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Any oncological treatment must aim at maximizing patient survival with the best quality of life and not merely at eliminating the tumor. Since the liver has a vital function, any radical treatment severely compromising liver function will result in a shortening of life expectancy, rather than a prolongation. Furthermore, even non-severe liver damage may imply the risk of impeding the opportunity to receive further effective therapies. This is particularly important in the case of hepatocellular carcinoma (HCC), since this tumor is associated with underlying cirrhosis in the majority of patients, and cirrhosis itself is not only a potentially lethal disease and independent prognostic factor in HCC, but it also makes liver function fragile. Accordingly, some information about liver dysfunction is included in most staging systems for HCC, in an attempt to suggest treatments that the functional liver reserve can adequately tolerate. Unfortunately, the prediction of functional liver damage in the case of antitumor treatments is very challenging and still suboptimal in any given specific patient. Moreover, while the assessment of the functional reserve has been sufficiently elucidated in the surgical setting to avoid postoperative liver failure, it has instead been less clarified for non-surgical therapies, despite the fact that this aspect appears to have become of particular relevance today, when several lines of effective non-surgical treatments, including systemic therapies, have become available. The present article will a) critically review the implications of the assessment of liver functional reserve in patients with HCC, b) illustrate the different available tools to assess the liver functional reserve and c) discuss the role of functional assessment in the setting of each type of non-surgical therapy for HCC.

Metabolic dysfunction–associated fatty liver disease (MAFLD) is nowadays considered the liver manifestation of metabolic syndrome. Its prevalence is increasing worldwide in parallel to the epidemic of diabetes and obesity. MAFLD includes a wide spectrum of liver injury including simple steatosis and non-alcoholic steatohepatitis (NASH) that may lead to serious complications such as liver cirrhosis and liver cancer. The complexity of its pathophysiology and the intricate mechanisms underlying disease progression explains the huge variety of molecules targeting diverse biological mechanisms that have been tested in preclinical and clinical settings in the last two decades. Thanks to the large number of clinical trials of the last few years, most of them still ongoing, the pharmacotherapy scenario of MAFLD is rapidly evolving. The three major components of MAFLD, steatosis, inflammation, and fibrosis seem to be safely targeted with different agents at least in a large proportion of patients. Likely, in the next few years more than one drug will be approved for the treatment of MAFLD at different disease stages. The aim of this review is to synthesize the characteristics and the results of the most advanced clinical trials for the treatment of NASH to evaluate the recent advances of pharmacotherapy in this disease.

Introduction: The combination of easy-to-obtain validated biomarkers is interesting in the prognostic evaluation of patients at cardiovascular risk in a precision medicine scenario. The evaluation of the effect modification of insulin resistance and liver fibrosis with the Triglyceride-Glucose index (TyG) and Fibrosis-4 index (FIB4) might provide prognostic information in patients at cardiovascular risk. Patients and methods: A retrospective cohort study was performed with 2055 patients recruited in the Vascular Metabolic CUN cohort. The studied outcome was the incidence rate of major cardiovascular events (MACE). The Systematic Coronary Risk Evaluation (SCORE), FIB4 and TyG indexes were calculated according to validated formulas. Results: FIB4 and TyG showed a synergistic interaction using validated cut-offs for both indexes in the prediction of MACE (Hazard ratio (HR) 1.05 CI95% 1.01–1.08) which remained after adjustment by age, sex, SCORE subgroup, presence of diabetes, or previous MACE using standardized cut-off (HR 2.29 CI95% 1.33–3.94). Finally, a subgroup with significant TyG and FIB4 showed a higher cardiovascular risk in the study population (adjusted HR 3.34 CI 95% 1.94–5.77). Conclusion: The combined interpretation of TyG and FIB4 indexes might have a potential predictive value of major cardiovascular events.

Little is known about the mutational landscape of advanced hepatocellular carcinoma (HCC), and predictive biomarkers of response to systemic therapies are lacking. We aimed to describe the mutational landscape of advanced HCC and to identify predictors of primary resistance to systemic therapies using circulating tumor DNA (ctDNA). We prospectively enrolled 121 patients between October 2015 and January 2019. We performed targeted ultra-deep sequencing of 25 genes and Digital Droplet PCR of TERT promoter, including sequential samples throughout treatment. Primary endpoint was progression-free survival (PFS) stratified by mutation profiles in ctDNA. Secondary endpoints were overall survival and objective response rate. The most frequent mutations in ctDNA of advanced HCC were TERT promoter (51%), TP53 (32%), CTNNB1 (17%), PTEN (8%), AXIN1, ARID2, KMT2D, and TSC2 (each 6%). TP53 and CTNNB1 mutations were mutually exclusive. Patients with mutations in the PI3K/MTOR pathway had significantly shorter PFS than those without these mutations after tyrosine kinase inhibitors (2.1 vs 3.7 months, p

International audience; The analysis of circulating tumor cells (CTCs) in blood is a powerful noninvasive alternative to conventional tumor biopsy. Inertial-based separation is a promising highthroughput, marker-free sorting strategy for the enrichment and isolation of CTCs. Here, we present and validate a double spiral microfluidic device that efficiently isolates CTCs with a fine-tunable cutoff value of 9 μm and a separation range of 2 μm. We designed the device based on computer simulations that introduce a novel, customized inertial force term, and provide practical fabrication guidelines. We validated the device using calibration beads, which allowed us to refine the simulations and redesign the device. Then we validated the redesigned device using blood samples and a murine model of metastatic breast cancer. Finally, as a proof of principle, we tested the device using peripheral blood from a patient with hepatocellular carcinoma, isolating more than 17 CTCs/ml, with purity/removal values of 96.03% and 99.99% of white blood cell and red blood cells, respectively. These results confirmhighly efficient CTC isolation with a stringent cut-off value and better separationresults than the state of the art.

AIMS Access to tissue in patients with hepatocellular carcinoma (HCC) is limited compared to other malignancies, particularly at advanced stages. This has precluded a thorough characterisation of molecular drivers of HCC dissemination, particularly in relation to distant metastases. Biomarker assessment is restricted to early stages, and paired primary-metastatic comparisons between samples from the same patient are difficult. METHODS AND RESULTS We report the evaluation of 88 patients with HCC who underwent autopsy, including multiregional sampling of primary and metastatic sites totalling 230 nodules analysed. The study included morphological assessment, immunohistochemistry and mutation status of the TERT promoter, the most frequently mutated gene in HCC. We confirm a strong predilection of HCC for lung dissemination, including subclinical micrometastases (unrecognised during imaging and macroscopic examinations) in 30% of patients with disseminated disease. Size of dominant tumour nodule; multinodularity; macrovascular invasion; high histological, nuclear and architectural grades; and cellular crowding were associated with the presence of extrahepatic metastasis. Among the immunohistochemistry markers tested, metastatic nodules had significantly higher K19 and EpCAM expression than primary liver tumours. Morphological and immunohistochemical features showed that metastatic HCC could be traced back to the primary tumour, sometimes to a specific hepatic nodule. CONCLUSIONS This study suggests limited heterogeneity in metastatic sites compared to primary tumour sites.

ObjectiveSurveillance tools for early cancer detection are suboptimal, including hepatocellular carcinoma (HCC), and biomarkers are urgently needed. Extracellular vesicles (EVs) have gained increasing scientific interest due to their involvement in tumour initiation and metastasis; however, most extracellular RNA (exRNA) blood-based biomarker studies are limited to annotated genomic regions.DesignEVs were isolated with differential ultracentrifugation and integrated nanoscale deterministic lateral displacement arrays (nanoDLD) and quality assessed by electron microscopy, immunoblotting, nanoparticle tracking and deconvolution analysis. Genome-wide sequencing of the largely unexplored small exRNA landscape, including unannotated transcripts, identified and reproducibly quantified small RNA clusters (smRCs). Their key genomic features were delineated across biospecimens and EV isolation techniques in prostate cancer and HCC. Three independent exRNA cancer datasets with a total of 479 samples from 375 patients, including longitudinal samples, were used for this study.ResultsExRNA smRCs were dominated by uncharacterised, unannotated small RNA with a consensus sequence of 20 nt. An unannotated 3-smRC signature was significantly overexpressed in plasma exRNA of patients with HCC (pConclusionThese findings directly lead to the prospect of a minimally invasive, blood-only, operator-independent clinical tool for HCC surveillance, thus highlighting the potential of unannotated smRCs for biomarker research in cancer.

Cancer testis antigens (CTAs) are an extensive gene family with a unique expression pattern restricted to germ cells, but aberrantly reactivated in cancer tissues. Studies indicate that the expression (or re-expression) of CTAs within the MAGE-A family is common in hepatocellular carcinoma (HCC). However, no systematic characterization has yet been reported. The aim of this study is to perform a comprehensive profile of CTA de-regulation in HCC and experimentally evaluate the role of MAGEA3 as a driver of HCC progression. The transcriptomic analysis of 44 multi-regionally sampled HCCs from 12 patients identified high intra-tumor heterogeneity of CTAs. In addition, a subset of CTAs was significantly overexpressed in histologically poorly differentiated regions. Further analysis of CTAs in larger patient cohorts revealed high CTA expression related to worse overall survival and several other markers of poor prognosis. Functional analysis of MAGEA3 was performed in human HCC cell lines by gene silencing and in a genetic mouse model by overexpression of MAGEA3 in the liver. Knockdown of MAGEA3 decreased cell proliferation, colony formation and increased apoptosis. MAGEA3 overexpression was associated with more aggressive tumors in vivo. In conclusion MAGEA3 enhances tumor progression and should be considered as a novel therapeutic target in HCC., Author summary Hepatocellular carcinoma (HCC) is one of the leading causes of cancer related death worldwide. Several recurrent driver genes of HCC have been established, yet no targeted drug against these drivers has been approved for the treatment of HCC. Novel targets for therapeutic development are desperately needed. This study aims to identify novel drivers of tumor progression from low-grade tumors to high-grade, aggressive malignancies. We collected multiple biopsies of single tumors from 12 patients and performed RNA-sequencing. We hypothesized that we could identify important genes to HCC progression by identifying differentially up-regulated genes in high-grade regions compared to low-grade regions of the same tumor. Interestingly, we found that one family of genes was recurrently over-expressed in the most aggressive regions of the tumors: cancer testis antigens (CTAs). Here we show that X-chromosome located CTAs, and especially MAGEA3, are associated with markers of poor prognosis in HCC. Experimentally, inhibiting expression of MAGEA3 in HCC cell lines decreases the ability for cells to proliferate and causes cells to undergo cell death. In mice, over-expressing MAGEA3 in the liver along with known HCC drivers causes mice to succumb to disease faster. Changes in RNA and protein levels within HCC cells after MAGEA3 inhibition suggests that maintenance of Survivin, an apoptosis inhibiting protein, plays an important role in how MAGEA3 drives HCC progression. Overall, our study identified the CTA MAGEA3 as important for HCC progression and a novel therapeutic target.

Liquid biopsy, the molecular analysis of tumor components released into the bloodstream, has emerged as a noninvasive and resourceful means to access genomic information from cancers. Most data derived from translational studies showcase its numerous potential clinical applications. However, data from experimental models are scarce, and little is known about the underlying mechanisms and factors controlling the release of circulating tumor DNA (ctDNA) and cells (CTCs). This study aimed to model liquid biopsy in hepatocellular carcinoma xenografts and to study the dynamics of release of ctDNA and CTCs; this included models of intratumoral heterogeneity (ITH) and metastatic disease. We quantified ctDNA by quantitative polymerase chain reaction (PCR) targeting human long interspersed nuclear element group 1; targeted mutation analysis was performed with digital droplet PCR. CTCs were traced by flow cytometry. Results demonstrated the feasibility of detecting ctDNA, including clone-specific mutations, as well as CTCs in blood samples of mice. In addition, the concentration of ctDNA and presence of tumor-specific mutations reflected tumor progression, and detection of CTCs was associated with metastases. Our ITH model suggested differences in the release of DNA fragments impacted by the cell-clone origin and the treatment. Conclusion: These data present new models to study liquid biopsy and its underlying mechanisms and highlighted a clone-dependent release of ctDNA into the bloodstream.

The morphological changes that occur in the central nervous system of patients with severe acute intermittent porphyria (AIP) have not yet been clearly established. The aim of this work was to analyse brain involvement in patients with severe AIP without epileptic seizures or clinical posterior reversible encephalopathy syndrome, as well as in a mouse model receiving or not liver-directed gene therapy aimed at correcting the metabolic disorder. We conducted neuroradiologic studies in 8 severely affected patients (6 women) and 16 gender- and age-matched controls. Seven patients showed significant enlargement of the cerebral ventricles and decreased brain perfusion was observed during the acute attack in two patients in whom perfusion imaging data were acquired. AIP mice exhibited reduced cerebral blood flow and developed chronic dilatation of the cerebral ventricles even in the presence of slightly increased porphyrin precursors. While repeated phenobarbital-induced attacks exacerbated ventricular dilation in AIP mice, correction of the metabolic defect using liver-directed gene therapy restored brain perfusion and afforded protection against ventricular enlargement. Histological studies revealed no signs of neuronal loss but a denser neurofilament pattern in the periventricular areas, suggesting compression probably caused by imbalance in cerebrospinal fluid dynamics. In conclusion, severely affected AIP patients exhibit cerebral ventricular enlargement. Liver-directed gene therapy protected against the morphological consequences of the disease seen in the brain of AIP mice. The observational study was registered at Clinicaltrial.gov as NCT02076763.

Little is known about the mutational landscape of advanced hepatocellular carcinoma (HCC), and predictive biomarkers of response to systemic therapies are lacking. We aimed to describe the mutational landscape of advanced HCC and to identify predictors of primary resistance to systemic therapies using circulating tumor DNA (ctDNA). We prospectively enrolled 121 patients between October 2015 and January 2019. We performed targeted ultra-deep sequencing of 25 genes and Digital Droplet PCR of TERT promoter, including sequential samples throughout treatment. Primary endpoint was progression-free survival (PFS) stratified by mutation profiles in ctDNA. Secondary endpoints were overall survival and objective response rate. The most frequent mutations in ctDNA of advanced HCC were TERT promoter (51%), TP53 (32%), CTNNB1 (17%), PTEN (8%), AXIN1, ARID2, KMT2D, and TSC2 (each 6%). TP53 and CTNNB1 mutations were mutually exclusive. Patients with mutations in the PI3K/MTOR pathway had significantly shorter PFS than those without these mutations after tyrosine kinase inhibitors (2.1 vs 3.7 months, p 0.001), but not after immune checkpoint inhibition (CPI). WNT pathway mutations were not associated with PFS, overall survival, or objective response after CPI. Serial profiling of ctDNA in a subset correlated with treatment response. Mutation profiling of ctDNA in advanced HCC shows similar mutation frequencies for known HCC drivers compared to early stages and identifies predictive biomarkers of response to systemic therapies.

BackgroundHepatocellular carcinoma (HCC) is among the deadliest malignancies and surveillance tools for early detection are suboptimal. Extracellular vesicles (EVs) have gained increasing scientific interest due to their involvement in tumor initiation and metastasis, however, most extracellular RNA (exRNA) biomarker studies are limited to annotated genomic regions.MethodsEVs were isolated with ultracentrifugation and nanoDLD and quality assessed by electron microscopy, immunoblotting, nanoparticle tracking, and deconvolution analysis. We performed genome-wide small exRNA sequencing, including unannotated transcripts. We identified small RNA clusters (smRCs) and delineated their key genomic features across biospecimens (blood, urine, tissue) and EV isolation techniques. A 3-smRC signature for early HCC detection was trained and validated in two independent cohorts.ResultsEV-derived smRCs were dominated by uncharacterized, unannotated small RNA and uniformly tiled across the genome with a consensus sequence of 20bp. A 3-smRC signature was significantly overexpressed in circulating EVs of HCC patients compared to controls at risk or patients with non-HCC malignancies (pConclusionAn exRNA-based 3-smRC signature from plasma detects early stage HCC, which directly leads to the prospect of a minimally-invasive, blood-only, operator-independent surveillance biomarker.One sentence summaryWe employ a novel, data-driven approach to identify and characterize small RNA clusters from unannotated loci in extracellular vesicle-derived RNA across different cancer types, isolation techniques, and biofluids, facilitating discovery of a robust biomarker for detection of early stage liver cancer.

// Leire Neri 1 , Marta Lasa 1 , Alberto Elosegui-Artola 2 , Delia D'Avola 3, 4 , Beatriz Carte 1 , Cristina Gazquez 1 , Sara Alve 5 , Pere Roca-Cusachs 2, 6 , Mercedes Inarrairaegui 3, 4 , Jose Herrero 3, 4 , Jesus Prieto 1, 3 , Bruno Sangro 3, 4 and Rafael Aldabe 1, 4 1 Gene Therapy and Regulation of Gene Expression Program, Centro de Investigacion Medica Aplicada, Universidad de Navarra, Pamplona, Spain 2 Institute for Bioengineering of Catalonia, Barcelona, Spain 3 Liver Unit, Clinica Universidad de Navarra, Centro de Investigacion Biomedica en Red en el Area Tematica de Enfermedades Hepaticas y Digestivas (Ciberehd), Pamplona, Spain 4 Instituto de Investigacion Sanitaria de Navarra (IdiSNA), Pamplona, Spain 5 Department of Biology, CBMA-Centre of Molecular and Environmental Biology, University of Minho, Campus de Gualtar, Braga, Portugal 6 University of Barcelona, Barcelona, Spain Correspondence to: Rafael Aldabe, email: raldabe@unav.es Keywords: tropomyosin, CDK2, focal adhesions, cell-cell junctions, cell cycle arrest Received: December 21, 2016 Accepted: April 04, 2017 Published: April 21, 2017 ABSTRACT The identification of new targets for systemic therapy of hepatocellular carcinoma (HCC) is an urgent medical need. Recently, we showed that hNatB catalyzes the N-α-terminal acetylation of 15% of the human proteome and that this action is necessary for proper actin cytoskeleton structure and function. In tumors, cytoskeletal changes influence motility, invasion, survival, cell growth and tumor progression, making the cytoskeleton a very attractive antitumor target. Here, we show that hNatB subunits are upregulated in in over 59% HCC tumors compared to non-tumor tissue and that this upregulation is associated with microscopic vascular invasion. We found that hNatB silencing blocks proliferation and tumor formation in HCC cell lines in association with hampered DNA synthesis and impaired progression through the S and the G2/M phases. Growth inhibition is mediated by the degradation of two hNatB substrates, tropomyosin and CDK2, which occurs when these proteins lack N-α-terminal acetylation. In addition, hNatB inhibition disrupts the actin cytoskeleton, focal adhesions and tight/adherens junctions, abrogating two proliferative signaling pathways, Hippo/YAP and ERK1/2. Therefore, inhibition of NatB activity represents an interesting new approach to treating HCC by blocking cell proliferation and disrupting actin cytoskeleton function.

Cardiovascular (CV) diseases are recognized longterm causes of death after liver transplantation (LT). The objective of this multicenter study was to analyze the prevalence and the evolution of CV risk factors and CV morbidity and mortality in 1819 LT recipients along 5 years after LT. The influence of baseline variables on survival, morbidity, and mortality was studied. There was a continuous and significant increase of the prevalence of all the CV risk factors (except smoking) after LT. CV diseases were the fourth cause of mortality in the 5 years after LT, causing 12% of deaths during the follow-up. Most CV events (39%) occurred in the first year after LT. Preexisting CV risk factors such as age, pre-LT CV events, diabetes, metabolic syndrome, and hyperuricemia, and mycophenolate-free immunosuppressive therapy, increased post-LT CV morbidity and mortality. The development of new-onset CV risk factors after LT, such as dyslipidemia and obesity, independently affected late CV morbidity and mortality. Tacrolimus and steroids increased the risk of posttransplant diabetes, whereas cyclosporine increased the risk of arterial hypertension, dyslipidemia, and metabolic syndrome. In conclusion, CV complications and CV mortality are frequent in LT recipients. Preexisting CV risk factors, immunosuppressive drugs, but also the early new onset of obesity and dyslipidemia after LT play an important role on late CV complications. A strict metabolic control in the immediate post-LT period is advisable for improving CV risk of LT recipients. Liver Transplantation 23 498-509 2017 AASLD.

BACKGROUND Immunosuppression increases the risk of malignancy in liver transplant recipients. The potential impact of mycophenolate mofetil monotherapy on this risk has not been studied. MATERIAL AND METHODS The incidence and risk factors for de novo malignancies of 392 liver transplant recipients with a survival higher than 3 months and a mean follow-up of 8.5 years were studied. RESULTS De novo malignancies were diagnosed in 126 patients (32.1%) (64 non-melanoma skin cancer and 81 other malignancies). Sixty-nine patients (18.1%) stopped receiving calcineurin inhibitors and were maintained on mycophenolate mofetil monotherapy. The proportion of time on mycophenolate mofetil monotherapy (obtained after dividing the time on monotherapy by the time until diagnosis of neoplasia/last follow-up) was independently associated with a lower risk of de novo malignancy (HR: 0.16, 95% CI: 0.05-0.48; P=0.001), non-melanoma skin cancer (HR: 0.17, 95% CI: 0.03-0.79; P=0.024), and other malignancies (HR: 0.23, 95% CI: 0.07-0.77; P=0.017). Older age and male sex were also associated with a higher risk of malignancy, and transplantation for hepatocellular carcinoma increased the risk of non-melanoma skin cancer. CONCLUSIONS Mycophenolate mofetil monotherapy is associated with a lower risk of cancer in liver transplant recipients compared with maintenance immunosuppression with calcineurin inhibitors.

Background & Aims Acute intermittent porphyria (AIP) results from porphobilinogen deaminase (PBGD) haploinsufficiency, which leads to hepatic over-production of the neurotoxic heme precursors porphobilinogen (PBG) and delta-aminolevulinic acid (ALA) and the occurrence of neurovisceral attacks. Severe AIP is a devastating disease that can only be corrected by liver transplantation. Gene therapy represents a promising curative option. The objective of this study was to investigate the safety of a recombinant adeno-associated vector expressing PBGD (rAAV2/5- PBGD ) administered for the first time in humans for the treatment of AIP. Methods In this phase I, open label, dose-escalation, multicenter clinical trial, four cohorts of 2 patients each received a single intravenous injection of the vector ranging from 5×10 11 to 1.8×10 13 genome copies/kg. Adverse events and changes in urinary PBG and ALA and in the clinical course of the disease were periodically evaluated prior and after treatment. Viral shedding, immune response against the vector and vector persistence in the liver were investigated. Results Treatment was safe in all cases. All patients developed anti-AAV5 neutralizing antibodies but no cellular responses against AAV5 or PBGD were observed. There was a trend towards a reduction of hospitalizations and heme treatments, although ALA and PBG levels remained unchanged. Vector genomes and transgene expression could be detected in the liver one year after therapy. Conclusions rAAV2/5- PBGD administration is safe but AIP metabolic correction was not achieved at the doses tested in this trial. Notwithstanding, the treatment had a positive impact in clinical outcomes in most patients. Lay summary Studies in an acute intermittent porphyria (AIP) animal model have shown that gene delivery of PBGD to hepatocytes using an adeno-associated virus vector (rAAV2/5- PBG ) prevent mice from suffering porphyria acute attacks. In this phase I, open label, dose-escalation, multicenter clinical trial we show that the administration of rAAV2/5- PBGD to patients with severe AIP is safe but metabolic correction was not achieved at the doses tested; the treatment, however, had a positive but heterogeneous impact on clinical outcomes among treated patients and 2 out of 8 patients have stopped hematin treatment. Clinical trial number The observational phase was registered at Clinicaltrial.gov as NCT 02076763. The interventional phase study was registered at EudraCT as n° 2011-005590-23 and at Clinicaltrial.gov as NCT02082860.

Introduction: Acute Intermittent porphyria (AIP) is a rare autosomal dominant disease caused by mutations in the gene encoding hydroxymethylbilane synthase (HMBS), previously called porphobilinogen deaminase (PBGD), the third enzyme in the heme synthesis pathway. Activation of the first enzyme of this pathway, delta-aminolevulinic acid synthase-1 (ALAS1) in the context of porphobilinogen deaminase (PBGD) deficiency leads to accumulation of the neurotoxic molecules, aminolevulinic acid (ALA) and porphobilinogen (PBG), and precipitates acute porphyria attacks. The fact that liver transplantation resolves the disease indicates that gene-complementation or gene-silencing approaches targeting the hepatocytes might correct or attenuate the biochemical and clinical manifestations of AIP.Areas covered: Remarkable progress has been made in the development of genetic treatments for AIP, in particular the use of gene transfer vectors such as AAV-based vectors to introduce the therapeutic gene into hepatocyte...

Recombinant adeno-associated viral vectors (rAAV) currently constitute a real therapeutic strategy for the sustained correction of diverse genetic conditions. Though a wealth of preclinical and clinical studies have been conducted with rAAV, the oncogenic potential of these vectors is still controversial, particularly when considering liver-directed gene therapy. Few preclinical studies and the recent discovery of incomplete wild-type AAV2 genomes integrated in human hepatocellular carcinoma biopsies have raised concerns on rAAV safety. In the present study, we have characterized the integration of both complete and partial rAAV2/5 genomes in nonhuman primate tissues and clinical liver biopsies from a trial aimed to treat acute intermittent porphyria. We applied a new multiplex linear amplification-mediated polymerase chain reaction (PCR) assay capable of detecting integration events that are originated throughout the rAAV genome. The integration rate was low both in nonhuman primates and patient's samples. Importantly, no integration clusters or events were found in genes previously reported to link rAAV integration with hepatocellular carcinoma development, thus showing the absence of genotoxicity of a systemically administered rAAV2/5 in a large animal model and in the clinical context.

Hypermetabolism in cirrhosis is associated with a high risk of complications and mortality. However, studies about underlying mechanisms are usually focussed on isolated potential determinants and specific etiologies, with contradictory results. We aimed at investigating differences in nutrition, metabolic hormones, and hepatic function between hypermetabolic and nonhypermetabolic men with cirrhosis of the liver.We prospectively enrolled 48 male cirrhotic inpatients. We evaluated their resting energy expenditure (REE) and substrate utilization by indirect calorimetry, body composition by dual-energy X-ray absorptiometry, liver function, and levels of major hormones involved in energy metabolism by serum sample tests. Patients with ascites, specific metabolic disturbances, and hepatocellular carcinoma were excluded.REE and REE adjusted per fat-free mass (FFM) were significantly increased in cirrhotic patients. Overall, 58.3% of cirrhotic patients were classified as hypermetabolic. Groups did not differ significantly in age, etiology of cirrhosis, liver function, presence of ascites, use of diuretics, β-blockers, or presence of transjugular intrahepatic portosystemic shunts. Hypermetabolic cirrhotic patients had lower weight, BMI (P0.05), nonprotein respiratory quotient (P0.01), leptin (P0.05), and leptin adjusted per fat mass (FM) (P0.05), but higher FFM% (P0.05) and insulin resistance [homeostatic model assessment-insulin resistance (HOMA-IR)] (P0.05). Only HOMA-IR, leptin/FM, and FFM% were independently related to the presence of hypermetabolism.Hypermetabolic cirrhotic men are characterized by lower weight, higher FFM%, insulin resistance, and lower leptin/FM when compared with nonhypermetabolic men. HOMA-IR, FFM%, and leptin/FM were independently associated with hypermetabolism, and may serve as easily detectable markers of this condition in daily clinical practice.

Patients with hepatocellular carcinoma (HCC) release tumor cells to the bloodstream, which can be detected using cell surface markers. Despite numerous reports suggest a direct correlation between the number of circulating tumor cells (CTCs) and poor clinical outcomes, few studies have provided a thorough molecular characterization of CTCs. Due to the limited access to tissue samples in patients at advanced stages of HCC, it is crucial to develop new technologies to identify HCC cancer drivers in routine clinical conditions. Here, we describe a method that sequentially combines image flow cytometry and high density single-cell mRNA sequencing to identify CTCs in HCC patients. Genome wide expression profiling of CTCs using this approach demonstrates CTC heterogeneity and helps detect known oncogenic drivers in HCC such as IGF2. This integrated approach provides a novel tool for biomarker development in HCC using liquid biopsy.

Several studies have shown that some liver transplant recipients may tolerate immunosuppression withdrawal. Mechanisms and biomarkers of tolerance are not well known. Methods Twenty-four LT patients with immunosuppression side-effects underwent progressive immunosuppression withdrawal. Peripheral lymphocyte populations and secretion of cytokines were analyzed at baseline and during withdrawal until tolerance (n = 15) or rejection (n = 9), as well as 3 months after tolerance achievement or rejection resolution (as follow-up). Immunological markers were compared among groups. Results The percentages of CD3 + CD4 + cells progressively decreased in both groups. CD3 + CD8 + cells gradually increased in tolerant patients. B lymphocytes gradually decreased in tolerant and initially in non-tolerant patients, reverting at rejection. Regulatory T cells progressively increased until rejection in non-tolerants, decreasing to basal levels after renewing immunosuppression; no significant changes were found in tolerant patients. The percentages and absolute counts of natural killer cells significantly increased in both groups, being more evident in tolerant patients. The secretion of several cytokines was higher in non-tolerant patients when rejection was diagnosed. Conclusions The greater increase of natural killer cells in tolerant patients suggests their potential role in the tolerance phenomenon.

Cellular components of solid tumors including DNA are released into the bloodstream, but data on circulating-free DNA (cfDNA) in hepatocellular carcinoma (HCC) are still scarce. This study aimed at analyzing mutations in cfDNA and their correlation with tissue mutations in patients with HCC. We included 8 HCC patients treated with surgical resection for whom we collected paired tissue and plasma/serum samples. We analyzed 45 specimens, including multiregional tumor tissue sampling (n = 24), peripheral blood mononuclear cells (PMBC, n = 8), plasma (n = 8) and serum (n = 5). Ultra-deep sequencing (5500× coverage) of all exons was performed in a targeted panel of 58 genes, including frequent HCC driver genes and druggable mutations. Mutations detected in plasma included known HCC oncogenes and tumor suppressors (e.g., TERT promoter, TP53, and NTRK3) as well as a candidate druggable mutation (JAK1). This approach increased the detection rates previously reported for mutations in plasma of HCC patients. A thorough characterization of cis mutations found in plasma confirmed their tumoral origin, which provides definitive evidence of the release of HCC-derived DNA fragments into the bloodstream. This study demonstrates that ultra-deep sequencing of cfDNA is feasible and can confidently detect somatic mutations found in tissue; these data reinforce the role of plasma DNA as a promising minimally invasive tool to interrogate HCC genetics.

The FibroScan(®) XL probe has been specifically designed for obese patients to measure liver stiffness by transient elastography, but it has not been well tested in non-obese patients. The aim of this study was to compare the M and XL FibroScan(®) probes in a series of unselected obese (body mass index above 30 kg/m(2)) and non-obese patients with chronic liver disease. Two hundred and fifty-four patients underwent a transient elastography examination with both the M and XL probes. The results obtained with the two probes were compared in the whole series and in obese (n=82) and non-obese (n=167) patients separately. The reliability of the examinations was assessed using the criteria defined by Castera et al. The proportion of reliable exams was significantly higher when the XL probe was used (83% versus 73%; P=.001). This significance was maintained in the group of obese patients (82% versus 55%; P

BackgroundPost-treatment contralateral hemiliver hypertrophy has created an interest in lobar liver radioembolization (RE) as a pre-surgery tool.MethodsLiver and spleen volumes and function were studied in 83 patients submitted to partial liver volume RE at 4–8 weeks (T1), 10–26 weeks (T2), and >26 weeks (T3) after RE.ResultsMore than half of the patients had cirrhosis with hepatocellular carcinoma. The main finding was a progressive increase in the volume of the spared hemiliver (mean absolute increase at T3: 230ml). The percentage of patients in whom the baseline ratio of spared volume to total liver volume was

Acute intermittent porphyria (AIP) results from haplo-insufficient activity of porphobilinogen deaminase (PBGD) and is characterized clinically by life-threatening, acute neurovisceral attacks. To date, liver transplantation is the only curative option for AIP. The aim of the present preclinical nonhuman primate study was to determine the safety and transduction efficacy of an adeno-associated viral vector encoding PBGD (recombinant AAV serotype 5-codon-optimized human porphobilinogen deaminase, rAAV5-cohPBGD) administered intravenously as part of a safety program to start a clinical study in patients with AIP. Macaques injected with either 1 × 10(13) or 5 × 10(13) vector genomes/kg of clinical-grade rAAV5-cohPBGD were monitored by standardized clinical parameters, and vector shedding was analyzed. Liver transduction efficacy, biodistribution, vector integration, and histopathology at day 30 postvector administration were determined. There was no evidence of acute toxicity, and no adverse effects were observed. The vector achieved efficient and homogenous hepatocellular transduction, reaching transgenic PBGD expression levels equivalent to 50% of the naturally expressed PBGD mRNA. No cellular immune response was detected against the human PBGD or AAV capsid proteins. Integration site analysis in transduced liver cells revealed an almost random integration pattern supporting the good safety profile of rAAV5-cohPBGD. Together, data obtained in nonhuman primates indicate that rAAV5-cohPBGD represents a safe therapy to correct the metabolic defect present in AIP patients.

Recipients of liver transplantation (LT) may develop immunological tolerance. Factors predictive of tolerance are not clearly understood. Transplant recipients with normal liver function tests and without active viral hepatitis or autoimmune disease who presented with side effects of immunosuppression or a high risk of de novo malignancies were selected to participate in this prospective study. Twenty-four patients fulfilled the inclusion criteria and, therefore, underwent a gradual reduction of immunosuppression. Tolerance was defined as normal liver function tests after immunosuppression withdrawal. Basal clinical and immunological characteristics, including lymphocyte counts and subpopulations (T, B, natural killer, CD4+, CD8+, and regulatory T cells) and the phytohemagglutinin stimulation index (SI), were compared for tolerant and nontolerant patients. Fifteen of the 24 patients (62.5%) were tolerant at a median of 14 months (interquartile range = 8.5-22.5 months) after complete immunosuppression withdrawal. Tolerant patients had a longer median interval between transplantation and inclusion in the study (156 for tolerant patients versus 71 months for nontolerant patients, P = 0.003) and a lower median SI (7.49 for tolerant patients versus 41.73 for nontolerant patients, P = 0.01). We identified 3 groups of patients with different probabilities of tolerance: in the first group (n = 7 for an interval > 10 years and an SI 10 years and an SI > 20 or an interval 20), 29% reached tolerance. In conclusion, a high proportion of select LT recipients can reach tolerance over the long term. Two simple basal variables—the time from transplantation and the SI—may help to identify these patients. Liver Transpl 19:937–944, 2013. © 2013 AASLD.

Radioembolization (RE)-induced liver disease (REILD) has been defined as jaundice and ascites appearing 1 to 2 months after RE in the absence of tumor progression or bile duct occlusion. Our aims were to study the incidence of REILD in a large cohort of patients and the impact of a series of changes introduced in the processes of treatment design, activity calculation, and the routine use of ursodeoxycholic acid and low-dose steroids (modified protocol). Between 2003 and 2011, 260 patients with liver tumors treated by RE were studied (standard protocol: 75, modified protocol: 185). REILD appeared only in patients with cirrhosis or in noncirrhosis patients exposed to systemic chemotherapy prior to RE. Globally, the incidence of REILD was reduced in the modified protocol group from 22.7% to 5.4% and the incidence of severe REILD from 13.3% to 2.2% (P0.0001). Treatment efficacy was not jeopardized since 3-month disease control rates were virtually identical in both groups (66.7% and 67.2%, P=0.93). Exposure to chemotherapy in the 2-month period following RE and being treated by the standard protocol were independent predictors of REILD among noncirrhosis patients. In cirrhosis, the presence of a small liver (total volume1.5 L), an abnormal bilirubin (1.2 mg/dL), and treatment in a selective fashion were independently associated with REILD.REILD is an uncommon but relevant complication that appears when liver tissue primed by cirrhosis or prior and subsequent chemotherapy is exposed to the radiation delivered by radioactive microspheres. We designed a comprehensive treatment protocol that reduces the frequency and the severity of REILD.

Hepatocarcinogenesis and tumor growth may result in the deregulation of antigen expression in liver cells that can be recognized by the immune system. The immune system plays a major role in controlling tumor progression, and tumor growth depends on the development of escape mechanisms from immunological surveillance. Several clinical evidences suggest that in patients with liver cancer a strong immune response is associated to better prognosis. In the last 20 years, different strategies aimed to enhance the immune response against liver cancer have been tested in the setting of clinical trials with encouraging results. These included the use of cytokines aimed at enhancing the T-cell response, active immunization using peptide or DC platforms, the injection of genetically modified viruses and more recently, the administration of antibodies that interact with immune checkpoint molecules that stimulate the T cell response by suppressing the activity of T regulatory cells or by preventing T cell exhaustion. The combination of immunotherapeutic agents with antiangiogenic drugs may plausibly achieve a stronger antitumoral activity compared to single agents. Ongoing studies will shortly address the safety and the efficacy of such strategies.

Immune checkpoints are membrane-bound molecules that provide fine-tuning of the complex process of immune response against cancer. From a therapeutic perspective, interaction with checkpoint molecules has revolutionized the field of cancer therapy. Attention has focused in two players, CTLA-4 and PD-1. The former is essential for the activation of CD4+ T cells and the priming phase of the immune response while the latter inhibits CD4+ and CD8+ T cell activation. In patients with hepatocellular carcinoma, there is substantial evidence that supports a relevant role of immune checkpoints in the absence of an effective antitumor immune response. These negative signals can be blocked with the use of monoclonal antibodies. While CTLA-4 blockade first produced encouraging signals of antitumor activity, PD-1 blockade results in consistent tumor responses and prolonged stabilizations that lead to substantial prolongation of survival among patients with advanced tumors. This has fueled the interest in developing better therapeutic strategies through combinations of immune-oncology agents and combinations of such agents with targeted agents that may act synergistically.

Background: Hepatocellular carcinoma (HCC) is the sixth most common cancer in the world and the third most common cause of cancer-related mortality. Despite the widespread implementation of surveillance programs, more than half of the patients with HCC are diagnosed at late stages, when curative treatments (resection or liver transplantation) cannot be applied. For patients presenting with unresectable, non-transplantable HCC and a relatively preserved liver function, locoregional transarterial therapies are the main therapeutic options. Therefore, locoregional transarterial treatments play a key role in the management of HCC. The concept of catheter transarterial therapies is to selectively deliver anticancer treatments to liver tumors. This is possible due to the unique double irrigation of the liver and the predominantly arterial irrigation of liver tumors. Method: An exhaustive analysis of the literature, focused mainly on the last 25 years, has been carried out. Results: This review discusses the scientific evidence supporting the use of transarterial therapies for patients with HCC, including chemoembolization with conventional materials or drug-eluting beads, and internal radiation procedures such as the injection of radiation-loaded microspheres. Conclusion: Transarterial chemoembolization has been shown to improve the survival of those patients with unresectable HCC who have a preserved liver function and a low tumor burden. Recently developed devices and procedures, particularly drug-eluting beads and radioactive microspheres, may further improve the clinical outcome of patients receiving transarterial therapies. The combination with antiangiogenic agents is an appealing approach that should be explored.

Hepatocellular carcinoma (HCC) is often fatal due to local growth inside the liver and its unique arterial vascularization provides the basis for transarterial therapies. Around 35% of patients are diagnosed at stages in which transarterial therapies are indicated as first-line therapy; many others are treated after recurrence or progression to surgery or percutaneous ablation. However, the scientific evidence supporting the use of transarterial therapy is heterogeneous and certainly weak for several subgroups. New developments have emerged in the last decade.This review discusses the scientific evidence supporting the use of transarterial therapies for patients with HCC, including chemoembolization with conventional materials or drug-eluting beads, and internal radiation procedures such as the injection radioactive lipiodol or radioembolization with (90)Y-loaded microspheres. The literature on clinical development of transarterial therapies for HCC has been reviewed since 1990.Transarterial chemoembolization has been shown to improve the survival of those patients with unresectable, mostly viral-related HCC who have a preserved liver function and low tumor burden. Recently developed devices and procedures, particularly drug-eluting beads and radioactive microspheres, may further improve the clinical outcome of patients receiving transarterial therapies. Combination with antiangiogenic agents is an appealing approach that should be explored.

Liver transplant recipients have an increased risk of malignancy. Smoking is related to some of the most frequent causes of posttransplant malignancy. The incidence and risk factors for the development of neoplasia related to smoking (head and neck, lung, esophageal, and kidney and urinary tract carcinomas) were studied in 339 liver transplant recipients. Risk factors for the development of smoking-related neoplasia were also studied in 135 patients who had a history of smoking so that it could be determined whether smoking withdrawal was associated with a lower risk of malignancy. After a mean follow-up of 7.5 years, 26 patients were diagnosed with 29 smoking-related malignancies. The 5- and 10-year actuarial rates were 5% and 13%, respectively. In multivariate analysis, smoking and older age were independently associated with a higher risk of malignancy. In the smoker subgroup, the variables related to a higher risk of malignancy were active smoking and older age. In conclusion, smoking withdrawal after liver transplantation may have a protective effect against the development of neoplasia.

The Barcelona Clinic Liver Cancer (BCLC) staging system recommends first-line therapy for each tumor stage. We evaluated the effect of compliance with BCLC treatment allocation on the prognosis of patients with hepatocellular carcinoma (HCC). We retrospectively analyzed 359 consecutive, newly diagnosed HCC patients treated in our Liver Unit during a 14-year period. For each stage, survival was compared according to whether treatment matched the BCLC recommendation. We also compared the survival of patients in the same BCLC stage who received different treatments, and patients in different BCLC stages receiving the same treatment. BCLC-A patients treated with radical therapies (66%) survived longer (117 vs. 20 months; p

Background and aims: Clonal evolution of a tumor ecosystem depends not only on somatic mutations driving uncontrolled growth, but on a full array of selection pressures, principally immune and resource mediated. We aimed at mapping the spatio-temporal interactions between cancer and immune cells in hepatocellular carcinoma (HCC) by quantifying regional adaptive immune editing, and how this impacts clonal evolution. Methods: We integrated data from RNAseq, targeted DNA sequencing, SNP array and histological evaluation from multiple regions of 10 HCC treatment-naive surgical specimens (55 samples, median of 4 tumor and 2 non-tumor regions per patient), 6 of whom with Hepatitis B virus infection (HBV). Immune cells were assessed with immunofluorescence for T (CD3) and B (CD19) markers. MAGEA3 was down-regulated in 3 liver cancer cell lines using shRNA and proliferation assessed with the MTS assay. Analyses included: intra-tumoral and HBV differential gene expression, prediction of immunogenicity of expressed mutations (i.e., tumor neoantigens) and HBV antigens, T and B cell receptor sequencing and survival and network analysis on the liver cancer TCGA dataset. Results & Conclusions: There is a tumor-driven adaptive immune response contributing to HCC heterogeneity, mainly recruited by subclonal mutations as compared to an interplay between clonal mutations and HBV epitopes. Indeed, we found different regional configurations of tumor infiltrating lymphocytes with higher density of Tertiary Lymphoid Structures in areas enriched in highly immunogenic tumor neoantigens. Furthermore, regional differences in gene expression of heterogeneous tumors can capture stronger prognostic signals than best-in-class single biopsy based predictors tested on TCGA LIHC HCC data. This demonstrates that the breadth of molecular states in a single tumor can easily approach that of a large population sample of tumors. We also found evidence that some tumors' spatiotemporal expression profile is directly correlated with HBV expression, including the extreme case where some regions of a HBV infected patient have zero HBV expression, suggesting regional selection of infected clones via resource constraints. Finally we found that some tumoral evolution is dominated by cancer testis antigen (CTA) dysregulation led by MAGEA3/6, which we determined is a key causal driver of a major regulatory subnetwork from a Bayesian gene interaction network we inferred from TCGA LIHC HCC data. Hypothesizing a oncodriver role for MAGEA3, we confirmed that MAGEA3 downregulation has anti-tumoral effects in HCC experimental models. Citation Format: Bojan Losic, Amanda J. Craig, Sebastiao N. Martins-Filho, Carlos Villacorta-Martin, Nicholas Akers, Xintong Chen, Mehmet E. Ahsen, Ismail Labgaa, Delia D'Avola, Sergio A. Lira, Glaucia C. Furtado, Ashley Stueck, Stphen C. Ward, Maria I. Fiel, Ganesh Gunasekaran, Daniela Sia, Eric E. Schadt, Myron Schwartz, Josep M. Llovet, Swan Thung, Gustavo Stolovitzky, Augusto Villanueva. Deciphering the impact of immune editing on liver cancer clonal evolution using immunogenomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1189.