Chronic hepatitis B virus (HBV) infected patients have an almost 100-fold increased risk to develop hepatocellular carcinoma (HCC). HCC is the fifth most common and third most deadly cancer worldwide. Up to 50% of newly diagnosed HCC cases are attributed to HBV infection. Early detection improves survival and can be achieved through regular screening. Six-monthly abdominal ultrasound, either alone or in combination with alpha-fetoprotein serum levels, has been widely endorsed for this purpose. Both techniques however yield limited diagnostic accuracy, which is not improved when they are combined. Alternative circulating or histological markers to predict or diagnose HCC are therefore urgently needed. Recent advances in systems biology technologies have enabled the identification of several new putative circulating biomarkers. Although results from studies assessing combinations of these biomarkers are promising, evidence for their clinical utility remains low. In addition, most of the studies conducted so far show limitations in design. Attention must be paid for instance to different ethnicities and different etiologies when studying biomarkers for hepatocellular carcinoma. This review provides an overview on the current understandings and recent progress in the field of diagnostic and predictive circulating biomarkers for hepatocellular carcinoma in chronically infected HBV patients and discusses the future prospects.

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and ranks third as cancer-related death cause due to a 5-year survival of only 15%[1]. Moreover, at a time of decreasing overall cancer-related deaths due to an immense progress in cancer diagnostics and treatment options, mortality from hepatocellular carcinoma is increasing[1,2].

Chronic hepatitis B virus (HBV) infection is a major risk factor for HCC development. Prospective cohort studies have revealed an up to 100-fold increased risk for HCC in chronically infected HBV patients[3]. Up to 50% of newly diagnosed HCC cases are attributed to HBV infection, due to both direct and indirect oncogenic effects of the virus[2,4-7]. Integration of HBV DNA in the human genome may result in genomic instability, while inflammation-related oxidative stress, caused by immunological responses, may contribute indirectly to HCC development[6-9].

Four clinical phases can be distinguished during the natural course of a HBV infection: an immune-tolerance phase, an immune active phase, an inactive carrier phase and a hepatitis B e antigen (HBeAg) negative phase[2,10]. Patients in the immune active phase and the HBeAg negative phase are at increased risk for progression towards fibrosis and ultimately the development of cirrhosis, which is a major risk factor for HCC[11,12].

Several years to decades are needed for HCC to develop in a HBV infected liver[13]. Early diagnosis of HCC in HBV patients is challenging but is proven to result in an improved long-term survival due to an increased chance to detect tumors at a resectable stage[14-19].

Importantly, also a significant number of HBV patients develop HCC in a non-cirrhotic liver[20]. Current guidelines therefore advise 6-monthly abdominal ultrasound (US) surveillance for HCC in advanced fibrosis or cirrhotic HBV patients and in non-cirrhotic patients depending on ethnic background and age[21-25]. The technique, however, faces a disappointing 63% sensitivity to detect HCC and is hampered by inter- and intra-observer variability[20]. Finding biomarkers to better predict or diagnose HCC therefore remains an important clinical and research priority.

Serum alpha-fetoprotein (AFP) levels are widely used for HCC screening and diagnostics, but the clinical utility to rule out or detect HCC is still a matter of debate. The protein lacks sensitivity and specificity to detect HCC. The recent improvement of systems biology techniques, such as proteomics and genomics, has enabled the identification of several new putative biomarkers[26,27]. This review provides an overview of diagnostic and predictive serum biomarkers for HBV-associated hepatocellular carcinoma and discusses future prospects.

An overview of the discussed diagnostic circulating biomarkers with their respective sensitivities and specificities to detect HCC is displayed in Table 1. An overview of their cellular origin is displayed in Figure 1.

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Figure 1 Cellular origin of the discussed predictive and diagnostic biomarkers in a physiological and oncological setting. Predictive biomarkers are displayed in italics. PIVKA-II: Protein induced by vitamin K absence; AFP: Alpha-fetoprotein; GPC3: Glypican-3; SCCA: Squamous cell carcinoma antigen; DKK1: Dickkopf-1 protein; miRNA: MicroRNA; COMP: Cartilage oligomeric matrix protein; GP73: Glycoprotein-73; sPD-1: Soluble programmed death-1; Gamma-GT: Gamma-glutamyltransferase.

Three strategies can be applied when assessing the long-term risk for HCC. Firstly, clinical risk scores, e.g., REACH-B and PAGE-B, can be calculated based on viral and host-related (e.g., age and gender) clinical parameters. Most of these models have however been developed in Asian populations and lack validation in non-Asian populations[112,113]. HBeAg positivity and HBV DNA levels above 1 million copies/mL are associated with a 4- and 11-fold increased HCC-risk during 8 and 11 years of follow-up respectively[114-116]. Hepatitis B surface antigen (HBsAg) levels above 1000 IU/mL are accompanied with an up to 6.5-fold increased HCC risk in men and an up to 11-fold increased risk in women within 15 years[117]. HBsAg levels are suggested to be especially useful in case of low HBV DNA levels[118,119].

Secondly, genome-wide association studies have enabled the linkage of genetic variants to specific disease outcomes. Single nucleotide polymorphisms (SNPs) in a wide range of genes, including the Interleukin-21 and the CRP-gene, have been associated with an increased susceptibility for HCC over a variable time course[120-124]. Increasing evidence indicates that SNP’s in the STAT4, MDM2 and HFE gene, determined on whole blood, are germline risk factors for HCC[125,126]. On the other hand, also somatically acquired mutations, e.g., in the TP53 gene, have been associated with an increased risk for HCC[127]. All together these findings are strongly suggestive for interindividual differences in the genetic predisposition for HCC development, a predisposition that can be boosted by additional somatic mutations.

Thirdly, circulating biomolecules would be ideal as a non-invasive, predictive biomarker for HCC. An overview on the discussed predictive biomarkers including their respective increase in HCC risk is displayed in Table 2. The clinical utility of Gamma-Glutamyl Transferase (Gamma-GT) Iso-enzyme II was first evaluated as a predictive HCC marker in 1992. Patients showing persistently elevated levels of Gamma-GT Iso-enzyme II at presentation had a 86.7% risk to develop HCC within 10 years’ time[128]. Gamma-GT levels above 41 U/L and AFP-levels > 5 ng/mL have later been associated with an 8-fold increased risk for HCC in a large HBV cohort followed up for 6 years[129]. The usefulness of AFP-levels for HCC prediction has, however, been assessed in several other studies with contradictory results[128-130].

Cartilage oligomeric matrix protein (COMP) is an extracellular matrix protein involved in tissue genesis and remodeling[131]. The protein is released into the circulation upon cartilage damage[132]. Overexpression of the protein in serum from HCC patients suggested that serum COMP levels reflected an individual’s fibrosis stage and subsequent risk for HCC[133]. A recent study in Greece supports this hypothesis: COMP positivity (> 15 U/L) was associated with a 3-fold increased HCC risk during a median follow-up of 8 years[132,134].

In a study of 27 serum cytokines and growth factors, interleukin-6 (IL-6) levels were found to predict cancer development within a timeframe of 8 to 11 years with moderate accuracy[135]. Levels above 7 pg/mL were associated with a 3-fold increased HCC risk. As IL-6 induces C-reactive protein (CRP), the potential value of CRP in HCC risk prediction was assessed, but turned out to be disappointing[136,137]. Recently, soluble programmed death-1 (sPD-1), a soluble form of the membrane-bound programmed death 1 on T cells with a largely unknown function was put forward as a marker[138,139]. sPD-1 levels above 637.6 pg/mL at baseline reflected a 2-fold increased risk to develop HCC during a median follow-up time of 20 years, when compared to sPD-1 levels below 117.3 pg/mL[138].

Despite its disappointing sensitivity and specificity, AFP still remains the most widely used serum HCC biomarker. Some newly discovered circulating biomarkers, e.g., AFP-l3, DCP and microRNA’s show promising potential for implementation in clinical practice. However, only GP73 strongly outperforms AFP in terms of diagnostic accuracy. Large variations in sensitivity and specificity are noticed between different studies assessing the same biomarker (Table 1).

Due to the heterogeneity of HCC, one single biomarker with 100% sensitivity and specificity in all HCC cases will be hard to find. A more rational approach to increase the diagnostic accuracy might be the combination of different biomarkers[41,52,55,62,65,72,140-142]. The most recent APASL guidelines indeed recommend the combined use of AFP, AFP-l3 and DCP in HCC screening[22,142]. In favor of this approach, a meta-analysis showed that combined testing of GP73 and AFP increased the pooled sensitivity without decreasing the specificity to detect hepatocellular carcinoma. The pooled sensitivity and specificity were 87% and 85% respectively when biomarkers were combined, compared to 77% and 91% for GP73 and 62% and 84% for AFP when used alone[72].

All currently identified circulating biomarkers and their combinations definitely need more validation studies. Most of the biomarker discovery studies have been performed in cohorts of a few 100 patients. The majority of identified markers has so far not been subject of large, external validation studies[100-103,108]. Five subsequent steps are to be followed in cancer biomarker discovery. The first step is the implementation of preclinical exploratory studies. Step 2 is the development of a clinical assay. Step 3 involves retrospective studies, step 4 prospective studies and step 5 large, randomized controlled trials. Biomarkers identified in step 1 must pass all other steps before they can be termed validated biomarkers[143]. So far, only AFP has reached step 5[15].

In addition, the studies that have been conducted over the last decades are hampered by limitations in their study design. As an example the patient cohorts for HCC biomarker discovery studies are often heterogeneous regarding liver disease etiology and ethnicity. In their paper, da Costa et al[144] proved the need to validate biomarkers in different ethnic populations. They investigated the potential of osteopontin and latent Transforming Growth Factor beta binding-protein in HCC diagnosis in separate cohorts in Gambia, Korea, Thailand and France. The sensitivity and specificity of both markers differed (> 10%) among ethnicities. The onset of HCC occurs at a median age of 45 in sub-Saharan African people, whereas a mean age of 52 to 65 has been observed in the rest of the world[145,146]. In addition, HCC incidence varies among HBV and hepatitis C virus (HCV) patients, highlighting the importance of homogeneous patient cohorts. Future biomarker discovery and validation studies should therefore distinguish between different ethnicities and etiologies as this most probably explains the variation in sensitivities and specificities noticed between studies assessing the same biomarker (Table 1).

The inclusion of clinical parameters into biomarker scores could increase their performance. One study demonstrated that incorporation of age into combined models of biomarker testing significantly improved the diagnostic performance for HCC[140].

From a clinical point of view, however, predictive serum biomarkers would be preferred over diagnostic biomarkers to tailor HCC surveillance according to the individual needs. Proteomic approaches are encouraging, but also need a validation in larger cohorts[147,148]. Expression of Heat-shock Protein 27 was e.g., detected in 90% of sera from HCC patients and in 0% of sera from non-HCC patients, which seems promising[148]. Other groups have focused on genomics and have identified a gene signature in liver tissue of HCV infected patients predictive of HCC development[149,150]. It could be of interest to identify corresponding secretory biomarkers in blood.

Monitoring HCC development in chronic hepatitis B patients based on serum biomarkers remains challenging. During recent years, new predictive and diagnostic circulating biomarkers have been proposed. Combinations of these biomarkers show a higher potential for implementation in clinical practice, but large validation studies in homogeneous ethnic and etiological populations are urgently needed to unequivocally demonstrate their clinical utility.

The authors thank professor Benedicte Y De Winter for the critical revision and language editing of the manuscript.