NASH limits anti-tumour surveillance in immunotherapy-treated HCC.

Hepatocellular carcinoma (HCC) can have viral or non-viral causes1-5. Non-alcoholic steatohepatitis (NASH) is an important driver of HCC. Immunotherapy has been approved for treating HCC, but biomarker-based stratification of patients for optimal response to therapy is an unmet need6,7. Here we report the progressive accumulation of exhausted, unconventionally activated CD8+PD1+ T cells in NASH-affected livers. In preclinical models of NASH-induced HCC, therapeutic immunotherapy targeted at programmed death-1 (PD1) expanded activated CD8+PD1+ T cells within tumours but did not lead to tumour regression, which indicates that tumour immune surveillance was impaired. When given prophylactically, anti-PD1 treatment led to an increase in the incidence of NASH-HCC and in the number and size of tumour nodules, which correlated with increased hepatic CD8+PD1+CXCR6+, TOX+, and TNF+ T cells. The increase in HCC triggered by anti-PD1 treatment was prevented by depletion of CD8+ T cells or TNF neutralization, suggesting that CD8+ T cells help to induce NASH-HCC, rather than invigorating or executing immune surveillance. We found similar phenotypic and functional profiles in hepatic CD8+PD1+ T cells from humans with NAFLD or NASH. A meta-analysis of three randomized phase III clinical trials that tested inhibitors of PDL1 (programmed death-ligand 1) or PD1 in more than 1,600 patients with advanced HCC revealed that immune therapy did not improve survival in patients with non-viral HCC. In two additional cohorts, patients with NASH-driven HCC who received anti-PD1 or anti-PDL1 treatment showed reduced overall survival compared to patients with other aetiologies. Collectively, these data show that non-viral HCC, and particularly NASH-HCC, might be less responsive to immunotherapy, probably owing to NASH-related aberrant T cell activation causing tissue damage that leads to impaired immune surveillance. Our data provide a rationale for stratification of patients with HCC according to underlying aetiology in studies of immunotherapy as a primary or adjuvant treatment.

Alcoholic Foamy Degeneration, an Entity Resembling Alcohol-Associated Hepatitis: Diagnosis, Prognosis, and Molecular Profiling.

BACKGROUND & AIMS: Alcoholic foamy degeneration (AFD) is a condition with similar clinical presentation to alcohol-associated hepatitis (AH), but with a specific histologic pattern. Information regarding the prevalence and prognosis of AFD is scarce and there are no tools for a noninvasive diagnosis. METHODS: A cohort of patients admitted to the Hospital Clinic of Barcelona for clinical suspicion of AH who underwent liver biopsy was included. Patients were classified as AFD, AH, or other findings, according to histology. Clinical features, histology, and genetic expression of liver biopsy specimens were analyzed. The accuracy of National Institute on Alcohol Abuse and Alcoholism criteria and laboratory parameters for differential diagnosis were investigated. RESULTS: Of 230 patients with a suspicion of AH, 18 (8%) met histologic criteria for AFD, 184 (80%) had definite AH, and 28 (12%) had other findings. In patients with AFD, massive steatosis was more frequent and the fibrosis stage was lower. AFD was characterized by down-regulation of liver fibrosis and inflammation genes and up-regulation of lipid metabolism and mitochondrial function genes. Patients with AFD had markedly better long-term survival (100% vs 57% in AFD vs AH; P = .002) despite not receiving corticosteroid treatment, even in a model for end-stage liver disease-matched sensitivity analysis. Serum triglyceride levels had an area under the receiver operating characteristic of 0.886 (95% CI, 0.807-0.964) for the diagnosis of AFD, whereas the National Institute on Alcohol Abuse and Alcoholism criteria performed poorly. A 1-step algorithm using triglyceride levels of 225 mg/dL (sensitivity, 0.77; specificity, 0.90; and Youden index, 0.67) is proposed for differential diagnosis. CONCLUSIONS: AFD in the setting of suspicion of AH is not uncommon. A differential diagnosis is important because prognosis and treatment differ largely. Triglyceride levels successfully identify most patients with AFD and may be helpful in decision making.

Restrospective reappraisal of the prognostic classification of spitzoid melanocytic neoplasms after BRAF and NRAS mutation characterisation: a single institution experience.

AIMS: The current WHO classification of melanocytic tumours excludes neoplasms showing BRAF or NRAS mutations from the Spitz category. This study aimed to review and reclassify atypical melanocytic tumours with spitzoid morphological features diagnosed between 2009 and 2021 in our hospital after expanding the molecular profile, including BRAF and NRAS mutations in all cases. METHODS AND RESULTS: A total of 71 neoplasms showing spitzoid features (Spitz-like) and atypia were included. The risk of progression of tumours was first studied by integrating the morphology, immunohistochemistry (p16, Ki67, HMB45 and PRAME) and fluorescence in-situ hybridisation (FISH) results (melanoma multiprobe and 9p21). In a second step, after expanding the molecular study, including BRAF and NRAS mutational status, the neoplasms were finally classified into four subgroups: atypical Spitz tumour (AST, n = 45); BRAF-mutated naevus/low-grade melanocytoma with spitzoid morphology (BAMS, n = 2); Spitz melanoma (SM, n = 14); and BRAF or NRAS mutated melanoma with spitzoid features (MSF, n = 10). Follow-up of patients revealed uneventful results for AST and BAMS. Only one SM presented lymph node metastasis after 134 months. Conversely, patients with MSF showed an unfavourable outcome: three developed lymph node metastases after a mean time of 22 months, with one patient presenting distant metastasis and dying of the disease 64 months from diagnosis. The progression-free survival showed significant differences between the four groups of spitzoid tumours (P < 0.001) and between both melanoma subtypes (P = 0.012). CONCLUSIONS: The classification and prognostication of atypical neoplasms with spitzoid features requires the integration of histomorphology with the molecular investigation of tumours, which should include BRAF and NRAS mutational status.

Interspecies transcriptomic comparison identifies a potential porto-sinusoidal vascular disorder rat model suitable for in vivo drug testing.

BACKGROUND: Porto-sinusoidal vascular disorder (PSVD) involves a group of rare vascular liver diseases of unknown aetiology that may lead to the development of portal hypertension and its life-threatening complications. Its pathophysiology is not well understood, and animal models described to date do not fully recapitulate human disease. METHODS: We developed three different PSVD rat models by either immunosensitization (repetitive intraportal LPS or intramuscular spleen extract injections) or toxic (Selfox: combination of FOLFOX and a selenium-enriched diet) treatment and characterized them at haemodynamic, histological, biochemical and transcriptional levels. We compared these results to human data. RESULTS: All three models developed significant portal hypertension, while only the LPS and the Selfox models displayed PSVD-specific and nonspecific histological alterations in the absence of cirrhosis. Transcriptional comparison between rat models and human data showed that both LPS and Selfox models recapitulate the main transcriptional alterations observed in humans, especially regarding haemostasis, oxidative phosphorylation and cell cycle regulation. Reproducibility and feasibility was higher for the Selfox model. CONCLUSIONS: The Selfox rat model faithfully reproduces the main alterations described in PSVD. Its use as a preclinical model for drug testing in progressing PSVD can be a significant step forward towards the development of new therapeutic targets for this rare condition.

Combined WNT-activated deep-penetrating/plexiform melanocytoma: insights into clinicopathological and molecular characterization.

BACKGROUND: A combined deep-penetrating tumour redefined as WNT-activated deep-penetrating/plexiform melanocytoma (DPM), may pose challenging clinical and histological diagnoses. OBJECTIVES: To review the clinicopathological characteristics of combined DPMs and characterize the molecular profile of atypical and malignant forms. METHODS: The study included 51 patients with combined DPMs diagnosed at the Hospital Clinic of Barcelona and the University of Florence between 2012 and 2020. Clinical data, dermoscopy images (when available) and histological characteristics were reviewed. Immunohistochemistry for ß-catenin, LEF1, HMB45, Ki67, p16 and PRAME (preferentially expressed antigen in melanoma) was performed. Atypical forms underwent next-generation sequencing (NGS) panel analysis, including driver genes implicated in DPMs, TERT-promoter (p) mutations and the investigation of the 9p21 locus via fluorescence in situ hybridization. RESULTS: Among the 51 patients (32 females and 19 males, age range 4-74 years), 68% with available clinical data (15/22) were initially suspected of having melanoma. Except for one patient, complete excision resulted in no recurrences or metastases. One patient who had an incompletely excised combined DPM developed a lymph node melanoma metastasis 10 years later. In the 51 patients, 10 samples (20%) showed atypical histological features; 7 (14%) exhibited a significant loss of p16 expression; and 2 (4%) showed a high-proliferative index (Ki67 over 5%). NGS analysis in 11 patients revealed a double mutation BRAFV600E and exon 3 CTNNB1; no TERTp mutations were detected. CONCLUSIONS: Clinical suspicion of melanoma is common in combined DPMs, but malignant progression is infrequent in tumours lacking high-grade atypia or proliferation. These findings are congruent with the consideration of these lesions as intermediate-grade tumours or melanocytomas.

Feasibility and Impact of Embedding an Extended DNA and RNA Tissue-Based Sequencing Panel for the Routine Care of Patients with Advanced Melanoma in Spain.

Targeted NGS allows a fast and efficient multi-gene analysis and the detection of key gene aberrations in melanoma. In this study, we aim to describe the genetic alterations in a series of 87 melanoma cases using the oncomine focus assay (OFA), relate these results with the clinicopathological features of the patients, and compare them with our previous study results in which we used a smaller panel, the oncomine solid tumor (OST) DNA kit. Patients diagnosed with advanced melanoma at our center from 2020 to 2022 were included and DNA and RNA were extracted for sequencing. Common mutated genes were BRAF (29%), NRAS (28%), ALK, KIT, and MAP2K1 (5% each). Co-occurring mutations were detected in 29% of the samples, including BRAF with KIT, CTNNB1, EGFR, ALK, HRAS, or MAP2K1. Amplifications and rearrangements were detected in 5% of cases. Only BRAF mutation showed a significant statistical association with sun exposure. For patients with a given genetic profile, the melanoma survival and recurrence-free survival rates were equivalent, but not for stage and LDH values. This expanded knowledge of molecular alterations has helped to more comprehensively characterize our patients and has provided relevant information for deciding the best treatment strategy.

Inflamed and non-inflamed classes of HCC: a revised immunogenomic classification.

OBJECTIVE: We previously reported a characterisation of the hepatocellular carcinoma (HCC) immune contexture and described an immune-specific class. We now aim to further delineate the immunogenomic classification of HCC to incorporate features that explain responses/resistance to immunotherapy. DESIGN: We performed RNA and whole-exome sequencing, T-cell receptor (TCR)-sequencing, multiplex immunofluorescence and immunohistochemistry in a novel cohort of 240 HCC patients and validated our results in other cohorts comprising 660 patients. RESULTS: Our integrative analysis led to define: (1) the inflamed class of HCC (37%), which includes the previously reported immune subclass (22%) and a new immune-like subclass (15%) with high interferon signalling, cytolytic activity, expression of immune-effector cytokines and a more diverse T-cell repertoire. A 20-gene signature was able to capture ~90% of these tumours and is associated with response to immunotherapy. Proteins identified in liquid biopsies recapitulated the inflamed class with an area under the ROC curve (AUC) of 0.91; (2) The intermediate class, enriched in TP53 mutations (49% vs 29%, p=0.035), and chromosomal losses involving immune-related genes and; (3) the excluded class, enriched in CTNNB1 mutations (93% vs 27%, p<0.001) and PTK2 overexpression due to gene amplification and promoter hypomethylation. CTNNB1 mutations outside the excluded class led to weak activation of the Wnt-ßcatenin pathway or occurred in HCCs dominated by high interferon signalling and type I antigen presenting genes. CONCLUSION: We have characterised the immunogenomic contexture of HCC and defined inflamed and non-inflamed tumours. Two distinct CTNNB1 patterns associated with a differential role in immune evasion are described. These features may help predict immune response in HCC.

Adding Inflammatory Markers and Refining National Institute on Alcohol Abuse and Alcoholism Criteria Improve Diagnostic Accuracy for Alcohol-associated Hepatitis.

BACKGROUND & AIMS: Although histology is considered the gold standard for diagnosis of alcohol-associated hepatitis (AH), it is not required for entry into therapeutic studies if patients meet National Institute on Alcohol Abuse and Alcoholism (NIAAA) consensus criteria for probable AH. Our aim was to assess the diagnostic accuracy of NIAAA criteria against liver biopsy and to explore new criteria to enhance diagnostic accuracy for AH. METHODS: A total of 268 consecutive patients with alcohol-related liver disease with liver biopsy were prospectively included: 210 and 58 in the derivation and validation cohorts, respectively. NIAAA criteria and histological diagnosis of alcoholic steatohepatitis (ASH) were independently reviewed by clinical investigators and pathologists from Hospital Clínic and Mayo Clinic. Using biopsy-proven ASH as gold standard we determined diagnostic capability of NIAAA criteria and proposed the new improved criteria. RESULTS: In the derivation cohort, diagnostic accuracy of NIAAA for AH was modest (72%) due to low sensitivity (63%). Subjects who did not meet NIAAA with ASH at liver biopsy had lower 1-year survival compared with subjects without ASH (70% vs 90%; P < .001). NIAAAm-CRP criteria, created by adding C-reactive protein and modifying the variables of the original NIAAA, had higher sensitivity (70%), accuracy (78%), and specificity (83%). Accuracy was also higher in a sensitivity analysis in severe AH (74% vs 65%). In the validation cohort, NIAAAm-CRP and NIAAA criteria had a sensitivity of 56% vs 52% and an accuracy of 76% vs 69%, respectively. CONCLUSION: NIAAA criteria are suboptimal for the diagnosis of AH. The proposed NIAAAm-CRP criteria may improve accuracy for noninvasive diagnosis of AH in patients with alcohol-related liver disease.

Lack of endothelial autophagy does not impair liver regeneration after partial hepatectomy in mice.

Liver sinusoidal endothelial cells (LSEC) are key elements in regulating the liver response to injury and regeneration. While endothelial autophagy is essential to protect endothelial cells from injury-induced oxidative stress and fibrosis, its role in liver regeneration has not been elucidated. This study was intended to investigate the role of endothelial autophagy in liver regeneration in the context of partial hepatectomy (PHx). Analysis of autophagy levels in rat LSEC after PHx indicated a tendency to decrease activity the first 2 days after surgery. PHx performed in mice with impaired endothelial autophagy (Atg7flox/flox ;VE-Cadherin-Cre+ ) and their littermate controls showed no differences neither in liver-to-body weight ratio, histological analysis, hepatocyte proliferation nor vascular integrity during the first 7 days after PH and liver regeneration was completely achieved. Our results indicate that endothelial autophagy does not play an essential role in the coordination of the liver regeneration process after PHx.

Immunomodulatory Effects of Lenvatinib Plus Anti-Programmed Cell Death Protein 1 in Mice and Rationale for Patient Enrichment in Hepatocellular Carcinoma.

BACKGROUND AND AIMS: Lenvatinib is an effective drug in advanced HCC. Its combination with the anti-PD1 (programmed cell death protein 1) immune checkpoint inhibitor, pembrolizumab, has generated encouraging results in phase Ib and is currently being tested in phase III trials. Here, we aimed to explore the molecular and immunomodulatory effects of lenvatinib alone or in combination with anti-PD1. APPROACH AND RESULTS: We generated three syngeneic models of HCC in C57BL/6J mice (subcutaneous and orthotopic) and randomized animals to receive placebo, lenvatinib, anti-PD1, or combination treatment. Flow cytometry, transcriptomic, and immunohistochemistry analyses were performed in tumor and blood samples. A gene signature, capturing molecular features associated with the combination therapy, was used to identify a subset of candidates in a cohort of 228 HCC patients who might respond beyond what is expected for monotherapies. In mice, the combination treatment resulted in tumor regression and shorter time to response compared to monotherapies (P < 0.001). Single-agent anti-PD1 induced dendritic and T-cell infiltrates, and lenvatinib reduced the regulatory T cell (Treg) proportion. However, only the combination treatment significantly inhibited immune suppressive signaling, which was associated with the TGFß pathway and induced an immune-active microenvironment (P < 0.05 vs. other therapies). Based on immune-related genomic profiles in human HCC, 22% of patients were identified as potential responders beyond single-agent therapies, with tumors characterized by Treg cell infiltrates, low inflammatory signaling, and VEGFR pathway activation. CONCLUSIONS: Lenvatinib plus anti-PD1 exerted unique immunomodulatory effects through activation of immune pathways, reduction of Treg cell infiltrate, and inhibition of TGFß signaling. A gene signature enabled the identification of ~20% of human HCCs that, although nonresponding to single agents, could benefit from the proposed combination.

Molecular characterisation of hepatocellular carcinoma in patients with non-alcoholic steatohepatitis.

BACKGROUND AND AIMS: Non-alcoholic steatohepatitis (NASH)-related hepatocellular carcinoma (HCC) is increasing globally, but its molecular features are not well defined. We aimed to identify unique molecular traits characterising NASH-HCC compared to other HCC aetiologies. METHODS: We collected 80 NASH-HCC and 125 NASH samples from 5 institutions. Expression array (n = 53 NASH-HCC; n = 74 NASH) and whole exome sequencing (n = 52 NASH-HCC) data were compared to HCCs of other aetiologies (n = 184). Three NASH-HCC mouse models were analysed by RNA-seq/expression-array (n = 20). Activin A receptor type 2A (ACVR2A) was silenced in HCC cells and proliferation assessed by colorimetric and colony formation assays. RESULTS: Mutational profiling of NASH-HCC tumours revealed TERT promoter (56%), CTNNB1 (28%), TP53 (18%) and ACVR2A (10%) as the most frequently mutated genes. ACVR2A mutation rates were higher in NASH-HCC than in other HCC aetiologies (10% vs. 3%, p <0.05). In vitro, ACVR2A silencing prompted a significant increase in cell proliferation in HCC cells. We identified a novel mutational signature (MutSig-NASH-HCC) significantly associated with NASH-HCC (16% vs. 2% in viral/alcohol-HCC, p = 0.03). Tumour mutational burden was higher in non-cirrhotic than in cirrhotic NASH-HCCs (1.45 vs. 0.94 mutations/megabase; p <0.0017). Compared to other aetiologies of HCC, NASH-HCCs were enriched in bile and fatty acid signalling, oxidative stress and inflammation, and presented a higher fraction of Wnt/TGF-ß proliferation subclass tumours (42% vs. 26%, p = 0.01) and a lower prevalence of the CTNNB1 subclass. Compared to other aetiologies, NASH-HCC showed a significantly higher prevalence of an immunosuppressive cancer field. In 3 murine models of NASH-HCC, key features of human NASH-HCC were preserved. CONCLUSIONS: NASH-HCCs display unique molecular features including higher rates of ACVR2A mutations and the presence of a newly identified mutational signature. LAY SUMMARY: The prevalence of hepatocellular carcinoma (HCC) associated with non-alcoholic steatohepatitis (NASH) is increasing globally, but its molecular traits are not well characterised. In this study, we uncovered higher rates of ACVR2A mutations (10%) - a potential tumour suppressor - and the presence of a novel mutational signature that characterises NASH-related HCC.

Liver CPT1A gene therapy reduces diet-induced hepatic steatosis in mice and highlights potential lipid biomarkers for human NAFLD.

The prevalence of nonalcoholic fatty liver disease (NAFLD) has increased drastically due to the global obesity pandemic but at present there are no approved therapies. Here, we aimed to revert high-fat diet (HFD)-induced obesity and NAFLD in mice by enhancing liver fatty acid oxidation (FAO). Moreover, we searched for potential new lipid biomarkers for monitoring liver steatosis in humans. We used adeno-associated virus (AAV) to deliver a permanently active mutant form of human carnitine palmitoyltransferase 1A (hCPT1AM), the key enzyme in FAO, in the liver of a mouse model of HFD-induced obesity and NAFLD. Expression of hCPT1AM enhanced hepatic FAO and autophagy, reduced liver steatosis, and improved glucose homeostasis. Lipidomic analysis in mice and humans before and after therapeutic interventions, such as hepatic AAV9-hCPT1AM administration and RYGB surgery, respectively, led to the identification of specific triacylglyceride (TAG) specie (C50:1) as a potential biomarker to monitor NAFFLD disease. To sum up, here we show for the first time that liver hCPT1AM gene therapy in a mouse model of established obesity, diabetes, and NAFLD can reduce HFD-induced derangements. Moreover, our study highlights TAG (C50:1) as a potential noninvasive biomarker that might be useful to monitor NAFLD in mice and humans.

Molecular classification and therapeutic targets in extrahepatic cholangiocarcinoma.

BACKGROUND & AIMS: Cholangiocarcinoma (CCA), a deadly malignancy of the bile ducts, can be classified based on its anatomical location into either intrahepatic (iCCA) or extrahepatic (eCCA), each with different pathogenesis and clinical management. There is limited understanding of the molecular landscape of eCCA and no targeted therapy with clinical efficacy has been approved. We aimed to provide a molecular classification of eCCA and identify potential targets for molecular therapies. METHODS: An integrative genomic analysis of an international multicenter cohort of 189 eCCA cases was conducted. Genomic analysis included whole-genome expression, targeted DNA-sequencing and immunohistochemistry. Molecular findings were validated in an external set of 181 biliary tract tumors from the ICGC. RESULTS: KRAS (36.7%), TP53 (34.7%), ARID1A (14%) and SMAD4 (10.7%) were the most prevalent mutations, with ∼25% of tumors having a putative actionable genomic alteration according to OncoKB. Transcriptome-based unsupervised clustering helped us define 4 molecular classes of eCCA. Tumors classified within the Metabolic class (19%) showed a hepatocyte-like phenotype with activation of the transcription factor HNF4A and enrichment in gene signatures related to bile acid metabolism. The Proliferation class (23%), more common in patients with distal CCA, was characterized by enrichment of MYC targets, ERBB2 mutations/amplifications and activation of mTOR signaling. The Mesenchymal class (47%) was defined by signatures of epithelial-mesenchymal transition, aberrant TGFß signaling and poor overall survival. Finally, tumors in the Immune class (11%) had a higher lymphocyte infiltration, overexpression of PD-1/PD-L1 and molecular features associated with a better response to immune checkpoint inhibitors. CONCLUSION: An integrative molecular characterization identified distinct subclasses of eCCA. Genomic traits of each class provide the rationale for exploring patient stratification and novel therapeutic approaches. LAY SUMMARY: Targeted therapies have not been approved for the treatment of extrahepatic cholangiocarcinoma. We performed a multi-platform molecular characterization of this tumor in a cohort of 189 patients. These analyses revealed 4 novel transcriptome-based molecular classes of extrahepatic cholangiocarcinoma and identified ∼25% of tumors with actionable genomic alterations, which has potential prognostic and therapeutic implications.

An Immune Gene Expression Signature Associated With Development of Human Hepatocellular Carcinoma Identifies Mice That Respond to Chemopreventive Agents.

BACKGROUND & AIMS: Cirrhosis and chronic inflammation precede development of hepatocellular carcinoma (HCC) in approximately 80% of cases. We investigated immune-related gene expression patterns in liver tissues surrounding early-stage HCCs and chemopreventive agents that might alter these patterns to prevent liver tumorigenesis. METHODS: We analyzed gene expression profiles of nontumor liver tissues from 392 patients with early-stage HCC (training set, N = 167 and validation set, N = 225) and liver tissue from patients with cirrhosis without HCC (N = 216, controls) to identify changes in expression of genes that regulate the immune response that could contribute to hepatocarcinogenesis. We defined 172 genes as markers for this deregulated immune response, which we called the immune-mediated cancer field (ICF). We analyzed the expression data of liver tissues from 216 patients with cirrhosis without HCC and investigated the association between this gene expression signature and development of HCC and outcomes of patients (median follow-up, 10 years). Human liver tissues were also analyzed by histology. C57BL/6J mice were given a single injection of diethylnitrosamine (DEN) followed by weekly doses of carbon tetrachloride to induce liver fibrosis and tumorigenesis. Mice were then orally given the multiple tyrosine inhibitor nintedanib or vehicle (controls); liver tissues were collected and histology, transcriptome, and protein analyses were performed. We also analyzed transcriptomes of liver tissues collected from mice on a choline-deficient high-fat diet, which developed chronic liver inflammation and tumors, orally given aspirin and clopidogrel or the anti-inflammatory agent sulindac vs mice on a chow (control) diet. RESULTS: We found the ICF gene expression pattern in 50% of liver tissues from patients with cirrhosis without HCC and in 60% of nontumor liver tissues from patients with early-stage HCC. The liver tissues with the ICF gene expression pattern had 3 different features: increased numbers of effector T cells; increased expression of genes that suppress the immune response and activation of transforming growth factor ß signaling; or expression of genes that promote inflammation and activation of interferon gamma signaling. Patients with cirrhosis and liver tissues with the immunosuppressive profile (10% of cases) had a higher risk of HCC (hazard ratio, 2.41; 95% confidence interval, 1.21-4.80). Mice with chemically induced fibrosis or diet-induced steatohepatitis given nintedanib or aspirin and clopidogrel down-regulated the ICF gene expression pattern in liver and developed fewer and smaller tumors than mice given vehicle. CONCLUSIONS: We identified an immune-related gene expression pattern in liver tissues of patients with early-stage HCC, called the ICF, that is associated with risk of HCC development in patients with cirrhosis. Administration of nintedanib or aspirin and clopidogrel to mice with chronic liver inflammation caused loss of this gene expression pattern and development of fewer and smaller liver tumors. Agents that alter immune regulatory gene expression patterns associated with carcinogenesis might be tested as chemopreventive agents in patients with cirrhosis.

Ductular reaction and hepatocyte ballooning identify patients with fibrosing cholestatic hepatitits after liver transplantation.

INTRODUCTION: Diagnosis of severe hepatitis C recurrence is based on analytical and histological criteria but there is little information about their correlation. AIM: To assess the accuracy of laboratory criteria for the diagnosis of fibrosing cholestatic hepatitis (FCH). PATIENTS AND METHODS: Retrospective analysis of prospectively collected data form HCV positive patients who underwent liver transplantation (LT) between 2000 and 2014 in two European university hospitals. Patients were classified according to laboratory criteria such as FCH, cholestatic hepatitis (CH) and non-cholestatic acute hepatitis (NCAH). Histological characteristics were also evaluated. RESULTS: Seventy patients with acute HCV recurrence within the first year after LT with an available liver biopsy were included in the study. Most patients were male (70%) with a median age of 58 years (50-64) and infected with genotype 1b (71.4%). Median time from LT to diagnosis of recurrence was 2.96 months (2.1-5.3). Thirty-nine patients were classified as FCH, 21 as CH and 10 as NCAH. Marked hepatocyte ballooning and ductular reaction were associated with the presence of FCH with an OR of 4.66 (p=0.047) and 20.58 (p=0.025), respectively. Considering liver biopsy as the gold standard, the sensitivity, specificity, positive and negative predictive values of the analytical criteria were 0.8, 0.5, 0.3 and 0.9, respectively. However, correlation between histological and analytical criteria was poor (k=0.033). DISCUSSION: Analytical criteria may be used to rule out the presence of FCH, but a biopsy is mandatory to confirm the diagnosis. Ductular reaction and hepatocyte ballooning were independent predictors of FCH.

Molecular portrait of high alpha-fetoprotein in hepatocellular carcinoma: implications for biomarker-driven clinical trials.

The clinical utility of serum alpha-fetoprotein (AFP) in patients with hepatocellular carcinoma (HCC) is widely recognised. However, a clear understanding of the mechanisms of AFP overexpression and the molecular traits of patients with AFP-high tumours are not known. We assessed transcriptome data, whole-exome sequencing data and DNA methylome profiling of 520 HCC patients from two independent cohorts to identify distinct molecular traits of patients with AFP-high tumours (serum concentration > 400 ng/ml), which represents an accepted prognostic cut-off and a predictor of response to ramucirumab. Those AFP-high tumours (18% of resected cases) were characterised by significantly lower AFP promoter methylation (p < 0.001), significant enrichment of progenitor-cell features (CK19, EPCAM), higher incidence of BAP1 oncogene mutations (8.5% vs 1.6%) and lower mutational rates of CTNNB1 (14% vs 30%). Specifically, AFP-high tumours displayed significant activation of VEGF signalling (p < 0.001), which might provide the rationale for the reported benefit of ramucirumab in this subgroup of patients.

Impaired endothelial autophagy promotes liver fibrosis by aggravating the oxidative stress response during acute liver injury.

BACKGROUND & AIMS: Endothelial dysfunction plays an essential role in liver injury, yet the phenotypic regulation of liver sinusoidal endothelial cells (LSECs) remains unknown. Autophagy is an endogenous protective system whose loss could undermine LSEC integrity and phenotype. The aim of our study was to investigate the role of autophagy in the regulation of endothelial dysfunction and the impact of its manipulation during liver injury. METHODS: We analyzed primary isolated LSECs from Atg7control and Atg7endo mice as well as rats after CCl4 induced liver injury. Liver tissue and primary isolated stellate cells were used to analyze liver fibrosis. Autophagy flux, microvascular function, nitric oxide bioavailability, cellular superoxide content and the antioxidant response were evaluated in endothelial cells. RESULTS: Autophagy maintains LSEC homeostasis and is rapidly upregulated during capillarization in vitro and in vivo. Pharmacological and genetic downregulation of endothelial autophagy increases oxidative stress in vitro. During liver injury in vivo, the selective loss of endothelial autophagy leads to cellular dysfunction and reduced intrahepatic nitric oxide. The loss of autophagy also impairs LSECs ability to handle oxidative stress and aggravates fibrosis. CONCLUSIONS: Autophagy contributes to maintaining endothelial phenotype and protecting LSECs from oxidative stress during early phases of liver disease. Selectively potentiating autophagy in LSECs during early stages of liver disease may be an attractive approach to modify the disease course and prevent fibrosis progression. LAY SUMMARY: Liver endothelial cells are the first liver cell type affected after any kind of liver injury. The loss of their unique phenotype during injury amplifies liver damage by orchestrating the response of the liver microenvironment. Autophagy is a mechanism involved in the regulation of this initial response and its manipulation can modify the progression of liver damage.

Portal pressure and liver stiffness measurements in the prediction of fibrosis regression after sustained virological response in recurrent hepatitis C.

Sustained virological response (SVR) improves survival in post-liver transplant (LT) recurrent hepatitis C. However, the impact of SVR on fibrosis regression is not well defined. In addition, the performance of noninvasive methods to evaluate the presence of fibrosis and portal hypertension (PH) post-SVR has been scarcely evaluated. We aimed to investigate the degree of fibrosis regression (decrease ≥1 METAVIR stage) after-SVR and its associated factors in recurrent hepatitis C, as well as the diagnostic capacity of noninvasive methods in the assessment of liver fibrosis and PH after viral clearance. We evaluated 112 hepatitis C virus-infected LT recipients who achieved SVR between 2001 and 2015. A liver biopsy was performed before treatment and 12 months post-SVR. Hepatic venous pressure gradient (HVPG), liver stiffness measurement (LSM), and Enhanced Liver Fibrosis (ELF) score were also determined at the same time points. Sixty-seven percent of the cohort presented fibrosis regression: 43% in recipients with cirrhosis and 72%-85% in the remaining stages (P = 0.002). HVPG, LSM, and ELF significantly decreased post-SVR. Liver function significantly improved, and survival was significantly better in patients achieving fibrosis regression. Baseline HVPG and LSM as well as decompensations before therapy were independent predictors of fibrosis regression. One year post-SVR, LSM had a high diagnostic accuracy to discard the presence of advanced fibrosis (AF) and clinically significant PH (AUROC, 0.902 and 0.888). CONCLUSION: In conclusion, SVR post-LT induces fibrosis regression in most patients, leading to significant clinical benefits. Pretreatment HVPG and LSM are significant determinants of the likelihood of fibrosis regression. Finally, LSM accurately predicts the presence of AF and PH 1 year after SVR and thus can be used to determine monitoring strategies. (Hepatology 2018;67:1683-1694).

Nuclear IGF-1R predicts chemotherapy and targeted therapy resistance in metastatic colorectal cancer.

BACKGROUND: Although chemotherapy is the cornerstone treatment for patients with metastatic colorectal cancer (mCRC), acquired chemoresistance is common and constitutes the main reason for treatment failure. Monoclonal antibodies against insulin-like growth factor-1 receptor (IGF-1R) have been tested in pre-treated mCRC patients, but results have been largely deceiving. METHODS: We analysed time to progression, overall survival, and the mutational status of RAS, BRAF and nuclear p-IGF-1R expression by immunohistochemistry, in 470 metastatic CRC patients. The effect of IGF-1R activation and distribution was also assessed using cellular models of CRC and RNAi for functional validation. RESULTS: Nuclear IGF-1R increased in metastatic tumours compared to paired untreated primary tumours, and significantly correlated with poor overall survival in mCRC patients. In vitro, chemo-resistant cell lines presented significantly higher levels of IGF-1R expression within the nuclear compartment, and PIAS3, a protein implicated also in the sumoylation process of intranuclear proteins, contributed to IGF-1R nuclear sequestration, highlighting the essential role of PIAS3 in this process. Intriguingly, we observed that ganitumab, an IGF-1R blocking-antibody used in several clinical trials, and dasatinib, an SRC inhibitor, increased the nuclear localisation of IGF-1R. CONCLUSIONS: Our study demonstrates that IGF-1R nuclear location might lead to chemotherapy and targeted agent resistance.