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Diagnostic value of circulating cell-free DNA levels for hepatocellular carcinoma

  • Linlin Yan
    Affiliations
    Department of Infectious Disease, Center for Liver Disease, Peking University First Hospital, No. 8, Xishiku Street, Xicheng District, Beijing 100034, China
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  • Yanhui Chen
    Affiliations
    Institute of Infectious Diseases, Beijing Ditan Hospital, Capital Medical University, Jingshundongjie 8, Beijing 100015, China

    Beijing Key Laboratory of Emerging Infectious Diseases, Beijing 100015, China

    Genecast Precision Medicine Technology Institute, Huayuanbeilu 35, Beijing 100089, China
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  • Jiyuan Zhou
    Affiliations
    Department of Infectious Disease, Center for Liver Disease, Peking University First Hospital, No. 8, Xishiku Street, Xicheng District, Beijing 100034, China
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  • Hong Zhao
    Affiliations
    Department of Infectious Disease, Center for Liver Disease, Peking University First Hospital, No. 8, Xishiku Street, Xicheng District, Beijing 100034, China
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  • Henghui Zhang
    Correspondence
    Corresponding author at: Institute of Infectious Diseases, Beijing Ditan Hospital, Capital Medical University, Jingshundongjie 8, Beijing 100015, China.
    Affiliations
    Institute of Infectious Diseases, Beijing Ditan Hospital, Capital Medical University, Jingshundongjie 8, Beijing 100015, China

    Beijing Key Laboratory of Emerging Infectious Diseases, Beijing 100015, China

    Genecast Precision Medicine Technology Institute, Huayuanbeilu 35, Beijing 100089, China
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  • Guiqiang Wang
    Correspondence
    Corresponding author at: Department of Infectious Disease, Center for Liver Disease, Peking University First Hospital, No. 8, Xishiku Street, Xicheng District, Beijing 100034, China.
    Affiliations
    Department of Infectious Disease, Center for Liver Disease, Peking University First Hospital, No. 8, Xishiku Street, Xicheng District, Beijing 100034, China

    The Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Zhejiang University, Hangzhou, Zhejiang, China
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Open AccessPublished:December 08, 2017DOI:https://doi.org/10.1016/j.ijid.2017.12.002

      Highlights

      • Plasma circulating cell-free DNA (cfDNA) levels are significantly elevated in hepatocellular carcinoma (HCC) patients.
      • Age and cfDNA levels were found to be independently associated with HCC.
      • The combination of cfDNA with age and alpha-fetoprotein performed best in diagnosing HCC.

      Abstract

      Objectives

      Circulating cell-free DNA (cfDNA) is a potential biomarker for tumor diagnosis. Hepatocyte damage is a characteristic component of the pathobiology of hepatocellular carcinoma (HCC), which would be expected to result in substantial leakage of cfDNA into the circulation. However, the diagnostic value of cfDNA levels for HCC remains unclear.

      Methods

      Plasma samples were collected from 24 HCC patients and 62 hepatitis B virus-related liver fibrosis patients. Plasma cfDNA levels were quantified by Qubit method.

      Results

      Plasma cfDNA levels were associated with the degree of liver inflammation, body mass index, and alpha-fetoprotein (AFP) level, but were not associated with fibrosis stages. Plasma cfDNA levels were significantly higher in HCC patients than in non-HCC patients. Multivariate analysis revealed that age and cfDNA, rather than AFP, were independent predictors of HCC. The HCC index, a combination model including age, cfDNA, and AFP, had an area of 0.98 (95% confidence interval 0.92–1.00) under the receiver operating characteristics curve for the diagnosis of HCC at the cut-off value of 0.61, with 87.0% sensitivity and 100% specificity. The diagnostic power of the HCC index was superior to that of cfDNA alone and AFP alone.

      Conclusions

      These results suggest that the combination of cfDNA with age and AFP could improve the diagnostic performance for HCC.

      Keywords

      Introduction

      Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third leading cause of mortality from cancer worldwide, with an estimated 700 000 new cases of HCC globally in 2008 (
      • Ferlay J.
      • Shin H.R.
      • Bray F.
      • Forman D.
      • Mathers C.
      • Parkin D.M.
      Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008.
      ,
      • El-Serag H.B.
      • Rudolph K.L.
      Hepatocellular carcinoma: epidemiology and molecular carcinogenesis.
      ). The highest incidence rates of HCC are found in East and Southeast Asia and Central and West Africa, regions with high hepatitis B virus (HBV) prevalence, and approximately 60–65% of HCC develops with an underlying HBV infection (
      • El-Serag H.B.
      • Rudolph K.L.
      Hepatocellular carcinoma: epidemiology and molecular carcinogenesis.
      ,
      • Pisani P.
      • Parkin D.M.
      • Munoz N.
      • Ferlay J.
      Cancer and infection: estimates of the attributable fraction in 1990.
      ). HBV infection-induced non-controllable inflammation and malignant transformation has brought great clinical challenges.
      Most patients are diagnosed with HCC at late stages, after the best period for surgical resection (
      • Jemal A.
      • Siegel R.
      • Ward E.
      • Hao Y.
      • Xu J.
      • Thun M.J.
      Cancer statistics.
      ,
      • Tanase A.M.
      • Dumitrascu T.
      • Dima S.
      • Grigorie R.
      • Marchio A.
      • Pineau P.
      • et al.
      Influence of hepatitis viruses on clinicopathological profiles and long-term outcome in patients undergoing surgery for hepatocellular carcinoma.
      ). The key to treating HCC and achieving good outcomes is early detection and diagnosis (
      • Llovet J.M.
      • Burroughs A.
      • Bruix J.
      Hepatocellular carcinoma.
      ,
      • Yuen M.F.
      • Cheng C.C.
      • Lauder I.J.
      • Lam S.K.
      • Ooi C.G.
      • Lai C.L.
      Early detection of hepatocellular carcinoma increases the chance of treatment: Hong Kong experience.
      ). Currently, alpha-fetoprotein (AFP), imaging technologies, and histology are the predominant methods for HCC screening (
      • Benson 3rd, A.B.
      • Abrams T.A.
      • Ben-Josef E.
      • Bloomston P.M.
      • Botha J.F.
      • Clary B.M.
      • et al.
      NCCN clinical practice guidelines in oncology: hepatobiliary cancers.
      ). However, the AFP assay lacks adequate sensitivity and specificity in patients without typical AFP concentrations (
      • Farinati F.
      • Marino D.
      • De Giorgio M.
      • Baldan A.
      • Cantarini M.
      • Cursaro C.
      • et al.
      Diagnostic and prognostic role of alpha-fetoprotein in hepatocellular carcinoma: both or neither?.
      ), imaging technologies typically detect only tumors greater than 1 cm in diameter, and tissue biopsy is invasive and may increase the risk of needle metastasis (
      • El-Serag H.B.
      • Marrero J.A.
      • Rudolph L.
      • Reddy K.R.
      Diagnosis and treatment of hepatocellular carcinoma.
      ,
      • Maluccio M.
      • Covey A.
      Recent progress in understanding, diagnosing, and treating hepatocellular carcinoma.
      ). Therefore, non-invasive and more effective biomarkers for HCC are urgently needed.
      Circulating cell-free DNA (cfDNA), an extracellular DNA that is thought to be released into the bloodstream by tumor apoptotic and/or necrotic cells (
      • Choi J.J.
      • Reich 3rd, C.F.
      • Pisetsky D.S.
      Release of DNA from dead and dying lymphocyte and monocyte cell lines in vitro.
      ), has been detected in many types of cancer (
      • Alix-Panabieres C.
      • Schwarzenbach H.
      • Pantel K.
      Circulating tumor cells and circulating tumor DNA.
      ). Changes in the quantity of circulating DNA reflect the tumor burden (
      • Thierry A.R.
      • Mouliere F.
      • Gongora C.
      • Ollier J.
      • Robert B.
      • Ychou M.
      • et al.
      Origin and quantification of circulating DNA in mice with human colorectal cancer xenografts.
      ), and cfDNA methylation alterations are potential biomarkers for the detection of cancer (
      • Wen L.
      • Li J.
      • Guo H.
      • Liu X.
      • Zheng S.
      • Zhang D.
      • et al.
      Genome-scale detection of hypermethylated CpG islands in circulating cell-free DNA of hepatocellular carcinoma patients.
      ,
      • Zhao Y.
      • Xue F.
      • Sun J.
      • Guo S.
      • Zhang H.
      • Qiu B.
      • et al.
      Genome-wide methylation profiling of the different stages of hepatitis B virus-related hepatocellular carcinoma development in plasma cell-free DNA reveals potential biomarkers for early detection and high-risk monitoring of hepatocellular carcinoma.
      ). Furthermore, the levels of cfDNA also reflect non-tumor-specific physiological and pathological processes (
      • Swaminathan R.
      • Butt A.N.
      Circulating nucleic acids in plasma and serum: recent developments.
      ), such as inflammatory diseases or tissue trauma (
      • Fleischhacker M.
      • Schmidt B.
      Circulating nucleic acids (CNAs) and cancer–a survey.
      ). These biological characteristics indicate that cfDNA could be considered for diagnostic application in human diseases, because differential forms of cfDNA are likely to be present in these patients (
      • Schwarzenbach H.
      • Hoon D.S.
      • Pantel K.
      Cell-free nucleic acids as biomarkers in cancer patients.
      ).
      Recent studies have reported the diagnostic efficiency of cfDNA levels for the detection of HCC. Norio et al. indicated that the diagnostic power of cfDNA was superior to that of AFP in hepatitis C virus-related HCC (
      • Iizuka N.
      • Sakaida I.
      • Moribe T.
      • Fujita N.
      • Miura T.
      • Stark M.
      • et al.
      Elevated levels of circulating cell-free DNA in the blood of patients with hepatitis C virus-associated hepatocellular carcinoma.
      ). However, elevated cfDNA levels can be detected in multiple cancers and are not HCC-specific. So, a quantitative analysis of cfDNA only has limited diagnostic value for the detection of HCC. It is proposed that cfDNA in combination with a clinical index, such as AFP, may improve the accuracy of HCC diagnosis.
      Thus, the present study was designed to quantitate the plasma levels of cfDNA in HBV-related liver fibrosis and HCC patients using a simple Qubit method, and to evaluate the diagnostic efficiency of the combined analysis of cfDNA with a clinical index for HCC screening.

      Methods

      Patients and samples

      A total of 86 patients were enrolled in this study, including 62 patients with HBV-related liver fibrosis and 24 patients with HCC. The 62 liver fibrosis patients were selected randomly from the China HepB Related Fibrosis Assessment Research cohort supported by the China Mega-project for Infectious Diseases. Inclusion and exclusion criteria have been described previously (
      • Deng Y.Q.
      • Zhao H.
      • Ma A.L.
      • Zhou J.Y.
      • Xie S.B.
      • Zhang X.Q.
      • et al.
      Selected cytokines serve as potential biomarkers for predicting liver inflammation and fibrosis in chronic hepatitis b patients with normal to mildly elevated aminotransferases.
      ). All of the selected liver fibrosis patients underwent a liver biopsy; the degree of inflammation and the fibrosis stage were assessed according to the Ishak criteria (
      • Ishak K.
      • Baptista A.
      • Bianchi L.
      • Callea F.
      • De Groote J.
      • Gudat F.
      • et al.
      Histological grading and staging of chronic hepatitis.
      ). The detailed clinical trial protocol has been registered at clinicaltrials.gov (NCT01962155) and chictr.org (ChiCTR-DDT-13003724).
      The 24 HCC patients were included from those who had been diagnosed with HCC by imaging and pathological evaluations at the Peking Union Medical College Hospital and Henan Cancer Hospital. The clinical stage of HCC was evaluated on the basis of the Barcelona clinic liver cancer (BCLC) classification system. Clinical and biochemical data were documented. This study was approved by the Ethics Committee of Peking University First Hospital. Written informed consent was obtained from each patient for the use of their clinical data and specimens in research.
      Plasma samples were collected before therapy or surgery. Peripheral blood was collected in ethylenediaminetetraacetic acid (EDTA)-containing tubes and centrifuged at 1600 g for 10 min within 2 h of collection. The supernatants were transferred into new microtubes and centrifuged again at 16 000 g for 10 min to completely remove cell debris. The plasma was immediately stored at −80 °C until use.

      Laboratory tests

      The hematological and biochemical parameters, including platelet count (PLT) and levels of AFP, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), cholinesterase (CHE), and albumin, were tested routinely using standard methods in local hospitals.

      Extraction and quantification of plasma cfDNA

      The MagMAX Cell-Free DNA Isolation Kit (Life Technologies, USA) was used for the isolation of cfDNA from 1 ml of plasma sample. For each sample, the level of extracted cfDNA was quantified using a Qubit dsDNA HS Assay Kit (Life Technologies, USA). All protocols were carried out in accordance with the manufacturer’s instructions.

      Statistical analysis

      Quantitative variables were presented as the mean ± standard deviation (SD) and categorical variables were presented as proportions. The Student t-test and Mann–Whitney U-test were used to analyze differences between two groups of normally and non-normally distributed variables, respectively. The Chi-square test was used for the comparison of categorical variables. The correlation between plasma cfDNA levels and different variables was analyzed by Spearman rank correlation test. A multivariate backward logistic regression analysis was performed to determine the independent variables of HCC. Receiver operating characteristics (ROC) curves were used to assess the discriminative power of cfDNA, AFP, and the HCC index. The predictive performance of variables was expressed as the area under the ROC curve (AUROC), sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV). Statistical analyses were performed using SPSS 16.0 software (SPSS, Inc., Chicago, IL, USA). Values of P< 0.05 were considered statistically significant.

      Results

      Demographic and clinical characteristics of all patients

      A total of 62 HBV-related liver fibrosis patients and 24 HCC patients were enrolled in this study. The general characteristics of the subjects are shown in Table 1. The liver fibrosis patients were significantly younger than the HCC patients (p< 0.0001). The proportion of males was higher than that of females in both groups (74.2% and 79.2%, respectively). The liver fibrosis group and HCC group had statistically different laboratory results for ALT (p = 0.006). In addition, the liver fibrosis group was divided into different inflammation and fibrosis subgroups according to the histological activity index (HAI) and Ishak criteria.
      Table 1Demographic and clinical characteristics of all patients.
      Fibrosis (n = 62)HCC (n = 24)p-Value
      Age, mean ± SD, years38.7 ± 10.157.7 ± 8.7<0.0001
      Male, n (%)46 (74.2)19 (79.2)0.7822
      BMI, ≥24 kg/m2, n (%)19 (31.1)
       Unknown1 (1.6)
      ALT, mean ± SD, U/l122.2 ± 156.884.2 ± 141.30.006
      AFP, n (%)>0.9999
       <20 ng/ml48 (81.4)12 (52.2)
       ≥20 ng/ml11 (18.6)11 (47.8)
       Unknown3 (4.8)1 (4.2)
      HAI, n (%)
       0–425 (40.3)
       5–618 (29.0)
       ≥719 (30.6)
      Fibrosis stage (Ishak), n (%)
       F0–224 (38.7)
       F3–420 (32.3)
       F5–618 (29.0)
      Liver cirrhosis, yes, n (%)13 (56.5)
       Unknown1 (4.2)
      BCLC stage, n (%)
       A14 (58.3)
       B8 (33.3)
       C2 (8.3)
      HCC, hepatocellular carcinoma; SD, standard deviation; BMI, body mass index; ALT, alanine aminotransferase; AFP, alpha fetoprotein; HAI, histological activity index; BCLC: Barcelona clinic liver cancer.

      Associations between plasma cfDNA levels and clinical characteristics

      Among liver fibrosis patients, there was no association between plasma cfDNA levels and fibrosis stage (p = 0.986, rho = 0.002, Table 2). However, plasma cfDNA levels were positively associated with HAI (p = 0.033, rho = 0.271, Table 2). Patients with an HAI score ≥10 (severe inflammation) showed significantly elevated plasma cfDNA levels in comparison to patients with an HAI score <10 (23.31 ± 10.20 ng/ml vs. 14.34 ± 9.16 ng/ml, p = 0.0096, Figure 1a). In addition, plasma cfDNA levels were associated with body mass index (BMI) (p = 0.001, rho = 0.413, Table 2; p = 0.0008, Figure 1b) and AFP (p = 0.028, rho = 0.287, Table 2; p = 0.0016, Figure 1c). The cfDNA levels were significantly higher in plasma from HCC patients than in plasma from liver fibrosis patients (64.0 ± 49.2 ng/ml vs. 15.5 ± 9.7 ng/ml, p< 0.0001, Figure 1d).
      Table 2Associations between plasma cfDNA levels and clinical characteristics in liver fibrosis patients.
      Spearman’s rhop-Value
      Age (years)0.0160.903
      BMI (kg/m2)0.4130.001
      ALT (U/l)0.1590.217
      AST (U/l)0.1670.194
      ALP (U/l)0.1890.147
      CHE (KU/l)0.040.769
      Albumin (g/l)−0.2320.07
      PLT (×109/l)0.1530.236
      AFP (ng/ml)0.2870.028
      HAI0.2710.033
      Fibrosis stages (Ishak criteria)0.0020.986
      cfDNA, circulating cell-free DNA; BMI, body mass index; ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALP, alkaline phosphatase; CHE, cholinesterase; PLT, platelet counts; AFP, alpha fetoprotein; HAI, histological activity index.
      Figure 1
      Figure 1Associations between plasma cfDNA levels and clinical characteristics. The associations between plasma cfDNA levels and (a) histological activity index (HAI), (b) body mass index (BMI; BMI ≥24 kg/m2 is the cut-off point for overweight in China), (c) alpha-fetoprotein (AFP) levels (AFP <20 ng/ml is clinically normal), and (d) hepatocellular carcinoma (HCC).

      cfDNA is an independent predictor of HCC

      For liver fibrosis and HCC patients, age (57.7 ± 8.7 years vs. 38.7 ± 10.1 years, p< 0.0001), AFP (1397.6 ± 2484.2 ng/ml vs. 19.0 ± 44.5 ng/ml, p = 0.021), and plasma cfDNA levels (64.0 ± 49.2 ng/ml vs. 15.5 ± 9.7 ng/ml, p< 0.0001) were significantly higher in HCC patients than in liver fibrosis patients (Table 3). Multivariate analysis revealed that age (p = 0.006) and cfDNA (p = 0.023), but not AFP (p = 0.24), were independent predictors of HCC (Table 3). Therefore, the HCC index − a combination model − was developed to diagnose HCC by backward logistic regression analysis, using age, cfDNA, and AFP as variables: Hx = 0.246 × age + 0.006 × AFP + 0.098 × cfDNA−16.126; HCC index = exp(Hx)/(1 + exp(Hx)).
      Table 3Univariate and multivariate analysis of cfDNA and clinical parameters with fibrosis and HCC.
      Univariate analysisp-ValueMultivariate analysisp-Value
      Fibrosis (n = 62)HCC (n = 24)Exp (B) (95% CI)
      Age, mean ± SD, years38.7 ± 10.157.7 ± 8.7<0.00011.293 (1.076–1.554)0.006
      Male, n (%)46 (74.2%)19 (79.2%)0.7822
      ALT, mean ± SD, U/l122.2 ± 156.884.2 ± 141.30.0060.977 (0.939–1.016)0.238
      AFP, mean ± SD, ng/ml19.0 ± 44.51397.6 ± 2484.20.0211.014 (0.991–1.037)0.24
      cfDNA, mean ± SD, ng/ml15.5 ± 9.764.0 ± 49.2<0.00011.115 (1.015–1.225)0.023
      cfDNA, circulating cell-free DNA; HCC, hepatocellular carcinoma; CI, confidence interval; SD, standard deviation; ALT, alanine aminotransferase; AFP, alpha fetoprotein.

      The diagnostic power of the HCC index is superior to that of cfDNA and AFP

      The HCC index had an AUROC of 0.98 (95% confidence interval 0.92–1.00) for the diagnosis of HCC, with 87.0% sensitivity, 100% specificity, 100% PPV, and 95.2% NPV at the optimal cut-off value of 0.61 (Figure 2a, Table 4). The plasma cfDNA assay had an AUROC of 0.82 (95% confidence interval 0.73–0.90), with 62.5% sensitivity, 93.6% specificity, 79.0% PPV, and 86.6% NPV at the cut-off of 30 ng/ml (Figure 2b, Table 4). AFP had an AUROC of 0.67 (95% confidence interval 0.55–0.77), with 47.8% sensitivity, 93.2% specificity, 73.3% PPV, and 82.1% NPV at the cut-off of 80.5 ng/ml (Figure 2c, Table 4). The HCC index showed the least overlap between liver fibrosis and HCC patients (see Supplementary Material, Figure S1). The diagnostic performance of the HCC index for HCC was superior to that of the cfDNA assay alone (Z statistic = 2.835, p = 0.0046) and AFP assay alone (Z statistic = 4.073, p< 0.0001) (see Supplementary Material, Table S1).
      Figure 2
      Figure 2Receiver operating characteristics curve analysis: (a) hepatocellular carcinoma (HCC) index, (b) cfDNA, and (c) alpha-fetoprotein (AFP) had area under the receiver operating characteristics curve (AUROC) values of 0.98 (95% confidence interval 0.92–1.00), 0.82 (95% confidence interval 0.73–0.90), and 0.67 (95% confidence interval 0.55–0.77), respectively, in discriminating liver fibrosis and HCC patients.
      Table 4Area under the receiver operating characteristics curve of HCC index, cfDNA, and AFP for HCC screening.
      HCC indexcfDNAAFP
      AUROC (95% CI)0.98 (0.92–1.00)0.82 (0.73–0.90)0.67 (0.55–0.77)
      Accuracy (%)96.384.980.5
      Cut-off value0.613048.8
      Sensitivity (%)87.062.547.8
      Specificity (%)100.093.693.2
      PPV (%)100.079.073.3
      NPV (%)95.286.682.1
      HCC, hepatocellular carcinoma; cfDNA, circulating cell-free DNA; AFP, alpha fetoprotein; AUROC, area under the receiver operating characteristics curve; CI, confidence interval; PPV, positive predictive value; NPV, negative predictive value.

      Discussion

      Hepatocellular carcinoma (HCC) develops mainly from underlying chronic liver diseases such as chronic hepatitis B (CHB), fibrosis, or cirrhosis. Patients would benefit from the identification of HCC at an early stage, when treatment can significantly reverse the progress of disease and prolong the survival time. It is therefore of importance to identify biomarkers that will enable the early detection of HCC. However, current HCC screening methods such as AFP present unsatisfactory performance due to the lack of sensitivity and specificity (
      • Farinati F.
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      • De Giorgio M.
      • Baldan A.
      • Cantarini M.
      • Cursaro C.
      • et al.
      Diagnostic and prognostic role of alpha-fetoprotein in hepatocellular carcinoma: both or neither?.
      ). Given the higher levels of cfDNA in cancers, including HCC (
      • Alix-Panabieres C.
      • Schwarzenbach H.
      • Pantel K.
      Circulating tumor cells and circulating tumor DNA.
      ,
      • Huang Z.H.
      • Li L.H.
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      Quantitative analysis of plasma circulating DNA at diagnosis and during follow-up of breast cancer patients.
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      ), it was proposed that cfDNA in combination with another clinical index may be a reliable approach for cancer screening. The present study examined the relationship between cfDNA levels and clinical characteristics in patients with HBV-related liver fibrosis and assessed the diagnostic value of the cfDNA combination model for HCC.
      Increasing evidence has demonstrated the relationship between cfDNA and inflammatory diseases (
      • Atamaniuk J.
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      • Fodinger M.
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      Analysing cell-free plasma DNA and SLE disease activity.
      ,
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      Apoptotic cell-free DNA promotes inflammation in haemodialysis patients.
      ,
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      • Carpenter S.B.
      • Keogh L.
      • Doyle B.
      • Martin C.
      • et al.
      TLR9 provokes inflammation in response to fetal DNA: mechanism for fetal loss in preterm birth and preeclampsia.
      ). The cytotoxic T-lymphocyte-mediated inflammatory process is central to resisting HBV infection, which also results in hepatocyte injury (
      • Chisari F.V.
      • Ferrari C.
      Hepatitis B virus immunopathogenesis.
      ). The present study found that cfDNA levels were positively associated with HAI, but not associated with ALT, AST, ALP, or viral load (data not shown, r = −0.160, p = 0.214), which suggests that immune-induced hepatocyte injury might contribute to the shedding of cfDNA into the bloodstream, although the underlying mechanism remains to be determined in further research.
      In this study, it was found that plasma cfDNA levels were positively correlated with BMI in liver fibrosis patients. Previous studies have suggested that obesity disrupts the balance of the adipose tissue microenvironment, causing apoptosis and/or necrosis of adipocytes (
      • Cinti S.
      • Mitchell G.
      • Barbatelli G.
      • Murano I.
      • Ceresi E.
      • Faloia E.
      • et al.
      Adipocyte death defines macrophage localization and function in adipose tissue of obese mice and humans.
      ,
      • Murano I.
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      • Castellucci M.
      • et al.
      Dead adipocytes, detected as crown-like structures, are prevalent in visceral fat depots of genetically obese mice.
      ) and subsequently increasing the release of cfDNA into the circulation (
      • Nishimoto S.
      • Fukuda D.
      • Higashikuni Y.
      • Tanaka K.
      • Hirata Y.
      • Murata C.
      • et al.
      Obesity-induced DNA released from adipocytes stimulates chronic adipose tissue inflammation and insulin resistance.
      ). Obesity-induced cfDNA release is supported by the present study finding that the increase in plasma cfDNA levels was associated with BMI. In addition, results of the correlation test also revealed that cfDNA levels were associated with AFP levels, suggesting that the increase in plasma cfDNA and AFP levels might have additive effects. Therefore, it was hypothesized that the combination of cfDNA and AFP might improve the diagnostic efficiency for HCC, which was also confirmed by the results of the HCC index in this study.
      It was found in this study that cfDNA levels were significantly higher in HCC patients than in liver fibrosis patients. Multivariate analysis revealed that age and cfDNA, rather than AFP, were independent predictors of HCC. The HCC index as a combination biomarker model including age, cfDNA, and AFP was constructed given the positive correlation between cfDNA and AFP. This index presented the optimal performance for HCC screening, with the least overlap between liver fibrosis and HCC patients (see Supplementary Material, Figure S1). The diagnostic parameters were AUROC 0.98, sensitivity 87.0%, and specificity 100%. The AUROC for the cfDNA assay alone was 0.82 and for the AFP assay alone was 0.67; these values are in line with those of previous studies (
      • Ren N.
      • Qin L.X.
      • Tu H.
      • Liu Y.K.
      • Zhang B.H.
      • Tang Z.Y.
      The prognostic value of circulating plasma DNA level and its allelic imbalance on chromosome 8p in patients with hepatocellular carcinoma.
      ,
      • Piciocchi M.
      • Cardin R.
      • Vitale A.
      • Vanin V.
      • Giacomin A.
      • Pozzan C.
      • et al.
      Circulating free DNA in the progression of liver damage to hepatocellular carcinoma.
      ). Unfortunately the sensitivity of the cfDNA assay alone remains relatively low, hence the independent application of cfDNA is not recommended. However, the diagnostic performance can be improved when cfDNA is used in combination with age and AFP. The HCC index gives support to the clinical application of cfDNA in combination with a clinical index for the diagnosis of HCC, which may be more accurate and cost-effective.
      In conclusion, plasma levels of cfDNA were elevated not only in HCC patients, but also in liver fibrosis patients with severe inflammation, suggesting that an increased cfDNA level is not specific for a defined stage of the HCC process. Therefore, cfDNA as a single biomarker is not sufficiently robust for HCC screening. However, the findings of the present study support cfDNA as a valuable marker in HCC screening when used in combination with age and AFP. These findings provide new insights into the application of cfDNA combination biomarker models for the diagnosis of HCC. As the number of cases included in this study was small, additional investigations should be performed to confirm the results.

      Ethical approval

      This study was approved by the local ethics committee of Peking University First Hospital.

      Conflict of interest

      No conflicts of interest are declared by any of the authors.

      Acknowledgements

      This work was supported by the China Mega-Project for Infectious Diseases (grant numbers 2013ZX10002005 , 2012ZX10002006 , 2013ZX10002004 , and 2012ZX10005005 ) and the Project of Beijing Science and Technology Committee (grant number D121100003912002 ). The authors thank the members of the China HepB Related Fibrosis Assessment Research Group for assisting with patient inclusion and data acquisition. The authors thank Xinyuan Zhang, Wanning Yang, and Xiaoxi Pan at GeneCrab Biotechnology Co., Ltd for their expert technical assistance.

      Appendix A. Supplementary data

      The following is Supplementary data to this article:

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