HCV-induced HCC development is a multi-step process that may progress over 20-40 years and involves a number of steps: establishment of chronic HCV infection, chronic hepatic inflammation, progressive liver fibrosis, initiation of neoplastic clones accompanied by irreversible somatic genetic/epigenetic alterations, and progression of the malignant clones in a carcinogenic tissue microenvironment (Fig. 1). Each step of HCV-induced hepatocarcinogenesis is a potential target for therapeutic intervention or chemoprevention.12 Unlike HBV which can integrate into the host genome leading to potential direct carcinogenic activity, HCV is a RNA virus with limited integration of its genetic material into the host's genome. Therefore, the carcinogenic potential of HCV is generally assumed to be linked to indirect mechanisms, although the lack of a convenient in vitro model system to study biology is a major obstacle for the understanding of the mechanisms linking HCV infection, inflammation and carcinogenesis.1314

The "field effect" or "field cancerization" refers to the cirrhotic milieu/microenvironment that allows initiation and promotion of neoplastic clones by facilitating genetic aberrations and cellular transformation.15 Chronic damage to hepatocytes, such as HCV infection, leads to a release of inflammatory and fibrotic mediators such as reactive oxygen species, cell death signals, hedgehog ligands and nucleotides.16 A complex series of mechanisms centering on the hepatic stellate cell, mediated through intracellular inflammasome activation, the nuclear receptor family, (farsenoid-X-receptor, peroxisome proliferator-activated receptors and others) and other transcriptional events contribute to stellate cell activation. The activated hepatic stellate cell promotes liver scarring through proliferation, contractility, fibrogenesis, matrix degradation and inflammatory signaling. HCV broadly infects hepatocytes, monocytes, lymphocytes and other secretory cells, and contributes to stellate cell activation. HCV core and nonstructural proteins stimulate profibrogenic mediators such as TGF-beta.17 HCV infection induces TGFB1 through reactive oxygen species (ROS) production, p38 MAPK, JNK, ERK, and NF-kappaB pathways.18 Platelet-derived growth factor (PDGF) is the most potent mitogenic signal, inducing expression of beta PDGF receptor expression in stellate cells together with other cell surface receptors of growth signaling such as integrins.19 Transgenic mice expressing PDGF-C develop liver fibrosis and HCC,20 and an acyclic retinoid, peretinoin, represses fibrosis and HCC development in the model.21 Severity of liver fibrosis is tightly correlated with increasing risk of HCC in patients with chronic HCV infection, suggesting that cirrhosis-driven carcinogenesis is the major mechanism in the development of HCV-related HCC.622

Clinical data suggest the role of HCV viral factors in disease progression and HCC development. For example, genotype 3 HCV has a worse response to DAAs, increased steatosis, and increased fibrosis progression rate,2324 whilst, although the evidence is conflicting, HCC risk seems to be dependent on viral genotype and potentially increased in subjects with HCV genotype 3.252627 Experimental data also seem to suggest that HCV proteins interact with cellular proliferation and survival pathways leading to an increased risk of HCC. Over-expression of HCV proteins, promotes cellular proliferation, transformation, and/or tumor formation in mice, suggesting the direct contribution of viral proteins to activating oncogenic molecular pathways.28293031 The viral core protein inhibits tumor suppressor genes TP53, TP73, and RB1 as well as negative regulator of cell cycle such as CDKN1A.32333435 NS3 and NS5A also inhibit TP53,3637 and NS5B inhibited RB1.38 HCV core, E2, NS5A, and NS5B activate cellular proliferative RAF/MAPK/ERK kinase pathways and E2F1 pathway, which are associated with more aggressive biological phenotype of HCC tumors.3438394041 HCV proteins such as core are known to induce generation of ROS and transactivate MAPK and AP1 pathways.42 Insulin-like growth factor signaling is activated via IGF1R in early stage HCV-related HCC.43 NS5A was found to be involved in activation of PI3K/AKT and β-catenin/WNT pathways, and evasion from apoptosis by caspase-3 inhibition.44 NS5A inhibits TGF-beta signaling by preventing nuclear translocation of Smad proteins, resulting in downregulation of the tumor suppressor CDKN1A.45

Interferon pathway activation is a well-known innate immune response to HCV infection, and recent studies have elucidated its role in anti-tumor immunity.46 The HCV core protein inhibits the NF-kappaB mediated immune responses47 whereas the JNK pathway, activated in non-parenchymal liver cells by proinflammatory signals such as ROS, generates an inflammatory hepatic microenvironment that supports HCC development.48 Viral proteins also appear to subvert innate immune pathways via the suppression of innate immunity, inhibition of natural killer cells and impairment of antigen-presenting cells by viral proteins.495051

Clinically, the interaction of HCV, steatosis and the metabolic syndrome has suggested a modulation of metabolic pathways by chronic HCV infection.5253 In addition, HCV-related HCC is often accompanied by histological steatosis within the tumor and nontumoral liver.54 Expression of core protein in transgenic mice induces steatosis, insulin resistance and HCC, whereas HCV core protein co-localizes with apolipoprotein A2 on the surface of triglycerides, suggesting its association with lipid metabolism.3155 HCV core protein suppresses microsomal triglyceride transfer protein activity and interferes with hepatic assembly and secretion of triglyceride-rich very low density lipoproteins, further contributing to steatosis.56 HCV core protein interacts with RXR-alpha and peroxisome proliferator-activated receptor-alpha (PPAR-alpha), and modulates cell differentiation, proliferation and fatty acid transport and catabolism in mice.57 In the presence of HCV core induced mitochondrial dysfunction, PPAR-alpha exacerbates steatosis, induces oxidative stress, and increases cell growth signals.58

Activating somatic mutations in telomerase reverse-transcriptase promoter is a frequent early neoplastic event in HCC with mixed etiologies including HCV.59 Hepatocyte proliferation is generally decreased at the stage of cirrhosis after many rounds of regeneration accompanied by telomere shortening that triggers cellular senescence though ATM, TP53, and CDKN1A as a safeguard to prevent carcinogenesis.60 HCV core protein overcomes stressinduced hepatocyte senescence by down-regulating CDKN2A expression via DNA methylation.61

A multi-kinase inhibitor, sorafenib, is the only drug currently approved for advanced HCC.62 Sorafenib showed anti-HCC effect by blocking paracrine hepatocyte growth factor from stromal cells in response to vascular endothelial growth factor-A secreted from HCC cells.63 Interestingly it improved portal hypertension in cirrhotic patients, supposedly due to its anti-angiogenic activity.64 Epidermal growth factor (EGF) is a mitogen involved in cellular growth, proliferation, differentiation, and carcinogenesis. In rodent models of cirrhosis-driven HCC, pharmacological inhibition with a small molecule EGF receptor (EGFR) inhibitor, erlotinib, regressed fibrosis and inhibited HCC development65 despite no inhibition of EGF pathway in the tumors, suggesting that the HCC preventive effect was through action on the cirrhotic tissue microenvironment that supports initiation of neoplastic clones. In contrast, another small molecule EGFR inhibitor, gefitinib, suppressed growth of initiated HCC clones in rats.66 EGFR was recently identified as a co-factor for HCV cellular entry, and erlotinib inhibited HCV infection, suggesting its role as a potential anti-HCV drug.6768

Various host polymorphisms associated with the immune system have been associated with HCV-induced HCC. A Japanese genome-wide association study comparing HCV-related HCC patients with chronic hepatitis C patients identified a single nucleotide polymorphism (SNP) in MHC class I polypeptide-related sequence A (MICA - rs2596542), which is involved in response of dendritic cells to type-I interferon in chronic hepatitis C.6970 Another SNP in the MICA promoter (rs2596538) was associated with increased serum soluble MICA protein.71 A subsequent study in Caucasian hepatitis C patients in Switzerland did not replicate the association with HCC for this locus, but for a nearby locus in HCP5 (rs2244546), suggesting that the MICA/HCP5 region contains a potential susceptibility locus.72 An IL28B variant (rs12979860), initially identified as an interferon response predictor, may be associated with increased risk of HCV-related HCC.7374

SNPs in genes associated with metabolic functions have also been weakly associated with HCV-induced HCC. Although some teams reported an association between HFE gene mutations, in particular H63D, and increased risk of HCC,75 these findings were not replicated in other studies.76 In patients with chronic hepatitis C with advanced fibrosis, positive association between liver iron deposition and higher incidence of HCC and poor prognosis was nevertheless observed.77 A SNP in the patatin-like phospholipase domain-containing protein 3 (PNPLA3) gene (rs738409) associated with alcoholic and non-alcoholic steatohepatitis may have weak association with HCV-related HCC.78

Considering the high prevalence of HCV-induced cirrhosis, the population at risk of development of HCC, requiring surveillance according to clinical guidelines, is huge and likely unmanageable. For instance, despite guidelines recommending HCC surveillance in subjects with cirrhosis,79 most patients at risk of HCC in the US do not receive recommended regular surveillance. Only 12% of cirrhotic HCV patients had routine annual surveillance in one US Veterans Affairs series and only 2% of HCV patients who developed HCC had previous appropriate screening in another series.8081 Additionally, in a population-based US study, less than 20% of patients with cirrhosis who developed HCC received regular surveillance.82 Growing numbers of early-stage, asymptomatic cirrhotics identified by non-invasive fibrosis detection methods such as elastography may also add to the HCC screening burden.83 Therefore prognostic indicators are being actively developed to stratify HCV subjects, as well as patients with other liver diseases, into clearly defined risk groups to enable effective clinical management of the patients.12

A number of non-invasive and invasive clinical markers and systems have been proposed to assess risk of HCC in subjects with HCV, in particular in patients with HCV-induced cirrhosis (Table 1). Scores identifying subjects at risk of developing HCC after SVR have also been reported8485 although these scores require further validation. This is especially relevant in this era of highly effective DAAs where the burden of HCV-related complications might shift to subjects who have achieved SVR, but remain at high risk of liver-related complications. Overall, although clinical variable-based prediction models for HCC development have been explored, their performance is limited and none of them has been established in practice.8687

Molecular biomarkers of HCC risk and/or poor prognosis may enable further enrichment of the high-risk population and may significantly contribute to improvement of early HCC detection. Numerous germline SNPs have been reported as HCC risk variants, although very few of them are replicated in independent patient series/cohorts.88 The EGF 61^*G allele (rs4444903) was associated with HCC risk in a prospective cohort of patients with HCVrelated advanced fibrosis or cirrhosis (Table 2),8990 while a SNP in an antioxidant enzymes, myeloperoxidase, (MPO-463^*G, rs2333227) was associated with HCC risk in another prospective study.91 A 186-gene-expression signature was associated with HCC risk in prospectively followed patients with early-stage HCV-related cirrhosis (HR=2.65).92 The gene signature has proven prognostic not only for HCC recurrence but also for liver disease progression, HCC development and overall survival in subjects with early-stage HCV cirrhosis.929394 The signature was present in the liver of rodent models of fibrosis/cirrhosis-driven HCC, and the poor prognosis pattern of the signature was reversed in association with the HCC chemopreventive effect of an FDA-approved EGFR inhibitor, erlotinib,65 which is now being tested in a phase 1 trial with the gene signature as a companion biomarker (ClinicalTrials.gov, NCT02273362). Circulating cells or biomolecules such as miRNAs may be alternative sources to obtain similar molecular information less invasively.95 In addition, molecular imaging of collagen could potentially be used to monitor fibrosis regression, which may correlate with decreased HCC risk.96

One possible role of molecular risk stratification of subjects with HCV-induced liver disease could be chemopreventive trial enrichment with subjects at high risk of HCC.12 Until now, several large phase 3 clinical trials have failed to show the usefulness of chemopreventive strategies in HCC despite the enrollment of thousands of patients and a follow-up time approaching decade(s).979899 Enrichment of high-risk patients with the use of HCC risk biomarkers and/or prognostic indices is critical to increase HCC incidence and keep required sample size and study duration within practically feasible range. HCC risk biomarker-based clinical trial enrichment will drastically lower the bar to conduct cancer chemoprevention trials by substantially reducing required sample size and the duration of follow-up comparable to oncology trials enrolling advanced-stage cancer patients.100