Amelioration of Liver Injury by Continuously Targeted Intervention against TNFRp55 in Rats with Acute-on-Chronic Liver Failure

Background

Acute-on-chronic liver failure (ACLF) is an acute deterioration of established liver disease. Blocking the TNF (tumor necrosis factor)/TNFR (tumor necrosis factor receptor) 1 pathway may reduce hepatocyte apoptosis/necrosis, and subsequently decrease mortality during development of ACLF. We demonstrated that a long-acting TNF antagonist (soluble TNF receptor: IgG Fc [sTNFR:IgG-Fc]) prevented/reduced development of acute liver failure by blocking the TNF/TNFR1 (TNFRp55) pathway. However, it is still unclear if sTNFR:IgG-Fc can inhibit hepatocyte damage during development of ACLF.

Methodology

Chronic liver disease (liver fibrosis/cirrhosis) was induced in Wistar rats by repeatedly challenging with human serum albumin (HSA), and confirmed by histopathology. ACLF was induced with D-galactosamine (D-GalN)/lipopolysaccharide (LPS) i.p. in the rats with chronic liver disease. Serum and liver were collected for biochemical, pathological and molecular biological examinations.

Principal Findings

Reduced mortality was observed in sTNFR:IgG-Fc treated ACLF rats, consistent with reduced interleukin (IL)-6 levels in serum and liver, as well as reduced hepatic caspase-3 activity, compared to that of mock treated group. Reduced hepatic damage was confirmed with histopathology in the sTNFR:IgG-Fc treated group, which is consistent with reduced Bcl-2 and Bax, at mRNA and protein levels, but increased hepatocyte proliferation (PCNA). This is also supported by the findings that caspase-3 production was up-regulated significantly in ACLF group compared to the mock treated group. Moreover, up-regulated caspase-3 was inhibited following sTNFR:IgG-Fc treatment. Finally, there was up-regulation of hepatic IL-22R in sTNFR:IgG-Fc treated ACLF rats.

Conclusions

sTNFR:IgG-Fc improved survival rate during development of ACLF via ameliorating liver injury with a potential therapeutic value.     

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.     

Global Gene Expression Profiling Reveals SPINK1 as a Potential Hepatocellular Carcinoma Marker

Background

Liver cirrhosis is the most important risk factor for hepatocellular carcinoma (HCC) but the role of liver disease aetiology in cancer development remains under-explored. We investigated global gene expression profiles from HCC arising in different liver diseases to test whether HCC development is driven by expression of common or different genes, which could provide new diagnostic markers or therapeutic targets.

Methodology and Principal Findings

Global gene expression profiling was performed for 4 normal (control) livers as well as 8 background liver and 7 HCC from 3 patients with hereditary haemochromatosis (HH) undergoing surgery. In order to investigate different disease phenotypes causing HCC, the data were compared with public microarray repositories for gene expression in normal liver, hepatitis C virus (HCV) cirrhosis, HCV-related HCC (HCV-HCC), hepatitis B virus (HBV) cirrhosis and HBV-related HCC (HBV-HCC). Principal component analysis and differential gene expression analysis were carried out using R Bioconductor. Liver disease-specific and shared gene lists were created and genes identified as highly expressed in hereditary haemochromatosis HCC (HH-HCC) were validated using quantitative RT-PCR. Selected genes were investigated further using immunohistochemistry in 86 HCC arising in liver disorders with varied aetiology. Using a 2-fold cut-off, 9 genes were highly expressed in all HCC, 11 in HH-HCC, 270 in HBV-HCC and 9 in HCV-HCC. Six genes identified by microarray as highly expressed in HH-HCC were confirmed by RT qPCR. Serine peptidase inhibitor, Kazal type 1 (SPINK1) mRNA was very highly expressed in HH-HCC (median fold change 2291, p = 0.0072) and was detected by immunohistochemistry in 91% of HH-HCC, 0% of HH-related cirrhotic or dysplastic nodules and 79% of mixed-aetiology HCC.

Conclusion

HCC, arising from diverse backgrounds, uniformly over-express a small set of genes. SPINK1, a secretory trypsin inhibitor, demonstrated potential as a diagnostic HCC marker and should be evaluated in future studies.     

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.     

Immunopositivity for Histone MacroH2A1 Isoforms Marks Steatosis-Associated Hepatocellular Carcinoma

Background

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Prevention and risk reduction are important and the identification of specific biomarkers for early diagnosis of HCC represents an active field of research. Increasing evidence indicates that fat accumulation in the liver, defined as hepatosteatosis, is an independent and strong risk factor for developing an HCC. MacroH2A1, a histone protein generally associated with the repressed regions of chromosomes, is involved in hepatic lipid metabolism and is present in two alternative spliced isoforms, macroH2A1.1 and macroH2A1.2. These isoforms have been shown to predict lung and colon cancer recurrence but to our knowledge, their role in fatty-liver associated HCC has not been investigated previously.

Methods

We examined macroH2A1.1 and macroH2A1.2 protein expression levels in the liver of two murine models of fat-associated HCC, the high fat diet/diethylnistrosamine (DEN) and the phosphatase and tensin homolog (PTEN) liver specific knock-out (KO) mouse, and in human liver samples of subjects with steatosis or HCC, using immunoblotting and immunohistochemistry.

Results

Protein levels for both macroH2A1 isoforms were massively upregulated in HCC, whereas macroH2A1.2 was specifically upregulated in steatosis. In addition, examination of human liver samples showed a significant difference (p<0.01) in number of positive nuclei in HCC (100% of tumor cells positive for either macroH2A1.1 or macroH2A1.2), when compared to steatosis (<2% of hepatocytes positive for either isoform). The steatotic areas flanking the tumors were highly immunopositive for macroH2A1.1 and macroH2A1.2.

Conclusions

These data obtained in mice and humans suggest that both macroH2A1 isoforms may play a role in HCC pathogenesis and moreover may be considered as novel diagnostic markers for human 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.     

The progression of liver fibrosis is related with overexpression of the miR-199 and 200 families

Background

Chronic hepatitis C (CH) can develop into liver cirrhosis (LC) and hepatocellular carcinoma (HCC). Liver fibrosis and HCC development are strongly correlated, but there is no effective treatment against fibrosis because the critical mechanism of progression of liver fibrosis is not fully understood. microRNAs (miRNAs) are now essential to the molecular mechanisms of several biological processes. In order to clarify how the aberrant expression of miRNAs participates in development of the liver fibrosis, we analyzed the liver fibrosis in mouse liver fibrosis model and human clinical samples.

Methodology

In a CCL₄-induced mouse liver fibrosis model, we compared the miRNA expression profile from CCL₄ and olive oil administrated liver specimens on 4, 6, and 8 weeks. We also measured expression profiles of human miRNAs in the liver biopsy specimens from 105 CH type C patients without a history of anti-viral therapy.

Principle Findings

Eleven mouse miRNAs were significantly elevated in progressed liver fibrosis relative to control. By using a large amount of human material in CH analysis, we determined the miRNA expression pattern according to the grade of liver fibrosis. We detected several human miRNAs whose expression levels were correlated with the degree of progression of liver fibrosis. In both the mouse and human studies, the expression levels of miR-199a, 199a*, 200a, and 200b were positively and significantly correlated to the progressed liver fibrosis. The expression level of fibrosis related genes in hepatic stellate cells (HSC), were significantly increased by overexpression of these miRNAs.

Conclusion

Four miRNAs are tightly related to the grade of liver fibrosis in both human and mouse was shown. This information may uncover the critical mechanism of progression of liver fibrosis. miRNA expression profiling has potential for diagnostic and therapeutic applications.     

Protective effect of branched chain amino acids on hindlimb suspension-induced muscle atrophy in growing rats

Purpose

The effect of BCAA (branched chain amino acid) administration on muscle atrophy during growth phases is not well known. We investigated whether BCAA administration can prevent the muscle atrophy induced by hindlimb suspension in growing male rats.

Methods

Male Wistar rats were assigned to 1 of 2 groups (n = 7/group): hindlimb suspension and hindlimb suspension with oral BCAA administration (600 mg·kg⁻¹·day⁻¹, valine 1: leucine 2: isoleucine 1). After 14 days of hindlimb suspension, the weight and mRNA levels of the soleus muscle were measured.

Results

BCAA administration prevented a decrease in soleus muscle weight. BCAA administration attenuated atrogin-1 and MuRF1 mRNA expression, which has been reported to play a pivotal role in muscle atrophy.

Conclusion

BCAA could serve as an effective supplement for the prevention or treatment of muscle atrophy, especially atrophy caused by weightlessness.     

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.     

Circulating microRNA-200 Family as Diagnostic Marker in Hepatocellular Carcinoma

Goals

In this clinical study, we aimed to evaluate the role of circulating microRNA-200 family as a non-invasive tool to identify patients with cirrhosis-associated hepatocellular carcinoma (HCC).

Background

Prognosis of HCC remains poor with increasing incidence worldwide, mainly related to liver cirrhosis. So far, no reliable molecular targets exist for early detection of HCC at surgically manageable stages. Recently, we identified members of the microRNA-200 family as potential diagnostic markers of cirrhosis-associated HCC in patient tissue samples. Their value as circulating biomarkers for HCC remained undefined.

Methods

Blood samples and clinicopathological data of consecutive patients with liver diseases were collected prospectively. Expression of the microRNA-200 family was investigated by qRT-PCR in blood serum samples of 22 HCC patients with and without cirrhosis. Serum samples of patients with non-cancerous chronic liver cirrhosis (n = 22) and of healthy volunteers (n = 15) served as controls.

Results

MicroRNA-141 and microRNA-200a were significantly downregulated in blood serum of patients with HCC compared to liver cirrhosis (p<0.007) and healthy controls (p<0.002). MicroRNA-141 and microRNA-200a could well discriminate patients with cirrhosis-associated HCC from healthy volunteers with area under the receiver-operating characteristic curve (AUC) values of 0.85 and 0.82, respectively. Additionally, both microRNAs could differentiate between HCC and non-cancerous liver cirrhosis with a fair accuracy.

Conclusions

Circulating microRNA-200 family members are significantly deregulated in patients with HCC and liver cirrhosis. Further studies are necessary to confirm the diagnostic value of the microRNA-200 family as accurate serum marker for cirrhosis-associated HCC.     

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).     

The PNPLA3 rs738409 148M/M genotype is a risk factor for liver cancer in alcoholic cirrhosis but shows no or weak association in hepatitis C cirrhosis

Background

An isoleucine>methionine mutation at position 148 in the PNPLA3 gene (p.I148M, rs738409) has recently been identified as a susceptibility factor for liver damage in steatohepatitis. Here, we studied whether the PNPLA3 rs738409 polymorphism also affects predisposition to hepatocellular carcinoma (HCC).

Methods

We compared distributions of PNPLA3 genotypes in 80 and 81 Caucasian patients with alcoholic and hepatitis C virus (HCV)-associated HCC to 80 and 81 age- and sex-matched patients with alcohol-related and HCV-related cirrhosis without HCC, respectively. PNPLA3 genotypes in 190 healthy individuals from the same population served as reference. Potential confounders obesity, diabetes, HCV genotype and HBV co-infection were controlled by univariate and multivariate logistic regression with forward variable selection.

Results

PNPLA3 genotypes were in Hardy-Weinberg equilibrium for all study groups. The frequency of the 148M allele was significantly (p<0.001) increased in alcoholic cirrhosis with (53.7%) and without HCC (36.2%) but was not different between healthy controls (22.9%) and patients with cirrhosis (25.3%; p = 0.545) and HCC (30.2%; p = 0.071) due to hepatitis C. HCC risk was highest in 148M/M homozygous patients with alcoholic liver disease (odds ratio (OR) 16.8 versus healthy controls; 95% confidence interval (CI) 6.68–42.43, p<0.001). Finally, multivariate regression confirmed 148M/M homozygosity (OR 2.8; 95%-CI: 1.24–6.42; p = 0.013) as HCC risk factor in alcoholic cirrhosis. In HCV-related cirrhosis only HCV genotype 1 was confirmed as a HCC risk factor (OR 4.2; 95%-CI: 1.50–11.52; p = 0.006).

Conclusion

The PNPLA3 148M variant is a prominent risk factor for HCC in patients with alcoholic cirrhosis, while its effects are negligible in patients with cirrhosis due to HCV. This polymorphism provides an useful tool to identify individuals with particularly high HCC risk in patients with alcoholic liver disease that should be taken into account in future HCC prevention studies.     

Detailed Analysis of Temporal Features on Contrast Enhanced Ultrasound May Help Differentiate Intrahepatic Cholangiocarcinoma from Hepatocellular Carcinoma in Cirrhosis

Aim

To verify if detailed analysis of temporal enhancement patterns on contrast enhanced ultrasound (CEUS) may help differentiate intrahepatic cholangiocarcinoma (ICC) from hepatocellular carcinoma (HCC) in cirrhosis.

Methods

Thirty three ICC and fifty HCC in cirrhosis were enrolled in this study. The contrast kinetics of ICC and HCC was analyzed and compared.

Results

Statistical analysis did not reveal significant difference between ICC and HCC in the time of contrast first appearance and arterial peak maximum time. ICC displayed much earlier washout than that of HCC (47.93±26.45 seconds vs 90.86±31.26 seconds) in the portal phase, and most ICC (87.9%) showed washout before 60 seconds than HCC (16.0%). Much more ICC (78.8%) revealed marked washout than HCC (12.0%) while most HCC (88.0%) showed mild washout or no washout in late part of the portal phase (90–120 seconds). Twenty six out of thirty three ICC (78.8%) demonstrated both early washout(<60seconds) and marked washout in late part of the portal phase, whereas, only six of fifty HCC (12.0%)showed these temporal enhancement features (p = 0.000).When both early washout and marked washout in the portal phase are taken as diagnostic criterion for ICC, the diagnostic sensitivity, specificity, positive predictive value, negative predictive value and accuracy were 78.8%,88.0%,81.3%,86.3%,and 84.3% respectively by CEUS.

Conclusions

Analysis of detailed temporal enhancement features on CEUS is helpful differentiate ICC from HCC in cirrhosis.If a nodule in cirrhotic liver displays hyper-enhancement in the arterial phase followed by early and marked washout in the portal phase, the nodule is highly suspicious of ICC rather than HCC.     

Large Variations in Risk of Hepatocellular Carcinoma and Mortality in Treatment Na?ve Hepatitis B Patients: Systematic Review with Meta-Analyses

Background

The complications to chronic hepatitis B (HBV) include incidence of hepatocellular carcinoma (HCC) and mortality. The risk of these complications may vary in different patient groups.

Aim

To estimate the incidence and predictors of HCC and in untreated HBV patients.

Methods

Systematic review with random effects meta-analyses of randomized controlled trials and observational studies. Results are expressed as annual incidence (events per 100 person-years) with 95% confidence intervals. Subgroup and sensitivity analyses of patient and study characteristics were performed to identify common risk factors.

Results

We included 68 trials and studies with a total of 27,584 patients (264,919 person-years). In total, 1,285 of 26,687 (5%) patients developed HCC and 730 of 12,511 (6%) patients died. The annual incidence was 0.88 (95% CI, 0.76–0.99) for HCC and 1.26 (95% CI, 1.01–1.51) for mortality. Patients with cirrhosis had a higher risk of HCC (incidence 3.16; 95% CI, 2.58–3.74) than patients without cirrhosis (0.10; 95% CI, 0.02–0.18). The risk of dying was also higher for patients with than patients without cirrhosis (4.89; 95% CI, 3.16–6.63; and 0.11; 95% CI, 0.09–0.14). The risk of developing HCC increased with HCV coinfection, older age and inflammatory activity. The country of origin did not clearly predict HCC or mortality estimates.

Conclusions

Cirrhosis was the strongest predictor of HCC incidence and mortality. Patients with HBV cirrhosis have a 31-fold increased risk of HCC and a 44-fold increased mortality compared to non-cirrhotic patients. The low incidence rates should be taken into account when considering HCC screening in non-cirrhotic patients.

Trial Registration

Prospero CRD42013004764     

Cirrhosis,Liver Transplantation and HIV Infection Are Risk Factors Associated with Hepatitis E Virus Infection

Background

Acute and chronic hepatitis E have been associated with high mortality and development of cirrhosis, particularly in solid-organ recipients and patients infected by human immunodeficiency virus. However, data regarding the epidemiology of hepatitis E in special populations is still limited.

Aims

Investigate seroprevalence and possible factors associated with HEV infection in a large cohort of immunosuppressed patients.

Methods

Cross-sectional study testing IgG anti-HEV in serum samples from 1373 consecutive individuals: 332 liver-transplant, 296 kidney-transplant, 6 dual organ recipients, 301 non-transplanted patients with chronic liver disease, 238 HIV-infected patients and 200 healthy controls.

Results

IgG anti-HEV was detected in 3.5% controls, 3.7% kidney recipients, 7.4% liver transplant without cirrhosis and 32.1% patients who developed post-transplant cirrhosis (p<0.01). In patients with chronic liver disease, IgG anti-HEV was also statistically higher in those with liver cirrhosis (2% vs 17.5%, p<0.01). HIV-infected patients showed an IgG anti-HEV rate of 9.2%, higher than those patients without HIV infection (p<0.03). Multivariate analysis showed that the factors independently associated with anti-HEV detection were liver cirrhosis, liver transplantation and HIV infection (OR: 7.6, 3.1 and 2.4). HCV infection was a protective factor for HEV infection (OR: 0.4).

Conclusions

HEV seroprevalence was high in liver transplant recipients, particularly those with liver cirrhosis. The difference in anti-HEV prevalence between Liver and Kidney transplanted cases suggests an association with advanced liver disease. Further research is needed to ascertain whether cirrhosis is a predisposing factor for HEV infection or whether HEV infection may play a role in the pathogeneses of cirrhosis.     

Protein Profiling in Hepatocellular Carcinoma by Label-Free Quantitative Proteomics in Two West African Populations

Background

Hepatocellular Carcinoma is the third most common cause of cancer related death worldwide, often diagnosed by measuring serum AFP; a poor performance stand-alone biomarker. With the aim of improving on this, our study focuses on plasma proteins identified by Mass Spectrometry in order to investigate and validate differences seen in the respective proteomes of controls and subjects with LC and HCC.

Methods

Mass Spectrometry analysis using liquid chromatography electro spray ionization quadrupole time-of-flight was conducted on 339 subjects using a pooled expression profiling approach. ELISA assays were performed on four significantly differentially expressed proteins to validate their expression profiles in subjects from the Gambia and a pilot group from Nigeria. Results from this were collated for statistical multiplexing using logistic regression analysis.

Results

Twenty-six proteins were identified as differentially expressed between the three subject groups. Direct measurements of four; hemopexin, alpha-1-antitrypsin, apolipoprotein A1 and complement component 3 confirmed their change in abundance in LC and HCC versus control patients. These trends were independently replicated in the pilot validation subjects from Nigeria. The statistical multiplexing of these proteins demonstrated performance comparable to or greater than ALT in identifying liver cirrhosis or carcinogenesis. This exercise also proposed preliminary cut offs with achievable sensitivity, specificity and AUC statistics greater than reported AFP averages.

Conclusions

The validated changes of expression in these proteins have the potential for development into high-performance tests usable in the diagnosis and or monitoring of HCC and LC patients. The identification of sustained expression trends strengthens the suggestion of these four proteins as worthy candidates for further investigation in the context of liver disease. The statistical combinations also provide a novel inroad of analyses able to propose definitive cut-offs and combinations for evaluation of performance.     

Protein expression profiling of nuclear membrane protein reveals potential biomarker of human hepatocellular carcinoma

Background

Complex molecular events lead to development and progression of liver cirrhosis to HCC. Differentially expressed nuclear membrane associated proteins are responsible for the functional and structural alteration during the progression from cirrhosis to carcinoma. Although alterations/ post translational modifications in protein expression have been extensively quantified, complementary analysis of nuclear membrane proteome changes have been limited. Deciphering the molecular mechanism that differentiate between normal and disease state may lead to identification of biomarkers for carcinoma.

Results

Many proteins displayed differential expression when nuclear membrane proteome of hepatocellular carcinoma (HCC), fibrotic liver, and HepG2 cell line were assessed using 2-DE and ESI-Q-TOF MS/MS. From the down regulated set in HCC, we have identified for the first time a 15 KDa cytochrome b5A (CYB5A), ATP synthase subunit delta (ATPD) and Hemoglobin subunit beta (HBB) with 11, 5 and 22 peptide matches respectively. Furthermore, nitrosylation studies with S-nitrosocysteine followed by immunoblotting with anti SNO-cysteine demonstrated a novel and biologically relevant post translational modification of thiols of CYB5A in HCC specimens only. Immunofluorescence images demonstrated increased protein S-nitrosylation signals in the tumor cells and fibrotic region of HCC tissues. The two other nuclear membrane proteins which were only found to be nitrosylated in case of HCC were up regulated ATP synthase subunit beta (ATPB) and down regulated HBB. The decrease in expression of CYB5A in HCC suggests their possible role in disease progression. Further insight of the functional association of the identified proteins was obtained through KEGG/ REACTOME pathway analysis databases. String 8.3 interaction network shows strong interactions with proteins at high confidence score, which is helpful in characterization of functional abnormalities that may be a causative factor of liver pathology.

Conclusion

These findings may have broader implications for understanding the mechanism of development of carcinoma. However, large scale studies will be required for further verification of their critical role in development and progression of HCC.     

Diabetes Mellitus Is Associated with Hepatocellular Carcinoma: A Retrospective Case-Control Study in Hepatitis Endemic Area

Background

A number of case-control patient studies have been conducted to investigate the association between diabetes mellitus (DM) and hepatocellular carcinoma (HCC). Despite some controversial reports, it has been suggested that DM is associated with HCC. The previous studies on this subject vary in the selection of populations, sample sizes, methodology, and analysis results. Therefore, it is necessary to further delineate the involvement of DM, together with other related risk factors, in HCC with large sample size and strict analysis methodology.

Methods

We conducted a hospital-based retrospective case-control study at Perking Union Medical College Hospital, China. A total of 1,568 patients with liver diseases were enrolled in the statistical study to evaluate the association of DM and other risk factors with HCC. Among these patients, 716 of them were diagnosed with benign liver diseases, and 852 patients were diagnosed as HCC. We utilized binary logistic regression and stepwise logistic regression to investigate the associations among DM, hypertension, fatty liver, cirrhosis, gallstone, HBV infection, HCV infection, and HCC.

Results

Statistical analysis through the stepwise regression model indicated that the prevalence of DM, male gender, cirrhosis, HCV infection, or HBV infection is higher in the HCC patient group compared to the control group. However, the prevalence of gallstone is negatively associated with HCC cases. DM co-exists with HBV infection, male gender, and age in the HCC cases. Binary logistic regression analysis suggested that DM may synergize with HBV infection in HCC development.

Conclusion

DM is strongly associated with the increased risk of HCC regardless of the prevalence of HBV infection, HCV infection, cirrhosis, male gender, and age. However, the synergistic interaction between DM and HBV in HCC occurrence is significant. Therefore, DM patients with HBV infection represent a very high HCC risk population and should be considered for HCC close surveillance program.