Clinical, histological and molecular profiling of different stages of alcohol-related liver disease.

OBJECTIVE: Alcohol-related liver disease (ALD) ranges from never-decompensated ALD (ndALD) to the life-threatening decompensated phenotype, known as alcohol-related hepatitis (AH). A multidimensional study of the clinical, histological and molecular features of these subtypes is lacking. DESIGN: Two large cohorts of patients were recruited in an international, observational multicentre study: a retrospective cohort of patients with ndALD (n=110) and a prospective cohort of patients with AH (n=225). Clinical, analytical, immunohistochemistry and hepatic RNA microarray analysis of both disease phenotypes were performed. RESULTS: Age and mean alcohol intake were similar in both groups. AH patients had greater aspartate amino transferase/alanine amino transferase ratio and lower gamma-glutamyl transferase levels than in ndALD patients. Patients with AH demonstrated profound liver failure and increased mortality. One-year mortality was 10% in ndALD and 50% in AH. Histologically, steatosis grade, ballooning and pericellular fibrosis were similar in both groups, while advanced fibrosis, Mallory-Denk bodies, bilirubinostasis, severe neutrophil infiltration and ductular reaction were more frequent among AH patients. Transcriptome analysis revealed a profound gene dysregulation within both phenotypes when compare to controls. While ndALD was characterised by deregulated expression of genes involved in matrisome and immune response, the development of AH resulted in a marked deregulation of genes involved in hepatocyte reprogramming and bile acid metabolism. CONCLUSIONS: Despite comparable alcohol intake, AH patients presented with worse liver function compared with ndALD patients. Bilirubinostasis, severe fibrosis and ductular reaction were prominent features of AH. AH patients exhibited a more profound deregulation of gene expression compared with ndALD patients.

Adiponectin, Leptin, and IGF-1 Are Useful Diagnostic and Stratification Biomarkers of NAFLD.

Background: Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease where liver biopsy remains the gold standard for diagnosis. Here we aimed to evaluate the role of circulating adiponectin, leptin, and insulin-like growth factor 1 (IGF-1) levels as non-invasive NAFLD biomarkers and assess their correlation with the metabolome. Materials and Methods: Leptin, adiponectin, and IGF-1 serum levels were measured by ELISA in two independent cohorts of biopsy-proven obese NAFLD patients and healthy-liver controls (discovery: 38 NAFLD, 13 controls; validation: 194 NAFLD, 31 controls) and correlated with clinical data, histology, genetic parameters, and serum metabolomics. Results: In both cohorts, leptin increased in NAFLD vs. controls (discovery: AUROC 0.88; validation: AUROC 0.83; p < 0.0001). The leptin levels were similar between obese and non-obese healthy controls, suggesting that obesity is not a confounding factor. In the discovery cohort, adiponectin was lower in non-alcoholic steatohepatitis (NASH) vs. non-alcoholic fatty liver (AUROC 0.87; p < 0.0001). For the validation cohort, significance was attained for homozygous for PNPLA3 allele c.444C (AUROC 0.63; p < 0.05). Combining adiponectin with specific serum lipids improved the assay performance (AUROC 0.80; p < 0.0001). For the validation cohort, IGF-1 was lower with advanced fibrosis (AUROC 0.67, p < 0.05), but combination with international normalized ratio (INR) and ferritin increased the assay performance (AUROC 0.81; p < 0.01). Conclusion: Serum leptin discriminates NAFLD, and adiponectin combined with specific lipids stratifies NASH. IGF-1, INR, and ferritin distinguish advanced fibrosis.

Pseudomalignant osteoblastoma of the odontoid process.

INTRODUCTION: The anterior elements of the spine, particularly the odontoid processes, are a rare location for osteoblastomas. Pseudomalignant osteoblastomas are themselves rare histologic types and are also extremely rare in this location. Most osteoblastomas are Enneking stage 2 lesions; less frequently, they can be more aggressive with extra-capsular extension (Enneking stage 3). En bloc resection is recommended for aggressive lesions, but the literature is less clear regarding the approach to stage 2 tumors, particularly those with pseudomalignant histologic features. CASE REPORT: A 6-year-old male child presented with a type III pathologic fracture of the odontoid. The fracture healed but upon 6-month follow-up CT scanning, an expansile lesion was detected. Surgical biopsy revealed an osteoblastoma which was treated with intralesional excision. Meanwhile, the excised specimen showed histological features of a pseudomalignant osteoblastoma. Despite this diagnosis, no further treatment was undertaken. At a 10-year follow-up, the patient was free from pain and had full range of motion of the cervical spine; no recurrence was detected. CONCLUSION: This unique case of odontoid osteoblastoma illustrates that malignant behavior may not be predicted only by the presence of pseudomalignant features on histology.

Prognostic significance of AKT/mTOR signaling in advanced neuroendocrine tumors treated with somatostatin analogs.

INTRODUCTION: Somatostatin analogs (SSAs) are used as part of standard treatment for advanced neuroendocrine tumors (NETs). The mechanisms behind the antiproliferative action of SSAs remain largely unknown, but a connection with the mammalian target of rapamycin (mTOR) signaling pathway has been suggested. Our purpose was to evaluate the activation status of the AKT/mTOR pathway in advanced metastatic NETs and identify biomarkers of response to SSA therapy. PATIENTS AND METHODS: Expression of phosphatase and tensin homolog (PTEN), phosphorylated (p)-AKT(Ser473), and p-S6(Ser240/244) was evaluated using immunohistochemistry in archival paraffin samples from 23 patients. Expression levels were correlated with clinicopathological parameters and progression-free survival under treatment with SSAs. RESULTS: A positive association between p-AKT and p-S6 expression was identified (P = 0.01) and higher expression of both markers was observed in pancreatic NETs. AKT/mTOR activation was observed without the loss of PTEN expression. Tumors showing AKT/mTOR signaling activation progressed faster when treated with SSAs: higher expression of p-AKT or p-S6 predicted a median progression-free survival of 1 month vs 26.5 months for lower expression (P = 0.02). CONCLUSION: Constitutive activation of the AKT/mTOR pathway was associated with shorter time-to-progression in patients undergoing treatment with SSAs. Larger case series are needed to validate whether p-AKT(Ser473) and p-S6(Ser240/244) can be used as prognostic markers of response to therapy with SSAs.

How adiponectin, leptin, and ghrelin orchestrate together and correlate with the severity of nonalcoholic fatty liver disease.

BACKGROUND: Adipose tissue contributes to nonalcoholic fatty liver disease (NAFLD), being a source of fatty acids and cytokines such as leptin and adiponectin, and regulating ghrelin production. Their role in NAFLD pathogenesis remains controversial. We aimed to study the influence of those cytokines on the severity of NAFLD. METHODS: Morbidly obese individuals with biopsy-proven NAFLD were recruited. The NAFLD activity score was applied to liver histology. Serum concentrations of adiponectin, leptin, and ghrelin were determined. RESULTS: Eighty-two patients were included, 13% with nonalcoholic steatohepatitis (NASH). Hypertriglyceridemia (P=0.018) and metabolic syndrome (P=0.040) were independent factors associated with NASH. Leptin associated positively and ghrelin associated negatively with BMI; adiponectin associated negatively with the waist to hip ratio. Adiponectin associated negatively with insulin resistance, hypertension, and metabolic syndrome; ghrelin associated positively with diabetes mellitus. Adiponectin below 23 ng/ml associated with NASH (odds ratio 12.95, P<0.001). Leptin increased progressively (P=0.032) and adiponectin decreased (P=0.004) with increasing severity of steatosis. Also, leptin increased progressively with more severe fibrosis (P=0.053). A formula incorporating the three cytokines yielded an AUROC of 0.789 (P=0.002), a sensitivity of 81.8%, and a specificity of 76.1% for NASH. CONCLUSION: An imbalance in adiponectin, leptin, and ghrelin seems to be associated with more severe NAFLD. A formula combining the three cytokines showed good accuracy for NASH.

Liver and muscle in morbid obesity: the interplay of fatty liver and insulin resistance.

INTRODUCTION: Nonalcoholic fatty liver disease (NAFLD) can be seen as a manifestation of overnutrition. The muscle is a central player in the adaptation to energy overload, and there is an association between fatty-muscle and -liver. We aimed to correlate muscle morphology, mitochondrial function and insulin signaling with NAFLD severity in morbid obese patients. METHODS: Liver and deltoid muscle biopsies were collected during bariatric surgery in NAFLD patients. NAFLD Activity Score and Younossi's classification for nonalcoholic steatohepatitis (NASH) were applied to liver histology. Muscle evaluation included morphology studies, respiratory chain complex I to IV enzyme assays, and analysis of the insulin signaling cascade. A healthy lean control group was included for muscle morphology and mitochondrial function analyses. RESULTS: Fifty one NAFLD patients were included of whom 43% had NASH. Intramyocellular lipids (IMCL) were associated with the presence of NASH (OR 12.5, p<0.001), progressive hepatic inflammation (p = 0.029) and fibrosis severity (p = 0.010). There was a trend to an association between IMCL and decreased Akt phosphorylation (p = 0.059), despite no association with insulin resistance. In turn, hepatic steatosis (p = 0.015) and inflammation (p = 0.013) were associated with decreased Akt phosphoryation. Citrate synthase activity was lower in obese patients (p = 0.047) whereas complex I (p = 0.040) and III (p = 0.036) activities were higher, compared with controls. Finally, in obese patients, complex I activity increased with progressive steatosis (p = 0.049) and with a trend with fibrosis severity (p = 0.056). CONCLUSIONS: In morbid obese patients, presence of IMCL associates with NASH and advanced fibrosis. Muscle mitochondrial dysfunction does not appear to be a major driving force contributing to muscle fat accumulation, insulin resistance or liver disease. Importantly, insulin resistance in muscle might occur at a late point in the insulin signaling cascade and be associated with IMCL and NAFLD severity.

Impaired renal function in morbid obese patients with nonalcoholic fatty liver disease.

INTRODUCTION AND AIMS: Obesity is a common risk factor for nonalcoholic fatty liver disease (NAFLD) and chronic kidney disease (CKD). NAFLD and CKD have been associated in many epidemiological studies. We hypothesize that more severe liver disease, namely nonalcoholic steatohepatitis (NASH), is related with further renal impairment. We aimed to evaluate if changes in renal function were present in morbid obese patients with NAFLD. METHODS: Prospective and consecutive recruitment of morbid obese patients with biopsy proven NAFLD obtained during bariatric surgery. Renal function was evaluated with CKD-Epidemiology Collaboration estimated glomerular filtration rate (eGFR). Plasmatic adiponectin, leptin and active ghrelin concentrations were determined. RESULTS: One hundred and forty-eight patients were included of whom 25% had NASH and 75% simple steatosis. NASH patients were older, with higher body mass index and had more frequently metabolic syndrome and lower eGFR (97 ± 22 vs 106 ± 16 ml/min/1.73(2), P = 0.035). NASH conferred an odds ratio (OR) 3.0 (1.3-7.0) for eGFR < 90 and OR 9.7 (1.0-96.4) for eGFR < 60 ml/min/1.73(2). eGFR < 90 ml/min/1.73(2) associated with aspartate aminotransferase [OR 2.9 (1.1-7.6)] and γ-glutamyl transpeptidase elevation [OR 3.0 (1.3-7.2)], NASH [OR 3.0 (1.3-7.0)], any lobular inflammatory activity [OR 3.0 (1.3-7.0)] and severe fibrosis [OR 3.4 (1.1-10.8)]. Neither eGFR nor liver histology was associated with adipokines levels. CONCLUSIONS: In morbid obese patients, NASH, particularly lobular inflammation and advanced fibrosis, associates with mild decreases in eGFR, suggesting a common inflammatory link between liver and renal lesion.

Bile acid levels are increased in the liver of patients with steatohepatitis.

BACKGROUND/AIMS: The pathogenesis of steatohepatitis remains largely unknown; however, bile acids may play a role as potential mediators of liver damage. The aim of this study was to characterize bile acid profiles in liver tissue of patients with steatohepatitis. METHODS: Bile acid composition was determined by gas-liquid chromatography in liver tissue from patients with nonalcoholic steatohepatitis (NASH; n=15), patients with alcoholic steatohepatitis (ASH; n=14), and controls (n=8). Liver biopsies were graded for steatosis, inflammation, and fibrosis. RESULTS: Bile acids were moderately increased in liver tissue of steatohepatitis patients compared with controls (P<0.05). Deoxycholic, chenodeoxycholic, and cholic acids were elevated by 92, 64, and 43%, respectively, in patients with steatohepatitis (P<0.05). Cholic acid was the prevailing bile acid in NASH patients and in controls. More hydrophobic bile acid species were elevated in ASH patients compared with controls (P<0.05). Significant correlations were found in NASH patients between hepatic chenodeoxycholic acid and fibrosis, and between cholic acid and trihydroxy/dihydroxy bile acids and inflammation (P<0.05). In patients with ASH, cholic acid and trihydroxy/dihydroxy bile acids were correlated with steatosis (P<0.01). CONCLUSION: This study shows a distinct pattern of bile acids in the liver of patients with steatohepatitis. Further, the association between bile acids and histological liver injury suggests an association of specific bile acids and disease progression, possibly through bile acid-induced liver injury.

Apoptosis and Bcl-2 expression in the livers of patients with steatohepatitis.

OBJECTIVES: Apoptosis may play a role in the pathogenesis of alcoholic (ASH) and non-alcoholic steatohepatitis (NASH). In this study, we investigated the modulation of apoptosis-related liver proteins in steatohepatitis. METHODS: Hepatocyte apoptosis was evaluated by the TUNEL assay in liver tissue of 12 patients with NASH, 12 with ASH and in histologically normal controls. In addition, caspase-3 processing was evaluated by immunoblot analysis. Expression of death receptors, Bcl-2 family members, and NF-kappaB inhibitor (IkappaB) were determined by western blot. Liver biopsies were also graded for inflammation and fibrosis. RESULTS: Apoptotic hepatocytes were markedly increased in NASH (P<0.05) and ASH (P<0.001) as compared to controls. Active caspase-3 was also elevated in steatohepatitis (P<0.01), coinciding with upregulation of pro-apoptotic Bax (P<0.001). Further, production of tumour necrosis factor-receptor 1 was increased up to 4-fold (P<0.05). Degradation of IkappaB increased >70% in steatohepatitis (P<0.001). Notably, Bcl-2 was also strongly expressed (>100-fold; P<0.001). These data were significantly correlated with relative degrees of portal and lobular inflammation. CONCLUSION: The results show that liver injury in NASH and ASH is associated with apoptosis and NF-kappaB activation. Anti-apoptotic Bcl-2 is strongly expressed, probably reflecting an adaptive response to obesity or alcohol-related stress.

Oxidative stress in chronic hepatitis C: a preliminary study on the protective effects of antioxidant flavonoids.

BACKGROUND/AIMS: Oxidative stress is involved in chronic hepatitis C, and efforts have been made to influence the disease process with antioxidants. The present study evaluates the protective effects of a phenol-rich processed grain food with superoxide-scavenging properties (trade name antioxidant biofactor AOB). METHODOLOGY: Thirty patients participated in this placebo-controlled double-blind pilot study. AOB was taken orally by fifteen patients for 3 mo at the recommended daily dose of 3x2 sachets, containing 3 g of powder each. Another fifteen patients received a herbal extract with practically no superoxide scavenging properties as a placebo. Oxidative stress biomarkers, aminotransferase levels and viral load were evaluated immediately before and after treatment. RESULTS: AOB treatment considerably improved the antioxidant defenses. Also ALT and AST decreased in 11 of the 15 patients (-11% to -65%, mean -22%, p<0.05). The effects of placebo were not significant. Viral load remained unchanged. Control biopsies were not done after the short interval of 3 mo. There were no adverse effects. After the 3-mo treatment with AOB or placebo, 16 of the 30 patients received conventional antiviral treatment (pegylated interferon alpha and ribavirin). A sustained response was observed in 5 of 9 AOB pretreated patients six mo after discontinuation of the 12-mo antiviral therapy. The 7 patients pretreated with placebo were all non-responders. CONCLUSIONS: These preliminary results are encouraging to conduct more extensive clinical studies combining antioxidant with antiviral treatment in hepatitis C.