitis C virus (HCV) therapies are available for patients with decompensated cirrhosis, setting the stage for potentially reversing decompensation and avoiding liver transplantation.

• Approximately ~20% of treated patients on the waiting list achieve sufficient improvement in their liver disease, allowing for inactivation or delisting within a year of starting treatment.

• Patients with a Model for End-stage Liver Disease (MELD) score <20 and an absence of moderate to severe portal hypertensive complications are most likely to achieve inactivation or delisting status with HCV treatment.

• Pre-transplant treatment increases life expectancy if the baseline MELD score is ≤23 to 27 (depending on median MELD at transplant in the region) but decreased at higher MELD scores, supporting a recommendation not to treat those with higher MELD scores.

• Long-term outcomes of those who are delisted for improvement are lacking but in the short- term most delisted patients remain without indications for liver transplant.

INTRODUCTION HCV – SESSION 6 The ability to offer a cure to patients infected with chronic hepatitis C virus (HCV) infection, even those with decompensated cirrhosis, has provided the opportunity to treat wait-listed patients with the goal of avoiding liver transplantation. The potential benefits of direct-acting antivirals (DAA) in patients with decompensated cirrhosis include reversal of decompensation (recompensation) and avoidance of liver transplant, lower rates of hepatocellular carcinoma (HCC), and lower rates of wait-list mortality. The potential risks include worsening decompensation, HCC and/or death despite achieving cure and the possibility of Model for End-stage Liver Disease (MELD) purgatory. It is the latter that is of greatest concern for wait-listed patients – HCV treatment leading to insufficient clinical improvement but improved MELD score and reduced access to transplant.

While historically treatment prior to transplant was viewed as highly desirable because treatment post-transplant was challenging and efficacy reduced, the currently approved DAA therapies are safe, highly effective and simple to use in the post-transplant setting thereby significantly reducing the impetus to treat HCV pre-transplant solely for this reason. Moreover, sustained virological response (SVR) rates in patients with decompensated cirrhosis are reduced compared to patients with compensated cirrhosis, and patients who fail therapy may have resistance profiles that make retreatment challenging. In contrast, after transplantation,

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when liver and renal function are typically better than in patients on the waiting list, DAA treatment options are greater (i.e. protease inhibitors can be used) and treatment success high [1]. Thus, the decision to treat patients with decompensated cirrhosis with the intent of achieving delisting requires an individualised consideration of the risks and benefits.

KEY QUESTIONS WHAT IS THE LIKELIHOOD OF HCV TREATMENT ACHIEVING SUFFICIENT CLINICAL IMPROVEMENT TO ALLOW REMOVAL FROM THE WAITING LIST? Prospective studies are lacking, but several retrospective studies have yielded estimates of success in delisting patients treated with DAA therapy (Table 1). Belli et al. examined a multicentre European transplant registry of 103 HCV-infected patients, with baseline median MELD of 16 and decompensated cirrhosis, treated with sofosbuvir-based DAA therapy and followed for median 52 weeks. The cumulative incidence of delisting at 24, 48 and 60 weeks was 9, 10 and 19%, respectively. Inactivation was achieved in 33% [2]. In an Italian study of 90 wait-listed patients with MELD ≥15 and Child-Pugh ≥B followed for median 48 weeks, 17% achieved MELD <15 and Child-Pugh-A (criterion judged to compatible with inactivation). Patients with a 2-point decline in MELD within the first 4 weeks of treatment were most likely (65%) to meet deactivation criteria at 48 weeks [3]. In a retrospective study from 18 centres in Spain of 122 DAA treated patients with decompensated cirrhosis without HCC on the waiting list, the cumulative probability of delisting was 27% with a median time from treatment start to delisting of 50 weeks (IQR 39-63) [4]. Of note, standard criteria for delisting were not used but all but 2/29 delisted had compensated cirrhosis and MELD <15. In a preliminary report from a French cohort of 77 wait-listed patients with decompensated cirrhosis without HCC, 16% of patients were delisted with a mean follow-up of 68 weeks [5]. Finally, in the only US data (data from SOLAR-1, SOLAR-2, GU-US-334-0125 and ASTRAL-4), of 622 patients with decompensated cirrhosis 19.8% Child-Pugh-C or 80.2% Child-Pugh-B clinically improved to Child-Pugh-A status (compatible with inactivation/delisted if wait-listed) achieved in 26.6%

SESSION 6 – HCV – SESSION 6 patients at 36 weeks [6]. Thus, the overall likelihood of improving to the point of deactivation or delisting is ~20% within the first year after treatment start.

Table 1. Summary of retrospective studies on the success in delisting patients treated with DAA therapy and baseline predictors. Author, N Percent Median time Predictors at Predictors on Region delisting or to delisting/ baseline treatment [Ref.] inactivation activation Belli, 100 19% Inactivation 26 MELD ∆MELD Europe [2] (17-38) weeks ∆Albumin Delisting 52 (37-59) weeks Martini, 90 17.3% n/a MELD ∆MELD Italy [3] Absence of (first 4 weeks) HE ∆Albumin ∆Bilirubin ∆INR

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Author, N Percent Median time Predictors at Predictors on Region delisting or to delisting/ baseline treatment [Ref.] inactivation activation Pascasio, 122 27% Delisted 50 None, but only ∆MELD Spain [4] (IQR = 39-63) patients with (baseline to weeks MELD <20 EOT) were delisted Coilly, 77 16% n/a Child-Pugh Not evaluated France [5] Mean follow-up score 68 weeks Jiang, US/ 622 36% (Child- n/a BMI <25 Not evaluated Europe [6] Pugh B Median follow-up Absence of baseline) 36 weeks HE Absence 12% (Child- of ascites ALT Pugh C >1.5 U LN baseline) Albumin >3.5 g/dl

ALT, alanine aminotransferase; BMI, body mass index; EOT, end of treatment; HE, hepatic encephalopathy; INR, international normalisation ratio; IQR, interquartile ratio; MELD, Model for End-stage Liver Disease; n/a, not applicable; ULN, the upper limit of normal.

WHAT IS THE LIKELIHOOD OF REMOVING PATIENTS FROM THE TRANSPLANT LIST AFTER ANTI-HCV TREATMENT ACCORDING TO MELD AND OTHER SCORES? Since only one in five treated patients is likely to improve to the point of removal from the waiting list and the remainder may be at risk of MELD purgatory, being able to predict those likely to improve to the point of removal versus those unlikely to do so, is critical. Several HCV – SESSION 6 studies have highlighted that patients with improvements in MELD, MELD components and albumin during treatment, especially early on, are more likely to achieve improvements that lead to wait-list inactivation/delisting [2-4] (Table 1). In one study, 65% of patients with a 2-point decline in MELD score within the first 4 weeks of treatment were able to achieve the endpoint of reactivation [3]. The time to greatest improvement in MELD is 12 weeks, whereas improvements in the Child-Pugh score are slower [2, 3]. Thus, clinical improvements lag behind MELD score improvements. However, from the standpoint of guiding decisions on who to treat, the best predictors would be prior to treatment, rather than those during or after treatment.

The factors most consistently associated with baseline characteristics predictive of delisting, inactivation or return to Child-Pugh-A status, are lower baseline MELD or Child-Pugh score and absence of portal hypertensive complications (ascites/encephalopathy). In the Spanish study from Pascasio and colleagues, 50% of patients with baseline MELD <16 achieved inactivation status, 20% of those with baseline MELD 16-20 and none with baseline MELD >20 [4]. In the multicentre European registry study, the median baseline MELD score among delisted patients was 13.5 (range 9-24) [2]. In the largest study of Child-Pugh-B and C patients from Jiang and colleagues, a 5-point scale including BMI <25, absence of encephalopathy, absence of ascites, alanine aminotransferase (ALT) >1.5 upper limit of normal (ULN) and albumin

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>3.5 g/dl were shown to predict the likelihood of achieving Child-Pugh-A status [6]. For each point increase from 0 to 5, the likelihood of achieving Child-Pugh-A status increased from <5% to ~50% at 12 months post-treatment [6]. Collectively, these results suggest treatment is best undertaken in those with MELD <20 and the absence of significant ascites or hepatic encephalopathy [7].

WHAT IS THE PROGNOSIS OF PATIENTS TREATED PRE-TRANSPLANT? WHAT ARE THE CONSEQUENCES OF MELD PURGATORY? The majority of patients who are treated achieve an improvement in MELD, though the magnitude of the improvement is modest with follow-up periods of approximately one year [8-10]. For example, among 122 wait-listed patients with median MELD score of 14 (IQR 12-17), 60% had lower, 9% no change and 31% higher MELD score between baseline and the last follow-up [4]. The median improvement in MELD was 3 points (range 1-5) [4]. In a multicentre study with 528 patients with Child-Pugh-B/C cirrhosis who achieved SVR12 and followed for a median of 255 days, 330 (62.5%) remained in MELD purgatory, which was defined as having Child-Pugh-B or C cirrhosis with MELD <15 [6]. Baseline albumin ≤3.5 g/ dl, ascites or encephalopathy were associated with MELD purgatory [6].

Treatment of wait-listed patients is associated with a reduction in the incidence of decompensating events – in one study, from 18% during the first 6 months after start of treatment to 8% in the subsequent 6-15 months [4]. In a study of patients with HCC, DAA therapy was associated with a reduction in wait-list dropout due to tumour progression and/or death [11] and another study found that treated wait-listed patients with HCC were made more amenable to curative interventions (resection, radiofrequency ablation) [4]. These HCC studies highlight that clinically relevant improvements in liver function occur with viral eradication.

Decision analyses comparing treatment pre-transplant to post-transplant indicate that pre-transplant treatment is more cost-effective in both patients with HCC and those with decompensated cirrhosis but with results influenced by SVR rates, utility of patients post-

SESSION 6 – HCV – SESSION 6 liver transplantation, liver transplantation costs, and baseline MELD score in patients with decompensated cirrhosis [12]. Life expectancy is increased with HCV treatment before transplant if the baseline MELD was ≤27 but decreased at higher MELD scores. For example, in the US where median MELD scores at liver transplant varies by United Network of Organ Sharing (UNOS) region, the threshold MELD score to treat HCV pre-liver transplantation varied between 23 and 27 across the different UNOS regions [13].

IS DELISTING A MEANINGFUL ENDPOINT FOR STUDYING DAA THERAPY AMONG WAIT-LISTED PATIENTS? Limited data suggest that liver complications after delisting are infrequent – with HCC being the most commonly reported complication. Pascasia et al. followed 29 delisted patients for a median of 88 weeks after delisting and found 6 (21%) had clinical events: 2 with de novo HCC, 1 with variceal bleeding and 2 with mild ascites. Only 2 of the 6 required re-listing [4]. In the multicentre European registry study, of the 34 patients delisted, none died of liver-related complications in follow-up [2]. While additional longitudinal studies of delisted/inactivated patients are desirable to accurately characterise risks of liver complications, the available data suggest most remain without the need for liver transplantation.

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Delisting criteria are not standardised across transplant centres and thus not as ideal as a treatment endpoint. Transplant centres may opt to inactivate rather than delist in case the patient should develop HCC or decompensation in the future. Thus, studies that capture both delisting and inactivation are better. However, additional criteria such as achievement of Child- Pugh-A status, MELD <15 and resolution of ascites and hepatic encephalopathy would also be important to include, in order to capture meaningful improvements with treatment. The reason for delisting/inactivation is also important – as worsening liver complications or non- HCV-related comorbidities or reasons may lead to delisting.

WHAT IS THE BEST TIMING FOR HCV TREATMENT IN PATIENTS WITH AN INDICATION FOR LIVER TRANSPLANTATION? In patients with an indication for transplantation, the decision to treat should be made by the transplant team after listing. Those with high MELD score (>20) or moderate to severe symptoms of portal hypertension have a low likelihood of delisting and thus, generally should not be treated. Rather, efforts to increase their access to liver transplant through use of living donors or anti-HCV-positive donors should be made. For those with the potential for delisting/ deactivation, treatment should be started as soon as possible to maximise the opportunity for improvement. Available data suggests treatment benefits, in terms of MELD improvement, occurs within the first 1-3 months of treatment initiation, and clinical improvements take longer [2-6]. The pretreatment discussion with the patient regarding the timing of treatment and anticipated outcomes needs to be taken into account for the timeline for improvement. References

[1] Chen T, Terrault NA. Perspectives on treating hepatitis C infection in the liver transplantation setting. Curr Opin Organ Transplant. 2016;21(2):111-9.

[2] Belli LS, Berenguer M, Cortesi PA, Strazzabosco M, Rockenschaub SR, Martini S, et al. Delisting of liver transplant candidates with chronic hepatitis C after viral eradication: A European study. J Hepatol. 2016;65(3):524-31.

[3] Martini S, Donato MF, Mazzarelli C, Rendina M, Visco-Comandini U, Fili D, et HCV – SESSION 6 al. The Italian compassionate use of sofosbuvir in HCV patients waitlisted for liver transplantation: A national real-life experience. Liver Int. 2017. [Epub ahead of print].

[4] Pascasio JM, Vinaixa C, Ferrer MT, Colmenero J, Rubin A, Castells L, et al. Clinical outcomes of patients undergoing antiviral therapy while awaiting liver transplantation. J Hepatol. 2017;67(6):1168-76.

[5] Coilly A, Pageaux G, Hous-sel-Debry P, Duvoux C, Radenne S, De Ledinghen V, et al. Improving liver function and delisting of patients awaiting liver transplantation for HCV cirrhosis: do we ask too much to DAA? Hepatology. 2015;62:254A–8A.

[6] Jiang Z, El-Sherif O, Tapper E, Charlton M, Manns M, Afdhal N, et al. Predictive features of clinical improvement or deterioration for patients with decompensated hepatitis C cirrhosis after direct acting antiviral therapy. Hepatology. 2017;66(1 Suppl):573A.

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[7] Terrault NA, Berenguer M, Strasser SI, Gadano A, Lilly L, Samuel D, et al. International Liver Transplantation Society Consensus Statement on Hepatitis C Management in Liver Transplant Recipients. Transplantation. 2017;101(5):956-67.

[8] Manns M, Samuel D, Gane EJ, Mutimer D, McCaughan G, Buti M, et al. Ledipasvir and sofosbuvir plus ribavirin in patients with genotype 1 or 4 hepatitis C virus infection and advanced liver disease: a multicentre, open-label, randomised, phase 2 trial. Lancet Infect Dis. 2016;16(6):685-97.

[9] Charlton M, Everson GT, Flamm SL, Kumar P, Landis C, Brown RS, Jr., et al. Ledipasvir and Sofosbuvir Plus Ribavirin for Treatment of HCV Infection in Patients With Advanced Liver Disease. Gastroenterology. 2015;149(3):649-59.

[10] Poordad F, Schiff ER, Vierling JM, Landis C, Fontana RJ, Yang R, et al. Daclatasvir with Sofosbuvir and Ribavirin for Hepatitis C Virus Infection Patients with Advanced Cirrhosis or Post-transplant Recurrence. Hepatology. 2016;63(5):1493-505.

[11] Huang AC, Mehta N, Dodge J, Yao F, Terrault N. Direct-acting Antivirals Do Not Increase the Risk of Hepatocellular Carcinoma Recurrence after Local-Regional Therapy or Liver Transplant Waitlist Dropout. Hepatology. 2018. [In press].

[12] Ahmed A, Gonzalez SA, Cholankeril G, Perumpail RB, McGinnis J, Saab S, et al. Treatment of patients waitlisted for liver transplant with all-oral direct-acting antivirals is a cost-effective treatment strategy in the United States. Hepatology. 2017;66(1):46-56.

[13] Chhatwal J, Samur S, Kues B, Ayer T, Roberts MS, Kanwal F, et al. Optimal timing of hepatitis C treatment for patients on the liver transplant waiting list. Hepatology. 2017;65(3):777-88. SESSION 6 – HCV – SESSION 6

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HCV TREATMENT IN THE IMMUNOSUPPRESSED PATIENT

Vincenzo Boccaccio1, Alessio Aghemo1,2* 1Humanitas Research Hospital, Department of Internal Medicine, Via Alessandro Manzoni 56, 20089, Rozzano (MI); 2Humanitas University, Via Rita Levi Montalcini, 4, 20090, Pieve Emanuele (MI), Italy. E-mail address: [email protected]

TAKE-HOME MESSAGE • Clinical management of HCV patients treated with direct-acting antivirals and immunosuppressive agents can be challenging and requires expertise in the choice of antiviral regimen as well as in the accurate evaluation of possible drug-drug interactions with concomitant therapy.

The immunopathogenesis of HCV infection is complex and the relationship between infection and immunosuppression seems quite peculiar. Although HCV-related hepatocytolysis is classically interpreted as secondary to the attack by cytotoxic T lymphocytes against infected cells, the liver disease is usually exacerbated and evolves more rapidly in immunosuppressed patients. This mainly occurs during the immunosuppression state, and not at the reconstitution of the host response after therapy discontinuation [1].

Immunosuppression might be expected to reduce host-mediated inflammatory pathways that lead to liver damage; it may also reduce immune defences against direct virally mediated liver injury [2, 3].

Despite that, some immunosuppressive regimens have been widely and safely used, even in the interferon (IFN)-era, in the setting of chronic HCV infection. In fact, steroids used in patients SESSION 6 – HCV – SESSION 6 with hepatitis C/autoimmune hepatitis overlap syndrome have led to clinical, biochemical and histologic improvements, despite an increase in viral load. Inhibitors of tumour necrosis factor-alpha appear to be safe in patients with HCV and do not affect hepatocellular carcinoma (HCC) onset rate in psoriatic patients treated with these drugs. Finally, among patients with inflammatory bowel disease and HCV infection who are on immunosuppressive therapy, the rate of fibrosis progression is similar to rates reported in patients not on immunosuppressive therapy [2, 3].

On the other hand, if we compare the average time and range of years necessary for the establishment of end-stage chronic liver disease under physiological conditions and in particular categories of immunocompromised patients (i.e., transplanted patients, HIV-co- infected subjects, patients with hypogammaglobulinemia), a clear difference appears. Time intervals range from an average period of 30 years necessary to reach the end-stage disease in normal conditions to an average interval of 2 years after orthotopic liver transplantation (OLT) [1].

The field of immunosuppression and HCV infection is complicated further both by the different behaviour observed in different situations and/or by contrasting data obtained in the same conditions. Several unanswered questions remain, such as the opportunity to modify

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treatment schedules in the setting of post-transplant follow-up, as well as by the intrinsic tendency of chronic HCV infection in itself to lead to immunological disorders [1].

Recent advances in antiviral therapy based on all-oral, IFN-free regimens with direct-acting antiviral agents (DAAs), has resulted in significant improvements in safety and sustained virological response (SVR) rates. However, outside of the organ transplant context, limited data are available on the safety and efficacy in patients undergoing concomitant immunosuppressive therapy. Although clinical trials have focused on specific populations of immunocompromised patients, including those with HIV co-infection and post-liver or -kidney transplantation, patients undergoing other forms of immunosuppressive drug therapy in the context of cancer chemotherapy and the treatment of autoimmune conditions have generally been excluded [2].

WHAT HCV DRUGS CAN BE USED IN IMMUNOSUPPRESSED PATIENTS? EASL recommendations for the treatment of post-OLT recurrence of HCV infection [4]

• Patients with post-OLT recurrence of HCV genotype 1, 4, 5 or 6 infection without cirrhosis (F0-F3), with compensated (Child-Pugh A) cirrhosis or with decompensated (Child-Pugh B or C) cirrhosis should be treated with the fixed-dose combination of sofosbuvir (SOF) and ledipasvir (LDV), or the combination of SOF and daclatasvir (DCV) for 12 weeks with daily weight-based ribavirin (RBV), without the need for immunosuppressant drug dose adjustments (with the exception of everolimus, which requires strict drug monitoring).

• Patients with post-OLT recurrence of HCV genotype 2 without cirrhosis (F0-F3), with compensated (Child-Pugh A) cirrhosis or with decompensated (Child-Pugh B or C) cirrhosis should be treated with the combination of SOF and DCV for 12 weeks with daily weight-based RBV, without the need for immunosuppressant drug dose adjustments (with the exception of everolimus, which requires strict drug monitoring).

• Patients with post-transplant recurrence of HCV genotype 3 should be treated with the SESSION 6 – HCV – SESSION 6 combination of SOF and DCV for 24 weeks regardless of the stage of liver disease, with daily weight-based RBV, without the need for immunosuppressant drug dose adjustments (with the exception of everolimus, which requires strict drug monitoring).

• Patients with post-transplant recurrence of all HCV genotypes without cirrhosis (F0-F3), with compensated (Child-Pugh A) cirrhosis or with decompensated (Child-Pugh B or C) cirrhosis could be treated with the fixed-dose combination of SOF and velpatasvir (VEL) for 12 weeks (24 weeks in patients with genotype 3 and decompensated Child-Pugh B or C cirrhosis) with daily weight-based RBV.

• In patients with decompensated cirrhosis, RBV can be started at a dose of 600 mg daily and the dose subsequently adjusted depending on tolerance.

• Patients with contraindications to the use of RBV or with poor tolerance to RBV on treatment should receive the fixed-dose combination of SOF and LDV (genotypes 1, 4, 5 or 6), the fixed-dose combination of SOF and VEL (all genotypes), or the combination of SOF and DCV (all genotypes) for 24 weeks without RBV.

• The need for RBV in post-OLT patients without cirrhosis or with compensated (Child-Pugh A cirrhosis) has not been demonstrated and needs further exploration.

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EASL recommendations for the treatment of solid organ transplant recipients [4]

• Patients infected with HCV genotype 1, 4, 5 or 6 infection should be treated with the fixed- dose combination of SOF and LDV, the fixed-dose combination of SOF and VEL, or the combination of SOF and DCV according to the general recommendations, without the need for immunosuppressant drug dose adjustments (with the exception of everolimus, which requires strict drug monitoring).

• Patients infected with HCV genotype 2 should be treated with the fixed-dose combination of SOF and VEL or the combination of SOF and DCV according to the general recommendations, without the need for immunosuppressant drug dose adjustments (with the exception of everolimus, which requires strict drug monitoring).

• Patients infected with HCV genotype 3 should be treated with the fixed-dose combination of SOF and VEL or the combination of SOF and DCV according to the general recommendations, without the need for immunosuppressant drug dose adjustments (with the exception of everolimus, which requires strict drug monitoring). Regimens not yet included in the EASL recommendations

• 12 weeks of the RBV-free regimen glecaprevir/pibrentasvir (GP) were safe and effective in patients without cirrhosis with genotype 1 to 6, naïve or experienced, who underwent liver or renal transplant. No major dose adjustments in the concomitant immunosuppressive regimens were required [5].

• 12 weeks of the RBV-free regimen SOF/VEL were highly effective in liver transplant recipients with recurrent chronic HCV infection, genotype 1 to 4, including patients with cirrhosis and previous treatment failure. No significant changes in the dose of the concomitant immunosuppressive regimens were needed for rejection or drug-drug interactions [6].

• No data are currently available on the efficacy and safety of the fixed-dose combination regimen SOF/VEL/voxilaprevir in the setting of transplant recipients or immunosuppressed patients. SESSION 6 – HCV – SESSION 6

1) HOW SHOULD ORGAN RECIPIENTS BE TREATED FOR THEIR HCV? EASL recommendations for the treatment of post-transplant recurrence of HCV infection [4]

All patients with post-transplant recurrence of HCV infection should be considered for therapy.

• Treatment should be initiated early after liver transplantation, ideally as early as possible when the patient is stabilised (generally after the first 3 months post-transplant), because the SVR12 rates decrease in patients with advanced post-transplant liver disease.

• Acute cholestatic hepatitis or the presence of moderate to extensive fibrosis or portal hypertension one-year after transplantation predict rapid disease progression and graft loss and suggest urgent antiviral treatment.

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EASL recommendations for the treatment of solid organ transplant recipients [4]

• Solid organ transplant recipients, including kidney, heart, lung, pancreas or small bowel recipients should be treated for their HCV infection after transplantation, provided that their life expectancy exceeds one year.

POST-OLT HCV RECURRENCE: RBV YES OR NO? The usefulness of RBV in DAA-combinations is a topic of ongoing debate. Particularly in post-OLT patients, undergoing long lasting immunosuppressive treatment with calcineurin inhibitors (CNIs), tolerability of RBV-including regimens is poor. Data obtained from well- defined RBV-free DAA-based OLT-cohorts are sparse and do not support the benefit of RBV- addition to treatment combinations based on second-generation DAAs, neither in terms of efficacy nor in shortening treatment duration. Consequently, RBV-free treatment with second- generation DAAs-based combinations might be considered the best option in the vast majority of transplanted patients.

In a recent paper, Beinhardt et al. carried out a retrospective analysis on 62 OLT recipients who received RBV-free treatment with second-generation DAA-combinations (SOF/DCV, SOF/ SMV, SOF/LDV or paritaprevir/ritonavir, ombitasvir, and dasabuvir [PrOD]), with different treatment durations (longer in treatment-experienced and patients with cirrhosis), and with at least 96 weeks of follow-up after treatment cessation. RBV-free DAA-combinations for the treatment of HCV recurrence after OLT were highly efficacious and well-tolerated, leading to durable viral eradication. However, SVR achievement was not necessarily translated into benefits in terms of long-term clinical outcome, as 10% of patients died although achieving SVR12 [7].

KIDNEY TRANSPLANTATION

SESSION 6 – HCV – SESSION 6 DAAs in kidney transplantation (KT) recipients: experience and efficacy

To date, there have been two prospective clinical trials using DAAs in KT recipients. Colombo et al. [8] randomised 114 adult patients at least 6 months after KT to receive 12 or 24 weeks of SOF/LDV fixed-dose combination therapy. 15% of patients had compensated cirrhosis, and 69% were treatment-naïve. All patients had estimated glomerular filtration rate (eGFR) ≥40 ml/min at the time of screening. All 114 patients were cured. In the MAGELLAN-2 trial [5], 80 OLT recipients and 20 KT recipients received GP. Patients had genotype 1 to 6 infection, and all but two patients (98%) were cured.

Several centres have retrospectively reported experience with DAAs in a total of 287 KT recipients chronically infected with HCV. Though a mix of genotypes and regimens were used, nearly all used SOF-based DAA regimens (94%). Success rates have been outstanding, with only 11 treatment failures reported in the 287 patients reported (97% cured); 2 of the treatment failures were successfully retreated with an alternative DAA regimen and achieved a cure [9].

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DAAs in KT recipients: safety

Overall, DAAs have been remarkably well-tolerated in the KT population. In the trial by Colombo et al., although 18% required adjustment in immunosuppressant medications, there were no documented episodes of acute rejection [8]. In the MAGELLAN-2 study of GP in liver and KT recipients, only one patient discontinued therapy early due to an adverse event (AE). There were no cases of KT rejection among the 20 KT recipients participating in this study [5].

The 10 retrospective series of KT recipients treated with DAAs reported higher rates of anaemia (0-33%), generally correlated with the administering RBV. A significant interaction was pointed out between ritonavir and CNIs, making this regimen more challenging to use in the post-transplant period. On average, 97 of 287 patients (34%) in the retrospective series were reported to require CNI dose adjustments during or shortly after DAA therapy. Despite this, acute rejection was only reported in 8 KT recipients (3%) across all studies [9]. When to treat: before or after KT

There is considerable debate about the timing of DAA therapy in patients on the KT wait-list. Waiting until after KT allows patients the opportunity to accept a kidney from a donor with HCV infection, thereby substantially shortening KT wait time, improving organ allocation, and lowering costs. Recent studies suggest that HCV-infected KT recipients accepting HCV- infected kidneys may have higher rates of graft survival than those accepting uninfected donor kidneys, possibly mediated by their decreased time undergoing dialysis prior to transplant. Nearly all wait-listed end-stage renal disease patients are predicted to live longer with KT than on dialysis; this benefit extends to HCV-infected patients. As the wait-list continues to grow, in many regions, expected time on the wait-list exceeds 5 years. Thus, strategies to increase organ utilisation and shortening wait time are critical. Today, many centres that utilise kidneys from HCV-infected donors expect to transplant HCV-infected patients within 6 months. Thus, according to current recommendations, treatment of HCV should be delayed until the post- transplant period for any patient who is waiting for a deceased donor transplant and willing to accept a HCV-infected donor organ, provided they are not at risk of clinically significantly worsening liver disease. Patients who are not candidates for KT, who are listed at transplant centres that do not use HCV-infected kidneys, patients with living donors, those with significant extrahepatic manifestations of HCV such as cryoglobulinemic vasculitis, those who HCV – SESSION 6 are at significant risk of progressive liver disease prior to KT, and those with strong personal preference to be treated on dialysis should undergo DAA therapy on dialysis. A case by-case decision on the best timing for treatment is advisable, bearing in mind the potential benefits of eradicating HCV as soon as possible, while recognising that access to HCV-infected deceased donor kidneys would significantly reduce time on dialysis. For patients who need to be treated for HCV in the post-transplant period the optimal timeframe to initiate DAA therapy after transplant is still debatable. Approximately one-third of patients required adjustment of CNIs dosage to remain within the target range. Because of this, some have advocated waiting until at least 6 months after transplant, when the risk of acute rejection is decreased and patients may be less sensitive to lower CNIs levels. Moreover, waiting 6 months also allows confirmation that the patient was not “superinfected” with a new HCV genotype from the donor, a rare but important consideration to ensure that the appropriate treatment regimen has been selected. However, with the approval of new pan-genotypic HCV regimens, such as SOF/VEL and GP, this concern should disappear. On the other hand, waiting 6 months may be too late to prevent post-transplant complications associated with HCV. The increased risk of post-transplant diabetes was apparent in the early post-transplant period, within 6 months, suggesting that there may be some urgency in eradicating HCV immediately after transplant to prevent the adverse outcomes associated with the virus [9].

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LUNG TRANSPLANTATION Only case reports are available in this particular setting. D’Ambrosio et al. showed safety and efficacy of SOF-based regimens in the treatment of three patients with chronic HCV infection who underwent lung transplantation (LuT) caused by end-stage pulmonary disease. No significant modification in type and dosage of immunosuppressant agents were required during treatment.

The optimal timing for antiviral treatment is unclear. Pre-transplant HCV eradication might help prevent liver disease development and/or progression, potentially occurring long-time after transplantation, and could simplify patients’ clinical management. Moreover, HCV cure in LuT candidates could have a positive impact as it could enlarge the number of LuT candidates given that HCV still represents a relative contraindication to receive a LuT in most of the transplant centres. Although international recommendations support this position, in some centres the decision-making process is based on a histological evaluation of the severity of liver disease. Recently, international guidelines for the selection of LuT patients have changed their position, and finally HCV patients are considered as potential LuT recipients in the absence of cirrhosis or contraindication to anti-HCV therapies [10].

HEART TRANSPLANTATION A recent paper Liu et al. showed the safety and efficacy of antiviral treatment with DAAs (LDV/ SOF or DCV/SOF) in 12 heart transplant recipients affected with chronic HCV infection. All patients successfully completed the assigned treatment without experiencing serious AEs or deaths. No dose modifications were required for the most part of immunosuppressive drugs co-administered, just close monitoring of serum everolimus concentrations was required given the possible increase in serum levels of everolimus through the inhibition of P-glycoprotein due to DCV or LDV co-administration [11].

On the basis of this short experience, the authors suggested that healthcare providers should be active to treat these patients to reduce the recipients’ mortality and morbidity. The cardiac

SESSION 6 – HCV – SESSION 6 surgeons may also expand the use of HCV-infected heart donors to increase the organ pool for the uninfected heart recipients because treatment with LDV/SOF or DCV/SOF was also efficacious and safe for recipients with newly acquired HCV infection from HCV-infected donors.

2) HOW SHOULD PATIENTS ON ANTICANCER CHEMOTHERAPY OR IMMUNOSUPPRESSIVE DRUGS BE TREATED FOR THEIR HCV? Antiviral treatment with DAAs in patients in chemotherapy

Economides et al. [12] prospectively evaluated the safety of the concomitant use of DAAs and chemotherapy in 21 cancer patients with chronic HCV infection, showing that new IFN- free regimens can be safely and effectively used along with selected chemotherapeutic agents. Patients were treated with one of the following antiviral regimens: SOF/LDV ± RBV, SOF and RBV, SOF and SMV, SOF and DCV. Neoplastic comorbidities included solid tumours (breast cancer, HCC, cholangiocarcinoma, oesophageal, colon ad pharyngeal cancer) as well as haematological malignancies (multiple myeloma, myelodysplastic syndrome, acute

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PARIS, FRANCE myelogenous leukaemia, diffuse large B cell lymphoma, follicular lymphoma, Waldestrom macroglobulinemia and mycosis fungoides/T cell lymphoma), which were treated with several classes of chemotherapies (topoisomerase inhibitors; alkylating agents; antitumor antibiotics; targeted therapy; hormone therapy; antimetabolites; antiangiogenic agents; immunosuppressive agents; proteasome inhibitors).

Antiviral treatment was provided to prevent delays in the administration of chemotherapy in HCV-infected cancer patients. In some cases, DAAs could be started in the attempt to normalise liver function before starting chemotherapy for concomitant neoplasms. In fact, even in patients with advanced cirrhosis, liver function parameters have been shown to improve when HCV replication is successfully blocked. The normalisation of liver enzymes would grant these patients access to clinical trials with various chemotherapeutic agents, from which now they are frequently excluded. DAA therapy can be also started in cancer patients to halt liver disease progression. Moreover, patients with HCV-associated malignancies including HCC and non-Hodgkin lymphoma (NHL) frequently receive maintenance chemotherapy for their cancers (e.g. rituximab in patients with lymphoma). In some cases, this cancer treatment is temporarily suspended during DAA combination therapy, to avoid overlapping toxicities or possible drug-to-drug interactions (DDIs). This could lead to cancer recurrence.

The most serious AEs observed in this study were mainly attributed to either chemotherapeutic agents or ribavirin. This was confirmed by the fact that AEs improved after the necessary adjustments of chemotherapy and RBV treatment. One of the concerns of DDIs occurring in patients with underlying liver disease is acute decompensation. The patients in this study did not have any clinically significant DDIs, mainly because of the close follow-up and preventive assessment of potential DDIs before administration of the treatment regimens [12]. DAAs and mixed cryoglobulinemia (MC)

Limited evidence is currently available investigating the treatment with DAAs in patients with HCV-related MC, and results are difficult to interpret. Although cases of remission of multiorgan involvement and disappearance of serum cryoglobulins are described, just after initiating DAA therapy (a wide broad of therapeutic regimens were used in this setting, including SOF, SMF, DCV, PrOD), rituximab co-administration may in some cases contribute to the favourable response, particularly in polyneuropathy, which is known to require at least HCV – SESSION 6 6-9 months to partially revert. Similar controversial evidence has described the reduction in proteinuria and, concomitantly, eGFR improvement in patients achieving SVR. Moreover, in some cases, serum cryoglobulins persists despite viral eradication, so SVR may lead to no clinical improvement.

In conclusion, more studies are needed to establish the ideal regimen and treatment duration of therapy with DAAs for chronic HCV and coincident MC. Meanwhile, interest in rituximab therapy in the most severe patients with MC remains unchanged. Besides its immunomodulatory effect, rituximab also plays an important role by depleting CD19-positive B cells, known to be HCV reservoirs. Improving the effects on vasculitis treatment and HCV eradication using a combination of DAAs and rituximab can be envisaged [13]. DAAs and non-Hodgkin lymphoma

Among the spectrum of HCV-related extrahepatic manifestations, one of the better characterised associations was found with low-grade B cell non-Hodgkin lymphomas (B-NHL). The management of HCV-positive NHL patients is based on the treatment of NHL with standard chemotherapy. Some researchers document the possibility of treating primary splenic lymphomas and in general low-grade (marginal zone) lymphomas in HCV

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patients with pegylated interferon (PegIFN)-based antiviral regimens as a first-line therapy. According to that, the PegIFN plus RBV regimen was shown to lead remission in HCV-related low-grade NHL, in which SVR was achieved. Preliminary data on IFN-free DAA therapy in indolent HCV-positive NHL seem to confirm their activity in inducing lymphoma regression. Nonetheless, chemotherapy remains the only possible choice for more severe B-NHLs. Based on this evidence, all national and international guidelines on the prescription of the new DAAs in HCV-positive subjects included in the population to be treated patients with haematological disorders, such as cryoglobulinemic syndrome and B-NHLs.

In a recent observational study, Persico et al. [14] aimed to evaluate the safety and efficacy of concomitant antiviral treatment with DAA and chemotherapy for diffuse large B cell lymphomas (DLBCL) in HCV-infected patients. Twenty HCV genotype 1b patients, underwent concomitant chemotherapy for DLBCL (with one or more of the following regimens: rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone) and antiviral treatment with SOF/LDV. Only one patient had a serious AE, none of the patients discontinued treatment. Only one patient died in the course of therapy, due to a non-treatment related event. All the patients, except one, reached the end of antiviral treatment and experienced an SVR.

In conclusion, DAA treatment in concomitance with chemotherapy has resulted to be safe and effective in inducing remission of aggressive NHL in HCV patients. References

References in bold are required reading

[1] Zignego AL, Giannini C, Gragnani L, Piluso A, Fognani E. Hepatitis C virus infection in the immunocompromised host: a complex scenario with variable clinical impact. J Transl Med. 2012 Aug 3;10:158.

[2] Ooka K, Lim JK. Treatment of hepatitis C in patients undergoing immunosuppressive drug therapy. J Clin Transl Hepatol. 2016 Sep 28;4(3):206-227.

[3] Einav S, Koziel MJ. Immunopathogenesis of hepatitis C virus in the immunosuppressed host. Transpl Infect Dis 2002;4:85–92. SESSION 6 – HCV – SESSION 6 [4] European Association for the Study of the Liver. EASL Recommendations on Treatment of Hepatitis C 2016. J Hepatol. 2017 Jan;66(1):153-194.

[5] Reau N, Kwo PY, Ree S, et al. Magellan-2: Safety and efficacy of Glecaprevir/Pibrentasvir in liver or renal transplant adults with chronic hepatitis C genotype 1-6 infection. EASL 2017, Abs. LBO-03.

[6] Agarwal K, Castells L, Müllhaupt B, et al. Sofosbuvir/Velpatasvir for 12 weeks in genotype 1-4 HCV-infected transplant recipients. AASLD 2017, Abs. 1069.

[7] Beinhardt S, Al-Zoairy R, Kozbial K, et al. Long-term follow-up of ribavirin-free DAA- based treatment in HCV recurrence after orthotopic liver transplantation. Liver Int. 2017 Dec 2. doi: 10.1111/liv.13652.

[8] Colombo M, Aghemo A, Liu H, et al. Treatment With Ledipasvir-Sofosbuvir for 12 or 24 Weeks in Kidney Transplant Recipients With Chronic Hepatitis C Virus Genotype 1 or 4 Infection: A Randomized Trial. Ann Intern Med. 2017 Jan 17;166(2):109-117.

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[9] Chute DF, Chung RT, Sise ME. Direct-acting antiviral therapy for hepatitis C virus infection in the kidney transplant recipient. Kidney Int. 2018 Jan 9. pii: S0085- 2538(17)30807-4.

[10] D’Ambrosio R, Aghemo A, Rossetti V, Carrinola R, Colombo M. Sofosbuvir-based regimens for the treatment of hepatitis C virus in patients who underwent lung transplant: case series and review of the literature. Liver Int. 2016 Nov;36(11):1585-1589.

[11] Liu CH, Chen YS, Wang SS, et al. Sofosbuvir-based Interferon-Free Direct Acting Antiviral Regimens for Heart Transplant Recipients With Chronic Hepatitis C Virus Infection. Clin Infect Dis. 2018 Jan 6;66(2):289-292.

[12] Economides MP, Mahale P, Kyvernitakis A, et al. Concomitant use of direct-acting antivirals and chemotherapy in hepatitis C virus-infected patients with cancer. Aliment Pharmacol Ther. 2016 Dec;44(11-12):1235-1241.

[13] Roccatello D, Sciascia S, Rossi D, et al. The challenge of treating hepatitis C virus- associated cryoglobulinemic vasculitis in the era of anti-CD20 monoclonal antibodies and direct antiviral agents. Oncotarget. 2017 Jun 20;8(25):41764-41777.

[14] Persico M, Aglitti A, Caruso R, et al. Efficacy and safety of new direct antiviral agents in hepatitis C virus-infected patients with diffuse large B-cell non-Hodgkin’s lymphoma. Hepatology. 2018 Jan;67(1):48-55. SESSION 6 – HCV – SESSION 6

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PROS AND CONS OF TRANSPLANTING HCV-INFECTED ORGANS

María Trapero-Marugán1,4, Marina Berenguer2,3,4* 1Liver Unit- Hospital Universitario Puerta de Hierro, Majadahonda, Madrid, Spain. IDIPHISA. Autonomous University of Madrid, Dept. of Medicine, Madrid, Spain; 2Liver Transplantation and Hepatology Unit – La Fe University Hospital, Avenida Fernando Abril Martorell, 106 (Torre F5) – 46026 Valencia, Spain; 3University of Valencia, Dept of Medicine, Valencia, Spain; 4CIBERehd (Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas), Avenida Monforte de Lemos, 3-5 (Pabellón 11, planta 0) – 28029 Madrid, Spain. E-mail address: [email protected]

TAKE-HOME MESSAGES • Serologically positive anti-hepatitis C virus (HCV) donors may harbour active viral infection (viraemia positive) or not (following spontaneous or treatment-induced viral clearance).

• Accepting grafts from serologically positive anti-HCV donors, even in the presence of viraemia, is one of the few means to increase access to liver transplantation.

• Transmission of HCV infection is the rule if the donor is viraemic; in contrast, transmission of HCV infection is very rare if the donor is anti-HCV-positive, nucleic acid test (NAT) negative; some cases of transmission have occurred in cases of high risk donors acutely infected at the time of organ retrieval.

• With the availability of recent extremely effective direct-acting antiviral agents, treatment of HCV infection following transplantation results in viral eradication in nearly 100% of cases.

• Assessing graft quality is essential when accepting these grafts.

SESSION 6 – HCV – SESSION 6 • Recipients at high risk of poor outcome in the absence of liver transplantation (high Model for End-stage Liver Disease (MELD) score, high risk hepatocellular carcinoma etc.) should be the target of anti-HCV/NAT positive organs; in these instances, informed consent is mandatory.

INTRODUCTION Liver disease caused by hepatitis C virus (HCV) is the main indication for liver transplantation (LT) in adults in most countries including the United States (US) and within Europe. Globally, it is estimated that 71.1 million people had chronic HCV infection in 2015 with 1.7 million new HCV infections that year [1]. Without treatment, 15-25% of chronically infected individuals will progress to cirrhosis, with concomitant increased risk for hepatocellular carcinoma and will need a liver transplant. Importantly, to date, a large number of individuals remain with no or limited access to treatment (<10% in the US and Canada, and <16% in Europe). There is also a large number of people unaware that they are infected with HCV (50% in the US, >60% in Europe), leading to ongoing HCV transmission. In fact, annually, an estimated 700,000 persons with chronic HCV infection die untreated, potentially becoming a source of organ donors.

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For years, hepatologists and liver transplant surgeons were reluctant to accept grafts from infected donors due to the high risk of HCV transmission, universal in case of positive viremia, and subsequent development of severe graft hepatitis and graft loss. With the advent of new extremely effective therapies against HCV, also in the liver transplant population, that fear is progressively decreasing, and an increasing number of HCV-positive grafts are being used, particularly in HCV-positive recipients.

In this review, the pros and cons of transplanting HCV-infected organs will be reviewed, taking into consideration recent Consensus documents [2].

EVOLUTION OF HCV-POSITIVE DONATION In the 1990s, grafts from anti-HCV-positive donors were not accepted for LT. During these early years, studies were mostly focused on assessing the risk of HCV transmission when anti-HCV-positive organs were used in transplantation, and effective and non-toxic therapies against HCV infection were mostly lacking.

Due to the increasing gap between potential donors and recipients, different centres started using anti-HCV-positive grafts, particularly in HCV-positive recipients at high risk of dying on the waiting list. These studies demonstrated that (i) only one strain dominates after LT; (ii) this dominance is determined according to both donor and recipient genotypes; (iii) overall survival of HCV-positive recipients is not affected by the HCV positivity of the donor [3]; (iv) survival of HCV-negative recipients is impaired when anti-HCV-positive donors are used. In addition, graft hepatitis in HCV-positive recipients was found to be no different in terms of severity and progression whether the graft came from a positive or negative donor [3]. More careful analysis of these studies and large registries demonstrate that donor age was a significant risk factor for aggressive graft hepatitis and overall survival when using HCV-positive donors in HCV-positive recipients, as previously demonstrated in HCV-positive recipients receiving grafts from uninfected donors with a cut-off donor age of 45-50 years in most studies.

Based on these findings and the increasing successful use of pegylated interferon (IFN) plus ribavirin in the transplant setting, the use of anti-HCV-positive organs expanded growing from 2.4% in the 1990s to 6-10% in the years 2000-2013. Overall, 5.7% (n = 1,930) of grafts HCV – SESSION 6 from anti-HCV-positive donors were used from 1995 to 2013 in the US based on data from the Scientific Registry of Transplant Recipients (SRTR).

In more recent years, with the advent of the new direct-acting antiviral agents (DAA), the use of HCV-positive organs has substantially increased, but the number of those discarded is still relevant. In a recent study from the SRTR encompassing 25,566 HCV-positive deceased donor LT performed between 2005 and 2015, HCV-positive recipients were 61% more likely to receive an HCV-positive donor after 2010 (early DAA/IFN era) and almost three times more likely after 2013 (IFN-free DAA era) than before 2010. Furthermore, the number of discarded livers, while still being greater in HCV-positive vs. HCV-negative donors, decreased over time so that it was 3 times higher from 2005 to 2010, 2.2 times higher after 2010 and 1.7 times higher after 2013 [4]. In the UK, between 2000 and 2015, 244 HCV-positive potential donors were identified from the UK Transplant Registry, and only 76 (31%) proceeded to transplant donation. Most (69%) offered organs were declined because of positive serology although their quality was similar to that of other transplanted organs.

The use of anti-HCV-positive organs into HCV-negative recipients, particularly in the case of viraemic donors, is the more recent approach which, has demonstrated to be successful

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in the setting of kidney transplantation, significantly reducing the time on the waiting list without compromising outcome by successfully treating recipients after transplantation with new DAAs [5]. While there is not much data in the setting of LT, because these organs tend to be from younger and more optimal donors, this practice is likely to be embraced, particularly in areas with substantial organ shortage and high wait-list mortality.

Interestingly, while the possibility to use anti-HCV-positive organs has progressively increased, the number of LT performed in HCV-positive recipients has also decreased, particularly in the most recent years, concomitant with the introduction of DAA. In the UK, it fell from 21% in 2008 to 12% in 2015 (17% overall throughout the period). In the US, data from the SRTR database showed that the adjusted incidences of waitlisting for decompensated cirrhosis in HCV patients decreased by 5% in the protease inhibitor (PI) era and 32% in the DAA era compared to the IFN era. Similar data was reported recently from a single centre study in Spain, where the incidence of waitlisting for HCV patients decreased by 19.5% in the DAA era compared to the IFN era and by 15% when compared to the PI era.

HCV-POSITIVE DONORS: CONSIDERATION OF VIROLOGIC TERMINOLOGY Historically, HCV-positive donors were identified based only on serologic testing (that detect antibodies within 2 to 6 months after exposure) without differentiating those who were viraemic (nucleic acid testing [NAT] positive), and thus at universal risk of transmitting the infection, from those who were non-viraemic, and thus not infectious. An HCV antibody positive/NAT negative serologic status may be found in individuals who have spontaneously cleared the virus, a situation which occurs in 15-25% of cases or in those successfully treated with antiviral therapy, a situation that approaches 100% with current DAA. In addition, although extremely rare with the use of third-generation ELISA tests, which have a specificity of 92-100% a positive serology with negative viremia may result from a false positive HCV antibody test.

Given the increasing number of individuals being successfully treated with new DAAs, it is

SESSION 6 – HCV – SESSION 6 conceivable to predict a future reduction of viraemic donors at very high risk of transmitting infection and an increase in the number of serologically positive non-viraemic donors. Conceptually, these organs are not at risk of transmitting HCV infection. In a recent study, 26 consecutive HCV antibody (n = 25) or NAT (n = 1) negative transplant recipients received a liver graft from HCV antibody positive but serum NAT negative donors between March 2016 and March 2017, HCV transmission (defined as positive HCV-polymerase chain reaction test by 3 months following transplantation) occurred in 4 (16%) cases, at a median follow-up of 11 months. Interestingly, drug overdose, which was listed as the cause of death in 15 (60%) of the donors, was the cause of death in these 4 particular cases raising the hypothesis that these organs had been harvested just at the time of viral infection. Thus, while NAT testing has a significant value with regard to HCV transmission, increased risk behaviour and particularly cause of death may also need to be taken into consideration. Currently, 16.9% of HCV-positive recipients receive an HCV serologically positive organ, and only 50% are HCV viraemic [6].

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PROS FOR TRANSPLANTING HCV-INFECTED LIVER ORGANS Increased access to liver transplantation The increasing gap between donors and recipients and subsequent death on the waiting list is the main reason to accept organs from serologically and virologically positive donors. While transmission is the norm in these cases, and until recently treatment of HCV infection in the transplant recipient was toxic and relatively ineffective, the use of DAA has completely changed this scenario allowing for viral eradication in more than 98% of cases, particularly if therapy is initiated before advanced graft cirrhosis has developed [7]. Even in the setting of fibrosing cholestatic hepatitis, new oral antivirals are extremely effective, and it is very rare now to lose a graft to HCV-related disease. A similar changing approach has been used in the past with the use of cytomegalovirus, Epstein-Barr virus and anti-core positive organs. Indeed, the development of effective antiviral drugs in these settings has allowed for the use of these organs in specific circumstances, with progressive expansion of indications. Successful attempt in kidney transplantations Experience in kidney transplantation with HCV viraemic organs has resulted in a significant reduction in the waiting list and survival advantage [5]. The percentage of patients awaiting kidney transplantation increased in the US from 11.4% in 2010 to 15.7% in 2015. Unfortunately, over 4,000 HCV-positive kidney organs were discarded between 2005 and 2014 in the US. In the most recent trial of transplantation of HCV-infected kidneys into uninfected recipients, 10 HCV-positive genotype 1 donors were used into HCV-negative kidney recipients. The median time from eligibility on the waiting list for HCV-positive kidneys to kidney transplantation was very short (58 days, IQ range: 53 to 100). All recipients were successfully treated with oral antivirals with an acceptable graft function at 6 months of follow-up. Examples like this and others in lung and heart recipients are likely to increase progressively in the near future. Outcomes from HCV-positive donors are similar to HCV-negative donors While overall past studies have revealed similar outcomes, both in terms of disease severity of graft hepatitis and graft/patient survival [3], except when old donors were utilised, not all studies differentiated between serological and virologically positive donation. SESSION 6 – HCV – SESSION 6 Increased organ quality Recent studies from the US reveal a 200% increase in overdose deaths (“opioid epidemic”) since 2000 markedly in the young people segment population. The number of intravenous drug users (IVDU) that contribute to the organ donor pool in the US has increased more than 50% from 2012 to 2017. While overall, the rate of HCV infection is higher in this population, due to disposable syringes use, HCV sero-positivity in younger IVDU (less than 30 years old) prevalence is lower than in older IVDU (30% vs. 70-80% in recent surveys).

IVDU organ donors are younger (less than 30 years old) and less obese. Furthermore, donation after cardiac death occurs less frequently in this setting. Direct-acting antiviral agents The number of HCV patients with cirrhosis on the LT waiting list that can and have already been delisted due to clinical improvement following successful viral eradication with new DAA is increasing. In a European multicentre study, one out of 4 treated candidates was shown to improve to the point that LT was no longer indicated [2]. While still under evaluation, the point of no return seems to increase with the Model for End-stage Liver disease (MELD) score so that the probability of achieving significant clinical improvement is almost null in those with

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a MELD score greater than 20 [2]. Furthermore, liver volume adjusted to body surface area may also become a suitable non-invasive tool to predict clinical improvement following DAA therapies, as recently suggested in a small study.

Overall, data from large registries and single centre studies have shown that the LT epidemiology has substantially changed with the increasing use of DAA. Indeed, the number of patients undergoing LT with active HCV infection has significantly declined as the proportion of patients wait-listed for LT for decompensated HCV cirrhosis has decreased and the proportion of wait-listed HCV-positive patients delisted for clinical improvement has increased [2].

Reducing the need for LT among HCV-infected patients with cirrhosis will not only have significant consequences from an individual patient point of view but also in terms of organ sparing. But mostly, the first and clearer consequence of the use of DAAs is the decreasing number of HCV-infected candidates in any given waiting list, reducing the chance to use HCV-positive organs into HCV-positive recipients.

CONS FOR TRANSPLANTING HCV-INFECTED ORGANS HCV infection is universal

HCV transmission will occur universally in cases of anti-HCV-positive viremia donors. All recipients who accept the use of these organs need to be adequately informed. Based on OPTN data as of December 5, 2014, 4.4% of deceased donors with organs recovered for transplantation in 2013 had a positive HCV antibody test [8]. Transmission of a different HCV genotype

While some genotypes were less likely to respond to first-generation DAAs, recent pan- genotypic combinations seem to be equally efficacious with all genotypes. Regardless, testing for genotype after LT before treatment is mandatory as HCV genotype may have changed in those previously infected.

SESSION 6 – HCV – SESSION 6 Legal implications of HCV transmission: access to new DAA

Because of legal implications related to transmission of a new infection into a recipient, potential recipients need to sign an informed document after receiving all the pertinent information regarding the risk of transmission, the possibility to develop an early and aggressive course of HCV disease (particularly fibrosing cholestatic hepatitis), and the actual response rates with new DAAs. Most importantly, the recipient needs to be aware of the possibilities to use these new DAAs in a timely manner if needed.

Unfortunately, access to DAA regimens remains limited, expensive and, in some countries, insurance companies refuse to cover the cost of treatment for this indication. Worse graft quality

While continuously improved histology was the norm following viral eradication with IFN- based therapy, data from some studies revealed though that a substantial percentage continues to have advanced fibrosis despite HCV cure. Thus, in a report of 70 liver biopsies from anti- HCV-positive non-viraemic individuals, only 7% had a normal liver, and 82% continued to have a fibrosis stage greater than 2. While the residual impact of prior HCV infection may be the cause, one needs to consider current extremely frequent comorbidities such as obesity, diabetes and/or alcohol consumption. Thus, an adequate evaluation of HCV-positive organs

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PARIS, FRANCE is mandatory in LT. While grafts with advanced fibrosis are declining and those with no or mild fibrosis are typically accepted, it is still unclear whether those with moderate fibrosis should be accepted for transplantation. Future data on fibrosis progression following early post-transplant therapy with new DAA is needed before liberally accepting these grafts.

Assessment of the quality of the graft has been typically done with visual inspection and histological examination generally by frozen section either at the time of organ evaluation or during the backtable. A frozen section is a quick assessment that allows establishing changes in liver architecture, the presence of steatosis, inflammation or necrosis of hepatocytes. While precise staging of fibrosis requires a trichrome stain, advanced fibrosis is generally apparent and can be visualised more easily with the use of polarised light. Transient elastography (FibroScan®) is currently being used to assess fibrosis both in the immune competent and immune suppressed individual. Whether it is a valuable tool to assess fibrosis in donors is still under evaluation (www.Clinicaltrials.gov NCT02460250). DAA limitations: Risk of DAA failure, drug-drug interactions, DAA-induced complications

While extremely low and possibly close to 0% with new generation DAAs, there remains a risk of DAA failure. Furthermore, in the setting of advanced renal failure and infection with non- genotype 1, treatment options are extremely limited as sofosbuvir is not approved in patients with chronic kidney disease stage IV or V [2].

Drug-drug interactions may limit the use of certain DAA, particularly in HIV-HCV co- infected patients under highly active antiretroviral therapy (HAART).

Finally, while supporting data is limited, a few studies have suggested that DAA therapy may increase the risk of donor-specific antibody formation and rejection.

Anti-HCV + donor

High risk donor

Yes No SESSION 6 – HCV – SESSION 6

NAT testing SVR or spontaneous clearance or false anti-HCV (+) Negative Positive Offer to any recipient (HCV negative or positive) SVR or Liver biopsy spontaneous clearance** Stage ≥F2 Stage

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CONSIDERATIONS AND FUTURE In summary, the pros of using HCV-positive donors clearly outweigh the cons [9, 10]. But certain measures need to be considered before using these grafts, including:

1. Informed consent.

2. NAT evaluation in patients at risk of HCV infection.

3. Histological assessment of graft quality. New techniques like elastography, or even liquid biopsy may soon become available for this purpose.

While more data is being acquired on the safety of using viraemic organs in uninfected recipients, the use of these organs is an acceptable practice in patients at high risk of dying on the waiting list. In one study, the benefit in terms of life expectancy was observed with MELD scores greater than 20 with the maximum benefit observed in those with a MELD score higher than 28 [11]. In contrast, in the absence of guaranteed or delayed access to HCV therapy, these organs should not be offered to non-viraemic recipients with lower MELD scores. References

References in bold are required reading

[1] Polaris Observatory HCV Collaborators. Global prevalence and genotype distribution of hepatitis C virus infection in 2015: a modelling study. Lancet Gastroenterol Hepatol 2017;2(3):161-76.

[2] Belli LS, Duvoux C, Berenguer M, Berg T, Coilly A, Colle I, et al. ELITA consensus statements on the use of DAAs in liver transplant candidates and recipients. J Hepatol 2017;67(3):585-602.

[3] Coilly A, Samuel D. Pros and Cons: Usage of organs from donors infected with hepatitis C virus – Revision in the direct-acting antiviral era. J Hepatol 2016;64(1):226-31.

[4] Bowring MG, Kucirka LM, Massie AB, Luo X, Cameron A, Sulkowski M, et al. Changes

SESSION 6 – HCV – SESSION 6 in Utilization and Discard of Hepatitis C-Infected Donor Livers in the Recent Era. Am J Transplant 2017;17(2):519-27.

[5] Goldberg DS, Abt PL, Reese PP. Transplanting HCV-Infected Kidneys into Uninfected Recipients. N Engl J Med 2017;377(11):1105.

[6] Levitsky J, Formica RN, Bloom RD, Charlton M, Curry M, Friedewald J, et al. The American Society of Transplantation Consensus Conference on the Use of Hepatitis C Viremic Donors in Solid Organ Transplantation. Am J Transplant 2017;17(11):2790-802.

[7] Terrault NA, McCaughan GW, Curry MP, Gane E, Fagiuoli S, Fung JYY, et al. International Liver Transplantation Society Consensus Statement on Hepatitis C Management in Liver Transplant Candidates. Transplantation 2017;101(5):945-55.

[8] Theodoropoulos N, Nowicki MJ, Chinchilla-Reyes C, Dionne S, Jaramillo A, Mone T, et al. Deceased organ donor screening for human immunodeficiency virus, hepatitis B, and hepatitis C viruses: Discordant serology and nucleic acid testing results. Transpl Infect Dis 2017.

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[9] Verna EC, Goldberg DS. Hepatitis C viremic donors for hepatitis C nonviremic liver transplant recipients: Ready for prime time? Liver Transpl 2018;24(1):12-4.

[10] Selzner N, Berenguer M. Should hepatitis C positive donor be used in Hepatitis C Negative Recipients for Liver Transplantation? Liver Transplantation 2018 [Epub ahead of print].

[11] Chhatwal J, Samur S, Kues B, Ayer T, Roberts MS, Kanwal F, et al. Optimal timing of hepatitis C treatment for patients on the liver transplant waiting list. Hepatology 2017;65(3):777-88. SESSION 6 – HCV – SESSION 6

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