An atlas of cells in the human tonsil.

Palatine tonsils are secondary lymphoid organs (SLOs) representing the first line of immunological defense against inhaled or ingested pathogens. We generated an atlas of the human tonsil composed of >556,000 cells profiled across five different data modalities, including single-cell transcriptome, epigenome, proteome, and immune repertoire sequencing, as well as spatial transcriptomics. This census identified 121 cell types and states, defined developmental trajectories, and enabled an understanding of the functional units of the tonsil. Exemplarily, we stratified myeloid slan-like subtypes, established a BCL6 enhancer as locally active in follicle-associated T and B cells, and identified SIX5 as putative transcriptional regulator of plasma cell maturation. Analyses of a validation cohort confirmed the presence, annotation, and markers of tonsillar cell types and provided evidence of age-related compositional shifts. We demonstrate the value of this resource by annotating cells from B cell-derived mantle cell lymphomas, linking transcriptional heterogeneity to normal B cell differentiation states of the human tonsil.

Histological predictors of aggressive recurrence of hepatocellular carcinoma after liver resection.

BACKGROUND AND AIMS: Assessment of recurrence risk after liver resection (LR) is critical in hepatocellular carcinoma (HCC), particularly with the advent of effective adjuvant therapy. The aim of the study was to analyze the clinical and pathological factors associated with recurrence, aggressive recurrence, and survival after LR. METHOD: Retrospective study in which all single HCC (BCLC-0/A) patients treated with LR between February 2000 and November 2020 were included. The main clinical variables were recorded. Histological features were blindly evaluated by two independent pathologists. Aggressive recurrence was defined as those that exceeded the Milan criteria at 1st recurrence. RESULTS: A total of 218 patients were included (30% BCLC 0 and 70% BCLC A), median (IQR) tumor size of 28 (19-42mm). The prevalence of microvascular invasion and/or satellitosis (mVI/S) was 39%, with a kappa-index between both pathologists of 0.8. After a median follow-up of 49 (23-85) months, 61/218 (28%) patients died, 32/218 (15%) underwent LT, 127 (58%) developed HCC recurrence. The prevalence of aggressive recurrence was 35% (44/127 Milan-out, with 20 cases at advanced stage), and the 5-year survival was 81%. The presence of mVI/S was the only independent predictor of recurrence [HR:1.83 (1.28-2.61), p<0.001], aggressive recurrence [HR:3.31(1.74-6.29), p<0.001] and mortality [HR:2.23(1.27- 3.91), p:0.005]. The presence of MTM was significantly associated with a higher prevalence of mVI/S, Edmonson Steiner grade III-IV, AFP values and vessels that encapsulate tumor clusters, but MTM was not significantly associated with recurrence, aggressive recurrence, or OS. CONCLUSION: The presence of mVI/S was the only independent risk factor for aggressive recurrence and mortality. This has important implications for early-stage patient management, especially in the setting of adjuvant immunotherapy or ab initio LT.

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.

Single-pixel imaging through non-homogeneous turbid media with adaptive illumination.

The presence of scattering media limits the quality of images obtained by optical systems. Single-pixel imaging techniques based on structured illumination are highly tolerant to the presence of scattering between the object and the sensor, but very sensitive when the scattering medium is between the light source and the object. This makes it difficult to develop single-pixel imaging techniques for the case of objects immersed in scattering media. We present what we believe to be a new system for imaging objects through inhomogeneous scattering media in an epi-illumination configuration. It works in an adaptive way by combining diffuse optical imaging (DOI) and single pixel imaging (SPI) techniques in two stages. First, the turbid media is characterized by projecting light patterns with an LED array and applying DOI techniques. Second, the LED array is programmed to project light only through the less scattering areas of the media, while simultaneously using a digital micromirror device (DMD) to project light patterns onto the target using Hadamard basis coding functions. With this adaptive technique, we are able to obtain images of targets through two different scattering media with better quality than using conventional illumination. We also show that the system works with fluorescent targets.

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.

Anticancer drugs are the first cause of drug-induced liver injury in a reference hospital.

Drug-induced liver injury (DILI) is a challenging liver disorder for hepatologists. We aimed to assess the pattern and causes of DILI in a tertiary hospital. We registered prospectively all patients referred with suspicion of DILI from 2018 to 2023. A total of 106 patients fulfilled the diagnostic criteria (30 caused by paracetamol were excluded; total number 76). The pattern of liver injury was hepatocellular in 55 (72%). Drugs causing DILI were antineoplastic (26%), antibiotics (24%), analgesics (12%), and recreational drugs (9%). Regarding clinical outcomes, 39 (51%) required hospitalization and 7 (9%) underwent a liver transplantation or died from acute liver injury. We identified 126 additional patients with DILI due to immune check-point inhibitors who were not referred to a liver disease specialist. Antineoplastic drugs have become the first cause of DILI in hospitals. A multidisciplinary approach and specific educational tools to increase DILI awareness are needed among different specialists.

Delayed immune-related hepatitis after 24 months of pembrolizumab treatment: a case report and literature review.

Immune checkpoint inhibitors targeting the programmed cell death protein 1 (PD-1) pathway have revolutionized cancer immunotherapy by enhancing the immune system's ability to combat cancer cells. However, this innovative approach comes with a distinctive set of challenges, as these therapies can lead to immune-related adverse events (irAEs) due to their mechanism of action. The most common irAEs involve the skin, gastrointestinal tract, liver, endocrine system, and lungs. These events can range from mild skin rashes to severe colitis, pneumonitis, or even autoimmune organ damage. These adverse effects usually appear with an average of 5-15 weeks from the start of treatment depending on the affected organ. This article presents a case report of a delayed related-mediated hepatitis, after 24 months of treatment with pembrolizumab and almost 3 months after its termination, and a review of the scientific literature on cases of delayed immune-related hepatitis caused by anti-PD1. This case highlights the importance of monitoring patients treated with immune checkpoint inhibitors after cessation as a growing number of patients stop treatment due to achieving durable responses.

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.

Stimulation of soluble guanylate cyclase exerts antiinflammatory actions in the liver through a VASP/NF-κB/NLRP3 inflammasome circuit.

Soluble guanylate cyclase (sGC) catalyzes the conversion of guanosine triphosphate into cyclic guanosine-3',5'-monophosphate, a key second messenger in cell signaling and tissue homeostasis. It was recently demonstrated that sGC stimulation is associated with a marked antiinflammatory effect in the liver of mice with experimental nonalcoholic steatohepatitis (NASH). Here, we investigated the mechanisms underlying the antiinflammatory effect of the sGC stimulator praliciguat (PRL) in the liver. Therapeutic administration of PRL exerted antiinflammatory and antifibrotic actions in mice with choline-deficient l-amino acid-defined high-fat diet-induced NASH. The PRL antiinflammatory effect was associated with lower F4/80- and CX3CR1-positive macrophage infiltration into the liver in parallel with lower Ly6CHigh- and higher Ly6CLow-expressing monocytes in peripheral circulation. The PRL antiinflammatory effect was also associated with suppression of hepatic levels of interleukin (IL)-1ß, NLPR3 (NACHT, LRR, and PYD domain-containing protein 3), ASC (apoptosis-associated speck-like protein containing a caspase-recruitment domain), and active cleaved-caspase-1, which are components of the NLRP3 inflammasome. In Kupffer cells challenged with the classical inflammasome model of lipopolysaccharide plus adenosine triphosphate, PRL inhibited the priming (expression of Il1b and Nlrp3) and blocked the release of mature IL-1ß. Mechanistically, PRL induced the protein kinase G (PKG)-mediated phosphorylation of the VASP (vasodilator-stimulated phosphoprotein) Ser239 residue which, in turn, reduced nuclear factor-κB (NF-κB) activity and Il1b and Nlrp3 gene transcription. PRL also reduced active cleaved-caspase-1 levels independent of pannexin-1 activity. These data indicate that sGC stimulation with PRL exerts antiinflammatory actions in the liver through mechanisms related to a PKG/VASP/NF-κB/NLRP3 inflammasome circuit.

Liver cancer risk after HCV cure in patients with advanced liver disease without non-characterized nodules.

BACKGROUND & AIMS: Recognition of non-characterized liver nodules (NCLN) prior to direct-acting antivirals (DAAs) is associated with increased hepatocellular carcinoma (HCC) risk in patients with HCV. The risk of HCC has not been defined in F3/F4 patients in whom NCLN have been ruled-out before starting DAAs and at sustained virological response (SVR). This study aimed to estimate HCC incidence in this population. METHODS: We performed a prospective study including HCV-infected patients with F3/F4 fibrosis, without a history of HCC, and who achieved SVR after DAAs. Patients were only included if they had undergone ultrasound imaging that excluded the presence of HCC/NCLN within 30 days after SVR. All patients were evaluated every 6 months until developing primary liver cancer, death or withdrawal of informed consent. HCC incidence was expressed per 100 patient-years (/100PY). Adherence to screening program was calculated every 6 months for the first 48 months. RESULTS: A total of 185 patients (63/122, F3/F4) were included. Among those with cirrhosis, 92% were Child-Pugh A and 42.7% had clinically significant portal hypertension (CSPH). Albumin-bilirubin score was 1 in 84.9% and 2 in 15.1% of patients, respectively. The median clinical and radiologic follow-up was 52.4 months and 48 months, respectively. Ten patients developed HCC: HCC incidence was 1.46/100PY (95% CI 0.79-2.71) in the whole cohort, 2.24/100PY (95% CI 1.21-4.17) in F4 only and 3.63/100PY (95% CI 1.95-6.74) in patients with CSPH. No HCC was registered in patients with F3. Median time between SVR and HCC occurrence was 28.1 months; 12 non-primary liver cancers were also identified. CONCLUSIONS: Patients with cirrhosis without NCLN at SVR remain at risk of HCC development. The absence of HCC in patients with F3 reinforces their marginal cancer risk, but prospective studies are needed to exclude them from screening programs. LAY SUMMARY: Patients with HCV-related cirrhosis, without non-characterized liver nodules at sustained virologic response, remain at risk of hepatocellular carcinoma despite viral cure. However, the cancer risk after successful direct-acting antiviral treatment is marginal in patients with F3 fibrosis without non-characterized liver nodules. If confirmed in larger prospective studies, current screening recommendations may need to be revisited in this group of patients.

Nestin as a diagnostic and prognostic marker for combined hepatocellular-cholangiocarcinoma.

BACKGROUND & AIMS: Combined hepatocellular-cholangiocarcinoma (cHCC-CCA) is a rare primary liver cancer (PLC) associated with a poor prognosis. Given the challenges in its identification and its clinical implications, biomarkers are critically needed. We aimed to investigate the diagnostic and prognostic value of the immunohistochemical expression of Nestin, a progenitor cell marker, in a large multicentric series of PLCs. METHODS: We collected 461 cHCC-CCA samples from 32 different clinical centers. Control cases included 368 hepatocellular carcinomas (HCCs) and 221 intrahepatic cholangiocarcinomas (iCCAs). Nestin immunohistochemistry was performed on whole tumor sections. Diagnostic and prognostic performances of Nestin expression were determined using receiver-operating characteristic curves and Cox regression modeling. RESULTS: Nestin was able to distinguish cHCC-CCA from HCC with AUCs of 0.85 and 0.86 on surgical and biopsy samples, respectively. Performance was lower for the distinction of cHCC-CCA from iCCA (AUCs of 0.59 and 0.60). Nestin, however, showed a high prognostic value, allowing identification of the subset of cHCC-CCA ("Nestin High", >30% neoplastic cells with positive staining) associated with the worst clinical outcome (shorter disease-free and overall survival) after surgical resection and liver transplantation, as well as when assessment was performed on biopsies. CONCLUSION: We show in different clinical settings that Nestin has diagnostic value and that it is a useful biomarker to identify the subset of cHCC-CCA associated with the worst clinical outcome. Nestin immunohistochemistry may be used to refine risk stratification and improve treatment allocation for patients with this highly aggressive malignancy. LAY SUMMARY: There are different types of primary liver cancers (i.e. cancers that originate in the liver). Accurately identifying a specific subtype of primary liver cancer (and determining its associated prognosis) is important as it can have a major impact on treatment allocation. Herein, we show that a protein called Nestin could be used to refine risk stratification and improve treatment allocation for patients with combined hepatocellular carcinoma, a rare but highly aggressive subtype of primary liver cancer.

Liver Stiffness by Transient Elastography to Detect Porto-Sinusoidal Vascular Liver Disease With Portal Hypertension.

BACKGROUND AND AIMS: Porto-sinusoidal vascular liver disease (PSVD) is a rare cause of portal hypertension. PSVD is still often misdiagnosed as cirrhosis, emphasizing the need to improve PSVD diagnosis strategies. Data on liver stiffness measurement using transient elastography (TE-LSM) in PSVD are limited. The aim of this study was to evaluate the accuracy of TE-LSM to discriminate PSVD from cirrhosis in patients with signs of portal hypertension. APPROACH AND RESULTS: Retrospective multicenter study comparing TE-LSM in patients with PSVD, according to Vascular Liver Disease Interest Group criteria, with patients with compensated biopsy-proven cirrhosis associated with alcohol (n = 117), HCV infection (n = 110), or NAFLD (n = 46). All patients had at least one sign of portal hypertension among gastroesophageal varices, splenomegaly, portosystemic collaterals, history of ascites, or platelet count < 150 × 109 /L. The 77 patients with PSVD included in the test cohort had lower median TE-LSM (7.9 kPa) than the patients with alcohol-associated, HCV-related, and NAFLD-related cirrhosis (33.8, 18.2, and 33.6 kPa, respectively; P < 0.001). When compared with cirrhosis, a cutoff value of 10 kPa had a specificity of 97% for the diagnosis of PSVD with a 85% positive predictive value. A cutoff value of 20 kPa had a sensitivity of 94% for ruling out PSVD with a 97% negative predictive value. Of the patients, 94% were well-classified. Even better results were obtained in a validation cohort including 78 patients with PSVD. CONCLUSIONS: This study including a total of 155 patients with PSVD and 273 patients with cirrhosis demonstrates that TE-LSM < 10 kPa strongly suggests PSVD in patients with signs of portal hypertension. Conversely, when TE-LSM is >20 kPa, PSVD is highly unlikely.

Albumin protects the liver from tumor necrosis factor α-induced immunopathology.

Besides its oncotic power, albumin exerts pleiotropic actions, including binding, transport, and detoxification of endogenous and exogenous molecules, antioxidant activity, and modulation of immune and inflammatory responses. In particular, recent studies have demonstrated that albumin reduces leukocyte cytokine production. Here, we investigated whether albumin also has the ability to protect tissues from the damaging actions of these inflammatory mediators. We circumscribed our investigation to tumor necrosis factor (TNF) α, which exemplifies the connection between immunity and tissue injury. In vivo experiments in analbuminemic mice showed that these mice exhibit a more pronounced response to a model of TNFα-mediated liver injury induced by the administration of lipopolysaccharide (LPS) and D-galactosamine (D-gal). A tissue protective action against LPS/D-gal liver injury was also observed during the administration of human albumin to humanized mice expressing the human genes for albumin and neonatal Fc receptor (hAlb+/+ /hFcRn+/+ ) with preestablished carbon tetrachloride (CCl4 )-induced early cirrhosis. The cytoprotective actions of albumin against TNFα-induced injury were confirmed ex vivo, in precision-cut liver slices, and in vitro, in primary hepatocytes in culture. Albumin protective actions were independent of its scavenging properties and were reproduced by recombinant human albumin expressed in Oryza sativa. Albumin cytoprotection against TNFα injury was related to inhibition of lysosomal cathepsin B leakage accompanied by reductions in mitochondrial cytochrome c release and caspase-3 activity. These data provide evidence that in addition to reducing cytokines, the albumin molecule also has the ability to protect tissues against inflammatory injury.

Adult diffuse midline gliomas H3 K27-altered: review of a redefined entity.

INTRODUCTION: Diffuse midline glioma (DMG) H3 K27-altered is a type of high-grade gliomas first recognized as a new entity in the 2016 World Health Organization Classification of Central Nervous System (CNS) Tumors as DMG H3 K27M-mutant, recently renamed in the new 2021 WHO classification. The aim of this review is to describe the characteristics of diffuse midline gliomas H3 K27-altered in the adult population. METHODS: We performed a review of the current literature regarding the genetic, clinical, imaging characteristics and management of diffuse midline gliomas H3 K27-altered in adult patients. RESULTS: The 2021 WHO classification now designates the previously recognized DMG H3K27M-mutant as DMG H3 K27-altered, recognizing the alternative mechanisms by which the pathogenic pathway can be altered. Thus, the diagnostic criteria for this entity consist of diffuse growth pattern, midline anatomic location, and H3 K27-specific neuroglial mutations. DMGs' characteristic midline location makes them difficult to surgically resect and biopsy, carrying high mortality and morbidity rates, with median survival ranging from 9 to 12 months in adult patients. CONCLUSION: The diagnosis of DMGs H3 K27-altered in adult patients should be considered upon neurological symptoms associated with an infiltrative midline brain tumor detected on imaging. Future studies are necessary to continue refining their characteristics in this age group.

LiverScreen project: study protocol for screening for liver fibrosis in the general population in European countries.

BACKGROUND: The development of liver cirrhosis is usually an asymptomatic process until late stages when complications occur. The potential reversibility of the disease is dependent on early diagnosis of liver fibrosis and timely targeted treatment. Recently, the use of non-invasive tools has been suggested for screening of liver fibrosis, especially in subjects with risk factors for chronic liver disease. Nevertheless, large population-based studies with cost-effectiveness analyses are still lacking to support the widespread use of such tools. The aim of this study is to investigate whether non-invasive liver stiffness measurement in the general population is useful to identify subjects with asymptomatic, advanced chronic liver disease. METHODS: This study aims to include 30,000 subjects from eight European countries. Subjects from the general population aged ≥ 40 years without known liver disease will be invited to participate in the study either through phone calls/letters or through their primary care center. In the first study visit, subjects will undergo bloodwork as well as hepatic fat quantification and liver stiffness measurement (LSM) by vibration-controlled transient elastography. If LSM is ≥ 8 kPa and/or if ALT levels are ≥1.5 x upper limit of normal, subjects will be referred to hospital for further evaluation and consideration of liver biopsy. The primary outcome is the percentage of subjects with LSM ≥ 8kPa. In addition, a health economic evaluation will be performed to assess the cost-effectiveness and budget impact of such an intervention. The project is funded by the European Commission H2020 program. DISCUSSION: This study comes at an especially important time, as the burden of chronic liver diseases is expected to increase in the coming years. There is consequently an urgent need to change our current approach, from diagnosing the disease late when the impact of interventions may be limited to diagnosing the disease earlier, when the patient is asymptomatic and free of complications, and the disease potentially reversible. Ultimately, the LiverScreen study will serve as a basis from which diagnostic pathways can be developed and adapted to the specific socio-economic and healthcare conditions in each country. TRIAL REGISTRATION: This study is registered on Clinicaltrials.gov ( NCT03789825 ).

Limited tumour progression beyond Milan criteria while on the waiting list does not result in unacceptable impairment of survival.

BACKGROUND & AIMS: Defining optimum management of patients progressing beyond Milan criteria on the waiting list is a controversial topic. Our aim was to determine whether the policy of allowing a limited progression beyond enlistment criteria permits acceptable post-transplant outcomes in terms of survival and recurrence. METHODS: Patients with hepatocellular carcinoma included on the waiting list for orthotopic liver transplantation (OLT) between January 1989 and December 2016 were analysed. Tumour features were assessed at inclusion on the waiting list, before OLT and at explant pathology. Patients were retained on the waiting list despite exceeding enlistment criteria if not presenting with macrovascular invasion, extrahepatic spread or cancer-related symptoms. RESULTS: A total of 495 patients constituted the target population. Comparison between the Milan-in (n = 434) and Milan-out (n = 61) groups showed statistically significant differences in: largest tumour size; BCLC stage; patients treated before OLT; alpha-fetoprotein, and time on the waiting list. Milan-out patients showed a significantly higher number of poorly differentiated nodules, satellitosis and microscopic vascular invasion. The 1-, 3-, 5- and 10-year survival rate was 89.6%, 82.5%, 75%, and 55.5%, vs. 83.6%, 70.5%, 65.5%, and 53.9% for Milan-in/Milan-out patients, respectively. Recurrence rates at 1, 3, 5 and 10 years were 1.2%, 3.3%, 5.5%, and 10.8% vs. 7.1% 14.5%, 23%, and 23% for Milan-in and Milan-out patients, respectively (p <0.01). CONCLUSION: This study shows that although limited tumour progression without reaching major adverse predictors (vascular invasion, extrahepatic spread, cancer symptoms) has an expected impact on recurrence rate, overall survival remains above the minimum proposed benchmark of 65% at 5 years. The clinically relevant increase in tumour recurrence must be considered when analysing the benefit of this approach in the face of limited organ supply. LAY SUMMARY: When considering orthotopic liver transplantation for patients with hepatocellular carcinoma, optimum results are achieved when transplanting patients within the Milan criteria. However, the most appropriate strategy for patients who progress beyond these criteria while on the waiting list is still unclear. Herein, we show that transplantation is associated with acceptable overall survival in select patients who progress beyond the Milan criteria, although recurrence rates were notably higher. Therefore, the assessment of transplantation viability in these patients must consider the availability of organs and the impact on other patient categories.

Co-expression gene network analysis reveals novel regulatory pathways involved in porto-sinusoidal vascular disease.

BACKGROUND & AIMS: Porto-sinusoidal vascular disease (PSVD) is a rare vascular liver disease of unknown etiology that causes portal hypertension. It usually affects young individuals and shortens live expectancy. The deregulated pathways involved in PSVD development are unknown and therefore we lack curative treatments. The purpose of this study was to integrate transcriptomic and clinical data by comprehensive network-based modeling in order to uncover altered biological processes in patients with PSVD. METHODS: We obtained liver tissue samples from 20 consecutive patients with PSVD and 21 sex- and age-matched patients with cirrhosis and 13 histologically normal livers (HNL) (initial cohort) and performed transcriptomic analysis. Microarray data were analyzed using weighted gene correlation network analysis to identify clusters of highly correlated genes differently expressed in patients with PSVD. We next evaluated the molecular pathways enriched in patients with PSVD and the core-related genes from the most significantly enriched pathways in patients with PSVD. Our main findings were validated using RNA sequencing in a different cohort of PSVD, cirrhosis and HNL (n = 8 for each group). RESULTS: Patients with PSVD have a distinctive genetic profile enriched mainly in canonical pathways involving hemostasis and coagulation but also lipid metabolism and oxidative phosphorylation. Serpin family (SERPINC1), the apolipoproteins (APOA, APOB, APOC), ATP synthases (ATP5G1, ATP5B), fibrinogen genes (FGB, FGA) and alpha-2-macroglobulin were identified as highly connective genes that may have an important role in PSVD pathogenesis. CONCLUSION: PSVD has a unique transcriptomic profile and we have identified deregulation of pathways involved in vascular homeostasis as the main pathogenic event of disease development. LAY SUMMARY: Porto-sinusoidal vascular disease is a rare but life-shortening disease that affects mainly young people. Knowledge of the disrupted pathways involved in its development will help to identify novel therapeutic targets and new treatments. Using a systems biology approach, we identify that pathways regulating endothelial function and tone may act as drivers of porto-sinusoidal vascular disease.

Interaction between metabolic syndrome and alcohol consumption, risk factors of liver fibrosis: A population-based study.

BACKGROUND AND AIMS: Alcohol and metabolic syndrome (MS) coexist frequently as cofactors of liver disease. Previous studies suggest a deleterious effect of MS in advanced alcohol-related liver disease (ArLD). However, it is unknow whether MS can increase the risk of liver fibrosis in early stages of ArLD. The aim of this study was to investigate the effect of MS on liver fibrosis in subjects with alcohol consumption from a population-based cohort. METHODS: The number of subjects include 1760(58%) of 3014 who were randomly selected from the community consumed alcohol and were classified as current drinkers, divided in moderate (n = 1222) or high-risk drinkers (n = 275) (>21 units/week men, >14 units/week women for high-risk drinkers), or former drinkers (n = 263). Liver fibrosis was estimated by measuring liver stiffness(LS) with transient elastography (TE). RESULTS: Prevalence of significant LS using cutoff values of TE of 8 and 9.1kPa was increased in high-risk compared with moderate or former drinkers and lifetime abstainers. In subjects with alcohol consumption, LS was associated with male gender, AST, ALT, years of consumption, and MS. In high-risk drinkers, MS and intensity of consumption were the only factors associated with significant LS (OR 3.7 and 4.6 for LS ≥ 8 kPa and 3.9 and 9.2 kPa for LS ≥ 9.1 kPa, respectively). Presence of significant liver fibrosis in the liver biopsy was higher among high-risk as compared with moderate or former drinkers. CONCLUSION: MS increases the risk of liver fibrosis in subjects with alcohol consumption. Among high-risk drinkers, only MS and consumption of high amount of alcohol are associated with risk of liver fibrosis.

Autoimmune biomarkers in porto-sinusoidal vascular disease: Potential role in its diagnosis and pathophysiology.

BACKGROUND AND AIMS: Porto-sinusoidal vascular disease (PSVD) is a rare disease that requires excluding cirrhosis and other causes of portal hypertension for its diagnosis because it lacks a specific diagnostical test. Although it has been occasionally associated with autoimmune diseases, the pathophysiology of PSVD remains unknown. The aim of this study was to evaluate the potential role of autoimmunity in the pathophysiology and diagnosis of PSVD. METHODS: Thirty-seven consecutive patients with PSVD and 39 with cirrhosis matched by gender, signs of portal hypertension and liver function were included (training set). By using Indirect Immunofluorescence, ELISA and slot-blot methods data 22 autoantibodies were identified in patients with PSVD and cirrhosis. Presence of anti-endothelial cells antibodies (AECA) was assayed by a cell-based ELISA. Thirty-one PSVD, 40 cirrhosis patients, 15 patients with splenomegaly associated with haematological disease and 14 healthy donors were included in a validation set. FINDINGS: The proportion of patients with at least one positive antibody was statistically significantly higher in patients with PSVD compared with cirrhosis (92% vs 56%; P < .01). Specifically, AECA were significantly more frequent in PSVD than in cirrhosis (38% vs 15%; P = .013). Results were confirmed in the validation set. In the overall population, presence of AECA had a 63% positive predictive value for diagnosing PSVD and a 71% negative predictive value, with a specificity of 94% when the 1/16 level is used as cut-off. AECA positive serum samples react with a 68-72 kDa protein of human liver endothelial sinusoidal cells.

Pancreatic Insufficiency in Patients Under Sorafenib Treatment for Hepatocellular Carcinoma.

GOALS: To describe the occurrence of malabsorption (MA) in hepatocellular carcinoma (HCC) patients under sorafenib, the potential relationship with pancreatic insufficiency (PI), and the role of pancreatic enzymes supplementation. BACKGROUND: With the increasing options of second-line systemic therapies for HCC, the recognition of drug intolerance using practical tools is crucial. It has been proposed that a MA syndrome could be due to sorafenib-induced pancreatic dysfunction. STUDY: All sorafenib-treated patients with suspicion of MA (defined as decreased stool consistency lasting >4 wk or presenting ≥10% body weight loss without HCC progression) were prospectively evaluated by serum markers, endoscopy, and imaging techniques. RESULTS: We evaluated 81 sorafenib-treated patients and 21 developed MA suspicion (85.7% male, 81.5% Child-Pugh A, 52.4% BCLC-B, and 47.6% BCLC-C) within a median 5.9 months after starting sorafenib. The median treatment duration, follow-up, and overall survival after MA suspicion were 5.9, 20.3, and 20.3 months, respectively. Nine of them (42.9%) presented hyperparathyroidism secondary to vitamin D deficiency and 8 with PI. A gradual decrease in pancreatic volume of up to 19% was observed among patients with PI. Six of the 8 patients with PI received pancreatic enzymes, with complete recovery from MA symptoms and stabilization of pancreatic volume. CONCLUSIONS: We validated the association between MA and PI in 10% of sorafenib-treated patients. Pancreatic enzymes supplementation successfully led to symptomatic recovery. Awareness of this adverse event can help in the management of sorafenib irrespective of cancer type and likely, of other tyrosine kinase inhibitors for HCC patients.