Distinct Distribution of Ectopically Expressed Histone Variants H2A.Bbd and MacroH2A in Open and Closed Chromatin Domains

Background

It becomes increasingly evident that nuclesomes are far from being identical to each other. This nucleosome diversity is due partially to the existence of histone variants encoded by separate genes. Among the known histone variants the less characterized are H2A.Bbd and different forms of macroH2A. This is especially true in the case of H2A.Bbd as there are still no commercially available antibodies specific to H2A.Bbd that can be used for chromatin immunoprecipitation (ChIP).

Methods

We have generated HeLa S3 cell lines stably expressing epitope-tagged versions of macroH2A1.1, H2A.Bbd or canonical H2A and analyzed genomic distribution of the tagged histones using ChIP-on-chip technique.

Results

The presence of histone H2A variants macroH2A1.1 and H2A.Bbd has been analyzed in the chromatin of several segments of human chromosomes 11, 16 and X that have been chosen for their different gene densities and chromatin status. Chromatin immunoprecipitation (ChIP) followed by hybridization with custom NimbleGene genomic microarrays demonstrated that in open chromatin domains containing tissue-specific along with housekeeping genes, the H2A.Bbd variant was preferentially associated with the body of a subset of transcribed genes. The macroH2A1.1 variant was virtually absent from some genes and underrepresented in others. In contrast, in closed chromatin domains which contain only tissue-specific genes inactive in HeLa S3 cells, both macroH2A1.1 and H2A.Bbd histone variants were present and often colocalized.

Conclusions

Genomic distribution of macro H2A and H2A.Bbd does not follow any simple rule and is drastically different in open and closed genomic domains.     

IgM-linked SerpinB3 and SerpinB4 in sera of patients with chronic liver disease

Background

Epidemiological studies indicate that a growing number of cirrhotic patients will develop hepatocellular carcinoma (HCC) in the next decade. Recent findings have demonstrated that Squamous cell carcinoma antigen 1 (SCCA1) and 2 (SCCA2) isoforms, now classified as serpinB3 and serpinB4, are over-expressed in HCC, but not in normal liver. As reported, high levels of circulating SCCA-IgM immunocomplexes in patients with cirrhosis are significantly associated with HCC development.

Aim

To ascertain whether IgM-linked SCCA isoforms circulate in patients with chronic liver disease, compared to total SCCA-IgM levels.

Methodology and Findings

79 patients with chronic liver disease were studied, including 17 patients with chronic hepatitis, 36 patients with cirrhosis and 26 with HCC. 28 blood donors were used as control. Monoclonal antibodies against serpinB3 and serpinB4 were used as catcher antibodies to set up specific ELISA assays, while total SCCA-IgM immunocomplexes were detected by commercially available ELISA assay. Overall, the results revealed a better diagnostic sensitivity of total SCCA-IgM assay, compared to both serpinB3 and serpinB4 IgM-linked assays. SerpinB4-IgM median values obtained with SCC103 antibody were moderately higher in patients with cirrhosis than in those with HCC, median values: 0.168 (IQR 0.140–0.427) vs. 0.140 (IQR 0.140–0.278), (p = 0.177). A trend toward decreasing serpinB4-IgM/serpinB3-IgM median ratio was observed in patients with advanced liver disease, being 1.08 in patients with HCC, 1.10 in patients with cirrhosis and 1.40 in patients with chronic hepatitis (p = 0.079).

Conclusions

IgM-linked SCCA isoforms in serum of patients with chronic liver diseases were quantified for the first time. Although the number of patients was limited, this preliminary study reveals that the relative balance of the two serpin isoforms is altered in HCC and it is characterized by a lower serpinB4-IgM/serpinB3-IgM ratio, determined by lower serpinB4 levels.     

Identification of histone deacetylase 3 as a biomarker for tumor recurrence following liver transplantation in HBV-associated hepatocellular carcinoma

Background

Recent studies have shown that high expression levels of class I histone deacetylases (HDACs) correlate with malignant phenotype and poor prognosis in some human tumors. However, the expression patterns and prognostic role of class I HDAC isoforms in hepatocellular carcinoma (HCC) remain unclear.

Methodology/Principal Findings

The expression patterns and clinical significance of class I HDAC isoforms were assessed by immunohistochemistry in a cohort of 43 hepatitis B virus-associated HCC patients treated with liver transplantation. In addition, the effects of HDAC inhibition on HCC cell behavior were investigated by knockdown of the HDAC isoform with short interfering RNA. Class I HDACs were highly expressed in a subset of HCCs with positivity for HDAC1 in 51.2%, HDAC2 in 48.8%, and HDAC3 in 32.6% of cases. The expression levels of HDAC isoforms were significantly associated with the proliferation index of HCC. Kaplan-Meier curves showed that a high expression level of HDAC2 or HDAC3 implicated significantly reduced recurrence-free survival. Cox proportional hazards model analysis revealed HDAC3 overexpression was an unfavorable independent prognostic factor (P = 0.002; HR 3.907). In vitro, inhibition of HDAC2 and HDAC3, but not HDAC1, suppressed proliferation and the invasiveness of liver cancer cells.

Conclusions/Significance

Our findings demonstrate that HDAC3 plays a significant role in regulating tumor cell proliferation and invasion, and it could be served as a candidate biomarker for predicting the recurrence of hepatitis B virus-associated HCC following liver transplantation and a potential therapeutic target.     

Linkage Specific Fucosylation of Alpha-1-Antitrypsin in Liver Cirrhosis and Cancer Patients: Implications for a Biomarker of Hepatocellular Carcinoma

Background

We previously reported increased levels of protein-linked fucosylation with the development of liver cancer and identified many of the proteins containing the altered glycan structures. One such protein is alpha-1-antitrypsin (A1AT). To advance these studies, we performed N-linked glycan analysis on the five major isoforms of A1AT and completed a comprehensive study of the glycosylation of A1AT found in healthy controls, patients with hepatitis C- (HCV) induced liver cirrhosis, and in patients infected with HCV with a diagnosis of hepatocellular carcinoma (HCC).

Methodology/Principal Findings

Patients with liver cirrhosis and liver cancer had increased levels of triantennary glycan-containing outer arm (α-1,3) fucosylation. Increases in core (α-1,6) fucosylation were observed only on A1AT from patients with cancer. We performed a lectin fluorophore-linked immunosorbent assay using Aleuria Aurantia lectin (AAL), specific for core and outer arm fucosylation in over 400 patients with liver disease. AAL-reactive A1AT was able to detect HCC with a sensitivity of 70% and a specificity of 86%, which was greater than that observed with the current marker of HCC, alpha-fetoprotein. Glycosylation analysis of the false positives was performed; results indicated that these patients had increases in outer arm fucosylation but not in core fucosylation, suggesting that core fucosylation is cancer specific.

Conclusions/Significance

This report details the stepwise change in the glycosylation of A1AT with the progression from liver cirrhosis to cancer and identifies core fucosylation on A1AT as an HCC specific modification.     

Diagnostic Performance of a Rapid Magnetic Resonance Imaging Method of Measuring Hepatic Steatosis

Objectives

Hepatic steatosis is associated with an increased risk of developing serious liver disease and other clinical sequelae of the metabolic syndrome. However, visual estimates of steatosis from histological sections of biopsy samples are subjective and reliant on an invasive procedure with associated risks. The aim of this study was to test the ability of a rapid, routinely available, magnetic resonance imaging (MRI) method to diagnose clinically relevant grades of hepatic steatosis in a cohort of patients with diverse liver diseases.

Materials and Methods

Fifty-nine patients with a range of liver diseases underwent liver biopsy and MRI. Hepatic steatosis was quantified firstly using an opposed-phase, in-phase gradient echo, single breath-hold MRI methodology and secondly, using liver biopsy with visual estimation by a histopathologist and by computer-assisted morphometric image analysis. The area under the receiver operating characteristic (ROC) curve was used to assess the diagnostic performance of the MRI method against the biopsy observations.

Results

The MRI approach had high sensitivity and specificity at all hepatic steatosis thresholds. Areas under ROC curves were 0.962, 0.993, and 0.972 at thresholds of 5%, 33%, and 66% liver fat, respectively. MRI measurements were strongly associated with visual (r² = 0.83) and computer-assisted morphometric (r² = 0.84) estimates of hepatic steatosis from histological specimens.

Conclusions

This MRI approach, using a conventional, rapid, gradient echo method, has high sensitivity and specificity for diagnosing liver fat at all grades of steatosis in a cohort with a range of liver diseases.     

Gene Expression Profiling Unravels Cancer-Related Hepatic Molecular Signatures in Steatohepatitis but Not in Steatosis

Background

Pathogenesis and factors for determining progression of alcoholic and non-alcoholic steatosis to steatohepatitis with risk of further progression to liver cirrhosis and cancer are poorly understood. In the present study, we aimed to identify potential molecular signatures for discrimination of steatohepatitis from steatosis.

Methodology and Results

Global microarray gene expression analysis was applied to unravel differentially expressed genes between steatohepatitis compared to steatosis and control samples. For functional annotation as well as the identification of disease-relevant biological processes of the differentially expressed genes the gene ontology (GO) database was used. Selected candidate genes (n = 46) were validated in 87 human liver samples from two sample cohorts by quantitative real-time PCR (qRT-PCR). The GO analysis revealed that genes down-regulated in steatohepatitis were mainly involved in metabolic processes. Genes up-regulated in steatohepatitis samples were associated with cancer progression and proliferation. In surgical liver resection samples, 39 genes and in percutaneous liver biopsies, 30 genes were significantly up-regulated in steatohepatitis. Furthermore, immunohistochemical investigation of human liver tissue revealed a significant increase of AKR1B10 protein expression in steatohepatitis.

Conclusions

The development of steatohepatitis is characterized by distinct molecular changes. The most striking examples in this respect were KRT23 and AKR1B10, which we found to be highly differentially expressed in steatohepatitis compared to steatosis and normal liver. We propose that KRT23 and AKR1B10 may serve as future potential biomarkers for steatohepatitis as well as markers for progression to HCC.     

The Predictive Value of Golgi Protein 73 in Differentiating Benign from Malignant Liver Tumors

Introduction

In the work up of primary solid liver lesions it is essential to differentiate correctly between benign and malignant tumors, such as hepatocellular adenoma (HCA) and hepatocellular carcinoma (HCC) respectively. A promising new marker to detect HCC is Golgi Protein 73 (GP73). Studies comparing patients with HCC and cirrhosis with normal controls suggested that GP73 is specific for patients with HCC; however, patients with other liver tumors were not included. We therefore studied the predictive value of GP73 in differentiating between solid benign and malignant liver tumors.

Materials and Methods

This study included 264 patients: 88 patients with HCC, 88 with hepatocellular adenoma (HCA), and 88 with focal nodal hyperplasia (FNH). A blood sample was collected from each patient to measure GP73 levels using a quantitative ELISA assay and differences in outcome between subgroups were compared. The receiver operating characteristic (ROC) curve, sensitivity and specificity of GP73 were calculated and compared to alpha-fetoprotein (AFP) levels.

Results

When comparing malignant and benign liver tumors the area under ROC was 0.701 and 0.912 for GP73 and AFP respectively. Test characteristics revealed a sensitivity of 60% for GP73 and 65% for AFP; in addition the specificity was 77% for GP73 and 96% for AFP.

Conclusion

Although the literature suggests that GP73 is a valuable serum marker in patients with HCC, the serum concentration may also be increased in patients with solid benign liver tumors. Therefore, a GP73 assay is less suitable for discriminating between primary malignant and benign tumors of the liver.     

A New Model to Estimate Prognosis in Patients with Hepatocellular Carcinoma after Yttrium-90 Radioembolization

Aims

The current prognostic model to estimate the survival in hepatocellular carcinoma (HCC) patients treated with transarterial hepatic selective internal radiotherapy (SIRT) is not fully characterized. The aim of this study was to establish a new scoring model including assessment of both tumor responses and therapy-induced systemic changes in HCC patients to predict survival at an early time point post-SIRT.

Methods and materials

Between 2008 and 2012, 149 HCC patients treated with SIRT were included into this study. CT images and biomarkers in blood tested at one month post-SIRT were analyzed and correlated with clinical outcome. Tumor responses were assessed by RECIST 1.1, mRECIST, and Choi criteria. Kaplan-Meier methods were used to estimate survival curves. Cox regression was used in uni- and multivariable survival analyses and in the establishment of a prognostic model.

Results

A multivariate proportional hazards model was created based on the tumor response, the number of tumor nodules, the score of the model for end stage liver disease (MELD), and the serum C-reactive protein levels which were independent predictors of survival in HCC patients at one month post-SIRT. This prognostic model accurately differentiated the outcome of patients with different risk scores in this cohort (P<0.001). The model also had the ability to assign a predicted survival probability for individual patients.

Conclusions

A new model to predict survival of HCC patients mainly based on tumor responses and therapy-induced systemic changes provides reliable prognosis and accurately discriminates the survival at an early time point after SIRT in these patients.     

Absence of Perilipin 2 Prevents Hepatic Steatosis,Glucose Intolerance and Ceramide Accumulation in Alcohol-Fed Mice

Background

Perilipin 2 (Plin2) is a lipid droplet protein that has roles in both lipid and glucose homeostasis. An increase in Plin2 in liver is associated with the development of steatosis, glucose intolerance, and ceramide accumulation in alcoholic liver disease. We investigated the role of Plin2 on energy balance and glucose and lipid homeostasis in wildtype and Plin2 knockout (Plin2KO) mice chronically fed a Lieber-DeCarli liquid ethanol or control diet for six weeks.

Methods

We performed in vivo measurements of energy intake and expenditure; body composition; and glucose tolerance. After sacrifice, liver was dissected for histology and lipid analysis.

Results

We found that neither genotype nor diet had a significant effect on final weight, body composition, or energy intake between WT and Plin2KO mice fed alcohol or control diets. Additionally, alcohol feeding did not affect oxygen consumption or carbon dioxide production in Plin2KO mice. We performed glucose tolerance testing and observed that alcohol feeding failed to impair glucose tolerance in Plin2KO mice. Most notably, absence of Plin2 prevented hepatic steatosis and ceramide accumulation in alcohol-fed mice. These changes were related to downregulation of genes involved in lipogenesis and triglyceride synthesis.

Conclusions

Plin2KO mice chronically fed alcohol are protected from hepatic steatosis, glucose intolerance, and hepatic ceramide accumulation, suggesting a critical pathogenic role of Plin2 in experimental alcoholic liver disease.     

Hedgehog signaling antagonist promotes regression of both liver fibrosis and hepatocellular carcinoma in a murine model of primary liver cancer

Objective

Chronic fibrosing liver injury is a major risk factor for hepatocarcinogenesis in humans. Mice with targeted deletion of Mdr2 (the murine ortholog of MDR3) develop chronic fibrosing liver injury. Hepatocellular carcinoma (HCC) emerges spontaneously in such mice by 50–60 weeks of age, providing a model of fibrosis-associated hepatocarcinogenesis. We used Mdr2^−/− mice to investigate the hypothesis that activation of the hedgehog (Hh) signaling pathway promotes development of both liver fibrosis and HCC.

Methods

Hepatic injury and fibrosis, Hh pathway activation, and liver progenitor populations were compared in Mdr2^−/− mice and age-matched wild type controls. A dose finding experiment with the Hh signaling antagonist GDC-0449 was performed to optimize Hh pathway inhibition. Mice were then treated with GDC-0449 or vehicle for 9 days, and effects on liver fibrosis and tumor burden were assessed by immunohistochemistry, qRT-PCR, Western blot, and magnetic resonance imaging.

Results

Unlike controls, Mdr2^−/− mice consistently expressed Hh ligands and progressively accumulated Hh-responsive liver myofibroblasts and progenitors with age. Treatment of aged Mdr2-deficient mice with GDC-0449 significantly inhibited hepatic Hh activity, decreased liver myofibroblasts and progenitors, reduced liver fibrosis, promoted regression of intra-hepatic HCCs, and decreased the number of metastatic HCC without increasing mortality.

Conclusions

Hh pathway activation promotes liver fibrosis and hepatocarcinogenesis, and inhibiting Hh signaling safely reverses both processes even when fibrosis and HCC are advanced.     

Serum Autoantibody Measurement for the Detection of Hepatocellular Carcinoma

Background

Individuals with liver disease, and especially those with Hepatitis B or C, are at an increased risk of developing hepatocellular carcinoma (HCC) which is the third most common cause of cancer-related death worldwide. Inadequate screening tests largely account for presentation of advanced tumours and high mortality rates. Early detection of HCC amongst high-risk groups is paramount in improving prognosis. This research aimed to further characterise the previously described humoral immune response raised to tumour-associated antigens (TAAs) in the serum of patients with HCC.

Methods

Serum from 96 patients with confirmed HCC, 96 healthy controls matched for age and sex, 78 patients with confirmed liver cirrhosis and 91 patients with confirmed chronic liver disease were analysed for the presence of IgG autoantibodies raised to 41 recombinant TAAs/antigen fragments by ELISA.

Results

Varying autoantibody specificities (97–100%) and sensitivities (0–10%) were observed to individual TAAs. A 21-antigen panel achieved a specificity of 92% and sensitivity of 45% for the detection of HCC. This same panel identified 21% of 169 high-risk controls as having elevated autoantibody levels. A reproducible panel of 10 antigens achieved a specificity of 91% and sensitivity of 41% in HCC. 15% of 152 high-risk controls gave positive results with this panel.

Conclusions

This minimally invasive blood test has the potential to offer advantages over currently available tools for the identification of HCC amongst pre-disposed patients. Results are comparable to current gold standards in HCC (Ultrasonography) and to similar tests in other cancers (EarlyCDT-Lung).     

Serum fetuin A and chemerin levels correlate with hepatic steatosis and regional adiposity in maintenance hemodialysis patients

Background

A deficiency of fetuin A is linked to cardiovascular calcification and mortality in dialysis patients. But, high levels of fetuin A and chemerin correlate with hepatic steatosis and regional adiposity in general population. The association between hepatic steatosis and fetuin A/chemerin levels in hemodialysis (HD) remains unclear.

Methods

We performed a cross-sectional, observational study; 216 prevalent HD patients from a single center were enrolled. Baseline serum fetuin A, chemerin levels, conicity index and anthropometric parameters were checked. Presence of hepatic steatosis was qualified by liver ultrasound and quantified by the hepato-renal index (HRI); central obesity defined by waist circumference (WC). ROC analyses and multivariate logistic regression analyses for prediction of hepatic steatosis and central obesity on the basis of fetuin A/chemerin levels, anthropometric parameters, and other relevant covariates were performed.

Results

Data from 103 women and 113 men (mean age 60±12 years) were analyzed. Eighty subjects had hepatic steatosis and their HRIs were significantly higher than those without hepatic steatosis (P<0.001). Serum fetuin A levels were positively associated with HRIs (P<0.001) and chemerin levels (P<0.001). Fetuin A, chemerin and WC were predictors for hepatic steatosis and central obesity by ROC curve. In multivariate logistic regression analysis, fetuin A and WC independently predicted hepatic steatosis defined by HRIs. And chemerin predicted central obesity and regional adiposity after covariate adjustments (all P<0.05).

Conclusions

Serum fetuin A levels were higher in prevalent HD patients with hepatic steatosis, and positively correlated with chemerin levels. Chemerin levels predicted central obesity as well as regional adiposity in the HD patients.     

Comparison of Liver Fat Indices for the Diagnosis of Hepatic Steatosis and Insulin Resistance

Context

Hepatic steatosis, defined as increased hepatocellular lipid content (HCL), associates with visceral obesity and glucose intolerance. As exact HCL quantification by ¹H-magnetic resonance spectroscopy (¹H-MRS) is not generally available, various clinical indices are increasingly used to predict steatosis.

Objective

The purpose of this study was to test the accuracy of NAFLD liver fat score (NAFLD-LFS), hepatic steatosis index (HSI) and fatty liver index (FLI) against ¹H-MRS and their relationships with insulin sensitivity and secretion.

Design, Setting and Participants

Ninety-two non-diabetic, predominantly non-obese humans underwent clinical examination, ¹H-MRS and an oral glucose tolerance test (OGTT) to calculate insulin sensitivity and β-cell function. Accuracy of indices was assessed from the area under the receiver operating characteristic curve (AROC).

Results

Median HCL was 2.49% (0.62;4.23) and correlated with parameters of glycemia across all subjects. NAFLD-LFS, FLI and HSI yielded AROCs of 0.70, 0.72, and 0.79, respectively, and related positively to HCL, insulin resistance, fasting and post-load β-cell function normalized for insulin resistance. Upon adjustment for age, sex and HCL, regression analysis revealed that NAFLD-LFS, FLI and HSI still independently associated with both insulin sensitivity and β-cell function.

Conclusion

The tested indices offer modest efficacy to detect steatosis and cannot substitute for fat quantification by ¹H-MRS. However, all indices might serve as surrogate parameters for liver fat content and also as rough clinical estimates of abnormal insulin sensitivity and secretion. Further validation in larger collectives such as epidemiological studies is needed.     

Metformin Prevents and Reverses Inflammation in a Non-Diabetic Mouse Model of Nonalcoholic Steatohepatitis

Background

Optimal treatment for nonalcoholic steatohepatitis (NASH) has not yet been established, particularly for individuals without diabetes. We examined the effects of metformin, commonly used to treat patients with type 2 diabetes, on liver pathology in a non-diabetic NASH mouse model.

Methodology/Principal Findings

Eight-week-old C57BL/6 mice were fed a methionine- and choline-deficient plus high fat (MCD+HF) diet with or without 0.1% metformin for 8 weeks. Co-administration of metformin significantly decreased fasting plasma glucose levels, but did not affect glucose tolerance or peripheral insulin sensitivity. Metformin ameliorated MCD+HF diet-induced hepatic steatosis, inflammation, and fibrosis. Furthermore, metformin significantly reversed hepatic steatosis and inflammation when administered after the development of experimental NASH.

Conclusions/Significance

These histological changes were accompanied by reduced hepatic triglyceride content, suppressed hepatic stellate cell activation, and the downregulation of genes involved in fatty acid metabolism, inflammation, and fibrogenesis. Metformin prevented and reversed steatosis and inflammation of NASH in an experimental non-diabetic model without affecting peripheral insulin resistance.     

IL28B Gene Polymorphisms and US Liver Fatty Changes in Patients Who Spontaneously Cleared Hepatitis C Virus Infection

Background

Recent clinical studies have shown that the presence of CC genotype in the rs12979860 region of IL28B gene is associated with an increase in the probability of spontaneous clearance of hepatitis C virus (HCV). Moreover, IL28B polymorphism seems to influence the probability of developing liver steatosis in chronic HCV patients.

Aims

The aims of our clinical study were 1) to verify the distribution of IL28B genotypes (CC, CT or TT) among subjects with spontaneous clearance of HCV infection and 2) to examine the correlation between IL28B polymorphism and hepatic steatosis among these subjects.

Methods and patients

We enrolled 41 subjects with spontaneous resolution of HCV infection (detectable serum anti-HCV but undetectable HCV-RNA) and 134 healthy controls from the same geographical area. The IL28B single-nucleotide polymorphism (SNP) rs12979860 was genotyped by using a Pyrosequencing™ technique. The presence of steatosis was assessed by liver biopsy or ultrasound examination in the 41 study subjects.

Results

CC, CT and TT-genotypes of the SNP rs1979860 were found in 66%, 24% and 10% of the subjects who spontaneously cleared HCV and in 31%, 54% and 15% of controls, respectively (p = 0.0003). Among the study subjects, females with CC-genotype were significantly more represented (p = 0.02). Hepatic steatosis did not correlate with IL28B genotype (p = 0,14) but only with a high body mass index (BMI) value (p = 0.03).

Conclusions

Female subjects carrying IL28B CC-genotype are significantly more represented among Italian patients who spontaneously cleared HCV infection. In addition, among these subjects, the presence of liver steatosis does not correlate with IL28B genotype but is solely related to the occurrence of high BMI. Thus, the association between IL28B polymorphism and steatosis in chronic HCV patients requires the presence of active HCV replication to occur, while in subjects who have cleared the infection, the mechanism(s) inducing liver steatosis are independent from IL28B profile.     

Circulating fibroblast growth factor 21 levels are closely associated with hepatic fat content: a cross-sectional study

Background and Aims

Fibroblasts growth factor 21 (FGF21), a liver-secreted endocrine factor involved in regulating glucose and lipid metabolism, has been shown to be elevated in patients with non-alcoholic fatty liver disease (NAFLD). This study aimed to evaluate the quantitative correlation between serum FGF21 level and hepatic fat content.

Methods

A total of 138 subjects (72 male and 66 female) aged from 18 to 65 years with abnormal glucose metabolism and B-ultrasonography diagnosed fatty liver were enrolled in the study. Serum FGF21 levels were determined by an in-house chemiluminescence immunoassay and hepatic fat contents were measured by proton magnetic resonance spectroscopy.

Results

Serum FGF21 increased progressively with the increase of hepatic fat content, but when hepatic fat content increased to the fourth quartile, FGF21 tended to decline. Serum FGF21 concentrations were positively correlated with hepatic fat content especially in subjects with mild/moderate hepatic steatosis (r = 0.276, p = 0.009). Within the range of hepatic steatosis from the first to third quartile, FGF21 was superior to any other traditional clinical markers including ALT to reflect hepatic fat content. When the patients with severe hepatic steatosis (the fourth quartile) were included, the quantitative correlation between FGF21 and hepatic fat content was weakened.

Conclusions

Serum FGF21 was a potential biomarker to reflect the hepatic fat content in patients with mild or moderate NAFLD. In severe NAFLD patients, FGF21 concentration might decrease due to liver inflammation or injury.     

A Metabolomic Analysis of Two Intravenous Lipid Emulsions in a Murine Model

Background

Parenteral nutrition (PN), including intravenous lipid administration, is a life-saving therapy but can be complicated by cholestasis and liver disease. The administration of intravenous soy bean oil (SO) has been associated with the development of liver disease, while the administration of intravenous fish oil (FO) has been associated with the resolution of liver disease. The biochemical mechanism of this differential effect is unclear. This study compares SO and FO lipid emulsions in a murine model of hepatic steatosis, one of the first hits in PN-associated liver disease.

Methods

We established a murine model of hepatic steatosis in which liver injury is induced by orally feeding mice a PN solution. C57BL/6J mice were randomized to receive PN alone (a high carbohydrate diet (HCD)), PN plus intravenous FO (Omegaven®; Fresenius Kabi AG, Bad Homburg VDH, Germany), PN plus intravenous SO (Intralipid®; Fresenius Kabi AG, Bad Homburg v.d.H., Germany, for Baxter Healthcare, Deerfield, IL), or a chow diet. After 19 days, liver tissue was harvested from all animals and subjected to metabolomic profiling.

Results

The administration of an oral HCD without lipid induced profound hepatic steatosis. SO was associated with macro- and microvesicular hepatic steatosis, while FO largely prevented the development of steatosis. 321 detectable compounds were identified in the metabolomic analysis. HCD induced de novo fatty acid synthesis and oxidative stress. Both FO and SO relieved some of the metabolic shift towards de novo lipogenesis, but FO offered additional advantages in terms of lipid peroxidation and the generation of inflammatory precursors.

Conclusions

Improved lipid metabolism combined with reduced oxidative stress may explain the protective effect offered by intravenous FO in vivo.     

Association of Hepatitis B Virus Pre-S Deletions with the Development of Hepatocellular Carcinoma in Qidong,China

Background/Aim

To investigate the roles of mutations in pre-S and S regions of hepatitis B virus (HBV) on the progression of hepatocellular carcinoma (HCC) in Qidong, China.

Methods

We conducted an age matched case-control study within a cohort of 2387 male HBV carriers who were recruited from August, 1996. The HBV DNA sequence in pre-S/S regions was successfully determined in 96 HCC cases and 97 control subjects. In addition, a consecutive series of samples from 11 HCC cases were employed to evaluate the pre-S deletion patterns before and after the occurrence of HCC.

Results

After adjustment for age, history of cigarette smoking and alcohol consumption, HBeAg positivity, pre-S deletions, pre-S2 start codon mutations, and T53C mutation were significantly associated with HCC, showing adjusted odds ratios (ORs) from 1.914 to 3.199. HCC patients also had a lower frequency of T31C mutation in pre-S2 gene, compared with control subjects (0.524; 95% CI 0.280-0.982). HBV pre-S deletions were clustered mainly in the 5′ end of pre-S2 region. Multivariate analysis showed that pre-S deletions and pre-S2 start codon mutations were independent risk factors for HCC. The OR (95% CI) were 2.434 (1.063–5.573) and 3.065 (1.099–8.547), respectively. The longitudinal observation indicated that the pre-S deletion mutations were not acquired at the beginning of HBV infection, but that the mutations occurred during the long course of liver disease.

Conclusion

Pre-S deletions and pre-S2 start codon mutations were independently associated with the development of HCC. The results also provided direct evidence that pre-S deletion mutations were not acquired from the beginning of infection but arose de novo during the progression of liver disease.     

Does 18F-FDG Positron Emission Tomography-Computed Tomography Have a Role in Initial Staging of Hepatocellular Carcinoma?

Background and Aim

The utility of fluorine-18 fluorodeoxyglucose (¹⁸F-FDG) positron emission tomography-computed tomography (PET/CT) in initial staging of hepatocellular carcinoma (HCC) has yet to be fully explored. We assessed the usefulness of ¹⁸F-FDG PET/CT in initial staging of HCC.

Methods

A total of 457 consecutive patients initially diagnosed with HCC at Seoul National University Hospital between 2006 and 2012 were evaluated retrospectively to assess the impact of ¹⁸F-FDG PET/CT on staging and compliancy with Milan criteria, relative to dynamic CT of liver and chest x-ray.

Results

Seven among the 457 patients studied showed a shift in Barcelona Clinic Liver Cancer [BCLC] stage (A→C: 6 patients; B→C: 1 patient) and 5 patients who had originally met Milan criteria no longer qualified. ¹⁸F-FDG PET/CT had value in initial staging of early (stage A) or intermediate (stage B) HCC, as determined by dynamic CT of liver and BCLC or AJCC classifications, whereas BCLC stage 0 and stage C tumors were unchanged (P<0.001). ¹⁸F-FDG PET/CT disclosed additional metastases in patients with American Joint Committee on Cancer [AJCC] T2 (2.7%), T3a (5.3%), and T3b (4.8%) classifications.

Conclusions

In initial staging of HCC, ¹⁸F-FDG PET/CT provided additional information, impacting the patients with BCLC (stages A and B) and AJCC (T2 and T3) classifications. Its use might be thus appropriate for these patient subsets, especially if hepatic resection or liver transplantation is planned.     

PNPLA3 I148M Polymorphism,Clinical Presentation,and Survival in Patients with Hepatocellular Carcinoma

Background & Aims

Aim of this study was to evaluate whether the PNPLA3 I148M polymorphism, previously associated with hepatocellular carcinoma (HCC) risk, influences the clinical presentation of HCC and survival.

Methods

we considered 460 consecutive HCC patients referred to tertiary care centers in Northern Italy, 353 with follow-up data.

Results

Homozygosity for PNPLA3 148M at risk allele was enriched in HCC patients with alcoholic liver disease or nonalcoholic fatty liver disease (ALD&NAFLD: relative risk 5.9, 95% c.i. 3.5–9.9; other liver diseases: relative risk 1.9, 95% c.i. 1.1–3.4). In ALD&NAFLD patients, the PNPLA3 148M allele was associated with younger age, shorter history of cirrhosis, less advanced (Child A) cirrhosis at HCC diagnosis, and lower HCC differentiation grade (p<0.05). Homozygosity for PNPLA3 148M was associated with reduced survival in the overall series (p = 0.009), and with a higher number of HCC lesions at presentation (p = 0.007) and reduced survival in ALD&NAFLD patients (p = 0.003; median survival 30, 95% c.i. 20–39 vs. 45, 95% c.i. 38–52 months), but not in those with HCC related to other etiologies (p = 0.86; 48, 95% c.i. 32–64 vs. 55, 95% c.i. 43–67 months). At multivariate Cox regression analysis, homozygosity for PNPLA3 148M was the only negative predictor of survival in ALD&NAFLD patients (HR of death 1.57, 95% c.i. 1.12–2.78).

Conclusions

PNPLA3 148M is over-represented in ALD&NAFLD HCC patients, and is associated with occurrence at a less advanced stage of liver disease in ALD&NAFLD. In ALD&NAFLD, PNPLA3 148M is associated with more diffuse HCC at presentation, and with reduced survival.