Liver disease in germline mutations of telomere-related genes: Prevalence, clinical, radiological, pathological features, outcome, and risk factors.

BACKGROUND AND AIM: Germline mutations of telomere-related genes (TRG) induce multiorgan dysfunction, and liver-specific manifestations have not been clearly outlined. We aimed to describe TRG mutations-associated liver diseases. APPROACH AND RESULTS: Retrospective multicenter analysis of liver disease (transaminases > 30 IU/L and/or abnormal liver imaging) in patients with TRG mutations. Main measurements were characteristics, outcomes, and risk factors of liver disease in a TRG mutations cohort. The prevalence of liver disease was compared to a community-based control group (n = 1190) stratified for age and matched 1:3 for known risk factors of liver disease. Among 132 patients with TRG mutations, 95 (72%) had liver disease, with associated lung, blood, skin, rheumatological, and ophthalmological TRG diseases in 82%, 77%, 55%, 39%, and 30% of cases, respectively. Liver biopsy was performed in 52/95 patients, identifying porto-sinusoidal vascular disease in 48% and advanced fibrosis/cirrhosis in 15%. After a follow-up of 21 months (12-54), ascites, hepato-pulmonary syndrome, variceal bleeding, and HCC occurred in 14%, 13%, 13%, and 2% of cases, respectively. Five-year liver transplantation-free survival was 69%. A FIB-4 score ≥ 3·25 and ≥1 risk factor for cirrhosis were associated with poor liver transplantation-free survival. Liver disease was more frequent in patients with TRG mutations than in the paired control group [80/396, (20%)], OR 12.9 (CI 95%: 7.8-21.3, p < 0.001). CONCLUSIONS: TRG mutations significantly increase the risk of developing liver disease. Although symptoms may be mild, they may be associated with severe disease. Porto-sinusoidal vascular disease and cirrhosis were the most frequent lesions, suggesting that the mechanism of action is multifactorial.

Pathologist Computer-Aided Diagnostic Scoring of Tumor Cell Fraction: A Swiss National Study.

Tumor cell fraction (TCF) estimation is a common clinical task with well-established large interobserver variability. It thus provides an ideal test bed to evaluate potential impacts of employing a tumor cell fraction computer-aided diagnostic (TCFCAD) tool to support pathologists' evaluation. During a National Slide Seminar event, pathologists (n = 69) were asked to visually estimate TCF in 10 regions of interest (ROIs) from hematoxylin and eosin colorectal cancer images intentionally curated for diverse tissue compositions, cellularity, and stain intensities. Next, they re-evaluated the same ROIs while being provided a TCFCAD-created overlay highlighting predicted tumor vs nontumor cells, together with the corresponding TCF percentage. Participants also reported confidence levels in their assessments using a 5-tier scale, indicating no confidence to high confidence, respectively. The TCF ground truth (GT) was defined by manual cell-counting by experts. When assisted, interobserver variability significantly decreased, showing estimates converging to the GT. This improvement remained even when TCFCAD predictions deviated slightly from the GT. The standard deviation (SD) of the estimated TCF to the GT across ROIs was 9.9% vs 5.8% with TCFCAD (P < .0001). The intraclass correlation coefficient increased from 0.8 to 0.93 (95% CI, 0.65-0.93 vs 0.86-0.98), and pathologists stated feeling more confident when aided (3.67 ± 0.81 vs 4.17 ± 0.82 with the computer-aided diagnostic [CAD] tool). TCFCAD estimation support demonstrated improved scoring accuracy, interpathologist agreement, and scoring confidence. Interestingly, pathologists also expressed more willingness to use such a CAD tool at the end of the survey, highlighting the importance of training/education to increase adoption of CAD systems.

Liver Stiffness by Transient Elastography to Detect Porto-Sinusoidal Vascular Liver Disease With Portal Hypertension.

BACKGROUND AND AIMS: Porto-sinusoidal vascular liver disease (PSVD) is a rare cause of portal hypertension. PSVD is still often misdiagnosed as cirrhosis, emphasizing the need to improve PSVD diagnosis strategies. Data on liver stiffness measurement using transient elastography (TE-LSM) in PSVD are limited. The aim of this study was to evaluate the accuracy of TE-LSM to discriminate PSVD from cirrhosis in patients with signs of portal hypertension. APPROACH AND RESULTS: Retrospective multicenter study comparing TE-LSM in patients with PSVD, according to Vascular Liver Disease Interest Group criteria, with patients with compensated biopsy-proven cirrhosis associated with alcohol (n = 117), HCV infection (n = 110), or NAFLD (n = 46). All patients had at least one sign of portal hypertension among gastroesophageal varices, splenomegaly, portosystemic collaterals, history of ascites, or platelet count < 150 × 109 /L. The 77 patients with PSVD included in the test cohort had lower median TE-LSM (7.9 kPa) than the patients with alcohol-associated, HCV-related, and NAFLD-related cirrhosis (33.8, 18.2, and 33.6 kPa, respectively; P < 0.001). When compared with cirrhosis, a cutoff value of 10 kPa had a specificity of 97% for the diagnosis of PSVD with a 85% positive predictive value. A cutoff value of 20 kPa had a sensitivity of 94% for ruling out PSVD with a 97% negative predictive value. Of the patients, 94% were well-classified. Even better results were obtained in a validation cohort including 78 patients with PSVD. CONCLUSIONS: This study including a total of 155 patients with PSVD and 273 patients with cirrhosis demonstrates that TE-LSM < 10 kPa strongly suggests PSVD in patients with signs of portal hypertension. Conversely, when TE-LSM is >20 kPa, PSVD is highly unlikely.

The diagnostic performance of a simulated "short" gadoxetic acid-enhanced MRI protocol is similar to that of a conventional protocol for the detection of colorectal liver metastases.

OBJECTIVES: To compare the performance of standard and simulated short gadoxetic acid-enhanced MRI protocols for the detection of colorectal liver metastases (CRLM). METHODS: From 2008 to 2017, 67 patients (44 men (66%); mean age 65 ± 11 years old) who underwent gadoxetic acid-enhanced MRI during the initial work-up for colorectal cancer were included. Exams were independently reviewed by two readers blinded to clinical data in two reading sessions: (1) all acquired sequences (standard "long" protocol) and (2) only T2-weighted, diffusion-weighted, and hepatobiliary phase images (simulated "short" protocol). Readers characterized detected lesions using a 5-point scale (1-certainly benign to 5-certainly malignant). A lesion was considered a CRLM when the score was ≥ 3. The reference standard was histopathology or 12-month imaging follow-up. Chi-square, Student's t, and McNemar tests were used for comparisons. RESULTS: A total of 486 lesions including 331 metastases (68%) were analyzed. The metastasis detection rate was 86.1% (95% CI 82-89.4)-86.7% (82.6-90) and 85.8% (81.6-89.2)-87% (82.9-90.2) with the short and long protocols, respectively (p > 0.99). Among detected lesions, 92.1% (89.1-94.4)-94.8% (92.2-96.6) and 84.6% (80.8-87.7)-88.8% (85.4-91.5) were correctly classified with the short and long protocols, respectively (p = 0.13 and p = 0.10). The results remained unchanged when lesions scored ≥ 4 were considered as CRLM. CONCLUSION: The diagnostic performance of a simulated short gadoxetic enhanced-MR protocol including T2-weighted, diffusion-weighted, and hepatobiliary phase appears similar to that of a standard long protocol including dynamic phase images. Since this protocol shortens the duration of MR examination, it could facilitate the evaluation of patients with colorectal liver metastases. KEY POINTS: • The detection rate of colorectal metastases with a simulated, short, MRI protocol was similar to that of a standard protocol. • The performance of both protocols for the differentiation of metastases and benign lesions appears to be similar. • A short MR imaging protocol could facilitate the evaluation of patients with colorectal liver metastases.

Deficient Natural Killer Cell NKp30-Mediated Function and Altered NCR3 Splice Variants in Hepatocellular Carcinoma.

The activating natural cytotoxicity receptor NKp30 is critical for natural killer (NK) cell function and tumor immune surveillance. The natural cytotoxicity receptor-3 (NCR3) gene is transcribed into several splice variants whose physiological relevance is still incompletely understood. In this study, we investigated the role of NKp30 and its major ligand B7 homolog 6 (B7-H6) in patients with hepatocellular carcinoma (HCC). Peripheral blood NK cell phenotype was skewed toward a defective/exhausted immune profile with decreased frequencies of cells expressing NKp30 and natural killer group 2, member D and an increased proportion of cells expressing T-cell immunoglobulin and mucin-domain containing-3. Moreover, NKp30-positive NK cells had a reduced expression of NCR3 immunostimulatory splice variants and an increased expression of the inhibitory variant in patients with advanced tumor, resulting in deficient NKp30-mediated functionality. Tumor-infiltrating lymphocytes showed a prevalent inhibitory NKp30 isoform profile, consistent with decreased NKp30-mediated function. Of note, there were significant differences in the cytokine milieu between the neoplastic and the surrounding non-neoplastic tissue, which may have further influenced NKp30 function. Exposure of NK cells to B7-H6-expressing HCC cells significantly down-modulated NKp30, that was prevented by small interfering RNA-mediated knockdown, suggesting a role for this ligand in inhibiting NKp30-mediated responses. Interestingly, B7-H6 expression was reduced in HCC tissue and simultaneously augmented as a soluble form in HCC patients, particularly those with advanced staging or larger nodule size. Conclusion: These findings provide evidence in support of a role of NKp30 and its major ligand in HCC development and evolution.

Abdominal Surgery in Patients With Idiopathic Noncirrhotic Portal Hypertension: A Multicenter Retrospective Study.

In patients with idiopathic noncirrhotic portal hypertension (INCPH), data on morbidity and mortality of abdominal surgery are scarce. We retrospectively analyzed the charts of patients with INCPH undergoing abdominal surgery within the Vascular Liver Disease Interest Group network. Forty-four patients with biopsy-proven INCPH were included. Twenty-five (57%) patients had one or more extrahepatic conditions related to INCPH, and 16 (36%) had a history of ascites. Forty-five procedures were performed, including 30 that were minor and 15 major. Nine (20%) patients had one or more Dindo-Clavien grade ≥ 3 complication within 1 month after surgery. Sixteen (33%) patients had one or more portal hypertension-related complication within 3 months after surgery. Extrahepatic conditions related to INCPH (P = 0.03) and history of ascites (P = 0.02) were associated with portal hypertension-related complications within 3 months after surgery. Splenectomy was associated with development of portal vein thrombosis after surgery (P = 0.01). Four (9%) patients died within 6 months after surgery. Six-month cumulative risk of death was higher in patients with serum creatinine ≥ 100 µmol/L at surgery (33% versus 0%, P < 0.001). An unfavorable outcome (i.e., either liver or surgical complication or death) occurred in 22 (50%) patients and was associated with the presence of extrahepatic conditions related to INCPH, history of ascites, and serum creatinine ≥ 100 µmol/L: 5% of the patients with none of these features had an unfavorable outcome versus 32% and 64% when one or two or more features were present, respectively. Portal decompression procedures prior to surgery (n = 10) were not associated with postoperative outcome. Conclusion: Patients with INCPH are at high risk of major surgical and portal hypertension-related complications when they harbor extrahepatic conditions related to INCPH, history of ascites, or increased serum creatinine.

Tolerogenic properties of liver macrophages in non-alcoholic steatohepatitis.

BACKGROUND & AIMS: Our understanding of non-alcoholic fatty liver disease (NAFLD) pathogenesis is improving, but there is still limited data on the function of resident liver macrophages in this context, especially when considering their contribution in dampening liver inflammation. METHODS: Liver macrophages were studied in mouse models of prolonged diet-induced liver steatohepatitis and carbon tetrachloride-induced liver injury. We assessed liver macrophages phenotype and costimulatory/inhibitory properties upon exposure to lipopolysaccharide or interleukin 4. We did phagocytosis and antigen presentation assays to investigate liver macrophages function as scavengers and immune response initiators. Using immunofluorescence staining, we further determined, in human liver tissue of patients with simple steatosis, non-alcoholic steatohepatitis and chronic hepatitis B infection, the expression of the co-inhibitory protein CD274 (Programmed-death ligand 1) and major histocompatibility complex (MHC) class II. RESULTS: Both in humans and mice, within chronically inflamed fatty livers, liver macrophages acquired immunomodulatory properties by reducing the expression of MHC class II, and by enhancing co-inhibitory signalling. Liver macrophages circumscribed endotoxin-mediated inflammatory response by upregulating anti-inflammatory genes arginase 1 and interleukin-10. While hepatic macrophages isolated from mice with normal livers were capable of achieving endotoxin tolerance, our results indicated an impairment of this protective mechanism in the presence NASH-like parenchymal abnormalities. CONCLUSIONS: Liver macrophages can achieve endotoxin tolerance, but in the chronically inflamed fatty liver, while they acquire an immunomodulatory phenotype, liver macrophages fail to dampen immune-mediated damage. Therefore, loss of tolerogenicity induced by ongoing liver insult may be a mechanism contributing to the worsening of NAFLD.

Combination of Gene Expression Signature and Model for End-Stage Liver Disease Score Predicts Survival of Patients With Severe Alcoholic Hepatitis.

BACKGROUND & AIMS: Patients with severe alcoholic hepatitis (AH) have a high risk of death within 90 days. Corticosteroids, which can cause severe adverse events, are the only treatment that increases short-term survival. It is a challenge to predict outcomes of patients with severe AH. Therefore, we developed a scoring system to predict patient survival, integrating baseline molecular and clinical variables. METHODS: We obtained fixed liver biopsy samples from 71 consecutive patients diagnosed with severe AH and treated with corticosteroids from July 2006 through December 2013 in Brussels, Belgium (derivation cohort). Gene expression patterns were analyzed by microarrays and clinical data were collected for 180 days. We identified gene expression signatures and clinical data that are associated with survival without liver transplantation at 90 and 180 days after initiation of corticosteroid therapy. Findings were validated using liver biopsies from 48 consecutive patients with severe AH treated with corticosteroids, collected from March 2010 through February 2015 at hospitals in Belgium and Switzerland (validation cohort 1) and in liver biopsies from 20 patients (9 received corticosteroid treatment), collected from January 2012 through May 2015 in the United States (validation cohort 2). RESULTS: We integrated data on expression patterns of 123 genes and the model for end-stage liver disease (MELD) scores to assign patients to groups with poor survival (29% survived 90 days and 26% survived 180 days) and good survival (76% survived 90 days and 65% survived 180 days) (P < .001) in the derivation cohort. We named this assignment system the gene signature-MELD (gs-MELD) score. In validation cohort 1, the gs-MELD score discriminated patients with poor survival (43% survived 90 days) from those with good survival (96% survived 90 days) (P < .001). The gs-MELD score also discriminated between patients with a poor survival at 180 days (34% survived) and a good survival at 180 days (84% survived) (P < .001). The time-dependent area under the receiver operator characteristic curve for the score was 0.86 (95% confidence interval 0.73-0.99) for survival at 90 days, and 0.83 (95% confidence interval 0.71-0.96) for survival at 180 days. This score outperformed other clinical models to predict survival of patients with severe AH in validation cohort 1. In validation cohort 2, the gs-MELD discriminated patients with a poor survival at 90 days (12% survived) from those with a good survival at 90 days (100%) (P < .001). CONCLUSIONS: We integrated data on baseline liver gene expression pattern and the MELD score to create the gs-MELD scoring system, which identifies patients with severe AH, treated or not with corticosteroids, most and least likely to survive for 90 and 180 days.

Late hepatitis B reactivation following direct-acting antiviral-based treatment of recurrent hepatitis C in an anti-HBc-positive liver transplant recipient.

Direct-acting antivirals (DAAs) have changed the landscape of hepatitis C virus (HCV) treatment, but chronic hepatitis C (CHC) remains a leading indication for liver transplantation (LT). Hepatitis B virus (HBV) reactivation has been reported in HBV-HCV-coinfected patients treated with DAAs. We report on a case of late HBV reactivation after DAA-based treatment of recurrent hepatitis C in an antibody against hepatitis B core antigen (anti-HBc)-positive LT recipient. (Hepatology 2018;67:791-793).

[Abdominal surgery in patients with portal hypertension†: clinical implication and management]. / Chirurgie abdominale chez les malades avec hypertension portale†: implications et prise en charge.

Abdominal surgery is sometimes needed in patients with portal hypertension. The indication may be related to the underlying liver disease, including liver resection for liver cancer and parietal surgery. Surgery may also be performed for another indication, unrelated to the liver disease. Portal hypertension increases both morbidity and mortality after abdominal surgery, and it should be taken into account when planning surgery timing and surgical strategy. This article provides an overview of the main etiologies of portal hypertension, and the post-operative outcomes after liver resection and non-hepatic abdominal surgery, underlining the importance of a multidisciplinary approach.

Une chirurgie abdominale est parfois nécessaire chez les patients atteints d'hypertension portale. Son indication peut être en lien avec une complication de la maladie du foie, comme une résection de tumeur hépatique ou une chirurgie pariétale, ou avec une affection non liée à la pathologie hépatique. L'hypertension portale augmente la morbidité et la mortalité de cette chirurgie, et l'indication ainsi que la stratégie doivent être définies avec soin. Cet article décrit les causes d'hypertension portale, les risques associés à sa présence lors d'une résection hépatique ou d'une chirurgie abdominale non hépatique. Enfin, il propose une approche multidisciplinaire, associant les chirurgiens, les hépatologues, les anatomopathologistes et les anesthésistes-réanimateurs.

Effects of the gut-liver axis on ischaemia-mediated hepatocellular carcinoma recurrence in the mouse liver.

BACKGROUND & AIMS: There is growing evidence that liver graft ischemia-reperfusion (I/R) is a risk factor for hepatocellular carcinoma (HCC) recurrence, but the mechanisms involved are unclear. Herein, we tested the hypothesis that mesenteric congestion resulting from portal blood flow interruption induces endotoxin-mediated Toll-like receptor 4 (Tlr4) engagement, resulting in elevated liver cancer burden. We also assessed the role of remote ischemic preconditioning (RIPC) in this context. METHODS: C57Bl/6j mice were exposed to standardized models of liver I/R injury and RIPC, induced by occluding the hepatic and femoral blood vessels. HCC was induced by injecting RIL-175 cells into the portal vein. We further evaluated the impact of the gut-liver axis (lipopolysaccharide (LPS)-Tlr4 pathway) in this context by studying mice with enhanced (lipopolysaccharide infusion) or defective (Tlr4-/- mice, gut sterilization, and Tlr4 antagonist) Tlr4 responses. RESULTS: Portal triad clamping provoked upstream mesenteric venous engorgement and increased bacterial translocation, resulting in aggravated tumor burden. RIPC prevented this mechanism by preserving intestinal integrity and reducing bacterial translocation, thereby mitigating HCC recurrence. These observations were linked to the LPS-Tlr4 pathway, as supported by the high and low tumor burden displayed by mice with enhanced or defective Tlr4 responses, respectively. CONCLUSIONS: Modulation of the gut-liver axis and the LPS-Tlr4 response by RIPC, gut sterilization, and Tlr4 antagonism represents a potential therapeutic target to prevent I/R lesions, and to alleviate HCC recurrence after liver transplantation and resection. LAY SUMMARY: Cancer recurrence can occur after liver resection or liver transplantation for hepatocellular carcinoma (HCC). This study suggests that intestinal venous congestion, which often occurs during liver surgery, favors the translocation of gut-derived bacterial products in the portal vein, thereby facilitating cancer recurrence by enhancing the signaling of Toll-like receptor 4 in the liver. Using a mouse model of HCC recurrence, we show that strategies that (i) reduce bacterial translocation (by gut decontamination, or by protecting the intestine from venous ischemia damage) or (ii) inhibit Tlr4 signaling in the liver, could reduce cancer recurrence.

Chimeric liver transplantation reveals interspecific graft remodelling.

BACKGROUND & AIMS: A major limitation in the field of liver transplantation is the shortage of transplantable organs. Chimeric animals carrying human tissue have the potential to solve this problem. However, currently available chimeric organs retain a high level of xenogeneic cells, and the transplantation of impure organs needs to be tested. METHODS: We created chimeric livers by injecting Lewis rat hepatocytes into C57Bl/6Fah-/-Rag2-/-Il2rg-/- mice, and further transplanted them into newly weaned Lewis rats (45 ±â€¯3 g) with or without suboptimal immunosuppression (tacrolimus 0.6 mg/kg/day for 56 or 112 days). Control donors included wild-type C57Bl/6 mice (xenogeneic) and Lewis rats (syngeneic). RESULTS: Without immunosuppression, recipients of chimeric livers experienced acute rejection, and died within 8 to 11 days. With immunosuppression, they all survived for >112 days with normal weight gain compared to syngeneic controls, while all xenogeneic controls died within 98 days due to rejection with Banff scores >6 (p = 0.0014). The chimeric grafts underwent post-transplant remodelling, growing by 670% on average. Rat hepatocytes fully replaced mouse hepatocytes starting from day 56 (absence of detectable mouse serum albumin, histological clearance of mouse hepatocytes). In addition, rat albumin levels reached those of syngeneic recipients. Four months after transplantation of chimeric livers, we observed the development of diffuse mature rat bile ducts through transdifferentiation of hepatocytes (up to 72% of cholangiocytes), and patchy areas of portal endothelium originating from the host (seen in one out of five recipients). CONCLUSIONS: Taken together, these data demonstrate the efficacy of transplanting rat-to-mouse chimeric livers into rats, with a high potential for post-transplant recipient-oriented graft remodelling. Validation in a large animal model is still needed. LAY SUMMARY: Chimeric animals are composed of cells from different species. Chimeric animals carrying human tissue have the potential to increase the availability of transplantable organs. We transplanted rat-to-mouse liver grafts into newly weaned rats. The chimeric grafts underwent post-transplant remodelling with rat hepatocytes replacing all mouse hepatocytes within 56 days. In addition, we observed the post-transplant development of diffuse mature rat bile ducts through the transformation of hepatocytes, and patchy areas of portal endothelium originating from the host. These data demonstrate the efficacy of transplanting rat-to-mouse chimeric livers into rats, with a high potential for post-transplant graft remodelling.

Ethylene vinyl alcohol copolymer for occlusion of specific portal branches during preoperative portal vein embolisation with n-butyl-cyanoacrylate.

OBJECTIVES: To evaluate the safety and efficacy of ethylene vinyl alcohol copolymer (EVOH) injection for selective occlusion of portal branches considered at risk for non-target embolisation during preoperative portal vein embolisation (PVE). METHODS: Twenty-nine patients (mean age, 57 ± 17 years) submitted to PVE with n-butyl-cyanoacrylate (NBCA) and additional EVOH for selected portal branches were retrospectively analysed. Indications for the use of EVOH and the selected portal branches were evaluated. Degree of hypertrophy of the future liver remnant (FLR) and kinetic growth were assessed by CT volumetry performed before and 3-6 weeks after PVE. Clinical outcome and histopathological analysis of portal veins occluded with EVOH were reviewed. RESULTS: EVOH was indicated intraoperatively for embolisation of selected portal branches that the operator reported at risk to provoke non-target embolisation with NBCA. Indications for the use of EVOH were embolisation of segment IV (n = 21), embolisation of segmental portal branches with early bifurcation (n = 7) and PVE in a 1-year-old girl with cystic hamartomas. All targeted portal branches were successfully embolised. There were no cases with non-target embolisation by EVOH. The degree of hypertrophy of the FLR was 14.3 ± 8.1% and the kinetic growth rate was 2.7 ± 1.8% per week. CONCLUSION: EVOH is safe and effective for embolisation of selected portal vein branches considered at risk for non-target embolisation. KEY POINTS: • EVOH is another effective liquid embolic agent for preoperative PVE. • EVOH is relatively simple to handle with a minimal risk of non-target embolisation. • During PVE, some portal branches considered complicated to occlude with NBCA may be efficiently embolised with EVOH.

Reversibility of chemotherapy-related liver injury.

BACKGROUND & AIMS: Chemotherapy-associated liver injury (CALI) increases the risk of liver resection and may prejudice further surgery and chemotherapy. The reversibility of CALI is therefore important; however, no data concerning this are available. This study aimed to retrospectively analyze the reversibility of CALI in patients undergoing liver resection for colorectal metastases. METHODS: All resections of colorectal liver metastases after oxaliplatin and/or irinotecan-based chemotherapy were included. First, liver resections were stratified by time between end of chemotherapy and hepatectomy and several possible cut-off values tested. CALI prevalence in various groups was compared. Second, CALI in the two specimens from each patient who had undergone repeat liver resections without interval chemotherapy were compared. RESULTS: Overall, 524 liver resections in 429 patients were analyzed. The median interval chemotherapy-surgery was 56days (15-1264). CALI prevalence did not differ significantly between groups with a chemotherapy-surgery interval <270days. Grade 2-3 sinusoidal dilatation (SOS, 19.4% vs. 40.0%, p=0.022) and nodular regenerative hyperplasia (NRH, 6.5% vs. 20.1%, p=0.063) occurred less frequently in patients with an interval >270days (n=31); prevalence of steatosis and steatohepatitis was similar in all groups. A chemotherapy-surgery interval >270days was an independent protector against Grade 2-3 SOS (p=0.009). Forty-seven patients had repeat liver resection without interval chemotherapy. CALI differed between surgeries only for a chemotherapy-surgery interval >270days (n=15), Grade 2-3 SOS having regressed in 4/5 patients and NRH in 7/8; whereas steatosis and steatohepatitis had persisted. CONCLUSIONS: CALI persists for a long time after chemotherapy. SOS and NRH regress only after nine months without chemotherapy, whereas steatosis and steatohepatitis persist. LAY SUMMARY: The patients affected by colorectal liver metastases often receive chemotherapy before liver resection, but chemotherapy causes liver injuries that may increase operative risks and reduce tolerance to further chemotherapy. The authors analyzed the reversibility of the liver injuries after the chemotherapy interruption. Liver injuries persist for a long time after chemotherapy. Sinusoidal dilatation and nodular regenerative hyperplasia regress only nine months after the end of chemotherapy, whereas steatosis and steatohepatitis persist even after this long interval.

Inhibiting poly ADP-ribosylation increases fatty acid oxidation and protects against fatty liver disease.

BACKGROUND & AIMS: To date, no pharmacological therapy has been approved for non-alcoholic fatty liver disease (NAFLD). The aim of the present study was to evaluate the therapeutic potential of poly ADP-ribose polymerase (PARP) inhibitors in mouse models of NAFLD. METHODS: As poly ADP-ribosylation (PARylation) of proteins by PARPs consumes nicotinamide adenine dinucleotide (NAD+), we hypothesized that overactivation of PARPs drives NAD+ depletion in NAFLD. Therefore, we assessed the effectiveness of PARP inhibition to replenish NAD+ and activate NAD+-dependent sirtuins, hence improving hepatic fatty acid oxidation. To do this, we examined the preventive and therapeutic benefits of the PARP inhibitor (PARPi), olaparib, in different models of NAFLD. RESULTS: The induction of NAFLD in C57BL/6J mice using a high-fat high-sucrose (HFHS)-diet increased PARylation of proteins by PARPs. As such, increased PARylation was associated with reduced NAD+ levels and mitochondrial function and content, which was concurrent with elevated hepatic lipid content. HFHS diet supplemented with PARPi reversed NAFLD through repletion of NAD+, increasing mitochondrial biogenesis and ß-oxidation in liver. Furthermore, PARPi reduced reactive oxygen species, endoplasmic reticulum stress and fibrosis. The benefits of PARPi treatment were confirmed in mice fed with a methionine- and choline-deficient diet and in mice with lipopolysaccharide-induced hepatitis; PARP activation was attenuated and the development of hepatic injury was delayed in both models. Using Sirt1hep-/- mice, the beneficial effects of a PARPi-supplemented HFHS diet were found to be Sirt1-dependent. CONCLUSIONS: Our study provides a novel and practical pharmacological approach for treating NAFLD, fueling optimism for potential clinical studies. LAY SUMMARY: Non-alcoholic fatty liver disease (NAFLD) is now considered to be the most common liver disease in the Western world and has no approved pharmacological therapy. PARP inhibitors given as a treatment in two different mouse models of NAFLD confer a protection against its development. PARP inhibitors may therefore represent a novel and practical pharmacological approach for treating NAFLD.

Visualization of hepatitis E virus RNA and proteins in the human liver.

BACKGROUND & AIMS: Although hepatitis E constitutes a substantial disease burden worldwide, surprisingly little is known about the localization of hepatitis E virus (HEV) in the human liver. We therefore aimed to visualize HEV RNA and proteins in situ. METHODS: A panel of 12 different antibodies against HEV open reading frame (ORF) 1-3 proteins was evaluated for immunohistochemistry (IHC) and two probes for in situ hybridization (ISH) in formalin-fixed, paraffin-embedded (FFPE) HuH7 cells transfected with HEV ORF1-3 expression vectors. IHC (and partly ISH) were then applied to Hep293TT cells replicating infectious HEV and liver specimens from patients with hepatitis E (n=20) and controls (n=134). RESULTS: Whereas ORF1-3 proteins were all detectable in transfected, HEV protein-expressing cells, only ORF2 and 3 proteins were traceable in cells replicating infectious HEV. Only the ORF2-encoded capsid protein was also unequivocally detectable in liver specimens from patients with hepatitis E. IHC for ORF2 protein revealed a patchy expression in individual or grouped hepatocytes, generally stronger in chronic compared to acute hepatitis. Besides cytoplasmic and canalicular, ORF2 protein also displayed a hitherto unknown nuclear localization. Positivity for ORF2 protein in defined areas correlated with HEV RNA detection by ISH. IHC was specific and comparably sensitive as PCR for HEV RNA. CONCLUSIONS: ORF2 protein can be reliably visualized in the liver of patients with hepatitis E, allowing for sensitive and specific detection of HEV in FFPE samples. Its variable subcellular distribution in individual hepatocytes of the same liver suggests a redistribution of ORF2 protein during infection and interaction with nuclear components. LAY SUMMARY: The open reading frame (ORF) 2 protein can be used to visualize the hepatitis E virus (HEV) in the human liver. This enabled us to discover a hitherto unknown localization of the HEV ORF2 protein in the nucleus of hepatocytes and to develop a test for rapid histopathologic diagnosis of hepatitis E, the most common cause of acute hepatitis worldwide.

Peribiliary Gland Dilatation in Cirrhosis: Relationship with Liver Failure and Stem Cell/Proliferation Markers.

BACKGROUND AND AIMS: Dilated peribiliary glands (PBG) in patients with cirrhosis are often an incidental finding although their significance and physiopathology remain unclear. We aimed to identify clinical factors associated with dilated PBG and to perform a detailed morphometric assessment of dilated PBG in cirrhotic patients undergoing liver transplantation (LT). METHODS: All consecutive cirrhotic patients undergoing LT at our institution between October 2006 and October 2011 were assessed for inclusion. Ten non-cirrhotic patients were included as controls. We performed morphometrical assessment of PBG, assessed baseline clinical factors associated with dilated PBG, immunohistochemistry staining with CK-19, MiB-1 and EpCAM, and radiological assessment of all available cases. RESULTS: Seventy-one patients met the inclusion criteria, 24% had PBG dilatation of >1000 µm. On multivariable analysis, MELD (OR 1.11 per unit increase in MELD, p = 0.004) was the only significant factor associated with dilated PBG. Compared to PBG < 1000 µm, large PBG had a higher proportion of EpCAM-positive (69 vs. 28%, p < 0.001) and MiB-1-positive lining cells (2.8 vs. 0.55%, p = 0.036). Computed tomography and magnetic resonance imaging had high specificity but low sensitivity for the diagnosis of dilated PBG > 1000 µm (specificity 90-100%, sensitivity 25-29%). CONCLUSIONS: Dilated PBGs are a common finding in explants of cirrhotic subjects undergoing LT and are associated with liver failure although diagnostic performance of cross-sectional imaging is inconstant. The high number of proliferative and EpCAM-positive cells lining the PBG may suggest a role of PBG in organ repair during liver failure.

Quantification of HER2 by Targeted Mass Spectrometry in Formalin-Fixed Paraffin-Embedded (FFPE) Breast Cancer Tissues.

The ability to accurately quantify proteins in formalin-fixed paraffin-embedded tissues using targeted mass spectrometry opens exciting perspectives for biomarker discovery. We have developed and evaluated a selectedreaction monitoring assay for the human receptor tyrosine-protein kinase erbB-2 (HER2) in formalin-fixed paraffin-embedded breast tumors. Peptide candidates were identified using an untargeted mass spectrometry approach in relevant cell lines. A multiplexed assay was developed for the six best candidate peptides and evaluated for linearity, precision and lower limit of quantification. Results showed a linear response over a calibration range of 0.012 to 100 fmol on column (R(2): 0.99-1.00).The lower limit of quantification was 0.155 fmol on column for all peptides evaluated. The six HER2 peptides were quantified by selected reaction monitoring in a cohort of 40 archival formalin-fixed paraffin-embedded tumor tissues from women with invasive breast carcinomas, which showed different levels of HER2 gene amplification as assessed by standard methods used in clinical pathology. The amounts of the six HER2 peptides were highly and significantly correlated with each other, indicating that peptide levels can be used as surrogates of protein amounts in formalin-fixed paraffin-embedded tissues. After normalization for sample size, selected reaction monitoring peptide measurements were able to correctly predict 90% of cases based on HER2 amplification as defined by the American Society of Clinical Oncology and College of American Pathologists. In conclusion, the developed assay showed good analytical performance and a high agreement with immunohistochemistry and fluorescence in situ hybridization data. This study demonstrated that selected reaction monitoring allows to accurately quantify protein expression in formalin-fixed paraffin-embedded tissues and represents therefore a powerful approach for biomarker discovery studies. The untargeted mass spectrometry data is available via ProteomeXchange whereas the quantification data by selected reaction monitoring is available on the Panorama Public website.

Nonalcoholic Steatohepatitis Is Associated With Increased Mortality in Obese Patients Undergoing Bariatric Surgery.

BACKGROUND & AIMS: Bariatric surgery is associated with improved outcomes in subjects with severe obesity. We investigated the prognostic relevance of nonalcoholic steatohepatitis (NASH) and liver gene expression patterns in patients undergoing bariatric surgery. METHODS: We performed a retrospective analysis of 492 subjects who underwent gastric bypass bariatric surgery at a single center in Switzerland from January 1997 through December 2004; routine perioperative liver biopsies were collected, analyzed histologically, and RNA was isolated. We collected data on overall survival and clinical and biochemical parameters and compared these with data from propensity score-matched subjects participating in the third National Health and Nutrition Examination Survey (NHANES III). We used liver biopsies to identify bariatric surgery patients with NASH; NHANES III participants with NASH were identified based on a hyperechogenic liver at ultrasound and increased alanine transaminase levels. We analyzed a 32-gene signature associated with NAFLD severity in the liver tissues collected from 47 bariatric surgery patients with NASH, and assessed its prognostic features using nearest template prediction and survival analysis. RESULTS: At baseline, the median body mass index of patients who underwent bariatric surgery was 43.6 kg/m2; based on histologic findings, 12% had NASH and 16% had fibrosis. During a median follow-up of 10.2 years after the surgery, 4.2% of the subjects died. In multivariable Cox regression, the presence of NASH (hazard ratio [HR], 2.9; P = .02) and arterial hypertension (HR, 3.9; P = .02) were associated with overall mortality. When bariatric surgery patients were matched with NHANES III participants, bariatric surgery reduced the risk of death during the follow-up period (HR, 0.54; P = .04). However, bariatric surgery patients with NASH did not have a reduced risk of death compared with NHANES III participants with NASH (HR, 0.90; P = .85). We identified an expression pattern of 32 genes in liver tissues from patients with NASH that was associated with increased risk of death in multivariable analysis (HR, 7.7; P = .045). CONCLUSIONS: Histologically proven NASH is associated with increased risk of death within a median follow-up of 10.2 years after bariatric surgery, compared with patients who undergo bariatric surgery without NASH. The survival benefit of bariatric surgery in subjects with NASH may be reduced. A 32-gene expression pattern identified patients with NASH who underwent bariatric surgery and had shorter survival times.

Efficient nonarterialized mouse liver transplantation using 3-dimensional-printed instruments.

Because of the wide availability of genetically modified animals, mouse orthotopic liver transplantation is often preferred over rat liver transplantation. We present a simplified mouse liver transplantation technique and compare transplantation outcomes with versus without hepatic artery anastomosis. Instruments for liver implantation were designed and printed with a 3-dimensional (3D) printer. The suprahepatic vena cava anastomosis was performed with a 10-0 running suture. The vena porta and infrahepatic vena cava were joined on extraluminal cuffs, using the 3D-printed device for spatial alignment and stabilization. The hepatic artery was reconstructed in half of the recipients using intraluminal stents. Liver function tests (3, 7, and 28 days) and histology (7 and 28 days) were assessed after transplantation. We performed 22 consecutive syngeneic C57BL/6 mouse orthotopic liver transplantations. The median portal clamping time was 12.5 ± 1.5 minutes. The survival rate at 4 weeks was 100% for both arterialized and nonarterialized recipients (n = 7, 4 recipients of each group being killed for early histology at day 7). Liver function tests at 3, 7, and 28 days were similar between arterialized versus nonarterialized groups. Liver parenchyma demonstrated only irrelevant abnormalities in both groups. The proposed device allows for a shorter clamping time compared with the published literature. Using this technique, the artery does not need to be anastomosed, with no impact on graft and recipient outcomes. The device is available for 3D printing. Liver Transplantation 22 1688-1696 2016 AASLD.