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BackgroundNon-alcoholic fatty liver disease (NAFLD) is a complex, multifactorial disease affected by diet, lifestyle and genetics. Proinflammatory cytokines like IL-1β and IL-6 have been shown to be elevated in non-alcoholic steatohepatitis (NASH).AimTo investigate the relationship between IL1B and IL6 gene polymorphisms and histological features of NAFLD in the NASH CRN cohort.MethodsA total of 604 adult (≥18 years) non-Hispanic Caucasians with biopsy-proven NAFLD were genotyped for the following SNPs: IL1B, rs16944, rs1143634; IL6, rs1800795, rs10499563. Logistic regression was used to examine the relationship between genotype and a definitive diagnosis and advanced histological features of NASH after controlling for the following variables selected a priori: age, sex, diabetes, obesity and HOMA-IR level.ResultsThe IL6 rs10499563 C allele was independently associated with the presence of definitive NASH, and increased ballooning and Mallory bodies. The IL1B rs1143634 TT genotype was associated with advanced fibrosis and increased Mallory bodies. The IL6 rs1800795 C allele was associated with not only increased risk for severe steatosis, >66% but also decreased risk for advanced fibrosis and lobular inflammation and Mallory body formation.ConclusionsThese results suggest that common variants in the IL6 and IL1B genes may increase susceptibility for NASH and confer a higher risk of hepatic parenchymal damage including increased ballooning, increased Mallory bodies, and bridging fibrosis or cirrhosis. In contrast, the IL6 rs1800795 C allele may confer a higher risk for steatosis, but less parenchymal damage. Our findings support the development of therapeutics aimed at IL-1β and IL-6 suppression.

Background: Metabolic dysfunction-associated steatotic liver disease (MASLD) is common in people with HIV (PWH). The morphological spectrum of MASLD compared to matched controls and of the correlation between the NAFLD activity score (NAS) and fibrosis stage in PWH remains unknown., Methods: Overall, 107 liver biopsies from PWH with MASLD (MASLD-PWH) were matched to 107 biopsies from individuals with MASLD and without HIV (MASLD controls) on age at biopsy, race/ethnicity, sex, type 2 diabetes, body mass index (BMI) and alanine aminotransferase (ALT) level. Biopsies were scored using NAS., Results: Compared to MASLD-controls, MASLD-PWH had lower steatosis grade (OR: 0.65, 95% CI: (0.47-0.90), p = 0.01), lower lobular inflammation grade (OR: 0.55, 95% CI: (0.34-0.89), p = 0.02), less portal inflammation (OR: 0.42, 95% CI: (0.25-0.72), p = 0.002) and less ballooned hepatocytes (OR: 0.60, 95% CI: (0.41-0.88), p = 0.01). Thus, NAS was lower in MASLD-PWH (OR: 0.69, 95% CI: (0.56-0.85), p < 0.001) than in MASLD controls. There was a trend towards lower prevalence of steatohepatitis in MASLD-PWH (OR: 0.84, 95% CI: (0.68-1.03), p = 0.09). A multivariate analysis demonstrated that MASLD-PWH cases had significantly less steatosis (OR: 0.66, p = 0.03), portal inflammation (OR: 0.34, p = 0.001) and ballooned hepatocytes (OR: 0.55, p = 0.01), yet higher stage fibrosis (OR: 1.42, p = 0.03) compared to MASLD controls., Conclusion: The NAS and histological drivers of fibrosis (e.g. inflammation and hepatocyte ballooning) are less pronounced in MASLD-PWH, and yet fibrosis stage was generally higher when compared to matched controls with MASLD without HIV. This suggests HIV-specific factors beyond hepatic necroinflammation may contribute to fibrosis progression in MASLD-PWH., (© 2024 The Author(s). Alimentary Pharmacology & Therapeutics published by John Wiley & Sons Ltd. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

Goal : The early diagnosis and treatment of hepatitis is essential to reduce hepatitis-related liver function deterioration and mortality. One component of the widely-used Ishak grading system for the grading of periportal interface hepatitis is based on the percentage of portal borders infiltrated by lymphocytes. Thus, the accurate detection of lymphocyte-infiltrated periportal regions is critical in the diagnosis of hepatitis. However, the infiltrating lymphocytes usually result in the formation of ambiguous and highly-irregular portal boundaries, and thus identifying the infiltrated portal boundary regions precisely using automated methods is challenging. This study aims to develop a deep-learning-based automatic detection framework to assist diagnosis. Methods : The present study proposes a framework consisting of a Structurally-REfined Deep Portal Segmentation module and an Infiltrated Periportal Region Detection module based on heterogeneous infiltration features to accurately identify the infiltrated periportal regions in liver Whole Slide Images. Results : The proposed method achieves 0.725 in F1-score of lymphocyte-infiltrated periportal region detection. Moreover, the statistics of the ratio of the detected infiltrated portal boundary have high correlation to the Ishak grade (Spearman's correlations more than 0.87 with p-values less than 0.001) and medium correlation to the liver function index aspartate aminotransferase and alanine aminotransferase (Spearman's correlations more than 0.63 and 0.57 with p-values less than 0.001). Conclusions : The study shows the statistics of the ratio of infiltrated portal boundary have correlation to the Ishak grade and liver function index. The proposed framework provides pathologists with a useful and reliable tool for hepatitis diagnosis., Competing Interests: VI.No conflict of interest., (© 2024 The Authors.)

Subtyping hepatic adenomas is important for patient management due to differing complication risks. Immunohistochemical staining with C-reactive protein (CRP) and serum amyloid-A (SAA) is widely accepted as a surrogate for molecular classification to identify inflammatory hepatocellular adenomas. Limited data, however, has been published on how these 2 stains compare for sensitivity. We conducted a large, multicenter, retrospective study to examine the sensitivity and staining characteristics of CRP and SAA in inflammatory hepatic adenomas, with focal nodular hyperplasia (FNHs) as a control group. Inflammatory adenomas were identified in 133 patients (average age 37 years, 109 were female). In all, 69.9% of cases were resection specimens and 90.2% of all cases showed positive staining for both CRP and SAA; 10 (7.5%) were positive for CRP only and 3 (2.3%) were positive for SAA only. CRP was more sensitive than SAA (97.74% vs. 92.48%, P -value = 0.0961) and showed more extensive and intense staining, with a significantly higher modified H-score ( P <0.001). Focal nodular hyperplasia can also show positive CRP and SAA staining but with a lower modified H-score ( P <0.0001). Based on beta-catenin and glutamine synthetase staining, 26 of inflammatory adenomas also had beta-catenin activation (19.5%). All 3 cases with positive SAA and negative CRP staining were beta-catenin activated. In contrast, the proportion of cases that were CRP positive and SAA negative was similar regardless of beta-catenin activation. The data affirms the strategy of using both CRP and SAA immunostains for hepatic adenoma subtyping and raises the awareness of the highly variable nature of SAA staining characteristics., Competing Interests: The authors declare no conflict of interest., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Liver fibrosis staging has many challenges, including the large number of proposed staging systems, the heterogeneity of the histopathologic changes of many primary liver diseases, and the potential for slight differences in histologic interpretation to significantly affect clinical management. This review focuses first on fibrosis regression. Following this, each of the major categories of liver disease is discussed in regard to (1) appropriate fibrosis staging systems, (2) emerging concepts, (3) current clinical indications for liver biopsy, (4) clinical decisions determined by fibrosis stage, and (5) histologic challenges and pitfalls related to staging., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Hepatocellular carcinoma (HCC) is one of the most fatal cancers in the world. There is an urgent need to understand the molecular background of HCC to facilitate the identification of biomarkers and discover effective therapeutic targets. Published transcriptomic studies have reported a large number of genes that are individually significant for HCC. However, reliable biomarkers remain to be determined. In this study, built on max-linear competing risk factor models, we developed a machine learning analytical framework to analyze transcriptomic data to identify the most miniature set of differentially expressed genes (DEGs). By analyzing 9 public whole-transcriptome datasets (containing 1184 HCC samples and 672 nontumor controls), we identified 5 critical differentially expressed genes (DEGs) (ie, CCDC107, CXCL12, GIGYF1, GMNN, and IFFO1) between HCC and control samples. The classifiers built on these 5 DEGs reached nearly perfect performance in identification of HCC. The performance of the 5 DEGs was further validated in a US Caucasian cohort that we collected (containing 17 HCC with paired nontumor tissue). The conceptual advance of our work lies in modeling gene-gene interactions and correcting batch effect in the analytic framework. The classifiers built on the 5 DEGs demonstrated clear signature patterns for HCC. The results are interpretable, robust, and reproducible across diverse cohorts/populations with various disease etiologies, indicating the 5 DEGs are intrinsic variables that can describe the overall features of HCC at the genomic level. The analytical framework applied in this study may pave a new way for improving transcriptome profiling analysis of human cancers., Competing Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2023.)

Hepatocellular adenomas (HCAs) are benign liver neoplasms which most commonly present in women in their reproductive age. In men, they are rare and have a higher risk of malignant transformation to hepatocellular carcinoma (HCC). Here we present our multicenter experience with HCA in men in the United States. A total of 27 HCA cases were included, with a mean age of presentation of 37 years (range, 9-69 years) and a mean size of 6.8 cm (range, 0.9-18.5 cm). Based on the 2019 World Health Organization classification, the most common subtype identified was inflammatory HCA (IHCA; 10 cases, 37.0%) followed by unclassified HCA (UHCA; 7 cases, 25.9%), HNF1A-inactivated HCA (H-HCA; 6 cases, 22.2%), β-catenin-activated IHCA (b-IHCA; 3 cases, 11.1%), and β-catenin-activated HCA (b-HCA; 1 case, 3.7%). Six additional cases diagnosed as hepatocellular neoplasm of uncertain malignant potential (HUMP) were also included in the study. These cases presented in a mean age of 46 years (range, 17-64 years) and a size of 10.8 cm (range, 4.2-16.5 cm). We evaluated the significance of androgen receptor (AR) expression by immunohistochemistry (IHC); of the 16 cases with materials available, 8 were considered positive using the Allred score system (2 IHCA, 2 H-HCA, 1 UHCA, and 3 HUMP). Of the total cases, 12 were diagnosed on biopsies, for which follow-up information is available for 7, and none of them show evidence of malignant transformation. Of the 21 resection cases, a concomitant well-differentiated HCC within the same lesion was identified in 5 cases (23.8%), which were diagnosed as HCA (n = 4) or HUMP (n = 1). Overall, 15% of cases in our entire cohort of HCA and HUMP showed concomitant HCC, while none of the 7 biopsy cases showed any malignant transformation on follow-up (range, 22-160 months; mean, 61.8 months)., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Paroxysmal nocturnal hemoglobinuria (PNH), a rare acquired hematologic disorder, can be treated with C5 inhibitors (C5i) such as eculizumab or ravulizumab. This retrospective study is the first to describe real-world treatment patterns and changes in hematologic PNH-monitoring laboratory tests among C5i-treated US patients. Data were extracted from TriNetX Dataworks Network and included patients with a PNH diagnosis between 1 January 2010, and 20 August 2021. Patients were stratified into three cohorts based on their C5i usage: eculizumab, ravulizumab (prior eculizumab), and ravulizumab (eculizumab naïve). Hematological markers (hemoglobin [Hb], lactate dehydrogenase [LDH], and absolute reticulocyte count [ARC]) and relevant clinical events (e.g., breakthrough hemolysis [BTH], complement-amplifying conditions [CAC], thrombosis, infection, and all-cause mortality) were captured any time within 12 months post-index treatment. Of the 143 (eculizumab), 43 (ravulizumab, prior eculizumab), and 33 (ravulizumab, eculizumab naïve) patients, mean age across cohorts was 42-51 years, 55-61% were female, 63-73% were White, and 33-40% had aplastic anemia. Among all cohorts 12 months post-C5i treatment, 50-82% remained anemic, 8-32% required ≥1 transfusion, and 13-59% had BTH, of which 33%-54% had CACs. Additionally, thrombosis was seen in 7-15% of patients, infection in 20-25%, and mortality in 1-7%. These findings suggest many C5i-treated patients experience suboptimal disease control.

Cholangiocarcinoma represents the second most frequent type of primary liver cancer that develops through a multistep histopathologic sequence. Dysplasia in the biliary tract epithelium is a precursor lesion of cholangiocarcinoma. This review provides a practical overview of bile duct dysplasia in relation to invasive carcinoma, covering clinicopathological features, diagnostic criteria, differential diagnosis, useful testing modalities, and challenges in daily practice. The key features of biliary intraepithelial neoplasia, intraductal papillary neoplasm, intraductal tubulopapillary neoplasm, and mucinous cystic neoplasm are described. Important differential diagnoses are included. Common pitfalls in histopathologic interpretation of bile duct biopsies and frozen sections are discussed., (Copyright © 2022. Published by Elsevier Inc.)

It has been postulated that inflammasomes, in particular the NLRP3 (NLR family pyrin domain containing 3) inflammasome, mediate the necroinflammation and fibrosis that characterize nonalcoholic steatohepatitis (NASH) by engaging innate immune responses. We aimed to investigate the impact of genetic deletion or pharmacologic inhibition of the NLRP3 inflammasome on experimental steatohepatitis. Global Nlrp3 KO (expected to inhibit the NLRP3 inflammasome) or Casp1 KO (expected to inhibit all inflammasomes) mice were compared to wild type controls after 6 months on a high-fat, high-cholesterol (HFHC, 1% cholesterol) diet known to induce fibrosing steatohepatitis. Additionally, wildtype mice on a HFHC diet (0.75% or 0.5% cholesterol) for 6 months were either treated or not treated with an oral, pharmacologic inhibitor of Nlrp3 (MCC950) that was delivered in the drinking water (0.3 mg/ml). We found that genetic deletion or pharmacologic inhibition of the NLRP3 inflammasome did not ameliorate any of the histological components of fibrosing NASH in HFHC-fed mice. Collectively, these results do not support NLRP3 inhibition as a potential target for human NASH., Competing Interests: Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier Inc.)

Objectives: Primary biliary cholangitis (PBC) and autoimmune hepatitis (AIH) can be difficult to distinguish in end-stage liver disease. Previous studies have shown that immunoglobulin G (IgG) and immunoglobulin M (IgM) immunostaining can differentiate AIH from PBC in needle core biopsy specimens, and we seek to extend these data to cirrhotic liver explants, in which the histology of AIH or PBC may be indiscernible., Methods: Clinical data were reviewed for 20 patients with PBC cirrhosis and 16 with AIH cirrhosis. Immunohistochemistry for IgM and IgG was performed on representative blocks of explanted livers. Three high-power fields with the highest concentration of IgG- and IgM-positive plasma cells were counted and compared., Results: The average number of IgM-positive plasma cells was significantly higher in PBC explants (7.3) than in AIH (1.8) (P = .001). There was no significant difference in the average number of IgG-positive plasma cells in PBC (2.5) and AIH (2.8) (P = .8). The IgG/IgM ratio was more likely to be less than 1.0 in PBC (17/20, 85%) compared with AIH (7/16, 44%) (P = .01)., Conclusions: Our study demonstrates that the absolute number of IgM plasma cells is greater in explants of cirrhotic PBC compared with AIH. These findings may be helpful in the evaluation of cryptogenic cirrhosis., (© The Author(s) 2022. Published by Oxford University Press on behalf of American Society for Clinical Pathology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Dysregulation of expression of functional genes and pathways plays critical roles in the etiology and progression of hepatocellular carcinoma (HCC). Next generation-based RNA sequencing (RNA-seq) offers unparalleled power to comprehensively characterize HCC at the whole transcriptome level. In this study, 17 fresh-frozen HCC samples with paired non-neoplastic liver tissue from Caucasian patients undergoing liver resection or transplantation were used for RNA-seq analysis. Pairwise differential expression analysis of the RNA-seq data was performed to identify genes, pathways, and functional terms differentially regulated in HCC versus normal tissues. At a false discovery rate (FDR) of 0.10, 13% (n = 4335) of transcripts were up-regulated and 19% (n = 6454) of transcripts were down-regulated in HCC versus non-neoplastic tissue. Eighty-five Kyoto Encyclopedia of Genes and Genomes pathways were differentially regulated (FDR, <0.10), with almost all pathways (n = 83) being up-regulated in HCC versus non-neoplastic tissue. Among the top up-regulated pathways was oxidative phosphorylation (hsa00190; FDR, 1.12E-15), which was confirmed by Database for Annotation, Visualization, and Integrated Discovery (DAVID) gene set enrichment analysis. Consistent with potential oxidative stress due to activated oxidative phosphorylation, DNA damage-related signals (e.g., the up-regulated hsa03420 nucleotide excision repair [FDR, 1.14E-04] and hsa03410 base excision repair [FDR, 2.71E-04] pathways) were observed. Among down-regulated genes (FDR, <0.10), functional terms related to cellular structures (e.g., cell membrane [FDR, 3.05E-21] and cell junction [FDR, 2.41E-07], were highly enriched, suggesting compromised formation of cellular structure in HCC at the transcriptome level. Interestingly, the olfactory transduction (hsa04740; FDR, 1.53E-07) pathway was observed to be down-regulated in HCC versus non-neoplastic tissue, suggesting impaired liver chemosensory functions in HCC. Our findings suggest oxidative phosphorylation and the associated DNA damage may be the major driving pathologic feature in HCC., (© 2022 The Authors. Hepatology Communications published by Wiley Periodicals LLC on behalf of American Association for the Study of Liver Diseases.)

Purpose: Mismatch repair (MMR) immunohistochemistry (IHC) is frequently used to inform prognosis, select (immuno-)therapy, and identify patients for heritable cancer syndrome testing. However, false-negative and false-positive MMR IHC interpretations have been described., Materials and Methods: Following identification of discordant MMR IHC and DNA-based microsatellite instability testing in a patient with colorectal carcinoma, we retrospectively reviewed institutional archives to identify patient samples with similar discrepancies., Results: We report a patient with metastatic colorectal carcinoma who initially received immunotherapy on the basis of apparent isolated loss of MLH1 by IHC; notably, MLH1 promoter hypermethylation was negative. Subsequent evaluation of neoplastic tissue on a DNA-based targeted next-generation sequencing panel demonstrated microsatellite stability, low tumor mutational burden, and a benign MLH1 variant, MLH1 p.V384D, accompanied by loss of heterozygosity. The constellation of findings and repeat MLH1 IHC demonstrating retained expression using a different antibody-clone, supported reclassification of the neoplasm as MMR-proficient. Immunotherapy was discontinued, and cytotoxic chemotherapy was initiated. This index case of apparent discordance between MMR IHC and DNA-based microsatellite instability prompted a retrospective review of institutional archives to identify patient samples with similar discrepancies. Further evaluation of neoplasms harboring MLH1 p.V384D with loss of heterozygosity revealed systematic antibody-dependent interference. The review also identified a second IHC-interference candidate, MLH1 p.A441T., Conclusion: This study confirms that rare germline polymorphisms can result in incorrect IHC results, potentially affecting selection of optimal therapy and the decision to pursue germline testing. This case further highlights the need for expert molecular pathologic review and communication between clinical and molecular oncology teams.

Subtyping of hepatocellular adenoma (HCA) is an important task in practice as different subtypes may have different clinical outcomes and management algorithms. Definitive subtyping is currently dependent on immunohistochemical and molecular testing. The association between some morphologic/clinical features and HCA subtypes has been reported; however, the predictive performance of these features has been controversial. In this study, we attempted machine learning based methods to select an efficient and parsimonious set of morphologic/clinical features for differentiating a HCA subtype from the others, and then assessed the performance of the selected features in identifying the correct subtypes. We first examined 50 liver HCA resection specimens collected at the University of Washington and Kobe University/Kings College London, including HNF1α-mutated HCA (H-HCA) (n = 16), inflammatory HCA (I-HCA) (n = 20), beta-catenin activated HCA (β-HCA) (n = 8), and unclassified HCA (U-HCA) (n = 6). Twenty-six morphologic/clinical features were assessed. We used LASSO (least absolute shrinkage and selection operator) to select key features that could differentiate a subtype from the others. We further performed SVM (support vector machine) analysis to assess the performance (sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy) of the selected features in HCA subtyping in an independent cohort of liver resection samples (n = 20) collected at the University of Wisconsin-Madison. With some overlap, different combinations of morphologic/clinical features were selected for each subtype. Based on SVM analysis, the selected features classified HCA into correct subtypes with an overall accuracy of at least 80%. Our findings are useful for initial diagnosis and subtyping of HCA, especially in clinical settings without access to immunohistochemical and molecular assays., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Hepatic adenomas occur most commonly in women between the ages of 20 and 40 years, but rarely they occur in older aged persons, including those 60 years of age or older. This group of adenomas, however, has not been systemically examined. Twenty-six hepatic adenomas in persons 60 years of age or older were studied, along with a control group of 50 hepatic adenomas in persons aged 30 to 39. Hepatic adenomas in persons 60 or more years of age were found in 21 women and 5 men, while the control group had 44 women and 6 men. Subtyping the adenomas in persons 60 years or older showed the following results: 18 HNF1A-inactivated adenomas (69%), 4 inflammatory adenomas (15%), and 4 unclassified adenomas (15%). In contrast, the control group showed a significantly different pattern (P=0.003), with a greater percentage of inflammatory adenomas (28, 56%), fewer HNF1A-inactivated adenomas (8, 16%), and more unclassified adenomas (14, 28%). Atypia and malignant transformation within the hepatic adenomas was studied next. Of the hepatic adenomas in persons age 60 or greater, 3 (12%) showed atypical histologic features, and 6 (23%) had a malignant transformation. In contrast, for hepatic adenomas in the control group, only 4 (8%) adenomas showed atypical histologic features, and 3 (6%) had undergone malignant transformation. In addition, the hepatic adenomas that were atypical or showed early malignant transformation were less likely to have beta-catenin activation in patients over 60 (2/9 cases) compared with those between 30 and 39 years (5/7 cases). Myxoid change and heavy lipofuscin deposition were also more common in adenomas in older aged persons. In conclusion, hepatic adenomas in persons 60 years of age or older are enriched for HNF1A-inactivated adenomas and have a higher frequency of malignant transformation. Malignant transformation, however, is less likely to develop through activation of the beta-catenin pathway., Competing Interests: Conflicts of Interest and Source of Funding: M.S.T. received grant support: P50 CA210964. The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Aims: The prevalence of non-alcoholic fatty liver disease (NAFLD) and its progression to non-alcoholic steatohepatitis (NASH) is higher in postmenopausal women than men. The aim of this study was to determine the molecular mechanisms underlying this sexual dimorphism in NAFLD., Methods: A total of 24 frozen liver samples of both sexes (normal and NAFLD/NASH) were used in this study. Total RNAseq was first used to identify differentially expressed genes (DEGs) between samples. Enrichment analysis of Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Reactome were used to analyze biological pathways. RT 2 profiler polymerase chain reaction (PCR) arrays were used to identify genes associated with the biological pathways. Immunoblotting was used to validate protein expression of certain genes., Results: We identified 4362 genes that are differentially expressed between NAFLD/NASH and normal samples; of those 745 genes were characterized as sex specific in NAFLD/NASH. Multiple pathway analysis platforms showed that Wnt-signaling is a candidate shared for a common biological pathway-associated with NAFLD/NASH. Using Wnt pathway focused PCR array we identified many genes involved in canonical pathway (Wnt/β-catenin activation) such as CTNNB1, c-Myc and CCND2 are overexpressed in female cases, whereas these genes are either not detected or downregulated in male cases. Immunoblot analysis validated the expression of CTNNB1 in female cases but not in male protein samples., Conclusions: Our study suggests, for the first time, that the activation of canonical Wnt signaling could be one of the main pathways associated with sexual dimorphism in NAFLD and NASH., (© 2022 Japan Society of Hepatology.)

Proprotein convertase subtilisin/kexin type 9 (Pcsk9) binds to hepatic low-density lipoprotein receptor (LDLR) and induces its internalization and degradation. Pcsk9 inhibition increases LDLR expression by hepatocytes, which causes increased uptake of circulating LDL, thereby reducing plasma LDL-cholesterol. However, by increasing the uptake of LDL by the liver, Pcsk9 inhibition increases the exposure of the liver to cholesterol, which may result in higher risk of steatohepatitis and ever carcinogenesis. We compared Pcsk9-/- knockout (KO) mice and appropriate wild-type (WT) controls of the same strain assigned to a high-fat (15%, wt/wt) diet for 9 months supplemented with 0.25%, 0.5%, or 0.75% dietary cholesterol. Pcsk9 KO mice on a high-fat, high-cholesterol diet exhibited higher levels of hepatic free cholesterol loading and hepatic cholesterol crystallization than their WT counterparts. Pcsk9 KO mice developed crown-like structures of macrophages surrounding cholesterol crystal-containing lipid droplets and hepatocytes, exhibited higher levels of apoptosis, and developed significantly more hepatic inflammation and fibrosis consistent with fibrosing steatohepatitis, including 5-fold and 11-fold more fibrosis at 0.5% and 0.75% dietary cholesterol, respectively. When injected with diethylnitrosamine, a hepatic carcinogen, early-in-life Pcsk9 KO mice were more likely to develop liver cancer than WT mice. Conclusion: Pcsk9 KO mice on high-cholesterol diets developed increased hepatic free cholesterol and cholesterol crystals and fibrosing steatohepatitis with a higher predisposition to liver cancer compared with WT mice. Future studies should evaluate whether patients on long-term treatment with anti-PSCK9 monoclonal antibodies are at increased risk of hepatic steatosis, steatohepatitis or liver cancer, while accounting for concurrent use of statins., (© 2022 The Authors. Hepatology Communications published by Wiley Periodicals LLC on behalf of the American Association for the Study of Liver Diseases.)

Objectives: Histopathologic evaluation of bile biopsies for biliary strictures is frequently challenging and is affected by interobserver disagreement. Reliable ancillary tests that can help differentiate benign from malignant are not available. This study aimed to evaluate whether DNA content abnormalities detected by flow cytometry on formalin-fixed, paraffin-embedded (FFPE) tissue can help differentiate benign/reactive, dysplastic from malignant cell populations in bile duct biopsies., Methods: We performed DNA flow cytometry on 30 FFPE bile duct biopsies in 5 well-defined diagnostic categories: (1) negative for dysplasia (NED), (2) low-grade dysplasia (LGD), (3) high-grade dysplasia (HGD), (4) carcinoma (CA), and (5) indefinite for dysplasia (IND)., Results: Abnormal DNA content was detected in 0 NED, 5 LGD (62.5%), 2 HGD (33.3%), 3 CA (60%), and 4 IND (80%) samples. As a diagnostic marker, the estimated sensitivity, specificity, positive predictive value, and negative predictive value were 63%, 100%, 100%, and 50%, respectively, for diagnosing HGD or CA., Conclusions: DNA flow cytometry analysis is a useful ancillary test for the interpretation of bile duct biopsies. DNA content abnormalities, when correlated with histologic findings, will not only help confirm the morphologic impression but also identify patients who are at a higher risk of developing malignancy., (© American Society for Clinical Pathology, 2021. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Aims: Centrizonal hepatocyte dropout has been described in diverse liver pathologies, including viral hepatitis, venous outflow obstruction, and allograft cellular rejection. However, its clinical significance remains uncertain., Methods and Results: We designed a clinicopathological study of 206 allograft liver biopsies with centrizonal hepatocyte dropout. Centrizonal hepatocyte dropout was associated most frequently with cellular rejection (n = 62), asymptomatic/protocol biopsies (n = 56), immediate post-transplantation biopsies (n = 21), biliary obstruction (n = 14), and viral hepatitis (n = 13). The differential diagnosis is informed by timing post-transplantation, biliary imaging and laboratory test results. 'Cholestatic' and 'hepatocytic' laboratory patterns were associated with biliary obstruction and cellular rejection, respectively. A mixed pattern peaking after biopsy was observed in viral hepatitis cases. In the context of cellular rejection, dropout was not associated with the time interval to normalisation of serum alanine aminotransferase (ALT), but was associated with shorter transplant-free survival (hazard ratio 4, P = 0.01) than that of histological severity-matched controls. In time zero allograft biopsies, time to ALT normalisation was prolonged (median, 15 versus 11 days, P = 0.002) in allografts with centrizonal dropout, with no effect on retransplant-free survival., Conclusions: Centrizonal hepatocyte dropout has low clinicopathological diagnostic specificity. However, it correlates with adverse clinical outcomes in allograft cellular rejection and time zero biopsies., (© 2021 John Wiley & Sons Ltd.)

Gallbladder carcinoma is an uncommon malignancy with an overall 5-year survival of less than 5%. Gallbladder carcinoma has been strongly linked with cholelithiasis and chronic inflammation. Case reports and series have described cholecystitis with acute (neutrophilic) inflammation in association with gallbladder carcinoma, although a clear relationship to patient outcome has not been established. Our series included 8 cases of gallbladder carcinoma with high tumor-associated neutrophils (>25 per high power field) that were associated with shorter patient survival (Cox regression coefficient 6.2, p = 0.004) than age- and stage-matched controls. High tumor-associated neutrophils were not associated with gallbladder rupture/perforation or increased bacterial load measured by 16S PCR. Neutrophilic inflammation with gallbladder carcinoma correlates to shorter survival, independent of patient age and stage of carcinoma. The findings suggest that the degree of neutrophilic inflammation may have prognostic significance in specimens from patients with gallbladder carcinoma after cholecystectomy. Further studies with larger case numbers are needed to confirm and generalize these findings., (Copyright © 2021. Published by Elsevier Inc.)

Hepatocellular adenomas are rare in children. A large study focused on pediatric patients has not been undertaken. A natural language search was performed at 5 institutions for hepatocellular adenomas in patients younger than 21 years old. Clinical characteristics as well as immunohistochemical staining profile was reviewed and adenomas subtyped per standard classification. Patients were divided into prepubescent and postpubescent age group. Thirty-one patients were included. Eleven (35%) were male and 10 (32%) were prepubescent. Fifteen (54%) of 28 patients with known clinical histories had adenomas associated with a syndrome. The percentage of the different adenoma subtypes was: 16% β-catenin activated, 10% combined inflammatory and β-catenin activated, 29% HFN1α-inactivated, 35% inflammatory, and 10% unclassified subtype by immunohistochemical staining. Interestingly 53% of patients with syndromes were male, while 85% of patients in the nonsyndromic group were female. The total number of β-catenin activated tumors was greater in the syndromic group (5/15, 33%) and prepubescent group (5/10, 50%) than in the nonsyndromic group (2/13, 16%) and postpubescent group (3/21, 14%), P=0.4 and 0.07, respectively. Inflammatory type adenoma was more frequent in the postpubescent (10/21, 48%) than in the prepubescent group (1/10, 10%), P=0.06, trending toward significance. Pediatric patients with hepatocellular adenomas frequently have syndromes, especially in the prepubescent group. In patients with syndromes a greater percentage of adenomas were β-catenin activated. In patients without a known syndrome the distribution of hepatocellular adenoma subtypes appears similar to adults., Competing Interests: Conflicts of Interest and Source of Funding: The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC) are biliary tract pathologies with increased risk of HCC, although HCC is more commonly associated with viral hepatitis and steatohepatitis. HCC risk stratification in PBC/PSC populations may help select patients for surveillance. We hypothesized that metabolic syndrome associated diagnoses and co-morbid nonalcoholic fatty liver disease (NAFLD) may be risk factors for HCC in patients with PBC and PSC. We undertook a multi-institutional case control study of PSC (19 cases, 38 controls) and PBC (39 cases and controls) patients with advanced fibrosis, matched for known HCC risk factors of age and sex, who had native liver explant or resection specimens. In the PSC population, HCC risk was significantly associated with multiple metabolic syndrome associated diagnoses (OR 13, p = 0.02), hyperlipidemia (OR 29, p = 0.03), and obesity (OR 6.8, p = 0.01). In the PBC cohort, only type 2 diabetes was a risk factor for HCC (OR 4.7, p = 0.03). In the PSC cohort, thick fibrous septae were associated with HCC risk (OR 3.4, p = 0.04). No other pathologic features of the nonneoplastic liver were significantly associated with HCC, including features of NAFLD such as macrovesicular steatosis, pericellular fibrosis, and steatohepatitis. Metabolic syndrome associated diagnoses, specifically type 2 diabetes among PBC patients, is associated with HCC risk in patients with biliary type cirrhosis. However, we found no evidence that HCC risk is related to co-morbid NAFLD, indicating a likely distinct mechanism of metabolic syndrome-associated carcinogenesis in these populations., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Aims: Combined hepatocellular-cholangiocarcinoma (cHCC-CCA) is an uncommon hepatic malignancy with a poor outcome. The 2019 World Health Organization (WHO) classification modified the definition and discarded the subtypes with stem cell features. However, the differences among cHCC-CCA, hepatocellular carcinoma (HCC), HCC with stem cell/progenitor features (HCCscf) and intrahepatic cholangiocarcinoma (iCCA) remain undetermined. The aim of this study was to investigate the characteristics of cHCC-CCA in comparison with those of other primary liver cancers by utilising the updated WHO classification., Methods and Results: We retrospectively analysed 64 cHCC-CCA patients and 55 HCCscf patients from December 2007 to May 2018. Propensity score matching was conducted to compare these with HCC and iCCA patients. Clinicopathological characteristics, event-free survival and overall survival were evaluated with multivariate Cox proportional hazard regression. During a median follow-up of 55.9 months, cHCC-CCA patients had significantly poorer survival than HCCscf patients, and survival intermediate between that of HCC patients and that of iCCA patients. Hepatitis B virus (HBV) infection and high levels of tumour-infiltrating lymphocytes (TILs) were associated with favourable survival in cHCC-CCA patients. In the multivariate analysis, poor hepatic reserve, absence of HBV infection, stage IV disease and low levels of TILs were significant negative prognostic factors in cHCC-CCA patients. After being pooled with other primary liver cancers, cHCC-CCA and iCCA resulted in the worse survival., Conclusions: cHCC-CCA patients have survival intermediate between that of HCC patients and iCCA patients, and HBV infection and high levels of TILs predict favourable survival. Our study provides clinical correlations for the new 2019 WHO classification., (© 2021 John Wiley & Sons Ltd.)

Immune checkpoint inhibitors (ICIs) represent a promising therapy for many types of cancer. However, only a portion of patients respond to this therapy and some patients develop clinically significant immune-mediated liver injury caused by immune checkpoint inhibitors (ILICI), an immune-related adverse event (irAE) that may require the interruption or termination of treatment and administration of systemic corticosteroids or other immunosuppressive agents. Although the incidence of ILICI is lower with monotherapy, the surge in combining ICIs with chemotherapy, targeted therapy, and combination of different ICIs has led to an increase in the incidence and severity of ILICI - a major challenge for development of effective and safe ICI therapy. In this review, we highlight the importance and contribution of the liver microenvironment to ILICI by focusing on the emerging roles of resident liver cells in modulating immune homeostasis and hepatocyte regeneration, two important decisive factors that dictate the initiation, progression, and recovery from ILICI. Based on the proposed contribution of the liver microenvironment on ICILI, we discuss the clinical characteristics of ILICI in patients with preexisting liver diseases, as well as the challenges of identifying prognostic biomarkers to guide the clinical management of severe ILICI. A better understanding of the liver microenvironment may lead to novel strategies and identification of novel biomarkers for effective management of ILICI., (© 2021. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

Aims: Gallbladder carcinomas usually present in advanced stages and has a dismal prognosis despite modern imaging techniques and aggressive surgical intervention. Identification of biologic markers for early diagnosis and improved therapeutic strategies is thus of paramount importance. S100P has been identified in a variety of malignant neoplasms of the gastrointestinal and pancreaticobiliary systems, but it is not yet known if S100P expression is associated with clinically-relevant characteristics of gall bladder carcinoma. The aims of the present study were: 1) to investigate the relationship between S100P expression and histological type, grade, tumor-node-metastasis stage, presence of vascular invasion, perineural invasion and necrosis; and 2) to evaluate for any S100P-defined difference in the risk for tumor recurrence or death., Method: Immunostains for S100P were performed on 4 tissue microarray blocks containing 91 cases of gall bladder carcinoma., Result: The intensity of S100P staining was significantly associated with pathological T stage 4 (p = 0. 0238). Staining intensity ≥3 in ≥25% tumor cells was associated with pathological T stage 4 (p = 0.0005). A higher S100P immunoreactivity score (IRS) was significantly associated with higher TNM stage (p = 0.0341). Age (p = 0.0485), presence of vascular invasion (p = 0.0359), pathological T stage (p = 0.0291) and TNM stage (p = 0.0153) were significantly associated with tumor recurrence. Intense S100P reactivity was associated with decreased overall survival [hazard ratio = 9.614; 95% confidence interval (CI), 1.873-49.338; p = 0.0067]., Conclusion: Our findings indicate that S100P over-expression is a potential prognostic marker for gall bladder carcinoma and is significantly associated with advanced tumor stage and poorer survival., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background/aims: Glycogen synthesis and storage are normal hepatocyte functions. However, glycogenosis, defined as excess hepatocyte glycogen visible by routine H&E light microscopy, has not been well characterized in nonalcoholic fatty liver disease (NAFLD)., Methods: Glycogenosis in NAFLD liver biopsies was graded as "none", "focal" (in <50% of hepatocytes), or "diffuse" (in ≥50% of hepatocytes). Clinical and pathological variables associated with glycogenosis were assessed. 2047 liver biopsies were prospectively analysed., Results: In adults and children, any glycogenosis was present in 54% of cases; diffuse glycogenosis was noted in approximately 1/3 of cases. On multiple logistic regression analysis, adults with glycogenosis tended to be older (P = .003), female (P = .04), have higher serum glucose (P = .01), and use insulin (P = .02). Adults tended to have lower steatosis scores (P = .006) and lower fibrosis stages (P = .005); however, unexpectedly, they also tended to have more hepatocyte injury including ballooning (P = .003). On multiple logistic regression analysis, paediatric patients with glycogenosis were more likely to be Hispanic (P = .03), have lower body weight (P = .002), elevated triglycerides (P = .001), and a higher fasting glucose (P = .007). Paediatric patients with glycogenosis also had less steatosis (P < .001) than those without., Conclusions: Glycogenosis is common in adult and paediatric NAFLD, and is associated with clinical features of insulin resistance. Glycogenosis is important to recognize histologically because it may be misinterpreted as ballooning, and when diffuse, confusion with glycogen storage disorders or glycogenic hepatopathy must be avoided. The newly observed dichotomous relationship between glycogenosis and increased liver cell injury but decreased steatosis and fibrosis requires further study., (© 2020 The Authors. Liver International published by John Wiley & Sons Ltd.)

Our aims were to assess performance of duodenal intraepithelial lymphocyte counting for diagnosis of Helicobacter pylori (H. pylori) gastritis, and effects of eradication therapy on intraepithelial lymphocytosis. Paired duodenal and gastric biopsies from subjects with a pathologic diagnosis of H. pylori gastritis were reviewed. Higher duodenal intraepithelial lymphocyte counts were observed in 40 subjects with H. pylori gastritis (26 ± 5 per villus) than 52 subjects negative for H. pylori (12 ± 2 per villus). After successful eradication therapy, duodenal lymphocytes were indistinguishable from H. pylori-negative subjects, whereas they remained elevated after failed eradication therapy. This study confirms previous reports of increased duodenal intraepithelial lymphocytes in patients with concurrent Helicobacter pylori gastritis. Intraepithelial lymphocyte counts of > 15 per villus or > 10 per 100 enterocytes were predictive of infection. Duodenal lymphocytosis decreases significantly after successful eradication therapy but remains elevated when treatment fails.

Hepatocellular neoplasms can develop in multiple genetic metabolic disorders. While there have been rare case reports, clinical and pathological characterizations have not been systematically performed. We conducted a retrospective study in 9 patients with these rare genetic metabolic disorders, including glycogen storage disease type 1, ornithine carbamyl transferase deficiency, hereditary tyrosinemia type 1, and Navajo neurohepatopathy, who developed hepatocellular neoplasms. Our results show that steatosis is a common finding in both tumor (6/9 cases, 67%) and background liver parenchyma (8/9 cases, 89%), underlying a possible role for steatosis in tumorigenesis in these genetic metabolic disorders. Our findings also raise a consideration of underlying genetic metabolic disorder when young patients with hepatocellular neoplasm show steatosis in both the tumor and background liver., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in North America and Europe, with increasing prevalence in other regions of the world. Its spectrum encompass steatosis, non-alcoholic steatohepatitis (NASH), fibrosis and cirrhosis. It is considered as the manifestation of metabolic syndrome in liver, and its development and progression is influenced by complex interaction of environmental and genetic factors. In this review we discuss the histopathological features, differential diagnoses, and the commonly used grading and staging systems of NAFLD. NAFLD associated with other diseases, histological changes after therapeutic intervention and recurrence or occurrence of NAFLD after liver transplantation are also addressed., Competing Interests: Declaration of Competing Interest The authors have no conflicts of interest relevant to this article., (Copyright © 2020. Published by Elsevier B.V.)

Intraductal biopsy is commonly used for preoperative evaluation of the etiology of biliary strictures. Interpretation of intraductal biopsies is frequently challenging. The diagnosis often suffers from interobserver disagreement, which has not been studied in the literature. We sought to assess interobserver concordance in the interpretation of intraductal biopsies. Eighty-five biopsies were retrieved, falling into five diagnostic categories: negative for dysplasia (NED), indefinite for dysplasia (IND), low-grade dysplasia (LGD), high-grade dysplasia (HGD), and carcinoma (CA). Eight gastrointestinal pathologists blindly reviewed all the slides. Agreement among pathologists was analyzed using Fleiss κ and weighted concordance coefficient S∗. A face-to-face consensus/training session was held to discuss the classification criteria, followed by a second round review. The overall interobserver agreement was fair in the first round review (κ = 0.39; S∗ = 0.56) and improved to moderate in the second round review (κ = 0.48; S∗ = 0.69). The agreement before and after consensus meeting was substantial to nearly perfect for CA (κ = 0.65, S∗ = 0.83; and κ = 0.80, S∗ = 0.91), fair for HGD (κ = 0.28, S∗ = 0.69; and κ = 0.40, S∗ = 0.63), and moderate for NED (κ = 0.47, S∗ = 0.50; and κ = 0.47, S∗ = 0.53). Agreement improved from fair to moderate for LGD (κ = 0.36, S∗ = 0.61; and κ = 0.49, S∗ = 0.71) and slight to fair for IND (κ = 0.16, S∗ = 0.51; and κ = 0.33, S∗ = 0.50). Compared with Hollande's fixed specimens, the agreement was higher in almost all diagnostic categories in formalin-fixed biopsies. Overall, interobserver concordance was improved after a consensus/training session. Interobserver reproducibility was high at the end of the diagnostic spectrum (CA) but fair to moderate for other diagnostic categories., (Copyright © 2020. Published by Elsevier Inc.)

Objectives: Our aim was to investigate the frequency of steatohepatitic morphology in hepatocellular adenoma (HCA) and correlate with its clinical parameters and risk factors underlying nonalcoholic fatty liver disease (NAFLD) or nonalcoholic steatohepatitis (NASH)., Methods: We examined a series of 41 liver resection specimens diagnosed with HCA for steatohepatitic changes. Background nonneoplastic liver was also evaluated. Clinical records were reviewed for risk factors of NAFLD/NASH., Results: Six steatohepatitic HCAs (SH-HCAs) were identified, with an overall prevalence of six (14.6%) of 41, of which three were HNF1α inactivated and three were inflammatory, but none were β-catenin mutated. Five of the six patients with SH-HCA had at least one known risk factor for NAFLD/NASH, including obesity (n = 4; 66.7%), diabetes (n = 5; 83.3%), hypertension (n = 3; 50%), and dyslipidemia (n = 1; 16.7%). Compared with the patients without SH-HCA, the patients with SH-HCA had a higher frequency of type 2 diabetes, obesity, and hypertension. Of the six SH-HCAs, background nonneoplastic liver showed significant steatosis in three (50%) cases and steatohepatitic changes in one (16.7%) case., Conclusions: Approximately 15% of HCAs in our series demonstrated steatohepatitic changes. Lack of such morphology in β-catenin-mutated subtype suggests reassurance in this morphologic variant of HCA., (© American Society for Clinical Pathology, 2020. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Objectives: To assess the concordance and performance characteristics of Helicobacter pylori laboratory tests compared with histopathology and to propose algorithms for the diagnosis of H pylori that minimize diagnostic error., Methods: H pylori diagnostics were reviewed from a 12-year period within a health system (2,560 cases). Analyses were performed to adjust diagnostic performance based on treatment and consensus histopathologic diagnoses among pathologists. Markers of access to care, including test cancellation frequency and turnaround time, were assessed. Costs and performance of candidate noninvasive testing algorithms were modeled as a function of disease prevalence., Results: Serum H pylori IgG demonstrated a higher sensitivity (0.94) than urea breath and stool antigen tests (0.64 and 0.61, respectively). Evidence of an advantage in access to care for serology included a lower cancellation rate. Interobserver variability was higher (κ = 0.34) among pathologists for cases with a discordant laboratory test than concordant cases (κ = 0.56). A model testing algorithm utilizing serology for first-time diagnoses minimizes diagnostic error., Conclusions: Although H pylori serology has modestly lower specificity than other noninvasive tests, the superior sensitivity and negative predictive value in our population support its use as a noninvasive test to rule out H pylori infection. Reflexive testing with positive serology followed by either stool antigen or urea breath test may optimize diagnostic accuracy in low-prevalence populations., (© American Society for Clinical Pathology, 2020. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background & Aims: Obesity and type 2 diabetes increase hepatocellular carcinoma (HCC) incidence in humans and accelerate diethylnitrosamine (DEN)-induced hepatocarcinogenesis in mice. We investigated whether exercise reduces HCC development in obese/diabetic Alms1 mutant (foz/foz) mice and studied protective mechanisms., Methods: We measured HCC development in DEN-injected male foz/foz and wild-type (WT) littermates housed with or without an exercise wheel from week 4 until 12 or 24 weeks, and in foz/foz mice pair-fed to WT littermates. We also studied HCC development in DEN-injected Jnk1 -/- .foz/foz mice generated by cross breeding, as well as their genetic controls. Dysplastic hepatocytes were identified by glutathione-S-transferase pi form (GST-pi) immunohistochemistry, liver nodules were counted, and HCC was analysed by histopathology., Results: Exercising foz/foz mice maintained similar weight as WT mice up to 10 weeks, but then gained weight and were obese by 24 weeks; a similar body weight profile was obtained by pair-feeding foz/foz mice to WT. At 12 weeks, livers of exercising foz/foz mice exhibited fewer GST-pi positive hepatocytes than sedentary counterparts; by 24 weeks, fewer exercising foz/foz mice developed HCC (15% vs. 64%, p <0.05). Conversely, pair-feeding foz/foz mice failed to reduce HCC incidence. In these insulin-resistant foz/foz mice, exercise failed to activate hepatic AMPK or Akt/mTORC1. Instead, it improved insulin sensitivity, ameliorated steatosis and liver injury, activated p53 to increase p27 expression, and prevented JNK activation. This was associated with suppression of hepatocellular proliferation. DEN-injected Jnk1 -/- .foz/foz mice failed to develop liver tumours or HCC at 24 weeks., Conclusions: Direct effects of exercise dampen proliferation of dysplastic hepatocytes to reduce 3-month dysplastic foci and 6-month incidence of DEN-induced HCC in obese, insulin-resistant mice. The effects of exercise that potentially slow hepatocarcinogenesis include p53-mediated induction of p27 and prevention of JNK activation., Lay Summary: Fatty liver disease commonly occurs alongside obesity and diabetes, contributing to rapidly increasing rates of liver cancer throughout the world. Herein, we show that exercise reduces the incidence and progression of hepatocellular carcinoma in mouse models. The effect of exercise on cancer risk was shown to be independent of changes in weight. Exercise could be a protective mechanism against liver cancer in at-risk individuals., Competing Interests: Conflict of interests The authors have no conflict of interest to disclose. Please refer to the accompanying ICMJE disclosure forms for further details., (Copyright © 2020 European Association for the Study of the Liver. All rights reserved.)

Combined hepatocellular-cholangiocarcinoma (cHCC-CCA) is a primary liver tumor with neoplastic components of both hepatocytic and cholangiocytic differentiation. This unique neoplasm is gaining increasing recognition due to the intriguing pathology, tumor biology, and clinical behavior. It also poses challenges in diagnosis, treatment, and research, largely because of its histological and phenotypic diversity that lead to confusion in terminology and classification. There have been efforts attempting to unify the terminology of this neoplasm recently. Advances in investigation in various aspects have also been made. This review aims to update the terminology, classification, and clinical and pathological characteristics of cHCC-CCA., Competing Interests: Both authors have no conflicts of interest to declare., (Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.)

Until recently, 10% of hepatocellular adenomas (HCAs) remained unclassified (UHCA). Among the UHCAs, the sonic hedgehog HCA (shHCA) was defined by focal deletions that fuse the promoter of Inhibin beta E chain with GLI1. Prostaglandin D2 synthase was proposed as immunomarker. In parallel, our previous work using proteomic analysis showed that most UHCAs constitute a homogeneous subtype associated with overexpression of argininosuccinate synthase (ASS1). To clarify the use of ASS1 in the HCA classification and avoid misinterpretations of the immunohistochemical staining, the aims of this work were to study (1) the link between shHCA and ASS1 overexpression and (2) the clinical relevance of ASS1 overexpression for diagnosis. Molecular, proteomic, and immunohistochemical analyses were performed in UHCA cases of the Bordeaux series. The clinico-pathological features, including ASS1 immunohistochemical labeling, were analyzed on a large international series of 67 cases. ASS1 overexpression and the shHCA subgroup were superimposed in 15 cases studied by molecular analysis, establishing ASS1 overexpression as a hallmark of shHCA. Moreover, the ASS1 immunomarker was better than prostaglandin D2 synthase and only found positive in 7 of 22 shHCAs. Of the 67 UHCA cases, 58 (85.3%) overexpressed ASS1, four cases were ASS1 negative, and in five cases ASS1 was noncontributory. Proteomic analysis performed in the case of doubtful interpretation of ASS1 overexpression, especially on biopsies, can be a support to interpret such cases. ASS1 overexpression is a specific hallmark of shHCA known to be at high risk of bleeding. Therefore, ASS1 is an additional tool for HCA classification and clinical diagnosis., (© 2020 The Authors. Hepatology Communications published by Wiley Periodicals, Inc., on behalf of the American Association for the Study of Liver Diseases.)

Aims: Immune check-point inhibitors are known to cause immune-mediated adverse liver injury, but our knowledge is mainly based on cases treated with ipilimumab or nivolumab., Methods and Results: Clinicopathological features of 10 patients with hepatobiliary adverse reactions caused by second-generation drugs, pembrolizumab (n = 6) and atezolizumab (n = 4), were reviewed. Liver dysfunction developed during a median period of 3.5 weeks after administration of the check-point inhibitor (3 days-1 year). Antinuclear antibodies were detected in two patients at a low titre (1/80), and serum IgG concentrations were also only mildly elevated in two patients. Liver biopsies showed panlobular hepatitis (n = 5), cholangiopathic changes (n = 2), granulomatous injury (n = 2) and bland cholestasis (n = 1). Two cases of cholangiopathy (both pembrolizumab-treated) showed diffuse sclerosing cholangitis on imaging, and one also presented lymphocytic cholangitis resembling primary biliary cholangitis on biopsy. In two atezolizumab-treated cases, Küpffer cells were hyperplastic and aggregated, forming microgranulomas. Confluent necrosis and eosinophilic or plasma cell infiltration were rare. On immunostaining, the ratio of CD8 + /CD4 + cells was 12.2 ± 5.1, which was significantly higher than that in autoimmune hepatitis (2.7 ± 1.1; P < 0.001) or idiosyncratic drug-induced liver injury (5.0 ± 1.1; P = 0.014). All patients responded to steroid therapy, but it was less effective in patients with sclerosing cholangitis., Conclusions: Pembrolizumab and atezolizumab manifested not only lobular hepatitis but also sclerosing cholangitis, lymphocytic duct injury and granulomatous hepatitis, probably representing various impaired cellular functions in CD8 + lymphocytes and macrophages due to blockage of PD-1-PD-L1 interaction., (© 2019 John Wiley & Sons Ltd.)

Pancreatic neuroendocrine tumors (PanNETs) are rare, and prediction of aggressive characteristics, such as recurrence and metastasis and prognosis of PanNETs remain difficult. Nectins are cell adhesion molecules that regulate the formation of adherens and tight junctions. In this study, we investigated the clinicopathological significance of nectin-3 expression in patients with PanNETs. Immunohistochemical analysis of nectin-3 expression was performed on 78 cases of PanNET. Low nectin-3 expression in the membrane (positive ratio ≤25%) was observed in 62 cases (79.5%) and was significantly correlated with larger tumor size (>20 mm; P = 0.003), G2/G3 tumors (P = 0.025), higher Ki67 labeling index (≥3%; P = 0.009), lymphatic involvement (P = 0.047), advanced pT-factor (T2-T4; P = 0.003), lymph node metastasis (P = 0.006), advanced Union for International Cancer Control/American Joint Committee on Cancer-stage (Stage II-IV; P = 0.001), advanced ENETS stage (Stage IIa-IV; P = 0.001), nonfunctioning tumors (P = 0.002), and a shorter disease-free survival (P = 0.019). However, there was no significant correlation between nectin-3 expression in the membrane and/or cytoplasm and the clinicopathological parameters. The present results suggest that decreased nectin-3 expression in the membrane is associated with increased tumor aggressiveness of PanNETs. Clinically, immunohistochemical analysis of nectin-3 may help predict tumor aggressiveness for PanNETs., (© 2019 Japanese Society of Pathology and John Wiley & Sons Australia, Ltd.)

Background and Aims: Gastric intestinal metaplasia (GIM) is an important precursor lesion to gastric cancer (GC), the second leading cause of cancer death worldwide. There exist few data regarding the prevalence of, risk factors for, and clinical practice patterns regarding GIM in the United States. Furthermore, there are currently no U.S. guidelines regarding screening/surveillance for GIM., Methods: All consecutive upper endoscopic procedures from 2 academic medical centers in Seattle between 1999 and 2014 were reviewed. Demographic, clinical, and endoscopic covariates were recorded at time of endoscopy. Procedures with gastric biopsy were matched to final the histologic diagnoses, including the presence of Helicobacter pylori. Cases of GIM and dysplasia were recorded and compared with non-GIM controls using univariate and multivariable regression. Surveillance patterns for cases of GIM were recorded., Results: Data from 36,799 upper endoscopies, 17,710 gastric biopsies, 2073 cases of GIM, 43 cases of dysplasia, and 78 cases of GC were captured. The point prevalence of GIM was 11.7% in patients who underwent gastric biopsy. Non-white race (P < .001), increasing age (P < .001), and presence of H pylori (P < .001) were associated with GIM. If GIM was present, increasing age (P < .001) and male gender (P < .001) were associated with progression, and the presence of H pylori (P < .001) was inversely associated with progression to dysplasia/GC. Few cases of GIM/dysplasia/GC were identified during procedures for GIM screening/surveillance. Only 16% of patients with a diagnosis of GIM received a recommendation for surveillance., Conclusions: There is a high prevalence of GIM among non-white and Hispanic Americans. Risk factors for development of GIM may be distinct from the risk factors for progression to GC., (Copyright © 2020 American Society for Gastrointestinal Endoscopy. Published by Elsevier Inc. All rights reserved.)

Gastric intestinal metaplasia (GIM) is a known premalignant condition of the human stomach along the pathway to gastric cancer (GC). Histologically, GIM represents the replacement of normal gastric mucosa by mucin-secreting intestinal mucosa. Helicobacter pylori infection is the most common etiologic agent of GIM development worldwide. The prevalence of GIM is heterogeneous among different regions of the world and correlates with the population endemicity of H. pylori carriage, among other environmental factors. GC remains the third leading cause of cancer-related mortality globally. GIM is usually diagnosed by upper endoscopy with biopsy, and histologic scoring systems have been developed to risk-stratify patients at highest risk for progression to GC. Several recent endoscopic imaging modalities may improve the optical detection of GIM and early GC. Appropriate surveillance of GIM may be cost effective and represents an opportunity for the early diagnosis and therapy of GC. Certain East Asian nations have established population-level programs for the screening and surveillance of GIM; guidelines regarding GIM surveillance have also recently been published in Europe. By contrast, few data exist regarding the appropriateness of surveillance of GIM in the United States. In this review, we discuss the pathogenesis, epidemiology, diagnosis, and management of GIM with an emphasis on the role of appropriate endoscopic surveillance.

Liver injury triggered by immune checkpoint inhibitors has been increasingly seen in clinical practice, and the incidence is likely to rise further in the next several years because of expanded indications for cancer immunotherapy. Tissue damage driven by disrupted immune tolerance against self-antigens is called an immune-related adverse event (irAE). irAEs in the liver histologically presents panlobular hepatitis (∼70%), isolated central zonal necrosis (∼20%), primarily granulomatous hepatitis (∼5%), and other minor forms of tissue injury (∼5%). Infiltrating cells are mainly lymphocytes and occasional eosinophils. Unlike classic autoimmune hepatitis (AIH), plasma cell infiltration is not conspicuous. Immunostaining reveals a large number of CD8+ T lymphocytes and a markedly smaller number of CD4+ cells or CD20+ B lymphocytes. The unique CD3+/CD20+ and CD4+/CD8+ ratios shifted in favor of CD8+ cytotoxic T lymphocytes are helpful to discriminate irAEs from other conditions (e.g., AIH, idiosyncratic drug-induced liver injury). Another hepatobiliary manifestation of irAEs is sclerosing cholangitis clinically characterized by elevations of biliary enzymes, diffuse duct wall thickening, and duct dilatation. Lymphocytic infiltration can be observed by endoscopic biopsies from the thick extrahepatic bile ducts, and liver needle biopsies may also show severe lymphocytic cholangitis resembling primary biliary cholangitis. An important differential diagnosis of irAEs is previously asymptomatic or subclinical liver disease unmasked by cancer immunotherapy, which is often challenging and requires close clinicopathological correlations., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Primary liver cancer ranks among the leading causes of cancer death worldwide. Risk factors are closely linked to inflammation, such as viral hepatitis and alcoholic as well as non-alcoholic steatohepatitis. Among the pathways involved in the pathogenesis of malignant liver tumors, dysregulation of NF-κB signaling plays a prominent role. It provides a link between inflammation and cancer. To examine the role of NF-κB in a MYC-induced model of hepatocellular carcinoma we deleted NEMO (IKKγ) specifically from hepatocytes. NEMO deletion accelerated tumor development and shortened survival, suggesting a tumor-suppressive function of NF-κB signaling. We observed increased proliferation, inflammation and fibrosis, as well as activation of MAPK and STAT signaling. Importantly, deletion of NEMO modified the tumor phenotype from hepatocellular carcinoma to combined hepatocellular cholangiocarcinoma. The intrahepatic cholangiocarcinoma tumor component showed increased expression of progenitor markers such as Sox9 and reduced expression of mature hepatic markers such as CPS1. In both cases tumorigenesis was reversible by turning off MYC expression. To our knowledge this is the first mouse model of combined hepatocellular cholangiocarcinoma and may provide insights into the development of this rare malignant tumor., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Hepatocyte nuclear factor 4-alpha (HNF4α) is a highly conserved member of nuclear receptor superfamily of ligand-dependent transcription factors that is expressed in liver and gastrointestinal organs (pancreas, stomach, and intestine). In liver, HNF4α is best known for its role as a master regulator of liver-specific gene expression and essential for adult and fetal liver function. Dysregulation of HNF4α expression has been associated with many human diseases such as ulcerative colitis, colon cancer, maturity-onset diabetes of the young, liver cirrhosis, and hepatocellular carcinoma. However, the precise role of HNF4α in the etiology of these human pathogenesis is not well understood. Limited information is known about the role of HNF4α isoforms in liver and gastrointestinal disease progression. There is, therefore, a critical need to know how disruption of the expression of these isoforms may impact on disease progression and phenotypes. In this review, we will update our current understanding on the role of HNF4α in human liver and gastrointestinal diseases. We further provide additional information on possible use of HNF4α as a target for potential therapeutic approaches., Competing Interests: Conflict-of-interest statement: No potential conflicts of interest. No financial support.

Appendix pathology represents uncommonly encountered specimens with unique diagnostic challenges. To delineate common knowledge gaps, extramural consults submitted to seven institutions between 2016-2017 were reviewed. All appendix consults were resections (100%, n = 43), and the majority were directed for consultation by the originating pathologist (95%, n = 41) with no additional studies performed by the consultant (65%, n = 28). This study was dominated by inquiries related to low grade appendiceal mucinous neoplasms (44%, n = 19) and goblet cell carcinoid related neoplasms (19%, n = 8). Of the 43 appendiceal consults, 19 were submitted by the contributing pathologist as low grade appendiceal mucinous neoplasm, but only half of these were diagnosed by the consultant as such (n = 9). Low grade appendiceal mucinous neoplasm-related consultation themes included diverticular disease, criteria for invasion, high grade atypia, extra-appendiceal mucin, and staging. Examples of major disagreements that were downgraded included consults submitted as low grade appendiceal mucinous neoplasm and diagnosed by the consultant as serrated polyp (n = 3), appendicitis (n = 1), and benign appendix (n = 1). Examples of major disagreements-upgraded included cases submitted as low grade appendiceal mucinous neoplasm and diagnosed by the consultant as low grade appendiceal mucinous neoplasm with high-risk features (n = 2) and mucinous adenocarcinoma (n = 2). One case contained both a major disagreement-upgrade (low grade appendiceal mucinous neoplasm changed to high grade appendiceal mucinous neoplasm) and a major disagreement-downgrade (pT3 changed to Tis). Of the 15 cases diagnosed by the consultants as low grade appendiceal mucinous neoplasm, submitted diagnoses included low grade appendiceal mucinous neoplasm (n = 9), adenocarcinoma (n = 5), and one case was submitted without a diagnosis. For goblet cell carcinoid-related consults, the usual inquiry related to distinguishing goblet cell carcinoid from goblet cell carcinoid with adenocarcinoma (adenocarcinoma ex-goblet cell carcinoid). Of the 38 overall consults with a submitted diagnosis, 53% (n = 20) were disagreements, and most of these were major disagreements-downgraded (n = 13).

Congestive hepatopathy is a complication of right heart failure and chronically elevated right heart pressure. Histologic findings include sinusoidal dilatation, centrilobular hepatocellular plate atrophy, and fibrosis. We performed a validation study of a recently proposed scoring system (0 to 4 scale) for congestive hepatic fibrosis on 38 liver biopsies. Glutamine synthetase immunohistochemistry was also performed, and loss of centrizonal immunoreactivity correlated with increasing fibrosis score (P<0.01). Interobserver concordance for congestive hepatic fibrosis score based on Masson trichrome stain was initially fair (Fleiss κ=0.28, weighted concordance coefficient=0.60) and significantly improved (κ=0.40, weighted concordance coefficient=0.66) following a multiheaded microscope training session and inclusion of glutamine synthetase immunohistochemistry. Average congestive hepatic fibrosis score correlated with significantly higher right atrial pressure, severity of right atrial dilation, presence of right ventricular dilation, elevated serum alanine aminotransferase, platelet counts, prothrombin time, and model for end-stage liver disease score. In conclusion, the congestive hepatic fibrosis scoring system is reproducible among pathologists and correlates with clinical and laboratory markers of congestive hepatopathy.

Nonalcoholic steatohepatitis (NASH) arises from a variable interplay between environmental factors and genetic determinants that cannot be completely replicated in animals. Notwithstanding, preclinical models are needed to understand NASH pathophysiology and test mechanism-based therapies. Among several mouse models of NASH, some exhibit the key pathophysiologic as well as histopathologic criteria for human NASH, whereas others may be useful to address specific questions. Models based on overnutrition with adipose restriction/inflammation and metabolic complications, particularly insulin resistance, may be most useful to investigate critical etiopathogenic factors. In-depth pathologic description is required for all models. Some models demonstrate hepatocyte ballooning, which can be confused with microvesicular steatosis, whereas demonstration of an inflammatory infiltrate and pattern of liver fibrosis compatible with human NASH is desirable in models used for pharmacologic testing. When mice with specific genetic strains or mutations that cause overeating consume a diet enriched with fat, modest amounts of cholesterol, and/or simple sugars ("Western diet"), they readily develop obesity with liver disease similar to human NASH, including significant fibrosis. Purely dietary models, such as high-fat/high-cholesterol, Western diet, and choline-deficient, amino acid-defined, are similarly promising. We share concern about using models without weight gain, adipose pathology, or insulin resistance/hyperinsulinemia and with inadequate documentation of liver pathology. NASH-related fibrosis is a key endpoint in trials of possible therapies. When studied for this purpose, NASH models should be reproducible and show steatohepatitis (ideally with ballooning) and at least focal bridging fibrosis, while metabolic factors/disordered lipid partitioning should contribute to etiopathogenesis. Because murine models are increasingly used to explore pharmacologic therapies for NASH, we propose a minimum set of requirements that investigators, drug companies, and journals should consider to optimize their translational value., (© 2018 by the American Association for the Study of Liver Diseases.)

We have previously demonstrated that iron overload in hepatic reticuloendothelial system cells (RES) is associated with severe nonalcoholic steatohepatitis (NASH) and advanced fibrosis in patients with nonalcoholic fatty liver disease (NAFLD). Recruited myeloid-derived macrophages have gained a pivotal position as drivers of NASH progression and fibrosis. In this study, we used bone marrow-derived macrophages (BMDM) from C57Bl6 mice as surrogates for recruited macrophages and examined the effect of iron on macrophage polarization. Treatment with iron (ferric ammonium citrate, FAC) led to increased expression levels of M1 markers: CCL2, CD14, iNOS, IL-1β, IL-6, and TNF-α; it also increased protein levels of CD68, TNF-α, IL-1β, and IL-6 by flow cytometry. This effect could be reversed by desferrioxamine, an iron chelator. Furthermore, iron loading of macrophages in the presence of IL-4 led to the down-regulation of M2 markers: arginase-1, Mgl-1, and M2-specific transcriptional regulator, KLF4. Iron loading of macrophages with IL-4 also resulted in reduced phosphorylation of STAT6, another transcriptional regulator of M2 activation. Dietary iron overload of C57Bl6 mice led to hepatic macrophage M1 activation. Iron overload also stimulated hepatic fibrogenesis. Histologic analysis revealed that iron overload resulted in steatohepatitis. Furthermore, NAFLD patients with hepatic RES iron deposition had increased hepatic gene expression levels of M1 markers, IL-6, IL-1β, and CD40 and reduced gene expression of an M2 marker, TGM2, relative to patients with hepatocellular iron deposition pattern. We conclude that iron disrupts the balance between M1/M2 macrophage polarization and leads to macrophage-driven inflammation and fibrogenesis in NAFLD., (©2019 Society for Leukocyte Biology.)