Taofic Mounajjed

Absence of the intestinal microbiota exacerbates hepatobiliary disease in a murine model of primary sclerosing cholangitis

Primary sclerosing cholangitis (PSC) is a chronic, idiopathic, fibroinflammatory cholangiopathy. The role of the microbiota in PSC etiopathogenesis may be fundamentally important, yet remains obscure. We tested the hypothesis that germ‐free (GF) mutltidrug resistance 2 knockout (mdr2−/−) mice develop a distinct PSC phenotype, compared to conventionally housed (CV) mdr2−/− mice. Mdr2−/− mice (n = 12) were rederived as GF by embryo transfer, maintained in isolators, and sacrificed at 60 days in parallel with age‐matched CV mdr2−/− mice. Serum biochemistries, gallbladder bile acids, and liver sections were examined. Histological findings were validated morphometrically, biochemically, and by immunofluorescence microscopy (IFM). Cholangiocyte senescence was assessed by p16INK4a in situ hybridization in liver tissue and by senescence‐associated β‐galactosidase staining in a culture‐based model of insult‐induced senescence. Serum biochemistries, including alkaline phosphatase, aspartate aminotransferase, and bilirubin, were significantly higher in GF mdr2−/− (P < 0.01). Primary bile acids were similar, whereas secondary bile acids were absent, in GF mdr2−/− mice. Fibrosis, ductular reaction, and ductopenia were significantly more severe histopathologically in GF mdr2−/− mice (P < 0.01) and were confirmed by hepatic morphometry, hydroxyproline assay, and IFM. Cholangiocyte senescence was significantly increased in GF mdr2−/− mice and abrogated in vitro by ursodeoxycholic acid (UDCA) treatment. Conclusions: GF mdr2−/− mice exhibit exacerbated biochemical and histological features of PSC and increased cholangiocyte senescence, a characteristic and potential mediator of progressive biliary disease. UDCA, a commensal microbial metabolite, abrogates senescence in vitro. These findings demonstrate the importance of the commensal microbiota and its metabolites in protecting against biliary injury and suggest avenues for future studies of biomarkers and therapeutic interventions in PSC. (Hepatology 2016;63:185–196)

Integrative Genomic Analysis of Cholangiocarcinoma Identifies Distinct IDH-Mutant Molecular Profiles

Summary Cholangiocarcinoma (CCA) is an aggressive malignancy of the bile ducts, with poor prognosis and limited treatment options. Here, we describe the integrated analysis of somatic mutations, RNA expression, copy number, and DNA methylation by The Cancer Genome Atlas of a set of predominantly intrahepatic CCA cases and propose a molecular classification scheme. We identified an IDH mutant-enriched subtype with distinct molecular features including low expression of chromatin modifiers, elevated expression of mitochondrial genes, and increased mitochondrial DNA copy number. Leveraging the multi-platform data, we observed that ARID1A exhibited DNA hypermethylation and decreased expression in the IDH mutant subtype. More broadly, we found that IDH mutations are associated with an expanded histological spectrum of liver tumors with molecular features that stratify with CCA. Our studies reveal insights into the molecular pathogenesis and heterogeneity of cholangiocarcinoma and provide classification information of potential therapeutic significance.

Glypican-3 expression in gastrointestinal and pancreatic epithelial neoplasms

Glypican-3 (GPC3) is a plasma membrane-bound proteoglycan that can be overexpressed in certain malignancies but has been particularly linked to hepatocellular carcinoma (HCC). GPC3 is currently used as an immunohistochemical marker for HCC, but its expression in epithelial neoplasms of the gastrointestinal (GI) tract and pancreas, a common source of liver metastasis, has not been studied in detail. In this study, we examined GPC3 immunoreactivity in 98 neoplasms of the GI tract including 30 adenocarcinomas (ADCA), 29 squamous cell carcinomas (esophageal and anal), and 39 neuroendocrine carcinomas, and 60 neoplasms of the pancreas including 22 ADCA, 26 pancreatic neuroendocrine neoplasms, and 12 pancreatic acinar cell carcinomas. Two control groups of 32 HCCs and 16 intrahepatic cholangiocarcinomas were also stained with GPC3. Although most (7/12, 58.5%) acinar cell carcinomas were GPC3 positive, pancreatic ADCA and neuroendocrine neoplasms were GPC3 negative. In addition, 27.5%, (8/29) of squamous cell carcinomas, 20% (6/30) of ADCA, and 2.5% (1/39) of neuroendocrine carcinomas of the GI tract were immunoreactive for GPC3. HCC was positive for GPC3 in 75% (24/32) of cases but cholangiocarcinoma was negative. While significant correlation between GPC3 positivity and poor differentiation was observed in HCC only, GPC3 expression did not correlate with tumor size. In conclusion, 14% of GI tract and pancreatic carcinomas/neoplasms (particularly pancreatic acinar cell carcinoma) can express GPC3 by immunohistochemistry. As these tumors commonly metastasize to the liver, this offers a potential pitfall in differentiating between HCC and metastatic carcinoma when evaluating tumors involving the liver.

The Liver in Celiac Disease Clinical Manifestations, Histologic Features, and Response to Gluten-Free Diet in 30 Patients

Descriptive reports of liver histologic features in celiac disease (CD) are sparse, and the effect of a gluten-free diet (GFD) on the course of liver injury is poorly understood. We reviewed liver biopsy specimens in 30 patients with CD and performed immunostains for IgG, IgG4, IgM, and IgA. Subsequent liver biochemical tests and compliance with the GFD were recorded. Of the patients, 19 had autoimmune-mediated liver disease (AILD; autoimmune hepatitis, 9; primary sclerosing cholangitis, 7; and primary biliary cirrhosis, 3). The remaining 11 patients had cryptogenic hepatitis (5), hepatitis C (2), steatohepatitis (2), sarcoidosis (1), and T-cell lymphoma (1). The liver disease diagnosis preceded the CD diagnosis in all groups except steatohepatitis. Although 82% of patients without AILD had symptomatic CD, only 26% of patients with AILD had such symptoms. The pathology of the specific liver disease was not atypical in histologic features or IgG/IgM ratios. While GFD improved cryptogenic hepatitis, it did not seem to affect AILD. We propose that AILD and cryptogenic hepatitis in patients with CD represent distinct clinical, histologic, and immunohistochemical entities rather than 2 ends of a spectrum of liver injury.

Telangiectatic variant of hepatic adenoma: clinicopathologic features and correlation between liver needle biopsy and resection.

Telangiectatic hepatic adenoma (THA) is a benign neoplasm treated by resection. The role of liver needle biopsy in identifying THA before resection has not been evaluated. We identified 55 patients who have undergone resection for hepatic adenoma (HA), THA, or focal nodular hyperplasia (FNH) after needle biopsy. Needle biopsies and resections were evaluated for the following: (1) abortive portal tracts; (2) sinusoidal dilatation; (3) ductular reaction; (4) inflammation; (5) aberrant naked vessels; (6) nodules, fibrous septa, and/or central stellate scar. THA diagnosis was made if the lesion had the first 4 criteria and lacked criterion 6. Most patients (36 of 55), including patients with THA (12 of 16), had multiple lesions (0.2 to 14.4 cm). Patients with THA showed no difference in age, body mass index, prevalence of diabetes or glucose intolerance, or presence of oral contraceptive (OCP) use from patients with HA or FNH, but patients with THA had longer periods of OCP use than patients with HA. Thirty-one percent of THAs had tumor hemorrhage. Of sampled THAs, 27% showed steatosis compared with 76% of sampled HAs (P<0.05). All resected HAs and FNHs were correctly diagnosed on needle biopsy. Of 14 patients with resected THA, 3 histologic patterns were noted on needle biopsy: (1) All THA criteria and naked vessels were present in 6 patients (43%). (2) Consistent with HA: naked vessels only were present in 4 patients (29%). (3) Suggestive of THA: some but not all THA criteria were present in 4 patients (29%). No needle biopsy of a THA was misdiagnosed as FNH. Although evaluation of resection specimens is the gold standard for diagnosis of THA, liver needle biopsy is a useful diagnostic tool that leads to adequate treatment.

Distinguishing between Hepatic Inflammation and Fibrosis with MR Elastography

Purpose To investigate the utility of magnetic resonance (MR) elastography-derived mechanical properties in the discrimination of hepatic inflammation and fibrosis in the early stages of chronic liver diseases. Materials and Methods All studies were approved by the institutional animal care and use committee. A total of 187 animals were studied, including 182 mice and five pigs. These animals represented five different liver diseases with a varying combination and extent of hepatic inflammation, fibrosis, congestion, and portal hypertension. Multifrequency three-dimensional MR elastography was performed, and shear stiffness, storage modulus, shear loss modulus, and damping ratio were calculated for all animals. Necroinflammation, fibrosis, and portal pressure were either histologically scored or biochemically and physically quantified in all animals. Two-sided Welch t tests were used to evaluate mean differences between disease and control groups. Spearman correlation analyses were used to evaluate the relationships between mechanical parameters and quantitative fibrosis extent (hydroxyproline concentration) and portal pressure. Results Liver stiffness and storage modulus increased with progressively developed fibrosis and portal hypertension (mean stiffness at 80 Hz and 48-week feeding, 0.51 kPa ± 0.12 in the steatohepatitis group vs 0.29 kPa ± 0.01 in the control group; P = .02). Damping ratio and shear loss modulus can be used to distinguish inflammation from fibrosis at early stages of disease, even before the development of histologically detectable necroinflammation and fibrosis (mean damping ratio at 80 Hz and 20-week feeding, 0.044 ± 0.012 in the steatohepatitis group vs 0.014 ± 0.008 in the control group; P < .001). Damping ratio and liver stiffness vary differently with respect to cause of portal hypertension (ie, congestion- or cirrhosis-induced hypertension). These differentiation abilities have frequency-dependent variations. Conclusion Liver stiffness and damping ratio measurements can extend hepatic MR elastography to potentially enable assessment of necroinflammatory, congestive, and fibrotic processes of chronic liver diseases.

Model to predict survival after surgical resection of intrahepatic cholangiocarcinoma: the Mayo Clinic experience

BACKGROUND The 7th edition of the American Joint Committee on Cancer (AJCC) staging system has recently been validated and shown to predict survival in patients with intrahepatic cholangiocarcinoma (ICC). The present study attempted to investigate the validity of these findings. METHODS A single-centre, retrospective cohort study was conducted. Histopathological restaging of disease subsequent to primary surgical resection was carried out in all consecutive ICC patients. Overall survival was compared using Kaplan-Meier estimates and log-rank tests. RESULTS A total of 150 patients underwent surgery, 126 (84%) of whom met the present studys inclusion criteria. Of these 126 patients, 68 (54%) were female. The median length of follow-up was 4.5 years. The median patient age was 58 years (range: 24-79 years). Median body mass index was 27 kg/m(2) (range: 17-46 kg/m(2) ). Staging according to the AJCC 7th edition categorized 33 (26%) patients with stage I disease, 27 (21%) with stage II disease, five (4%) with stage III disease, and 61 (48%) with stage IVa disease. The AJCC 7th edition failed to accurately stratify survival in the current cohort; analysis revealed significantly worse survival in those with microvascular invasion, tumour size of >5 cm, grade 4 disease, multiple tumours and positive lymph nodes (P < 0.001). A negative resection margin was associated with improved survival (P < 0.001). CONCLUSIONS The AJCC 7th edition did not accurately predict survival in patients with ICC. A multivariable model including tumour size and differentiation in addition to the criteria used in the AJCC 7th edition may offer a more accurate method of predicting survival in patients with ICC.

Activation of the transforming growth factor-β/SMAD transcriptional pathway underlies a novel tumor-promoting role of sulfatase 1 in hepatocellular carcinoma.

In vitro studies have proposed a tumor suppressor role for sulfatase 1 (SULF1) in hepatocellular carcinoma (HCC); however, high expression in human HCC has been associated with poor prognosis. The reason underlying this paradoxical observation remains to be explored. Using a transgenic (Tg) mouse model overexpressing Sulf1 (Sulf1‐Tg), we assessed the effects of SULF1 on the diethylnitrosamine model of liver carcinogenesis. Sulf1‐Tg mice show a higher incidence of large and multifocal tumors with diethylnitrosamine injection compared to wild‐type mice. Lung metastases were found in 75% of Sulf1‐Tg mice but not in wild‐type mice. Immunohistochemistry, immunoblotting, and reporter assays all show a significant activation of the transforming growth factor‐β (TGF‐β)/SMAD transcriptional pathway by SULF1 both in vitro and in vivo. This effect of SULF1 on the TGF‐β/SMAD pathway is functional; overexpression of SULF1 promotes TGF‐β‐induced gene expression and epithelial–mesenchymal transition and enhances cell migration/invasiveness. Mechanistic analyses demonstrate that inactivating mutation of the catalytic site of SULF1 impairs the above actions of SULF1 and diminishes the release of TGF‐β from the cell surface. We also show that SULF1 expression decreases the interaction between TGF‐β1 and its heparan sulfate proteoglycan sequestration receptor, TGFβR3. Finally, using gene expression from human HCCs, we show that patients with high SULF1 expression have poorer recurrence‐free survival (hazard ratio 4.1, 95% confidence interval 1.9‐8.3; P = 0.002) compared to patients with low SULF1. We also found strong correlations of SULF1 expression with TGF‐β expression and with several TGF‐β‐related epithelial–mesenchymal transition genes in human HCC. Conclusion: Our study proposes a novel role of SULF1 in HCC tumor progression through augmentation of the TGF‐β pathway, thus defining SULF1 as a potential biomarker for tumor progression and a novel target for drug development for HCC. (Hepatology 2015;61:1269–1283)

A longitudinal study of whole body, tissue, and cellular physiology in a mouse model of fibrosing NASH with high fidelity to the human condition

The sequence of events that lead to inflammation and fibrosing nonalcoholic steatohepatitis (NASH) is incompletely understood. Hence, we investigated the chronology of whole body, tissue, and cellular events that occur during the evolution of diet-induced NASH. Male C57Bl/6 mice were assigned to a fast-food (FF; high calorie, high cholesterol, high fructose) or standard-chow (SC) diet over a period of 36 wk. Liver histology, body composition, mitochondrial respiration, metabolic rate, gene expression, and hepatic lipid content were analyzed. Insulin resistance [homeostasis model assessment-insulin resistance (HOMA-IR)] increased 10-fold after 4 wk. Fibrosing NASH was fully established by 16 wk. Total hepatic lipids increased by 4 wk and remained two- to threefold increased throughout. Hepatic triglycerides declined from sixfold increase at 8 wk to threefold increase by 36 wk. In contrast, hepatic cholesterol levels steadily increased from baseline at 8 wk to twofold by 36 wk. The hepatic immune cell population altered over time with macrophages persisting beyond 16 wk. Mitochondrial oxygen flux rates of FF mice diet were uniformly lower with all the tested substrates (13-276 pmol·s-1·ml-1 per unit citrate synthase) than SC mice (17-394 pmol·s-1·ml-1 per unit citrate synthase) and was accompanied by decreased mitochondrial:nuclear gene copy number ratios after 4 wk. Metabolic rate was lower in FF mice. Mitochondrial glutathione was significantly decreased at 24 wk in FF mice. Expression of dismutases and catalase was also decreased in FF mice. The evolution of NASH in the FF diet-induced model is multiphasic, particularly in terms of hepatic lipid composition. Insulin resistance precedes hepatic inflammation and fibrosis. Mitochondrial dysfunction and depletion occur after the histological features of NASH are apparent. Collectively, these observations provide a unique overview of the sequence of changes that coevolve with the histological evolution of NASH.NEW & NOTEWORTHY This study demonstrates in a first of kind longitudinal analysis, the evolution of nonalcoholic steatohepatitis (NASH) on a fast-food diet-induced model. Key findings include 1) hepatic lipid composition changes in a multiphasic fashion as NASH evolves; 2) insulin resistance precedes hepatic inflammation and fibrosis, answering a longstanding chicken-and-egg question regarding the relationship of insulin resistance to liver histology in NASH; and 3) mitochondrial dysfunction and depletion occur after the histological features of NASH are apparent.

Hepatocellular Neoplasms Arising in Association With Androgen Use.

Correlation between androgen use and hepatocellular neoplasia is well established. However, there are no detailed studies of the histopathology and immunohistochemical/molecular profile of these tumors. We studied 9 patients with androgen-associated hepatocellular neoplasms. In addition to histology, immunostains for liver fatty acid–binding protein, &bgr;-catenin, glutamine synthetase, C-reactive protein, and serum amyloid A were utilized for tumor subtyping. Molecular testing using Solid Tumor Targeted Cancer Panel was performed on 3 cases. The neoplasms were predominantly seen in male individuals (7/9). Two patients (22%) had multifocal lesions. All lesions had architectural and 4/9 had cytologic atypia. Cholestasis was present in 6/9 cases. Reticulin was focally disrupted in 5/9 cases. Given the clinical setting, all lesions were classified as well-differentiated hepatocellular neoplasms of uncertain malignant potential. In cases with follow-up (6/9 cases, 67%), there were no recurrences or metastases. On the basis of the immunoprofile, 7 (78%) cases were &bgr;-catenin activated (including 1 hepatic adenoma with concurrent hepatocyte nuclear factor 1&agr; inactivation) and 2 (22%) had inflammatory phenotype. Somatic mutations in CTNNB1 were detected in all 3 tested cases (all &bgr;-catenin activated by immunostain), all involving exon-3. Our data indicate that androgen-associated hepatocellular neoplasms most often develop in male individuals and always show some degree of atypia and/or focal reticulin disruption. Most are &bgr;-catenin activated, often harboring CTNNB1 exon-3 mutations, and a minority is inflammatory type. Although &bgr;-catenin and inflammatory pathways likely play a role in pathogenesis, the heterogenous molecular profile suggests there are other (yet to be characterized) primary oncogenic mechanisms in this unique tumor type.