Marco Maggioni

Homozygosity for the patatin-like phospholipase-3/adiponutrin i148m polymorphism influences liver fibrosis in patients with nonalcoholic fatty liver disease

Inherited factors play a major role in the predisposition to nonalcoholic fatty liver disease (NAFLD), and the rs738409 C→G polymorphism of PNPLA3/adiponutrin, encoding for the isoleucine‐to‐methionine substitution at residue 148 (I148M) protein variant, has recently been recognized as a major determinant of liver fat content. However, the effect of the rs738409 polymorphism on the severity of liver fibrosis in patients with NAFLD is still unknown. In this study, we considered 253 Italian patients, 179 healthy controls, and 71 family trios with an affected child with NAFLD. Analyses were replicated in 321 patients from the United Kingdom. The rs738409 polymorphism was determined by TaqMan assays. Liver histology was scored according to Kleiner et al. Hepatic expression of genes regulating liver damage was assessed by real‐time polymerase chain reaction in 52 patients. The rs738409 GG genotype was more prevalent in patients than in controls (14% versus 3%, adjusted odds ratio [OR] = 3.29, 95% confidence interval [CI] = 1.8‐6.9), and in the family study, the G allele was overtransmitted to affected children (P = 0.001). In Italian and United Kingdom patients, adiponutrin genotype influenced alanine aminotransferase levels and the severity of steatosis. Adiponutrin genotype was associated with the expression of genes involved in the steatosis‐related liver damage, including the proapoptotic molecule Fas ligand. In the whole series combined, adiponutrin genotype was associated with steatosis grade >1 (OR = 1.35, 95% CI = 1.04‐1.76), nonalcoholic steatohepatitis (OR = 1.5, 95% CI = 1.12‐2.04), and fibrosis stage >1 (OR = 1.5, 95% CI = 1.09‐2.12), independent of age, body mass index, and diabetes. Adiponutrin genotype demonstrated a dose effect with heterozygote risk intermediate between CC and GG homozygotes. Conclusion: In patients with NAFLD, adiponutrin rs738409 C→G genotype, encoding for I148M, is associated with the severity of steatosis and fibrosis and the presence of nonalcoholic steatohepatitis. (Hepatology 2010;51:1209–1217)

Patatin-Like Phospholipase Domain-Containing 3 I148M Polymorphism, Steatosis, and Liver Damage in Chronic Hepatitis C

Steatosis has been reported to negatively influence the natural history of chronic hepatitis C (CHC), but controversy remains over its causal role due to the confounding effect of adiposity, insulin resistance, and diabetes. The rs738409 C>G patatin‐like phospholipase domain‐containing 3 (PNPLA3) single nucleotide polymorphism (SNP), encoding for the I148M protein variant, influences liver fat without affecting insulin resistance and body composition. The aim of this study was to evaluate the effect of the rs738409 CG genotype on liver fat and fibrosis in CHC patients. We also explored the possible effect of PNPLA3 genotype on other steatosis‐related complications, namely, treatment failure and hepatocellular carcinoma (HCC) development. To this end we considered two independent series of 325 and 494 CHC patients with available DNA and liver biopsy followed at tertiary referral centers in northern Italy. The rs738409 genotype was determined by a Taqman assay. The rs738409 GG genotype, observed in 10% of patients, was associated with steatosis independently of age, sex, body mass index (BMI), diabetes, alcohol intake, and viral genotype (odds ratio [OR] 1.90, 95% confidence interval [CI] 1.4‐2.7; P < 0.001). The association with rs738409 genotype was confirmed for severe steatosis, was independent of alanine aminotransferase (ALT) and gamma‐glutamyl transferase (GGT) values, and was observed in all viral genotypes but the 3. The rs738409 GG genotype was associated with fibrosis stage and cirrhosis (OR 1.47, 95% CI 1.2‐1.9; P = 0.002), treatment response (n = 470; OR 0.63, 95% CI 0.4‐0.8; P = 0.006), and HCC occurrence (n = 325; OR 2.16, 95% CI 1.3‐3.6; P = 0.002), independently of confounders. Conclusion: The rs738409 PNPLA3 genotype influences steatosis development in CHC and is independently associated with cirrhosis and other steatosis‐related clinical outcomes, such as lack of response to antiviral treatment and possibly HCC. (HEPATOLOGY 2011)

Lack of CD 29 (β1 integrin) and CD 54 (ICAM-1) adhesion molecules in intravascular lymphomatosis*

Intravascular Lymphomatosis (IL) is a rare and usually aggressive form of non-Hodgkins lymphoma characterized by the growth of neoplastic cells within vascular lumina that usually presents with skin or central nervous system (CNS) involvement. The mechanism(s) for the selective intravascular growth of this neoplasm remain(s) unexplained. We now report clinical and immunohistologic data on surgical material from 6 cases of IL; in 4 of 6 cases, autopsies were performed. Our IL cases shared the following features: (1) B-cell lineage; (2) lack of skin involvement at presentation; (3) aggressive behavior; and (4) lack of extravascular lymphomatous masses; in addition, 1 case had an associated gastric low-grade MALT lymphoma. We studied by immunohistochemistry formalin-fixed, paraffin-embedded sections with monoclonal antibodies to molecules known to be involved in lymphocyte and endothelial adhesion phenomena, that is, CD29 (beta1 integrin subunit), CD43 (leukosialin), CD44 (H-CAM), CD54 (ICAM-1), embryonal N-CAM (e-NCAM), and EMA (episialin). In all cases, the surfaces of IL aggregates reacted for CD44 but were consistently negative for CD29; also absent was CD54. Conversely, the integrity of the endothelial cells was underscored by their even reactivity for CD29, CD44, and CD54. Given that CD29 is currently regarded as critical for lymphocyte trafficking in general and for transvascular migration in particular, and CD54 is also involved in transvascular lymphocyte migration, we conclude that their consistent absence in IL may contribute to its intravascular and disseminated distribution pattern. The rather frequent association of IL with various conventional lymphomas is known; yet, one of our cases appears to be the first report of IL associated with a low-grade MALT lymphoma.

Transmembrane 6 superfamily member 2 gene variant disentangles nonalcoholic steatohepatitis from cardiovascular disease

Excess hepatic storage of triglycerides is considered a benign condition, but nonalcoholic steatohepatitis (NASH) may progress to fibrosis and promote atherosclerosis. Carriers of the TM6SF2 E167K variant have fatty liver as a result of reduced secretion of very‐low‐density lipoproteins (VLDLs). As a result, they have lower circulating lipids and reduced risk of myocardial infarction. In this study, we aimed to assess whether TM6SF2 E167K affects liver damage and cardiovascular outcomes in subjects at risk of NASH. Liver damage was evaluated in 1,201 patients who underwent liver biopsy for suspected NASH; 427 were evaluated for carotid atherosclerosis. Cardiovascular outcomes were assessed in 1,819 controls from the Swedish Obese Subjects (SOS) cohort. Presence of the inherited TM6SF2 E167K variant was determined by TaqMan assays. In the liver biopsy cohort, 188 subjects (13%) were carriers of the E167K variant. They had lower serum lipid levels than noncarriers (P < 0.05), had more‐severe steatosis, necroinflammation, ballooning, and fibrosis (P < 0.05), and were more likely to have NASH (odds ratio [OR]: 1.84; 95% confidence interval [CI]: 1.23‐2.79) and advanced fibrosis (OR, 2.08; 95% CI: 1.20‐3.55), after adjustment for age, sex, body mass index, fasting hyperglycemia, and the I148M PNPLA3 risk variant. However, E167K carriers had lower risk of developing carotid plaques (OR, 0.49; 95% CI: 0.25‐0.94). In the SOS cohort, E167K carriers had higher alanine aminotransferase ALT and lower lipid levels (P < 0.05), as well as a lower incidence of cardiovascular events (hazard ratio: 0.61; 95% CI: 0.39‐0.95). Conclusions: Carriers of the TM6SF2 E167K variant are more susceptible to progressive NASH, but are protected against cardiovascular disease. Our findings suggest that reduced ability to export VLDLs is deleterious for the liver. (Hepatology 2015;61:506‐514)

Autoantibodies against nuclear pore complexes are associated with more active and severe liver disease in primary biliary cirrhosis

BACKGROUND/AIMS Antibodies against nuclear pore complexes (NPCs) have been detected in primary biliary cirrhosis (PBC), but their clinical relevance is still unsettled. METHODS We tested sera from 171 consecutive PBC patients and 230 control subjects (149 with autoimmune or viral liver diseases, 28 with systemic lupus erythematosus, and 53 healthy) by immunoblotting for antibodies against purified human NPCs. RESULTS Antibodies to NPCs were detected in 27% of the patients with PBC, were highly specific (97%), and were not associated with antimitochondrial antibodies. Their prevalence was higher in symptomatic patients (36 vs. 16%, P < 0.01) and was associated (P < 0.001) with more severe disease, as assessed by the presence of cirrhosis or its complications (13% prevalence in patients without cirrhosis, 31% in uncomplicated, and 54% in complicated cirrhosis), or by the application of the Mayo prognostic model (12% in the lowest, 21% in the median, 47% in the highest score tertile). Positive patients had higher levels of serum bilirubin (2.2 +/- 3.7 vs. 1.0 +/- 1.1 mg/dl, P < 0.01) and more marked inflammatory infiltrates on liver biopsy (P < 0.05). CONCLUSIONS Autoantibodies to NPCs are more prevalent in PBC patients than in controls and are strongly associated with more active and severe disease.

Incidence and risk factors for non-alcoholic steatohepatitis: prospective study of 5408 women enrolled in Italian tamoxifen chemoprevention trial.

Abstract Objective To assess the incidence, cofactors, and excess risk of development of non-alcoholic fatty liver disease, including non-alcoholic steatohepatitis, attributable to tamoxifen in women. Design Prospective, randomised, double blind, placebo controlled trial. Setting and participants 5408 healthy women who had had hysterectomies, recruited into the Italian tamoxifen chemoprevention trial from 58 centres in Italy. Intervention Women were randomly assigned to receive tamoxifen (20 mg daily) or placebo for five years. Main outcome measure Development of non-alcoholic fatty liver disease in all women with normal baseline liver function who showed at least two elevations of alanine aminotransferase (≥ 1.5 times upper limit of normal) over a six month period. Results During follow up, 64 women met the predefined criteria: 12 tested positive for hepatitis C virus, and the remaining 52 were suspected of having developed non-alcoholic fatty liver disease (34 tamoxifen, 18 placebo)—hazard ratio = 2.0 (95% confidence interval 1.1 to 3.5; P = 0.04). In all 52 women ultrasonography confirmed the presence of fatty liver. Other factors associated with the development of non-alcoholic fatty liver disease included overweight (2.4, 1.2 to 4.8), obesity (3.6, 1.7 to 7.6), hypercholesterolaemia (3.4, 1.4 to 7.8), and arterial hypertension (2.0, 1.0 to 3.8). Twenty women had liver biopsies: 15 were diagnosed as having mild to moderate steatohepatitis (12 tamoxifen, 3 placebo), and five had fatty liver alone (1 tamoxifen, 4 placebo). No clinical, biochemical, ultrasonic, or histological signs suggestive of progression to cirrhosis were observed after a median follow up of 8.7 years. Conclusions Tamoxifen was associated with higher risk of development of non-alcoholic steatohepatitis only in overweight and obese women with features of metabolic syndrome, but the disease, in both the tamoxifen and the placebo group, after 10 years of follow up seems to be indolent.

Inhibitors of apoptosis proteins (IAPs) expression and their prognostic significance in hepatocellular carcinoma

BackgroundSimilarly to other tumor types, an imbalance between unrestrained cell proliferation and impaired apoptosis appears to be a major unfavorable feature of hepatocellular carcinoma (HCC). The members of IAP family are key regulators of apoptosis, cytokinesis and signal transduction. IAP survival action is antagonized by specific binding of Smac/DIABLO and XAF1. This study aimed to investigate the gene and protein expression pattern of IAP family members and their antagonists in a series of human HCCs and to assess their clinical significance.MethodsRelative quantification of IAPs and their antagonist genes was assessed by quantitative Real Time RT-PCR (qPCR) in 80 patients who underwent surgical resection for HCC. The expression ratios of XIAP/XAF1 and of XIAP/Smac were also evaluated. Survivin, XIAP and XAF1 protein expression were investigated by immunohistochemistry. Correlations between mRNA levels, protein expression and clinicopathological features were assessed. Follow-up data were available for 69 HCC patients. The overall survival analysis was estimated according to the Kaplan-Meier method.ResultsSurvivin and Livin/ML-IAP mRNAs were significantly over-expressed in cancer tissues compared to non-neoplastic counterparts. Although Survivin immunoreactivity did not correlate with qPCR data, a significant relation was found between higher Survivin mRNA level and tumor stage, tumor grade and vascular invasion.The mRNA ratio XIAP/XAF1 was significantly higher in HCCs than in cirrhotic tissues. Moreover, high XIAP/XAF1 ratio was an indicator of poor prognosis when overall survival was estimated and elevated XIAP immunoreactivity was significantly associated with shorter survival.ConclusionOur study demonstrates that alterations in the expression of IAP family members, including Survivin and Livin/ML-IAP, are frequent in HCCs. Of interest, we could determine that an imbalance in XIAP/XAF1 mRNA expression levels correlated to overall patient survival, and that high XIAP immunoreactivity was a poor prognostic factor.

Statin use and non-alcoholic steatohepatitis in at risk individuals

BACKGROUND & AIMS Excess hepatic free cholesterol contributes to the pathogenesis of non-alcoholic steatohepatitis, and statins reduce cholesterol synthesis. Aim of this study was to assess whether statin use is associated with histological liver damage related to steatohepatitis. METHODS The relationship between statin use, genetic risk factors, and liver damage was assessed in a multi-center cohort of 1201 European individuals, who underwent liver biopsy for suspected non-alcoholic steatohepatitis. RESULTS Statin use was recorded in 107 subjects, and was associated with protection from steatosis, NASH, and fibrosis stage F2-F4, in a dose-dependent manner (adjusted p<0.05 for all). In 100 treated patients matched 1:1 for modality of recruitment, gender, presence of IFG or type 2 diabetes, PNPLA3 I148M risk alleles, TM6SF2 E167K variant, age, and BMI, statin use remained associated with protection from steatosis (OR 0.09, 95% C.I. 0.01-0.32; p=0.004), steatohepatitis (OR 0.25, 95% C.I. 0.13-0.47; p<0.001), and fibrosis stage F2-F4 (OR 0.42, 95% C.I. 0.20-0.8; p=0.017). Results were confirmed in a second analysis, where individuals were matched within recruitment center (p<0.05 for all). The protective effect of statins on steatohepatitis was stronger in subjects not carrying the I148M PNPLA3 risk variant (p=0.02 for interaction), as statins were negatively associated with steatohepatitis in patients negative (p<0.001), but not in those positive for the I148M variant (p=n.s.). CONCLUSIONS Statin use was associated with protection towards the full spectrum of liver damage in individuals at risk of non-alcoholic steatohepatitis. However, the I148M PNPLA3 risk variant limited this beneficial effect.

Transarterial chemoembolization with epirubicin-eluting beads versus transarterial embolization before liver transplantation for hepatocellular carcinoma.

PURPOSE To retrospectively compare radiologic tumor response and degree of necrosis in explanted livers after chemoembolization with epirubicin-loaded DC Bead versus bland embolization in patients on a transplant waiting list. MATERIALS AND METHODS From 2003 to 2007, 49 patients with hepatocellular carcinoma (HCC) underwent transplantation at a single center. Sixteen patients were treated with bland embolization (n = 8) with 100-300-microm Embosphere particles or chemoembolization with epirubicin-loaded 100-300-microm DC Bead particles (n = 8) every other month until complete tumor devascularization. Computed tomography was performed every 3 months until recurrence. Explanted livers were analyzed to evaluate the degree of necrosis in the nodules. After orthotopic liver transplantation (OLT), patients were followed up for survival and disease status. RESULTS The groups were comparable for baseline characteristics. Most patients had Child-Pugh class A disease. Solitary HCC was found in 75% of patients. Mean target lesion size was 32 mm +/- 15.4. Chemoembolization with drug-eluting beads achieved complete necrosis in 77% of lesions whereas bland embolization achieved complete necrosis in 27.2% of lesions. There was a significant difference between bland embolization and chemoembolization with DC Bead with regard to histologic necrosis (P = .043). No significant treatment-related complications were observed for either group. Fifteen patients are alive with no tumor recurrence. CONCLUSIONS Chemoembolization with drug-eluting beads before OLT achieved higher rates of complete histologic response than bland embolization, with no serious adverse events observed. Because of the retrospective data analyses and small sample size, further studies are warranted to confirm these promising results.

A proteomic approach to differentiate histologically classified stable and unstable plaques from human carotid arteries

OBJECTIVES By using proteomics we isolated and identified proteins that were expressed/retained in stable and unstable human carotid artery atherosclerotic plaques. METHODS The criteria for plaque instability were the presence of a thin fibrous cap or fissured cap covering the foamy or necrotic core, and the presence of overt, hemorrhagic, ulcerated or thrombotic plaques. Proteins were extracted from finely minced endarterectomy specimens (19 stable and 29 unstable plaques) and separated by two-dimensional gel electrophoresis. Coomassie Blue-stained gels were analysed using PD-Quest software. RESULTS A total of 57 distinct spots corresponding to 33 different proteins were identified by matrix assisted laser desorption/ionization mass spectrometry using the NCBI database. Most of the spots were present in both types of extracts, although significantly (p<0.05) differing in abundance between them. Compared to stable plaque, unstable ones showed reduced abundance of: protective enzymes SOD3 and GST, small heat shock proteins HSP27 and HSP20, annexin A10, and Rho GDI. In unstable plaques the more abundant proteins were: ferritin light subunit, SOD 2 and fibrinogen fragment D. For fibrinogen fragment D, the increased levels in unstable versus stable plaques was confirmed by Western blot analysis. CONCLUSIONS Since many of the differentially expressed proteins are known to have a functional role in inflammation and oxidative stress, we speculate that they may be involved in events relating to plaque stability.