Circulating Cell-free DNA Species Affect the Risk of Hepatocellular Carcinoma in Treated Chronic Hepatitis B Patients

Alkistis Papatheodoridi; Antonios Chatzigeorgiou; Lampros Chrysavgis; Panagiotis Lembessis; Alessandro Loglio; Floriana Facchetti; Evangelos Cholongitas; Michael Koutsilieris; Pietro Lampertico; George Papatheodoridis


J Viral Hepat. 2021;28(3):464-474. 

In This Article

Abstract and Introduction


Hepatocellular carcinoma (HCC) may still develop in chronic hepatitis B (CHB) patients even under effective long-term oral antiviral therapy, but its pathogenesis in the setting of long-standing inhibition of viral replication has not been completely elucidated. We investigated whether species of circulating cell-free DNA (cfDNA) may be involved in the process of hepatocarcinogenesis in treated CHB patients. Serum samples were obtained from HBeAg-negative CHB patients with (HCC cases, n = 37) or without HCC development during the first 5 years of oral antiviral therapy (controls, n = 74). HCC cases and controls were matched 1:2 for age, sex and platelets. Determination of different circulating cfDNA species (before HCC diagnosis in HCC cases) including total cfDNA quantity, levels of Alu repeat DNA and RNase P coding DNA, copies of mitochondrial DNA and levels of 5-methyl-2'-deoxycytidine as an indicator of DNA methylation was performed. HCC cases compared with controls had higher median levels of Alu247 (123 vs 69 genomic equivalent, p = .042) and RNase P coding DNA (68 vs 15 genomic equivalent, p < .001). In contrast, median cfDNA concentration, Alu115 levels, Alu247/Alu115 ratio as an index of DNA integrity and mitochondrial DNA copies did not differ significantly between HCC cases and controls. Receiver operating characteristic curve analysis showed that levels RNase P coding DNA offered good prediction of subsequent HCC development (c-statistic: 0.80, p < .001). In conclusion, serum levels of RNase P coding DNA are increased years before HCC diagnosis and could be potentially helpful in the prediction of the HCC risk in treated HBeAg-negative CHB patients.


Chronic infection with hepatitis B virus (HBV) represents a global health problem associated with increased morbidity and mortality.[1,2] Over the last two decades, oral antiviral therapy with a nucleos(t)ide analogue (NA), which represents the treatment of choice for the majority of patients with chronic hepatitis B (CHB), has improved their outcomes and overall survival.[1–3] However, CHB patients treated with long-term NA therapy do not achieve HBV eradication and only a small minority of them may achieve functional cure defined as HBsAg seroclearance.[1,2] Therefore, they remain at some risk for the development of hepatocellular carcinoma (HCC), as its incidence is decreased but not eliminated under NA therapy.[4,5] Thus, HCC currently represents the main serious complication of diagnosed and treated CHB patients and the only factor that affects liver-related mortality in this setting.[3–6]

For the above reasons, the understanding of the pathogenesis of hepatocarcinogenesis in CHB remains of great interest, particularly in NA-treated patients. Several groups have recently focused on the assessment of the HCC risk in CHB patients mostly treated with NAs.[7,8] The main factors affecting the incidence of HCC in NA-treated CHB patients seem to be key parameters related to the host, such as age and sex, as well as the severity of liver disease expressed by platelets or presence of cirrhosis.[7,8] On the other hand, it is well known that HBV proteins can affect several cellular activities and pathways that are involved in malignant transformation.[9,10]

Cell-free DNA (cfDNA) is mostly released by cell death. Raised concentrations of cfDNA have been reported in cancer patients,[11,12] as fragmented cfDNA derived from cancer cells and its changes are considered as potential surrogate markers for early detection, risk stratification and response monitoring in oncology.[13,14] In addition, increased levels of cfDNA isolated from serum or plasma have been reported in patients with HCC.[15–18] cfDNA consists of nuclear DNA (gene-coding and non-coding, repeated sequences) and mitochondrial DNA. In particular, RNase P is a single-copy gene that is frequently used as reference gene for gene-coding cfDNA.[19] Moreover, cfDNA Alu elements are over-represented in cfDNA[20,21] and the ratio of longer Alu elements of 247 base pairs (bp) to shorter elements of 115 bp, known as DNA integrity, seems to be increased in patients with cancer, as malignant cells release longer fragments of DNA compared with healthy apoptotic cells.[22,23] The levels of the circulating mitochondrial DNA are also proposed as a potential biomarker for early tumour detection.[24] Finally, epigenetic alterations including DNA methylation are a common feature of carcinogenesis.[25,26] Especially during hepatocarcinogenesis, HBV can induce global hypomethylation both in hepatic cells and in circulating DNA.[27,28]

In this study, we aimed to evaluate whether different species of serum circulating cfDNA may be involved in the process of hepatocarcinogenesis and could affect the risk of HCC development in CHB patients receiving oral antiviral therapy with an agent of high genetic barrier.