Molecular biology of cholangiocarcinoma and its implications for targeted therapy in patient management.

Cholangiocarcinoma (CCA) remains a devastating malignancy and a significant challenge to treat. The majority of CCA patients are diagnosed at an advanced stage, making the disease incurable in most cases. The advent of high-throughput genetic sequencing has significantly improved our understanding of the molecular biology underpinning cancer. The identification of 'druggable' genetic aberrations and the development of novel targeted therapies against them is opening up new treatment strategies. Currently, 3 targeted therapies are approved for use in CCA; Ivosidenib in patients with IDH1 mutations and Infigratinib/Pemigatinib in those with FGFR2 fusions. As our understanding of the biology underpinning CCA continues to improve it is highly likely that additional targeted therapies will become available in the near future. This is important, as it is thought up to 40 % of CCA patients harbour a potentially actionable mutation. In this review we provide an overview of the molecular pathogenesis of CCA and highlight currently available and potential future targeted treatments.

Predicting physiologically-relevant oxygen concentrations in precision-cut liver slices using mathematical modelling.

Precision cut liver slices represent an encouraging ex vivo method to understand the pathogenesis of liver disease alongside drug induced liver injury. Despite being more physiologically relevant compared to in vitro models, precision cut liver slices are limited by the availability of healthy human tissue and experimental variability. Internal oxygen concentration and media composition govern the longevity and viability of the slices during the culture period and as such, a variety of approaches have been taken to maximise the appropriateness of the internal oxygen concentrations across the slice. The aim of this study was to predict whether it is possible to generate a physiologically relevant oxygen gradient of 35-65mmHg across a precision cut liver slice using mathematical modelling. Simulations explore how the internal oxygen concentration changes as a function of the diameter of the slice, the position inside the well and the external incubator oxygen concentration. The model predicts that the desired oxygen gradient may be achieved using a 5mm diameter slice at atmospheric oxygen concentrations, provided that the slice is positioned at a certain height within the well of a 12-well plate.

Making use of transcription factor enrichment to identify functional microRNA-regulons.

microRNAs (miRNAs) are important modulators of messenger RNA stability and translation, controlling wide gene networks. Albeit generally modest on individual targets, the regulatory effect of miRNAs translates into meaningful pathway modulation through concurrent targeting of regulons with functional convergence. Identification of miRNA-regulons is therefore essential to understand the function of miRNAs and to help realise their therapeutic potential, but it remains challenging due to the large number of false positive target sites predicted per miRNA. In the current work, we investigated whether genes regulated by a given miRNA were under the transcriptional control of a predominant transcription factor (TF). Strikingly we found that for ~50% of the miRNAs analysed, their targets were significantly enriched in at least one common TF. We leveraged such miRNA-TF co-regulatory networks to identify pathways under miRNA control, and demonstrated that filtering predicted miRNA-target interactions (MTIs) relying on such pathways significantly enriched the proportion of predicted true MTIs. To our knowledge, this is the first description of an in- silico pipeline facilitating the identification of miRNA-regulons, to help understand miRNA function.

Preimplant Normothermic Liver Perfusion of a Suboptimal Liver Donated After Circulatory Death.

Livers retrieved after circulatory death are associated with an increased incidence of primary nonfunction, early allograft dysfunction, and biliary strictures. The authors report a case of preimplant normothermic perfusion of a suboptimal liver from a 57-year-old donor after circulatory death who had been hospitalized for 9 days; predonation alanine transaminase level was 63 IU/L, and the period from withdrawal of life-supporting treatment to circulatory arrest was 150 minutes. After 5 hours of static cold storage, the liver was subject to normothermic machine perfusion with a plasma-free red cell-based perfusate. Perfusate lactate level fell from 7.2 to 0.3 mmol/L within 74 minutes of ex situ perfusion, at which point perfusate alanine transaminase level was 1152 IU/L and urea concentration was 9.4 mmol/L. After 132 minutes, normothermic perfusion was stopped and implantation begun. After transplantation, the patient made an uneventful recovery and was discharged on day 8; liver biochemistry was normal by day 19 and has remained normal thereafter. Donor common bile duct excised at implantation showed preservation of peribiliary glands, and cholangiography 6 months posttransplantation showed no evidence of cholangiopathy. Preimplant ex situ normothermic perfusion of the liver appears to be a promising way to evaluate a marginal liver before transplantation and may modify the response to ischemia.

Adaptation to acetaminophen exposure elicits major changes in expression and distribution of the hepatic proteome.

Acetaminophen overdose is the leading cause of acute liver failure. One dose of 10-15 g causes severe liver damage in humans, whereas repeated exposure to acetaminophen in humans and animal models results in autoprotection. Insight of this process is limited to select proteins implicated in acetaminophen toxicity and cellular defence. Here we investigate hepatic adaptation to acetaminophen toxicity from a whole proteome perspective, using quantitative mass spectrometry. In a rat model, we show the response to acetaminophen involves the expression of 30% of all proteins detected in the liver. Genetic ablation of a master regulator of cellular defence, NFE2L2, has little effect, suggesting redundancy in the regulation of adaptation. We show that adaptation to acetaminophen has a spatial component, involving a shift in regionalisation of CYP2E1, which may prevent toxicity thresholds being reached. These data reveal unexpected complexity and dynamic behaviour in the biological response to drug-induced liver injury.

In situ normothermic regional perfusion for controlled donation after circulatory death--the United Kingdom experience.

Organs recovered from donors after circulatory death (DCD) suffer warm ischemia before cold storage which may prejudice graft survival and result in a greater risk of complications after transplant. A period of normothermic regional perfusion (NRP) in the donor may reverse these effects and improve organ function. Twenty-one NRP retrievals from Maastricht category III DCD donors were performed at three UK centers. NRP was established postasystole via aortic and caval cannulation and maintained for 2 h. Blood gases and biochemistry were monitored to assess organ function. Sixty-three organs were recovered. Forty-nine patients were transplanted. The median time from asystole to NRP was 16 min (range 10-23 min). Thirty-two patients received a kidney transplant. The median cold ischemia time was 12 h 30 min (range 5 h 25 min-18 h 22 min). The median creatinine at 3 and 12 months was 107 µmol/L (range 72-222) and 121 µmol/L (range 63-157), respectively. Thirteen (40%) recipients had delayed graft function and four lost the grafts. Eleven patients received a liver transplant. The first week median peak ALT was 389 IU/L (range 58-3043). One patient had primary nonfunction. Two combined pancreas-kidney transplants, one islet transplant and three double lung transplants were performed with primary function. NRP in DCD donation facilitates organ recovery and may improve short-term outcomes.

alpha(1)-Adrenoceptor antagonists prevent paracetamol-induced hepatotoxicity in mice.

BACKGROUND AND PURPOSE: Paracetamol, a major cause of acute liver failure (ALF) represents a significant clinical problem. Adrenoceptor stimulation or antagonism can modulate chemical-induced hepatotoxicity. We investigated the role of endogenous catecholamines and alpha(1)-adrenoceptors in the development of paracetamol- induced hepatotoxicity. EXPERIMENTAL APPROACH: Paracetamol (3.5 mmol kg(-1)) was administered to male CD-1 mice, with and without alpha(1)-adrenoceptor antagonists (prazosin, doxazosin, terazosin and tamsulosin; 35.7 micromol kg(-1)). Serum transaminases and hepatic glutathione (GSH) levels were assessed as markers of hepatic damage. Paracetamol bioactivation was assessed by covalent binding, hepatic and urinary conjugate formation and uridine glucuronosyltransferase activity. Plasma catecholamines levels and hepatic congestion were also analysed. KEY RESULTS: Plasma catecholamine levels were significantly elevated 5 h post paracetamol administration. Prazosin prevented hepatotoxicity when administered 1 h before a toxic paracetamol insult and importantly, when administered up to 1 h post paracetamol injection. Prazosin had no effect on paracetamol-induced depletion of hepatic GSH, paracetamol bioactivation or paracetamol-induced transcription of defence genes. Paracetamol toxicity is associated with marked accumulation of erythrocytes within hepatic sinusoids and prazosin completely prevented this accumulation. CONCLUSION AND IMPLICATIONS: Paracetamol-induced hepatocellular damage is associated with increased circulating catecholamines. alpha(1)-Adrenoceptor antagonists conferred complete protection from paracetamol -induced hepatotoxicity. Protection was associated with absence of hepatic erythrocyte accumulation. Increased catecholamine levels may contribute to the pathophysiology of paracetamol-induced hepatotoxicity by compromising hepatic perfusion. Protection against paracetamol toxicity by alpha(1) antagonists in mice has implications for therapeutic management of patients presenting with paracetamol overdose and ALF.