Severe acute liver disease in adults: Contemporary role of histopathology.

Liver biopsies have consistently contributed to our understanding of the pathogenesis and aetiologies of acute liver disease. As other diagnostic modalities have been developed and refined, the role of biopsy in the management of patients with acute liver failure (ALF), acute-on-chronic liver failure (ACLF) and acute hepatitis, including acute liver injury (ALI), has changed. Liver biopsy remains particularly valuable when first-line diagnostic algorithms fail to determine aetiology. Despite not being identified as a mandatory diagnostic tool in recent clinical guidelines for the management of ALF or ACLF, many centres continue to undertake biopsies given the relative safety of transjugular biopsy in this setting. Several studies have demonstrated that liver biopsy can provide prognostic information, particularly in the context of so-called indeterminate hepatitis, and is extremely useful in excluding conditions such as metastatic tumours that would preclude transplantation. In addition, its widespread use of percutaneous biopsies in cases of less severe acute liver injury, for example in the establishment of a diagnosis of acute presentation of autoimmune hepatitis or confirmation of a probable or definite drug-induced liver injury (DILI), has meant that many centres have seen a shift in the ratio of specimens they are receiving from patients with chronic to acute liver disease. Histopathologists therefore need to be equipped to deal with these challenging specimens. This overview provides an insight into the contemporary role of biopsies (as well as explant and autopsy material) in diagnosing acute liver disease. It outlines up-to-date clinical definitions of liver injury and considers recent recommendations for the diagnosis of AIH and drug-induced, autoimmune-like hepatitis (DI-AIH).

Genetic variation in TERT modifies the risk of hepatocellular carcinoma in alcohol-related cirrhosis: results from a genome-wide case-control study.

OBJECTIVE: Hepatocellular carcinoma (HCC) often develops in patients with alcohol-related cirrhosis at an annual risk of up to 2.5%. Some host genetic risk factors have been identified but do not account for the majority of the variance in occurrence. This study aimed to identify novel susceptibility loci for the development of HCC in people with alcohol related cirrhosis. DESIGN: Patients with alcohol-related cirrhosis and HCC (cases: n=1214) and controls without HCC (n=1866), recruited from Germany, Austria, Switzerland, Italy and the UK, were included in a two-stage genome-wide association study using a case-control design. A validation cohort of 1520 people misusing alcohol but with no evidence of liver disease was included to control for possible association effects with alcohol misuse. Genotyping was performed using the InfiniumGlobal Screening Array (V.24v2, Illumina) and the OmniExpress Array (V.24v1-0a, Illumina). RESULTS: Associations with variants rs738409 in PNPLA3 and rs58542926 in TM6SF2 previously associated with an increased risk of HCC in patients with alcohol-related cirrhosis were confirmed at genome-wide significance. A novel locus rs2242652(A) in TERT (telomerase reverse transcriptase) was also associated with a decreased risk of HCC, in the combined meta-analysis, at genome-wide significance (p=6.41×10-9, OR=0.61 (95% CI 0.52 to 0.70). This protective association remained significant after correction for sex, age, body mass index and type 2 diabetes (p=7.94×10-5, OR=0.63 (95% CI 0.50 to 0.79). Carriage of rs2242652(A) in TERT was associated with an increased leucocyte telomere length (p=2.12×10-44). CONCLUSION: This study identifies rs2242652 in TERT as a novel protective factor for HCC in patients with alcohol-related cirrhosis.

Human Precision-Cut Liver Slices: A Potential Platform to Study Alcohol-Related Liver Disease.

Alcohol-related liver disease (ALD) encompasses a range of pathological conditions that are complex to study at the clinical and preclinical levels. Despite the global burden of ALD, there is a lack of effective treatments, and mortality is high. One of the reasons for the unsuccessful development of novel therapies is that experimental studies are hindered by the challenge of recapitulating this multifactorial disorder in vitro, including the contributions of hepatotoxicity, impaired lipid metabolism, fibrosis and inflammatory cytokine storm, which are critical drivers in the pathogenesis of ALD in patients and primary targets for drug development. Here, we present the unique characteristics of the culture of human precision-cut liver slices (PCLS) to replicate key disease processes in ALD. PCLS were prepared from human liver specimens and treated with ethanol alone or in combination with fatty acids and lipopolysaccharide (FA + LPS) for up to 5 days to induce hepatotoxic, inflammatory and fibrotic events associated with ALD. Alcohol insult induced hepatocyte death which was more pronounced with the addition of FA + LPS. This mixture showed a significant increase in the cytokines conventionally associated with the prototypical inflammatory response observed in severe ALD, and interestingly, alcohol alone exhibited a different effect. Profibrogenic activation was also observed in the slices and investigated in the context of slice preparation. These results support the versatility of this organotypic model to study different pathways involved in alcohol-induced liver damage and ALD progression and highlight the applicability of the PCLS for drug discovery, confirming their relevance as a bridge between preclinical and clinical studies.

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.

Complexity of ballooned hepatocyte feature recognition: Defining a training atlas for artificial intelligence-based imaging in NAFLD.

BACKGROUND & AIMS: Histologically assessed hepatocyte ballooning is a key feature discriminating non-alcoholic steatohepatitis (NASH) from steatosis (NAFL). Reliable identification underpins patient inclusion in clinical trials and serves as a key regulatory-approved surrogate endpoint for drug efficacy. High inter/intra-observer variation in ballooning measured using the NASH CRN semi-quantitative score has been reported yet no actionable solutions have been proposed. METHODS: A focused evaluation of hepatocyte ballooning recognition was conducted. Digitized slides were evaluated by 9 internationally recognized expert liver pathologists on 2 separate occasions: each pathologist independently marked every ballooned hepatocyte and later provided an overall non-NASH NAFL/NASH assessment. Interobserver variation was assessed and a 'concordance atlas' of ballooned hepatocytes generated to train second harmonic generation/two-photon excitation fluorescence imaging-based artificial intelligence (AI). RESULTS: The Fleiss kappa statistic for overall interobserver agreement for presence/absence of ballooning was 0.197 (95% CI 0.094-0.300), rising to 0.362 (0.258-0.465) with a ≥5-cell threshold. However, the intraclass correlation coefficient for consistency was higher (0.718 [0.511-0.900]), indicating 'moderate' agreement on ballooning burden. 133 ballooned cells were identified using a ≥5/9 majority to train AI ballooning detection (AI-pathologist pairwise concordance 19-42%, comparable to inter-pathologist pairwise concordance of between 8-75%). AI quantified change in ballooned cell burden in response to therapy in a separate slide set. CONCLUSIONS: The substantial divergence in hepatocyte ballooning identified amongst expert hepatopathologists suggests that ballooning is a spectrum, too subjective for its presence or complete absence to be unequivocally determined as a trial endpoint. A concordance atlas may be used to train AI assistive technologies to reproducibly quantify ballooned hepatocytes that standardize assessment of therapeutic efficacy. This atlas serves as a reference standard for ongoing work to refine how ballooning is classified by both pathologists and AI. LAY SUMMARY: For the first time, we show that, even amongst expert hepatopathologists, there is poor agreement regarding the number of ballooned hepatocytes seen on the same digitized histology images. This has important implications as the presence of ballooning is needed to establish the diagnosis of non-alcoholic steatohepatitis (NASH), and its unequivocal absence is one of the key requirements to show 'NASH resolution' to support drug efficacy in clinical trials. Artificial intelligence-based approaches may provide a more reliable way to assess the range of injury recorded as "hepatocyte ballooning".

Senescence markers in focal nodular hyperplasia of the liver: pathogenic considerations on the basis of immunohistochemical results.

Focal nodular hyperplasia (FNH) is a polyclonal tumour-like hepatic lesion characterised by parenchymal nodules, connective tissue septa without interlobular bile ducts, pronounced ductular reaction and inflammation. It may represent a response to local arterial hyperperfusion and hyperoxygenation resulting in oxidative stress. We aimed at obtaining closer insight into the pathogenesis of FNH with its characteristic morphologic features. Immunohistochemistry and immunofluorescence microscopy was performed on FNH specimens using antibodies against keratins (K) 7 and 19, neural cell adhesion molecule (NCAM), lamin B1, senescence markers (CDK inhibitor 1/p21Cip1, CDK inhibitor /p16Ink4a, senescence-associated (SA) ß- galactosidase activity), proliferation markers (Ki-67, proliferating-cell nuclear antigen (PCNA)), and the abnormally phosphorylated histone γ-H2AX, indicating DNA double strand breaks; moreover SA ß- galactosidase activity was determined histochemically. Ductular metaplasia of hepatocytes indicated by K7 expression in the absence of K19 plays a major role in the development of ductular reaction in FNH. Moreover, the expression of senescence markers (p21Cip1, p16Ink4a, γ-H2AX, SA ß-galactosidase activity) in hepatocytes and cholangiocytes suggests that stress-induced cellular senescence contributes to fibrosis and inflammation via production of components of the senescence-associated secretory phenotype. Expression of proliferation markers (Ki-67, PCNA) was not enhanced in hepatocytes and biliary cells. Senescence and ductular metaplasia of hepatocytes may thus be involved in inflammation, fibrosis and apoptosis resistance. Hence, fibrosis, inflammation and reduced apoptotic cell death, rather than proliferation (hyperplasia) may be responsible for increased tissue mass and tumour-like appearance of FNH.

Consensus recommendations for histological criteria of autoimmune hepatitis from the International AIH Pathology Group: Results of a workshop on AIH histology hosted by the European Reference Network on Hepatological Diseases and the European Society of Pathology: Results of a workshop on AIH histology hosted by the European Reference Network on Hepatological Diseases and the European Society of Pathology.

BACKGROUND & AIMS: Diagnostic histological criteria for autoimmune hepatitis (AIH) have not been clearly established. Previously published criteria focused mainly on chronic AIH, in which inflammatory changes mainly occur in portal/periportal regions and may not be applicable to acute presentation of AIH, in which inflammatory changes are typically predominantly lobular in location. International consensus criteria for the diagnosis and assessment of disease severity in both acute and chronic AIH are thus urgently needed. METHODS: Seventeen expert liver pathologists convened at an international workshop and subsequently used a modified Delphi panel approach to establish consensus criteria for the histopathological diagnosis of AIH. RESULTS: The consensus view is that liver biopsy should remain standard for diagnosing AIH. AIH is considered likely, if there is a predominantly portal lymphoplasmacytic hepatitis with more than mild interface activity and/or more than mild lobular hepatitis in the absence of histological features suggestive of another liver disease. AIH is also considered likely if there is predominantly lobular hepatitis with or without centrilobular necroinflammation and at least one of the following features: portal lymphoplasmacytic hepatitis, interface hepatitis or portal-based fibrosis, in the absence of histological features suggestive of another liver disease. Emperipolesis and hepatocellular rosettes are not regarded as being specific for AIH. CONCLUSIONS: The criteria proposed in this consensus statement provide a uniform approach to the histological diagnosis of AIH, which is relevant for patients with an acute as well as a chronic presentation and to more accurately reflect the current understanding of liver pathology in AIH.

Development and prognostic relevance of a histologic grading and staging system for alcohol-related liver disease.

BACKGROUND & AIMS: The SALVE Histopathology Group (SHG) developed and validated a grading and staging system for the clinical and full histological spectrum of alcohol-related liver disease (ALD) and evaluated its prognostic utility in a multinational cohort of 445 patients. METHODS: SALVE grade was described by semiquantitative scores for steatosis, activity (hepatocellular injury and lobular neutrophils) and cholestasis. The histological diagnosis of steatohepatitis due to ALD (histological ASH, hASH) was based on the presence of hepatocellular ballooning and lobular neutrophils. Fibrosis staging was adapted from the Clinical Research Network staging system for non-alcoholic fatty liver disease and the Laennec staging system and reflects the pattern and extent of ALD fibrosis. There are 7 SALVE fibrosis stages (SFS) ranging from no fibrosis to severe cirrhosis. RESULTS: Interobserver κ-value for each grading and staging parameter was >0.6. In the whole study cohort, long-term outcome was associated with activity grade and cholestasis, as well as cirrhosis with very broad septa (severe cirrhosis) (p <0.001 for all parameters). In decompensated ALD, adverse short-term outcome was associated with activity grade, hASH and cholestasis (p = 0.038, 0.012 and 0.001, respectively), whereas in compensated ALD, hASH and severe fibrosis/cirrhosis were associated with decompensation-free survival (p = 0.011 and 0.001, respectively). On multivariable analysis, severe cirrhosis emerged as an independent histological predictor of long-term survival in the whole study cohort. Severe cirrhosis and hASH were identified as independent predictors of short-term survival in decompensated ALD, and also as independent predictors of decompensation-free survival in compensated ALD. CONCLUSION: The SALVE grading and staging system is a reproducible and prognostically relevant method for the histological assessment of disease activity and fibrosis in ALD. LAY SUMMARY: Patients with alcohol-related liver disease (ALD) may undergo liver biopsy to assess disease severity. We developed a system to classify ALD under the microscope by grading ALD activity and staging the extent of liver scarring. We validated the prognostic performance of this system in 445 patients from 4 European centers.

Genetic Variation in HSD17B13 Reduces the Risk of Developing Cirrhosis and Hepatocellular Carcinoma in Alcohol Misusers.

BACKGROUND AND AIMS: Carriage of rs738409:G in patatin-like phospholipase domain containing 3 (PNPLA3) is associated with an increased risk for developing alcohol-related cirrhosis and hepatocellular carcinoma (HCC). Recently, rs72613567:TA in hydroxysteroid 17-beta dehydrogenase 13 (HSD17B13) was shown to be associated with a reduced risk for developing alcohol-related liver disease and to attenuate the risk associated with carriage of PNPLA3 rs738409:G. This study explores the risk associations between these two genetic variants and the development of alcohol-related cirrhosis and HCC. APPROACH AND RESULTS: Variants in HSD17B13 and PNPLA3 were genotyped in 6,171 participants, including 1,031 with alcohol-related cirrhosis and HCC, 1,653 with alcohol-related cirrhosis without HCC, 2,588 alcohol misusers with no liver disease, and 899 healthy controls. Genetic associations with the risks for developing alcohol-related cirrhosis and HCC were determined using logistic regression analysis. Carriage of HSD17B13 rs72613567:TA was associated with a lower risk for developing both cirrhosis (odds ratio [OR], 0.79; 95% confidence interval [CI], 0.72-0.88; P = 8.13 × 10-6 ) and HCC (OR, 0.77; 95% CI, 0.68-0.89; P = 2.27 × 10-4 ), whereas carriage of PNPLA3 rs738409:G was associated with an increased risk for developing cirrhosis (OR, 1.70; 95% CI, 1.54-1.88; P = 1.52 × 10-26 ) and HCC (OR, 1.77; 95% CI, 1.58-1.98; P = 2.31 × 10-23 ). These associations remained significant after adjusting for age, sex, body mass index, type 2 diabetes, and country. Carriage of HSD17B13 rs72613567:TA attenuated the risk for developing cirrhosis associated with PNPLA3 rs738409:G in both men and women, but the protective effect against the subsequent development of HCC was only observed in men (ORallelic , 0.75; 95% CI, 0.64-0.87; P = 1.72 × 10-4 ). CONCLUSIONS: Carriage of variants in PNPLA3 and HSD17B13 differentially affect the risk for developing advanced alcohol-related liver disease. A genotypic/phenotypic risk score might facilitate earlier diagnosis of HCC in this population.

Long-term outcomes in patients with decompensated alcohol-related liver disease, steatohepatitis and Maddrey's discriminant function <32.

BACKGROUND & AIMS: Patients with alcoholic hepatitis and a modified Maddrey's discriminant function (mDF) <32 have a low risk of short-term mortality. However, few data exist concerning long-term outcomes. The aims of this study were to evaluate 5-year survival rates and to identify predictive factors for long-term prognosis in this patient population. METHODS: We studied patients from 2 centers who were admitted for hepatic decompensation (ascites, hepatic encephalopathy, or jaundice) and who had histological findings of steatohepatitis and an mDF <32. Clinical and biological parameters were recorded at the time of liver biopsy and alcohol consumption was recorded during follow-up. We performed Cox proportional hazard survival analysis to identify factors associated with 5-year survival. RESULTS: One hundred and twenty-one patients were included (male: 64%, mean age: 51.5 ± 10.3 years, presence of cirrhosis: 84%). The median model for end-stage liver disease and mDF scores were 14 (IQR 11.7-16.1) and 19 (IQR 11.1-24), respectively. During follow-up, 30% of the patients remained abstinent. Survival rates at 1, 6, 12, 24, and 60 months were 96.7 ± 1.6%, 90.1 ± 2.7%, 80.8 ± 3.6%, 69.9 ± 4.3%, and 50.7 ± 4.9%, respectively. The majority of deaths (80%) were liver related. In multivariable analysis, encephalopathy at baseline and alcohol abstinence were predictive of 5-year survival. The 5-year survival rates of patients without and with encephalopathy at baseline were 60.5 ± 5.8% and 29.7 ± 8.0%, respectively, and the 5-year survival rates of abstinent and non-abstinent patients were 74.0 ± 8.0% and 40.9 ± 5.8%, respectively. CONCLUSIONS: The mortality rate of patients with alcoholic hepatitis and an mDF <32 is around 50% at 5 years. Hepatic encephalopathy at baseline and lack of alcohol abstinence impair long-term prognosis. New treatment strategies, including measures to ensure abstinence, are required. LAY SUMMARY: Patients with alcoholic hepatitis that is of intermediate severity have a low risk of short-term mortality but not much is known regarding long-term outcomes for these patients. This study clearly indicates that patients with intermediate disease characteristics have poor long-term outcomes. The presence of hepatic encephalopathy at the time of diagnosis and the absence of alcohol abstinence during follow-up are factors that predict poor long-term mortality.

Histological demonstration of BSEP/ABCB11 inhibition in transient neonatal cholestasis: a case report.

BACKGROUND: Idiopathic or transient neonatal cholestasis (TNC) represents a group of cholestatic disorders with unidentified origin and remains a diagnosis of exclusion. Dysfunction of hepatobiliary transporters mediating excretion of biliary constituents from hepatocytes may play a central role in the pathogenesis of cholestasis. Despite variants of bile salt (BS) export pump (BSEP/ABCB11) have already been described in TNC, the pathogenic role of BSEP dysfunction in TNC remained so far elusive. CASE PRESENTATION: We report on a newly-identified heterozygous ABCB11 missense variant (c.1345G > A, p.Glu449Lys) which was associated with prolonged cholestasis in a term infant after a complicated neonatal period. Moreover, we show for the first time almost completely abolished BSEP expression on the hepatocellular membrane in TNC. CONCLUSION: This report demonstrates for the first time a close association between the prolonged cholestasis in infancy and impaired BSEP expression on the hepatocyte canalicular membrane in a heterozygous carrier of newly-identified ABCB11 variant.

Fibrosis and alcohol-related liver disease.

Histological fibrosis stage is one of the most important prognostic factors in compensated and decompensated alcohol-related liver disease (ALD). Morphological assessment of fibrosis is useful for patient stratification, enabling individualised management, and for evaluation of treatment effects in clinical studies. In contrast to most chronic liver diseases where fibrosis is portal-based, fatty liver disease (FLD) of alcoholic or non-alcoholic aetiology (NAFLD) is associated with a centrilobular pattern of injury which leads to perivenular fibrosis and/or pericellular fibrosis. Progression of FLD drives expansive pericellular fibrosis, linking vascular structures and paving the way for the development of cirrhosis. At the cirrhotic stage, ongoing tissue damage leads to increasing fibrosis severity due to parenchymal loss and proliferation of fibrous scars. Histologic fibrosis staging systems have been devised, based on topography and the extent of fibrosis, for most chronic liver diseases. The utility of histological staging is reflected in different risks associated with individual fibrosis stages which cannot be reliably distinguished by non-invasive fibrosis assessment. In contrast to NAFLD, ALD-specific staging systems that enable the standardised prognostication required for clinical management and trials are lacking. Although morphological similarities between NAFLD and ALD exist, differences in clinical and histological features may substantially limit the utility of established NAFLD-specific staging systems for prognostication in ALD. This review summarises morphological features of fibrosis in ALD and compares them to other chronic liver diseases, particularly NAFLD. ALD-related fibrosis is examined in the context of pathogenetic mechanisms of fibrosis progression, regression and clinical settings that need to be considered in future prognostically relevant ALD staging approaches.

Fibrolamellar carcinoma in the Carney complex: PRKAR1A loss instead of the classic DNAJB1-PRKACA fusion.

Fibrolamellar carcinomas are characterized by activation of protein kinase A, a kinase composed of catalytic and regulatory subunits. PRKACA encodes a catalytic subunit of protein kinase A, and almost all fibrolamellar carcinomas have a heterozygous 400-kb deletion that leads to the fusion of DNAJB1 and PRKACA. The resulting DNAJB1-PRKACA fusion transcript is believed to activate protein kinase A by dysregulation of the catalytic portion of the protein. In contrast, PRKAR1A encodes one of the regulatory subunits of protein kinase A. We hypothesized that loss of function of this regulatory unit could also lead to protein kinase A activation and thus to fibrolamellar carcinoma. Because PRKAR1A mutations underlie the Carney complex, we searched for liver tumors in individuals with the Carney complex. We identified 3 individuals with fibrolamellar carcinomas and a personal history of the Carney complex. All three tumors displayed the typical morphology of fibrolamellar carcinoma and were positive for arginase, cytokeratin 7, and cluster of differentiation 68. Fluorescence in situ hybridization was negative for PRKACA rearrangements. However, PRKAR1A sequencing identified pathogenic mutations in two of two cases with successful sequencing. In addition, all three cases were negative for PRKAR1A protein expression, consistent with inactivation of this key regulatory unit of protein kinase A. We also identified one additional fibrolamellar carcinoma in an individual without a documented history of the Carney complex who was negative for PRKACA rearrangements but had loss of PRKAR1A protein expression as well as PRKAR1A mutations. CONCLUSION: Fibrolamellar carcinoma can be part of the Carney complex; in this setting, fibrolamellar carcinomas have inactivating PRKAR1A mutations instead of the DNAJB1-PRKACA fusion gene found in sporadic fibrolamellar carcinomas, providing an alternate means for activation of protein kinase A. (Hepatology 2017).

Pseudoachalasia as First Manifestation of a Malignancy.

Pseudoachalasia is a condition in which symptoms, radiologic, endoscopic, and manometric findings mimick idiopathic achalasia. About 4% of patients with a typical constellation for idiopathic achalasia will turn out to have pseudoachalasia, posing a major diagnostic challenge. A large spectrum of underlying causes of pseudoachalasia has been described. However, in about 70% of affected patients, this condition is caused by a malignancy (mostly adenocarcinoma of the esophagogastric junction or cardia). We describe a 16-year-old high school student referred for management of achalasia who turned out to have pseudoachalasia due to adenocarcinoma of the cardia. He was cured with preoperative chemotherapy followed by radical surgery. Therapy of pseudoachalasia secondary to neoplasia is directed against the tumor or may be palliative to keep the lumen open. Other causes of pseudoachalasia include esophageal motility disturbances as a paraneoplastic phenomenon (e.g., with small cell lung cancer), post fundoplication or post bariatric surgery, in association with a thoracic aortic aneurysm, or with sarcoidosis or amyloidosis. Therapy is directed accordingly to eliminate or correct the underlying cause.

Lysyl oxidase-like protein 2 (LOXL2) modulates barrier function in cholangiocytes in cholestasis.

BACKGROUND & AIMS: The lysyl oxidase-like protein 2 (LOXL2) promotes stabilization of the extracellular matrix, chemotaxis, cell growth and cell mobility. We aimed to (i) identify stimuli of LOXL2 in cholangiopathies, (ii) characterize the effects of LOXL2 on biliary epithelial cells' (BECs) barrier function, (iii) compare LOXL2 expression in primary sclerosing cholangitis (PSC), primary biliary cholangitis, and disease controls, and (iv) to determine LOXL2 expression and its cellular sources in four mouse models of cholangiopathies. METHODS: Cultured murine BECs were challenged with well-known triggers of cellular senescence, hypoxia, phospholipid-deficient Abcb4-/- mouse bile and chenodeoxycholic acid and investigated for LOXL2, SNAIL1 and E-cadherin expression and transepithelial electrical resistance with and without LOX-inhibition. In vivo, LOXL2 expression was studied in PSC livers, and controls and mouse models. We compared LOXL2 serum levels in patients with PSC, secondary SC, primary biliary cholangitis, and controls. RESULTS: Cellular senescence, hypoxia, Abcb4-/- bile and chenodeoxycholic acid induced LOXL2 and SNAIL1 expression, repressed E-cadherin expression, and significantly reduced transepithelial electrical resistance in BECs. Notably, all of the pathological changes could be recovered via pharmacological LOX-inhibition. Mouse models showed induced LOXL2 expression in the portal region and in association with ductular reaction. LOXL2 serum levels were significantly elevated in patients with cholangiopathies. In PSC, LOXL2 expression was located to characteristic periductal onion skin-type fibrosis, ductular reaction, Kupffer cells, and fibrotic septa. Importantly, in PSC, LOXL2 overexpression was paralleled by E-cadherin loss in BECs from medium-sized bile ducts. CONCLUSIONS: Reactive BECs produce LOXL2, resulting in increased tight junction permeability, which can be ameliorated by pharmacological LOX-inhibition in vitro. Reactive BECs, portal myofibroblasts, and Kupffer cells are the main sources of LOXL2 in cholangiopathies. LAY SUMMARY: In this study, we investigate the role of lysyl oxidase-like protein 2 (LOXL2), an enzyme pivotal in the development of organ fibrosis, in the pathogenesis of cholangiopathies (diseases of bile ducts), such as primary sclerosing cholangitis. We found LOXL2 to be expressed in association with bile duct epithelial injury and uncovered mechanisms for its upregulation and the subsequent effects in vitro and in vivo. Our findings support testing of anti-LOXL2 treatment strategies for patients with primary sclerosing cholangitis.

Caspase-cleaved keratin-18 fragments increase during alcohol withdrawal and predict liver-related death in patients with alcoholic liver disease.

Noninvasive assessment of disease activity in patients with nonalcoholic fatty liver disease (NAFLD) and alcoholic liver disease (ALD) is still unsettled, but essential for the evaluation of disease progression. We here studied the association of total (M65) and caspase-cleaved (M30) serum keratin-18 fragments (n = 204) with histological parameters (n = 106) in heavy drinkers primarily admitted for alcohol withdrawal before and after alcohol detoxification. An age-, sex-, and fibrosis-stage matched NAFLD cohort (n = 30) was used for comparison. The prognostic value of M30 and M65 levels were assessed in an additional prospectively followed-up cohort of 230 patients with alcoholic cirrhosis (AC) using competing risk analyses. Among the histological parameters, both M30/65 correlated significantly and better than any other serum marker with apoptosis and liver damage, such as ballooning (r = 0.65; P < 0.001), followed by lobular inflammation (0.48; P < 0.001), steatosis (0.46; P < 0.001), but less with fibrosis (0.24; P < 0.001). Area under the receiver operating characteristics curves to detect ballooning, steatosis, or steatohepatitis (SH) were slightly better for M30 (P < 0.005). Optimal M30 cut-off values for mild and severe ballooning were 330 and 420 U/L, and 290 and 330 U/L for SH grades 1 and 2. No significant differences of M30/65 were found between the matched NAFLD and ALD cohort. In contrast to aspartate-amino-transferase and M65, M30 levels increased significantly from 391 to 518 U/L during alcohol detoxification. Moreover, levels of M30 and M65 predicted non-hepatocellular carcinoma liver-related mortality in patients with AC during a mean observation interval of 67.2 months. CONCLUSION: Our data suggest M30 as highly specific marker of liver apoptosis both in ALD and NAFLD. In addition, hepatocellular apoptosis, as determined by M30 levels, occurs during alcohol withdrawal, and survival data point toward a novel underestimated role of apoptosis in patients with ALD. (Hepatology 2017;66:96-107).

Histological parameters and alcohol abstinence determine long-term prognosis in patients with alcoholic liver disease.

BACKGROUND & AIMS: Few data exist on predictors of long-term prognosis in patients with alcoholic liver disease (ALD). Most studies have only assessed short-term prognosis in patients with advanced ALD. We aimed to assess the prognostic impact of clinical, biochemical and histological parameters on long-term prognosis in patients with early/compensated and decompensated ALD. METHODS: Consecutive patients (n=192) with biopsy-proven liver disease due to alcohol abuse were analyzed retrospectively. Prognostic factors were evaluated in patients with early/compensated ALD (n=60) and in patients with decompensated ALD (clinical decompensation and/or bilirubin >3mg/dl at entry) (n=132). Factors that predict long-term survival were identified using Cox regression models. RESULTS: Liver-related mortality at 5years was 13% in early/compensated and 43% in decompensated ALD. In early/compensated ALD patients, long-term prognosis was determined by fibrosis stage, but not by clinical or biochemical variables. Severe fibrosis (F3/4) was present in 52% and had a major impact on 10-year mortality (F3/4: 45% vs. F0-2: 0%, p<0.001). In contrast, in decompensated patients, a combination of clinical features (sex), biochemical markers of liver failure (bilirubin, international normalized ratio [INR]), and histological features (pericellular fibrosis) predicted long-term survival. During follow-up, abstinence from alcohol was an important predictor of survival in both early/compensated and decompensated ALD. CONCLUSION: Fibrosis stage is the main predictor of long-term survival in patients with early/compensated ALD, while clinical, biochemical and histological parameters predict survival in patients with decompensated disease. Promoting abstinence may improve survival in patients with both early and advanced ALD. LAY SUMMARY: In this study, we evaluated long-term outcome in 192 patients with alcoholic liver disease who underwent liver biopsy: 60 patients with early disease (no symptoms) and 132 patients with advanced disease (jaundice, complications of cirrhosis). Importantly, half of the patients with 'early' disease already had severe fibrosis or cirrhosis on liver histology and dismal outcome (45% mortality at 10years). Abstinence from alcohol improved the prognosis in both early and advanced stages of the disease.

Hepatocellular carcinomas with intracellular hyaline bodies have a poor prognosis.

BACKGROUND & AIMS: Mallory-Denk bodies (MDBs) and intracellular hyaline bodies (IHBs) are cytoplasmic inclusions found in a subset of hepatocellular carcinoma (HCC). MDBs are mainly composed of the intermediate filament proteins keratin (K) 8 and K18, the cellular stress- and adapter-protein sequestosome 1/p62 (p62) and ubiquitin, whereas IHBs consist of p62 and/or ubiquitin. Of note, cytoplasmic inclusions containing p62 can serve as markers of suppressed autophagy, which in turn has been associated with poor prognosis. The aim of this study was to evaluate the prognostic significance of p62-containing MDB and IHB in patients with HCC. METHODS: Ninety resected HCCs were assessed by H&E histology for MDB or IHB, and their presence was confirmed by immunohistochemistry using K8/18, p62 and ubiquitin antibodies. The prognostic impact of inclusions was assessed using Kaplan-Meier and multivariate Cox proportional model. RESULTS: Mallory-Denk bodies and/or IHB were found in about 50% of HCC. Both types of inclusions were seen in 21%, MDB only in 19% and IHB only in 10% of cases. The presence of MDB in tumours was associated with the steatohepatitic variant of HCC, which also showed fatty change, ballooning of tumour cells, MDBs, inflammation and pericellular fibrosis (P<.001). In contrast, IHBs were not associated with steatohepatitic morphology but were associated with significantly shorter overall survival (P=.006). Multivariate analysis revealed macroscopic vascular invasion (P=.045) and presence of IHB in HCC cells (P=.005) as independently associated with overall survival. CONCLUSIONS: Intracellular hyaline bodies and macroscopic vascular invasion identify a subset of HCC patients with poor prognosis.

Perspectives on NASH Histology: Cellular Ballooning.

Interpretation of liver biopsy in NAFLD can be challenging to distinguish histological NASH from non-NASH fatty liver - a broad dichotomy which carries significant prognostic and therapeutic implications and underlies the utility of many non-invasive tests. There is usually a reasonable degree of inter-observer agreement for some key parameters like steatosis, inflammation and fibrosis staging. However, the assessment of cellular ballooning can be a stumbling block even for experienced observers. Below, we recount some aspects of the history of histological definitions in NASH and propose specific methods to more objectively identify cellular ballooning in routine biopsy assessments.