Surgical Treatment and Outcome of Ovarian Cancer Patients With Liver Metastases: Experience of a Tertiary Hepatic and Peritoneal Surface Malignancy Center.

BACKGROUND/AIM: The aim of this study was to describe and evaluate the patterns, perioperative outcomes, and survival rates of patients subjected to hepatic resections for ovarian-derived liver metastasis as part of cytoreductive surgery with or without hyperthermic intraperitoneal chemotherapy (HIPEC). Furthermore, we investigated two subgroups of tumor patterns: hematogenous liver metastasis and infiltrative liver metastatic spread. PATIENTS AND METHODS: A retrospective study was conducted. Patients from a University Tertiary Hepatic and Peritoneal Surface Malignancy Center with primary or recurrent ovarian cancer, who underwent liver resection as part of cytoreductive surgery between January 1992 and December 2022, were included. RESULTS: Data from 35 patients were analyzed. Both median overall survival (OS) and disease-specific survival (DSS) were 24.97 months. In a multivariate setting, the combined effect of age, peritoneal carcinomatosis index, body mass index, hematogenous liver metastasis vs. infiltrative spread types, and HIPEC (HR=0.2372; 95%CI=0.0719-0.7823; p=0.0181) over OS was tested. Survival analysis revealed no differences between the two metastatic spread types (OS: p=0.9720; DSS: p=0.9610). Younger age (p=0.0301), splenectomy (p=0.0320), lesser omentectomy (p=0.0178), and right upper quadrant peritonectomy (p=0.0373) were more characteristic for those patients with infiltrative liver metastatic spread. CONCLUSION: Complete cytoreductive surgery, including hepatic resection is a feasible approach with or without additional HIPEC, which may provide survival benefit for patients with advanced and/or recurrent ovarian cancer. If metastatic and infiltrative liver involvement is suspected, liver-specific imaging is recommended.

Histological assessment of cardiac amyloidosis in patients undergoing transcatheter aortic valve replacement.

AIMS: Studies have reported a strongly varying co-prevalence of aortic stenosis (AS) and cardiac amyloidosis (CA). We sought to histologically determine the co-prevalence of AS and CA in patients undergoing transcatheter aortic valve replacement (TAVR). Consequently, we aimed to derive an algorithm to identify cases in which to suspect the co-prevalence of AS and CA. METHODS AND RESULTS: In this prospective, monocentric study, endomyocardial biopsies of 162 patients undergoing TAVR between January 2017 and March 2021 at the University Medical Centre Göttingen were analysed by one pathologist blinded to clinical data using haematoxylin-eosin staining, Elastica van Gieson staining, and Congo red staining of endomyocardial biopsies. CA was identified in only eight patients (4.9%). CA patients had significantly higher N-terminal pro-brain natriuretic peptide (NT-proBNP) levels (4356.20 vs. 1938.00 ng/L, P = 0.034), a lower voltage-to-mass ratio (0.73 vs. 1.46 × 10-2 mVm2/g, P = 0.022), and lower transaortic gradients (Pmean 17.5 vs. 38.0 mmHg, P = 0.004) than AS patients. Concomitant CA was associated with a higher prevalence of post-procedural acute kidney injury (50.0% vs. 13.1%, P = 0.018) and sudden cardiac death [SCD; P (log-rank test) = 0.017]. Following propensity score matching, 184 proteins were analysed to identify serum biomarkers of concomitant CA. CA patients expressed lower levels of chymotrypsin (P = 0.018) and carboxypeptidase 1 (P = 0.027). We propose an algorithm using commonly documented parameters-stroke volume index, ejection fraction, NT-proBNP levels, posterior wall thickness, and QRS voltage-to-mass ratio-to screen for CA in AS patients, reaching a sensitivity of 66.6% with a specificity of 98.1%. CONCLUSIONS: The co-prevalence of AS and CA was lower than expected, at 4.9%. Despite excellent 1 year mortality, AS + CA patients died significantly more often from SCD. We propose a multimodal algorithm to facilitate more effective screening for CA containing parameters commonly documented during clinical routine. Proteomic biomarkers may yield additional information in the future.

Hepatitis E virus infection of transplanted kidneys.

Immunocompromised patients are at risk of chronic hepatitis E (HEV) infection. Recurrent T cell and borderline rejections in a pediatric patient with high HEV copy numbers led us to study HEV infection within renal transplants. To investigate the frequency of renal HEV infection in transplanted patients, 15 samples from patients with contemporaneous diagnoses of HEV infection were identified at our center. Ten samples had sufficient residual paraffin tissue for immunofluorescence (IF) and RNA-fluorescence-in-situ-hybridization (RNA-FISH). The biopsy of the pediatric index patient was additionally sufficient for tissue polymerase chain reaction and electron microscopy. HEV RNA was detected in paraffin tissue of the index patient by tissue polymerase chain reaction. Subsequently, HEV infection was localized in tubular epithelial cells by IF, RNA-FISH, and electron microscopy. One additional biopsy from an adult was positive for HEV by RNA-FISH and IF. Focal IF positivity for HEV peptide was observed in 7 additional allografts. Ribavirin therapy was not successful in the pediatric index patient; after relapse, ribavirin is still administered. In the second patient, successful elimination of HEV was achieved after short-course ribavirin therapy. HEV infection is an important differential diagnosis for T cell rejection within transplanted kidneys. Immunostaining of HEV peptide does not necessarily prove acute infection. RNA-FISH seems to be a reliable method to localize HEV.

Targeting the microenvironment in the treatment of arteriovenous malformations.

Extracranial arteriovenous malformations (AVMs) are regarded as rare diseases and are prone to complications such as pain, bleeding, relentless growth, and high volume of shunted blood. Due to the high vascular pressure endothelial cells of AVMs are exposed to mechanical stress. To control symptoms and lesion growth pharmacological treatment strategies are urgently needed in addition to surgery and interventional radiology. AVM cells were isolated from three patients and exposed to cyclic mechanical stretching for 24 h. Thalidomide and bevacizumab, both VEGF inhibitors, were tested for their ability to prevent the formation of circular networks and proliferation of CD31+ endothelial AVM cells. Furthermore, the effect of thalidomide and bevacizumab on stretched endothelial AVM cells was evaluated. In response to mechanical stress, VEGF gene and protein expression increased in patient AVM endothelial cells. Thalidomide and bevacizumab reduced endothelial AVM cell proliferation. Bevacizumab inhibited circular network formation of endothelial AVM cells and lowered VEGF gene and protein expression, even though the cells were exposed to mechanical stress. With promising in vitro results, bevacizumab was used to treat three patients with unresectable AVMs or to prevent regrowth after incomplete resection. Bevacizumab controlled bleeding, pulsation, and pain over the follow up of eight months with no patient-reported side effects. Overall, mechanical stress increases VEGF expression in the microenvironment of AVM cells. The monoclonal VEGF antibody bevacizumab alleviates this effect, prevents circular network formation and proliferation of AVM endothelial cells in vitro. The clinical application of bevacizumab in AVM treatment demonstrates effective symptom control with no side effects.

Deep learning-based phenotyping reclassifies combined hepatocellular-cholangiocarcinoma.

Primary liver cancer arises either from hepatocytic or biliary lineage cells, giving rise to hepatocellular carcinoma (HCC) or intrahepatic cholangiocarcinoma (ICCA). Combined hepatocellular- cholangiocarcinomas (cHCC-CCA) exhibit equivocal or mixed features of both, causing diagnostic uncertainty and difficulty in determining proper management. Here, we perform a comprehensive deep learning-based phenotyping of multiple cohorts of patients. We show that deep learning can reproduce the diagnosis of HCC vs. CCA with a high performance. We analyze a series of 405 cHCC-CCA patients and demonstrate that the model can reclassify the tumors as HCC or ICCA, and that the predictions are consistent with clinical outcomes, genetic alterations and in situ spatial gene expression profiling. This type of approach could improve treatment decisions and ultimately clinical outcome for patients with rare and biphenotypic cancers such as cHCC-CCA.

Identification of DUSP4/6 overexpression as a potential rheostat to NRAS-induced hepatocarcinogenesis.

BACKGROUND: Upregulation of the mitogen-activated protein kinase (MAPK) cascade is common in hepatocellular carcinoma (HCC). Neuroblastoma RAS viral oncogene homolog (NRAS) is mutated in a small percentage of HCC and is hitherto considered insufficient for hepatocarcinogenesis. We aimed to characterize the process of N-Ras-dependent carcinogenesis in the liver and to identify potential therapeutic vulnerabilities. METHODS: NRAS V12 plasmid was delivered into the mouse liver via hydrodynamic tail vein injection (HTVI). The resulting tumours, preneoplastic lesions, and normal tissue were characterized by NanoString® gene expression analysis, Western Blot, and Immunohistochemistry (IHC). The results were further confirmed by in vitro analyses of HCC cell lines. RESULTS: HTVI with NRAS V12 plasmid resulted in the gradual formation of preneoplastic and neoplastic lesions in the liver three months post-injection. These lesions mostly showed characteristics of HCC, with some exceptions of spindle cell/ cholangiocellular differentiation. Progressive upregulation of the RAS/RAF/MEK/ERK signalling was detectable in the lesions by Western Blot and IHC. NanoString® gene expression analysis of preneoplastic and tumorous tissue revealed a gradual overexpression of the cancer stem cell marker CD133 and Dual Specificity Phosphatases 4 and 6 (DUSP4/6). In vitro, transfection of HCC cell lines with NRAS V12 plasmid resulted in a coherent upregulation of DUSP4 and DUSP6. Paradoxically, this upregulation in PLC/PRF/5 cells was accompanied by a downregulation of phosphorylated extracellular-signal-regulated kinase (pERK), suggesting an overshooting compensation. Silencing of DUSP4 and DUSP6 increased proliferation in HCC cell lines. CONCLUSIONS: Contrary to prior assumptions, the G12V NRAS mutant form is sufficient to elicit hepatocarcinogenesis in the mouse. Furthermore, the upregulation of the MAPK cascade was paralleled by the overexpression of DUSP4, DUSP6, and CD133 in vivo and in vitro. Therefore, DUSP4 and DUSP6 might fine-tune the excessive MAPK activation, a mechanism that can potentially be harnessed therapeutically.

Expression of G protein-coupled receptor GPR19 in normal and neoplastic human tissues.

Little is known about the expression of the orphan G protein-coupled receptor GPR19 at the protein level. Therefore, we developed a rabbit antibody, targeting human GPR19. After verification of the antibody specificity using GPR19-expressing cell lines and a GPR19-specific siRNA, the antibody was used for immunohistochemical staining of a variety of formalin-fixed, paraffin-embedded normal and neoplastic human tissue samples. In normal tissues, GPR19 expression was detected in a distinct cell population within the cortex, in single cells of the pancreatic islets, in intestinal ganglia, gastric chief cells, and in endocrine cells of the bronchial tract, the gastrointestinal tract, and the prostate. Among the 30 different tumour entities investigated, strong GPR19 expression was found in adenocarcinomas, typical and atypical carcinoids of the lung, and small cell lung cancer. To a lesser extent, the receptor was also present in large cell neuroendocrine carcinomas of the lung, medullary thyroid carcinomas, parathyroid adenomas, pheochromocytomas, and a subpopulation of pancreatic neuroendocrine neoplasms. In lung tumours, a negative correlation with the expression of the proliferation marker Ki-67 and a positive interrelationship with patient survival was observed. Overall, our results indicate that in adenocarcinomas and neuroendocrine tumours of the lung GPR19 may serve as a suitable diagnostic or therapeutic target.

Chemerin and Chemokine-like Receptor 1 Expression Are Associated with Hepatocellular Carcinoma Progression in European Patients.

The chemoattractant protein chemerin is protective in experimental hepatocellular carcinoma (HCC), and high expression in HCC tissues of Asian patients was related to a favorable prognosis. Studies from Asia found reduced expression of chemerin in HCC compared to para-tumor tissues while our previous analysis observed the opposite. Aim of this study was to correlate chemerin expression in HCC tissues with disease severity of European patients Hepatocyte chemerin protein expression was assessed by immunohistochemistry in HCC tissue of 383 patients, and was low in 24%, moderate in 49% and high in 27%. High chemerin protein in the HCC tissues was related to the T stage, vessel invasion, histologic grade, Union for International Cancer Control (UICC) stage and tumor size. Chemokine-like receptor 1 (CMKLR1) is a functional chemerin receptor. CMKLR1 protein in hepatocytes was low expressed in HCC tissues of 36%, moderate in tissues of 32% and high in 32% of the HCCs. Tumor CMKLR1 was associated with the T stage, vessel invasion, histologic grade and UICC stage. Notably, sex-specific analysis revealed that associations of chemerin and CMKLR1 expression with HCC progression were significant in males but not in females. The tumor chemerin and CMKLR1 protein expression were not related to steatosis, inflammation and fibrosis grades. In summary, chemerin as well as CMKLR1 protein were related to disease severity of European HCC patients, and this was significant in males. This observation is in contrast to Asian patients where higher chemerin in the tumors was protective. Current analysis provides evidence for ethnicity and sex-related differences of tumor expressed chemerin and HCC severity.

Phenotypic Characterization of Colorectal Liver Metastases: Capsule versus No Capsule and the Potential Role of Epithelial Mesenchymal Transition.

Background: Colorectal liver metastases (CRLM) can be encased in a fibrous capsule separating cancer from normal liver tissue, which correlates with increased patient survival. This study investigated the cellular and molecular components of capsule formation and the possible role of epithelial mesenchymal transition (EMT). Methods: From 222 patients with CRLM, 84 patients (37.8%) were categorized to have CRLM encased with a capsule. A total of 34 CRLM from 34 selected patients was analyzed in detail by EMT pathway-profiling and custom PCR arrays to identify differences in gene expression between CRLM with (n = 20) and without capsule (n = 14). In parallel, those 34 CRLM were used to analyze 16 gene products at the metastasis margin via immunohistochemistry. Results: Encapsulated CRLM showed an elevated expression of signal transduction pathways and effector molecules involved in EMT. E-cadherin and keratin-19 were more prevalent, and transcription as well as translation (immunohistochemistry) of pGSK-3-ß, SOX10, tomoregulin-1, and caldesmon were increased. By contrast, the loss of E-cadherin and the prevalence of snail-1 were increased in CRLM without capsule. Collagen I and III and versican were identified as capsule components with extracellular matrix fibers running concentrically around the malignant tissue and parallel to the invasive front. Caldesmon was also demonstrated as a capsule constituent. Conclusions: The fibrous capsule around CRLM can be produced by cells with mesenchymal characteristics. It functions as a protective border by both the features of fiber architecture and the inhibition of invasive growth through EMT recruiting mesenchymal cells such as myofibroblasts by transformation of surrounding epithelial or even carcinoma cells. By contrast, EMT demonstrated in non-encapsulated CRLM may lead to a more mesenchymal, mobile, and tissue-destructive carcinoma cell phenotype and facilitate malignant spread.

Aberrant fucosylation sustains the NOTCH and EGFR/NF-κB pathways and has a prognostic value in human intrahepatic cholangiocarcinoma.

BACKGROUND AND AIMS: Intrahepatic cholangiocarcinoma (iCCA) is a lethal malignancy, with increasing incidence worldwide and limited therapeutic options. Aberrant protein glycosylation is a hallmark of cancer. Here, we thoroughly investigated the possible involvement of fucosylation in cholangiocarcinogenesis. APPROACH AND RESULTS: We discovered that the levels of global fucosylation and members of the fucosylation pathway are ubiquitously upregulated in human iCCA tissues compared to nontumorous surrounding livers and normal biliary cells. In addition, total fucosylation levels correlate with poor patients' prognosis. Furthermore, fucosylation inhibition following 6-alkynylfucose (6AF) administration triggered a dose-dependent decrease in the proliferation and migration of iCCA cell lines. Notably, adding fucose to the cell medium annulled these effects. At the molecular level, 6AF administration or small interfering RNA-mediated silencing of GDP-L-fucose synthetase (FX) and the GDP-fucose transmembrane transporter (SLC35C1), both pivotal players of cellular fucosylation, decreased NOTCH activity, NOTCH1/Jagged1 interaction, NOTCH receptors, and related target genes in iCCA cell lines. In the same cells, EGFR, nuclear factor kappa-light-chain-enhancer of activated B cells p65, and Bcl-xL protein levels diminished, whereas IκBα (a critical cellular NF-κB inhibitor) increased after FX/SLC35C1 knockdown or 6AF administration. In the chick chorioallantoic membrane assay, 6AF treatment profoundly suppresses the growth of iCCA cells. CONCLUSIONS: Elevated global fucosylation characterizes human iCCA, contributing to cell growth and migration through the upregulation of the NOTCH and EGFR/NF-κB pathways. Thus, aberrant fucosylation is a novel pathogenetic player and a potential therapeutic target for human iCCA.

Anti-DNA-IgM Favors the Detection of NET-Associated Extracellular DNA.

During inflammatory responses, neutrophils enter the sites of attack where they execute various defense mechanisms. They (I) phagocytose microorganisms, (II) degranulate to release cytokines, (III) recruit various immune cells by cell-type specific chemokines, (IV) secrete anti-microbials including lactoferrin, lysozyme, defensins and reactive oxygen species, and (V) release DNA as neutrophil extracellular traps (NETs). The latter originates from mitochondria as well as from decondensed nuclei. This is easily detected in cultured cells by staining of DNA with specific dyes. However, in tissues sections the very high fluorescence signals emitted from the condensed nuclear DNA hamper the detection of the widespread, extranuclear DNA of the NETs. In contrast, when we employ anti-DNA-IgM antibodies, they are unable to penetrate deep into the tightly packed DNA of the nucleus, and we observe a robust signal for the extended DNA patches of the NETs. To validate anti-DNA-IgM, we additionally stained the sections for the NET-markers histone H2B, myeloperoxidase, citrullinated histone H3, and neutrophil elastase. Altogether, we have described a fast one-step procedure for the detection of NETs in tissue sections, which provides new perspectives to characterize neutrophil-associated immune reactions in disease.

Long-Term Simulation of Microgravity Induces Changes in Gene Expression in Breast Cancer Cells.

Microgravity changes the gene expression pattern in various cell types. This study focuses on the breast cancer cell lines MCF-7 (less invasive) and MDA-MB-231 (triple-negative, highly invasive). The cells were cultured for 14 days under simulated microgravity (s-µg) conditions using a random positioning machine (RPM). We investigated cytoskeletal and extracellular matrix (ECM) factors as well as focal adhesion (FA) and the transmembrane proteins involved in different cellular signaling pathways (MAPK, PAM and VEGF). The mRNA expressions of 24 genes of interest (TUBB, ACTB, COL1A1, COL4A5, LAMA3, ITGB1, CD44, VEGF, FLK1, EGFR, SRC, FAK1, RAF1, AKT1, ERK1, MAPK14, MAP2K1, MTOR, RICTOR, VCL, PXN, CDKN1, CTNNA1 and CTNNB1) were determined by quantitative real-time PCR (qPCR) and studied using STRING interaction analysis. Histochemical staining was carried out to investigate the morphology of the adherent cells (ADs) and the multicellular spheroids (MCSs) after RPM exposure. To better understand this experimental model in the context of breast cancer patients, a weighted gene co-expression network analysis (WGCNA) was conducted to obtain the expression profiles of 35 breast cell lines from the HMS LINCS Database. The qPCR-verified genes were searched in the mammalian phenotype database and the human genome-wide association studies (GWAS) Catalog. The results demonstrated the positive association between the real metastatic microtumor environment and MCSs with respect to the extracellular matrix, cytoskeleton, morphology, different cellular signaling pathway key proteins and several other components. In summary, the microgravity-engineered three-dimensional MCS model can be utilized to study breast cancer cell behavior and to assess the therapeutic efficacies of drugs against breast cancer in the future.

Early Subcellular Hepatocellular Alterations in Mice Post Hydrodynamic Transfection: An Explorative Study.

Hydrodynamic transfection (HT) or hydrodynamic tail vein injection (HTVi) is among the leading technique that is used to deliver plasmid genes mainly into the liver of live mice or rats. The DNA constructs are composed of coupled plasmids, while one contains the gene of interest that stably integrate into the hepatocyte genome with help of the other consisting sleeping beauty transposase system. The rapid injection of a large volume of DNA-solution through the tail vein induces an acute cardiac congestion that refluxed into the liver, mainly in acinus zone 3, also found through our EM study. Although, HT mediated hydrodynamic force can permeabilizes the fenestrated sinusoidal endothelium of liver, but the mechanism of plasmid incorporation into the hepatocytes remains unclear. Therefore, in the present study, we have hydrodynamically injected 2 mL volume of empty plasmid (transposon vector) or saline solution (control) into the tail vein of anesthetized C57BL/6J/129Sv mice. Liver tissue was resected at different time points from two animal group conditions, i.e., one time point per animal (1, 5, 10-20, 60 min or 24 and 48 hrs after HT) or multiple time points per animal (0, 1, 2, 5, 10, 20 min) and quickly fixed with buffered 4% osmium tetroxide. The tissues fed with only saline solution was also resected and fixed in the similar way. EM evaluation from the liver ultrathin sections reveals that swiftly after 1 min, the hepatocytes near to the central venule in the acinus zone 3 shows cytoplasmic membrane-bound vesicles. Such vesicles increased in both numbers and size to vacuoles and precisely often found in the proximity to the nucleus. Further, EM affirm these vacuoles are also optically empty and do not contain any electron dense material. Although, some of the other hepatocytes reveals sign of cell damage including swollen mitochondria, dilated endoplasmic reticulum, Golgi apparatus and disrupted plasma membrane, but most of the hepatocytes appeared normal. The ultrastructural findings in the mice injected with empty vector or saline injected control mice were similar. Therefore, we have interpreted the vacuole formation as nonspecific endocytosis without specific interactions at the plasma membrane.

Early γGT and bilirubin levels as biomarkers for regeneration and outcomes in damaged bile ducts after liver transplantation.

BACKGROUND: Early patient and allograft survival after liver transplantation (LT) depend primarily on parenchymal function, but long-term allograft success relies often on biliary-tree function. We examined parameters related to cholangiocyte damage that predict poor long-term LT outcomes after donation after brain death (DBD). METHODS: Sixty bile ducts (BD) were assessed by a BD damage-score and divided into groups with "major" BD-damage (n = 33) and "no relevant" damage (n = 27) during static cold storage. Patients with "major" BD damage were further investigated by measuring biliary excretion parameters in the first 14 days post-LT (followed-up for 60-months). RESULTS: Patients who received LT showing "major" BD damage had significantly worse long-term patient survival, versus grafts with "no relevant" damage (p = .03). When "major" BD damage developed, low bilirubin levels (p = .012) and high gamma-glutamyl transferase (GGT)/bilirubin ratio (p = .0003) were evident in the early post-LT phase (7-14 days) in patients who survived (> 60 months), compared to those who did not. "High risk" patients with bile duct damage and low GGT/bilirubin ratio had significantly shorter overall survival (p < .0001). CONCLUSIONS: Once "major" BD damage occurs, a high GGT/bilirubin ratio in the early post-operative phase is likely indicator of liver and cholangiocyte regeneration, and thus a harbinger of good overall outcomes. "Major" BD damage without markers of regeneration identifies LT patients that could benefit from future repair therapies.

A Pleomorphic Puzzle: Heterogeneous Pulmonary Vascular Occlusions in Patients with COVID-19.

Vascular occlusions in patients with coronavirus diseases 2019 (COVID-19) have been frequently reported in severe outcomes mainly due to a dysregulation of neutrophils mediating neutrophil extracellular trap (NET) formation. Lung specimens from patients with COVID-19 have previously shown a dynamic morphology, categorized into three types of pleomorphic occurrence based on histological findings in this study. These vascular occlusions in lung specimens were also detected using native endogenous fluorescence or NEF in a label-free method. The three types of vascular occlusions exhibit morphology of DNA rich neutrophil elastase (NE) poor (type I), NE rich DNA poor (type II), and DNA and NE rich (type III) cohort of eleven patients with six males and five females. Age and gender have been presented in this study as influencing variables linking the occurrence of several occlusions with pleomorphic contents within a patient specimen and amongst them. This study reports the categorization of pleomorphic occlusions in patients with COVID-19 and the detection of these occlusions in a label-free method utilizing NEF.

Role of Fibroblast Growth Factors in the Crosstalk of Hepatic Stellate Cells and Uveal Melanoma Cells in the Liver Metastatic Niche.

Hepatic metastasis is the critical factor determining tumor-associated mortality in different types of cancer. This is particularly true for uveal melanoma (UM), which almost exclusively metastasizes to the liver. Hepatic stellate cells (HSCs) are the precursors of tumor-associated fibroblasts and support the growth of metastases. However, the underlying mechanisms are widely unknown. Fibroblast growth factor (FGF) signaling is dysregulated in many types of cancer. The aim of this study was to analyze the pro-tumorigenic effects of HSCs on UM cells and the role of FGFs in this crosstalk. Conditioned medium (CM) from activated human HSCs significantly induced proliferation together with enhanced ERK and JNK activation in UM cells. An in silico database analysis revealed that there are almost no mutations of FGF receptors (FGFR) in UM. However, a high FGFR expression was found to be associated with poor survival for UM patients. In vitro, the pro-tumorigenic effects of HSC-CM on UM cells were abrogated by a pharmacological inhibitor (BGJ398) of FGFR1/2/3. The expression analysis revealed that the majority of paracrine FGFs are expressed by HSCs, but not by UM cells, including FGF9. Furthermore, the immunofluorescence analysis indicated HSCs as a cellular source of FGF9 in hepatic metastases of UM patients. Treatment with recombinant FGF9 significantly enhanced the proliferation of UM cells, and this effect was efficiently blocked by the FGFR1/2/3 inhibitor BGJ398. Our study indicates that FGF9 released by HSCs promotes the tumorigenicity of UM cells, and thus suggests FGF9 as a promising therapeutic target in hepatic metastasis.

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.

SARS-CoV-2 infects human cardiomyocytes promoted by inflammation and oxidative stress.

INTRODUCTION: The respiratory illness triggered by severe acute respiratory syndrome virus-2 (SARS-CoV-2) is often particularly serious or fatal amongst patients with pre-existing heart conditions. Although the mechanisms underlying SARS-CoV-2-related cardiac damage remain elusive, inflammation (i.e. 'cytokine storm') and oxidative stress are likely involved. METHODS AND RESULTS: Here we sought to determine: 1) if cardiomyocytes are targeted by SARS-CoV-2 and 2) how inflammation and oxidative stress promote the viral entry into cardiac cells. We analysed pro-inflammatory and oxidative stress and its impact on virus entry and virus-associated cardiac damage from SARS-CoV-2 infected patients and compared it to left ventricular myocardial tissues obtained from non-infected transplanted hearts either from end stage heart failure or non-failing hearts (donor group). We found that neuropilin-1 potentiates SARS-CoV-2 entry into human cardiomyocytes, a phenomenon driven by inflammatory and oxidant signals. These changes accounted for increased proteases activity and apoptotic markers thus leading to cell damage and apoptosis. CONCLUSION: This study provides new insights into the mechanisms of SARS-CoV-2 entry into the heart and defines promising targets for antiviral interventions for COVID-19 patients with pre-existing heart conditions or patients with co-morbidities.

The Hippo pathway effector TAZ induces intrahepatic cholangiocarcinoma in mice and is ubiquitously activated in the human disease.

BACKGROUND: Intrahepatic cholangiocarcinoma (iCCA) is a highly aggressive primary liver tumor with increasing incidence worldwide, dismal prognosis, and few therapeutic options. Mounting evidence underlines the role of the Hippo pathway in this disease; however, the molecular mechanisms whereby the Hippo cascade contributes to cholangiocarcinogenesis remain poorly defined. METHODS: We established novel iCCA mouse models via hydrodynamic transfection of an activated form of transcriptional coactivator with PDZ-binding motif (TAZ), a Hippo pathway downstream effector, either alone or combined with the myristoylated AKT (myr-AKT) protooncogene, in the mouse liver. Hematoxylin and eosin staining, immunohistochemistry, electron microscopy, and quantitative real-time RT-PCR were applied to characterize the models. In addition, in vitro cell line studies were conducted to address the growth-promoting roles of TAZ and its paralog YAP. RESULTS: Overexpression of TAZ in the mouse liver triggered iCCA development with very low incidence and long latency. In contrast, co-expression of TAZ and myr-AKT dramatically increased tumor frequency and accelerated cancer formation in mice, with 100% iCCA incidence and high tumor burden by 10 weeks post hydrodynamic injection. AKT/TAZ tumors faithfully recapitulated many of the histomolecular features of human iCCA. At the molecular level, the development of the cholangiocellular lesions depended on the binding of TAZ to TEAD transcription factors. In addition, inhibition of the Notch pathway did not hamper carcinogenesis but suppressed the cholangiocellular phenotype of AKT/TAZ tumors. Also, knockdown of YAP, the TAZ paralog, delayed cholangiocarcinogenesis in AKT/TAZ mice without affecting the tumor phenotype. Furthermore, human preinvasive and invasive iCCAs and mixed hepatocellular carcinoma/iCCA displayed widespread TAZ activation and downregulation of the mechanisms protecting TAZ from proteolysis. CONCLUSIONS: Overall, the present data underscore the crucial role of TAZ in cholangiocarcinogenesis.