Liquid Liver Biopsy for Disease Diagnosis and Prognosis

  • Desislava K. Tsoneva1,2,
  • Martin N. Ivanov2,3 and
  • Manlio Vinciguerra2,4,* 
 Author information
Journal of Clinical and Translational Hepatology 2023;11(7):1520-1541

DOI: 10.14218/JCTH.2023.00040


Liver diseases are a major burden worldwide, the scope of which is expected to further grow in the upcoming years. Clinically relevant liver dysfunction-related blood markers such as alanine aminotransferase and aspartate aminotransferase have limited accuracy. Nowadays, liver biopsy remains the gold standard for several liver-related pathologies, posing a risk of complication due to its invasive nature. Liquid biopsy is a minimally invasive approach, which has shown substantial potential in the diagnosis, prognosis, and monitoring of liver diseases by detecting disease-associated particles such as proteins and RNA molecules in biological fluids. Histones are the core components of the nucleosomes, regulating essential cellular processes, including gene expression and DNA repair. Following cell death or activation of immune cells, histones are released in the extracellular space and can be detected in circulation. Histones are stable in circulation, have a long half-life, and retain their post-translational modifications. Here, we provide an overview of the current research on histone-mediated liquid biopsy methods for liver diseases, with a focus on the most common detection methods.


Liquid biopsy, Histones, NAFLD/MAFLD, Chronic liver disease, Hepatocellular carcinoma


Chronic liver disease (CLD)

CLD is a clinical entity involving a process of progressive destruction and regeneration of the liver parenchyma. CLD refers to a hepatic disease that lasts over six months, and the related deteriorations include the decreased synthesis of clotting factors and other proteins, detoxification of harmful products of metabolism, and excretion of bile.1 Accordingly, CLD consists of a wide range of liver pathologies which include inflammation, liver cirrhosis, and hepatocellular carcinoma (HCC).1 CLD can be caused by viral infection, including hepatitis B and C, by alcohol misuse as in alcoholic fatty liver disease (AFLD), or by obesity/excessive nutrient intake as in nonalcoholic fatty liver disease (NAFLD). NAFLD is present when > 5% of hepatocytes are steatotic in patients who do not consume excessive alcohol consumption (< 20 g/day for women and < 30 g/day for men) and ranges in severity from simple steatosis (fat without significant hepatic inflammation or hepatocellular injury) to steatohepatitis (fat with hepatocellular injury and hepatic inflammation), through to advanced fibrosis and cirrhosis, which is a risk factor for HCC.2 AFLD broadly consists of three stages, each increasing in severity. Drinking a large amount of alcohol, even for just a few days, can lead to simple steatosis. Most individuals consuming > 40 g/day of alcohol per day develop simple steatosis; however, only a subset of individuals will develop more advanced disease over a longer period. Between 10–35% of individuals with alcohol-related steatosis who continue drinking heavily will develop it.3 The third stage of alcohol-related liver disease is cirrhosis, where healthy liver tissue has been replaced permanently by scar tissue, involving up to one in every five long-term heavy drinkers who will develop alcohol-related liver cirrhosis.3 AFLD also increases the risk of developing HCC. Chronic alcohol use of more than 80 g/day for more than 10 years increases the risk of HCC by approximately five-fold.4,5 In many patients, AFLD, NAFLD, and hepatitis infection can coexist and synergize to worsen CLD.5,6

The societal burden and the epidemiological figures of CLD are tremendous. An estimated 1.5 billion persons have CLD worldwide, with increasing prevalence and mortality.7 Cirrhosis is the eleventh leading cause of death, accounting for ∼2% of deaths in 2016.8 According to the results from the Global Burden of Diseases, Injuries, and Risk Factors Study 2017 on the burden of cirrhosis and its trends since 1990, by cause, sex, and age, for 195 countries and territories, CLD caused ∼1.32 million deaths in 2017, with approximately two-thirds inmen.9 In the last three decades, the epidemiology of CLD has changed,10 reflecting the implementation of large-scale hepatitis B vaccination and hepatitis C treatment programs, and CLD is nowadays more often the result of the increasing prevalence of the obesity- and/or metabolic syndrome-associated NAFLD, and increasing alcohol misuse, triggering AFLD.

As CLD is a leading cause of years of working-life loss, second only to ischemic heart disease in Europe, increasing attention has been given to policy actions for marketing, pricing, and taxation of alcohol and unhealthy foods.11


AFLD and NAFLD are CLDs that have similar pathological spectra, ranging from simple hepatic steatosis to steatohepatitis, liver cirrhosis, and HCC (Fig. 1A). In their initial to advanced stages, they are both characterized by the presence of hepatic steatosis, hepatocellular ballooning, and lobular inflammation with or without fibrosis. They are both frequently accompanied by extrahepatic complications, including cardiovascular disease, metabolic syndrome, and type 2 diabetes (T2DM). Among chronic drinkers and obese patients, about 35% and 65–85% develop CLD, respectively.12,13 Since the 1980s, there has been a tendency to separate NAFLD and AFLD as clinical entities. In particular, it was defined that NAFLD develops in the absence of known factors that cause fat accumulation such as alcohol consumption defined as < 30 g/day in men and < 20 g/day in women, viral liver disease, and hereditary disorders.14 Instead, AFLD is caused by heavy chronic alcohol consumption, defined as consumption of more than three standard drinks per day in men, and more than two drinks per day in women, or binge drinking defined as more than five standard drinks in men and more than four in women in a 2 h period.14 Upon liver biopsy, only two subtle histological changes may help to distinguish AFLD from NAFLD, as they are present in AFLD but not in NAFLD: (1) pronounced cholestasis (reduced or stopped bile flow), which is usually indicative of acute decompensation (hepatocyte keratin 7 immunostaining, in this case, becomes more intense), and (2) sclerosing hyaline necrosis, in which the central vein is almost completely obliterated.15 However, these features are not universal, and there is otherwise a substantial overlap in the histological findings between AFLD and NAFLD patients; in the absence of knowledge about the clinical history indicating the nature of the patient’s disease, the histology cannot be decisive for the diagnostic process. At the molecular level, AFLD and NAFLD implicate the involvement of common but also distinct cell signaling mechanisms in the liver parenchyma. A study from Sookoian et al.16 employing integrated omics and system biology approaches cross-comparing enrichment analyses showed that NAFLD is associated with pathways that include insulin signaling, caspases, and mitochondrial-related apoptosis, stress induction of heat shock proteins, cellular proliferation, hypoxia induction, and protein associated with epigenetic regulation. Conversely, AFLD is associated with a more reduced network of disease pathways, mostly focused on modulation of the immune response, toll-like receptor signaling, and cytokines.16 The same study postulated more pervasive systemic complications of NAFLD vs. AFLD, in particular at the cardiovascular level.16 A more recent comprehensive literature review indicated that NAFLD is regulated by the Nrf2/FXR/LXRα/RXR/SREBP-1c, PI3K/AKT/SREBP-1c, AMP-activated protein kinase (AMPK)/Sarcoendoplasmic reticulum Ca2+-ATPase 2b (SERCA2b), LILRB4/SHP1/TRAF6/NF-κB/MAPK, TXNIP/NLRP3, and TAZ/Ihh signaling pathways, while AFLD is regulated by the SIRT1/AMPK/Lipin-1, PI3K/AKT/Nrf2/PPARγ, p62/Nrf2/KEAP1, STING-IRF3-Bax, C3/CYP2E1/Gly-tRF/SIRT1 and LRP6/Wnt/β-catenin/CYP2E1 signaling pathways, respectively.17 Despite the molecular differences revealed by the mechanistic studies, it is clear that NAFLD and AFLD more often coexist and have overlapping physiopathology. This underscored the need for a change in nomenclature to account for the dual etiology of CLD, which is present in a likely significant proportion of patients with concurrent alcohol consumption and metabolic disturbances. Accordingly, in recent years, NAFLD was re-defined by experts from the European Liver Patients’ Association as metabolic (dysfunction)-associated fatty liver disease, or MAFLD, a more appropriate nomenclature encompassing clinical features independent of alcohol consumption: hepatic steatosis, in addition to one of the following three criteria, namely overweight/obesity, presence of T2DM, or evidence of metabolic dysregulation.18,19 Results from the large NHANES III study demonstrated that MAFLD has a closer association to all-cause and cause-specific mortality, compared with NAFLD, because it excluded participants with lower mortality risk and included participants with higher risk.20 In turn, MAFLD may coexist with other types of CLD, such as viral hepatitis. The relationship and the synergy between these two disease entities are outside the scope of this review and it has been summarized elsewhere.21

Liquid biopsies in CLD.
Fig. 1  Liquid biopsies in CLD.

(A) Mechanisms of NAFLD. NAFLD progression is marked by: the accumulation of fatty liver (steatosis, illustrated by fat droplets accumulation in the cells and increased collagen production in the extracellular space), inflammation (steatohepatitis, activated immune cells are shown in light gray); cirrhosis (necrosis, necrotic cells are shown in dark gray); HCC with cancer cells shown in green). (B) Liquid biopsy types reported for the respective stages indicated above. (C) Liquid biopsy analytes, acquired by peripheral blood collection, fractionation, and subsequent extraction. CTC, circulating tumor cells; cfDNA, cell-free DNA; circRNA, cicular RNA; lncRNA, long noncoding RNA; miRNA, microRNA. Obtained with BioRender software.

As mentioned, in a minority of NAFLD and AFLD (or MAFLD) patients, there is a progression to steatohepatitis, fibrosis, and ultimately HCC and liver failure (Fig. 1A). Steatohepatitis can begin to be symptomatic and patients may complain of fatigue, malaise, and dull right-upper-quadrant abdominal discomfort. The molecular mechanisms that combine to define the transition to steatohepatitis and progressive disease are complex.22 It is increasingly appreciated that this transition is multifactorial and in addition to genetic factors, alcohol or fat-induced hepatocyte damage, reactive oxygen species, and gut-derived microbial components result in steatosis and inflammatory cell (macrophage and neutrophil leukocyte) recruitment and activation in the liver, as reviewed elsewhere.23–26

There are still not many options in terms of Food and Drug Administration (FDA)-approved pharmacological or nutritional therapies for treating patients with MAFLD. For MAFLD patients, intake of pioglitazone, vitamin E, and abstinence from alcohol consumption are beneficial.27 Also, weight loss and healthy nutrition are common therapeutic strategies.28 In some instances, bariatric surgery may be recommended to achieve and maintain the necessary extent of weight loss required for therapeutic effect.29 Obeticholic acid , identified in 1999 and originally approved for the treatment of primary biliary cholangitis , is the only FDA-approved treatment for steatohepatitis.27 Obeticholic acid is a semisynthetic bile acid analog that has the chemical structure of 6α-ethyl-chenodeoxycholic acid. It is a FXR agonist which regulates the expression of transcription factors that reduce bile acid synthesis and hepatic steatosis, and its anti-inflammatory and anti-fibrotic properties have been demonstrated in several clinical trials.27 Many drugs are in various stages of research, but only a few have currently entered phases II and III, such as FGF-21, PPAR agonists, GLP-1 receptor agonists, THR-β agonists.30,31


Liver cirrhosis is highly prevalent worldwide. Its prevalence is increasing in middle-high developed regions and Eastern Europe, while it is decreasing in low-developed regions and Western Sub-Saharan Africa, as ranked by the sociodemographic index.32 It is an end-stage liver disease, where impaired liver function is caused by the formation of fibrosis consequent to damage.33 The latter can be in turn a consequence of different causes, such as obesity, MAFLD, high alcohol consumption, viral hepatitis B (HBV) and/or viral hepatitis C (HCV) infection, autoimmune diseases, cholestatic diseases, and iron or copper overload. Liver cirrhosis typically develops slowly over months or years, and over a long period of inflammation resulting in the substitution of the healthy liver parenchyma with fibrotic tissue and regenerative nodules, in turn, inducing portal hypertension.34 Liver cirrhosis evolves from an asymptomatic phase (compensated) to a symptomatic phase (decompensated). As cirrhosis worsens, symptoms may include itchiness, swelling in the lower legs, fluid build-up in the abdomen that can become spontaneously infected, jaundice, development of spider-like blood vessels in the skin, and dilated veins in the esophagus, stomach, or intestines. More serious complications, resulting in hospitalization, impaired quality of life, and high mortality, include hepatic encephalopathy and HCC. Notably, HCC is the leading cause of death in cirrhotic patients, with a yearly incidence of up to 6%.35 The severity of liver cirrhosis was commonly classified with the Child-Pugh score, which was used for a long time to determine patients who are candidates for liver transplantation, before being replaced by the European Foundation for the Study of Chronic Liver Failure Organ Failure score and by the Model for End-Stage Liver Disease in previous decades.36,37


Liver cancer is one of the most aggressive types of cancer, characterized by a lower than 20% 5-year overall survival rate and increasing disease burden and death incidence. HCC is by far the most common liver malignancy, representing approximately 90% of all cases. Among the most significant risk factors for the development of HCC are cirrhosis, HBV and HCV, chronic alcoholism, and NAFLD.38 There are several scoring systems developed to measure the risk of HCC development. However, these systems lack universal acceptance due to the variability of HCC etiology between geographic regions.39,40 HCC can be generally subdivided into two main classes: proliferative and nonproliferative, which exert distinct clinical features.41 Tumor burden/HCC staging is generally defined by the number and the size of nodules, the presence of vascular invasion, and extrahepatic spread.42,43 Clinical management includes surgical therapies, tumor ablation, transarterial therapies, and systemic therapies.44 HCC is usually diagnosed at an advanced stage when the disease has already spread further than the liver. The multikinase inhibitor Sorafenib was the first FDA-approved systemic drug to treat HCC and is currently a standard first-line therapy.45,46 Nevertheless, the increasing occurrence of drug resistance to Sorafenib in HCC patients47 and the generally very low numbers of targetable somatic mutations in HCC48 indicate the need for novel therapeutic strategies development and early detection biomarker identification. The poor prognosis of HCC has been linked to diagnostic delays,49 and failure to identify high-risk individuals owing to inadequate early detection screening methods.50,51

CLD diagnostic approaches

Patients with simple steatosis are considered at low risk of disease progression. Therefore, NAFLD/MAFLD detection at an early-stage is of utmost importance for appropriate preventive strategies. Noninvasive diagnostic tools are becoming increasingly significant for NAFLD staging and severity assessment: the European Association for the Study of the Liver (EASL);52 the American Association for the Study of Liver Disease (AASLD) and the Asian Pacific Association for the Study of the Liver (APASL) produced recent guidelines in this regard.53–55 In general, the diagnosis of NAFLD currently requires (1) evidence of hepatic steatosis by imaging or histology, (2) no significant alcohol consumption, (3) no competing causes of hepatic steatosis, and (4) no coexisting causes of CLD. Consistently, with the adoption of MAFLD new nomenclature, clinical practice guidelines have been debated by the APASL on MAFLD and have been recently produced by EASL, AASLD, and APASL.18,56,57 Although overall many similarities exist across guidelines, there are several key areas of guidelines from Europe, Asia, and the USA, including the definition of alcohol consumption, screening, fibrosis assessment, lifestyle intervention, and pharmacological intervention of MAFLD.53 Regardless, current imaging approaches present disadvantages. For instance, ultrasound is insensitive to mild steatosis.31 Computed tomography has low sensitivity and specificity, combined with patient exposure to ionizing radiation.31 Magnetic Resonance Imaging approaches have shown promising results in steatosis/nonalcoholicsteatohepatitis (NASH) detection.58 However, the requirement for trained operators, the high cost, and the inspection time limit their screening application in the clinic. The progression of simple steatosis to NASH poses an increased risk of the development of fibrosis, cirrhosis, and HCC,59,60 indicating the need for accurate methods for steatosis–NASH differentiation. To distinguish NASH from steatosis, current guidelines indicate the necessity of liver biopsy to confirm NASH diagnosis, severity grade, and the level of fibrosis.52,54,55 However, liver biopsies are inconvenient due to the invasiveness of the procedure, and they have limited heterogeneity assessment of the tissue sample since they represent a tiny fraction of the liver parenchyma.61 Current noninvasive diagnostic approaches for assessment of fibrotic stage and disease progression to cirrhosis aregenerally based on tissue stiffness quantification by tissue elastographyapproach, as recommended by the EASL; the AASLD and the APASL.62,63 However, tissue elastography approaches are marked by several drawbacks.64 Finally, HCC surveillance, in cirrhotic or noncirrhotic patients, currently remains an unmet need.65 In general, histopathological diagnosis upon liver biopsy remains a mainstay according to EASL,66 AASLD,67 and APASL guidelines.68 Noninvasive imaging strategies, such as Computed Tomography or Magnetic Resonance Imaging should be used first; ultrasound and FDG PET-scan, have limited sensitivity in particular for early-stage HCC.51,66 Therefore, there is an increasing interest in the identification of circulating HCC biomarkers for early detection.

Liquid Biopsy for Liver-Related Diseases

Liquid biopsy represents a minimally invasive, convenient, and cost-effective method of molecular diagnosis that can provide comprehensive information on the molecular landscapes of liver diseases and can represent approaches to overcome tumor heterogeneity and monitor them in real-time (Fig. 1B, C). A detailed description of the histone/nucleosome-independent liquid biopsy types is beyond the scope of the current review and is therefore briefly discussed below.

Circulating DNA

Cell-free DNA (cfDNA) fragments are shed into the circulation from dead cells both in healthy and diseased individuals (Table 1).69–97 However, a much larger amount of cfDNA is normally detected in cancer patients.98–100 Studies have found that cfDNA that originates from tumors, referred to as circulating tumor DNA (ctDNA) is notably shorter, compared with nonmutated cfDNA,69,101,102 and could be used to enrich tumor-derived fragments for further analysis on genomic and epigenomic level. Furthermore, targeted sequencing of ctDNA can detect tumor-associated mutations with high sensitivity, in cases without prior knowledge of their presence in the tumor tissue, indicating strong application for cancer diagnostics and patient stratification for targeted therapy.70,71,103 Positive ctDNA detection and gene analysis in HCC patients prior to surgery was also associated with an increased risk of early recurrence and extrahepatic metastasis.72 Furthermore, continuous assessment of ctDNA could inform on therapy response and disease progression.72,73 Study of cfDNA in liquid biopsies is highly focused on cancer diagnosis, therapy response, and prognosis. However, cfDNA has also shown promising results in NAFLD patient stratification and disease severity. Specifically, levels of 90bp and 222bp cfDNA fragments in the plasma of NAFLD patients, diagnosed with fatty liver, inflammation, and liver stiffness were significantly elevated, compared with healthy individuals, and correlated with disease severity.74 Similarly, levels of methylated cfDNA are significantly higher in nonfibrotic NAFLD patients, compared with NAFLD patients with confirmed fibrosis.75,76 Fibrosis was indicated as the strongest predictor of NAFLD progression and NAFLD-related mortality.104,105 Therefore, circulating cfDNA methylation may be a biomarker for therapy response and stratification of NAFLD patients at high risk of disease progression. However, despite the undeniable value of cfDNA in approaching liver-related disease, cfDNA has a short half-life106 and requires genetic differences to distinguish tumor-derived material.

Table 1

cfDNA in liver diseases

Liver-related pathologyTargetMethod of detectionLevel/resultSuggested functionReference
Pediatric NAFLDcfDNA methylationMethylamp global DNA methylation quantification↑ in NASH patients, compared with HC and NAFLD children without NASH, positive correlation with histological traitsRisk assessment, diagnosis77
NAFLDTotal cfDNA, gene-coding cfDNA, Alu repeat sequences, mitochondrial DNA copies, 5-methyl-2′-deoxycytidine in serumqPCR, RNAseP detection, ELISA↓ cfDNA and RNAse P coding DNA levels, ↑ levels of 5-methyl-2′-deoxycytidine in cirrhotic vs. noncirrhotic patientsSurveillance,monitoring disease progression78
Fibrosis in NAFLD and AFLDMethylation of the PPARγ gene promoter in plasmaPyrosequencingHypermethylation reflects a signature related to severe fibrosisDifferentiation of fibrosis stage75
Fibrosis in NAFLDcfDNA methylationDNA methylation array↑ in patients with nonsignificant fibrosis, compared with significant fibrosisDifferentiation of fibrosis stage76
NAFLDcfDNA levelsqPCR90bp cfDNA ↑ in NAFLD patients, compared with HC, correlation with disease severityDetection, staging74
Chronic Hepatitis C, PEG-IFN-alpha and ribavirin treatmentSerum cfDNA: Methylation of SOCS-1 promoter regionQuantitative methylation-specific polymerase chain reaction↑ SOCS-1 methylation post-treatment associated with better sustained virologic responseMonitoring of treatment response79
Liver cirrhosisPlasma circulating cfDNA, quantification and sequencingSomatic mutation analysis by NGS20 unique variants, including single nucleotide variation s or insertions and deletionsEarly detection80
Primary HCC in NAFLD patientsTERT mutation in serum cfDNAWild-type blocking polymerase chain reaction, Sanger sequencingPositivityEarly detection81
Nonviral liver cancer with fatty liver diseaseTERT C228TWild-type blocking polymerase chain reaction, Sanger sequencingPositivity rate ↑ compared with HBV and HCVEarly detection82
HCCPlasma cfDNA – size profileChromosome arm-level z-score analysis, qPCRShorter DNA fragments associated with tumor-associated copy number alterations,↑ mtDNA in HCC patients, compared with HC, HBV, and cirrhotic patientsDetection, profiling, monitoring69
HCCPlasma cfDNADeep sequencingDetection of somatic mutations in HCC-associated genesDetection, profiling, monitoring70
HCCPlasma cfDNATargeted sequencing with ultra-high coverage and molecular barcodingDetection of oncogenic mutations in HCCDetection, profiling, monitoring, prognosis71
Recurrent liver cancer post hepatectomy or liver transplantationPlasma cfDNAWhole-genome sequencing, Sanger sequencing, PCR, qPCR↑ ctDNA with disease progression, ctDNA as microscopic vascular invasion predictorDetection, profiling, monitoring, prognosis72
HCCUltra-deep sequencing, Droplet digital PCR of TERT promoter in cfDNASequencing, Droplet digital PCRMutation profiles predicting prognosis and therapy responseProfiling, monitoring, therapy response, prognosis73
HCCPlasma circulating Cell-free DNA integrityqPCR↓ cfDNA integrity than those with benign diseases and HCDiagnosis and surveillance83
HCCPlasma circulating cfDNA, quantification and sequencingSomatic mutation analysis by NGS↑ cfDNA levels post-therapy, associated with disease progression, 28 variants identified, including single nucleotide variationsor insertions and deletionsPrognosis, therapy response prediction84
HCCMethylation profiling of cfDNA, based on 2,321 tissue-based differentially methylated blocksDNA bisulfite sequencingMultilayer HCC screening distinguishing early-stage HCC from HC, asymptomatic Hepatitis B surface antigen + or cirrhotic patientsEarly detection85
HCCSerum cfDNA: HCCS1 promoter hypermethylationMethylation-specific polymerase chain reaction↑ HCCS1 methylation in HCC, compared with HC and CHB patients, positive correlation with tumor node metastasis stageDiagnosis, prognosis86
HCCSomatic copy number alterations in cfDNALow-pass sequencing1q+ and 8q+ : significantly associated with early cancer or high-grade dysplasiaDiagnosis, early detection, surveillance87
HCCCfDNANGS technology to acquire genome-wide 5-hmc,Nucleosome footprint, 5′ end motif and fragmentation profilesDistinct patterns/ landscapes compared with HC, Combining all four methods differentiated differentiate HCC from LCDiagnosis, surveillance88
HCCHCC-specific methylation marker panel, developed based on HCC tissueBisulfite sequencing, molecular-inversion (padlock) probes,prognostic prediction model401 selected markers discriminating HCC patients from HC and individuals with HBV/HCV infection, or fatty liverDetermine tumor burden, therapy response, and HCC stage, prognosis89
HCC in cirrhotic patientsPlasma cfDNAHCC blood test (epigenomics AG), DNA methylation panel established by NGSmSEPT9 positivity, methylation biomarker panelEarly detection, HCC surveillance90
HCC in cirrhotic patientscfDNA methylation profile of p16, SFRP1, LINE1Bisulfite modification, multiplex methylated PCRMethylation of p16, SFRP1, LINE1, and an overall increase in the number of aberrantly-methylated genes was associated with HCC developmentSurveillance91
HCC in HBV-diagnosed patientscfDNA methylationLow-pass whole-genome bisulfite sequencingcfDNA in intergenic and repeat regions, hypomethylation nearby HBV integration sites differentiates HCC patients from hepatic and cirrhotic patientsEarly detection, detecting minimal tumoral residual disease after surgical resection92
HCC, average/highrisk for HCC (viral hepatitis, cirrhosis)cfDNA fragmentationWhole-genome sequencingcfDNA fragmentation changes, exhibiting liver cancer-related genomic and chromatin alterationsHCC detection93
HCCcfDNA methylationDNA methylation datasets processing, machine learning modelMethylation signatures differentiated HCC patients from HC and patients with cirrhosis or with other types of cancers such as colorectal and breastDetection94
HCC and CHBcfDNA fragmentationNGSSomatic mutations, tumor-associated preferred DNA endsDiagnosis95
HBVcfDNA quantificationDuplex PCR↑ in HBV patients, in combination with other markers differentiate inflammation severityDiagnosis, assessment of liver injury96
Liver transplantationcfDNA quantificationqPCR↑ cfDNA levels post-transplantation associated with portal hepatitis and systemic inflammationPrognosis97

Circulating noncoding RNAs:long noncoding RNA(lncRNA), micro RNA(miRNA), and circular RNA(circRNA)

NcRNAs are endogenous RNA transcripts that do not code for a protein product. NcRNAs are highly abundant and relatively stable signaling molecules, crucial for gene expression regulation.107 The different types of ncRNAs use distinct regulatory mechanisms: lncRNAs are linear transcripts of > 200 nucleotides, which can function in cis or trans as signals, guides, decoys, scaffolds, or enhancers.108 miRNAs are the most abundant ncRNAs, exerting their inhibitory effect on mRNA stability and translation initiation by binding to DNA, RNA, and proteins.109,110 circRNAs is a covalently closed RNA that is produced by exon skipping or back-splicing of a precursor mRNA. circRNAs modulate various mechanisms such as transcription and translation by acting as transcriptional regulators, protein/RNA sponges, and templates.111 Research on circRNAs is limited. However, due to their closed conformation, circRNAs are highly stable, suggesting promising blood biomarker properties.111 NcRNAs are often deregulated and aberrantly expressed in liver diseases and in many cases are suggested to contribute to disease pathogenesis and disease progression.112,113

ncRNAs present in plasma or serum have shown promising results in distinguishing NAFLD and chronic hepatitis C (CHC)patients from healthy individuals (Table 2).114–134 Particularly, liver fibrosis was characterized by a distinct miRNA, lncRNA, and circRNA profile in circulation, correlating with staging.114–116,135 Furthermore, specific ncRNAs were shown to differentiate between patients with simple steatosis and NASH.115,117 Similarly, GAS5 lncRNA levels in plasma are decreased in patients with cirrhosis, compared with patients with advanced fibrosis.116 Changes in the levels of circulating miRNA have also been highly studied as potential HCC biomarkers.136 As an example, circulating miRNA-16 levels were decreased in the circulation of HCC patients and correlated with tumor size, and other clinical parameters.104,105 Importantly, circulating miRNA-16 was elevated in NAFLD and HCV patients,114 rendering miRNA-16 as a potential biomarker for disease progression. Similarly, plasma miRNA-21 expression was significantly elevated in HCC patients, discriminating HCC patients from healthy individuals and HCC patients from chronic hepatitis. Furthermore, levels of circulating miRNA-21 were decreased following tumor resection and correlated with post-operative tumor recurrence.118 While individual miRNAs have shown promising results, creating panels of several circulating miRNAs/lncRNAs is likely more robust, as indicated for HCC even in early-stage patients,119 cirrhosis, and acute liver failure (ACLF) (Table 2).120

Table 2

Circulating noncoding RNAs in liver diseases

Liver-related pathologyTargetMethod of detectionLevel/resultSuggested functionReference
NAFLDSerum miRNA-34a and miRNA-122qPCRHigher levels of miRNA-34a and miRNA-122 in NAFLD patients, compared with HCSurveillance, early detection121
NAFLDPostulated 18 different serum miRNAsSerum RNAseq analysismiRNA-192, -27b, -22, -197, and -30c were associated with NAFLD severity, but not with drug-induced liver injuryDetection, diagnosis122
Steatosis/NASHCirculating miRNAs in serummiRNA expression array and qPCRA panel of ↑ miRNAs in steatotic or NASH patients, compared with HC, association with increased cardiovascular disease risk and atherogenesis, differentiation of NASH from steatosis, miRNA-122 differentiated liver fibrosisSurveillance, diagnosis, prognosis115
CHBCirculating miRNAs in serummiRNA expression array and qPCRDifferential expression of miRNAs in CHB vs. HC and CHB vs. NASH vs. HCDetection of liver injury123
CHBSerum lincRNA-p21qPCR↓ lincRNA-p21 in CHB patients than in HC, negative correlation with fibrosis stage in CHB patientsDiagnosis, staging24
CHCSerum miRNA let-7a-5pqPCR↓ in CHC patients with cirrhosis, positive correlation with severity of fibrosisDiagnosis, staging125
CHC, NAFLDSerum miRNAsqPCR↑ miRNA-122, miRNA-34a, and miRNA-16 in NAFLD and CHC patients than in HC, miRNA-122 and miRNA-34a correlated with disease severityDetection, staging114
NASHPlasma lncRNA LeXisqPCR↑ LeXis in NASH patients than in steatotic individualsDiagnosis126
NASHSerum miRNAsSmall RNA sequencing and quantitative reverse transcription PCRmiRNA-21-5p, miRNA-151a-3p, miRNA-192-5p, and miRNA-4449 differentiated NASH from steatotic patientsDiagnosis117
NAFLD, fibrosisPlasma lncRNA GAS5qPCR↑ GAS5 in advanced fibrosis, ↓ in cirrhotic NAFLD patientsDiagnosis, monitoring of disease progression116
ACPlasma lncRNAs AK054921 and AK128652Global transcriptomic profiling by lncRNA microarray, qPCRLncRNA signature, specific for excessive drinkers, not found in HC and AC, ↑ AK128652 and AK054921, correlating with alcoholic cirrhosis severity and patient survivalDiagnosis, prognosis127
Cirrhosis, ACLFSerum miRNAsOpen arrayMiRNAs profiles differentiating cirrhosis disease progression, kidney or liver failure, poor outcomeMonitoring, prognosis120
HCCSerum miRNAsmiRNA expression array and qPCRmiRNA classifier differentiating HCC patients from non-HCC and at-risk patientsSurveillance, early detection128
HCCPlasma lncRNALncRNA microarrayHCC patients showed ↑ RP11–160H22.5, XLOC_014172, and LOC149086, compared with HC, association with metastasis, ↓ post-surgerySurveillance, metastasis prediction, monitoring of disease progression129
HCCPlasma ZFAS1qPCR↑ ZFAS1 in HCC patients, compared with HC, cirrhotic, or hepatitis B patientsDetection130
HCCPlasma miRNA-21qPCR↑ in HCC than in chronic hepatitis patients and HC. ↓ miRNA-21 post-surgery, correlating with a lower risk of recurrenceDetection, monitoring, prognosis118
HBV–Related HCCPlasma miRNAsMicroarray and qPCRMicroRNA panel differentiated HCC from HC, CHB, and cirrhosisSurveillance, early detection119
HCV and HCV-associated HCCSerum NEAT1 and TUG1 in HCVqPCR↓ NEAT1 and TUG1 in HCV and HCC, compared with HC,↓ TUG1 in HCC patients, compared with HCV and HCDiagnosis, monitoring of disease progression131
HCV-positive cirrhosis and HCCSerum miRNAsqPCRA panel of deregulated miRNAs in HCV-positive cirrhotic and HCV-positive HCC patientsSurveillance, early detection132
HCC, Sorafenib treatmentSerum miRNA-221 levelsqPCRPositive therapy response associated with ↓ miRNA-221 pretreatment levels and ↑ miRNA-221 post-treatmentTherapy response prediction and monitoring133
Biliary tract cancerPlasma miRNA-21qPCR↑ miRNA-21 in Biliary tract cancerpatients, compared with HC benign biliary disease patientsDetection, diagnosis134

Circulating HCC cells

Circulating tumor cells (CTCs) are tumor cells of primary or metastatic origin that are detected in the blood or lymphatic circulation following intravasation. CTCs have undergone epithelial-mesenchymal transition and are considered highly metastatic.137,138 To survive in the circulation CTCs can form clusters, increasing their metastatic potential, stemness features, and plasticity.139 CTCs are a scarce population of cells, carrying crucial features that can inform on the tumor characteristics and impact HCC diagnosis and treatment regimen.

The value of CTCs in the field of liquid biopsies is undisputed. CTC research has led to remarkable progress in the noninvasive diagnostic, prognostic, and therapy response monitoring of cancer, including HCC.140–142 Sequencing of CTCs isolated from metastatic HCC patients revealed liver cancer-characteristic mutations, including low-frequency variants.143 Furthermore, CTC count predicted poor prognosis and post-operative disease recurrence.143,144 PD-L1+ CTCs have been shown to distinguish between early and advanced stage HCC and are suggested as a promising biomarker for immunotherapy patient stratification and therapy response monitoring.145,146 Compared with ctDNA and circulating ncRNAs, CTCs can be analyzed at the genomic, epigenomic, transcriptomic, and proteomic levels as single cells or in bulk, providing a detailed scope of tumor-associated signatures.147 However, because of their limited presence in circulation, many clinically relevant challenges occur regarding their effective, pure, unbiased, and affordable capturing, which are critical for downstream analysis. CTCs can be detected based on several characteristics such as size, charge, density, and expression of cell-surface marker,148 even in vivo.149,150 The first and only FDA-approved CTC capturing method relies on the selection of EpCAM+ CTCs as EpCAM has been universally recognized as a CTC detection marker.151,152 Nevertheless, it has been increasingly appreciated that solely EpCAM+ expression could be insufficient for CTC enrichment153,154 as EpCAMlow/negative CTCs are missed. Furthermore, EpCAM-based detection of CTC was found highly inefficient in HCC with approximately 25% CTC detection rate.155 Instead, novel methods are focused on distinct hallmarks such as ploidy subtraction enrichment and immunostaining-fluorescence in situ hybridization , which has shown promising results in capturing CTCs expressing distinct biomarkers and establishing their prognostic value.156 Nevertheless, there is no perfect system for CTC caption, and future work is likely marked by the combined application of several methods.

Extracellular vesicles (EVs)

EVs are cargo-carrying particles, released from cells in the extracellular space, inducing crucial cell-cell signaling cascades in normal physiology or pathological processes.157 EVs vary in size, release mechanism, and the nature of the loaded cargo (protein, lipids, metabolites, and nucleic acids). The molecules found in EVs may carry essential information on the cell-of-origin or the underlying trigger of EV release. Increasing evidence implicates EVs as key players in the pathology of several liver diseases, including NAFLD, AFLD, viral hepatitis, and HCC.158 EVs can signal to cells in close proximity or be transported to distal sites to act as long-range signals.159 EVs have been purified from most mammalian cells and bodily fluids. Furthermore, cellular compounds transferred by EVs are protected from the hostile environment of the circulation and other degradation stimuli in the extracellular space, making them highly stable. Given that EVs are loaded with only a subset of molecules, which could be otherwise barely detected in the total volume of body fluids, EVs have emerged as a promising liquid biopsy approach (Table 3).160–177 Several EV-based biomarkers, including proteins and miRNAs, have been reported in the serum or plasma of people with liver disease. For instance, decreased levels of miRNA-718 in serum EVs of HCC patients were associated with aggressiveness and recurrence following liver transplantation,160 while high levels of EV-associated miRNA-21 correlated with cirrhosis and advanced tumor stage.161 Similarly, elevated levels of several circulating EV-associated miRNAs were found in alcoholic hepatitis (AH), ASH, and AFLD patients.178,179 EVs enriched with six sphingolipids were significantly elevated in AH patients, compared with healthy individuals, heavy drinkers, NASH patients, and alcoholic cirrhosis patients.158 Mitochondrial DNA (mtDNA) encapsulated in EVs was also shown to promote inflammation, which is in line with the EV-enclosed mtDNA in NASH patients.180 Furthermore, proteomic analysis of circulating EVs has identified differential proteomic profiles, differentiating precirrhotic NASH, cirrhotic NASH, and healthy individuals.162

Table 3

EVs in liver diseases

Liver-related pathologyTargetMethod ofdetectionLevel/resultSuggestedfunctionReference
NASHTotal circulating EVs and hepatocyte-derived EVsDifferential centrifugation and size-exclusion chromatography, flow cytometry, electron microscopy, western blotting, and dynamic light scatteringPositive correlation between NASH characteristics and total or hepatocyte-derived EVs, proteomic signatures differentiating precirrhotic/cirrhotic patients from HCSurveillance, detection, diagnosis162
NAFLD – pre and post-weight lossTotal EVs, hepatocyte-specific EVs, lipid and sphingolipids analysisDifferential ultracentrifugationand quantified by nanoparticle tracking analysis↓ total EVs and hepatocyte-specific EVs post weight loss, a positive correlation between hepatocyte-specific EVs and NAFLD clinical parametersDetection, diagnosis, monitoring163
ALD vs NAFLDProteomic analysis of serum extracellular vesiclesCentrifugation, Liquid chromatography-mass spectrometry, protein identification, and label-free quantification using the MaxQuant platformA panel of proteins differentiated between ALD and NAFLDDiagnosis164
NAFLD/NASH, CHCMicroparticles from immune cells in serumDifferential centrifugation, Fluorescence-activated cell scanningEV profiles correlating with inflammation severity and fibrotic stageSurveillance, early detection, diagnosis165
CHB, DeCiEVs and EV miRNA in serumCentrifugation, miRNA-seq, and qPCR arraysSevere liver injury associated with the highest concentration of EVs, compared with DeCi and HC patients, miRNAs as predictors of disease progressionSurveillance, early detection166
AIHEV-encapsulated miRNAs in serumMicroarray, digital PCR↑ EV-miRNA-557 in AIH, compared with patients with NASH, Primary biliary cholangitis, and HC that are correlated with relapseDiagnosis167
AHSphingolipids encapsulated in EVsТandem mass spectroscopy↑ EVs and EV sphingolipid cargo in AH patients, compared with HC, heavy drinkers, end-stage-liver disease, and DeCiDetection, survival prediction, monitoring168
CHCSoluble CD81 in the exosomal serum fractionDifferential centrifugation, immunoblotting, and densitometryPatients with CHC - ↑ CD81, associated with inflammation and fibrosis severity, cured CHC patients – CD81 levels, similar to HCDetection, diagnosis, monitoring169
CHCEV proteome in serumAffinity purification, shotgun and targeted proteomicsSAP and PPBP were ↓ with liver fibrosis severityDiagnosis, staging170
HCVExosome-encapsulated miRNA-19aExosome isolation (ExoQuick), qPCR↑ in HCV patients with fibrosis, compared withHC and fibrotic patients with non-HCV-related liver pathologyDiagnosis171
HCCMiRs in exosomes from serumUltracentrifuge, microarray↓ exosomal miRNA-718 in patients with tumor recurrence following liver transplantationMonitoring, prediction of recurrence160
HCC and CCAAnnexinV+ EpCAM+ CD147+ taMPs in serumDifferential centrifugation, Fluorescence-activated cell scanning↑ in HCC and CCA, differentiating nonliver cancers or other liver disorders. ↓ taMPs post tumor resectionDetection, diagnosis, monitoring172
HCC, CCA, Primary sclerosing cholangitisProteome of serum EVsNTA, mass spectrometryDifferentially expressed proteins in EVs, showing promising diagnostic capacity between the distinct groupsDiagnosis173
CCAEVs in human bileNanoparticle tracking analysis, qPCR miRNA arrays, qPCRDevelopment of biliary vesicle miRNA-based panel differentiating CCA from biliary obstruction and bile leak syndromesSurveillance, detection174
HCCExtracellular vesicle-derived lncRNAsqPCR based on differentially expressed lncRNAs in HCC tissue↑ EV-derived LINC00853 in all-stage HCC, including AFP-negative HCC, compared withHC, chronic hepatitis, and liver cirrhosisSurveillance, early detection175
HCCSerum exosomal microRNAqPCR↑ exosomal miRNA signature in HCC patients, compared with CHB and LC,↓ miRNA signature when compared with CHBSurveillance, early detection176
HCCmiRNA-21 in serum exosomesqPCR↑ in HCC than CHB or HC, correlating with cirrhosis and tumor stageDetection, diagnosis161
HCC recurrenceExosomal miRNAs in serumUltracentrifugation, microarray, qPCR↓ miRNA-718 levels correlated with HCC tumor aggressivenessMonitoring, prognosis160
Liver metastasis in CRCPlasma EVUltracentrifugation, tethered cationic lipoplex nanoparticlestechnologyCRC-derived sEVs with enriched microRNA-21-5p positively correlated with liver metastasisDetection, disease progression177

However, in line with the capturing-related challenges with CTCs, the detection and enrichment methods of EVs come with various limitations and factors affecting the yield.181,182 Despite the substantial progress in EV isolation, standardization of EV procedures and nomenclature is yet to progress, delaying their clinical applications.


Methods for isolation and analysis of liquid biopsies have rapidly evolved over the past few years, thus providing details on the development and progression of liver diseases, treatment strategies, and patient stratification. Their long half-life and stability in the bloodstream render histones and histone complexes valuable liquid biopsies. Anti-nucleosome antibodies are more than two-fold more sensitive, compared with antibodies against DNA.183 Furthermore, in contrast to CTC and EVs, circulating histones do not require complex extraction methods, which affect the downstream analysis. The purpose of the following sections of this review is to provide an overview of circulating histones as liquid biopsies and their application for the detection, prognosis, and monitoring of hepatic disease progression and outcomes.

Extracellular/circulating histones

DNA in eukaryotic cells is compacted into chromatin by wrapping around histone proteins, generating protein-DNA complexes referred to as nucleosomes. Each nucleosome comprises a histone octamer: one tetramer H2A-H2B, two H3-H4 dimers, and approximately 147 base pairs of DNA.184 Neighboring nucleosomes are connected through a short DNA stretch bound by histone H1, referred to as a linker histone creating the structure of chromatin.184 Nucleosomes are highly dynamic structures,185,186 regulating key cellular processes, including gene transcription, replication, and DNA repair through particularly ordered signaling cascades.186 Both core and linker histones can be epigenetically modified through post-translational modifications (PTMs), altering protein-protein and protein-DNA interactions, resulting in nucleosome occupancy or position changes, activation/suppression of gene expression, and cell division.185,187,188 Nucleosome function can also be altered on a structural level by the incorporation of histone variants, which have defined functions such as macroH2A1.2, H2AX, and H2AZ in DNA repair,189,190 or tissue-specific expression as the histone variants H3T and H3.5 in testicular cells.189 It is widely accepted that the correct organization and regulation of nucleosomes and chromatin are crucial for genome stability.185,191

Studies have shown that histones and histone complexes are also detected in the extracellular space and the bloodstream, following release by damaged cells and activated immune cells. Upon release, extracellular histones function as damage-associated molecular pattern molecules, exerting cytotoxic and pro-inflammatory activity.192–194 Neutrophils have been shown to utilize a distinct immune defense mechanism, referred to as neutrophil extracellular traps (NETs), in which histones, DNA, and other factors such as granule proteins are released in the extracellular space.195 NETs can cause a further histone release by inducing a specialized form of neutrophil cell death,195 resulting in a self-sustained inflammatory cascade and cell death.196–199 Furthermore, studies have shown that NETs are associated with pathogenesis and could act as effectors in disease maintenance or progression, as found in HCC.200–202 Importantly, histone H3 citrullination is an essential epigenetic change in NETs formation and it is widely utilized as NETs marker.203,204 Given that NETs have been suggested as potential biomarkers for disease prognosis and therapy response,205,206 accurate detection of extracellular histones is essential. Furthermore, while previously NETs have been considered neutrophil-specific, current research has found that other innate immune cells, including macrophages, mast cells, and eosinophils207 can fight pathogens by extracellular traps, further indicating the role of histones in the extracellular space.

An increasing number of studies suggest that circulating histones, post-translationally modified histones, and histone complexes are differentially detected in the plasma or serum of patients with liver-associated diseases, including HCC, indicating potential biomarker function.

Extracellular histones in liver diseases

Histones have been mostly studied as a component of NETs and have been studied in the context of liver disease for many years, most significantly concerning the epigenetic changes involved in liver pathogenesis and epigenetic-based therapeutics. The role of histones in liver disease is a vast topic and has been reviewed elsewhere.208–213 For this review, we will focus on the role of extracellular histones in liver pathology. Wang et al.214 recently showed that NETs are associated with cancer development and progression by modulating gene expression in naïve CD4+ T-cells in a TLR4-mediated manner, favoring Treg differentiation, resulting in repressed immunosurveillance in NASH mouse model and increased incidence of HCC development. Inhibition of NETs suppressed Treg activity and tumor burden in NASH-HCC models, directly linking histones with liver disease progression. Similarly, in the surgical stress murine model induced by liver ischemia-reperfusion injury, downregulation of NETs formation caused a reduction in the development and progression of metastatic liver disease.200 HCC is often caused by the progression of liver fibrosis to liver cirrhosis and subsequent cancer nodule formation. Recently, Wang et al.215 showed that induction of fibrosis in mice caused a drastic increase in circulating histones. Furthermore, direct histone treatment of LX2 human hepatic stellate cells caused activation of collagen I production and α-SMA through TLR4- MYD88 signaling. The administration of either noncoagulant heparin (NAHP) or TLR4-blocking antibody resulted in decreased Aminotransferase levels and histology-based liver injury score, suggesting histone inhibition as a potential therapeutic approach. However, it should be mentioned that no difference in extracellular histones was observed between carbon tetrachloride and carbon tetrachloride+ NAHP treated groups.215 Therefore, heparin could have exerted its liver protective function irrespective of coagulation and extracellular histone neutralization.

NETs have also been implicated as drivers of earlier stages of liver disease. Tanshinone IIA has been suggested to exert its anti-inflammatory and anti-steatotic effect in NASH-induced mice at least partially by regulating NETs.216 NET production is also drastically elevated in the circulation of AH patients and mice, together with a specific subpopulation of low-density neutrophils that exert defective properties. The authors showed that alcohol induces the activation of cultured human neutrophils, causing nonlytic NETs release in high-density neutrophils, which subsequently become low-density neutrophils with diminished homing capacity and clearance.217

Chen et al.218 further showed that extracellular H3 could induce ferroptosis in hepatic macrophages and ACLF model mice. Importantly, treatment with anti-H3 antibody suppressed cell damage and pro-inflammatory cytokine production in vitro and in vivo. Histone H4 has been also shown to directly induce hydrogen peroxide production in neutrophils in a calcium- and cell adhesion-dependent manner, resulting in degranulation and pro-inflammatory cytokine release. Mechanistically, the authors showed that histone H4 causes a prolonged increase in neutrophil intracellular calcium, membrane depolarization, and rapid permeabilization,219 suggesting a potential molecular mechanism behind the drastic neutrophil activation in liver diseases.220

Together, previous studies have indicated the role of histones in liver disease, the potential molecular mechanisms, and the promising therapeutic benefit of anti-histone therapy. Nevertheless, many questions remain, the main one being: can we use circulating extracellular histones as a biomarker for liver disease detection and monitoring in patients?

Methods of detection

Circulating histones and histone complexes can be detected by several methods, including enzyme-linked immunosorbent assay (ELISA), proteomic analysis, and imaging approaches including single-molecule imaging and ImageStream.


There are several ELISAs developed to detect histone subtypes, specific PTMs, or nucleosomes. In a study comparing patients with local, locally advanced, or metastatic prostate cancer (PC) following therapy by ELISA-based detection of specific plasma components, H3K27me3 levels were inversely correlated with metastatic PC and showed the ability to differentiate patients with localized and metastatic disease.221 A subsequent study utilized an ELISA-based assay consisting of antibody-mediated nucleosome immobilization and incorporation of antibodies detection histone variants or histone modifications of interest, to show the diagnostics potential of circulating nucleosomes in distinguishing PC patients from healthy controls or individuals with benign disease.222 The authors identified a panel of five nucleosome-associated marks in serum that achieved a higher disease-predictive score, compared with the common pancreatic tumor biomarker, carbohydrate antigen 19–9. The same assay was subsequently applied to the serum of people referred for colorectal cancer (CRC)-related endoscopic screening. Of the 12 epigenetic epitopes measured on circulating nucleosomes, the assay revealed two groups of four markers that differentiated early-stage CRC patients from healthy individuals, and healthy individuals from people with benign polyps, respectively.223 These results indicate that ELISA is a powerful approach, detecting differences in circulating histones and histone complexes between groups and could be used to assess their biomarker properties. Nevertheless, ELISA is characterized by antigen detection bias such as histone PTMs with high concentration due to the generally low sensitivity of the assay. Furthermore, while multiplexed ELISAs have been developed, increasing the assay significance and power as a detection and quantification approach, they are currently limited to the multiplexed detection of H3, H4, and post-translationally modified H3/H4.224,225


While ELISA is a time-efficient assay, providing multiplex opportunities that do not require a highly specialized scientist, ELISA has considerable bias in regards to antibody sensitivity and specificity, detection of known histone modifications, antigen concentration, and accessibility of antigen target.226 To tackle these limitations, one can choose to rely on proteomic analyses. Van den Ackerveken et al.227 developed an epigenetic profiling approach on circulating nucleosomes, based on intact H3.1-positive nucleosome capturing by immunoprecipitation, liquid chromatography, and tandem mass spectrometry. An alternative nucleosome isolation approach to immunoprecipitation is a previously described acid-based extraction, which applies trichloroacetic acid -mediated total protein precipitation, followed by histone extraction by 0.2 M H2SO4.228 Multiple reaction monitoring targeted mass spectrometry, involving heavy-isotope labeled Spike-In peptides,229 is another approach proven powerful in identifying and quantifying histone proteins230 and histone PTMs.231 Alternative MS approaches are isobaric tags for relative and absolute quantification and tandem mass tag MS, both of which have been previously applied in plasma proteome assessment.232 Overall, MS allows for an antibody-independent measurement of histone levels and a systemic nonbiased analysis of histone-associated PTMs.

Single-molecule imaging and Image stream

Recently, Fedyuk et al.233 developed a single-molecule imaging approach EPINUC that assesses the epigenetic signature of circulating nucleosomes, detecting individual histone PTMs and their combinatorial pattern on individual nucleosomes by total internal reflection microscopy. EPINUC differentiated patients with late-stage CRC from healthy individuals based on the epigenetic profile of circulating nucleosomes. EPINUC is yet to be utilized on other cancer types such as HCC and early-stage patients. ImageStream device combines the sensitivity of flow cytometry with the detailed phenotypic abilities of cellular imaging, allowing for the detection of multiple biomarkers and the acquisition of up to 12 channels. Furthermore, ImageStream is able to detect particles in the range of 1 µm to 20 nm as shown by analysis of calibration beads.234 ImageStream acquires a large number of images per sample, providing for fast collection of morphology-based and fluorescent signal-based data. Furthermore, ImageStream can be coupled with open-source artificial intelligence software, creating the possibility for a fully automated quantification. ImageStream has been proven valuable in the liquid biopsy field, especially in the detection and characterization of CTCs. Previously, the focus was on developing reliable methods for CTC extraction and quantification and determining their prognostic ability in terms of disease progression and therapy response. Current research aims to further increase and better estimate the clinical potential of CTCs by characterizing their features and behavior. ImageStream detects the expression of multiple markers on a single CTC, allowing for simultaneous positive and negative selection, as shown for esophageal, hepatocellular, thyroid, ovarian, and lung cancers.235,236

Similarly, an increasing number of studies indicate the value of ImageStream in detecting circulating histones and addressing their effect on the surrounding cells in various contexts. For instance, ImageStream was applied to assess NETs levels in both murine and human whole blood samples, based on the expression of NETs components, including positive staining for H3Cit,237,238 bypassing time-consuming analysis and potential bias. Similarly, anti-histone antibodies detecting histones H1, H2A, H2B, H3, and H4 were utilized as a marker of Eosinophil extracellular traps , allowing tracing the origin of Eosinophil extracellular traps -associated particles of interest as nuclear, rather than mitochondrial.239 ImageStream-mediated analysis showed that following trauma extracellular H4 exerts cytotoxic function on platelets, resulting in ballooning and H4-retaining microparticle secretion, which binds to leukocytes. These findings strongly suggest that ImageStream is a powerful tool not only to detect circulating histones but also to track extracellular histones-mediated cell-cell communication and the resulting intracellular changes. ImageStream-mediated detection of circulating histones has also been applied for liver-associated diseases that will be discussed in the following section.77,240 Nevertheless, ImageStream-based analysis of circulating histones in blood samples of HCC patients or animal models is currently lacking.

Circulating histones as markers and predictors of NAFLD/NASH

To decrease the risk of disease progression and determine the most optimal treatment approach, it is essential to discriminate between NAFLD and NASH patients. Current data on promising noninvasive NAFLD/NASH diagnostic approaches including imaging and biomarkers is scarce. Nevertheless, circulating histones and histone complexes have been indicated as potential NAFLD biomarkers. Circulating nucleosomes have been found elevated in obese individuals and correlated with fatty liver and poor metabolic health.241 Given that obesity and metabolic syndrome are among the main risk factors of NAFLD development, it could be hypothesized that nucleosomes render prognostic value. ImageStream has been recently applied to the serum and plasma of lean MAFLD240 and NAFLD77 patients, respectively. While high levels of circulating nucleosomes are reported in various diseases and conditions, including obesity-associated MAFLD,241 nucleosomes showed poor association with nonobese (lean or overweight) MAFLD or NASH. However, serum nucleosomes were found significantly elevated in grade 3 steatosis, compared with grade 1 lean MAFLD patients. Looking at histones onan individual level, circulating macroH2A1.1 and macroH2A1.2 histones, either as individual proteins or as a dimer with H2B, were significantly decreased in steatotic grade 3 lean/nonobese MAFLD patients, compared with grade 1. Conversely, H2A and H2A/H2B complex were significantly elevated in overweight MAFLD, but not in lean MAFLD patients. Together, circulating histones macroH2A1.2, H2B, and H4 were associated with MAFLD disease severity, further suggesting potential prognostic and patient stratification properties (Table 4).77,240-250 Interestingly, pediatric NAFLD patients were characterized by contrasting histone expression signature in circulation. Specifically, serum levels of macroH2A1.2 in NAFLD children were significantly elevated compared with healthy controls. However, no difference in circulating macroH2A1.2 was observed between children with or without NASH. Furthermore, macroH2A1.2 showed an inverse correlation, having the strongest correlation with early-stage steatosis and the weakest with NAFLD Activity Score (NAS).77 Together, these findings indicate the prognostic significance of macroH2A1.1 in early NAFLD stages but not in disease progression to NASH. It should be noted that high-risk individuals such as those with T2DM or metabolic syndrome were not included in the studies. It would be interesting to evaluate whether such a histone signature could differentiate between people at risk and those that have developed the initial stages of liver disease.

Table 4

Circulating histones and histone complexes in liver diseases

Liver-related pathologyTargetMethod of detectionLevel in circulationSuggested functionReference
Obesity, MAFLDNucleosomesELISAIncreasedDiagnosis, fatty liver, poor metabolic health241
Lean MAFLD grade 3 steatosisNucleosomesELISAIncreasedDisease/severity staging240
Lean/nonobese MAFLDmacroH2A1.1, macroH2A1.2ImageStreamDecreasedDiagnosis240
Overweight MAFLDH2B, H2A/H2B complexImageStreamIncreasedDiagnosis240
MAFLDmacroH2A1.2, H2B, and H4ImageStreamDecreasedDisease/severity staging240
Pediatric NAFLDmacroH2A1.2ImageStreamIncreasedDetection77
Pediatric NAFLDmacroH2A1.2ImageStreamDecreased in advanced steatotic childrenDisease/severity staging77
HCC post locoregional transarterial chemoembolization therapyNucleosomesELISAIncreasedDetection of a positive therapy response242
HCC post Sorafenib treatmentH3K27me3, H3K36me3ELISADecreasedDetection of apositive therapy response243
HCC post-Sorafenib treatmentH3K27me3/H3K36me3 ratioELISAIncreasedPrediction of therapy resistance and disease progression243
HCC post-RFANucleosomesELISATransiently increased and subsequently decreasedDetection of a positive therapy response244
HBV-related ACLFH4ELISAIncreasedDetection, prognosis245
Cirrhosis, ACLFNucleosomesELISAIncreased cirrhosis < ACLFDetection246
ACLF, grade I–IVNucleosomesELISAIncreased grade I/II < grade III/IVDetection, staging, prognosis247
ACLF (HBV-related), conventional therapy and QingchangliganNucleosomesELISADecreased nucleosomes and pro-inflammatory cytokines following conventional therapy + Qingchangligan, compared withconventional therapy aloneTherapeutic248
PGDNucleosomesELISATransiently increased and subsequently decreasedDetection, prognosis249
HepatectomyHistone H3ELISALow levels of post-surgical (24h) H3 levels were associated with delayed liver function recoveryPrognostic250

The progression of early-stage NAFLD to NASH is marked by the induction of inflammation, which among other pathways can be directly induced by NETs-associated histone release.193,251,252 In line with that, increased levels of NETs were detected in the circulation of patients diagnosed with NASH,201 cirrhosis, or HCC,253 compared with normal livers.

Recently developed DNA sequencing-coupled bioinformatic approaches indicated that the retained epigenetic profile on plasma/serum nucleosome-associated DNA could predict the tissue-of-origin of the circulating fragments. Sadeh et al.254 applied active marks-mediated chromatin immunoprecipitation of nucleosomal DNA on the plasma of patients with diverse liver-associated pathologies, including NAFLD/NASH patients. Interestingly, based on the region characterized by the active mark, the authors determined differential gene expression profiles that could be traced back to specific liver zones or condition-related transcriptional pathways.254 The method allows for noninvasive genetic-independent identification of differentially regulated pathways through unbiased analysis, which could potentially distinguish crucial clinical features of the disease. Nevertheless, such an approach requires extensive technology and trained personnel, making it currently challenging for potential routine clinical practice.

Circulating histones in liver dysfunction, transplantation, and HCC

An increasing number of studies are reporting the strong diagnostics and monitoring value of circulating nucleosomes in several solid malignancies.222,223,227,233 Nevertheless, no difference in nucleosome levels was found between chronic hepatitis B (CHB) and HCC,246,255 (Table 4) suggesting that HCC development following CHB is not associated with further elevated nucleosome content. This finding also indicates that solely measuring nucleosome levels is unable to distinguish HCC cases in high-risk individuals. The lack of specificity of circulating nucleosomes is further supported by the fact that nucleosomes in plasma and serum are elevated in several other types of cancer and benign conditions.256 However, elevated nucleosome content in the serum of HCC patients 24 h post locoregional transarterial chemoembolization therapy was found as an independent marker of therapy response, with elevated circulating nucleosomes 24 h post-therapy correlating with disease progression.242 Changes in nucleosome levels were also detected following hepatic radiofrequency ablation (RFA),244 which is also applied as a first-line HCC treatment.257,258 Specifically, a drastic transient increase in circulating nucleosomes was observed 24 h post-treatment, compared with the pre-RFA state, which correlated with liver damage and upregulated pro-inflammatory markers MPO, interleukin (IL)-6, and IL10. Interestingly, the values of circulating nucleosomes decreased back to pretreatment levels after four weeks.244 However, how these values correlate with the patient therapy response four weeks post-treatment was not addressed. Furthermore, the serum histone content in the HCC-induced mouse model was significantly increased, compared with HCC-free animals, and recapitulated the elevated extracellular histone levels in HCC tissues.228 More important, circulating histones in HCC rat serum had epigenetic signatures characteristic of both human and rat HCC tumor tissues, including hypo-acetylation at H4K16 and hypomethylation H4K20.228 Changes in post-translationally modified circulating histone levels from baseline to post-therapy were also shown to predict patient response and outcomes following Sorafenib treatment. A decrease of H3K27me3 and H3K36me3 post-treatment was associated with a detectable response to Sorafenib. Conversely, an increased H3K27me3/H3K36me3 ratio was correlated with therapy resistance and disease progression.243 It should be noted that a clear definition of a time point for post-treatment sample collection is not specified. Furthermore, changes in the post-translational modification levels are not addressed in the patients that showed stable disease at first visit, but progressive disease at follow-up assessments. Therefore, we cannot address how changes in the modifications of interest correlate with the disease alteration. These findings suggest that epigenetic profiling of circulating histones can be used as a patient stratification approach and therapy response biomarker. However, whether changes in circulating histone level post-therapy reflect disease progression or remission, and whether pretreatment levels of circulating histones are indicative of short/sustained therapeutic response is currently unclear as longitudinal studies are lacking.

Circulating H4 histones were drastically increased in ACLF patients, compared with healthy individuals/CHB/HBV-related liver cirrhosis.245 In line with these findings, nucleosomes were significantly elevated in the serum of ACLF patients246,247 and primary graft dysfunction (PGD),249 compared with healthy individuals and patients without PGD development, respectively. Blasi et al.246 also reported significantly elevated plasma nucleosome levels in patients with acutely decompensated cirrhosis (DeCi) , compared with healthy individuals, and in ACLF patients, compared with acutely DeCi. While cirrhotic patients were also included in the study of Wen et al.,247 patients were grouped as CLD, which includes inflammation, liver cirrhosis, and HCC. Importantly, circulating histones correlated with ACLF severity and predicted patient prognosis.245,247 Upon incubation of ACLF245,247and PGD249 from patient serum with human L02 hepatocytes and monocytic U937 cells, the authors found elevated L02 cell death and cytokine induction in U937 cells. Importantly, these effects were abrogated following heparin treatment, which binds histones,259–261 and anti-histone antibody administration.249 In ACLF mouse models, NAHP was able to diminish inflammation and liver injury,247 indicating (1) that histones exert their function irrespective of coagulation; and (2) the potential therapeutic value of anti-histone therapy in ACLF and liver transplantation-related dysfunction. In line with these findings, a subsequent study showed that a transiently increased nucleosome level followed by a partial decrease, instead of a decline back to baseline values, in patient plasma following liver transplantation was associated with early complications, including acute kidney injury, early allograft dysfunction, and decreased survival.262

Studies on individual circulating histones and histone PTMs are currently scarce compared with studies addressing nucleosomes in plasma or serum. Furthermore, while differences in plasma nucleosome levels have been detected between some patient groups, for example, cirrhosis vs. ACLF patients,246 and low vs. high-grade ACLF247), nucleosome levels vary substantially between the cohorts of patients with ACLF,246,247 and might be influenced by patient stratification, and the methodological approach, indicating the need for standardization.

Translational impact of liver liquid biopsy

Liquid biopsy is a continuously growing field, with new development in biomarker/s panel selection, methods, and ameliorated standardization approaches. It is essential to establish models that characterize the most suitable liquid biopsy type for a specific disease or condition, aiming at diagnosis, characterization, monitoring, or patient stratification.

Early detection of liver diseases is crucial for prognosis and patient quality of life. While currently applied enzyme biomarkers have long-established value for liver disease detection, they often present low sensitivity and specificity. For early-stage liver disease monitoring and detection, a biomarker should be suitable for routine screenings, fast, cost-effective, and easy to perform. Liquid biopsy biomarkers such as cfDNA-derived variants and epigenetic alterations, ncRNAs, and EVs have been shown to differentiate specific diseases not only from healthy individuals but also from patients with closely related diseases/conditions. Furthermore, compared with cfDNA and lncRNAs, EV isolation and analysis currently requires approaches that might not be applicable in every clinical laboratory. In comparison, while data suggest that circulating histones and histone complexes might be further studied as an early detection biomarker for several liver diseases, current research lack comparison with other liver pathologies.

Liver disease staging relies on tissue biopsy and imaging analysis. Liquid biopsy markers like circulating histones, nucleosomes, cfDNA, ncRNAs, and EVs are associated with liver disease severity and could provide diagnostic value at a relatively low cost and analysis time. EVs may contain important information regarding liver disease severity but require more elaborate processing and analysis. Furthermore, such noninvasive analysis could be clinically valuable when a set of detection biomarkers is developed to replace the need for invasive tissue biopsy.

The genetic signature is of utmost importance for appropriate diagnosis and treatment regimen definition, especially in liver malignancy diagnosis. To that end, CTCs and ctDNA have the greatest potential. Nevertheless, the concentration of CTCs and ctDNA in circulation is a crucial factor of such a liquid biopsy method. CTCs enter the circulation during intravasation, the initial stage of the metastatic cascade. Therefore, patients with a nonmetastatic early-stage of the respective malignant liver disease might not benefit from such analysis. However, ctDNA is released in circulation upon cell death. Cell turnover (proliferation and apoptosis) is often high in cancer, favoring the detection of ctDNA in plasma/serum of patients with localized disease. That allows for early detection and characterization of the disease phenotype such as the identification of potentially targetable markers and therefore, timely intervention through local or systemic treatment. It should be noted that advanced sequencing technology should be in place to perform such analysis. Alternatively, external institutions might be involved, which would likely increase the processing time. Furthermore, frequent genetic and epigenetic alterations, targetable mutations, or clinical-trial-relevant targets could be easily addressed by targeted polymerase chain reaction (PCR), which drastically decreases the processing time.

Circulating histones and post-translationally modified histones have shown promising results for treatment monitoring (surgical, conservative, or palliative). It is unclear whether specific histones, histones variants, or histones modifications might detect responses to particular treatments. However, changes in circulating nucleosomes, H3K27me3/H3K36me3, and histone H3 levels were shown to occur rapidly following RFA, Sorafenib therapy, and hepatectomy. Similarly, while the levels of circulating nucleosomes in plasma or serum lack sensitivity in detecting specific organ-specific diseases, they might be a valuable method for therapy response monitoring and disease progression surveillance. Subsequent studies should focus on acquiring data from longitudinal studies with large cohorts that would shed light on some essential questions such as (1) how the circulating histone levels correlate with disease progression and progression-free survival; and (2) could circulating histones be used for patient therapy stratification?


There is a growing need for minimally invasive biomarkers for the diagnosis, staging, prognosis, monitoring, and personalized management of liver diseases. Increasing evidence indicates the potential diagnostic, prognostic, and monitoring value of circulating nucleosomes, histones, and histone complexes in liver diseases. In this respect, histones joined DNA-based biomarkers in the market arena of epigenetic-based companion diagnostic tests (CDx), used as a companion to a therapeutic drug to determine its applicability to a specific patient.263 Nevertheless, results and suggested conclusions should be taken with caution, since data has been gathered from retrospective studies, some of which predominantly have a small sample size and/or lack high-risk groups. Furthermore, the currently nonstandardized circulating histone analysis and the interstudy variability pose challenges to the clinical application of circulating histones in liver disease assessment. Patients with early-stage liver diseases often do not show differential levels of circulating histones or histone complexes, limiting their value for disease diagnosis and prevention. While circulating histones have shown promising results for monitoring HCC therapy response and disease progression, current data on their potential in HCC detection is limited. Future studies should aim for large patient cohorts including various liver-related and liver-independent diseases. To increase the specificity of circulating histones, studies could focus on developing liver disease-predictive models combining several histone variants, complexes, and PTMs.



American Association for the Study of Liver Disease


Acute liver failure


Alcoholic fatty liver disease


Alcoholic hepatitis


Asian Pacific Association for the Study of the Liver




Cell-free DNA


Chronic hepatitis B


Chronic hepatitis C


Chronic liver disease


Colorectal cancer


Circulating tumor DNA


Circulating tumor cells


Decompensated cirrhosis


European Association for the Study of the Liver


Enzyme-linked immunosorbent assay


Extracellular vesicles


Food and Drug Administration


Viral hepatitis B, HC, Healthy controls


Hepatocellular carcinoma


Viral hepatitis C


Long noncoding RNAs




Mitochondrial DNA


Nonalcoholic fatty liver disease


Noncoagulant heparin


Nonalcoholic steatohepatitis


Nuclear enriched abundant transcript 1


Neutrophil extracellular traps


Next-generation sequencing


Prostate cancer




Primary graft dysfunction


Pro-platelet basic protein


Post-translational modifications


Radiofrequency ablation


Serum amyloid P component


Type 2 diabetes mellitus


Transarterial chemoembolization


Tumor-associated microparticles



This research was funded by the Ministry of Education and Science of Bulgaria under the National Scientific Program “Excellent Research and People for the Development of European Science” 2021 (VIHREN) of the Bulgarian National Science Fund, contract #KP-06-DV/4 from 15.12.2021; by the Bulgarian National Science Fund, contract #KP-06-N53/6 from 11.11.2021.

Conflict of interest

MV has been an editorial board member of Journal of Clinical and Translational Hepatology since 2022. The other authors have no conflict of interests related to this publication.

Authors’ contributions

Conceptualization (DKT, MV), writing-original draft preparation (DKT, MNI, MV), writing-review and editing (DKT, MI, MV). All authors have read and agreed to the published version of the manuscript.


  1. Sharma A, Nagalli S. StatPearls. Treasure Island (FL): StatPearls Publishing; 2023 View Article PubMed/NCBI
  2. Dyson JK, Anstee QM, McPherson S. Non-alcoholic fatty liver disease: a practical approach to treatment. Frontline Gastroenterol 2014;5(4):277-286 View Article PubMed/NCBI
  3. Seitz HK, Bataller R, Cortez-Pinto H, Gao B, Gual A, Lackner C, et al. Alcoholic liver disease. Nat Rev Dis Primers 2018;4(1):16 View Article PubMed/NCBI
  4. Joshi K, Kohli A, Manch R, Gish R. Alcoholic Liver Disease: High Risk or Low Risk for Developing Hepatocellular Carcinoma?. Clin Liver Dis 2016;20(3):563-580 View Article PubMed/NCBI
  5. Morgan TR, Mandayam S, Jamal MM. Alcohol and hepatocellular carcinoma. Gastroenterology 2004;127(5 Suppl 1):S87-S96 View Article PubMed/NCBI
  6. Li ZM, Kong CY, Zhang SL, Han B, Zhang ZY, Wang LS. Alcohol and HBV synergistically promote hepatic steatosis. Ann Hepatol 2019;18(6):913-917 View Article PubMed/NCBI
  7. Moon AM, Singal AG, Tapper EB. Contemporary Epidemiology of Chronic Liver Disease and Cirrhosis. Clin Gastroenterol Hepatol 2020;18(12):2650-2666 View Article PubMed/NCBI
  8. Cheemerla S, Balakrishnan M. Global Epidemiology of Chronic Liver Disease. Clin Liver Dis (Hoboken) 2021;17(5):365-370 View Article PubMed/NCBI
  9. GBD 2017 Cirrhosis Collaborators. The global, regional, and national burden of cirrhosis by cause in 195 countries and territories, 1990-2017: a systematic analysis for the Global Burden of Disease Study 2017. Lancet Gastroenterol Hepatol 2020;5(3):245-266 View Article PubMed/NCBI
  10. Younossi ZM, Stepanova M, Younossi Y, Golabi P, Mishra A, Rafiq N, et al. Epidemiology of chronic liver diseases in the USA in the past three decades. Gut 2020;69(3):564-568 View Article PubMed/NCBI
  11. Karlsen TH, Sheron N, Zelber-Sagi S, Carrieri P, Dusheiko G, Bugianesi E, et al. The EASL-Lancet Liver Commission: protecting the next generation of Europeans against liver disease complications and premature mortality. Lancet 2022;399(10319):61-116 View Article PubMed/NCBI
  12. Fabbrini E, Sullivan S, Klein S. Obesity and nonalcoholic fatty liver disease: biochemical, metabolic, and clinical implications. Hepatology 2010;51(2):679-689 View Article PubMed/NCBI
  13. Osna NA, Donohue TM, Kharbanda KK. Alcoholic Liver Disease: Pathogenesis and Current Management. Alcohol Res 2017;38(2):147-161 View Article PubMed/NCBI
  14. Idalsoaga F, Kulkarni AV, Mousa OY, Arrese M, Arab JP. Non-alcoholic Fatty Liver Disease and Alcohol-Related Liver Disease: Two Intertwined Entities. Front Med (Lausanne) 2020;7:448 View Article PubMed/NCBI
  15. Theise ND. Histopathology of alcoholic liver disease. Clin Liver Dis (Hoboken) 2013;2(2):64-67 View Article PubMed/NCBI
  16. Sookoian S, Pirola CJ. Systems biology elucidates common pathogenic mechanisms between nonalcoholic and alcoholic-fatty liver disease. PLoS One 2013;8(3):e58895 View Article PubMed/NCBI
  17. Zhang P, Wang W, Mao M, Gao R, Shi W, Li D, et al. Similarities and Differences: A Comparative Review of the Molecular Mechanisms and Effectors of NAFLD and AFLD. Front Physiol 2021;12:710285 View Article PubMed/NCBI
  18. Eslam M, Newsome PN, Sarin SK, Anstee QM, Targher G, Romero-Gomez M, et al. A new definition for metabolic dysfunction-associated fatty liver disease: An international expert consensus statement. J Hepatol 2020;73(1):202-209 View Article PubMed/NCBI
  19. Eslam M, Sanyal AJ, George J, International Consensus Panel. MAFLD: A Consensus-Driven Proposed Nomenclature for Metabolic Associated Fatty Liver Disease. Gastroenterology 2020;158(7):1999-2014.e1 View Article PubMed/NCBI
  20. Huang Q, Zou X, Wen X, Zhou X, Ji L. NAFLD or MAFLD: Which Has Closer Association With All-Cause and Cause-Specific Mortality?-Results From NHANES III. Front Med (Lausanne) 2021;8:693507 View Article PubMed/NCBI
  21. Wang X, Xie Q. Metabolic Dysfunction-associated Fatty Liver Disease (MAFLD) and Viral Hepatitis. J Clin Transl Hepatol 2022;10(1):128-133 View Article PubMed/NCBI
  22. Hardy T, Oakley F, Anstee QM, Day CP. Nonalcoholic Fatty Liver Disease: Pathogenesis and Disease Spectrum. Annu Rev Pathol 2016;11:451-496 View Article PubMed/NCBI
  23. Schuster S, Cabrera D, Arrese M, Feldstein AE. Triggering and resolution of inflammation in NASH. Nat Rev Gastroenterol Hepatol 2018;15(6):349-364 View Article PubMed/NCBI
  24. Hosseini N, Shor J, Szabo G. Alcoholic Hepatitis: A Review. Alcohol Alcohol 2019;54(4):408-416 View Article PubMed/NCBI
  25. Aron-Wisnewsky J, Vigliotti C, Witjes J, Le P, Holleboom AG, Verheij J, et al. Gut microbiota and human NAFLD: disentangling microbial signatures from metabolic disorders. Nat Rev Gastroenterol Hepatol 2020;17(5):279-297 View Article PubMed/NCBI
  26. Eslam M, George J. Genetic contributions to NAFLD: leveraging shared genetics to uncover systems biology. Nat Rev Gastroenterol Hepatol 2020;17(1):40-52 View Article PubMed/NCBI
  27. Peng C, Stewart AG, Woodman OL, Ritchie RH, Qin CX. Non-Alcoholic Steatohepatitis: A Review of Its Mechanism, Models and Medical Treatments. Front Pharmacol 2020;11:603926 View Article PubMed/NCBI
  28. Pouwels S, Sakran N, Graham Y, Leal A, Pintar T, Yang W, et al. Non-alcoholic fatty liver disease (NAFLD): a review of pathophysiology, clinical management and effects of weight loss. BMC Endocr Disord 2022;22(1):63 View Article PubMed/NCBI
  29. Sheka AC, Adeyi O, Thompson J, Hameed B, Crawford PA, Ikramuddin S. Nonalcoholic Steatohepatitis: A Review. JAMA 2020;323(12):1175-1183 View Article PubMed/NCBI
  30. Wattacheril J, Issa D, Sanyal A. Nonalcoholic Steatohepatitis (NASH) and Hepatic Fibrosis: Emerging Therapies. Annu Rev Pharmacol Toxicol 2018;58:649-662 View Article PubMed/NCBI
  31. Dufour JF, Anstee QM, Bugianesi E, Harrison S, Loomba R, Paradis V, et al. Current therapies and new developments in NASH. Gut 2022;71(10):2123-2134 View Article PubMed/NCBI
  32. Ye F, Zhai M, Long J, Gong Y, Ren C, Zhang D, et al. The burden of liver cirrhosis in mortality: Results from the global burden of disease study. Front Public Health 2022;10:909455 View Article PubMed/NCBI
  33. Qu W, Ma T, Cai J, Zhang X, Zhang P, She Z, et al. Liver Fibrosis and MAFLD: From Molecular Aspects to Novel Pharmacological Strategies. Front Med (Lausanne) 2021;8:761538 View Article PubMed/NCBI
  34. Ginès P, Krag A, Abraldes JG, Solà E, Fabrellas N, Kamath PS. Liver cirrhosis. Lancet 2021;398(10308):1359-1376 View Article PubMed/NCBI
  35. Trinchet JC, Bourcier V, Chaffaut C, Ait Ahmed M, Allam S, Marcellin P, et al. Complications and competing risks of death in compensated viral cirrhosis (ANRS CO12 CirVir prospective cohort). Hepatology 2015;62(3):737-750 View Article PubMed/NCBI
  36. Singal AK, Kamath PS. Model for End-stage Liver Disease. J Clin Exp Hepatol 2013;3(1):50-60 View Article PubMed/NCBI
  37. Jalan R, Perricone G, Moreau R, Arroyo V, Williams R. Acute-on-Chronic Liver Failure: A New Disease or an Old One Hiding in Plain Sight?. Clin Liver Dis (Hoboken) 2020;15(Suppl 1):S45-S51 View Article PubMed/NCBI
  38. Lambert MP, Paliwal A, Vaissière T, Chemin I, Zoulim F, Tommasino M, et al. Aberrant DNA methylation distinguishes hepatocellular carcinoma associated with HBV and HCV infection and alcohol intake. J Hepatol 2011;54(4):705-715 View Article PubMed/NCBI
  39. Suresh D, Srinivas AN, Kumar DP. Etiology of Hepatocellular Carcinoma: Special Focus on Fatty Liver Disease. Front Oncol 2020;10:601710 View Article PubMed/NCBI
  40. Burton A, Balachandrakumar VK, Driver RJ, Tataru D, Paley L, Marshall A, et al. Regional variations in hepatocellular carcinoma incidence, routes to diagnosis, treatment and survival in England. Br J Cancer 2022;126(5):804-814 View Article PubMed/NCBI
  41. Calderaro J, Ziol M, Paradis V, Zucman-Rossi J. Molecular and histological correlations in liver cancer. J Hepatol 2019;71(3):616-630 View Article PubMed/NCBI
  42. Kinoshita A, Onoda H, Fushiya N, Koike K, Nishino H, Tajiri H. Staging systems for hepatocellular carcinoma: Current status and future perspectives. World J Hepatol 2015;7(3):406-424 View Article PubMed/NCBI
  43. Bednarsch J, Czigany Z, Heise D, Joechle K, Luedde T, Heij L, et al. Prognostic evaluation of HCC patients undergoing surgical resection: an analysis of 8 different staging systems. Langenbecks Arch Surg 2021;406(1):75-86 View Article PubMed/NCBI
  44. de Lope CR, Tremosini S, Forner A, Reig M, Bruix J. Management of HCC. J Hepatol 2012;56(Suppl 1):S75-S87 View Article PubMed/NCBI
  45. Llovet JM, Ricci S, Mazzaferro V, Hilgard P, Gane E, Blanc JF, et al. Sorafenib in advanced hepatocellular carcinoma. N Engl J Med 2008;359(4):378-390 View Article PubMed/NCBI
  46. Kudo M, Finn RS, Qin S, Han KH, Ikeda K, Piscaglia F, et al. Lenvatinib versus sorafenib in first-line treatment of patients with unresectable hepatocellular carcinoma: a randomised phase 3 non-inferiority trial. Lancet 2018;391(10126):1163-1173 View Article PubMed/NCBI
  47. Tang W, Chen Z, Zhang W, Cheng Y, Zhang B, Wu F, et al. The mechanisms of sorafenib resistance in hepatocellular carcinoma: theoretical basis and therapeutic aspects. Signal Transduct Target Ther 2020;5(1):87 View Article PubMed/NCBI
  48. Zehir A, Benayed R, Shah RH, Syed A, Middha S, Kim HR, et al. Mutational landscape of metastatic cancer revealed from prospective clinical sequencing of 10,000 patients. Nat Med 2017;23(6):703-713 View Article PubMed/NCBI
  49. Patel N, Yopp AC, Singal AG. Diagnostic delays are common among patients with hepatocellular carcinoma. J Natl Compr Canc Netw 2015;13(5):543-549 View Article PubMed/NCBI
  50. Atiq O, Tiro J, Yopp AC, Muffler A, Marrero JA, Parikh ND, et al. An assessment of benefits and harms of hepatocellular carcinoma surveillance in patients with cirrhosis. Hepatology 2017;65(4):1196-1205 View Article PubMed/NCBI
  51. Tzartzeva K, Obi J, Rich NE, Parikh ND, Marrero JA, Yopp A, et al. Surveillance Imaging and Alpha Fetoprotein for Early Detection of Hepatocellular Carcinoma in Patients With Cirrhosis: A Meta-analysis. Gastroenterology 2018;154(6):1706-1718.e1 View Article PubMed/NCBI
  52. European Association for the Study of the Liver (EASL)., European Association for the Study of Diabetes (EASD)., European Association for the Study of Obesity (EASO). EASL-EASD-EASO Clinical Practice Guidelines for the management of non-alcoholic fatty liver disease. J Hepatol 2016;64(6):1388-1402 View Article PubMed/NCBI
  53. Ando Y, Jou JH. Nonalcoholic Fatty Liver Disease and Recent Guideline Updates. Clin Liver Dis (Hoboken) 2021;17(1):23-28 View Article PubMed/NCBI
  54. Wong VW, Chan WK, Chitturi S, Chawla Y, Dan YY, Duseja A, et al. Asia-Pacific Working Party on Non-alcoholic Fatty Liver Disease guidelines 2017-Part 1: Definition, risk factors and assessment. J Gastroenterol Hepatol 2018;33(1):70-85 View Article PubMed/NCBI
  55. Chitturi S, Wong VW, Chan WK, Wong GL, Wong SK, Sollano J, et al. The Asia-Pacific Working Party on Non-alcoholic Fatty Liver Disease guidelines 2017-Part 2: Management and special groups. J Gastroenterol Hepatol 2018;33(1):86-98 View Article PubMed/NCBI
  56. Eslam M, Sarin SK, Wong VW, Fan JG, Kawaguchi T, Ahn SH, et al. The Asian Pacific Association for the Study of the Liver clinical practice guidelines for the diagnosis and management of metabolic associated fatty liver disease. Hepatol Int 2020;14(6):889-919 View Article PubMed/NCBI
  57. Fouad YM, Gomaa A, El Etreby RM, AbdAllah M, Attia D. Editorial: The Metabolic (Dysfunction)-Associated Fatty Liver Disease (MAFLD) and Non-Alcoholic Fatty Liver Disease (NAFLD) Debate: Why the American Association for the Study of Liver Diseases (AASLD) and European Association for the Study of the Liver (EASL) Consensus Process is Not Representative. Med Sci Monit 2022;28:e938066 View Article PubMed/NCBI
  58. Dulai PS, Sirlin CB, Loomba R. MRI and MRE for non-invasive quantitative assessment of hepatic steatosis and fibrosis in NAFLD and NASH: Clinical trials to clinical practice. J Hepatol 2016;65(5):1006-1016 View Article PubMed/NCBI
  59. Ascha MS, Hanouneh IA, Lopez R, Tamimi TA, Feldstein AF, Zein NN. The incidence and risk factors of hepatocellular carcinoma in patients with nonalcoholic steatohepatitis. Hepatology 2010;51(6):1972-1978 View Article PubMed/NCBI
  60. Rinella ME, Sanyal AJ. Management of NAFLD: a stage-based approach. Nat Rev Gastroenterol Hepatol 2016;13(4):196-205 View Article PubMed/NCBI
  61. Arun J, Jhala N, Lazenby AJ, Clements R, Abrams GA. Influence of liver biopsy heterogeneity and diagnosis of nonalcoholic steatohepatitis in subjects undergoing gastric bypass. Obes Surg 2007;17(2):155-161 View Article PubMed/NCBI
  62. Yoshiji H, Nagoshi S, Akahane T, Asaoka Y, Ueno Y, Ogawa K, et al. Evidence-based clinical practice guidelines for liver cirrhosis 2020. Hepatol Res 2021;51(7):725-749 View Article PubMed/NCBI
  63. Shiha G, Ibrahim A, Helmy A, Sarin SK, Omata M, Kumar A, et al. Asian-Pacific Association for the Study of the Liver (APASL) consensus guidelines on invasive and non-invasive assessment of hepatic fibrosis: a 2016 update. Hepatol Int 2017;11(1):1-30 View Article PubMed/NCBI
  64. Ozturk A, Grajo JR, Dhyani M, Anthony BW, Samir AE. Principles of ultrasound elastography. Abdom Radiol (NY) 2018;43(4):773-785 View Article PubMed/NCBI
  65. Costentin CE, Layese R, Bourcier V, Cagnot C, Marcellin P, Guyader D, et al. Compliance With Hepatocellular Carcinoma Surveillance Guidelines Associated With Increased Lead-Time Adjusted Survival of Patients With Compensated Viral Cirrhosis: A Multi-Center Cohort Study. Gastroenterology 2018;155(2):431-442.e10 View Article PubMed/NCBI
  66. European Association for the Study of the Liver. EASL Clinical Practice Guidelines: Management of hepatocellular carcinoma. J Hepatol 2018;69(1):182-236 View Article PubMed/NCBI
  67. Heimbach JK, Kulik LM, Finn RS, Sirlin CB, Abecassis MM, Roberts LR, et al. AASLD guidelines for the treatment of hepatocellular carcinoma. Hepatology 2018;67(1):358-380 View Article PubMed/NCBI
  68. Tang H, Huang Y, Duan W, Li C, Meng X, Dong J. A concise review of current guidelines for the clinical management of hepatocellular carcinoma in Asia. Transl Cancer Res 2017;6(6):1214-1225 View Article PubMed/NCBI
  69. Jiang P, Chan CW, Chan KC, Cheng SH, Wong J, Wong VW, et al. Lengthening and shortening of plasma DNA in hepatocellular carcinoma patients. Proc Natl Acad Sci U S A 2015;112(11):E1317-E1325 View Article PubMed/NCBI
  70. Ng CKY, Di Costanzo GG, Tosti N, Paradiso V, Coto-Llerena M, Roscigno G, et al. Genetic profiling using plasma-derived cell-free DNA in therapy-naïve hepatocellular carcinoma patients: a pilot study. Ann Oncol 2018;29(5):1286-1291 View Article PubMed/NCBI
  71. Lee HW, Kim E, Cho KJ, Park HJ, Seo J, Lee H, et al. Applications of molecular barcode sequencing for the detection of low-frequency variants in circulating tumour DNA from hepatocellular carcinoma. Liver Int 2022;42(10):2317-2326 View Article PubMed/NCBI
  72. Ono A, Fujimoto A, Yamamoto Y, Akamatsu S, Hiraga N, Imamura M, et al. Circulating Tumor DNA Analysis for Liver Cancers and Its Usefulness as a Liquid Biopsy. Cell Mol Gastroenterol Hepatol 2015;1(5):516-534 View Article PubMed/NCBI
  73. von Felden J, Craig AJ, Garcia-Lezana T, Labgaa I, Haber PK, D’Avola D, et al. Mutations in circulating tumor DNA predict primary resistance to systemic therapies in advanced hepatocellular carcinoma. Oncogene 2021;40(1):140-151 View Article PubMed/NCBI
  74. Karlas T, Weise L, Kuhn S, Krenzien F, Mehdorn M, Petroff D, et al. Correlation of cell-free DNA plasma concentration with severity of non-alcoholic fatty liver disease. J Transl Med 2017;15(1):106 View Article PubMed/NCBI
  75. Hardy T, Zeybel M, Day CP, Dipper C, Masson S, McPherson S, et al. Plasma DNA methylation: a potential biomarker for stratification of liver fibrosis in non-alcoholic fatty liver disease. Gut 2017;66(7):1321-1328 View Article PubMed/NCBI
  76. Sun QF, Tang LJ, Wang MJ, Zhu PW, Li YY, Ma HL, et al. Potential Blood DNA Methylation Biomarker Genes for Diagnosis of Liver Fibrosis in Patients With Biopsy-Proven Non-alcoholic Fatty Liver Disease. Front Med (Lausanne) 2022;9:864570 View Article PubMed/NCBI
  77. Buzova D, Braghini MR, Bianco SD, Lo Re O, Raffaele M, Frohlich J, et al. Profiling of cell-free DNA methylation and histone signatures in pediatric NAFLD: A pilot study. Hepatol Commun 2022;6(12):3311-3323 View Article PubMed/NCBI
  78. Chrysavgis L, Papatheodoridi A, Cholongitas E, Koutsilieris M, Papatheodoridis G, Chatzigeorgiou A. Significance of Circulating Cell-Free DNA Species in Non-Alcoholic Fatty Liver Disease. Int J Mol Sci 2021;22(16):8849 View Article PubMed/NCBI
  79. Tseng KC, Chou JL, Huang HB, Tseng CW, Wu SF, Chan MW. SOCS-1 promoter methylation and treatment response in chronic hepatitis C patients receiving pegylated-interferon/ribavirin. J Clin Immunol 2013;33(6):1110-1116 View Article PubMed/NCBI
  80. Alunni-Fabbroni M, Weber S, Öcal O, Seidensticker M, Mayerle J, Malfertheiner P, et al. Circulating Cell-Free DNA Combined to Magnetic Resonance Imaging for Early Detection of HCC in Patients with Liver Cirrhosis. Cancers (Basel) 2021;13(3):521 View Article PubMed/NCBI
  81. Akuta N, Kawamura Y, Kobayashi M, Arase Y, Saitoh S, Fujiyama S, et al. TERT Promoter Mutation in Serum Cell-Free DNA Is a Diagnostic Marker of Primary Hepatocellular Carcinoma in Patients with Nonalcoholic Fatty Liver Disease. Oncology 2021;99(2):114-123 View Article PubMed/NCBI
  82. Akuta N, Kawamura Y, Suzuki F, Kobayashi M, Arase Y, Saitoh S, et al. Serum TERT C228T is an important predictor of non-viral liver cancer with fatty liver disease. Hepatol Int 2022;16(2):412-422 View Article PubMed/NCBI
  83. Huang A, Zhang X, Zhou SL, Cao Y, Huang XW, Fan J, et al. Plasma Circulating Cell-free DNA Integrity as a Promising Biomarker for Diagnosis and Surveillance in Patients with Hepatocellular Carcinoma. J Cancer 2016;7(13):1798-1803 View Article PubMed/NCBI
  84. Alunni-Fabbroni M, Rönsch K, Huber T, Cyran CC, Seidensticker M, Mayerle J, et al. Circulating DNA as prognostic biomarker in patients with advanced hepatocellular carcinoma: a translational exploratory study from the SORAMIC trial. J Transl Med 2019;17(1):328 View Article PubMed/NCBI
  85. Luo B, Ma F, Liu H, Hu J, Rao L, Liu C, et al. Cell-free DNA methylation markers for differential diagnosis of hepatocellular carcinoma. BMC Med 2022;20(1):8 View Article PubMed/NCBI
  86. Tian MM, Fan YC, Zhao J, Gao S, Zhao ZH, Chen LY, et al. Hepatocellular carcinoma suppressor 1 promoter hypermethylation in serum. A diagnostic and prognostic study in hepatitis B. Clin Res Hepatol Gastroenterol 2017;41(2):171-180 View Article PubMed/NCBI
  87. Fateen W, Johnson PJ, Wood HM, Zhang H, He S, El-Meteini M, et al. Characterisation of dysplastic liver nodules using low-pass DNA sequencing and detection of chromosome arm-level abnormalities in blood-derived cell-free DNA. J Pathol 2021;255(1):30-40 View Article PubMed/NCBI
  88. Chen L, Abou-Alfa GK, Zheng B, Liu JF, Bai J, Du LT, et al. Genome-scale profiling of circulating cell-free DNA signatures for early detection of hepatocellular carcinoma in cirrhotic patients. Cell Res 2021;31(5):589-592 View Article PubMed/NCBI
  89. Xu RH, Wei W, Krawczyk M, Wang W, Luo H, Flagg K, et al. Circulating tumour DNA methylation markers for diagnosis and prognosis of hepatocellular carcinoma. Nat Mater 2017;16(11):1155-1161 View Article PubMed/NCBI
  90. Lewin J, Kottwitz D, Aoyama J, deVos T, Garces J, Hasinger O, et al. Plasma cell free DNA methylation markers for hepatocellular carcinoma surveillance in patients with cirrhosis: a case control study. BMC Gastroenterol 2021;21(1):136 View Article PubMed/NCBI
  91. Huang Y, Wei L, Zhao RC, Liang WB, Zhang J, Ding XQ, et al. Predicting hepatocellular carcinoma development for cirrhosis patients via methylation detection of heparocarcinogenesis-related genes. J Cancer 2018;9(12):2203-2210 View Article PubMed/NCBI
  92. Zhang H, Dong P, Guo S, Tao C, Chen W, Zhao W, et al. Hypomethylation in HBV integration regions aids non-invasive surveillance to hepatocellular carcinoma by low-pass genome-wide bisulfite sequencing. BMC Med 2020;18(1):200 View Article PubMed/NCBI
  93. Foda ZH, Annapragada AV, Boyapati K, Bruhm DC, Vulpescu NA, Medina JE, et al. Detecting Liver Cancer Using Cell-Free DNA Fragmentomes. Cancer Discov 2023;13(3):616-631 View Article PubMed/NCBI
  94. Gonçalves E, Gonçalves-Reis M, Pereira-Leal JB, Cardoso J. DNA methylation fingerprint of hepatocellular carcinoma from tissue and liquid biopsies. Sci Rep 2022;12(1):11512 View Article PubMed/NCBI
  95. Jiang P, Sun K, Tong YK, Cheng SH, Cheng THT, Heung MMS, et al. Preferred end coordinates and somatic variants as signatures of circulating tumor DNA associated with hepatocellular carcinoma. Proc Natl Acad Sci U S A 2018;115(46):E10925-E10933 View Article PubMed/NCBI
  96. Xia WY, Gao L, Dai EH, Chen D, Xie EF, Yang L, et al. Liquid biopsy for non-invasive assessment of liver injury in hepatitis B patients. World J Gastroenterol 2019;25(29):3985-3995 View Article PubMed/NCBI
  97. Krenzien F, Katou S, Papa A, Sinn B, Benzing C, Feldbrügge L, et al. Increased Cell-Free DNA Plasma Concentration Following Liver Transplantation Is Linked to Portal Hepatitis and Inferior Survival. J Clin Med 2020;9(5):1543 View Article PubMed/NCBI
  98. Tokuhisa Y, Iizuka N, Sakaida I, Moribe T, Fujita N, Miura T, et al. Circulating cell-free DNA as a predictive marker for distant metastasis of hepatitis C virus-related hepatocellular carcinoma. Br J Cancer 2007;97(10):1399-1403 View Article PubMed/NCBI
  99. Valpione S, Gremel G, Mundra P, Middlehurst P, Galvani E, Girotti MR, et al. Plasma total cell-free DNA (cfDNA) is a surrogate biomarker for tumour burden and a prognostic biomarker for survival in metastatic melanoma patients. Eur J Cancer 2018;88:1-9 View Article PubMed/NCBI
  100. Hamfjord J, Guren TK, Dajani O, Johansen JS, Glimelius B, Sorbye H, et al. Total circulating cell-free DNA as a prognostic biomarker in metastatic colorectal cancer before first-line oxaliplatin-based chemotherapy. Ann Oncol 2019;30(7):1088-1095 View Article PubMed/NCBI
  101. Mouliere F, Chandrananda D, Piskorz AM, Moore EK, Morris J, Ahlborn LB, et al. Enhanced detection of circulating tumor DNA by fragment size analysis. Sci Transl Med 2018;10(466):eaat4921 View Article PubMed/NCBI
  102. Markus H, Chandrananda D, Moore E, Mouliere F, Morris J, Brenton JD, et al. Refined characterization of circulating tumor DNA through biological feature integration. Sci Rep 2022;12(1):1928 View Article PubMed/NCBI
  103. Mayo-de-Las-Casas C, Jordana-Ariza N, Garzón-Ibañez M, Balada-Bel A, Bertrán-Alamillo J, Viteri-Ramírez S, et al. Large scale, prospective screening of EGFR mutations in the blood of advanced NSCLC patients to guide treatment decisions. Ann Oncol 2017;28(9):2248-2255 View Article PubMed/NCBI
  104. Ekstedt M, Hagström H, Nasr P, Fredrikson M, Stål P, Kechagias S, et al. Fibrosis stage is the strongest predictor for disease-specific mortality in NAFLD after up to 33 years of follow-up. Hepatology 2015;61(5):1547-1554 View Article PubMed/NCBI
  105. Angulo P, Kleiner DE, Dam-Larsen S, Adams LA, Bjornsson ES, Charatcharoenwitthaya P, et al. Liver Fibrosis, but No Other Histologic Features, Is Associated With Long-term Outcomes of Patients With Nonalcoholic Fatty Liver Disease. Gastroenterology 2015;149(2):389-97.e10 View Article PubMed/NCBI
  106. Diehl F, Schmidt K, Choti MA, Romans K, Goodman S, Li M, et al. Circulating mutant DNA to assess tumor dynamics. Nat Med 2008;14(9):985-990 View Article PubMed/NCBI
  107. Esteller M. Non-coding RNAs in human disease. Nat Rev Genet 2011;12(12):861-874 View Article PubMed/NCBI
  108. Statello L, Guo CJ, Chen LL, Huarte M. Gene regulation by long non-coding RNAs and its biological functions. Nat Rev Mol Cell Biol 2021;22(2):96-118 View Article PubMed/NCBI
  109. Cai Y, Yu X, Hu S, Yu J. A brief review on the mechanisms of miRNA regulation. Genomics Proteomics Bioinformatics 2009;7(4):147-154 View Article PubMed/NCBI
  110. Breving K, Esquela-Kerscher A. The complexities of microRNA regulation: mirandering around the rules. Int J Biochem Cell Biol 2010;42(8):1316-1329 View Article PubMed/NCBI
  111. Geng X, Jia Y, Zhang Y, Shi L, Li Q, Zang A, et al. Circular RNA: biogenesis, degradation, functions and potential roles in mediating resistance to anticarcinogens. Epigenomics 2020;12(3):267-283 View Article PubMed/NCBI
  112. Yu F, Zheng J, Mao Y, Dong P, Li G, Lu Z, et al. Long non-coding RNA APTR promotes the activation of hepatic stellate cells and the progression of liver fibrosis. Biochem Biophys Res Commun 2015;463(4):679-685 View Article PubMed/NCBI
  113. DiStefano JK, Gerhard GS. Long Noncoding RNAs and Human Liver Disease. Annu Rev Pathol 2022;17:1-21 View Article PubMed/NCBI
  114. Cermelli S, Ruggieri A, Marrero JA, Ioannou GN, Beretta L. Circulating microRNAs in patients with chronic hepatitis C and non-alcoholic fatty liver disease. PLoS One 2011;6(8):e23937 View Article PubMed/NCBI
  115. Pirola CJ, Fernández Gianotti T, Castaño GO, Mallardi P, San Martino J, Mora Gonzalez Lopez Ledesma M, et al. Circulating microRNA signature in non-alcoholic fatty liver disease: from serum non-coding RNAs to liver histology and disease pathogenesis. Gut 2015;64(5):800-812 View Article PubMed/NCBI
  116. Han MH, Lee JH, Kim G, Lee E, Lee YR, Jang SY, et al. Expression of the Long Noncoding RNA GAS5 Correlates with Liver Fibrosis in Patients with Nonalcoholic Fatty Liver Disease. Genes (Basel) 2020;11(5):545 View Article PubMed/NCBI
  117. Kim TH, Lee Y, Lee YS, Gim JA, Ko E, Yim SY, et al. Circulating miRNA is a useful diagnostic biomarker for nonalcoholic steatohepatitis in nonalcoholic fatty liver disease. Sci Rep 2021;11(1):14639 View Article PubMed/NCBI
  118. Tomimaru Y, Eguchi H, Nagano H, Wada H, Kobayashi S, Marubashi S, et al. Circulating microRNA-21 as a novel biomarker for hepatocellular carcinoma. J Hepatol 2012;56(1):167-175 View Article PubMed/NCBI
  119. Zhou J, Yu L, Gao X, Hu J, Wang J, Dai Z, et al. Plasma microRNA panel to diagnose hepatitis B virus-related hepatocellular carcinoma. J Clin Oncol 2011;29(36):4781-4788 View Article PubMed/NCBI
  120. Blaya D, Pose E, Coll M, Lozano JJ, Graupera I, Schierwagen R, et al. Profiling circulating microRNAs in patients with cirrhosis and acute-on-chronic liver failure. JHEP Rep 2021;3(2):100233 View Article PubMed/NCBI
  121. Salvoza NC, Klinzing DC, Gopez-Cervantes J, Baclig MO. Association of Circulating Serum miR-34a and miR-122 with Dyslipidemia among Patients with Non-Alcoholic Fatty Liver Disease. PLoS One 2016;11(4):e0153497 View Article PubMed/NCBI
  122. López-Riera M, Conde I, Quintas G, Pedrola L, Zaragoza Á, Perez-Rojas J, et al. Non-invasive prediction of NAFLD severity: a comprehensive, independent validation of previously postulated serum microRNA biomarkers. Sci Rep 2018;8(1):10606 View Article PubMed/NCBI
  123. Zhang H, Li QY, Guo ZZ, Guan Y, Du J, Lu YY, et al. Serum levels of microRNAs can specifically predict liver injury of chronic hepatitis B. World J Gastroenterol 2012;18(37):5188-5196 View Article PubMed/NCBI
  124. Yu F, Zhou G, Huang K, Fan X, Li G, Chen B, et al. Serum lincRNA-p21 as a potential biomarker of liver fibrosis in chronic hepatitis B patients. J Viral Hepat 2017;24(7):580-588 View Article PubMed/NCBI
  125. Matsuura K, Aizawa N, Enomoto H, Nishiguchi S, Toyoda H, Kumada T, et al. Circulating let-7 Levels in Serum Correlate With the Severity of Hepatic Fibrosis in Chronic Hepatitis C. Open Forum Infect Dis 2018;5(11):ofy268 View Article PubMed/NCBI
  126. Park JG, Kim G, Jang SY, Lee YR, Lee E, Lee HW, et al. Plasma Long Noncoding RNA LeXis is a Potential Diagnostic Marker for Non-Alcoholic Steatohepatitis. Life (Basel) 2020;10(10):E230 View Article PubMed/NCBI
  127. Yang Z, Ross RA, Zhao S, Tu W, Liangpunsakul S, Wang L. LncRNA AK054921 and AK128652 are potential serum biomarkers and predictors of patient survival with alcoholic cirrhosis. Hepatol Commun 2017;1(6):513-523 View Article PubMed/NCBI
  128. Lin XJ, Chong Y, Guo ZW, Xie C, Yang XJ, Zhang Q, et al. A serum microRNA classifier for early detection of hepatocellular carcinoma: a multicentre, retrospective, longitudinal biomarker identification study with a nested case-control study. Lancet Oncol 2015;16(7):804-815 View Article PubMed/NCBI
  129. Tang J, Jiang R, Deng L, Zhang X, Wang K, Sun B. Circulation long non-coding RNAs act as biomarkers for predicting tumorigenesis and metastasis in hepatocellular carcinoma. Oncotarget 2015;6(6):4505-4515 View Article PubMed/NCBI
  130. Luo P, Liang C, Zhang X, Liu X, Wang Y, Wu M, et al. Identification of long non-coding RNA ZFAS1 as a novel biomarker for diagnosis of HCC. Biosci Rep 2018;38(4):BSR20171359 View Article PubMed/NCBI
  131. Mohyeldeen M, Ibrahim S, Shaker O, Helmy H. Serum expression and diagnostic potential of long non-coding RNAs NEAT1 and TUG1 in viral hepatitis C and viral hepatitis C-associated hepatocellular carcinoma. Clin Biochem 2020;84:38-44 View Article PubMed/NCBI
  132. Oksuz Z, Serin MS, Kaplan E, Dogen A, Tezcan S, Aslan G, et al. Serum microRNAs; miR-30c-5p, miR-223-3p, miR-302c-3p and miR-17-5p could be used as novel non-invasive biomarkers for HCV-positive cirrhosis and hepatocellular carcinoma. Mol Biol Rep 2015;42(3):713-720 View Article PubMed/NCBI
  133. Fornari F, Pollutri D, Patrizi C, La Bella T, Marinelli S, Casadei Gardini A, et al. In Hepatocellular Carcinoma miR-221 Modulates Sorafenib Resistance through Inhibition of Caspase-3-Mediated Apoptosis. Clin Cancer Res 2017;23(14):3953-3965 View Article PubMed/NCBI
  134. Kishimoto T, Eguchi H, Nagano H, Kobayashi S, Akita H, Hama N, et al. Plasma miR-21 is a novel diagnostic biomarker for biliary tract cancer. Cancer Sci 2013;104(12):1626-1631 View Article PubMed/NCBI
  135. Liu C, Hou X, Mo K, Li N, An C, Liu G, et al. Serum non-coding RNAs for diagnosis and stage of liver fibrosis. J Clin Lab Anal 2022;36(10):e24658 View Article PubMed/NCBI
  136. Ge W, Yu DC, Li QG, Chen X, Zhang CY, Ding YT. Expression of serum miR-16, let-7f, and miR-21 in patients with hepatocellular carcinoma and their clinical significances. Clin Lab 2014;60(3):427-434 View Article PubMed/NCBI
  137. Baccelli I, Schneeweiss A, Riethdorf S, Stenzinger A, Schillert A, Vogel V, et al. Identification of a population of blood circulating tumor cells from breast cancer patients that initiates metastasis in a xenograft assay. Nat Biotechnol 2013;31(6):539-544 View Article PubMed/NCBI
  138. Hodgkinson CL, Morrow CJ, Li Y, Metcalf RL, Rothwell DG, Trapani F, et al. Tumorigenicity and genetic profiling of circulating tumor cells in small-cell lung cancer. Nat Med 2014;20(8):897-903 View Article PubMed/NCBI
  139. Schuster E, Taftaf R, Reduzzi C, Albert MK, Romero-Calvo I, Liu H. Better together: circulating tumor cell clustering in metastatic cancer. Trends Cancer 2021;7(11):1020-1032 View Article PubMed/NCBI
  140. Bidard FC, Peeters DJ, Fehm T, Nolé F, Gisbert-Criado R, Mavroudis D, et al. Clinical validity of circulating tumour cells in patients with metastatic breast cancer: a pooled analysis of individual patient data. Lancet Oncol 2014;15(4):406-414 View Article PubMed/NCBI
  141. Qi LN, Xiang BD, Wu FX, Ye JZ, Zhong JH, Wang YY, et al. Circulating Tumor Cells Undergoing EMT Provide a Metric for Diagnosis and Prognosis of Patients with Hepatocellular Carcinoma. Cancer Res 2018;78(16):4731-4744 View Article PubMed/NCBI
  142. Lin D, Shen L, Luo M, Zhang K, Li J, Yang Q, et al. Circulating tumor cells: biology and clinical significance. Signal Transduct Target Ther 2021;6(1):404 View Article PubMed/NCBI
  143. Kelley RK, Magbanua MJ, Butler TM, Collisson EA, Hwang J, Sidiropoulos N, et al. Circulating tumor cells in hepatocellular carcinoma: a pilot study of detection, enumeration, and next-generation sequencing in cases and controls. BMC Cancer 2015;15:206 View Article PubMed/NCBI
  144. Sun YF, Xu Y, Yang XR, Guo W, Zhang X, Qiu SJ, et al. Circulating stem cell-like epithelial cell adhesion molecule-positive tumor cells indicate poor prognosis of hepatocellular carcinoma after curative resection. Hepatology 2013;57(4):1458-1468 View Article PubMed/NCBI
  145. Winograd P, Hou S, Court CM, Lee YT, Chen PJ, Zhu Y, et al. Hepatocellular Carcinoma-Circulating Tumor Cells Expressing PD-L1 Are Prognostic and Potentially Associated With Response to Checkpoint Inhibitors. Hepatol Commun 2020;4(10):1527-1540 View Article PubMed/NCBI
  146. Su K, Guo L, He K, Rao M, Zhang J, Yang X, et al. PD-L1 expression on circulating tumor cells can be a predictive biomarker to PD-1 inhibitors combined with radiotherapy and antiangiogenic therapy in advanced hepatocellular carcinoma. Front Oncol 2022;12:873830 View Article PubMed/NCBI
  147. Visal TH, den Hollander P, Cristofanilli M, Mani SA. Circulating tumour cells in the -omics era: how far are we from achieving the ‘singularity’?. Br J Cancer 2022;127(2):173-184 View Article PubMed/NCBI
  148. Sharma S, Zhuang R, Long M, Pavlovic M, Kang Y, Ilyas A, et al. Circulating tumor cell isolation, culture, and downstream molecular analysis. Biotechnol Adv 2018;36(4):1063-1078 View Article PubMed/NCBI
  149. Saucedo-Zeni N, Mewes S, Niestroj R, Gasiorowski L, Murawa D, Nowaczyk P, et al. A novel method for the in vivo isolation of circulating tumor cells from peripheral blood of cancer patients using a functionalized and structured medical wire. Int J Oncol 2012;41(4):1241-1250 View Article PubMed/NCBI
  150. He Y, Shi J, Shi G, Xu X, Liu Q, Liu C, et al. Using the New CellCollector to Capture Circulating Tumor Cells from Blood in Different Groups of Pulmonary Disease: A Cohort Study. Sci Rep 2017;7(1):9542 View Article PubMed/NCBI
  151. Cristofanilli M, Budd GT, Ellis MJ, Stopeck A, Matera J, Miller MC, et al. Circulating tumor cells, disease progression, and survival in metastatic breast cancer. N Engl J Med 2004;351(8):781-791 View Article PubMed/NCBI
  152. Alix-Panabières C, Pantel K. Challenges in circulating tumour cell research. Nat Rev Cancer 2014;14(9):623-631 View Article PubMed/NCBI
  153. Gosens MJ, van Kempen LC, van de Velde CJ, van Krieken JH, Nagtegaal ID. Loss of membranous Ep-CAM in budding colorectal carcinoma cells. Mod Pathol 2007;20(2):221-232 View Article PubMed/NCBI
  154. Wang L, Balasubramanian P, Chen AP, Kummar S, Evrard YA, Kinders RJ. Promise and limits of the CellSearch platform for evaluating pharmacodynamics in circulating tumor cells. Semin Oncol 2016;43(4):464-475 View Article PubMed/NCBI
  155. Gao Y, Fan WH, Song Z, Lou H, Kang X. Comparison of circulating tumor cell (CTC) detection rates with epithelial cell adhesion molecule (EpCAM) and cell surface vimentin (CSV) antibodies in different solid tumors: a retrospective study. PeerJ 2021;9:e10777 View Article PubMed/NCBI
  156. Wang L, Li Y, Xu J, Zhang A, Wang X, Tang R, et al. Quantified postsurgical small cell size CTCs and EpCAM(+) circulating tumor stem cells with cytogenetic abnormalities in hepatocellular carcinoma patients determine cancer relapse. Cancer Lett 2018;412:99-107 View Article PubMed/NCBI
  157. Yates AG, Pink RC, Erdbrügger U, Siljander PR, Dellar ER, Pantazi P, et al. In sickness and in health: The functional role of extracellular vesicles in physiology and pathology in vivo: Part I: Health and Normal Physiology: Part I: Health and Normal Physiology. J Extracell Vesicles 2022;11(1):e12151 View Article PubMed/NCBI
  158. Newman LA, Muller K, Rowland A. Circulating cell-specific extracellular vesicles as biomarkers for the diagnosis and monitoring of chronic liver diseases. Cell Mol Life Sci 2022;79(5):232 View Article PubMed/NCBI
  159. Eguchi A, Mulya A, Lazic M, Radhakrishnan D, Berk MP, Povero D, et al. Microparticles release by adipocytes act as “find-me” signals to promote macrophage migration. PLoS One 2015;10(4):e0123110 View Article PubMed/NCBI
  160. Sugimachi K, Matsumura T, Hirata H, Uchi R, Ueda M, Ueo H, et al. Identification of a bona fide microRNA biomarker in serum exosomes that predicts hepatocellular carcinoma recurrence after liver transplantation. Br J Cancer 2015;112(3):532-538 View Article PubMed/NCBI
  161. Wang H, Hou L, Li A, Duan Y, Gao H, Song X. Expression of serum exosomal microRNA-21 in human hepatocellular carcinoma. Biomed Res Int 2014;2014:864894 View Article PubMed/NCBI
  162. Povero D, Yamashita H, Ren W, Subramanian MG, Myers RP, Eguchi A, et al. Characterization and Proteome of Circulating Extracellular Vesicles as Potential Biomarkers for NASH. Hepatol Commun 2020;4(9):1263-1278 View Article PubMed/NCBI
  163. Nakao Y, Amrollahi P, Parthasarathy G, Mauer AS, Sehrawat TS, Vanderboom P, et al. Circulating extracellular vesicles are a biomarker for NAFLD resolution and response to weight loss surgery. Nanomedicine 2021;36:102430 View Article PubMed/NCBI
  164. Nguyen HQ, Lee D, Kim Y, Bang G, Cho K, Lee YS, et al. Label-free quantitative proteomic analysis of serum extracellular vesicles differentiating patients of alcoholic and nonalcoholic fatty liver diseases. J Proteomics 2021;245:104278 View Article PubMed/NCBI
  165. Kornek M, Lynch M, Mehta SH, Lai M, Exley M, Afdhal NH, et al. Circulating microparticles as disease-specific biomarkers of severity of inflammation in patients with hepatitis C or nonalcoholic steatohepatitis. Gastroenterology 2012;143(2):448-458 View Article PubMed/NCBI
  166. Liu M, Liu X, Pan M, Zhang Y, Tang X, Liu W, et al. Characterization and microRNA Expression Analysis of Serum-Derived Extracellular Vesicles in Severe Liver Injury from Chronic HBV Infection. Life (Basel) 2023;13(2):347 View Article PubMed/NCBI
  167. Abe K, Suzuki R, Fujita M, Hayashi M, Takahashi A, Ohira H. Circulating extracellular vesicle-encapsulated microRNA-557 induces a proinflammatory immune response and serves as a diagnostic or relapse marker in autoimmune hepatitis. Hepatol Res 2022;52(12):1034-1049 View Article PubMed/NCBI
  168. Sehrawat TS, Arab JP, Liu M, Amrollahi P, Wan M, Fan J, et al. Circulating Extracellular Vesicles Carrying Sphingolipid Cargo for the Diagnosis and Dynamic Risk Profiling of Alcoholic Hepatitis. Hepatology 2021;73(2):571-585 View Article PubMed/NCBI
  169. Welker MW, Reichert D, Susser S, Sarrazin C, Martinez Y, Herrmann E, et al. Soluble serum CD81 is elevated in patients with chronic hepatitis C and correlates with alanine aminotransferase serum activity. PLoS One 2012;7(2):e30796 View Article PubMed/NCBI
  170. Shirai K, Hikita H, Sakane S, Narumi R, Adachi J, Doi A, et al. Serum amyloid P component and pro-platelet basic protein in extracellular vesicles or serum are novel markers of liver fibrosis in chronic hepatitis C patients. PLoS One 2022;17(7):e0271020 View Article PubMed/NCBI
  171. Devhare PB, Sasaki R, Shrivastava S, Di Bisceglie AM, Ray R, Ray RB. Exosome-Mediated Intercellular Communication between Hepatitis C Virus-Infected Hepatocytes and Hepatic Stellate Cells. J Virol 2017;91(6):e02225-16 View Article PubMed/NCBI
  172. Julich-Haertel H, Urban SK, Krawczyk M, Willms A, Jankowski K, Patkowski W, et al. Cancer-associated circulating large extracellular vesicles in cholangiocarcinoma and hepatocellular carcinoma. J Hepatol 2017;67(2):282-292 View Article PubMed/NCBI
  173. Arbelaiz A, Azkargorta M, Krawczyk M, Santos-Laso A, Lapitz A, Perugorria MJ, et al. Serum extracellular vesicles contain protein biomarkers for primary sclerosing cholangitis and cholangiocarcinoma. Hepatology 2017;66(4):1125-1143 View Article PubMed/NCBI
  174. Li L, Masica D, Ishida M, Tomuleasa C, Umegaki S, Kalloo AN, et al. Human bile contains microRNA-laden extracellular vesicles that can be used for cholangiocarcinoma diagnosis. Hepatology 2014;60(3):896-907 View Article PubMed/NCBI
  175. Kim SS, Baek GO, Ahn HR, Sung S, Seo CW, Cho HJ, et al. Serum small extracellular vesicle-derived LINC00853 as a novel diagnostic marker for early hepatocellular carcinoma. Mol Oncol 2020;14(10):2646-2659 View Article PubMed/NCBI
  176. Sohn W, Kim J, Kang SH, Yang SR, Cho JY, Cho HC, et al. Serum exosomal microRNAs as novel biomarkers for hepatocellular carcinoma. Exp Mol Med 2015;47(9):e184 View Article PubMed/NCBI
  177. Shao Y, Chen T, Zheng X, Yang S, Xu K, Chen X, et al. Colorectal cancer-derived small extracellular vesicles establish an inflammatory premetastatic niche in liver metastasis. Carcinogenesis 2018;39(11):1368-1379 View Article PubMed/NCBI
  178. Saha B, Momen-Heravi F, Kodys K, Szabo G. MicroRNA Cargo of Extracellular Vesicles from Alcohol-exposed Monocytes Signals Naive Monocytes to Differentiate into M2 Macrophages. J Biol Chem 2016;291(1):149-159 View Article PubMed/NCBI
  179. Eguchi A, Lazaro RG, Wang J, Kim J, Povero D, Willliams B, et al. Extracellular vesicles released by hepatocytes from gastric infusion model of alcoholic liver disease contain a MicroRNA barcode that can be detected in blood. Hepatology 2017;65(2):475-490 View Article PubMed/NCBI
  180. Garcia-Martinez I, Santoro N, Chen Y, Hoque R, Ouyang X, Caprio S, et al. Hepatocyte mitochondrial DNA drives nonalcoholic steatohepatitis by activation of TLR9. J Clin Invest 2016;126(3):859-864 View Article PubMed/NCBI
  181. Mateescu B, Kowal EJ, van Balkom BW, Bartel S, Bhattacharyya SN, Buzás EI, et al. Obstacles and opportunities in the functional analysis of extracellular vesicle RNA - an ISEV position paper. J Extracell Vesicles 2017;6(1):1286095 View Article PubMed/NCBI
  182. Brennan K, Martin K, FitzGerald SP, O’Sullivan J, Wu Y, Blanco A, et al. A comparison of methods for the isolation and separation of extracellular vesicles from protein and lipid particles in human serum. Sci Rep 2020;10(1):1039 View Article PubMed/NCBI
  183. Gutiérrez-Adrianzén OA, Koutouzov S, Mota RM, das Chagas Medeiros MM, Bach JF, de Holanda Campos H. Diagnostic value of anti-nucleosome antibodies in the assessment of disease activity of systemic lupus erythematosus: a prospective study comparing anti-nucleosome with anti-dsDNA antibodies. J Rheumatol 2006;33(8):1538-1544 View Article PubMed/NCBI
  184. Luger K, Mäder AW, Richmond RK, Sargent DF, Richmond TJ. Crystal structure of the nucleosome core particle at 2.8 A resolution. Nature 1997;389(6648):251-260 View Article PubMed/NCBI
  185. Burgess RJ, Zhang Z. Histone chaperones in nucleosome assembly and human disease. Nat Struct Mol Biol 2013;20(1):14-22 View Article PubMed/NCBI
  186. Hogan AK, Foltz DR. Reduce, Retain, Recycle: Mechanisms for Promoting Histone Protein Degradation versus Stability and Retention. Mol Cell Biol 2021;41(6):e0000721 View Article PubMed/NCBI
  187. Bannister AJ, Kouzarides T. Regulation of chromatin by histone modifications. Cell Res 2011;21(3):381-395 View Article PubMed/NCBI
  188. Andonegui-Elguera MA, Cáceres-Gutiérrez RE, López-Saavedra A, Cisneros-Soberanis F, Justo-Garrido M, Díaz-Chávez J, et al. The Roles of Histone Post-Translational Modifications in the Formation and Function of a Mitotic Chromosome. Int J Mol Sci 2022;23(15):8704 View Article PubMed/NCBI
  189. Martire S, Banaszynski LA. The roles of histone variants in fine-tuning chromatin organization and function. Nat Rev Mol Cell Biol 2020;21(9):522-541 View Article PubMed/NCBI
  190. Giallongo S, Řeháková D, Biagini T, Lo Re O, Raina P, Lochmanová G, et al. Histone Variant macroH2A1.1 Enhances Nonhomologous End Joining-dependent DNA Double-strand-break Repair and Reprogramming Efficiency of Human iPSCs. Stem Cells 2022;40(1):35-48 View Article PubMed/NCBI
  191. Husmann D, Gozani O. Histone lysine methyltransferases in biology and disease. Nat Struct Mol Biol 2019;26(10):880-889 View Article PubMed/NCBI
  192. Xu J, Zhang X, Pelayo R, Monestier M, Ammollo CT, Semeraro F, et al. Extracellular histones are major mediators of death in sepsis. Nat Med 2009;15(11):1318-1321 View Article PubMed/NCBI
  193. Xu J, Zhang X, Monestier M, Esmon NL, Esmon CT. Extracellular histones are mediators of death through TLR2 and TLR4 in mouse fatal liver injury. J Immunol 2011;187(5):2626-2631 View Article PubMed/NCBI
  194. Allam R, Scherbaum CR, Darisipudi MN, Mulay SR, Hägele H, Lichtnekert J, et al. Histones from dying renal cells aggravate kidney injury via TLR2 and TLR4. J Am Soc Nephrol 2012;23(8):1375-1388 View Article PubMed/NCBI
  195. Block H, Rossaint J, Zarbock A. The Fatal Circle of NETs and NET-Associated DAMPs Contributing to Organ Dysfunction. Cells 2022;11(12):1919 View Article PubMed/NCBI
  196. Hakkim A, Fürnrohr BG, Amann K, Laube B, Abed UA, Brinkmann V, et al. Impairment of neutrophil extracellular trap degradation is associated with lupus nephritis. Proc Natl Acad Sci U S A 2010;107(21):9813-9818 View Article PubMed/NCBI
  197. Abrams ST, Zhang N, Manson J, Liu T, Dart C, Baluwa F, et al. Circulating histones are mediators of trauma-associated lung injury. Am J Respir Crit Care Med 2013;187(2):160-169 View Article PubMed/NCBI
  198. Barrero CA, Perez-Leal O, Aksoy M, Moncada C, Ji R, Lopez Y, et al. Histone 3.3 participates in a self-sustaining cascade of apoptosis that contributes to the progression of chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2013;188(6):673-683 View Article PubMed/NCBI
  199. Nakazawa D, Kumar SV, Marschner J, Desai J, Holderied A, Rath L, et al. Histones and Neutrophil Extracellular Traps Enhance Tubular Necrosis and Remote Organ Injury in Ischemic AKI. J Am Soc Nephrol 2017;28(6):1753-1768 View Article PubMed/NCBI
  200. Tohme S, Yazdani HO, Al-Khafaji AB, Chidi AP, Loughran P, Mowen K, et al. Neutrophil Extracellular Traps Promote the Development and Progression of Liver Metastases after Surgical Stress. Cancer Res 2016;76(6):1367-1380 View Article PubMed/NCBI
  201. van der Windt DJ, Sud V, Zhang H, Varley PR, Goswami J, Yazdani HO, et al. Neutrophil extracellular traps promote inflammation and development of hepatocellular carcinoma in nonalcoholic steatohepatitis. Hepatology 2018;68(4):1347-1360 View Article PubMed/NCBI
  202. Yang LY, Luo Q, Lu L, Zhu WW, Sun HT, Wei R, et al. Increased neutrophil extracellular traps promote metastasis potential of hepatocellular carcinoma via provoking tumorous inflammatory response. J Hematol Oncol 2020;13(1):3 View Article PubMed/NCBI
  203. Mauracher LM, Posch F, Martinod K, Grilz E, Däullary T, Hell L, et al. Citrullinated histone H3, a biomarker of neutrophil extracellular trap formation, predicts the risk of venous thromboembolism in cancer patients. J Thromb Haemost 2018;16(3):508-518 View Article PubMed/NCBI
  204. Nomura K, Miyashita T, Yamamoto Y, Munesue S, Harashima A, Takayama H, et al. Citrullinated Histone H3: Early Biomarker of Neutrophil Extracellular Traps in Septic Liver Damage. J Surg Res 2019;234:132-138 View Article PubMed/NCBI
  205. Ruiz-Limon P, Ladehesa-Pineda ML, Castro-Villegas MDC, Abalos-Aguilera MDC, Lopez-Medina C, Lopez-Pedrera C, et al. Enhanced NETosis generation in radiographic axial spondyloarthritis: utility as biomarker for disease activity and anti-TNF-α therapy effectiveness. J Biomed Sci 2020;27(1):54 View Article PubMed/NCBI
  206. Kaltenmeier CT, Yazdani H, van der Windt D, Molinari M, Geller D, Tsung A, et al. Neutrophil extracellular traps as a novel biomarker to predict recurrence-free and overall survival in patients with primary hepatic malignancies. HPB (Oxford) 2021;23(2):309-320 View Article PubMed/NCBI
  207. Pertiwi KR, de Boer OJ, Mackaaij C, Pabittei DR, de Winter RJ, Li X, et al. Extracellular traps derived from macrophages, mast cells, eosinophils and neutrophils are generated in a time-dependent manner during atherothrombosis. J Pathol 2019;247(4):505-512 View Article PubMed/NCBI
  208. Lee JH, Friso S, Choi SW. Epigenetic mechanisms underlying the link between non-alcoholic fatty liver diseases and nutrition. Nutrients 2014;6(8):3303-3325 View Article PubMed/NCBI
  209. Hardy T, Mann DA. Epigenetics in liver disease: from biology to therapeutics. Gut 2016;65(11):1895-1905 View Article PubMed/NCBI
  210. Moran-Salvador E, Mann J. Epigenetics and Liver Fibrosis. Cell Mol Gastroenterol Hepatol 2017;4(1):125-134 View Article PubMed/NCBI
  211. Del Campo JA, Gallego-Durán R, Gallego P, Grande L. Genetic and Epigenetic Regulation in Nonalcoholic Fatty Liver Disease (NAFLD). Int J Mol Sci 2018;19(3):911 View Article PubMed/NCBI
  212. Liu YR, Wang JQ, Huang ZG, Chen RN, Cao X, Zhu DC, et al. Histone deacetylase-2: A potential regulator and therapeutic target in liver disease (Review). Int J Mol Med 2021;48(1):131 View Article PubMed/NCBI
  213. Rodríguez-Sanabria JS, Escutia-Gutiérrez R, Rosas-Campos R, Armendáriz-Borunda JS, Sandoval-Rodríguez A. An Update in Epigenetics in Metabolic-Associated Fatty Liver Disease. Front Med (Lausanne) 2021;8:770504 View Article PubMed/NCBI
  214. Wang H, Zhang H, Wang Y, Brown ZJ, Xia Y, Huang Z, et al. Regulatory T-cell and neutrophil extracellular trap interaction contributes to carcinogenesis in non-alcoholic steatohepatitis. J Hepatol 2021;75(6):1271-1283 View Article PubMed/NCBI
  215. Wang Z, Cheng ZX, Abrams ST, Lin ZQ, Yates ED, Yu Q, et al. Extracellular histones stimulate collagen expression in vitro and promote liver fibrogenesis in a mouse model via the TLR4-MyD88 signaling pathway. World J Gastroenterol 2020;26(47):7513-7527 View Article PubMed/NCBI
  216. Xu L, Liu X, Jia T, Sun Y, Du Y, Wei S, et al. Tanshinone IIA Ameliorates Nonalcoholic Steatohepatitis in Mice by Modulating Neutrophil Extracellular Traps and Hepatocyte Apoptosis. Evid Based Complement Alternat Med 2022;2022:5769350 View Article PubMed/NCBI
  217. Cho Y, Bukong TN, Tornai D, Babuta M, Vlachos IS, Kanata E, et al. Neutrophil extracellular traps contribute to liver damage and increase defective low-density neutrophils in alcohol-associated hepatitis. J Hepatol 2023;78(1):28-44 View Article PubMed/NCBI
  218. Chen Q, Zhang Q, Cao P, Shi C, Zhang L, Wang L, et al. NOD2-mediated HDAC6/NF-κb signalling pathway regulates ferroptosis induced by extracellular histone H3 in acute liver failure. J Cell Mol Med 2022;26(21):5528-5538 View Article PubMed/NCBI
  219. Hsieh IN, Deluna X, White MR, Hartshorn KL. Histone H4 directly stimulates neutrophil activation through membrane permeabilization. J Leukoc Biol 2021;109(4):763-775 View Article PubMed/NCBI
  220. Liu K, Wang FS, Xu R. Neutrophils in liver diseases: pathogenesis and therapeutic targets. Cell Mol Immunol 2021;18(1):38-44 View Article PubMed/NCBI
  221. Deligezer U, Yaman F, Darendeliler E, Dizdar Y, Holdenrieder S, Kovancilar M, et al. Post-treatment circulating plasma BMP6 mRNA and H3K27 methylation levels discriminate metastatic prostate cancer from localized disease. Clin Chim Acta 2010;411(19-20):1452-1456 View Article PubMed/NCBI
  222. Bauden M, Pamart D, Ansari D, Herzog M, Eccleston M, Micallef J, et al. Circulating nucleosomes as epigenetic biomarkers in pancreatic cancer. Clin Epigenetics 2015;7:106 View Article PubMed/NCBI
  223. Rahier JF, Druez A, Faugeras L, Martinet JP, Géhénot M, Josseaux E, et al. Circulating nucleosomes as new blood-based biomarkers for detection of colorectal cancer. Clin Epigenetics 2017;9:53 View Article PubMed/NCBI
  224. Lillico R, Sobral MG, Stesco N, Lakowski TM. HDAC inhibitors induce global changes in histone lysine and arginine methylation and alter expression of lysine demethylases. J Proteomics 2016;133:125-133 View Article PubMed/NCBI
  225. Parira T, Figueroa G, Laverde A, Casteleiro G, Gomez Hernandez ME, Fernandez-Lima F, et al. Novel detection of post-translational modifications in human monocyte-derived dendritic cells after chronic alcohol exposure: Role of inflammation regulator H4K12ac. Sci Rep 2017;7(1):11236 View Article PubMed/NCBI
  226. Fegan JE, Yu RH, Islam EA, Schryvers AB. Development of a non-biased, high-throughput ELISA for the rapid evaluation of immunogenicity and cross-reactivity. J Immunol Methods 2021;493:113037 View Article PubMed/NCBI
  227. Van den Ackerveken P, Lobbens A, Turatsinze JV, Solis-Mezarino V, Völker-Albert M, Imhof A, et al. A novel proteomics approach to epigenetic profiling of circulating nucleosomes. Sci Rep 2021;11(1):7256 View Article PubMed/NCBI
  228. Reddy D, Khade B, Pandya R, Gupta S. A novel method for isolation of histones from serum and its implications in therapeutics and prognosis of solid tumours. Clin Epigenetics 2017;9:30 View Article PubMed/NCBI
  229. Bronsema KJ, Bischoff R, van de Merbel NC. High-sensitivity LC-MS/MS quantification of peptides and proteins in complex biological samples: the impact of enzymatic digestion and internal standard selection on method performance. Anal Chem 2013;85(20):9528-9535 View Article PubMed/NCBI
  230. García-Giménez JL, Romá-Mateo C, Carbonell N, Palacios L, Peiró-Chova L, García-López E, et al. A new mass spectrometry-based method for the quantification of histones in plasma from septic shock patients. Sci Rep 2017;7(1):10643 View Article PubMed/NCBI
  231. Biggar KK, Charih F, Liu H, Ruiz-Blanco YB, Stalker L, Chopra A, et al. Proteome-wide Prediction of Lysine Methylation Leads to Identification of H2BK43 Methylation and Outlines the Potential Methyllysine Proteome. Cell Rep 2020;32(2):107896 View Article PubMed/NCBI
  232. Keshishian H, Burgess MW, Gillette MA, Mertins P, Clauser KR, Mani DR, et al. Multiplexed, Quantitative Workflow for Sensitive Biomarker Discovery in Plasma Yields Novel Candidates for Early Myocardial Injury. Mol Cell Proteomics 2015;14(9):2375-2393 View Article PubMed/NCBI
  233. Fedyuk V, Erez N, Furth N, Beresh O, Andreishcheva E, Shinde A, et al. Multiplexed, single-molecule, epigenetic analysis of plasma-isolated nucleosomes for cancer diagnostics. Nat Biotechnol 2023;41(2):212-221 View Article PubMed/NCBI
  234. Headland SE, Jones HR, D’Sa AS, Perretti M, Norling LV. Cutting-edge analysis of extracellular microparticles using ImageStream(X) imaging flow cytometry. Sci Rep 2014;4:5237 View Article PubMed/NCBI
  235. Dent BM, Ogle LF, O’Donnell RL, Hayes N, Malik U, Curtin NJ, et al. High-resolution imaging for the detection and characterisation of circulating tumour cells from patients with oesophageal, hepatocellular, thyroid and ovarian cancers. Int J Cancer 2016;138(1):206-216 View Article PubMed/NCBI
  236. Ogle LF, Orr JG, Willoughby CE, Hutton C, McPherson S, Plummer R, et al. Imagestream detection and characterisation of circulating tumour cells - A liquid biopsy for hepatocellular carcinoma?. J Hepatol 2016;65(2):305-313 View Article PubMed/NCBI
  237. Gavillet M, Martinod K, Renella R, Harris C, Shapiro NI, Wagner DD, et al. Flow cytometric assay for direct quantification of neutrophil extracellular traps in blood samples. Am J Hematol 2015;90(12):1155-1158 View Article PubMed/NCBI
  238. Ginley BG, Emmons T, Lutnick B, Urban CF, Segal BH, Sarder P. Computational detection and quantification of human and mouse neutrophil extracellular traps in flow cytometry and confocal microscopy. Sci Rep 2017;7(1):17755 View Article PubMed/NCBI
  239. Lingblom C, Andersson K, Wennerås C. Kinetic studies of galectin-10 release from eosinophils exposed to proliferating T cells. Clin Exp Immunol 2021;203(2):230-243 View Article PubMed/NCBI
  240. Buzova D, Maugeri A, Liguori A, Napodano C, Lo Re O, Oben J, et al. Circulating histone signature of human lean metabolic-associated fatty liver disease (MAFLD). Clin Epigenetics 2020;12(1):126 View Article PubMed/NCBI
  241. Lo Re O, Maugeri A, Hruskova J, Jakubik J, Kucera J, Bienertova-Vasku J, et al. Obesity-induced nucleosome release predicts poor cardio-metabolic health. Clin Epigenetics 2019;12(1):2 View Article PubMed/NCBI
  242. Kohles N, Nagel D, Jüngst D, Durner J, Stieber P, Holdenrieder S. Relevance of circulating nucleosomes and oncological biomarkers for predicting response to transarterial chemoembolization therapy in liver cancer patients. BMC Cancer 2011;11:202 View Article PubMed/NCBI
  243. Salani F, Latarani M, Casadei-Gardini A, Gangadharannambiar P, Fornaro L, Vivaldi C, et al. Predictive significance of circulating histones in hepatocellular carcinoma patients treated with sorafenib. Epigenomics 2022;14(9):507-517 View Article PubMed/NCBI
  244. Gu T, Ge Y, Song Y, Fu Z, Zhang Y, Wang G, et al. Hepatic radiofrequency ablation causes an increase of circulating histones in patients with hepatocellular carcinoma. Scand J Clin Lab Invest 2015;75(7):621-627 View Article PubMed/NCBI
  245. Li X, Gou C, Yao L, Lei Z, Gu T, Ren F, et al. Patients with HBV-related acute-on-chronic liver failure have increased concentrations of extracellular histones aggravating cellular damage and systemic inflammation. J Viral Hepat 2017;24(1):59-67 View Article PubMed/NCBI
  246. Blasi A, Patel VC, Adelmeijer J, Azarian S, Aziz F, Fernández J, et al. Plasma levels of circulating DNA are associated with outcome, but not with activation of coagulation in decompensated cirrhosis and ACLF. JHEP Rep 2019;1(3):179-187 View Article PubMed/NCBI
  247. Wen Z, Lei Z, Yao L, Jiang P, Gu T, Ren F, et al. Circulating histones are major mediators of systemic inflammation and cellular injury in patients with acute liver failure. Cell Death Dis 2016;7(9):e2391 View Article PubMed/NCBI
  248. Ding L, Zhang X, Li L, Gou C, Luo X, Yang Y, et al. Qingchangligan formula alleviates acute liver injury by attenuating extracellular histone-associated inflammation. Biomed Pharmacother 2018;103:140-146 View Article PubMed/NCBI
  249. Li X, Gou C, Pang Y, Wang Y, Liu Y, Wen T. Extracellular histones are clinically associated with primary graft dysfunction in human liver transplantation. RSC Adv 2019;9(18):10264-10271 View Article PubMed/NCBI
  250. Novruzov N, Ersan V, Bayramov N, Otlu B, Aliyev E, Ince V, et al. Extracellular Histones H3 as a Prognostic Blood Marker for Delayed Liver Function Recovery After Donor Hepatectomy. Transplant Proc 2021;53(7):2305-2311 View Article PubMed/NCBI
  251. Wen Z, Liu Y, Li F, Ren F, Chen D, Li X, et al. Circulating histones exacerbate inflammation in mice with acute liver failure. J Cell Biochem 2013;114(10):2384-2391 View Article PubMed/NCBI
  252. Huang H, Tohme S, Al-Khafaji AB, Tai S, Loughran P, Chen L, et al. Damage-associated molecular pattern-activated neutrophil extracellular trap exacerbates sterile inflammatory liver injury. Hepatology 2015;62(2):600-614 View Article PubMed/NCBI
  253. Zenlander R, Havervall S, Magnusson M, Engstrand J, Ågren A, Thålin C, et al. Neutrophil extracellular traps in patients with liver cirrhosis and hepatocellular carcinoma. Sci Rep 2021;11(1):18025 View Article PubMed/NCBI
  254. Sadeh R, Sharkia I, Fialkoff G, Rahat A, Gutin J, Chappleboim A, et al. Author Correction: ChIP-seq of plasma cell-free nucleosomes identifies gene expression programs of the cells of origin. Nat Biotechnol 2021;39(5):642 View Article PubMed/NCBI
  255. Papatheodoridi A, Karakousis N, Lembessis P, Chatzigeorgiou A, Papatheodoridis GV. The Significance of Circulating Cell-Free DNA Markers in Chronic Hepatitis B Patients with Hepatocellular Carcinoma. Pathogens 2021;10(11):1524 View Article PubMed/NCBI
  256. Holdenrieder S, Stieber P, Bodenmüller H, Busch M, Fertig G, Fürst H, et al. Nucleosomes in serum of patients with benign and malignant diseases. Int J Cancer 2001;95(2):114-120 View Article PubMed/NCBI
  257. Lencioni R, Cioni D, Crocetti L, Franchini C, Pina CD, Lera J, et al. Early-stage hepatocellular carcinoma in patients with cirrhosis: long-term results of percutaneous image-guided radiofrequency ablation. Radiology 2005;234(3):961-967 View Article PubMed/NCBI
  258. Livraghi T, Meloni F, Di Stasi M, Rolle E, Solbiati L, Tinelli C, et al. Sustained complete response and complications rates after radiofrequency ablation of very early hepatocellular carcinoma in cirrhosis: Is resection still the treatment of choice?. Hepatology 2008;47(1):82-89 View Article PubMed/NCBI
  259. KENT PW, HICHENS M, WARD PF. Displacement fractionation of deoxyribonucleoproteins by heparin and dextran sulphate. Biochem J 1958;68(4):568-572 View Article PubMed/NCBI
  260. Demidenko O, Tsvetkova S. Action of heparin on protein fractions of isolated nuclei and on their DNA content. Histochemistry 1978;57(3):265-271 View Article PubMed/NCBI
  261. Wildhagen KC, García de Frutos P, Reutelingsperger CP, Schrijver R, Aresté C, Ortega-Gómez A, et al. Nonanticoagulant heparin prevents histone-mediated cytotoxicity in vitro and improves survival in sepsis. Blood 2014;123(7):1098-1101 View Article PubMed/NCBI
  262. Faria LC, Andrade AMF, Trant CGMC, Lima AS, Machado PAB, Porto RD, et al. Circulating levels of High-mobility group box 1 protein and nucleosomes are associated with outcomes after liver transplant. Clin Transplant 2020;34(7):e13869 View Article PubMed/NCBI
  263. Valla V, Alzabin S, Koukoura A, Lewis A, Nielsen AA, Vassiliadis E. Companion Diagnostics: State of the Art and New Regulations. Biomark Insights 2021;16:11772719211047763 View Article PubMed/NCBI
  • Journal of Clinical and Translational Hepatology
  • pISSN 2225-0719
  • eISSN 2310-8819
  • Copyright © 2022 JCTH. All Rights Reserved.
  • Published by Xia & He Publishing Inc.
  • Address: 14090 Southwest Freeway, Suite 300, Sugar Land, Texas 77478, USA
  • Email: service@xiahepublishing.com