Serum CXCL5 Detects Early Hepatocellular Carcinoma and Indicates Tumor Progression.

Chemokines or chemotactic cytokines play a pivotal role in the immune pathogenesis of liver cirrhosis and hepatocellular carcinoma (HCC). Nevertheless, comprehensive cytokine profiling data across different etiologies of liver diseases are lacking. Chemokines might serve as diagnostic and prognostic biomarkers. In our study, we analyzed serum concentrations of 12 inflammation-related chemokines in a cohort of patients (n = 222) with cirrhosis of different etiologies and/or HCC. We compared 97 patients with cirrhosis and treatment-naïve HCC to the chemokine profile of 125 patients with cirrhosis but confirmed absence of HCC. Nine out of twelve chemokines were significantly elevated in sera of cirrhotic patients with HCC compared to HCC-free cirrhosis controls (CCL2, CCL11, CCL17, CCL20, CXCL1, CXCL5, CXCL9, CXCL10, CXCL11). Among those, CXCL5, CXCL9, CXCL10, and CXCL11 were significantly elevated in patients with early HCC according to the Barcelona Clinic Liver Cancer (BCLC) stages 0/A compared to cirrhotic controls without HCC. In patients with HCC, CXCL5 serum levels were associated with tumor progression, and levels of CCL20 and CXCL8 with macrovascular invasion. Importantly, our study identified CXCL5, CXCL9, and CXCL10 as universal HCC markers, independent from underlying etiology of cirrhosis. In conclusion, regardless of the underlying liver disease, patients with cirrhosis share an HCC-specific chemokine profile. CXCL5 may serve as a diagnostic biomarker in cirrhotic patients for early HCC detection as well as for tumor progression.

Inflammatory Cytokines Associated with Diagnosis, Tumor Grade and Prognosis in Patients with Neuroendocrine Tumors.

Background and aims: Inflammatory cytokines represent diagnostic and prognostic biomarkers in manifold cancers. Recent data suggest a pivotal role of these cytokines in different biological processes involved in the development of neuroendocrine tumors (NETs). However, their role as biomarkers in NETs is only poorly understood. Methods: We analyzed serum concentrations of 13 inflammation-related cytokines at different time points in 43 patients with well-differentiated gastroenteropancreatic NETs (G1/G2) treated at Charité Berlin and compared them to 40 healthy controls. The results were correlated with clinical records. Results: Serum concentrations (Median (Interquartile Range (IQR)) in pg/mL) of IL-1ß (124 (82) vs. 68 (61) pg/mL; p = 0.0003), IL-6 (111(122) vs. 88 (32) pg/mL; p = 0.0086), IL-8 (1058 (768) vs. 210 (90) pg/mL; p < 0.0001), IL-18 (2936 (1723) vs. 1590 (704) pg/mL; p < 0.0001), and TNF (271 (260) vs. 42 (25) pg/mL; p < 0.0001) were significantly elevated in NET patients, whereas IL-10 (43 (44) vs. 105 (48) pg/mL; p < 0.0001) showed lower concentrations in NETs when compared to controls. Cytokine levels significantly correlated with tumor grade (IL-6; p = 0.0070), prevalence of distant metastasis (IL-18; p = 0.0313), and disease progression over time (IL-10; p = 0.0033) but not tumor location. Chromogranin A (CgA) and the NETest are currently used to monitor treatment response. A more accurate prediction could possibly be achieved by employing a subset of cytokines. Our data clearly warrants further functional investigation into the role of the immune response and cytokine release in NETs. Conclusion: A biologically plausible panel of cytokines might be added to the diagnostic and prognostic tools currently employed in patients with NETs. Combining different markers into a score would elevate diagnostic accuracy compared to single markers.

Studying metabolism with multi-organ chips: new tools for disease modelling, pharmacokinetics and pharmacodynamics.

Non-clinical models to study metabolism including animal models and cell assays are often limited in terms of species translatability and predictability of human biology. This field urgently requires a push towards more physiologically accurate recapitulations of drug interactions and disease progression in the body. Organ-on-chip systems, specifically multi-organ chips (MOCs), are an emerging technology that is well suited to providing a species-specific platform to study the various types of metabolism (glucose, lipid, protein and drug) by recreating organ-level function. This review provides a resource for scientists aiming to study human metabolism by providing an overview of MOCs recapitulating aspects of metabolism, by addressing the technical aspects of MOC development and by providing guidelines for correlation with in silico models. The current state and challenges are presented for two application areas: (i) disease modelling and (ii) pharmacokinetics/pharmacodynamics. Additionally, the guidelines to integrate the MOC data into in silico models could strengthen the predictive power of the technology. Finally, the translational aspects of metabolizing MOCs are addressed, including adoption for personalized medicine and prospects for the clinic. Predictive MOCs could enable a significantly reduced dependence on animal models and open doors towards economical non-clinical testing and understanding of disease mechanisms.

The APAC Score: A Novel and Highly Performant Serological Tool for Early Diagnosis of Hepatocellular Carcinoma in Patients with Liver Cirrhosis.

(1) Background: Surveillance of at-risk patients for hepatocellular carcinoma (HCC) is highly necessary, as curative treatment options are only feasible in early disease stages. However, to date, screening of patients with liver cirrhosis for HCC mostly relies on suboptimal ultrasound-mediated evaluation and α-fetoprotein (AFP) measurement. Therefore, we sought to develop a novel and blood-based scoring tool for the identification of early-stage HCC. (2) Methods: Serum samples from 267 patients with liver cirrhosis, including 122 patients with HCC and 145 without, were collected. Expression levels of soluble platelet-derived growth factor receptor beta (sPDGFRß) and routine clinical parameters were evaluated, and then utilized in logistic regression analysis. (3) Results: We developed a novel serological scoring tool, the APAC score, consisting of the parameters age, sPDGFRß, AFP, and creatinine, which identified patients with HCC in a cirrhotic population with an AUC of 0.9503, which was significantly better than the GALAD score (AUC: 0.9000, p = 0.0031). Moreover, the diagnostic accuracy of the APAC score was independent of disease etiology, including alcohol (AUC: 0.9317), viral infection (AUC: 0.9561), and NAFLD (AUC: 0.9545). For the detection of patients with (very) early (BCLC 0/A) HCC stage or within Milan criteria, the APAC score achieved an AUC of 0.9317 (sensitivity: 85.2%, specificity: 89.2%) and 0.9488 (sensitivity: 91.1%, specificity 85.3%), respectively. (4) Conclusions: The APAC score is a novel and highly accurate serological tool for the identification of HCC, especially for early stages. It is superior to the currently proposed blood-based algorithms, and has the potential to improve surveillance of the at-risk population.

The Role of miRNA in the Pathophysiology of Neuroendocrine Tumors.

Neuroendocrine tumors (NETs) represent a tumor group that is both rare and heterogeneous. Prognosis is largely determined by the tumor grading and the site of the primary tumor and metastases. Despite intensive research efforts, only modest advances in diagnostic and therapeutic approaches have been achieved in recent years. For patients with non-respectable tumor stages, prognosis is poor. In this context, the development of novel diagnostic tools for early detection of NETs and prediction of tumor response to therapy as well as estimation of the overall prognosis would greatly improve the clinical management of NETs. However, identification of novel diagnostic molecules is hampered by an inadequate understanding of the pathophysiology of neuroendocrine malignancies. It has recently been demonstrated that microRNA (miRNA), a family of small RNA molecules with an established role in the pathophysiology of quite different cancer entities, may also play a role as a biomarker. Here, we summarize the available knowledge on the role of miRNAs in the development of NET and highlight their potential use as serum-based biomarkers in the context of this disease. We discuss important challenges currently preventing their use in clinical routine and give an outlook on future directions of miRNA research in NET.

Lessons From Immune Checkpoint Inhibitor Trials in Hepatocellular Carcinoma.

The implementation of immune checkpoint inhibitors (ICI) into the clinical management of different malignancies has largely changed our understanding of cancer treatment. After having proven efficacy in different tumor entities such as malignant melanoma and lung cancer, ICI were intensively tested in the setting of hepatocellular carcinoma (HCC). Here they could achieve higher and more durable response rates compared to tyrosine-kinase inhibitors (TKI), that were sole standard of care for the last decade. Most recently, ICI treatment was approved in a first line setting of HCC, for cases not suitable for curative strategies. However, only a subset of patients benefits from ICI therapy, while others experience rapid tumor progression, worsening of liver function and poor prognosis. Efforts are being made to find immune characteristics that predict tumor responsiveness to ICI, but no reliable biomarker could be identified so far. Nevertheless, data convincingly demonstrate that combination therapies (such as dual inhibition of PD-L1 and VEGF) are more effective than the application of single agents. In this review, we will briefly recapitulate the current algorithms for systemic treatment, discuss available results from checkpoint inhibitor trials and give an outlook on future directions of immunotherapy in HCC.

From Liver Cirrhosis to Cancer: The Role of Micro-RNAs in Hepatocarcinogenesis.

In almost all cases, hepatocellular carcinoma (HCC) develops as the endpoint of a sequence that starts with chronic liver injury, progresses to liver cirrhosis, and finally, over years and decades, results in liver cancer. Recently, the role of non-coding RNA such as microRNA (miRNA) has been demonstrated in the context of chronic liver diseases and HCC. Moreover, data from a phase II trial suggested a potential role of microRNAs as therapeutics in hepatitis-C-virus infection, representing a significant risk factor for development of liver cirrhosis and HCC. Despite progress in the clinical management of chronic liver diseases, pharmacological treatment options for patients with liver cirrhosis and/or advanced HCC are still limited. With their potential to regulate whole networks of genes, miRNA might be used as novel therapeutics in these patients but could also serve as biomarkers for improved patient stratification. In this review, we discuss available data on the role of miRNA in the transition from liver cirrhosis to HCC. We highlight opportunities for clinical translation and discuss open issues applicable to future developments.

Hepatic macrophages in liver homeostasis and diseases-diversity, plasticity and therapeutic opportunities.

Macrophages, which are key cellular components of the liver, have emerged as essential players in the maintenance of hepatic homeostasis and in injury and repair processes in acute and chronic liver diseases. Upon liver injury, resident Kupffer cells (KCs) sense disturbances in homeostasis, interact with hepatic cell populations and release chemokines to recruit circulating leukocytes, including monocytes, which subsequently differentiate into monocyte-derived macrophages (MoMϕs) in the liver. Both KCs and MoMϕs contribute to both the progression and resolution of tissue inflammation and injury in various liver diseases. The diversity of hepatic macrophage subsets and their plasticity explain their different functional responses in distinct liver diseases. In this review, we highlight novel findings regarding the origins and functions of hepatic macrophages and discuss the potential of targeting macrophages as a therapeutic strategy for liver disease.

Serum levels of miR-223 but not miR-21 are decreased in patients with neuroendocrine tumors.

BACKGROUND AND AIMS: MicroRNAs (miRNAs) are profoundly involved into the pathophysiology of manifold cancers. Recent data suggested a pivotal role of miRNAs as biomarkers in different biological processes including carcinogenesis. However, their role in neuroendocrine tumors (NETs) is only poorly understood. METHODS: We determined circulating levels of miR-21 and miR-223 in 45 samples from patients with NET treated between 2010 and 2019 at our department and compared them to healthy controls. Results were correlated with clinical records. RESULTS: In the total cohort of Patients with NET, miR-223 presented significantly lower levels compared to healthy control samples. In contrast, levels of miR-21 indicated no significant changes between the two groups. Interestingly, despite being significantly downregulated in all NET patients, concentrations of miR-223 were independent of clinical or histopathological factors such as proliferation activity according to Ki-67 index, tumor grading, TNM stage, somatostatin receptor expression, presence of functional/ non-functional disease or tumor relapse. Moreover, in contrast to data from recent publications analyzing other tumor entities, levels of miR-223 serum levels did not reflect prognosis of patients with NET. CONCLUSION: Lower concentrations of circulating miR-223 rather reflect the presence of NET itself than certain tumor characteristics. The value of miR-223 as a biomarker in NET might be limited to diagnostic, but not prognostic purposes.

Current and emerging pharmacotherapeutic interventions for the treatment of liver fibrosis.

INTRODUCTION: Chronic liver disease is due to various causes of persistent liver damage and will eventually lead to the development of liver fibrosis. If no treatment is initiated, this condition may progress to cirrhosis and hepatocellular carcinoma. Current treatments comprise the elimination of the cause of injury, such as by lifestyle changes, alcohol abstinence, and antiviral agents. However, such etiology-driven therapy is often insufficient in patients with late-stage fibrosis/cirrhosis, therefore maintaining the need for efficient antifibrotic pharmacotherapeutic interventions. AREAS COVERED: The authors discuss the recent advances in the development of antifibrotic drugs, which target various pathways of the fibrogenesis process, including cell death, inflammation, gut-liver axis, and myofibroblast activation. Due to the significant burden of nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH), various agents which specifically target metabolic pathways and their related receptors/ligands have been developed. For some of them, e.g., obeticholic acid, advanced stage clinical trials indicate antifibrotic efficacy in NAFLD and NASH. EXPERT OPINION: Significant advances have been made in the development of novel antifibrotic pharmacotherapeutics. The authors expect that the development of combinatorial therapies, which combine compounds that target various pathways of fibrosis progression, will have a major impact as future etiology-independent therapies.

Controversies and Opportunities in the Use of Inflammatory Markers for Diagnosis or Risk Prediction in Fatty Liver Disease.

In the Western society, non-alcoholic fatty liver disease (NAFLD), characterized by the excessive accumulation of fat in the liver, represents the most common cause of chronic liver disease. If left untreated, approximately 15%-20% of patients with NAFLD will progress to non-alcoholic steatohepatitis (NASH), in which lobular inflammation, hepatocyte ballooning and fibrogenesis further contribute to a distorted liver architecture and function. NASH initiation has significant effects on liver-related mortality, as even the presence of early stage fibrosis increases the chances of adverse patient outcome. Therefore, adequate diagnostic tools for NASH are needed, to ensure that relevant therapeutic actions can be taken as soon as necessary. To date, the diagnostic gold standard remains the invasive liver biopsy, which is associated with several drawbacks such as high financial costs, procedural risks, and inter/intra-observer variability in histology analysis. As liver inflammation is a major hallmark of disease progression, inflammation-related circulating markers may represent an interesting source of non-invasive biomarkers for NAFLD/NASH. Examples for such markers include cytokines, chemokines or shed receptors from immune cells, circulating exosomes related to inflammation, and changing proportions of peripheral blood mononuclear cell (PBMC) subtypes. This review aims at documenting and critically discussing the utility of such novel inflammatory markers for NAFLD/NASH-diagnosis, patient stratification and risk prediction.

The miRFIB-Score: A Serological miRNA-Based Scoring Algorithm for the Diagnosis of Significant Liver Fibrosis.

Background: The current diagnosis of early-stage liver fibrosis often relies on a serological or imaging-based evaluation of the stage of fibrosis, sometimes followed by an invasive liver biopsy procedure. Novel non-invasive experimental diagnostic tools are often based on markers of hepatocyte damage, or changes in liver stiffness and architecture, which are late-stage characteristics of fibrosis progression, making them unsuitable for the diagnosis of early-stage liver fibrosis. miRNAs control hepatic stellate cell (HSC) activation and are proposed as relevant diagnostic markers. Methods: We investigated the possibility of circulating miRNAs, which we found to be dysregulated upon HSC activation, to mark the presence of significant liver fibrosis (F ≥ 2) in patients with chronic alcohol abuse, chronic viral infection (HBV/HCV), and non-alcoholic fatty liver disease (NAFLD). Results: miRNA-profiling identified miRNA-451a, miRNA-142-5p, Let-7f-5p, and miRNA-378a-3p to be significantly dysregulated upon in vitro HSC activation, and to be highly enriched in their extracellular vesicles, suggesting their potential use as biomarkers. Analysis of the plasma of patients with significant liver fibrosis (F ≥ 2) and no or mild fibrosis (F = 0-1), using miRNA-122-5p and miRNA-29a-3p as positive control, found miRNA-451a, miRNA-142-5p, and Let-7f-5p, but not miRNA-378a-3p, able to distinguish between the two patient populations. Using logistic regression analysis, combining all five dysregulated circulating miRNAs, we created the miRFIB-score with a predictive value superior to the clinical scores Fibrosis-4 (Fib-4), aspartate aminotransferase/alanine aminotransferase (AST/ALT) ratio, and AST to platelet ratio index (APRI). The combination of the miRFIB-score with circulating PDGFRß-levels further increased the predictive capacity for the diagnosis of significant liver fibrosis. Conclusions: The miRFIB- and miRFIBp-scores are accurate tools for the diagnosis of significant liver fibrosis in a heterogeneous patient population.

A PDGFRß-based score predicts significant liver fibrosis in patients with chronic alcohol abuse, NAFLD and viral liver disease.

BACKGROUND: Platelet Derived Growth Factor Receptor beta (PDGFRß) has been associated to hepatic stellate cell activation and has been the target of multiple therapeutic studies. However, little is known concerning its use as a diagnostic agent. METHODS: Circulating PDGFRß levels were analysed in a cohort of patients with liver fibrosis/cirrhosis due to chronic alcohol abuse, viral hepatitis, or non-alcoholic fatty liver disease (NAFLD). The diagnostic performance of PDGFRß as individual blood parameter, or in combination with other metabolic factors was evaluated. FINDINGS: sPDGFRß levels are progressively increased with increasing fibrosis stage and the largest difference was observed in patients with significant fibrosis, compared to no or mild fibrosis. The accuracy of sPDGFRß-levels predicting fibrosis could be increased by combining it with albumin levels and platelet counts into a novel diagnostic algorithm, the PRTA-score, generating a predictive value superior to Fib-4, APRI, and AST/ALT. The sPDGFRß levels and the PRTA-score are independent of liver disease aetiology, thus overcoming one of the major weaknesses of current non-invasive clinical and experimental scores. Finally, we confirmed the diagnostic value of sPDGFRß levels and the PRTA-score in an independent patient cohort with NAFLD which was staged for fibrosis by liver biopsy. INTERPRETATION: The PRTA-score is an accurate tool for detecting significant liver fibrosis in a broad range of liver disease aetiologies. FUND: Vrije Universiteit Brussel, the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Flanders) (HILIM-3D; SBO140045), and the Fund of Scientific Research Flanders (FWO).

Prospects in non-invasive assessment of liver fibrosis: Liquid biopsy as the future gold standard?

Liver fibrosis is the result of persistent liver injury, and is characterized by sustained scar formation and disruption of the normal liver architecture. The extent of fibrosis is considered as an important prognostic factor for the patient outcome, as an absence of (early) treatment can lead to the development of liver cirrhosis and hepatocellular carcinoma. Till date, the most sensitive and specific way for the diagnosis and staging of liver fibrosis remains liver biopsy, an invasive diagnostic tool, which is associated with high costs and discomfort for the patient. Over time, non-invasive scoring systems have been developed, of which the measurements of serum markers and liver stiffness are validated for use in the clinic. These tools lack however the sensitivity and specificity to detect small changes in the progression or regression of both early and late stages of fibrosis. Novel non-invasive diagnostic markers with the potential to overcome these limitations have been developed, but often lack validation in large patient cohorts. In this review, we will summarize novel trends in non-invasive markers of liver fibrosis development and will discuss their (dis-)advantages for use in the clinic.

Circulating ECV-Associated miRNAs as Potential Clinical Biomarkers in Early Stage HBV and HCV Induced Liver Fibrosis.

Introduction: Chronic hepatitis B (HBV) and C (HCV) virus infection is associated with the activation of hepatic stellate cells (HSCs) toward a myofibroblastic phenotype, resulting in excessive deposition of extracellular matrix, the development of liver fibrosis, and its progression toward cirrhosis. The gold standard for the detection and staging of liver fibrosis remains the liver biopsy, which is, however, associated with some mild and severe drawbacks. Other non-invasive techniques evade these drawbacks, but lack inter-stage specificity and are unable to detect early stages of fibrosis. We investigated whether circulating vesicle-associated miRNAs can be used in the diagnosis and staging of liver fibrosis in HBV and HCV patients. Methods: Plasma samples were obtained from 14 healthy individuals and 39 early stage fibrotic patients (F0-F2) with chronic HBV or HCV infection who underwent transient elastography (Fibroscan). Extracellular vesicles were extracted from the plasma and the level of miRNA-122, -150, -192, -21, -200b, and -92a was analyzed by qRT-PCR in total plasma and circulating vesicles. Finally, these same miRNAs were also quantified in vesicles extracted from in vitro activating primary HSCs. Results: In total plasma samples, only miRNA-200b (HBV: p = 0.0384; HCV: p = 0.0069) and miRNA-122 (HBV: p < 0.0001; HCV: p = 0.0007) were significantly up-regulated during early fibrosis. In circulating vesicles, miRNA-192 (HBV: p < 0.0001; HCV: p < 0.0001), -200b (HBV: p < 0.0001; HCV: p < 0.0001), -92a (HBV: p < 0.0001; HCV: p < 0.0001), and -150 (HBV: p = 0.0016; HCV: p = 0.004) displayed a significant down-regulation in both HBV and HCV patients. MiRNA expression profiles in vesicles isolated from in vitro activating primary mouse HSCs resembled the miRNA expression profile in circulating vesicles. Conclusion: Our analysis revealed a distinct miRNA expression pattern in total plasma and its circulating vesicles. The expression profile of miRNAs in circulating vesicles of fibrotic patients suggests the potential use of these vesicle-associated miRNAs as markers for early stages of liver fibrosis.

The role of miRNAs in stress-responsive hepatic stellate cells during liver fibrosis.

The progression of liver fibrosis and cirrhosis is associated with the persistence of an injury causing agent, leading to changes in the extracellular environment and a disruption of the cellular homeostasis of liver resident cells. Recruitment of inflammatory cells, apoptosis of hepatocytes, and changes in liver microvasculature are some examples of changing cellular environment that lead to the induction of stress responses in nearby cells. During liver fibrosis, the major stresses include hypoxia, oxidative stress, and endoplasmic reticulum stress. When hepatic stellate cells (HSCs) are subjected to such stress, they modulate fibrosis progression by induction of their activation toward a myofibroblastic phenotype, or by undergoing apoptosis, and thus helping fibrosis resolution. It is widely accepted that microRNAs are import regulators of gene expression, both during normal cellular homeostasis, as well as in pathologic conditions. MicroRNAs are short RNA sequences that regulate the gene expression by mRNA destabilization and inhibition of mRNA translation. Specific microRNAs have been identified to play a role in the activation process of HSCs on the one hand and in stress-responsive pathways on the other hand in other cell types (Table 2). However, so far there are no reports for the involvement of miRNAs in the different stress responses linked to HSC activation. Here, we review briefly the major stress response pathways and propose several miRNAs to be regulated by these stress responsive pathways in activating HSCs, and discuss their potential specific pro-or anti-fibrotic characteristics.

Integrative miRNA and Gene Expression Profiling Analysis of Human Quiescent Hepatic Stellate Cells.

Unveiling the regulatory pathways maintaining hepatic stellate cells (HSC) in a quiescent (q) phenotype is essential to develop new therapeutic strategies to treat fibrogenic diseases. To uncover the miRNA-mRNA regulatory interactions in qHSCs, HSCs were FACS-sorted from healthy livers and activated HSCs (aHSCs) were generated in vitro. MiRNA Taqman array analysis showed HSCs expressed a low number of miRNAs (n = 259), from which 47 were down-regulated and 212 up-regulated upon activation. Computational integration of miRNA and gene expression profiles revealed that 66% of qHSC-associated miRNAs correlated with more than 6 altered target mRNAs (17,28 ± 10,7 targets/miRNA) whereas aHSC-associated miRNAs had an average of 1,49 targeted genes. Interestingly, interaction networks generated by miRNA-targeted genes in qHSCs were associated with key HSC activation processes. Next, selected miRNAs were validated in healthy and cirrhotic human livers and miR-192 was chosen for functional analysis. Down-regulation of miR-192 in HSCs was found to be an early event during fibrosis progression in mouse models of liver injury. Moreover, mimic assays for miR-192 in HSCs revealed its role in HSC activation, proliferation and migration. Together, these results uncover the importance of miRNAs in the maintenance of the qHSC phenotype and form the basis for understanding the regulatory networks in HSCs.