Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD) represents the most frequent cause of hepatic disorder, and its progressive form defined as Metabolic Dysfunction-Associated Steatohepatitis (MASH) contributes to the development of fibrosis/cirrhosis and hepatocellular carcinoma (HCC). Today effective therapeutic strategies addressing MASH-related comorbidities, inflammation, and fibrosis are needed. The fibroblast growth factor (FGF) 19 and 21 and their fibroblast growth factor receptor/β-Klotho (KLB) complexes have recently emerged as promising druggable targets for MASLD. However, less is known regarding the causative association between KLB activity and advanced stages of liver disease.In the present narrative review, we aimed to provide an up-to-date picture of the role of the KLB co-receptor in MASLD development and progression. We performed a detailed analysis of recently published preclinical and clinical data to decipher the molecular mechanisms underlying KLB function and to correlate the presence of inherited or acquired KLB aberrancies with the predisposition towards MASLD. Moreover, we described ongoing clinical trials evaluating the therapeutic approaches targeting FGF19–21/FGFR/KLB in patients with MASLD and discussed the challenges related to their use. We furtherly described that KLB exhibits protective effects against metabolic disorders by acting in an FGF-dependent and independent manner thus triggering the hypothesis that KLB soluble forms may play a critical role in preserving liver health. Therefore, targeting KLB may provide promising strategies for treating MASLD, as supported by experimental evidence and ongoing clinical trials.

Introduction β-Klotho (KLB) gene encodes the hepatic co-receptor of fibroblast growth factor receptor 4 (FGFR4). We previously reported that the rs17618244 G>A KLB gene variant dampened KLB hepatic and plasma levels, leading to inflammation, ballooning and fibrosis both in pediatric and adult patients with nonalcoholic fatty liver disease (NAFLD). Hepatic stellate cells (HSCs) are directly involved in the fibrotic processes, playing a crucial role in the switching to severe forms of NAFLD. Aim To generate a new cell line of hepatic stellate cells (HSCs), responsible for hepatic fibrosis, which are deleted for KLB, by using Crispr/Cas9 and to investigate the impact of its deficiency on their pro-fibrotic phenotype, inflammation and oxidative stress. Materials and Methods We stably silenced KLB gene in LX2 cells (referred to as HSCs KLB−/−) by Crispr-Cas9 technology. Then, markers of activation, cellular stress, inflammation and proliferation have been investigated. Results KLB mRNA and protein levels were reduced in HSC KLB−/- cells (p<0.05). Markers of activation (i.e., aSMA, COL1A1/3A1/4A1, TGFb, PDGFRB, BAMBI) were increased in KLB−/− cells at both gene and protein levels. Accordingly, retinol synthesis (RALDH) was reduced (p<0.05), suggesting a more pro-fibrotic phenotype. Moreover, KLB deletion strongly induced oxidative stress, enhancing ROS/reactive nitrite species (RNS) and lactate production, aldehyde derivate concentrations (MDA) (p<0.05). These events triggered the release of pro-inflammatory cytokines (TNFα, IL1β and IL6) into the KLB−/- cultured media. Finally, KLB−/- cells also showed an elevated proliferative rate (p<0.05). Conclusions We generated, for the first time, a stable knock-out of KLB in HSCs. Our preliminary results outlined that KLB deficiency induced an enhanced pro-fibrotic phenotype with increased proliferation, inflammation and oxidative stress, thus possibly explaining the association between the rs17618244 KLB at-risk variant and more severe forms of liver damage. Altogether, these observations pointed KLB a novel target to prevent severe NAFLD.

Introduction Mitochondrial dysfunction is a key player in the transition from NASH to HCC. The knock-out (KO) of MBOAT7 and/or TM6SF2 hampers the mitochondrial dynamics in Hepg2 cells resulting in an enrichment of misshapen and failed mitochondria. The overexpression of MBOAT7 and/or TM6SF2 wild-type genes in KO models through lentiviral vectors decreases the number of damaged-globular mitochondria, while increases the normo-shaped ones. Aims To deepen the impact of PNPLA3/MBOAT7/TM6SF2 loss-of-function mutations on mitochondrial aberrances, we investigated the mitochondrial lifecycle and activity in KO cells overexpressed for the wild-type forms of MBOAT7 and/or TM6SF2. Method Mitochondrial lifecycle and activity were assessed through RT-PCR, Western Blot, Seahorse assay and immunohistochemistry. Results The overexpression of MBOAT7, TM6SF2 or both decreased PGC1α, the master regulator of mitobiogenesis, which was activated in KO cells in response to fusion-fission unbalance, boosted the expression of Mfn1/Mfn2 (mitochondrial outer membranes proteins involved in fusion) and OPA1 (inner mitochondrial membranes fusion protein) whereas decreased FIS1 and DRP1 (fission proteins) levels, thus re-establishing the mitochondrial turnover. Consistently, the mitophagy pathways (PINK/PARKIN/BNIP3/BNIP3-L/LC3/phospho-UBIQUITIN) increased after the MBOAT7 and/or TM6SF2 overexpression, prompting the disruption of damaged mitochondria. The balance of mitochondrial biogenesis is essential for organelles' homeostasis and activity. Indeed, the overexpressed models augmented the COX-I/SDHA ratio, COX-III, and citrate synthase activity alongside the oxygen consumption rate, recovering the OXPHOS capacity and Krebs cycle which were impaired in KO cells. Finally, lactate levels decreased after the upregulation of MBOAT7 and/or TM6SF2 wild-type genes together with the glycolytic/extracellular acidification rate, thus inhibiting the switch to anerobic glycolysis which was promoted in KO cells to trigger tumorigenesis. Conclusion Genetics impacts on mitochondrial maladaptation during NAFLD and the overexpression of MBOAT7 and/or TM6SF2 wild-type genes in KO HepG2 cells re-balances the mitochondrial lifecycle and turnover, thus ensuring the organelles' function and possibly reversing hepatocellular damage.

Background and AimsProgrammed cell death 1/programmed cell death-ligand 1 (PD-1/PDL-1) axis has been reported to modulate liver inflammation and progression to hepatocellular carcinoma (HCC) in patients with nonalcoholic fatty liver disease (NAFLD). Here, we examined whether the PDCD1 variation is associated with NAFLD severity in individuals with liver biopsy. MethodsWe examined the impact of PDCD1 gene variants on HCC, as robust severe liver disease phenotype in UK Biobank participants. The strongest genetic association with the rs13023138 G>C variation was subsequently tested for association with liver damage in 2889 individuals who underwent liver biopsy for suspected nonalcoholic steatohepatitis (NASH). Hepatic transcriptome was examined by RNA-Seq in a subset of NAFLD individuals (n = 121). Transcriptomic and deconvolution analyses were performed to identify biological pathways modulated by the risk allele. ResultsThe rs13023138 C>G showed the most robust association with HCC in UK Biobank (p = 5.28E-4, OR = 1.32, 95% CI [1.1, 1.5]). In the liver biopsy cohort, rs13023138 G allele was independently associated with severe steatosis (OR 1.17, 95% CI 1.02-1.34; p = .01), NASH (OR 1.22, 95% CI 1.09-1.37; p < .001) and advanced fibrosis (OR 1.26, 95% CI 1.06-1.50; p = .007). At deconvolution analysis, rs13023138 G>C allele was linked to higher hepatic representation of M1 macrophages, paralleled by upregulation of pathways related to inflammation and higher expression of CXCR6. ConclusionsThe PDCD1 rs13023138 G allele was associated with HCC development in the general population and with liver disease severity in patients at high risk of NASH.

Introduction: We previously reported that the rs17618244 G>A variant in the β-Klotho (KLB) gene, encoding the hepatic co-receptor of fibroblast growth factor receptor 4 (FGFR4), dampened KLB plasma levels, leading to inflammation, ballooning and fibrosis both in pediatric and adult patients with nonalcoholic fatty liver disease (NAFLD).

Non-alcoholic fatty liver disease (NAFLD) is characterized by an enhanced activation of the immune system, which predispose the evolution to nonalcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC). Resident macrophages and leukocytes exert a key role in the pathogenesis of NAFLD. In particular, CD4+ effector T cells are activated during the early stages of liver inflammation and are followed by the increase of natural killer T cells and of CD8+ T cytotoxic lymphocytes which contribute to auto-aggressive tissue damage. To counteract T cells activation, programmed cell death 1 (PD-1) and its ligand PDL-1 are exposed respectively on lymphocytes and liver cells' surface and can be targeted for therapy by using specific monoclonal antibodies, such as of Nivolumab, Pembrolizumab, and Atezolizumab. Despite the combination of Atezolizumab and Bevacizumab has been approved for the treatment of advanced HCC, PD-1/PD-L1 blockage treatment has not been approved for NAFLD and adjuvant immunotherapy does not seem to improve survival of patients with early-stage HCC. In this regard, different ongoing phase III trials are testing the efficacy of anti-PD-1/PD-L1 antibodies in HCC patients as first line therapy and in combination with other treatments. However, in the context of NAFLD, immune checkpoints inhibitors may not improve HCC prognosis, even worse leading to an increase of CD8+PD-1+ T cells and effector cytokines which aggravate liver damage. Here, we will describe the main pathogenetic mechanisms which characterize the immune system involvement in NAFLD discussing advantages and obstacles of anti PD-1/PDL-1 immunotherapy.

Nonalcoholic fatty liver disease (NAFLD) onset and its progression towards nonalcoholic steatohepatitis (NASH) features increased intestinal permeability and leaky gut, thereby favoring the escape of endotoxin [lipopolysaccharides (LPS)] from the gut to the liver. The aim of this study was to resemble the crosstalk between intestine and liver during NAFLD by using an in vitro model of co-culture system. Enterocytes (Caco-2) were seeded on Transwell filters (pore size: 0.4 μm) and cultured for 21 days to constitute a confluent monolayer, and then they were co-cultivated with hepatocytes (HepG2) for an additional 24 h. Caco-2 on the apical chamber were exposed to LPS and/or a mixture of palmitic and oleic acid (PAOA) for 24 h. FITC-4000 dextrans (FD4) permeability across Caco-2 monolayer was increased by the treatment of Caco-2 cells with PAOA and LPS, consistently with tight junction-associated proteins reduction. Caco-2 exposure to PAOA/LPS promoted ApoB, triglyceride (TG), and free fatty acid secretion in basolateral media. In turn, HepG2 co-cultured with Caco-2 exposed to LPS, PAOA, or both accumulated lipid droplets and increased intracellular TG content. Likewise, Caco-2 released pro-inflammatory cytokines in basolateral media. These events triggered endoplasmic reticulum (ER) and oxidative stress, enhancing reactive oxygen species (ROS), H2O2, aldehyde derivate production, and ROS-induced DNA damage in HepG2 cells. Hence, Caco-2/HepG2 co-culture system may faithfully reproduce the breach in the intestinal barrier integrity that occurs in NAFLD, thus resulting in the increased inflammatory response and ER and oxidative and stress, which promote the switch towards NASH.

Background and aims: Religious fasting during the holy month of Ramadan requires abstaining from food and liquids during the day.After sunset, it is allowed to consume food and drinks in defined periods.Fasting results in a reduced and timed calorie intake and can be regarded as a form of intermittent fasting.Type 2 diabetes (TD2) is a significant risk factor for the development of non-alcoholic steatohepatitis (NASH), the progressive form of non-alcoholic fatty liver disease (NAFLD).Recently several studies suggested some benefits of interval fasting for patients with TD2 and NASH.Here, we aimed to characterize the impact of a one-month interval fasting period on liver health, glucose and lipid metabolism in a cohort of T2D patients.Method: 20 muslim patients from a German metropolitan region were included.The status of the liver was assessed by Fibroscan®, including the measurement of the controlled attenuation parameter (CAP).Additionally to anthropometric measurements, patients' blood samples were collected at the beginning and end of the four-week fasting period to quantify key parameters of liver injury, lipid and glucose metabolism.Results: Following the fasting period transient elastography measurements (8.0 ± 1.2 vs. 8.6 ± 1.6 before/after fasting) and CAp values (311.4 ± 9.6 vs. 302.6 ± 10.4 before/after fasting) showed modest changes.In contrast, LFTs, apoptosis marker (M30), and adiponectin significantly decreased after the fasting period.Serum levels of triglycerides were lower following the fasting period.Glucose levels did not change significantly, but serum levels of C-peptide and insulin increased following the fasting period.In addition, the patients experienced a significant weight loss at the end of the fasting month.Conclusion: In this cohort of T2D patients, we demonstrated that a 4week intermittent fasting period resulted in an improvement in different serum parameters associated with glucose, liver, and lipid metabolism.

: Immune Checkpoint Inhibitors (ICIs) have remarkably modified the way solid tumors are managed, including breast cancer. Unfortunately, only a relatively small number of breast cancer patients significantly respond to these treatments. To maximize the immunotherapy benefit in breast cancer, several efforts are currently being put forward for the identification of i) the best therapeutic strategy (i.e. ICI monotherapy or in association with chemotherapy, radiotherapy, or other drugs); ii) optimal timing for administration (e.g. early/advanced stage of disease; adjuvant/ neoadjuvant setting); iii) most effective and reliable predictive biomarkers of response (e.g. tumor-infiltrating lymphocytes, programmed death-ligand 1, microsatellite instability associated with mismatch repair deficiency, and tumor mutational burden). In this article, we review the impacts and gaps in the characterization of immune-related biomarkers raised by clinical and translational research studies with immunotherapy treatments. Particular emphasis has been put on the documented evidence of significant clinical benefits of ICI in different randomized clinical trials, along with preanalytical and analytical issues in predictive biomarkers pathological assessment.

Background and aims: Non-alcoholic steatohepatitis (NASH), the inflammatory subtype of non-alcoholic fatty liver disease (NAFLD) associated with obesity and type2 diabetes mellitus (T2DM) can lead to advanced liver disease.There are limited data evaluating T cell subsets in multiple tissue compartments or following weight loss in NAFLD.We hypothesised that circulating and tissue T cells changed in advanced versus mild NASH.Method: Prospective samples were collected under ethicallyapproved protocols; peripheral blood from 37 NAFLD patients and 7 controls, with primary comparisons between advanced (elastography >9.7kPa or ≥F3) and mild (elastography <8.2kPa or ≤F2) NAFLD.A subset undergoing bariatric surgery (n = 21) underwent additional simultaneous sampling of liver, visceral (VAT) and subcutaneous adipose tissue (SAT) for flow cytometry immuno-profiling, with further blood samples after weight loss.Results: Peripheral blood MAIT cells were significantly reduced with greater activation in patients with advanced versus mild NAFLD or controls (0.8% vs 3.1%, 3.2% p < 0.05) although IFN gamma and TNF alpha expression was higher in all NAFLD MAIT cells (IL-17 in advanced) compared to control.In bariatric patients, Th1 (CD4 + CXCR3 + ) cells were more abundant in NASH vs no-NASH (33% vs 21% p < 0.05), particularly among patients with diabetes in whom pro-inflammatory Th1 and Th17 cells (CD4 + CD161 + ) expressed higher levels of liver homing CXCR6.After weight loss, the proportion of Th1, Th17 cells and activated (CD69 + ) CD8+ T cells reduced.The proportion of CXCR6 + Th1 and Th17 cells reduced, as did IFN gamma expressing CXCR6 + Th17 cells.In patients with T2DM NASH, intrahepatic Th17 cell numbers were higher and MAIT cells lower compared to other groups, with a similar reduction of MAIT cells in SAT.Conversely, SAT total T cell CXCR6 expression was higher in T2DM NASH than in other patient groups.In T2DM NASH, expression of tissue-retaining CD69 expression on Th1 cells was higher in the liver and lower in VAT.Conclusion: A peripheral and adipose tissue pro-inflammatory, liverhoming T cell phenotype is evident in patients with T2DM and NASH with improvement in this after weight loss.

Nonalcoholic fatty liver disease (NAFLD) is the major contributor to the global burden of chronic liver diseases and ranges from simple and reversible steatosis to nonalcoholic steatohepatitis (NASH), which may progress into cirrhosis and hepatocellular carcinoma (HCC). HCC represents the most common liver cancer, and it is a leading cause of death worldwide with an increasing trend for the future. Due to late diagnosis, non-responsiveness to systemic therapy, and high cancer heterogeneity, the treatment of this malignancy is challenging. To date, liver biopsy and ultrasound (US) are the gold standard procedures for HCC diagnosis and surveillance, although they are not suitable for mass screening. Therefore, it is impelling to find new, less invasive diagnostic strategies able to detect HCC at an early stage as well as monitor tumor progression and recurrence. Common and rare inherited variations that boost the switching from NASH to liver cancer may help to predict tumor onset. Furthermore, epigenetic changes which reflect intertumoral heterogeneity occur early in tumorigenesis and are highly stable under pathologic conditions. The severity of hepatic injuries can be detected through the analysis of cell circulating tumor DNAs (ctDNAs), microRNAs (miRNAs), and noncoding RNAs (ncRNAs), which are involved in several pathological processes that feature cancer, including cell growth, survival, and differentiation, thus representing appealing biomarkers for HCC. Therefore, this review discusses the current options for HCC surveillance, focusing on the role of genetic and epigenetic biomarkers as new strategies to refine HCC management.

Despite the remarkable advances in the diagnosis and treatment of breast cancer patients, the presence or development of metastasis remains an incurable condition. Bone is one of the most frequent sites of distant dissemination and negatively impacts on patient’s survival and overall frailty. The interplay between tumor cells and the bone microenvironment induces bone destruction and tumor progression. To date, the clinical management of bone metastatic breast cancer encompasses anti-tumor systemic therapies along with bone-targeting agents, aimed at slowing bone resorption to reduce the risk of skeletal-related events. However, their effect on patients’ survival remains controversial. Unraveling the biology that governs the interplay between breast neoplastic cells and bone tissue would provide means for the development of new therapeutic agents. This article outlines the state-of-the art in the characterization and targeting the bone metastasis in breast cancer, focusing on the major clinical and translational studies on this clinically relevant topic.

Despite extensive research efforts, advanced gastric cancer still has a dismal prognosis with conventional treatment options. Immune checkpoint inhibitors have revolutionized the treatment landscape for many solid tumors. Amongst gastric cancer subtypes, tumors with microsatellite instability and Epstein Barr Virus positive tumors provide the strongest rationale for responding to immunotherapy. Various predictive biomarkers such as mismatch repair status, programmed death ligand 1 expression, tumor mutational burden, assessment of tumor infiltrating lymphocytes and circulating biomarkers have been evaluated. However, results have been inconsistent due to different methodologies and thresholds used. Clinical implementation therefore remains a challenge. The role of immune checkpoint inhibitors in gastric cancer is emerging with data from monotherapy in the heavily pre-treated population already available and studies in earlier disease settings with different combinatorial approaches in progress. Immune checkpoint inhibitor combinations with chemotherapy (CT), anti-angiogenics, tyrosine kinase inhibitors, anti-Her2 directed therapy, poly (ADP-ribose) polymerase inhibitors or dual checkpoint inhibitor strategies are being explored. Moreover, novel strategies including vaccines and CAR T cell therapy are also being trialed. Here we provide an update on predictive biomarkers for response to immunotherapy with an overview of their strengths and limitations. We discuss clinical trials that have been reported and trials in progress whilst providing an account of future steps needed to improve outcome in this lethal disease.

Nonalcoholic fatty liver disease (NAFLD) is the commonest chronic liver disorder worldwide in line with the increasing prevalence of obesity and the metabolic syndrome [1]. It may progress to nonalcoholic steatohepatitis (NASH), which can lead to fibrosis, cirrhosis up to hepatocellular carcinoma (HCC) [1]. The inter-individual predisposition to develop NAFLD may be attributable to environmental and inherited factors [2]. Polymorphisms in genes regulate hepatic lipid remodeling, among which PNPLA3 I148M and TM6SF2 E167K increase the susceptibility to develop the entire spectrum of NAFLD [3, 4] and have been combined in polygenic risk score (PRS) to provide an indication of the risk long before the onset of clinical manifestations.

Muscle maintenance relies on a multidimensional biologic balance that is extremely delicate in breast cancer patients, particularly those with advanced-stage disease. The biology that underpins breast cancer tumorigenesis, tumor progression and response to pharmacotherapies can modify muscle homeostasis, resulting in volumetric muscle loss. This condition dramatically increases the overall patients' frailty, leading to reduced survival and impaired quality of life. Physical activity may potentially improve muscle health in these patients, providing that an optimal patients selection is performed. The understanding of volumetric muscle loss biology in breast cancer survivors, coupled with focused clinical studies, would allow for the implementation of individualized rehabilitation protocols.

The mismatch repair (MMR) system has a key role in supporting the DNA polymerase proofreading function and in maintaining genome stability. Alterations in the MMR genes are driving events of tumorigenesis, tumor progression, and resistance to therapy. These genetic scars may occur in either hereditary or sporadic settings, with different frequencies across tumor types. Appropriate characterization of the MMR status is a crucial task in oncologic pathology because it allows for both the tailored clinical management of cancer patients and surveillance of individuals at risk. The currently available MMR testing methods have specific strengths and weaknesses, and their application across different tumor types would require a tailored approach. This article highlights the indications and challenges in MMR status assessment for molecular pathologists, focusing on the possible strategies to overcome analytical and pre-analytical issues.