Transcriptomic characterization of cancer-testis antigens identifies MAGEA3 as a driver of tumor progression in hepatocellular carcinoma.

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.

Beneficial effect of ursodeoxycholic acid in patients with acyl-CoA oxidase 2 (ACOX2) deficiency-associated hypertransaminasemia.

BACKGROUND AND AIMS: A variant (p.Arg225Trp) of peroxisomal acyl-CoA oxidase 2 (ACOX2), involved in bile acid (BA) side-chain shortening, has been associated with unexplained persistent hypertransaminasemia and accumulation of C27-BAs, mainly 3α,7α,12α-trihydroxy-5ß-cholestanoic acid (THCA). We aimed to investigate the prevalence of ACOX2 deficiency-associated hypertransaminasemia (ADAH), its response to ursodeoxycholic acid (UDCA), elucidate its pathophysiological mechanism and identify other inborn errors that could cause this alteration. METHODS AND RESULTS: Among 33 patients with unexplained hypertransaminasemia from 11 hospitals and 13 of their relatives, seven individuals with abnormally high C27-BA levels (>50% of total BAs) were identified by high-performance liquid chromatography-mass spectrometry. The p.Arg225Trp variant was found in homozygosity (exon amplification/sequencing) in two patients and three family members. Two additional nonrelated patients were heterozygous carriers of different alleles: c.673C>T (p.Arg225Trp) and c.456_459del (p.Thr154fs). In patients with ADAH, impaired liver expression of ACOX2, but not ACOX3, was found (immunohistochemistry). Treatment with UDCA normalized aminotransferase levels. Incubation of HuH-7 hepatoma cells with THCA, which was efficiently taken up, but not through BA transporters, increased reactive oxygen species production (flow cytometry), endoplasmic reticulum stress biomarkers (GRP78, CHOP, and XBP1-S/XBP1-U ratio), and BAXα expression (reverse transcription followed by quantitative polymerase chain reaction and immunoblot), whereas cell viability was decreased (tetrazolium salt-based cell viability test). THCA-induced cell toxicity was higher than that of major C24-BAs and was not prevented by UDCA. Fourteen predicted ACOX2 variants were generated (site-directed mutagenesis) and expressed in HuH-7 cells. Functional tests to determine their ability to metabolize THCA identified six with the potential to cause ADAH. CONCLUSIONS: Dysfunctional ACOX2 has been found in several patients with unexplained hypertransaminasemia. This condition can be accurately identified by a noninvasive diagnostic strategy based on plasma BA profiling and ACOX2 sequencing. Moreover, UDCA treatment can efficiently attenuate liver damage in these patients.

Brain ventricular enlargement in human and murine acute intermittent porphyria.

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 analyze 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.

The importance of liver functional reserve in the non-surgical treatment of hepatocellular carcinoma.

The aim of any oncological treatment is not just to eliminate the tumour, but to maximise patient survival and quality of life. Since the liver has a vital function, any radical treatment that severely compromises liver function will result in a shortening of life expectancy, rather than a prolongation. Furthermore, even non-severe liver damage may prevent the delivery of further effective therapies. This is particularly important in the case of hepatocellular carcinoma (HCC), as it is associated with underlying cirrhosis in most patients - 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 and can be used to guide the selection of treatments that the functional liver reserve can tolerate. Unfortunately, the prediction of functional damage to the liver in the case of antitumor treatments is very challenging and still suboptimal in any given patient. Moreover, while the assessment of functional reserve can now be used to avoid postoperative liver failure in the surgical setting, its use has been less well clarified for non-surgical therapies, which is of particular relevance today, as 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 available tools to assess liver functional reserve and c) discuss the role of functional assessment for each type of non-surgical therapy for HCC.

Immunotherapy in hepatocellular carcinoma.

Hepatocellular carcinoma is a highly prevalent tumor worldwide and when it reaches an advanced stage, few systemic treatments are available to improve the survival of these patients. However, greater knowledge about the tumor microenvironment and the role of the immune system in the control of tumor progression has allowed the development of treatments targeting immune checkpoints, which result in encouraging tumor response rates and prolonged survival. Although most of these treatments are well tolerated, up to 20 % of patients may experience side effects derived from non-specific stimulation of the immune system. In the cirrhotic patient, the early diagnosis and treatment of such adverse events is particularly challenging. Therefore, the ongoing investigation on the use of these new therapies will allow us to better understand the profile of the patients who will benefit most.

Unannotated small RNA clusters associated with circulating extracellular vesicles detect early stage liver cancer.

OBJECTIVE: Surveillance 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. DESIGN: EVs 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. RESULTS: ExRNA 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 (p<0.01, n=157). An independent validation in a phase 2 biomarker case-control study revealed 86% sensitivity and 91% specificity for the detection of early HCC from controls at risk (n=209) (area under the receiver operating curve (AUC): 0.87). The 3-smRC signature was independent of alpha-fetoprotein (p<0.0001) and a composite model yielded an increased AUC of 0.93. CONCLUSION: These 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.

A phenotypical map of disseminated hepatocellular carcinoma suggests clonal constraints in metastatic sites.

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.

Cardiovascular morbidity and mortality after liver transplantation: The protective role of mycophenolate mofetil.

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.

Recombinant AAV Integration Is Not Associated With Hepatic Genotoxicity in Nonhuman Primates and Patients.

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.

Phase I open label liver-directed gene therapy clinical trial for acute intermittent porphyria.

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.

Pilot randomized trial of selective internal radiation therapy vs. chemoembolization in unresectable hepatocellular carcinoma.

BACKGROUND & AIMS: To compare selective internal radiation therapy (SIRT) with transarterial chemoembolization (TACE), the standard-of-care for intermediate-stage unresectable, hepatocellular carcinoma (HCC), as first-line treatment. METHODS: SIRTACE was an open-label multicenter randomized-controlled pilot study, which prospectively compared primarily safety and health-related quality of life (HRQoL) changes following TACE and SIRT. Patients with unresectable HCC, Child-Pugh ≤B7, ECOG performance status ≤2 and ≤5 liver lesions (≤20 cm total maximum diameter) without extrahepatic spread were randomized to receive either TACE (at 6-weekly intervals until tumour enhancement was not observed on MRI or disease progression) or single-session SIRT (yttrium-90 resin microspheres). RESULTS: Twenty-eight patients with BCLC stage A (32.1%), B (46.4%) or C (21.4%) received either a mean of 3.4 (median 2) TACE interventions (N = 15) or single SIRT (N = 13). Both treatments were well tolerated. Despite SIRT patients having significantly worse physical functioning at baseline, at week-12, neither treatment had a significantly different impact on HRQoL as measured by Functional Assessment of Cancer Therapy-Hepatobiliary total or its subscales. Both TACE and SIRT were effective for the local control of liver tumours. Best overall response-rate (RECIST 1.0) of target lesions were 13.3% and 30.8%, disease control rates were 73.3% and 76.9% for TACE and SIRT, respectively. Two patients in each group were down-staged for liver transplantation (N = 3) or radiofrequency ablation (N = 1). CONCLUSIONS: Single-session SIRT appeared to be as safe and had a similar impact on HRQoL as multiple sessions of TACE, suggesting that SIRT might be an alternative option for patients eligible for TACE.

Radioembolization of hepatocellular carcinoma activates liver regeneration, induces inflammation and endothelial stress and activates coagulation.

BACKGROUND & AIMS: Radioembolization may rarely induce liver disease resulting in a syndrome that is similar to veno-occlusive disease complicating bone marrow transplantation where inflammation, endothelial cell activation and thrombosis are likely involved. We hypothesized that similar mechanisms could be implicated in radioembolization-induced liver disease (REILD). Moreover, lobar radioembolization may induce hypertrophy of the non-treated hemiliver most probably by inducing liver regeneration. METHODS: In patients with hepatocellular carcinoma, we prospectively studied serum levels of markers of liver regeneration, oxidative stress, pro-inflammatory pathways, endothelial activation and coagulation parameters over 2 months after radioembolization. RESULTS: Although REILD did not occur among 14 treated patients, a decrease in effective liver blood flow was observed. Radioembolization was followed by a persistent increase in pro-inflammatory (interleukin 6 and 8) and oxidative stress (malondyaldehide) markers, an induction of endothelial injury markers (vW factor and PAI-1) and an activation of the coagulation cascade (factor VIII, PAI-1, D-Dimer) as well as a significant increase in factors related to liver regeneration (FGF-19 and HGF). CONCLUSION: Radioembolization activates liver regeneration, produces oxidative stress, activates inflammatory cytokines and induces endothelial injury with partial activation of the coagulation cascade. These findings may have implications in the pathogenesis, prevention and therapy of REILD and in the development of new therapies to enhance hypertrophy with a surgical perspective.

Prognostic factors and prevention of radioembolization-induced liver disease.

UNLABELLED: 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% (P<0.0001). 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 volume<1.5 L), an abnormal bilirubin (>1.2 mg/dL), and treatment in a selective fashion were independently associated with REILD. CONCLUSION: 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.

[Comparison of the M and XL FibroScan(®) probes to estimate liver stiffness by transient elastography]. / Comparación de las sondas M y XL para estimar la rigidez hepática por medio de elastografía de transición.

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 Castéra 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<.001), but not in the non-obese patients (84% versus 83%). Despite a high correlation between the stiffness values obtained with the two probes (R=.897; P<.001), and a high concordance in the estimation of fibrosis obtained with the two probes (Cronbach's alpha value: 0.932), the liver stiffness values obtained with the XL probe were significantly lower than those obtained with the M probe, both in the whole series (9.5 ± 9.1 kPa versus 11.3 ± 12.6 kPa; P<0.001) and in the obese and non-obese groups. In conclusion, transient elastography with the XL probe allows a higher proportion of reliable examinations in obese patients but not in non-obese patients. Stiffness values were lower with the XL probe than with the M probe.

Partial liver volume radioembolization induces hypertrophy in the spared hemiliver and no major signs of portal hypertension.

BACKGROUND: Post-treatment contralateral hemiliver hypertrophy has created an interest in lobar liver radioembolization (RE) as a pre-surgery tool. METHODS: Liver 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. RESULTS: More 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: 230 ml). The percentage of patients in whom the baseline ratio of spared volume to total liver volume was <40% dropped from 56.6% at baseline to 29.4% at T2 (P < 0.001). A significant and progressive increase in spleen volume but not in portal vein diameter was also observed. A small percentage of patients developed hypersplenism, mostly those without cirrhosis (16.0% at T2). Six patients (five with portal vein thrombosis, cirrhosis or both) developed signs of portal hypertension by T2. CONCLUSIONS: The present results warrant further studies to better elucidate the mechanism underlying this phenomenon of spared hemiliver hypertrophy and to investigate its role as an alternative to portal vein embolization in the management of patients with potentially resectable liver tumours.

Prognostic value of ultra-low-pass whole-genome sequencing of circulating tumor DNA in hepatocellular carcinoma under systemic treatment.

BACKGROUND/AIMS: New prognostic markers are needed to identify patients with hepatocellular carcinoma (HCC) who carry a worse prognosis. Ultra-low-pass whole-genome sequencing (ULP-WGS) (≤0.5× coverage) of cell-free DNA (cfDNA) has emerged as a low-cost promising tool to assess both circulating tumor DNA (ctDNA) fraction and large structural genomic alterations. Here, we studied the performance of ULP-WGS of plasma cfDNA to infer prognosis in patients with HCC. METHODS: Plasma samples were obtained from patients with HCC prior to surgery, locoregional or systemic therapy, and were analyzed by ULP-WGS of cfDNA to an average genome-wide fold coverage of 0.3x. ctDNA and copy number alterations (CNA) were estimated using the software package ichorCNA. RESULTS: Samples were obtained from 73 HCC patients at different BCLC stages (BCLC 0/A: n=37, 50.7%; BCLC B/C: n=36, 49.3%). ctDNA was detected in 18 out of 31 patients who received systemic treatment. Patients with detectable ctDNA showed significantly worse overall survival (median, 13.96 months vs not reached). ctDNA remained an independent predictor of prognosis after adjustment by clinical-pathologic features and type of systemic treatment (hazard ratio 7.69; 95%, CI 2.09-28.27). Among ctDNA-positive patients under systemic treatments, the loss of large genomic regions in 5q and 16q arms was associated with worse prognosis after multivariate analysis. CONCLUSION: ULP-WGS of cfDNA provides clinically relevant information about the tumor biology. The presence of ctDNA and the loss of 5q and 16q arms in ctDNA-positive patients are independent predictors of worse prognosis in patients with advanced HCC receiving systemic therapy.

Trial of complete weaning from immunosuppression for liver transplant recipients: factors predictive of tolerance.

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 < 20), 100% reached tolerance; in the second group (n = 10 for an interval > 10 years and an SI > 20 or an interval < 10 years and an SI < 20), 60% reached tolerance; and in the third group (n = 7 for an interval < 10 years and an SI > 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.

Metabolic dysfunction-associated fatty liver disease (MAFLD): an update of the recent advances in pharmacological treatment.

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.

Interactive Role of Surrogate Liver Fibrosis Assessment and Insulin Resistance on the Incidence of Major Cardiovascular Events.

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.

Design and validation of a tunable inertial microfluidic system for the efficient enrichment of circulating tumor cells in blood.

The analysis of circulating tumor cells (CTCs) in blood is a powerful noninvasive alternative to conventional tumor biopsy. Inertial-based separation is a promising high-throughput, 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 cut-off 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 confirm highly efficient CTC isolation with a stringent cut-off value and better separation results than the state of the art.