Review The oncogenic role of hepatitis delta virus in hepatocellular carcinoma Authors Marc Puigvehí, Carlos Moctezuma-Velázquez, Augusto Villanueva, Josep M. Llovet Correspondence augusto.villanueva@mssm.edu (A. Villanueva)josep.llovet@mountsinai.org (J.M. Llovet) (J. Llovet) Graphical abstract https://doi.org/10.1016/j.jhepr.2019.05.001 Review The oncogenic role of hepatitis delta virus in hepatocellular carcinoma Marc Puigvehí, 1,2,† Carlos Moctezuma-Velázquez, 1,† Augusto Villanueva, 1,3,6,* Josep M. Llovet 1,4,5,6,* Keywords: Hepatitis B virus; Summary liver cancer; HCC, co- Hepatitis delta virus (HDV) is a small defective virus that needs hepatitis B virus (HBV) to replicate infection; molecular patho- genesis; defective; superin- and propagate. HDV infection affects 20-40 million people worldwide and pegylated interferon fection. (PegIFN) is the only recommended therapy. There is limited data on the contribution of HDV infection to HBV-related liver disease or liver cancer. Evidence from retrospective and cohort Received 17 February 2019; studies suggests that HBV/HDV coinfection accelerates progression to cirrhosis and is associated received in revised form 18 April 2019; accepted 5 with an increased risk of hepatocellular carcinoma (HCC) development compared to HBV mono- May 2019; available online infection. Although the life cycle of HDV is relatively well known, there is only ancillary infor- 5 June 2019 mation on the molecular mechanisms that can drive specific HDV-related oncogenesis. No thorough reports on the specific landscape of mutations or molecular classes of HDV-related HCC have been published. This information could be critical to better understand the uniqueness, if any, of HDV-related HCC and help identify novel targetable mutations. Herein, we review the 1 Mount Sinai Liver Cancer Pro- evidence supporting an oncogenic role of HDV, the main reported mechanisms of HDV involve- gram, Division of Liver Diseases, ment and their impact on HCC development. Tisch Cancer Institute, Icahn School of Medicine at Mount © 2019 The Authors. Published by Elsevier B.V. on behalf of European Association for the Study of the Sinai, New York, NY, USA Liver (EASL). This is an open access article under the CC BY-NC-ND license (http://creativecommons. 2 Hepatology Section, Gastroen- org/licenses/by-nc-nd/4.0/). terology Department, Hospital del Mar, IMIM, Universitat Introduction surface antigen (HBsAg)-positive individuals is Autònoma de Barcelona, Barce- lona, Catalonia, Spain More than 40 years after its discovery,1 key features not a standard procedure, 6 HDV prevalence is 3 Division of Hematology and Medi- of hepatitis delta virus (HDV) infection remain unknown in many countries, resulting in a global cal Oncology, Department of Medi- unknown. This contrasts with other hepatotropic underestimation of HDV disease burden. To date, cine, Icahn School of Medicine at Mount Sinai, New York, USA viruses, such as hepatitis B (HBV) and hepatitis C few studies have evaluated the global burden of 4 Translational Research in Hepatic virus (HCV), which have been thoroughly investi- HDV, and available data are mostly biased Oncology, Liver Unit, IDIBAPS, gated and for which there are effective treatments. towards local reports from different regions in CIBERehd, Hospital Clínic, University specific cohorts. The World Health Organization of Barcelona, Barcelona, Catalonia, The lower prevalence of HDV compared to HBV or Spain HCV likely justified the limited research efforts dedi- (WHO) estimates that there are at least 20 million 5 Institució Catalana de Recerca i cated to HDV, even though HDV infection is highly people infected with HDV worldwide, which Estudis Avançats, Barcelona, Catalo- prevalent in certain countries. 2 HDV is a defective represents 5% of HBV carriers. 7 A recent meta- nia, Spain 6 Denotes co-senior authorship. virus that co-exists with HBV, and is related to the analysis including 182 articles from 61 countries † These authors contributed equally most severe form of liver failure attributable to estimated a pooled HDV prevalence of 10%, even to this work. chronic viral hepatitis. HDV is understood to accel- after excluding intravenous drug users and indivi- * Corresponding authors. erate the progression to cirrhosis, and it is consid- duals with high-risk sexual behaviour. 8 However, Addresses: Mount Sinai Liver ered a main driver of the malignant hepatocyte methodological errors regarding the definition of Cancer Program, Division of Liver Diseases, Icahn School of Medicine transformation. 3 HBV/HDV-coinfected patients HDV infection, extrapolation of data from the dif- at Mount Sinai, 1425 Madison seem to be at an increased risk of HCC development ferent cohorts, and selection bias have been Avenue, Box 11-23, New York, NY compared to HBV-monoinfected individuals, pointed out in this study. Thus, it is likely that 10029, USA; Tel.: +1 2126599503, although the evidence is still limited. Thus, HDV is such analysis represents an overestimation of fax: +1 212 849 2574, (JM. Llovet); or Division of Liver Diseases, Divi- not yet included on the list of oncogenic agents, the real prevalence. 9 In certain areas such as sion of Hematology and Medical whilst HBV and HCV are well defined carcinogens.4 Nigeria, Gabon, Benin, Mauritania, Cameroon, Oncology, Department of Medi- Herein, we will review the evidence available Senegal, Iran, Peru, the Western Brazilian Ama- cine, Liver Cancer Program, Tisch Cancer Institute, Graduate School regarding the oncogenic role and mechanisms of zon, the mountain regions of Colombia and Vene- of Biomedical Sciences, Icahn HDV-related carcinogenesis, as well as discussing zuela, Romania, Pakistan, and Tajikistan, the School of Medicine at Mount Sinai, the key unmet needs in HDV research. reported HDV prevalence exceeds 20%. 2,5,10,11 New York, NY 10029 USA (A. Vil- HDV is endemic in Punjab, Somalia, and Mongo- lanueva). E-mail address: augusto.villanue- HDV epidemiology lia, with an estimated prevalence of up to va@mssm.edu (A. Villanueva), The global prevalence of HDV is remarkably vari- 60–80% in HBV-infected patients. 12,13 Remark- josep.llovet@mountsinai.org able (Fig. 1). 5 As routine testing for HDV in HBV ably, these estimates of prevalence may be biased, (J.M. Llovet). Review as they are usually extrapolated from high-risk cohorts. HDV prevalence also varies across risk fac- Key points tors, with prevalences of 37% and 17% in intrave- • Hepatitis delta virus (HDV) is a small defective virus that needs hepatitis B virus (HBV) to nous drug users and people with high-risk sexual replicate and propagate behaviours, respectively. 8 • Initial data suggest that HDV accelerates the progression to cirrhosis and increases the risk Eight different HDV genotypes have been iden- of hepatocellular carcinoma (HCC) in patients with HBV tified, 14,15 with data suggesting different disease • Proposed mechanisms for HDV enhance HBV-related oncogenesis include activation of pathways related to inflammation and fibrosis courses depending on genotype. 16 Genotype 1, • Unlike HBV, there is limited data to support a direct oncogenic role of HDV in human the most common, has worldwide distribution hepatocarcinogenesis but is predominant in Europe, the Mediterranean countries, Iran, Turkey, and North America. Geno- circle replication. 21,22 HDV is a defective virus types 2 and 4 are found in Asia, genotype 3 has which needs the presence of HBV for infectivity only been described in South America, and geno- and assembly purposes, as it lacks its own envel- types 5-8 are found almost exclusively in Africa. 17 ope and uses HBsAg instead. 1 The preS1 domain Genotype 1 has been associated with worse out- of the large HBsAg (L-HBsAg) is necessary to infect comes than genotype 2, including progression to hepatocytes by binding to the sodium taurocholate cirrhosis and higher rates of HCC. 18,19 cotransporting polypeptide (NTCP) receptor, and the small HBsAg (S-HBsAg) is essential for HDV HDV pathogenesis assembly. Notably, HDV does not need active HBV Life cycle DNA synthesis, which is inhibited in patients HDV is a circular single-stranded negative-sense under effective anti-HBV treatment with nucleos RNA virus which encodes for a single protein, the (t)ide analogues (NUCs). HDV is able to replicate delta protein or delta antigen (HDAg). 5 The delta as long as the translation of these structural pro- antigen was discovered in Italy in 1977 by Rizzetto teins continues. Furthermore, integrated HBV and collaborators while examining liver biopsies of DNA can also provide the necessary envelope pro- chronic HBV patients with direct immunofluores- teins for HDV virions, independently of HBV repli- cence. They noticed that some HBV-infected cation. 23,24 In addition to its dependence on HBV, patients reacted and revealed a nuclear fluorescent HDV replication also needs a host (i.e. the hepato- pattern, which was initially thought to be a new cyte), as the virus does not code for an RNA poly- HBV antigen. 1 It was not until 1980 when they rea- merase but uses the host’s machinery. 2 The delta lised that it was a new RNA virus. 20 The HDV gen- protein is expressed in 2 isoforms with comple- ome spams between 1,672 and 1,697 base pairs mentary functions: the small form, called S-HDAg depending on the genotype, making it the smallest or p24 for its molecular weight of 24 kDa, regulates virus infecting humans. HDV shares more charac- the nuclear import of HDV ribonucleoproteins and teristics with plant virusoids than with other the replication process. The large form, called L- human pathogens, such as the circular configura- HDAg or p27, inhibits replication and participates tion of its RNA genome, its RNA self-cleavage (ribo- in virion assembly. 25 During HDV replication, zyme activity) property and its RNA to RNA rolling HDAg proteins form a ribonucleoprotein, which <5% 5-10% 10-20% 20-50% >50% No data Fig. 1. Global distribution of HDV infection among HBsAg carriers. HDV prevalence is highly different among different countries. The most prevalent areas are Punjab, the Amazon basin, Somalia, and Mongolia. In European countries, the highest prevalences are seen in Romania and Albania. HBsAg, hepatitis B virus surface antigen; HDV, hepatitis delta virus. JHEP Reports 2019 vol. 1 | 120–130 121 will later get a coat in the endoplasmic reticulum enhances HBV-related oncogenesis. L-HDAg has consisting of the 3 HBV HBsAg proteins. 16 Thus, been shown to activate c-Jun and to antagonize the outer envelope is the same for HBV and HDV, the inhibitory effect of c-Jun over TGF-β. 29 This which has crucial implications in the interaction effect could be synergistic with that of the HBx between the 2 viruses. 26 protein, which also activates these 2 signalling cas- cades. 31 Another proposed mechanism involves Potential HDV oncogenic mechanisms nuclear factor kappa B (NF-κB), a transcription fac- There is no data on the molecular alterations pre- tor with crucial roles in inflammation, immunity, sent in patients with HDV-related HCC. This is par- cell proliferation and apoptosis, and HCC develop- ticularly unfortunate for studies on genome-wide ment. 32 L-HDAg can induce NF-κB activation via mutation profiling, which could identify druggable tumor necrosis factor-α (TNF-α) stimulation 33 or mutations particularly enriched in these patients. oxidative stress. 34 L-HDAg has also been related HDV does not integrate into the genome and to the activation of other known oncogenic path- lacks the machinery required to propagate in the ways including the JAK-STAT pathway 35 (via acti- absence of HBV. Thus, a direct oncogenic mechan- vation of the signal transducer and activator of ism of HDV is unlikely. However, the interactions transcription 3 [STAT-3] downstream protein 34) between HDV and HBV could also help promote or c-Fos activation. 36 Another reported mechanism HCC development. 27 Preliminary data have indi- for HDV-related oncogenesis is downregulation of cated potential mechanisms by which HDV can glutathione S-transferase P1 (GSTP1), a tumour modify key signalling pathways related to fibrosis, suppressor gene. Transfection with S-HDAg in including epigenetic changes, immune response fetal hepatic cell lines inhibited GSTP1 expression modifications, specific dysregulation of long non- specifically by binding to its mRNA, which resulted coding RNAs (lncRNAs), and proteomic changes in accumulation of reactive oxygen species and (Fig. 2). Enhanced transforming growth factor-β increased apoptosis. 37 (TGF-β) signalling has been proposed as a mechan- Inactivation of tumour suppressor genes ism behind the accelerated liver disease in HBV/ through aberrant DNA methylation is frequent in HDV-coinfected patients. TGF-β is involved in HCC. 38 Some studies have evaluated the capacity multiple cellular processes, including growth, dif- of HDV to interfere with DNA methylation. For ferentiation, wound repair and apoptosis, with a example, the chaperon protein clusterin, which is major regulatory role in fibrosis and hepatocarci- involved in cell death regulation and frequently nogenesis.28 L-HDAg can activate the TGF-β path- overexpressed in HCC, 39 was found upregulated way, probably via the Smad3 protein, which through histone acetylation in HDV-infected cell could promote HCC development. 29 Since HBV lines. 40 Also, a small study in an HCC cell line over- can also upregulate TGF-β via the HBx protein, 30 expressing HDV found a mild increase in the levels this could be a mechanism by which HDV of methyltransferase 3b. 41 Other potential Cytoplasm HDV proteins TGF-βR TGF-βR s-HDAg Isoprenylated L-HDAg Non-isoprenylated L-HDAg JNK SMAD2 SMAD3 Nucleus P H3 JNK P SMAD2 SMAD2 SMAD3 SMAD3 P P P C-FOS C-JUN C -JUN SMAD4 A H A IκB SMAD4 3 P IκB Proteosomal NF-κB Clusterin degradation NF-κB STAT3 DNMT3b E2F1 IKK TRAF2 OS NF-κB NOX-4 TNF-αR Fig. 2. Potential mechanism of increased oncogenicity due to HDV. L-HDAg potentiates TGF-β and c-Jun signalling cascades. It can also activate NOX-4 and promote oxidative stress, which enhances NF-kB and STAT3 signalling. STAT3 promotes transcription of DNMT3b, which induces expression of the E2F1 transcription factor. L-HDAg potentiates NF-αB activation via TNF-α receptor by interacting with TRAF2. Finally, both L-HDAg and S-HDAg increase clusterin expression by promoting acetylation of histone 3. HDV, hepatitis delta virus; L-HDAg, large delta antigen; OS, oxidative stress; S-HDAg, small delta antigen. JHEP Reports 2019 vol. 1 | 120–130 122 Review epigenetic mechanisms involved long non-coding suppression. 50 HBV can also downregulate IFN RNAs (lncRNAs), including the deregulation of Y3 antiviral responses, and inhibit the transcription in HDV-related HCC. 42 Notably, dysregulation of of the MxA gene, but in the presence of HDV this lncRNAs is crucial in HDV replication. 43 In terms mechanism seems inefficient. 51 HDV replication of immune dysregulation, there is data on huma- may induce a strong type-I IFN signal, leading to nised mice showing a higher number of interferon an anti-HBV response. 52 This is further supported stimulated genes (ISGs) and cytokines such as TGF- by a model that compared HBV-monoinfected β and interleukin-28 in HBV/HDV-coinfected and HBV/HDV-coinfected human liver chimeric human hepatocytes compared to HBV- mice, in which a stronger and more sustained anti- monoinfected hepatocytes, suggesting enhanced viral response, including the activation of ISGs, was inflammation in HDV. 44 Finally, changes in the cel- observed in coinfected mice. 44 Similarly, in a lular proteome have also been linked to HDV infec- model of HepaRG cells, superinfection with HDV tion, including differential expression of 89 induced a strong type-I IFN response, with proteins, predominantly affecting DNA damage activation of ISGs (mainly RSAD2 and MxA), checkpoints and the cell cycle. 45 Most of this infor- which was associated with suppression of HBV mation comes from small studies, so a thorough replication. 53 evaluation of the key molecular features of HDV- HBV can also influence HDV, which would related HCC is eagerly awaited. explain why patients with chronic HBV/HDV coin- fection may have intermittent periods of HBV HDV infection and risk of HCC development replication. The HBsAg, which is different between HDV infection in patients with HBV the HBV genotypes, is essential for HDV infectivity HBV is invariably present in all HDV-infected and assembly, and also regulates the nuclear patients. The interaction between these 2 viruses export of HDV ribonucleoproteins. Hence, the is not completely understood but it seems to be number of hepatocytes that can be infected and reciprocal, and it may be crucial to the events the number of virions that successfully start repli- that lead to disease progression. Intriguingly, HDV cation after entry has been shown to be different may persist for several months after liver trans- between the variants, with genotypes B and D plantation without evidence of HBV replication. 46 being the most supportive. 54 Likewise, the packa- Besides, it has recently been shown that HDV repli- ging signal located in the C-terminal region of L- cation may persist even in the absence of HBV. 47 HDAg, which differs across HDV genotypes, may Two types of infection can be distinguished. The mediate its affinity to the HBsAg, determining the acquisition of both viruses at the same time (i.e. difference in assembly efficiency between geno- coinfection) is an important cause of severe hepati- types. 55 Hence, HDV genotype 1 can assemble tis, but only 5% of patients will develop a chronic and secrete more HDV virions than genotype 2, 56 infection, with the vast majority evolving to spon- which could explain why this genotype is asso- taneous clearance of HDV. 23 However, when HDV ciated with a more aggressive clinical course. 19 infection is transmitted in the setting of an already established chronic HBV infection (i.e. superinfec- Impact HBV/HDV in progression to cirrhosis tion), more than 90% of patients will evolve to Chronic hepatitis D promotes inflammatory cell chronic hepatitis, with a more severe course to cir- infiltration and progressive fibrosis, leading to rhosis. 23 Thus, to decipher the role of HDV in liver cirrhosis, as with other forms of chronic viral oncogenesis it is crucial to understand the interac- hepatitis. 16 Indeed, liver histology from HDV- tion between both viruses. Longitudinal studies infected patients is usually no different from have shown that the long-term interplay between that observed in other forms of viral hepatitis. 57 HDV and HBV is complex, with fluctuating levels Although HDV has been reported to have direct of HDV RNA and HBV DNA viremia over time and cytolytic effects, 58 it seems that liver damage in oscillating predominance at different time patients with HDV infection is mainly driven by points. 48,49 Importantly, in more than 50% of an immune-mediated process. HDV infection is cases, HBV activity is detected at different time associated with high levels of cytotoxic CD4+ T points, which has been associated with worse cells, as seen in HBV and HCV infections. 59 In con- prognosis. 48 trast to HBV, HDV induces a strong innate Several mechanisms have been proposed immune response via IFN β/λ stimulation after behind HDV-related suppression of HBV replica- being sensed by the pattern recognition receptor tion, including a direct effect of HDAg and the melanoma differentiation-associated protein 5 induction of an antiviral immune response. (MDA5), suggesting a different inflammatory Furthermore, it has been shown that both L-HDAg profile. 60 Notably, persistently high levels of and S-HDAg can repress the HBV enhancers Enh1 aspartate aminotransferase and alanine amino- and Enh2, and that L-HDAg can activate the tran- transferase have been described in HBV/HDV- scription of the myxovirus resistance-A (MxA) pro- coinfected patients, probably reflecting a high tein, an interferon (IFN)-κ inducible peptide that degree of inflammation in the liver and enhanced inhibits viral replication and contributes to HBV fibrosis formation. 61,62 JHEP Reports 2019 vol. 1 | 120–130 123 Cirrhosis and HCC are the final stages of chronic cirrhotic patients showed that HDV-infected liver inflammation, including HDV-related liver patients were younger (34 vs. 48 years) than disease. 63 Cirrhosis underlies HCC in 70–80% 64 of those with HBV monoinfection.79 Recent data con- cases, and thus, to decipher the oncogenic role of sistently show that patients with HBV/HDV coin- HDV it is necessary to evaluate its impact on cir- fection have higher odds of developing cirrhosis rhosis development. In viral hepatitis, progression than individuals with HBV monoinfection. For from chronic infection to cirrhosis depends on instance, a prospective study in Greece showed a numerous co-factors such as alcohol consumption, higher rate of liver-related events during follow- diabetes, or the existence of viral coinfection. 65,66 up among HBV/HDV-coinfected than HBV- HDV has repeatedly been included among these monoinfected individuals (20% vs. 8% at 4 years); co-factors that enhance the risk of cirrhosis. 67 the vast majority of events being cirrhosis devel- The natural history of HDV infection is shown in opment. 81 Another report from Australia found a Fig. 3. The delta antigen was initially discovered higher risk of cirrhosis progression in patients in patients with HBV infection who presented with HDV, 82 while a very recent study in Vietnam with a particularly severe disease course, 68,70-72 showed that HDV-infected patients were more including acute fulminant HBV hepatitis.69 Differ- likely to be Child-Pugh B or C than Child-Pugh A, ent studies have evaluated the potential role of indicating a predisposition towards advanced HDV as a catalyst of liver fibrosis in patients with liver disease in patients with HDV. 62 HBV coinfection. A summary of these studies is Altogether, these studies suggest that HDV is shown in Table 1. Additionally, a few studies associated with a more aggressive liver disease, have compared the risk of cirrhosis in HBV/HDV- leading to accelerated progression and an coinfected patients to that in HBV-monoinfected increased risk of cirrhosis. However, the patients. In a small study from Italy, progression mechanisms by which HDV hastens the pro- to cirrhosis was observed in 77% of HDV-infected gression to end-stage liver disease have not patients compared to 30% of HBV-monoinfected been elucidated. Accordingly, it is plausible patients over a follow-up period of 1–15 years. that HDV confers a higher risk of HCC develop- Strikingly, in 70% of these patients progression to ment than HBV monoinfection. Whether this is cirrhosis occurred in the first 2 years of follow- a result of enhanced inflammation/fibrosis up. 73 In another Japanese study with a median mediated by HDV or a direct oncogenic effect follow-up of 10 years, only 12% of 69 HBV/HDV- remains unknown. coinfected patients developed cirrhosis, but this was significantly higher than the 4% observed in Treatment of HDV infection HBV-monoinfected individuals. 74 Conversely, in 2 Treatment for HDV is currently limited to PegIFN studies performed in Taiwan, including 90 and 64 alpha 2a-2b for 48 weeks. Antiviral efficacy is patients with HDV, there was no increase in the modest, with virologic response rates ranging risk of cirrhosis development in HBV/HDV- between 17% and 47%. Furthermore, HDV relapse coinfected patients compared to HBV- after treatment occurs in more than 50% of respon- monoinfected patients. 75,76 Cirrhosis has been ders, 83 highlighting the need for new treatments. reported in 51–75% of histological samples in 2 For that purpose, surrogate markers that can be large series of HBV/HDV-coinfected patients from used to develop clinical endpoints have recently Italy. 77,78 Furthermore, a retrospective multi- been proposed. 84 Additionally, new drugs, such centric European study including 200 HBV- as inhibitors of viral entry into the hepatocyte, HBV infection HDV superinfection Normal liver Liver fibrosis Liver cirrhosis Hepatocellular carcinoma Chronic HBV infection Chronic HBV/HDV infection Estimated annual in <10% of patients in >90% of patients rate of HCC (2-5%) Increased in HBV-HDV compared 10 years with HBV mono-infection Progression risk factors: Genotype 1 infection, alcohol use Fig. 3. Natural history of HDV infection. Commonly, HDV infects hepatocytes already infected by HBV (i.e. superinfection). After that, 90% of patients will develop a chronic HBV/HDV infection with a faster evolution to cirrhosis in 10 years. Some risk factors, such as alcohol consumption or genotype 1 infection may accelerate liver disease development. HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HDV, hepatitis delta virus. JHEP Reports 2019 vol. 1 | 120–130 124 Table 1. Studies assessing the clinical course of patients with HDV infection Study Design Location HBV, n Age, mean ± SD Follow-up, Baseline Disease Limitations or median mean ± SD or cirrhosis, progression, HDV, n (range), yr median (range), yr n (%) n (%) Studies comparing HBV-monoinfected with HBV/HDV-coinfected patients Coghill, 201882 Retrospective Australia 370 40 (16–86) n.a. 83 (22) a 54/287 (19) Retrospective. Only 79 patients were tested for HDV RNA. Information 179 41 (16–69) n.a. 67 (37) 55/112 (49)⁎ about alcohol consumption not collected. No operational definition of cirrhosis. Manesis, 201381 Ambispective Greece 1,997 47.5 (35.1–57.9) 3.6 (3.3–3.9) 145 (7) a 142/1,852 (8) Ambispective. Only a subset of patients with HBV were tested for HDV, 81 43.1 (31.4–53.1) 4.2 (2.9–5.3) 16 (20)⁎ 12/65 (18) probably based on risk factors. HDV RNA was not tested. Cross, 200880 Cross–sectional England 882 35 (29–46) n.a. 109 (12) n.a. Retrospective and cross-sectional. No HDV RNA was tested. Susceptible 82 36 (30–47) 22 (27)⁎ n.a. to selection bias, being King´s College a tertiary referral center. Liaw, 200476 Retrospective Taiwan 64 n.a. 12.3 ± 6.4 (1–20) 0 a 11/64 (17) Retrospective. Sample size. 64 n.a. 8.2 ± 5.2 (1–21) 0 12/64 (19) Huo, 200075 Retrospective Taiwan 426 42 ± 15 5.7 ± 3.4 (1–17) 0 a 56/426 (13) Cirrhosis diagnosis was based on imaging features. Patients with acute JHEP Reports 2019 vol. 1 | 120–130 90 0 15/90 (17) decompensation that lead to death were excluded. Patients with a fol- low-up of less than 1 year were excluded. Fattovich, 200079 Retrospective Western 161 48 (11–78) 6.7 (0.5–16.5) 161 (100) b 31/161 (19) Competing outcomes were censored. No HDV RNA was determined. Europe 39 34 (13–28) 31 (100) 12/39 (30) HDV status was forced into the multivariate analysis. Tamura, 199374 Prospective Japan 1,058 n.a. 10.1 (3–18) n.a. a 43/1,058 (4) HDV RNA was not assessed. No multivariate analysis was performed. 69 n.a. n.a. 8/69 (12)⁎ Fattovich, 198773 Retrospective Italy 128 36 ± 13 4.7 ± 2.7 29 a 19/69 (28) No HDV RNA was determined. Some patients received corticosteroids 18 26 ± 8 5 ± 3.6 5 10/13 (77)⁎ during follow-up. Inclusion criteria included presence of transaminitis for 12 months. Patients with a follow-up of less than 1 year were excluded. Colombo, 198377 Retrospective Italy 142 37 n.a. (1–5) 31 (22) a 13/56 (23) HDV diagnosis was based on liver biopsies. Some patients 50 (8–72) 21 (42)⁎ 4/23 (17) received steroids during follow-up. No multivariate analysis. Studies in HBV/HDV coinfected patients Rosina, 199978 Retrospective. Italy 159 34 ± 11 6.5 ± 4.9 73 (46) n.a. No control group available. Patients with follow-up of less than 6 months were excluded. Govindarajan, 198672 Not clear USA 23 n.a. 2.5 (0.5–11) 7 (35) 6/16 (38) No other factors for progression were evaluated. Assessment for coinfection with HCV was not possible. Rizzetto, 198370 Retrospective Italy 137 34 (1–70) n.a. (2–6) 32 (23) 31/75 (41) Assessment for co–infection with HCV was not possible. Rizzetto, 197968 Retrospective Italy 63 35 (20–66) n.a. (1–4) n.a. 9/63 (14) Included cases with both acute and chronic presentations. Assessment Japan for coinfection with HCV was not possible. USA No other cofactors of progression of liver disease were assessed. Anti-HDV, antibodies against HDV; APRI, aspartate aminotransferase-to-platelet-ratio index; CAH, chronic active hepatitis; EIA, enzyme immunoassay; HBV, hepatitis B virus; HDAg, delta antigen; HDV, hepatitis delta virus; IF, immunofluor- escence; n.a., not available; OR, odds ratio; RIA, radioimmunoassay; RR, relative risk. *Statistically significant difference between HBV-monoinfected and HBV/HDV-coinfected patients a Progression to cirrhosis; b Decompensation. Review 125 inhibitors of HBsAg secretion, and virus assembly due to direct oncogenic viral effects such as DNA inhibitors are currently under investigation. 85 integration into the host genome, prolonged expression of the HBx regulatory protein or epige- Preliminary evidence of HDV as a risk factor for netic changes. 30 Compared to other risk factors, HCC development HBV-related tumours have a different genetic pro- The studies that evaluated the association file, with a higher rate of chromosomal alterations between HDV and HCC are generally small, retro- and TP53 mutations. 30 Apart from enhancing spective and have suboptimal designs. Thus, the fibrosis and inflammation, there is no evidence to extent of HDV involvement in HCC development suggest a direct oncogenic mechanism of HDV. is controversial. The unfailing presence of HBV in Studies comparing HCC incidence between HBV/ all HDV-infected hepatocytes leads one to ques- HDV-coinfected and HBV-monoinfected patients tion how HDV modifies the carcinogenic risk provide the best evidence from which to determine already imposed by HBV. Patients with HBV can the oncogenic potential of HDV (Table 2). Evidence develop HCC in the absence of cirrhosis. This is from these retrospective and cohort studies Table 2. Studies evaluating association between HDV and hepatocellular carcinoma. Authors & Study type Location HBV, n Follow–up HCC incidence in HBV Main limitations reference (range) HDV, n HCC incidence in HBV/HDV (%) Studies comparing HBV/HDV-coinfected vs. HBV-monoinfected Coghill, Case–control Australia 4,407 None 5.4% Case-control design. 201882 179 7.8% (RR 1.1; 95% CI 0.9–2.2; (3%) p = 0.17) Béguelin, Prospective Switzerland 771 8.7 (IQR 5– – HIV cohort. Only 73 (61%) patients 201787 119 13.8) years RR 9.3 (95% CI 3–28.6; p <0.001) presented HDV RNA+ (15%) Kushner, Retrospective United 2,175 Not stated – Low HDV prevalence and very low HDV 201588 States 73 (3%) Adjusted OR 2.1 (95% CI 1.1–3.9; testing in the cohort (8%) p = 0.025) Ji, 201286 Retrospective Sweden 9,162 None – Cross-sectional. Not clear definition of 323/ Acute OR 6.11 (2.77– 11.65) / Chronic acute vs. chronic HDV infection. 327 OR 3.90 (1.61–7.22) (4%) Cross, Cross– England 962 None 7.8% Cross–sectional (no follow-up, high risk 200880 sectional (London) 82 (9%) 9.7% (OR 1.34; 95% C.I. 0.62–2.91; of bias) p = ns) Fattovich, Retrospective Europe 200 80 (6–198) 2–4% Retrospective. Shows a trend without 200079 39 months 13% (RR 3.2; 95% CI 1–10; statistical significance (20%) p = 0.0523) Tamura, Prospective Japan 1,127 121 (36–216) 3% Old study performed in a small Japanese 199374 69 (6%) months 9% (p <0.01) region. Studies evaluating HCC incidence in HBV/HDV-coinfected Wranke, Retrospective Worldwide – None – Study design, no comparison 201891 1,576 1.9% Amougou, Case–control Cameroon – None – Study design, healthy controls 201695 24 OR 29.3 (95% CI 4.1–1231) compared to controls Romeo, Retrospective Italy – 9.5 years – Study design, no comparison 201489 193 OR 1.88 (95% CI 1.11–3.19; p = 0.019) in HDV–RNA+ Buti, Retrospective Spain – 6 years – Study design, no comparison 201194 158 3% Niro, Retrospective Italy – 8 years – Study design, no comparison 201092 126 1% HCC annual rate Romeo, Retrospective Italy – 28 years – Study design, no comparison 200990 299 2.8% HCC annual rate Gheorghe, Retrospective Romania – 10 years – Study design, no comparison 200593 166 12% HDV, hepatitis delta virus. JHEP Reports 2019 vol. 1 | 120–130 126 Review suggests that HBV/HDV coinfection accelerates pro- Box 1. Unmet needs in HDV-related HCC research. gression to cirrhosis and is associated with an • To catalogue the worldwide epidemiological distribution of HDV. increased risk of HCC development compared to • To standardize methods to detect and monitor HDV infection, such as the ones proposed by HBV monoinfection. 79,80,82,86–88 This increased risk the WHO. is difficult to estimate due to the nature of the data. • To understand the clinical course of HDV/HBV co-infection as opposed to HBV monoinfected A thorough European study including 200 patients patients, particularly in terms of their HCC risk. with HBV-related cirrhosis showed that those with • To dissect the contribution of HDV to HCC development in well-designed, prospective studies HBV/HDV coinfection had an estimated relative risk adjusting for well-known HCC risk factors (e.g., sex, family history, smoking, HBV genotype, (RR) of HCC development of 3.2 compared to those viral load, presence of basal core promoter and precore mutations, stage of liver disease, with HBV monoinfection over a follow-up of 6.6 HIV or HCV co-infections, amongst others). years. 79 Similar studies reported an increased • To perform a molecular characterization of HDV-related HCC, covering mutational landscape incidence of HCC from 3% to 9% in HBV/HDV- and signatures, DNA copy number alterations, gene expression de-regulation, immune profil- coinfected individuals compared to HBV- ing and epigenetic aberrations. monoinfected individuals. 74 The largest cohort • To develop new effective therapies to HDV. published to date is a population-based study per- formed in Sweden, including 9,160 patients with HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HDV, hepatitis delta virus; WHO, World Health Organization. HBV, 327 with chronic HDV infection and 323 with acute HDV-infection. 86 After adjusting for age, the authors found a significantly higher risk of HCC in patients with acute (RR 6.1; 95% CI 2.8–11.7) or low HCC annual incidence of 1.9%. 91 Smaller stu- chronic (RR 3.9; 95% CI 1.6–7.2) HDV-infection. 76 dies reported variable rates of HDV-related HCC Another large study including 2,175 American veter- ranging between 13% 92,93 and 3%94 after a median ans with HBV found a 2.9-fold higher incidence of follow-up of 5 to 10 years. Even considering the HCC in individuals with HDV, after adjusting for cir- suboptimal quality of the evidence, it is reasonable rhosis and HCV infection. 88 Conversely, in another to assume an association between HDV and HCC study including 962 consecutive HBV-infected development, likely due to the more aggressive patients, those with HBV/HDV coinfection (n = 82) underlying liver disease imposed by HDV. had a similar prevalence of HCC (9.7% vs. 7.8%) as those with HBV monoinfection80 A recent prospec- Conclusions and future directions tive study in a HIV cohort identified HDV as a strong Many questions remain unanswered regarding the predictor of HCC, with an RR of 9.3. 87 Although oncogenic role of HDV. It has been suggested that results were described for all patients with HDV, HDV accelerates the disease course, leading to cir- only 73/116 (63%) were RNA positive, which can rhosis and likely enhancing HCC development, impact on disease progression. 89 Finally, a case- compared to HBV monoinfection. However, studies control study in Australia reported no association evaluating HCC incidence in HBV/HDV-infected between HDV infection and HCC in a cohort of 179 patients are discordant and they mostly provide patients with HDV. 82 low levels of evidence. Furthermore, the potential There are also studies on HDV-related HCC that mechanisms underlying HDV-specific oncogenesis do not compare incidence rates in patients with are poorly understood. Overall, well-designed pro- HBV monoinfection. In a retrospective study from spective studies comparing cohorts of HBV/HDV- Italy, involving 299 HDV-infected patients over a coinfected and HBV-monoinfected individuals will follow-up of 28 years, 46 patients developed HCC, be key to determine the oncogenic capacity of which represents an annual rate of 2.8%. 90 HDV HDV. A summary of the actual unmet needs in RNA status was not associated with HCC develop- HDV research are shown in Box 1. Remarkably, in ment in this study. Years later, the same authors this context of unknown pathophysiological reported an association between HDV RNA mechanisms, molecular studies evaluating HDV- >600,000 copies/ml and progression to cirrhosis infected HCC samples will be a great resource to and HCC. 89 The biggest descriptive cohort of understand the singularities of this condition and HDV-infected patients published so far included identify novel targets for therapies in HDV- 1,576 patients from 19 countries and reported a infected patients. Financial support Waxman Cancer Research Foundation, Spanish National Health M. Puigvehi received a scholarship grant from Asociación Española para el Institute (SAF2016-76390), and the Generalitat de Catalunya/AGAUR Estudio del Hígado (AEEH). A. Villanueva was supported by the DoD (SGR-1358). Translational Team Science Award (CA150272P3). J.M. Llovet was supported by NCI (P30-CA196521), DoD Translational Team Science Conflicts of interest Award (CA150272P1), European Commission (EC)/Horizon 2020 Prof. Josep M. Llovet is receiving research support from Bayer Health- Program (HEPCAR, Ref. 667273-2), EIT Health (CRISH2, Ref. 18053), Accel- Care Pharmaceuticals, Eisai Inc, Bristol-Myers Squibb and Ipsen, and erator Award (CRUCK, AEEC, AIRC) (HUNTER, Ref. 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