Serum ferritin levels can predict long-term outcomes in patients with metabolic dysfunction-associated steatotic liver disease.

OBJECTIVE: Hyperferritinaemia is associated with liver fibrosis severity in patients with metabolic dysfunction-associated steatotic liver disease (MASLD), but the longitudinal implications have not been thoroughly investigated. We assessed the role of serum ferritin in predicting long-term outcomes or death. DESIGN: We evaluated the relationship between baseline serum ferritin and longitudinal events in a multicentre cohort of 1342 patients. Four survival models considering ferritin with confounders or non-invasive scoring systems were applied with repeated five-fold cross-validation schema. Prediction performance was evaluated in terms of Harrell's C-index and its improvement by including ferritin as a covariate. RESULTS: Median follow-up time was 96 months. Liver-related events occurred in 7.7%, hepatocellular carcinoma in 1.9%, cardiovascular events in 10.9%, extrahepatic cancers in 8.3% and all-cause mortality in 5.8%. Hyperferritinaemia was associated with a 50% increased risk of liver-related events and 27% of all-cause mortality. A stepwise increase in baseline ferritin thresholds was associated with a statistical increase in C-index, ranging between 0.02 (lasso-penalised Cox regression) and 0.03 (ridge-penalised Cox regression); the risk of developing liver-related events mainly increased from threshold 215.5 µg/L (median HR=1.71 and C-index=0.71) and the risk of overall mortality from threshold 272 µg/L (median HR=1.49 and C-index=0.70). The inclusion of serum ferritin thresholds (215.5 µg/L and 272 µg/L) in predictive models increased the performance of Fibrosis-4 and Non-Alcoholic Fatty Liver Disease Fibrosis Score in the longitudinal risk assessment of liver-related events (C-indices>0.71) and overall mortality (C-indices>0.65). CONCLUSIONS: This study supports the potential use of serum ferritin values for predicting the long-term prognosis of patients with MASLD.

SIRT5 rs12216101 T>G variant is associated with liver damage and mitochondrial dysfunction in patients with non-alcoholic fatty liver disease.

BACKGROUND & AIMS: Sirtuin 5, encoded by the SIRT5 gene, is a NAD+-dependent deacylase that modulates mitochondrial metabolic processes through post-translational modifications. In this study, we aimed to examine the impact of the SIRT5 rs12216101 T>G non-coding single nucleotide polymorphism on disease severity in patients with non-alcoholic fatty liver disease (NAFLD). METHODS: The rs12216101 variant was genotyped in 2,606 consecutive European patients with biopsy-proven NAFLD. Transcriptomic analysis, expression of mitochondrial complexes and oxidative stress levels were measured in liver samples from a subset of bariatric patients. Effects of SIRT5 pharmacological inhibition were evaluated in HepG2 cells exposed to excess free fatty acids. Mitochondrial energetics in vitro were investigated by high-performance liquid chromatography. RESULTS: In the whole cohort, the frequency distribution of SIRT5 rs12216101 TT, TG and GG genotypes was 47.0%, 42.3% and 10.7%, respectively. At multivariate logistic regression analysis adjusted for sex, age >50 years, diabetes, and PNPLA3 rs738409 status, the SIRT5 rs12216101 T>G variant was associated with the presence of non-alcoholic steatohepatitis (odds ratio 1.20, 95% CI 1.03-1.40) and F2-F4 fibrosis (odds ratio 1.18; 95% CI 1.00-1.37). Transcriptomic analysis showed that the SIRT5 rs12216101 T>G variant was associated with upregulation of transcripts involved in mitochondrial metabolic pathways, including the oxidative phosphorylation system. In patients carrying the G allele, western blot analysis confirmed an upregulation of oxidative phosphorylation complexes III, IV, V and consistently higher levels of reactive oxygen species, reactive nitrogen species and malondialdehyde, and lower ATP levels. Administration of a pharmacological SIRT5 inhibitor preserved mitochondrial energetic homeostasis in HepG2 cells, as evidenced by restored ATP/ADP, NAD+/NADH, NADP+/NADPH ratios and glutathione levels. CONCLUSIONS: The SIRT5 rs12216101 T>G variant, heightening SIRT5 activity, is associated with liver damage, mitochondrial dysfunction, and oxidative stress in patients with NAFLD. IMPACT AND IMPLICATIONS: In this study we discovered that the SIRT5 rs12216101 T>G variant is associated with higher disease severity in patients with non-alcoholic fatty liver disease (NAFLD). This risk variant leads to a SIRT5 gain-of-function, enhancing mitochondrial oxidative phosphorylation and thus leading to oxidative stress. SIRT5 may represent a novel disease modulator in NAFLD.

High-Risk HPV CISH Detection in Cervical Biopsies with Weak and/or Focal p16 Immunohistochemical Positivity.

In cervical biopsies, for diagnosis of Human Papilloma Virus (HPV) related conditions, the immunohistochemical staining for p16 has a diagnostic value only if diffusely and strongly positive, pattern named "block-like". "Weak and/or focal (w/f) p16 expression" is commonly considered nonspecific. In our previous study, we demonstrated the presence of high-risk HPV (hrHPV) DNA by LiPa method in biopsies showing w/f p16 positivity. The aim of the present study was to investigate the presence of hrHPV-DNA by CISH in the areas showing w/f p16 expression. We assessed the presence of hrHPV16, 18, 31, 33, 51 by CISH in a group of 20 cervical biopsies showing w/f p16 expression, some with increased Ki67, and in 10 cases of block-like expression, employed as control. The immunohistochemical p16 expression was also assessed by digital pathology. hrHPV-CISH nuclear positivity was encountered in 12/20 cases of w/f p16 expression (60%). Different patterns of nuclear positivity were identified, classified as punctate, diffuse and mixed, with different epithelial distributions. Our results, albeit in a limited casuistry, show the presence of HPV in an integrated status highlighted by CISH in w/f p16 positive cases. This could suggest the necessity of a careful follow-up of the patients with "weak" and/or "focal" immunohistochemical patterns of p16, mainly in cases of increased Ki67 cell proliferation index, supplemented with molecular biology examinations.

Caucasian lean subjects with non-alcoholic fatty liver disease share long-term prognosis of non-lean: time for reappraisal of BMI-driven approach?

OBJECTIVE: The full phenotypic expression of non-alcoholic fatty liver disease (NAFLD) in lean subjects is incompletely characterised. We aimed to investigate prevalence, characteristics and long-term prognosis of Caucasian lean subjects with NAFLD. DESIGN: The study cohort comprises 1339 biopsy-proven NAFLD subjects from four countries (Italy, UK, Spain and Australia), stratified into lean and non-lean (body mass index (BMI) 10 483 person-years), 4.7% of lean vs 7.7% of non-lean patients reported liver-related events (p=0.37). No difference in survival was observed compared with non-lean NAFLD (p=0.069). CONCLUSIONS: Caucasian lean subjects with NAFLD may progress to advanced liver disease, develop metabolic comorbidities and experience cardiovascular disease (CVD) as well as liver-related mortality, independent of longitudinal progression to obesity and PNPLA3 genotype. These patients represent one end of a wide spectrum of phenotypic expression of NAFLD where the disease manifests at lower overall BMI thresholds. LAY SUMMARY: NAFLD may affect and progress in both obese and lean individuals. Lean subjects are predominantly males, have a younger age at diagnosis and are more prevalent in some geographic areas. During the follow-up, lean subjects can develop hepatic and extrahepatic disease, including metabolic comorbidities, in the absence of weight gain. These patients represent one end of a wide spectrum of phenotypic expression of NAFLD.

NUPR1 protects liver from lipotoxic injury by improving the endoplasmic reticulum stress response.

Non-alcoholic fatty liver (NAFL) and related syndromes affect one-third of the adult population in industrialized and developing countries. Lifestyle and caloric oversupply are the main causes of such array of disorders, but the molecular mechanisms underlying their etiology remain elusive. Nuclear Protein 1 (NUPR1) expression increases upon cell injury in all organs including liver. Recently, we reported NUPR1 actively participates in the activation of the Unfolded Protein Response (UPR). The UPR typically maintains protein homeostasis, but downstream mediators of the pathway regulate metabolic functions including lipid metabolism. As increases in UPR and NUPR1 in obesity and liver disease have been well documented, the goal of this study was to investigate the roles of NUPR1 in this context. To establish whether NUPR1 is involved in these liver conditions we used patient-derived liver biopsies and in vitro and in vivo NUPR1 loss of functions models. First, we analyzed NUPR1 expression in a cohort of morbidly obese patients (MOPs), with simple fatty liver (NAFL) or more severe steatohepatitis (NASH). Next, we explored the metabolic roles of NUPR1 in wild-type (Nupr1+/+ ) or Nupr1 knockout mice (Nupr1-/- ) fed with a high-fat diet (HFD) for 15 weeks. Immunohistochemical and mRNA analysis revealed NUPR1 expression is inversely correlated to hepatic steatosis progression. Mechanistically, we found NUPR1 participates in the activation of PPAR-α signaling via UPR. As PPAR-α signaling is controlled by UPR, collectively, these findings suggest a novel function for NUPR1 in protecting liver from metabolic distress by controlling lipid homeostasis, possibly through the UPR.

A cholestatic pattern predicts major liver-related outcomes in patients with non-alcoholic fatty liver disease.

BACKGROUND & AIMS: NAFLD patients usually have an increase in AST/ALT levels, but cholestasis can also be observed. We aimed to assess in subjects with NAFLD the impact of the (cholestatic) C pattern on the likelihood of developing major liver-related outcomes (MALO). METHODS: Five hundred and eighty-two consecutive patients with biopsy-proven NAFLD or a clinical diagnosis of NAFLD-related compensated cirrhosis were classified as hepatocellular (H), C and mixed (M) patterns, by using the formula (ALT/ALT Upper Limit of Normal-ULN)/(ALP/ALP ULN). MALO were recorded during follow-up. An external cohort of 1281 biopsy-proven NAFLD patients was enrolled as validation set. RESULTS: H, M and C patterns were found in 153 (26.3%), 272 (46.7%) and 157 (27%) patients respectively. During a median follow-up of 78 months, only 1 (0.6%) patient with H pattern experienced MALO, whilst 15 (5.5%) and 38 (24.2%) patients in M and C groups had MALO. At multivariate Cox regression analysis, age >55 years (HR 2.55, 95% CI 1.17-5.54; p = .01), platelets <150 000/mmc (HR 0.14, 95% CI 0.06-0.32; p < .001), albumin <4 g/L(HR 0.62, 95% CI 0.35-1.08; p = .09), C versus M pattern (HR 7.86, 95% CI 1.03-60.1; p = .04), C versus H pattern(HR 12.1, 95% CI 1.61-90.9; p = .01) and fibrosis F3-F4(HR 35.8, 95% CI 4.65-275.2; p < .001) were independent risk factors for MALO occurrence. C versus M pattern(HR 14.3, 95% CI 1.90-105.6; p = .008) and C versus H pattern (HR 15.6, 95% CI 2.10-115.1; p = .0068) were confirmed independently associated with MALO occurrence in the validation set. The immunohistochemical analysis found a significantly higher prevalence of moderate-high-grade ductular metaplasia combined with low-grade ductular proliferation in C pattern when compared with the biochemical H pattern. Gene expression analysis showed a lower expression of NR1H3, RXRα and VCAM1 in patients with the C pattern. CONCLUSIONS: The presence of a cholestatic pattern in patients with NAFLD predicts a higher risk of MALO independently from other features of liver disease.

Diagnostic performance of 2D-shear wave elastography in the diagnosis of breast cancer: a clinical appraisal of cutoff values.

PURPOSE: To assess the role of 2D-shear wave elastography (2D-SWE) in differentiating benign from malignant focal breast lesions (FBLs), providing new vendor-specific cutoff values. METHODS: 158 FBLs (size: 3.5-50 mm) detected in 151 women (age: 21-87 years) were prospectively evaluated by means 2D-SWE. For each lesion, an expert radiologist assessed US BI-RADS category and calculated the following four 2D-SWE parameters: (1) elasticity maximum (Emax); (2) mean elasticity (Emean); (3) minimum elasticity (Emin); (4) elasticity ratio (Eratio). US-guided core-biopsy was considered as standard of reference for all the FBLs classified as BI-RADS 4 or 5. For each 2D-SWE parameter, the optimal cutoff value for a diagnostic test was calculated using the Youden method. Diagnostic performance of the US BI-RADS and 2D-SWE parameters was calculated accordingly. RESULTS: 83/158 (52.5%) FBLs were benign and 75/158 (47.5%) were malignant. Statistically significant higher stiffness values were observed in malignant FBLs for all 2D-SWE parameters than in benign ones (p < 0.001). 2D-SWE cutoff values were 82.6 kPa, 66.0 kPa and 53.6 kPa, respectively, for Emax, Emean, Emin and 330.8% for Eratio. The 2D-SWE parameter showing the best diagnostic accuracy was Emax (85.44%). Considering US BI-RADS 3 (n = 60) and 4a (n = 32) FBLs, Emax and Emean showed the best diagnostic accuracy (85.87% for both), without a statistically significant decrease in sensitivity (p = 0.7003 and p = 1, respectively). CONCLUSION: Our study provides new vendor-specific cutoff values for 2D-SWE, suggesting its possible clinical use in the adjunctive assessment of category US-BI-RADS 3 and 4a breast masses.

Long-term outcomes and predictive ability of non-invasive scoring systems in patients with non-alcoholic fatty liver disease.

BACKGROUND & AIMS: Non-invasive scoring systems (NSS) are used to identify patients with non-alcoholic fatty liver disease (NAFLD) who are at risk of advanced fibrosis, but their reliability in predicting long-term outcomes for hepatic/extrahepatic complications or death and their concordance in cross-sectional and longitudinal risk stratification remain uncertain. METHODS: The most common NSS (NFS, FIB-4, BARD, APRI) and the Hepamet fibrosis score (HFS) were assessed in 1,173 European patients with NAFLD from tertiary centres. Performance for fibrosis risk stratification and for the prediction of long-term hepatic/extrahepatic events, hepatocarcinoma (HCC) and overall mortality were evaluated in terms of AUC and Harrell's c-index. For longitudinal data, NSS-based Cox proportional hazard models were trained on the whole cohort with repeated 5-fold cross-validation, sampling for testing from the 607 patients with all NSS available. RESULTS: Cross-sectional analysis revealed HFS as the best performer for the identification of significant (F0-1 vs. F2-4, AUC = 0.758) and advanced (F0-2 vs. F3-4, AUC = 0.805) fibrosis, while NFS and FIB-4 showed the best performance for detecting histological cirrhosis (range AUCs 0.85-0.88). Considering longitudinal data (follow-up between 62 and 110 months), NFS and FIB-4 were the best at predicting liver-related events (c-indices>0.7), NFS for HCC (c-index = 0.9 on average), and FIB-4 and HFS for overall mortality (c-indices >0.8). All NSS showed limited performance (c-indices <0.7) for extrahepatic events. CONCLUSIONS: Overall, NFS, HFS and FIB-4 outperformed APRI and BARD for both cross-sectional identification of fibrosis and prediction of long-term outcomes, confirming that they are useful tools for the clinical management of patients with NAFLD at increased risk of fibrosis and liver-related complications or death. LAY SUMMARY: Non-invasive scoring systems are increasingly being used in patients with non-alcoholic fatty liver disease to identify those at risk of advanced fibrosis and hence clinical complications. Herein, we compared various non-invasive scoring systems and identified those that were best at identifying risk, as well as those that were best for the prediction of long-term outcomes, such as liver-related events, liver cancer and death.

Mild exacerbation of obesity- and age-dependent liver disease progression by senolytic cocktail dasatinib + quercetin.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is increasingly prevalent and represents a growing challenge in terms of prevention and treatment. A minority of affected patients develops inflammation, subsequently fibrosis, cirrhosis and hepatocellular carcinoma (HCC). HCC is a leading cause of cancer-related death. An increased number of senescent cells correlate with age-related tissue degeneration during NAFLD-induced HCC. Senolytics are promising agents that target selectively senescent cells. Previous studies showed that whereas a combination of the senolytic drugs dasatinib and quercetin (D + Q) reduced NAFLD in mice, D + Q lacked efficacy in removing doxorubicin-induced ß-gal-positive senescent cells in human HCC xenografted mice. Whether D + Q has an effect on the age-associated spectrum of NAFLD-inflammation-HCC remains unknown. METHODS: Here, we utilized an established model of age- and obesity-associated HCC, the low dose diethylnitrosamine (DEN)/high fat diet (HFD), a regimen promoting liver inflammation and tumorigenesis over a long period of 9 months. Four groups of mice each were created: group 1 included control untreated mice; group 2 included mice treated with D + Q; group 3 included mice undergoing the DEN/HFD protocol; group 4 included mice undergoing the DEN/HFD protocol with the administration of D + Q. At the end of the chemical/dietary regimen, we analyzed liver damage and cell senescence by histopathology, qPCR and immunoblotting approaches. RESULTS: Unexpectedly, D + Q worsened liver disease progression in the DEN/HFD mouse model, slightly increasing histological damage and tumorigenesis, while having no effect on senescent cells removal. CONCLUSIONS: In summary, using an animal model that fully recapitulates NAFLD, we demonstrate that these compounds are ineffective against age-associated NAFLD-induced HCC. Video Abstract.

Presence of Serum Antinuclear Antibodies Does Not Impact Long-Term Outcomes in Nonalcoholic Fatty Liver Disease.

INTRODUCTION: We investigated the longitudinal impact of antinuclear antibody (ANA) on clinical outcomes and survival in nonalcoholic fatty liver disease (NAFLD). METHODS: ANA were found in 16.9% of 923 biopsy-proven NAFLD patients, but none of them had histologic autoimmune hepatitis (AIH) or developed AIH after a mean follow-up of 106±50 months. RESULTS: Although ANA-positive cases had a higher prevalence of nonalcoholic steatohepatitis at baseline, the occurrence of liver-related events, hepatocellula carcinoma, cardiovascular events, extrahepatic malignancy, and overall survival were similar to ANA-negative. DISCUSSION: Once AIH has been ruled out, the long-term outcomes and survival are unaffected by the presence of ANA in patients with NAFLD.

Evaluating the association of serum ferritin and hepatic iron with disease severity in non-alcoholic fatty liver disease.

BACKGROUND & AIMS: Hyperferritinemia, with or without increased hepatic iron, represents a common finding in non-alcoholic fatty liver disease (NAFLD). However, it is unclear whether it reflects hepatic inflammation or true iron-overload and, in case the latter is confirmed, whether this influences disease progression. We therefore explored the association between serum ferritin, degree and pattern of hepatic iron deposition and liver disease severity in patients with NAFLD. METHODS: We selected 468 patients with biopsy-proven NAFLD from 2 European centres. Iron, hepatic and metabolic parameters were collected at the time of liver biopsy. Iron deposits in hepatocytes and reticuloendothelial cells were assessed and graded. Diagnosis of non-alcoholic steatohepatitis (NASH) and fibrosis staging were performed. RESULTS: A total of 122 (26%) patients had hyperferritinemia, whereas stainable hepatic iron was found in 116 (25%) patients (38% predominantly in hepatocytes, 20% in reticuloendothelial cells and 42% in both). Subjects with stainable hepatic iron, particularly those with a mixed pattern, had higher serum ferritin and transaminases but only a mixed pattern of iron deposition was among the variables significantly associated with presence of NASH. Serum ferritin was not associated with presence of NASH, however it increased with worsening fibrosis stage (F3 compared to F0-F1), and significantly decreased in stage F4. CONCLUSIONS: A mixed pattern of hepatic iron deposition is associated with the presence of steatohepatitis, while serum ferritin increases with worsening fibrosis up to pre-cirrhotic stage. In individual NAFLD patients, serum ferritin could be evaluated as part of non-invasive diagnostic panels but not on its own.

Immunomorphological Pattern of Molecular Chaperones in Normal and Pathological Thyroid Tissues and Circulating Exosomes: Potential Use in Clinics.

The thyroid is a major component of the endocrine system and its pathology can cause serious diseases, e.g., papillary carcinoma (PC). However, the carcinogenic mechanisms are poorly understood and clinical useful biomarkers are scarce. Therefore, we determined if there are quantitative patterns of molecular chaperones in the tumor tissue and circulating exosomes that may be useful in diagnosis and provide clues on their participation in carcinogenesis. Hsp27, Hsp60, Hsp70, and Hsp90 were quantified by immunohistochemistry in PC, benign goiter (BG), and normal peritumoral tissue (PT). The same chaperones were assessed in plasma exosomes from PC and BG patients before and after ablative surgery, using Western blotting. Hsp27, Hsp60, and Hsp90 were increased in PC in comparison with PT and BG but no differences were found for Hsp70. Similarly, exosomal levels of Hsp27, Hsp60, and Hsp90 were higher in PC than in BG, and those in PC were higher before ablative surgery than after it. Hsp27, Hsp60, and Hsp90 show distinctive quantitative patterns in thyroid tissue and circulating exosomes in PC as compared with BG, suggesting some implication in the carcinogenesis of these chaperones and indicating their potential as biomarkers for clinical applications.

Interferon lambda 4 rs368234815 TT>δG variant is associated with liver damage in patients with nonalcoholic fatty liver disease.

The interferon (IFN) lambda 3/4 (IFNL3/4) locus, influencing innate immunity regulation, has been associated with the severity of hepatitis and fibrosis progression during chronic hepatitis C infection, while contrasting results were reported in nonalcoholic fatty liver disease. In this study, we examined whether rs12979860 and the linked causal rs368234815 variant encoding for the alternative IFNL4 protein variant are associated with liver fibrosis and damage in a large multicenter cohort of patients at risk of nonalcoholic steatohepatitis. To clarify the mechanism, we also evaluated the impact on IFN-stimulated gene hepatic expression in a subset of patients. We considered 946 consecutive Italian individuals at risk of nonalcoholic steatohepatitis with liver histology evaluated according to Kleiner. The rs368234815 TT>δG, rs12979860 C>T, and patatin-like phospholipase-3 rs738409 C>G polymorphisms were genotyped; and IFN-stimulated gene hepatic expression (n = 16) was tested by TaqMan assays. We found that the rs368234815 TT allele was independently associated with severe F3-F4 fibrosis (odds ratio, 1.53; 95% confidence interval, 1.15-2.31; P = 0.005) and with severe (grade 2-3) lobular necroinflammation (odds ratio, 1.47; 95% confidence interval, 1.14-1.88; P = 0.002). The impact of rs368234815 on liver damage was generally more marked in nonobese individuals, where association with severe fibrosis, necroinflammation, and nonalcoholic steatohepatitis was observed (P < 0.05). IFN-stimulated genes were hypo-expressed in the liver of patients carrying the IFNL4 rs368234815 TT/TT genotype (P < 0.05). Similar results were observed when considering the rs12979860 polymorphism, which was in high linkage disequilibrium with rs368234815 (R2 = 0.87). CONCLUSION: The IFNL4 genotype is associated with severity of fibrosis in nonalcoholic fatty liver disease patients of European ancestry, likely by modulating the activation of innate immunity and necroinflammation. (Hepatology 2017;66:1885-1893).

Reactive hyperemia index (RHI) and cognitive performance indexes are associated with histologic markers of liver disease in subjects with non-alcoholic fatty liver disease (NAFLD): a case control study.

BACKGROUND: No study evaluated vascular health markers in subjects with non-alcoholic fatty liver disease (NAFLD) through a combined analysis of reactive hyperemia peripheral arterial tonometry (RH-PAT) and arterial stiffness indexes. AIM OF THE STUDY: We aimed to assess whether NAFLD and its histological severity are associated with impairment of arterial stiffness and RH-PAT indexes in a mixed cohort of patients with biopsy-proven NAFLD. MATERIALS AND METHODS: The Kleiner classification was used to grade NAFLD grade. Pulse wave velocity (PWV) and augmentation index (Aix) were used as markers of arterial stiffness, whereas endothelial function was assessed using reactive hyperemia index (RHI). The mini-mental state examination (MMSE) was administered to test cognitive performance. RESULTS: 80 consecutive patients with biopsy-proven NAFLD and 83 controls without fatty liver disease. NAFLD subjects showed significantly lower mean RHI, higher mean arterial stiffness indexes and lower mean MMSE score. Multivariable analysis after correction for BMI, dyslipidaemia, hypertension, sex, diabetes, age and cardiovascular disease showed that BMI, diastolic blood pressure and RHI are significantly associated to NAFLD. Simple linear regression analysis showed among non-alcoholic steatohepatitis (NASH) subjects a significant negative relationship between ballooning grade and MMSE and a significant positive association between Kleiner steatosis grade and augmentation index. CONCLUSIONS: Future research will be addressed to evaluate the relationship between inflammatory markers and arterial stiffness and endothelial function indexes in NAFLD subjects. These study will evaluate association between cardiovascular event incidence and arterial stiffness, endothelial and cognitive markers, and they will address the beneficial effects of cardiovascular drugs such as statins and ACE inhibitors on these surrogate markers in NAFLD subjects.

The severity of steatosis influences liver stiffness measurement in patients with nonalcoholic fatty liver disease.

UNLABELLED: In nonalcoholic fatty liver disease, the influence of severity of steatosis on liver stiffness measurement (LSM) is poorly studied and still debated. We assessed the impact of steatosis severity and its ultrasonographic (US) sign, severe bright liver echo pattern, on LSM values and on transient elastography accuracy for the diagnosis of liver fibrosis in a cohort of consecutive patients with nonalcoholic fatty liver disease. Patients (n = 253) were assessed by clinical, US, and histological (Kleiner score) features. Transient elastography was performed using the M probe. Among patients with low amounts of fibrosis (F0-F1 and F0-F2), median LSM values, expressed in kilopascals, were significantly higher in subjects with severe steatosis (≥66% at liver biopsy) compared to those without (F0-F1 6.9 versus 5.8, P = 0.04; F0-F2 7.4 versus 6.0, P = 0.001) as well as in patients with severe bright liver echo pattern on US compared to their counterparts (F0-F1 7.3 versus 5.6, P = 0.001; F0-F2 7.6 versus 6.0, P < 0.001). In subjects without significant fibrosis (F0-F1) and without severe fibrosis (F0-F2), a higher rate of false-positive LSM results was observed in patients with steatosis ≥66% compared to those without (F0-F1 23.6% versus 14.9%, F0-F2 33.3% versus 13.2%) and in patients with severe bright liver echo pattern on US (F0-F1 22.2% versus 15.4%, F0-F2 28.8% versus 15.6%) compared to their counterparts. CONCLUSIONS: In patients with nonalcoholic fatty liver disease, the presence of severe steatosis, detected by histology or by US, should always be taken into account in order to avoid overestimations of liver fibrosis assessed by transient elastography.

TM6SF2 rs58542926 is not associated with steatosis and fibrosis in large cohort of patients with genotype 1 chronic hepatitis C.

BACKGROUND & AIMS: We tested the putative association of the rs58542926 variant of TM6SF2, a recently described genetic determinant of nonalcoholic fatty liver disease, with steatosis and fibrosis in genotype 1(G1) chronic hepatitis C(CHC) patients. METHODS: A total of 694 consecutively biopsied Caucasian G1 CHC patients were genotyped for TM6SF2 rs58542926, IL28B rs12979860 and PNPLA3 rs738409. Steatosis was classified as absent (<5%), mild-moderate(5-29%) and severe(≥30%), Fibrosis was considered severe if=F3-F4. RESULTS: Carriers of TM6SF2 rs58542926 (6.3% of patients) exhibited lower serum levels of cholesterol (P = 0.04) and triglycerides (P = 0.01), but a similar distribution of steatosis severity (P = 0.63), compared to noncarriers. Prevalence and severity of steatosis were reduced in IL28B C allele carriers (P = 0.005) and elevated in PNPLA3 G allele carriers (P < 0.001). After adjustment for age, gender, body mass index and homoeostasis model assessment score, steatosis severity was independently associated with IL28B rs12979860 (odds ratio [OR] 0.69, 95% confidence interval [CI] 0.55-0.86, P = 0.001) and PNPLA3 rs738409 (OR 1.84, 95% CI 1.46-2.83, P < 0.001), but not TM6SF2 rs58542926 (OR 1.48, 95% CI 0.82-2.69, P = 0.19). Variants of TM6SF2 (30.9% vs. 25%, P = 0.40), IL28B and PNPLA3 were not directly associated with fibrosis severity, although variants of IL28B and PNPLA3 promoted steatosis (OR 1.36, 95% CI 1.06-1.75, P = 0.01) that in turn is associated with severe fibrosis. CONCLUSIONS: In G1 CHC patients, TM6SF2 rs58542926 does not affect the histological severity of liver damage. However, IL28B rs12979860 and PNPLA3 rs738409 modify steatosis.

Epicardial fat, cardiac geometry and cardiac function in patients with non-alcoholic fatty liver disease: association with the severity of liver disease.

BACKGROUND & AIMS: Non-alcoholic fatty liver disease (NAFLD) has been associated with increased cardiovascular risk, including coronary artery disease and cardiac dysfunction. In addition, recent evidence highlighted the possible role of epicardial fat as a new cardiometabolic risk factor. We tested the correlation between epicardial fat, alterations in cardiac geometry and function, and severity of liver damage, in patients with biopsy-proven NAFLD. METHODS: The anthropometric, biochemical and metabolic features were recorded in 147 consecutive biopsy-proven NAFLD cases (Kleiner score). Epicardial fat thickness was measured by echocardiography. RESULTS: Epicardial fat was higher in patients with severe vs. milder fibrosis (8.5 ± 3.0 vs. 7.2 ± 2.3 mm; p=0.006); this association was maintained at multivariate logistic regression analysis (OR 1.22, 95%C.I. 1.01-1.47; p=0.04) after correction for gender, age >50 years, visceral obesity, IFG/diabetes, non-alcoholic steatohepatitis and severe steatosis. Of note, 37.1% of patients with epicardial fat >7 mm (median value) had severe liver fibrosis, compared to 18.3% of the cases with lower epicardial fat (p=0.01). As for echocardiographic indices, after adjusting for cardiometabolic confounders, diastolic posterior-wall thickness (p=0.01), left ventricular mass (p=0.03), relative wall thickness (p=0.02), and left atrial volume (0.04), as well as ejection fraction (p=0.004), lower lateral TDI e' (p=0.009), E/A ratio (0.04) (cardiac geometry alterations and diastolic dysfunction) were linked to severe liver fibrosis. CONCLUSIONS: In patients with NAFLD, a higher epicardial fat thickness is associated with the severity of liver fibrosis, in keeping with a possible pathogenic role of ectopic fat depots in whole body organ damage. In addition, morphological and functional cardiac alterations are more pronounced according to the severity of fibrosis. Further studies are needed to validate our results.

The combination of liver stiffness measurement and NAFLD fibrosis score improves the noninvasive diagnostic accuracy for severe liver fibrosis in patients with nonalcoholic fatty liver disease.

BACKGROUND & AIMS: The accuracy of noninvasive tools for the diagnosis of severe fibrosis in patients with nonalcoholic fatty liver disease(NAFLD) in clinical practice is still limited. We aimed at assessing the diagnostic performance of combined noninvasive tools in two independent cohorts of Italian NAFLD patients. METHODS: We analysed data from 321 Italian patients(179 Sicilian-training cohort, and 142 northern Italy-validation cohort) with an histological diagnosis of NAFLD. Severe fibrosis was defined as fibrosis ≥ F3 according to Kleiner classification. The APRI, AST/ALT, BARD, FIB-4, and NFS scores were calculated according to published algorithms. Liver stiffness measurement(LSM) was performed by FibroScan. Cut-off points of LSM, NFS and FIB-4 for rule-in or rule-out F3-F4 fibrosis were calculated by the reported formulas. RESULTS: In the Sicilian cohort AUCs of LSM, NFS, FIB-4, LSM plus NFS, LSM plus FIB-4, and NFS plus FIB-4 were 0.857, 0.803, 0.790, 0.878, 0.888 and 0.807, respectively, while in the northern Italy cohort the corresponding AUCs were 0.848, 0.730, 0.703, 0.844, 0.850, and 0.733 respectively. In the training cohort, the combination of LSM plus NFS was the best performing strategy, providing false positive, false negative and uncertainty area rates of 0%,1.1% and 48% respectively. Similar results were obtained in the validation cohort with false positive, false negative and uncertainty area rates of 0%,7.3% and 40.8%. CONCLUSIONS: The combination of LSM with NFS, two complementary, easy-to-perform, and widely available tools, is able to accurately diagnose or exclude the presence of severe liver fibrosis, also reducing of about 50-60% the number of needed diagnostic liver biopsies.

Comparison of Histochemical Staining Methods and Correlation with Transient Elastography in Acute Hepatitis.

OBJECTIVE: To compare Masson's trichrome (MT), Sirius red (SR) and orcein staining in acute hepatitis (AH) and to correlate them with transient elastography (TE), a noninvasive method to assess hepatic fibrosis. METHODS: We evaluated liver stiffness by TE in a cohort of 34 consecutive patients and assessed MT-, SR- and orcein-stained biopsies using the METAVIR scoring system and digital image analysis (DIA). RESULTS: MT and SR both showed severe fibrosis (stage III-IV, DIA = 12.7%). Orcein showed absent or mild fibrosis (stage 0-II, DIA = 4.4%; p < 0.05). In 29/34 cases (85%), stiffness values were >12.5 kPa, in keeping with SR/MT but not with orcein results. CONCLUSIONS: Even though in AH true elastic fibrosis is typically absent or mild, TE shows elevated stiffness values, in keeping with SR/MT evaluations. If not properly evaluated in the clinical context, these results would lead to an overestimation of fibrosis. Orcein is the only staining able to evidence the absence of true elastic fibrosis, which is a typical feature of AH. This is the first study comparing different staining procedures performed on AH biopsies by DIA versus TE. © 2015 S. Karger AG, Basel.