A Macrophage-Pericyte Axis Directs Tissue Restoration via Amphiregulin-Induced Transforming Growth Factor Beta Activation.

The epidermal growth factor receptor ligand Amphiregulin has a well-documented role in the restoration of tissue homeostasis after injury; however, the mechanism by which Amphiregulin contributes to wound repair remains unknown. Here we show that Amphiregulin functioned by releasing bioactive transforming growth factor beta (TGF-ß) from latent complexes via integrin-αV activation. Using acute injury models in two different tissues, we found that by inducing TGF-ß activation on mesenchymal stromal cells (pericytes), Amphiregulin induced their differentiation into myofibroblasts, thereby selectively contributing to the restoration of vascular barrier function within injured tissue. Furthermore, we identified macrophages as a critical source of Amphiregulin, revealing a direct effector mechanism by which these cells contribute to tissue restoration after acute injury. Combined, these observations expose a so far under-appreciated mechanism of how cells of the immune system selectively control the differentiation of tissue progenitor cells during tissue repair and inflammation.

Accurate prediction of all-cause mortality in patients with metabolic dysfunction-associated steatotic liver disease using electronic health records.

INTRODUCTION AND OBJECTIVES: Despite the huge clinical burden of MASLD, validated tools for early risk stratification are lacking, and heterogeneous disease expression and a highly variable rate of progression to clinical outcomes result in prognostic uncertainty. We aimed to investigate longitudinal electronic health record-based outcome prediction in MASLD using a state-of-the-art machine learning model. PATIENTS AND METHODS: n=940 patients with histologically-defined MASLD were used to develop a deep-learning model for all-cause mortality prediction. Patient timelines, spanning 12 years, were fully-annotated with demographic/clinical characteristics, ICD-9 and -10 codes, blood test results, prescribing data, and secondary care activity. A Transformer neural network (TNN) was trained to output concomitant probabilities of 12-, 24-, and 36-month all-cause mortality. In-sample performance was assessed using 5-fold cross-validation. Out-of-sample performance was assessed in an independent set of n=528 MASLD patients. RESULTS: In-sample model performance achieved AUROC curve 0.74-0.90 (95% CI: 0.72-0.94), sensitivity 64%-82%, specificity 75%-92% and Positive Predictive Value (PPV) 94%-98%. Out-of-sample model validation had AUROC 0.70-0.86 (95% CI: 0.67-0.90), sensitivity 69%-70%, specificity 96%-97% and PPV 75%-77%. Key predictive factors, identified using coefficients of determination, were age, presence of type 2 diabetes, and history of hospital admissions with length of stay >14 days. CONCLUSIONS: A TNN, applied to routinely-collected longitudinal electronic health records, achieved good performance in prediction of 12-, 24-, and 36-month all-cause mortality in patients with MASLD. Extrapolation of our technique to population-level data will enable scalable and accurate risk stratification to identify people most likely to benefit from anticipatory health care and personalized interventions.

A data-driven approach to decode metabolic dysfunction-associated steatotic liver disease.

Metabolic dysfunction-associated steatotic liver disease (MASLD), defined by the presence of liver steatosis together with at least one out of five cardiometabolic factors, is the most common cause of chronic liver disease worldwide, affecting around one in three people. Yet the clinical presentation of MASLD and the risk of progression to cirrhosis and adverse clinical outcomes is highly variable. It, therefore, represents both a global public health threat and a precision medicine challenge. Artificial intelligence (AI) is being investigated in MASLD to develop reproducible, quantitative, and automated methods to enhance patient stratification and to discover new biomarkers and therapeutic targets in MASLD. This review details the different applications of AI and machine learning algorithms in MASLD, particularly in analyzing electronic health record, digital pathology, and imaging data. Additionally, it also describes how specific MASLD consortia are leveraging multimodal data sources to spark research breakthroughs in the field. Using a new national-level 'data commons' (SteatoSITE) as an exemplar, the opportunities, as well as the technical challenges of large-scale databases in MASLD research, are highlighted.

British Society of Gastroenterology guidelines for the diagnosis and management of cholangiocarcinoma.

These guidelines for the diagnosis and management of cholangiocarcinoma (CCA) were commissioned by the British Society of Gastroenterology liver section. The guideline writing committee included a multidisciplinary team of experts from various specialties involved in the management of CCA, as well as patient/public representatives from AMMF (the Cholangiocarcinoma Charity) and PSC Support. Quality of evidence is presented using the Appraisal of Guidelines for Research and Evaluation (AGREE II) format. The recommendations arising are to be used as guidance rather than as a strict protocol-based reference, as the management of patients with CCA is often complex and always requires individual patient-centred considerations.

Intestinal B cells license metabolic T-cell activation in NASH microbiota/antigen-independently and contribute to fibrosis by IgA-FcR signalling.

BACKGROUND & AIMS: The progression of non-alcoholic steatohepatitis (NASH) to fibrosis and hepatocellular carcinoma (HCC) is aggravated by auto-aggressive T cells. The gut-liver axis contributes to NASH, but the mechanisms involved and the consequences for NASH-induced fibrosis and liver cancer remain unknown. We investigated the role of gastrointestinal B cells in the development of NASH, fibrosis and NASH-induced HCC. METHODS: C57BL/6J wild-type (WT), B cell-deficient and different immunoglobulin-deficient or transgenic mice were fed distinct NASH-inducing diets or standard chow for 6 or 12 months, whereafter NASH, fibrosis, and NASH-induced HCC were assessed and analysed. Specific pathogen-free/germ-free WT and µMT mice (containing B cells only in the gastrointestinal tract) were fed a choline-deficient high-fat diet, and treated with an anti-CD20 antibody, whereafter NASH and fibrosis were assessed. Tissue biopsy samples from patients with simple steatosis, NASH and cirrhosis were analysed to correlate the secretion of immunoglobulins to clinicopathological features. Flow cytometry, immunohistochemistry and single-cell RNA-sequencing analysis were performed in liver and gastrointestinal tissue to characterise immune cells in mice and humans. RESULTS: Activated intestinal B cells were increased in mouse and human NASH samples and licensed metabolic T-cell activation to induce NASH independently of antigen specificity and gut microbiota. Genetic or therapeutic depletion of systemic or gastrointestinal B cells prevented or reverted NASH and liver fibrosis. IgA secretion was necessary for fibrosis induction by activating CD11b+CCR2+F4/80+CD11c-FCGR1+ hepatic myeloid cells through an IgA-FcR signalling axis. Similarly, patients with NASH had increased numbers of activated intestinal B cells; additionally, we observed a positive correlation between IgA levels and activated FcRg+ hepatic myeloid cells, as well the extent of liver fibrosis. CONCLUSIONS: Intestinal B cells and the IgA-FcR signalling axis represent potential therapeutic targets for the treatment of NASH. IMPACT AND IMPLICATIONS: There is currently no effective treatment for non-alcoholic steatohepatitis (NASH), which is associated with a substantial healthcare burden and is a growing risk factor for hepatocellular carcinoma (HCC). We have previously shown that NASH is an auto-aggressive condition aggravated, amongst others, by T cells. Therefore, we hypothesized that B cells might have a role in disease induction and progression. Our present work highlights that B cells have a dual role in NASH pathogenesis, being implicated in the activation of auto-aggressive T cells and the development of fibrosis via activation of monocyte-derived macrophages by secreted immunoglobulins (e.g., IgA). Furthermore, we show that the absence of B cells prevented HCC development. B cell-intrinsic signalling pathways, secreted immunoglobulins, and interactions of B cells with other immune cells are potential targets for combinatorial NASH therapies against inflammation and fibrosis.

Clinical relevance of biomarkers in cholangiocarcinoma: critical revision and future directions.

Cholangiocarcinoma (CCA) is a malignant tumour arising from the biliary system. In Europe, this tumour frequently presents as a sporadic cancer in patients without defined risk factors and is usually diagnosed at advanced stages with a consequent poor prognosis. Therefore, the identification of biomarkers represents an utmost need for patients with CCA. Numerous studies proposed a wide spectrum of biomarkers at tissue and molecular levels. With the present paper, a multidisciplinary group of experts within the European Network for the Study of Cholangiocarcinoma discusses the clinical role of tissue biomarkers and provides a selection based on their current relevance and potential applications in the framework of CCA. Recent advances are proposed by dividing biomarkers based on their potential role in diagnosis, prognosis and therapy response. Limitations of current biomarkers are also identified, together with specific promising areas (ie, artificial intelligence, patient-derived organoids, targeted therapy) where research should be focused to develop future biomarkers.

Noninvasive Detection of Ischemic Vascular Damage in a Pig Model of Liver Donation After Circulatory Death.

BACKGROUND AND AIMS: Liver graft quality is evaluated by visual inspection prior to transplantation, a process highly dependent on the surgeon's experience. We present an objective, noninvasive, quantitative way of assessing liver quality in real time using Raman spectroscopy, a laser-based tool for analyzing biomolecular composition. APPROACH AND RESULTS: A porcine model of donation after circulatory death (DCD) with normothermic regional perfusion (NRP) allowed assessment of liver quality premortem, during warm ischemia (WI) and post-NRP. Ten percent of circulating blood volume was removed in half of experiments to simulate blood recovery for DCD heart removal. Left median lobe biopsies were obtained before circulatory arrest, after 45 minutes of WI, and after 2 hours of NRP and analyzed using spontaneous Raman spectroscopy, stimulated Raman spectroscopy (SRS), and staining. Measurements were also taken in situ from the porcine liver using a handheld Raman spectrometer at these time points from left median and right lateral lobes. Raman microspectroscopy detected congestion during WI by measurement of the intrinsic Raman signal of hemoglobin in red blood cells (RBCs), eliminating the need for exogenous labels. Critically, this microvascular damage was not observed during WI when 10% of circulating blood was removed before cardiac arrest. Two hours of NRP effectively cleared RBCs from congested livers. Intact RBCs were visualized rapidly at high resolution using SRS. Optical properties of ischemic livers were significantly different from preischemic and post-NRP livers as measured using a handheld Raman spectrometer. CONCLUSIONS: Raman spectroscopy is an effective tool for detecting microvascular damage which could assist the decision to use marginal livers for transplantation. Reducing the volume of circulating blood before circulatory arrest in DCD may help reduce microvascular damage.

Guidelines for cellular and molecular pathology content in clinical trial protocols: the SPIRIT-Path extension.

The 2013 SPIRIT (Standard Protocol Items: Recommendations for Interventional Trials) Statement provides evidence-based recommendations for the minimum content to be included in a clinical trial protocol. Assessment of biospecimens is often required for trial eligibility or as part of an outcome evaluation, and precision molecular approaches are increasingly used in trial design. However, cellular and molecular pathology practices within trials have not been codified or formalised. We developed international consensus reporting guidelines for cellular and molecular pathology content in clinical trial protocols (the SPIRIT-Path extension) using an international Delphi process, which assesses candidate items generated from a previous systematic review, followed by an expert consensus meeting. 74 individuals from five continents responded, including clinicians, statisticians, laboratory scientists, patient advocates, funders, industry representatives, journal editors, and regulators. The SPIRIT-Path guidelines recommend 14 additional items (seven extensions to the SPIRIT checklist and seven elaborations) that should be addressed in trial protocols containing pathology content, alongside the SPIRIT 2013 Statement items. SPIRIT-Path recommends that protocols should document the individuals, processes, and standards for all cellular and molecular pathology components of the trial, including all stages of the specimen pathway and any digital pathology methods, with specific consideration of the value of trial data and biological tissues for additional translational studies.

TWEAK/Fn14 signalling promotes cholangiocarcinoma niche formation and progression.

BACKGROUND & AIMS: Cholangiocarcinoma (CCA) is a cancer of the hepatic bile ducts that is rarely resectable and is associated with poor prognosis. Tumour necrosis factor-like weak inducer of apoptosis (TWEAK) is known to signal via its receptor fibroblast growth factor-inducible 14 (Fn14) and induce cholangiocyte and myofibroblast proliferation in liver injury. We aimed to characterise its role in CCA. METHODS: The expression of the TWEAK ligand and Fn14 receptor was assessed immunohistochemically and by bulk RNA and single cell transcriptomics of human liver tissue. Spatiotemporal dynamics of pathway regulation were comprehensively analysed in rat and mouse models of thioacetamide (TAA)-mediated CCA. Flow cytometry, qPCR and proteomic analyses of CCA cell lines and conditioned medium experiments with primary macrophages were performed to evaluate the downstream functions of TWEAK/Fn14. In vivo pathway manipulation was assessed via TWEAK overexpression in NICD/AKT-induced CCA or genetic Fn14 knockout during TAA-mediated carcinogenesis. RESULTS: Our data reveal TWEAK and Fn14 overexpression in multiple human CCA cohorts, and Fn14 upregulation in early TAA-induced carcinogenesis. TWEAK regulated the secretion of factors from CC-SW-1 and SNU-1079 CCA cells, inducing polarisation of proinflammatory CD206+ macrophages. Pharmacological blocking of the TWEAK downstream target chemokine monocyte chemoattractant protein 1 (MCP-1 or CCL2) significantly reduced CCA xenograft growth, while TWEAK overexpression drove cancer-associated fibroblast proliferation and collagen deposition in the tumour niche. Genetic Fn14 ablation significantly reduced inflammatory, fibrogenic and ductular responses during carcinogenic TAA-mediated injury. CONCLUSION: These novel data provide evidence for the action of TWEAK/Fn14 on macrophage recruitment and phenotype, and cancer-associated fibroblast proliferation in CCA. Targeting TWEAK/Fn14 and its downstream signals may provide a means to inhibit CCA niche development and tumour growth. LAY SUMMARY: Cholangiocarcinoma is an aggressive, chemotherapy-resistant liver cancer. Interactions between tumour cells and cells that form a supportive environment for the tumour to grow are a source of this aggressiveness and resistance to chemotherapy. Herein, we describe interactions between tumour cells and their supportive environment via a chemical messenger, TWEAK and its receptor Fn14. TWEAK/Fn14 alters the recruitment and type of immune cells in tumours, increases the growth of cancer-associated fibroblasts in the tumour environment, and is a potential target to reduce tumour formation.

Alternatively activated macrophages promote resolution of necrosis following acute liver injury.

BACKGROUND & AIM: Following acetaminophen (APAP) overdose, acute liver injury (ALI) can occur in patients that present too late for N-acetylcysteine treatment, potentially leading to acute liver failure, systemic inflammation, and death. Macrophages influence the progression and resolution of ALI due to their innate immunological function and paracrine activity. Syngeneic primary bone marrow-derived macrophages (BMDMs) were tested as a cell-based therapy in a mouse model of APAP-induced ALI (APAP-ALI). METHODS: Several phenotypically distinct BMDM populations were delivered intravenously to APAP-ALI mice when hepatic necrosis was established, and then evaluated based on their effects on injury, inflammation, immunity, and regeneration. In vivo phagocytosis assays were used to interrogate the phenotype and function of alternatively activated BMDMs (AAMs) post-injection. Finally, primary human AAMs sourced from healthy volunteers were evaluated in immunocompetent APAP-ALI mice. RESULTS: BMDMs rapidly localised to the liver and spleen within 4 h of administration. Injection of AAMs specifically reduced hepatocellular necrosis, HMGB1 translocation, and infiltrating neutrophils following APAP-ALI. AAM delivery also stimulated proliferation in hepatocytes and endothelium, and reduced levels of several circulating proinflammatory cytokines within 24 h. AAMs displayed a high phagocytic activity both in vitro and in injured liver tissue post-injection. Crosstalk with the host innate immune system was demonstrated by reduced infiltrating host Ly6Chi macrophages in AAM-treated mice. Importantly, therapeutic efficacy was partially recapitulated using clinical-grade primary human AAMs in immunocompetent APAP-ALI mice, underscoring the translational potential of these findings. CONCLUSION: We identify that AAMs have value as a cell-based therapy in an experimental model of APAP-ALI. Human AAMs warrant further evaluation as a potential cell-based therapy for APAP overdose patients with established liver injury. LAY SUMMARY: After an overdose of acetaminophen (paracetamol), some patients present to hospital too late for the current antidote (N-acetylcysteine) to be effective. We tested whether macrophages, an injury-responsive leukocyte that can scavenge dead/dying cells, could serve as a cell-based therapy in an experimental model of acetaminophen overdose. Injection of alternatively activated macrophages rapidly reduced liver injury and reduced several mediators of inflammation. Macrophages show promise to serve as a potential cell-based therapy for acute liver injury.

The important role of the histopathologist in clinical trials: challenges and approaches to tackle them.

High-quality histopathology is essential for the success of clinical trials. Histopathologists have a detailed understanding of tumour biology and mechanisms of disease, as well as practical knowledge of optimal tissue handling and logistical service requirements for study delivery, such as biomarker evaluation, tissue acquisition and turnaround times. As such, histopathologist input is essential throughout every stage of research and clinical trials, from concept development and study design to trial delivery, analysis and dissemination of results. Patient recruitment to trials takes place among all healthcare settings, meaning that histopathologists make an invaluable contribution to clinical trials as part of their routine day-to-day work that often goes unrecognised. More complex evaluation of surgical specimens in the neoadjuvant setting and ever-expanding minimum data sets add to the workload of every histopathologist, not just academic pathologists in tertiary centres. This is occurring against a backdrop of increasing workload pressures and a worldwide shortage of histopathologists and biomedical scientists. Providing essential histopathology support for trials at grassroots level requires funding for adequate resources including histopathologist time, education and training, biomedical scientist and administrative support and greater recognition of the contribution made by histopathology. This paper will discuss the many ways in which histopathologists are involved in clinical trials and the challenges faced in meeting the additional demands posed by trial participation and potential ways to address this, with a special emphasis on the UK model and the Cellular-Molecular Pathology Initiative (CM-Path).

The STAT3-IL-10-IL-6 Pathway Is a Novel Regulator of Macrophage Efferocytosis and Phenotypic Conversion in Sterile Liver Injury.

The disposal of apoptotic bodies by professional phagocytes is crucial to effective inflammation resolution. Our ability to improve the disposal of apoptotic bodies by professional phagocytes is impaired by a limited understanding of the molecular mechanisms that regulate the engulfment and digestion of the efferocytic cargo. Macrophages are professional phagocytes necessary for liver inflammation, fibrosis, and resolution, switching their phenotype from proinflammatory to restorative. Using sterile liver injury models, we show that the STAT3-IL-10-IL-6 axis is a positive regulator of macrophage efferocytosis, survival, and phenotypic conversion, directly linking debris engulfment to tissue repair.

The clinical spectrum of Fontan-associated liver disease: results from a prospective multimodality screening cohort.

AIMS: Liver fibrosis and cirrhosis are a consequence of a Fontan physiology, and determine prognosis. It is unclear whether non-invasive assessment of liver pathology is helpful to provide clinically relevant information. The aims of this study were to assess the spectrum of Fontan-associated liver disease (FALD) and usefulness of non-invasive methods to assess biopsy confirmed liver fibrosis. METHODS AND RESULTS: Hepatic screening of consecutive patients consisted of a blood panel, ultrasonography, elastography, contrast-enhanced magnetic resonance imaging (MRI)/computed tomography (CT) scan, and liver biopsy (scored with Fontan specific fibrosis scores and collagen proportionate area; CPA). Fibrosis parameters, varices, ascites, and splenomegaly were measured on imaging. Thirty-eight of 49 referred patients (27 ± 6.6 years, 73.7% male) underwent the complete screening protocol. Liver fibrosis on biopsy was present in all patients, and classified as severe (Stages 3-4) in 68%. Median CPA was 22.5% (16.9-29.5) and correlated with individual fibrosis scores. ELF® and liver stiffness were elevated, but MELD-XI scores were low in all patients. Fibrosis severity neither correlated to ELF® and liver stiffness, nor to (semi-) quantitative fibrosis parameters on MRI/CT. Varices were present in 50% and hyperenhancing nodules in 25% of patients, both independent of fibrosis stage, but varices were associated with higher CPA values. CONCLUSION: The FALD spectrum includes both hepatic congestion and severe fibrosis, with signs of portal hypertension and hyperenhancing nodules as significant manifestations. Routine imaging, transient elastography, and serum biomarkers are unable to accurately assess severity of liver fibrosis in this cohort. Future research should focus on validating new diagnostic tools with biopsy as the reference standard.

11Beta-hydroxysteroid dehydrogenase-1 deficiency or inhibition enhances hepatic myofibroblast activation in murine liver fibrosis.

A hallmark of chronic liver injury is fibrosis, with accumulation of extracellular matrix orchestrated by activated hepatic stellate cells (HSCs). Glucocorticoids limit HSC activation in vitro, and tissue glucocorticoid levels are amplified by 11beta-hydroxysteroid dehydrogenase-1 (11ßHSD1). Although 11ßHSD1 inhibitors have been developed for type 2 diabetes mellitus and improve diet-induced fatty liver in various mouse models, effects on the progression and/or resolution of liver injury and consequent fibrosis have not been characterized. We have used the reversible carbon tetrachloride-induced model of hepatocyte injury and liver fibrosis to show that in two models of genetic 11ßHSD1 deficiency (global, Hsd11b1-/- , and hepatic myofibroblast-specific, Hsd11b1fl/fl /Pdgfrb-cre) 11ßHSD1 pharmacological inhibition in vivo exacerbates hepatic myofibroblast activation and liver fibrosis. In contrast, liver injury and fibrosis in hepatocyte-specific Hsd11b1fl/fl /albumin-cre mice did not differ from that of controls, ruling out 11ßHSD1 deficiency in hepatocytes as the cause of the increased fibrosis. In primary HSC culture, glucocorticoids inhibited expression of the key profibrotic genes Acta2 and Col1α1, an effect attenuated by the 11ßHSD1 inhibitor [4-(2-chlorophenyl-4-fluoro-1-piperidinyl][5-(1H-pyrazol-4-yl)-3-thienyl]-methanone. HSCs from Hsd11b1-/- and Hsd11b1fl/fl /Pdgfrb-cre mice expressed higher levels of Acta2 and Col1α1 and were correspondingly more potently activated. In vivo [4-(2-chlorophenyl-4-fluoro-1-piperidinyl][5-(1H-pyrazol-4-yl)-3-thienyl]-methanone administration prior to chemical injury recapitulated findings in Hsd11b1-/- mice, including greater fibrosis. CONCLUSION: 11ßHSD1 deficiency enhances myofibroblast activation and promotes initial fibrosis following chemical liver injury; hence, the effects of 11ßHSD1 inhibitors on liver injury and repair are likely to be context-dependent and deserve careful scrutiny as these compounds are developed for chronic diseases including metabolic syndrome and dementia. (Hepatology 2018;67:2167-2181).

Anatomical, histomorphological and molecular classification of cholangiocarcinoma.

Cholangiocarcinoma constitutes a heterogeneous group of malignancies that can emerge at any point of the biliary tree. Cholangiocarcinoma is classified into intrahepatic, perihilar and distal based on its anatomical location. Histologically, conventional perihilar/distal cholangiocarcinomas are mucin-producing adenocarcinomas or papillary tumours; intrahepatic cholangiocarcinomas are more heterogeneous and can be sub-classified according to the level or size of the displayed bile duct. Cholangiocarcinoma develops through multistep carcinogenesis and is preceded by dysplastic and in situ lesions. Definition and clinical significance of precursor lesions, including biliary intraepithelial neoplasia, intraductal papillary neoplasms of the bile duct, intraductal tubulopapillary neoplasms and mucinous cystic neoplasm, are discussed in this review. A main challenge in diagnosing cholangiocarcinoma is the fact that tumour tissue for histological examination is difficult to obtain. Thus, a major clinical obstacle is the establishment of the correct diagnosis at a tumour stage that is amenable to surgery which still represents the only curable therapeutic option. Current standards, methodology and criteria for diagnosis are discussed. Cholangiocarcinoma represents a heterogeneous tumour with regard to molecular alterations. In intrahepatic subtype, mainly two distinctive morpho-molecular groups can currently be discriminated. Large-duct type intrahepatic cholangiocarcinoma shows a high mutation frequency of oncogenes and tumour suppressor genes, such as KRAS and TP53 while Isocitrate Dehydrogenase 1/2 mutations and Fibroblast Growth Factor Receptor 2-fusions are typically seen in small-duct type tumours. It is most important to ensure the separation of the given anatomical subtypes and to search for distinct subgroups within the subtypes on a molecular and morphological basis.

Notch3 drives development and progression of cholangiocarcinoma.

The prognosis of cholangiocarcinoma (CC) is dismal. Notch has been identified as a potential driver; forced exogenous overexpression of Notch1 in hepatocytes results in the formation of biliary tumors. In human disease, however, it is unknown which components of the endogenously signaling pathway are required for tumorigenesis, how these orchestrate cancer, and how they can be targeted for therapy. Here we characterize Notch in human-resected CC, a toxin-driven model in rats, and a transgenic mouse model in which p53 deletion is targeted to biliary epithelia and CC induced using the hepatocarcinogen thioacetamide. We find that across species, the atypical receptor NOTCH3 is differentially overexpressed; it is progressively up-regulated with disease development and promotes tumor cell survival via activation of PI3k-Akt. We use genetic KO studies to show that tumor growth significantly attenuates after Notch3 deletion and demonstrate signaling occurs via a noncanonical pathway independent of the mediator of classical Notch, Recombinant Signal Binding Protein for Immunoglobulin Kappa J Region (RBPJ). These data present an opportunity in this aggressive cancer to selectively target Notch, bypassing toxicities known to be RBPJ dependent.

WT1 expression in vessels varies with histopathological grade in tumour-bearing and control tissue from patients with breast cancer.

BACKGROUND: The Wilms' tumour protein (WT1), which influences tumour development and angiogenesis, is a promising therapeutic target in breast cancer. We hypothesised that WT1 expression would vary in endothelial cells in distinct sub-classifications of breast cancer. METHODS: WT1 expression and vascular density were quantified by immunohistochemical analysis of human (n = 57) and murine breast cancers. Human tumours were sub-classified by histopathological grade, ER status and HER2 enrichment. RESULTS: WT1 was identified in endothelial (and epithelial and smooth muscle) cells in tumours and tumour-free tissues (controls) from patients and mice with breast cancer. WT1 expression was higher in tumours than in controls, but this was not due to increased endothelial WT1. Vascular WT1 in cancers decreased as histopathological grade increased. WT1 was higher in ER-positive versus ER-negative cancers. Strikingly, reduced WT1 expression in controls correlated with an increased Nottingham Prognostic Index score. CONCLUSIONS: Expression of WT1 is increased in breast cancers but this is not limited to the vascular compartment. The association between reduced WT1 in tumour-free tissue and poor prognosis suggests a protective role for WT1 in the healthy breast.

Hepatic elastin content is predictive of adverse outcome in advanced fibrotic liver disease.

AIMS: The aim of this study was to determine if elastin content in needle core native liver biopsies was predictive of clinical outcome in patients with chronic hepatitis C virus-related chronic liver disease. METHODS AND RESULTS: Elastin contents in liver biopsies were determined by image analysis, technically validated in an independent centre, and correlated with outcome in patients with advanced (Ishak stage ≥5) chronic hepatitis C virus-related chronic liver disease. Elastin was robustly quantified in an operator-independent and laboratory-independent manner, with very strong correlation of elastin staining measured with two methods of image classification (rs = 0.873, P < 0.00001). Elastin content (but not absolute scar content or Ishak stage) was predictive for future clinical outcomes. In a cohort of patients without sustained virological response, the median hepatic elastin content was 3.4%, and 17 patients (57%) progressed to a liver-related clinical outcome; 11 of the 15 patients (73%) with a hepatic elastin content of >3.4% progressed to a clinical outcome, as compared with only six of 15 (40%) with an elastin content of <3.4%. The difference in time to outcome was significant. CONCLUSIONS: We describe a simple and reproducible method for elastin quantification in liver biopsies that provides potentially valuable prognostic information to inform clinical management.

Extracellular matrix components indicate remodelling activity in different fibrosis stages of human non-alcoholic fatty liver disease.

AIMS: The composition of several important extracellular matrix components (ECM) has not yet been elucidated in human non-alcoholic fatty liver disease (NAFLD). We aim to investigate the proportion of hepatic stellate cells (HSCs) and activity of matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) in human NAFLD liver tissue with respect to severity of inflammation and fibrosis. METHODS AND RESULTS: Histopathological features were quantified by NAFLD activity score and grading assignment. The collagen proportionate area (CPA) was measured. Slides were stained with alpha-smooth muscle actin (α-SMA), as a marker of activated HSCs, and α-SMA was quantified digitally. Zymography was performed to measure the proteolytic activity of MMP-2 and MMP-9. TIMP-1 and TIMP-2 protein concentration was measured with enzyme-linked immunosorbent assay (ELISA). α-SMA was higher in severe fibrosis (6.3%, interquartile range 2.9-13.1) than mild and no fibrosis (median 1.1 and 0.9%, P < 0.001) and correlated strongly with CPA (Rs = 0.870, P < 0.001). ProMMP-2 activity in severe (4.1%, IQR 2.6-16.2) and mild fibrosis (2.7%, IQR 1.9-3.9) was higher than in no fibrosis (1.5%, (IQR 0.95-2.1); P = 0.001 and P = 0.046) and showed a moderate positive correlation with CPA (Rs = 0.495, P = 0.001). TIMP-1 and TIMP-2 were significantly higher in severe fibrosis than mild or no fibrosis. Both showed moderate correlation with CPA (TIMP-1: Rs = 0.471, P = 0.002 and TIMP-2: Rs = 0.325, P = 0.036). MMP-9 correlated as the only ECM component to inflammation severity. CONCLUSIONS: Advanced human NAFLD-fibrosis has a distinct ECM composition with increased HSCs and increased TIMP inhibition, but there is also ongoing remodelling activity of MMP-2.

Study protocol: HepaT1ca - an observational clinical cohort study to quantify liver health in surgical candidates for liver malignancies.

BACKGROUND: Accurate assessment of liver health prior to undertaking resectional liver surgery or chemoembolisation for primary and secondary cancers is essential for patient safety and optimal outcomes. LiverMultiScan™, an MRI-based technology, non-invasively quantifies hepatic fibroinflammatory disease, steatosis and iron content. We hypothesise that LiverMultiScan™can quantify liver health prior to surgery and inform the risk assessment for patients considering liver surgery or chemoembolization and seek to evaluate this technology in an operational environment. METHODS/DESIGN: HepaT1ca is an observational cohort study in two tertiary-referral liver surgery centres in the United Kingdom. The primary outcome is correlation between the pre-operative liver health assessment score (Hepatica score - calculated by weighting future remnant liver volume by liver inflammation and fibrosis (LIF) score) and the post-operative liver function composite integer-based risk (Hyder-Pawlik) score. With ethical approval and fully-informed consent, individuals considering liver surgery for primary or secondary cancer will undergo clinical assessment, blood sampling, and LiverMultiScan™multiparametric MRI before and after surgical liver resection or TACE. In nested cohorts of individuals undergoing chemotherapy prior to surgery, or those undergoing portal vein embolization (PVE) as an adjunct to surgery, an additional testing session prior to commencement of treatment will occur. Tissue will be examined histologically and by immunohistochemistry. Pre-operative liver health assessment scores and the post-operative risk scores will be correlated to define the ability of LiverMultiScan™to predict the risk of post-operative morbidity and mortality. Because technology performance in this setting is unknown, a pragmatic sample size will be used. For the primary outcome, n = 200 for the main cohort will allow detection of a minimum correlation coefficient of 0.2 with 5% significance and power of 80%. DISCUSSION: This study will refine the technology and clinical application of multiparametric MRI (including LiverMultiScan™), to quantify pre-existing liver health and predict post-intervention outcomes following liver resection. If successful, this study will advance the technology and support the use of multiparametric MRI as part of an enhanced pre-operative assessment to improve patient safety and to personalise operative risk assessment of liver surgery/non-surgical intervention. TRIAL REGISTRATION: This study is registered on ClinicalTrials.gov Identifier: NCT03213314 .