A proposal from the liver forum for the management of comorbidities in non-alcoholic steatohepatitis therapeutic trials.

The current document has been developed by the Liver Forum who mandated the NAFLD-Associated Comorbidities Working Group - a multistakeholder group comprised of experts from academic medicine, industry and patient associations - to identify aspects of diverse comorbidities frequently associated with non-alcoholic steatohepatitis (NASH) that can interfere with the conduct of therapeutic trials and, in particular, impact efficacy and safety results. The objective of this paper is to propose guidance for the management of relevant comorbidities in both candidates and actual participants in NASH therapeutic trials. We relied on specific guidelines from scientific societies, when available, but adapted them to the particulars of NASH trials with the aim of addressing multiple interacting requirements such as maintaining patient safety, reaching holistic therapeutic objectives, minimising confounding effects on efficacy and safety of investigational agents and allowing for trial completion. We divided the field of action into: first, analysis and stabilisation of the patient's condition before inclusion in the trial and, second, management of comorbidities during trial conduct. For the former, we discussed the concept of acceptable vs. optimal control of comorbidities, defined metabolic and ponderal stability prior to randomisation and weighed the pros and cons of a run-in period. For the latter, we analysed non-hepatological comorbid conditions for changes or acute events possibly occurring during the trial, including changes in alcohol consumption, in order to detail when specific interventions are necessary and how best to manage concomitant drug intake in line with methodological constraints. These recommendations are intended to act as a guide for clinical trialists and are open to further refinement when additional data become available.

Oncogenic Addiction of Fibrolamellar Hepatocellular Carcinoma to the Fusion Kinase DNAJB1-PRKACA.

PURPOSE: Gene fusions are drivers of many pediatric tumors. In fibrolamellar hepatocellular carcinoma (FLC), a fusion of DNAJB1 and PRKACA is the dominant recurrent mutation. Expression of the DNAJB1-PRKACA fusion gene in mice results in a tumor that recapitulates FLC. However, it is not known whether transient expression of DNAJB1-PRKACA is sufficient only to trigger tumor formation or whether ongoing expression is necessary for maintenance and progression. EXPERIMENTAL DESIGN: We screened short hairpin RNAs (shRNA) tiled over the fusion junction and identified several potent and specific candidates in vitro and two independent FLC patient-derived xenografts (PDX). RESULTS: We show that continued DNAJB1-PRKACA expression is not only required for continued tumor growth, but additionally its inhibition results in cell death. Inhibition of DNAJB1-PRKACA by an inducible shRNA in cells of PDX of FLC resulted in cell death in vitro. Induction of the shRNA inhibits FLC tumors growing in mice with no effect on xenografts from a hepatocellular carcinoma cell line engineered to express DNAJB1-PRKACA. CONCLUSIONS: Our results validate DNAJB1-PRKACA as the oncogene in FLC and demonstrate both a continued requirement for the oncogene for tumor growth as well as an oncogenic addiction that can be exploited for targeted therapies. We anticipate our approach will be useful for investigations of other fusion genes in pediatric cancers and spur development of precision therapies.

Targeting BCL-XL in fibrolamellar hepatocellular carcinoma.

Fibrolamellar hepatocellular carcinoma (FLC) is a rare and often lethal liver cancer with no proven effective systemic therapy. Inhibition of the antiapoptotic protein BCL-XL was found to synergize with a variety of systemic therapies in vitro using cells dissociated from patient-derived xenografts (PDX) of FLC or cells dissociated directly from surgical patient resections. As BCL-XL is physiologically expressed in platelets, prior efforts to leverage this vulnerability in other cancers have been hampered by severe thrombocytopenia. To overcome this toxicity, we treated FLC models with DT2216, a proteolysis targeting chimera (PROTAC) that directs BCL-XL for degradation via the von Hippel-Lindau (VHL) E3 ligase, which is minimally expressed in platelets. The combination of irinotecan and DT2216 in vitro on cells directly acquired from patients or in vivo using several xenografts derived from patients with FLC demonstrated remarkable synergy and at clinically achievable doses not associated with significant thrombocytopenia.

Human hepatocyte PNPLA3-148M exacerbates rapid non-alcoholic fatty liver disease development in chimeric mice.

Advanced non-alcoholic fatty liver disease (NAFLD) is a rapidly emerging global health problem associated with pre-disposing genetic polymorphisms, most strikingly an isoleucine to methionine substitution in patatin-like phospholipase domain-containing protein 3 (PNPLA3-I148M). Here, we study how human hepatocytes with PNPLA3 148I and 148M variants engrafted in the livers of broadly immunodeficient chimeric mice respond to hypercaloric diets. As early as four weeks, mice developed dyslipidemia, impaired glucose tolerance, and steatosis with ballooning degeneration selectively in the human graft, followed by pericellular fibrosis after eight weeks of hypercaloric feeding. Hepatocytes with the PNPLA3-148M variant, either from a homozygous 148M donor or overexpressed in a 148I donor background, developed microvesicular and severe steatosis with frequent ballooning degeneration, resulting in more active steatohepatitis than 148I hepatocytes. We conclude that PNPLA3-148M in human hepatocytes exacerbates NAFLD. These models will facilitate mechanistic studies into human genetic variant contributions to advanced fatty liver diseases.

Human liver organoids for disease modeling of fibrolamellar carcinoma.

Fibrolamellar carcinoma (FLC) is a rare, often lethal, liver cancer affecting adolescents and young adults, for which there are no approved therapeutics. The development of therapeutics is hampered by a lack of in vitro models. Organoids have shown utility as a model system for studying many diseases. In this study, tumor tissue and the adjacent non-tumor liver were obtained at the time of surgery. The tissue was dissociated and grown as organoids. We developed 21 patient-derived organoid lines: 12 from metastases, three from the liver tumor and six from adjacent non-tumor liver. These patient-derived FLC organoids recapitulate the histologic morphology, immunohistochemistry, and transcriptome of the patient tumor. Patient-derived FLC organoids were used in a preliminary high-throughput drug screen to show proof of concept for the identification of therapeutics. This model system has the potential to improve our understanding of this rare cancer and holds significant promise for drug testing and development.

Mouse characteristics that affect establishing xenografts from hepatocellular carcinoma patient biopsies in the United States.

BACKGROUND: Hepatocellular carcinoma (HCC) patient-derived xenograft (PDX) models hold potential to advance knowledge in HCC biology to help improve systemic therapies. Beside hepatitis B virus-associated tumors, HCC is poorly established in PDX. METHODS: PDX formation from fresh HCC biopsies were obtained and implanted intrahepatically or in subrenal capsule (SRC). Mouse liver injury was induced in immunodeficient Fah-/-  mice through cycling off nitisinone after HCC biopsy implantation, versus continuous nitisinone as non-liver injury controls. Mice with macroscopically detectable PDX showed rising human alpha1-antitrypsin (hAAT) serum levels, and conversely, no PDX was observed in mice with undetectable hAAT. RESULTS: Using rising hAAT as a marker for PDX formation, 20 PDX were established out of 45 HCC biopsy specimens (44%) reflecting the four major HCC etiologies most commonly identified at Memorial SloanKettering similar to many other institutions in the United States. PDX was established only in severely immunodeficient mice lacking lymphocytes and NK cells. Implantation under the renal capsule improved PDX formation two-fold compared to intrahepatic implantation. Two out of 18 biopsies required murine liver injury to establish PDX, one associated with hepatitis C virus and one with alcoholic liver disease. PDX tumors were histologically comparable to biopsy specimens and 75% of PDX lines could be passaged. CONCLUSIONS: Using cycling off nitisinone-induced liver injury, HCC biopsies implanted under the renal capsule of severely immunodeficient mice formed PDX with 57% efficiency as determined by rising hAAT levels. These findings facilitate a more efficient make-up of PDX for research into subset-specific HCC.

An Interesting Case of Isolated False-Reactive Hepatitis B Surface Antigen.

The standard serologic markers used to diagnose hepatitis B infection include hepatitis B surface antigen (HBsAg), hepatitis B surface antibody (anti-HBs), total hepatitis B core antibody (anti-HBc), and IgM antibody to hepatitis B core antigen (IgM anti-HBc). Different markers or combinations of markers are used to identify different phases of HBV infection and determine whether a patient has acute or chronic infection or immunity due to prior infection or vaccination or is seronegative and susceptible to future infection. Isolated HBsAg seropositivity is a peculiar serological pattern that requires investigation. Herein, we present a case of an asymptomatic female without a history of liver disease or evident risk factors for hepatitis, who underwent screening for infectious disease prior to resection of basal cell carcinoma involving her eyelid. The patient's laboratory testing showed positivity for HBsAg and the HIV 1/2 screen. To investigate, we performed serial dilutions, utilized heterophilicantibody blocking tubes, and repeated analysis using a different commercial assay (Abbott Architect i2000), all in support of a false-positive result attributed to a heterophilic antibody. Hence, we demonstrate that heterophilic antibody interference can result in isolated HBsAg positivity and recommend considering this form of interference in the differential where there is low clinical suspicion for viral infection.

Identification of Novel Therapeutic Targets for Fibrolamellar Carcinoma Using Patient-Derived Xenografts and Direct-from-Patient Screening.

To repurpose therapeutics for fibrolamellar carcinoma (FLC), we developed and validated patient-derived xenografts (PDX) from surgical resections. Most agents used clinically and inhibitors of oncogenes overexpressed in FLC showed little efficacy on PDX. A high-throughput functional drug screen found primary and metastatic FLC were vulnerable to clinically available inhibitors of TOPO1 and HDAC and to napabucasin. Napabucasin's efficacy was mediated through reactive oxygen species and inhibition of translation initiation, and specific inhibition of eIF4A was effective. The sensitivity of each PDX line inversely correlated with expression of the antiapoptotic protein Bcl-xL, and inhibition of Bcl-xL synergized with other drugs. Screening directly on cells dissociated from patient resections validated these results. This demonstrates that a direct functional screen on patient tumors provides therapeutically informative data within a clinically useful time frame. Identifying these novel therapeutic targets and combination therapies is an urgent need, as effective therapeutics for FLC are currently unavailable. SIGNIFICANCE: Therapeutics informed by genomics have not yielded effective therapies for FLC. A functional screen identified TOPO1, HDAC inhibitors, and napabucasin as efficacious and synergistic with inhibition of Bcl-xL. Validation on cells dissociated directly from patient tumors demonstrates the ability for functional precision medicine in a solid tumor.This article is highlighted in the In This Issue feature, p. 2355.

A Human Organoid Model of Aggressive Hepatoblastoma for Disease Modeling and Drug Testing.

Hepatoblastoma is the most common childhood liver cancer. Although survival has improved significantly over the past few decades, there remains a group of children with aggressive disease who do not respond to current treatment regimens. There is a critical need for novel models to study aggressive hepatoblastoma as research to find new treatments is hampered by the small number of laboratory models of the disease. Organoids have emerged as robust models for many diseases, including cancer. We have generated and characterized a novel organoid model of aggressive hepatoblastoma directly from freshly resected patient tumors as a proof of concept for this approach. Hepatoblastoma tumor organoids recapitulate the key elements of patient tumors, including tumor architecture, mutational profile, gene expression patterns, and features of Wnt/ß-catenin signaling that are hallmarks of hepatoblastoma pathophysiology. Tumor organoids were successfully used alongside non-tumor liver organoids from the same patient to perform a drug screen using twelve candidate compounds. One drug, JQ1, demonstrated increased destruction of liver organoids from hepatoblastoma tumor tissue relative to organoids from the adjacent non-tumor liver. Our findings suggest that hepatoblastoma organoids could be used for a variety of applications and have the potential to improve treatment options for the subset of hepatoblastoma patients who do not respond to existing treatments.

Administration of the Hyper-immune Bovine Colostrum Extract IMM-124E Ameliorates Experimental Murine Colitis.

BACKGROUND AND AIMS: Inflammatory bowel disease [IBD] is accompanied by lesions in the epithelial barrier, which allow translocation of bacterial products from the gut lumen to the host's circulation. IMM-124E is a colostrum-based product containing high levels of anti-E.coli-LPS IgG, and might limit exposure to bacterial endotoxins. Here, we investigated whether IMM-124E can ameliorate intestinal inflammation. METHODS: Acute colitis was induced in WT C57Bl/6J mice by administration of 2.5% dextran sodium sulphate [DSS] for 7 days. T cell transfer colitis was induced via transfer of 0.5 x 106 naïve T cells into RAG2-/- C57Bl/6J mice. IMM-124E was administered daily by oral gavage, either preventively or therapeutically. RESULTS: Treatment with IMM-124E significantly ameliorated colitis in acute DSS colitis and in T cell transfer colitis. Maximum anti-inflammatory effects were detected at an IMM-124E concentration of 100 mg/kg body weight, whereas 25 mg/kg and 500 mg/kg were less effective. Histology revealed reduced levels of infiltrating immune cells and less pronounced mucosal damage. Flow cytometry revealed reduced numbers of effector T helper cells in the intestine, whereas levels of regulatory T cells were enhanced. IMM-124E treatment reduced the DSS-induced increase of serum levels of lipopolysaccharide [LPS]-binding protein, indicating reduced systemic LPS exposure. CONCLUSIONS: Our results demonstrate that oral treatment with IMM-124E significantly reduces intestinal inflammation, via decreasing the accumulation of pathogenic T cells and concomitantly increasing the induction of regulatory T cells. Our study confirms the therapeutic efficacy of IMM-124E in acute colitis and suggests that administration of IMM-124E might represent a novel therapeutic strategy to induce or maintain remission in chronic colitis.

Glycosphingolipids Prevent APAP and HMG-CoA Reductase Inhibitors-mediated Liver Damage: A Novel Method for "Safer Drug" Formulation that Prevents Drug-induced Liver Injury.

Background and Aims: Acetaminophen (APAP) and HMG-CoA reductase inhibitors are common causes of drug-induced liver injury (DILI). This study aimed to determine the ability to reduce APAP- and statins-mediated liver injury by using formulations that combine glycosphingolipids and vitamin E. Methods: Mice were injected with APAP or with statins and treated before and after with ß-glucosylceramide (GC), with or without vitamin E. Mice were followed for changes in liver enzymes, liver histology, hepatic expression of JNK, STAT3 and caspase 3, as well as intrahepatic natural killer T cells (NKT) and the serum cytokine levels by flow cytometry. Results: Administration of GC before or after APAP alleviated the liver damage, as noted by a reduction of the liver enzymes, improvement in the liver histology and decreased hepatic caspase 3 expression. Beneficial effect was associated with a reduction of the intrahepatic NKT, JNK expression in the liver, and increased glutathione in the liver, and decreased TNF-α serum levels. Synergistic effect of co-administration of GC with vitamin E was observed. Similar protective effect of GC on statin-mediated liver damage was documented by a reduction in liver enzymes and improved liver histology, which was mediated by reduction of NKT, increased STAT3 expression in the liver, and reduced the TGF-ß and IL17 levels. Conclusions: ß-glycosphingolipids exert a hepatoprotective effect on APAP- and statins-mediated liver damage. Vitamin E exerted a synergistic effect to that of GC. The generation of "safer drug" formulations, which include an active molecule combined with a hepatoprotective adjuvant, may provide an answer to the real unmet need of DILI.

PSMD5 Inactivation Promotes 26S Proteasome Assembly during Colorectal Tumor Progression.

Protein degradation by the ubiquitin-proteasome system (UPS) is central to protein homeostasis and cell survival. The active 26S proteasome is a large protease complex consisting of a catalytic 20S subunit and 19S regulatory particles. Cancer cells are exposed to considerable protein overload due to high metabolic rates, reprogrammed energy metabolism, and aneuploidy. Here we report a mechanism that facilitates the assembly of active 26S proteasomes in malignant cells. Upon tumorigenic transformation of the gut epithelium, 26S proteasome assembly was significantly enhanced, but levels of individual subunits were not changed. This enhanced assembly of 26S proteasomes increased further with tumor progression and was observed specifically in transformed cells, but not in other rapidly dividing cells. Moreover, expression of PSMD5, an inhibitor of proteasome assembly, was reduced in intestinal tumors and silenced with tumor progression. Reexpression of PSMD5 in tumor cells caused decreased 26S assembly and accumulation of polyubiquitinated proteins. These results suggest that inhibition of cancer-associated proteasome assembly may provide a novel therapeutic strategy to selectively kill cancer cells.Significance: Enhanced cancer-associated proteasome assembly is a major stress response that allows tumors to adapt to and to withstand protein overload.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/13/3458/F1.large.jpg Cancer Res; 78(13); 3458-68. ©2018 AACR.

Non coding RNA analysis in fibrolamellar hepatocellular carcinoma.

Fibrolamellar hepatocellular carcinoma (FLC) is a rare primary liver cancer found in adolescents and young adults without underlying liver disease. A deletion of ~400 kD has been found in one copy of chromosome 19 in the tumor tissue of all patients tested. This produces a fusion of the genes DNAJB1 and PRKACA which, in turn, produces a chimeric transcript and protein. Transcriptomic analysis of the tumor has shown upregulation of various oncologically relevant pathways, including EGF/ErbB, Aurora Kinase A, pak21 and wnt. To explore other factors that may contribute to oncogenesis, we examined the microRNA (miRNA) and long non-coding RNA (lncRNA) expression in FLC. The non-coding RNA expression profile in tumor tissue samples is distinctly different from the adjacent normal liver and from other liver tumors. Furthermore, miRZip knock down or over expression of certain miRNAs led to changes in the levels of coding genes that recapitulated changes observed in FLC, suggesting mechanistically that the changes in the cellular levels of miRNA are not merely correlative. Thus, in addition to serving as diagnostic tools for FLC, non-coding RNAs may serve as therapeutic targets.

Fibrolamellar Carcinoma: Recent Advances and Unresolved Questions on the Molecular Mechanisms.

Fibrolamellar hepatocellular carcinoma (FLC) is a rare form of primary liver cancer that affects adolescents and young adults without underlying liver disease. Surgery remains the mainstay of therapy; however, most patients are either not surgical candidates or suffer from recurrence. There is no approved systemic therapy and the overall survival remains poor. Historically classified as a subtype of hepatocellular carcinoma (HCC), FLC has a unique clinical, histological, and molecular presentation. At the genomic level, FLC contains a single 400kB deletion in chromosome 19, leading to a functional DNAJB1-PRKACA fusion protein. In this review, we detail the recent advances in our understanding of the molecular underpinnings of FLC and outline the current knowledge gaps.

Intracranial metastasis in fibrolamellar hepatocellular carcinoma.

Fibrolamellar hepatocellular carcinoma (FLHCC) is a rare liver malignancy in adolescents and young adults. Surgery is the mainstay of therapy for primary and metastatic disease. Most patients relapse, with development of both local and distant metastases. Brain metastases from solid tumors are rare in the pediatric and young adult population. Here, we document three patients with brain metastases from FLHCC, confirmed by histology and molecular characterization of the chimeric fusion DNAJB1-PRKACA, each necessitating neurosurgical intervention. These observations highlight the ability of FLHCC to metastasize to the brain and suggest the need for surveillance neuroimaging for patients with advanced-stage disease.

DNAJB1-PRKACA fusion kinase interacts with ß-catenin and the liver regenerative response to drive fibrolamellar hepatocellular carcinoma.

A segmental deletion resulting in DNAJB1-PRKACA gene fusion is now recognized as the signature genetic event of fibrolamellar hepatocellular carcinoma (FL-HCC), a rare but lethal liver cancer that primarily affects adolescents and young adults. Here we implement CRISPR-Cas9 genome editing and transposon-mediated somatic gene transfer to demonstrate that expression of either the endogenous fusion protein or a chimeric cDNA leads to the formation of indolent liver tumors in mice that closely resemble human FL-HCC. Notably, overexpression of the wild-type PRKACA was unable to fully recapitulate the oncogenic activity of DNAJB1-PRKACA, implying that FL-HCC does not simply result from enhanced PRKACA expression. Tumorigenesis was significantly enhanced by genetic activation of ß-catenin, an observation supported by evidence of recurrent Wnt pathway mutations in human FL-HCC, as well as treatment with the hepatotoxin 3,5-diethoxycarbonyl-1,4-dihydrocollidine, which causes tissue injury, inflammation, and fibrosis. Our study validates the DNAJB1-PRKACA fusion kinase as an oncogenic driver and candidate drug target for FL-HCC, and establishes a practical model for preclinical studies to identify strategies to treat this disease.

Oral Administration of ß-Glucosylceramide for the Treatment of Insulin Resistance and Nonalcoholic Steatohepatitis: Results of a Double-Blind, Placebo-Controlled Trial.

ß-glucosylceramide (GC) is a naturally occurring glycosphingolipid that was shown to improve hepatic steatosis, steatohepatitis, and insulin resistance in animal models of nonalcoholic fatty liver disease. In this study, we evaluated the safety and efficacy of oral administration of GC in subjects with nonalcoholic steatohepatitis (NASH). Twenty-three patients with biopsy proven NASH were enrolled in a double-blind, placebo-controlled trial. Patients were orally administered daily with 7.5 mg of GC. Patients were followed for safety, liver enzymes, HbA1c, insulin sensitivity, lipid profile, hepatic fat content as measured by magnetic resonance imaging (MRI), and NASH score on liver biopsy. No treatment-related adverse events were observed during treatment. In a per protocol analysis of data, oral administration of GC decreased the hepatic fat content as measured by MRI in GC-treated compared with placebo. HbA1C decreased in patients treated with GC. GC treatment was associated with a milder decrease in the high-density lipoprotein serum levels. The beneficial effects were associated with a decrease in CD4 and NKT cell subsets of lymphocytes. Due to the small number of subjects enrolled, differences did reach statistical significance. Oral administration of GC is safe and biologically active in patients with NASH and insulin resistance.

Autophagy confers resistance to lipopolysaccharide-induced mouse hepatocyte injury.

During sepsis, bacterial products, particularly LPS, trigger injury in organs such as the liver. This common condition remains largely untreatable, in part due to a lack of understanding of how high concentrations of LPS cause cellular injury. In the liver, the lysosomal degradative pathway of autophagy performs essential hepatoprotective functions and is induced by LPS. We, therefore, examined whether hepatocyte autophagy protects against liver injury from septic levels of LPS. Mice with an inducible hepatocyte-specific knockout of the critical autophagy gene Atg7 were examined for their sensitivity to high-dose LPS. Increased liver injury occurred in knockout mice, as determined by significantly increased serum alanine aminotransferase levels, histological evidence of liver injury, terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate nick end-labeling, and effector caspase-3 and -7 activation. Hepatic inflammation and proinflammatory cytokine induction were unaffected by the decrease in hepatocyte autophagy. Although knockout mice had normal NF-κB signaling, hepatic levels of Akt1 and Akt2 phosphorylation in response to LPS were decreased. Cultured hepatocytes from knockout mice displayed a generalized defect in Akt signaling in response to multiple stimuli, including LPS, TNF, and IL-1ß. Akt activation mediates hepatocyte resistance to TNF cytotoxicity, and anti-TNF antibodies significantly decreased LPS-induced liver injury in knockout mice, indicating that the loss of autophagy sensitized to TNF-dependent liver damage. Hepatocyte autophagy, therefore, protects against LPS-induced liver injury. Conditions such as aging and steatosis that impair hepatic autophagy may predispose to poor outcomes from sepsis through this mechanism.

Transcriptomic characterization of fibrolamellar hepatocellular carcinoma.

Fibrolamellar hepatocellular carcinoma (FLHCC) tumors all carry a deletion of ∼ 400 kb in chromosome 19, resulting in a fusion of the genes for the heat shock protein, DNAJ (Hsp40) homolog, subfamily B, member 1, DNAJB1, and the catalytic subunit of protein kinase A, PRKACA. The resulting chimeric transcript produces a fusion protein that retains kinase activity. No other recurrent genomic alterations have been identified. Here we characterize the molecular pathogenesis of FLHCC with transcriptome sequencing (RNA sequencing). Differential expression (tumor vs. adjacent normal tissue) was detected for more than 3,500 genes (log2 fold change ≥ 1, false discovery rate ≤ 0.01), many of which were distinct from those found in hepatocellular carcinoma. Expression of several known oncogenes, such as ErbB2 and Aurora Kinase A, was increased in tumor samples. These and other dysregulated genes may serve as potential targets for therapeutic intervention.

Use of the methacetin breath test to classify the risk of cirrhotic complications and mortality in patients evaluated/listed for liver transplantation.

BACKGROUND & AIMS: The MELD score predicts short-term mortality in patients with cirrhosis; however, some patients with low scores develop complications and die unexpectedly. Consequently, we evaluated the diagnostic accuracy of the methacetin breath test (MBT), an assay of liver metabolic function, and the MELD score, to predict the risk of complications of cirrhosis and liver-related death. METHODS: One hundred sixty-five patients with cirrhosis received oral (13)C-methacetin; (13)CO2 was measured in expired breath (BreathID; Exalenz). The cumulative percent dose recovery of (13)CO2 at 20 min with a threshold of ⩽0.55% (high-risk) and >0.55% (low risk) most accurately predicted liver-related death and the risk of cirrhotic complications within one year. MELD thresholds of ⩾15 and ⩾19 were also examined to predict the same endpoints. RESULTS: Dose recovery ⩽0.55% and MELD ⩾19 both predicted liver-related death (HR 12.6 [95% CI 1.6-98.3]; p=0.016, and HR 5.5 [1.6-18.9]; p=0.007, respectively); MELD ⩾15 did not. Dose recovery ⩽0.55% (HR 1.9 [1.1-3.2]; p=0.03) also predicted the risk of ⩾1 complication(s), and was particularly able to foretell the risk of development/exacerbation of ascites (HR 4.7 [1.8-11.9]; p=0.001), which was not achieved by either MELD threshold. Finally, in patients with MELD <19, dose recovery ⩽0.55% predicted the risk of death (p=0.017), development of ⩾1 cirrhotic complication(s) (p=0.062), and development/exacerbation of ascites (p=0.0009). CONCLUSIONS: In this pilot study, methacetin breath testing predicted the risk of liver-related death and development/exacerbation of ascites more accurately than MELD ⩾15 or ⩾19. In patients with low MELD (<19points), MBT may be useful to identify patients in whom the frequency of clinical observation should be intensified.