PARP-1 selectively impairs KRAS-driven phenotypic and molecular features in intrahepatic cholangiocarcinoma.

OBJECTIVE: Intrahepatic cholangiocarcinoma (iCCA) is the second most common primary liver cancer with limited therapeutic options. KRAS mutations are among the most abundant genetic alterations in iCCA associated with poor clinical outcome and treatment response. Recent findings indicate that Poly(ADP-ribose)polymerase1 (PARP-1) is implicated in KRAS-driven cancers, but its exact role in cholangiocarcinogenesis remains undefined. DESIGN: PARP-1 inhibition was performed in patient-derived and established iCCA cells using RNAi, CRISPR/Cas9 and pharmacological inhibition in KRAS-mutant, non-mutant cells. In addition, Parp-1 knockout mice were combined with iCCA induction by hydrodynamic tail vein injection to evaluate an impact on phenotypic and molecular features of Kras-driven and Kras-wildtype iCCA. Clinical implications were confirmed in authentic human iCCA. RESULTS: PARP-1 was significantly enhanced in KRAS-mutant human iCCA. PARP-1-based interventions preferentially impaired cell viability and tumourigenicity in human KRAS-mutant cell lines. Consistently, loss of Parp-1 provoked distinct phenotype in Kras/Tp53-induced versus Akt/Nicd-induced iCCA and abolished Kras-dependent cholangiocarcinogenesis. Transcriptome analyses confirmed preferential impairment of DNA damage response pathways and replicative stress response mediated by CHK1. Consistently, inhibition of CHK1 effectively reversed PARP-1 mediated effects. Finally, Parp-1 depletion induced molecular switch of KRAS-mutant iCCA recapitulating good prognostic human iCCA patients. CONCLUSION: Our findings identify the novel prognostic and therapeutic role of PARP-1 in iCCA patients with activation of oncogenic KRAS signalling.

N-cadherin: A diagnostic marker to help discriminate primary liver carcinomas from extrahepatic carcinomas.

Distinguishing primary liver cancer (PLC), namely hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (iCCA), from liver metastases is of crucial clinical importance. Histopathology remains the gold standard, but differential diagnosis may be challenging. While absent in most epithelial, the expression of the adherens junction glycoprotein N-cadherin is commonly restricted to neural and mesenchymal cells, or carcinoma cells that undergo the phenomenon of epithelial-to-mesenchymal transition (EMT). However, we recently established N- and E-cadherin expression as hallmarks of normal hepatocytes and cholangiocytes, which are also preserved in HCC and iCCA. Therefore, we hypothesized that E- and/or N-cadherin may distinguish between carcinoma derived from the liver vs carcinoma of other origins. We comprehensively evaluated E- and N-cadherin in 3359 different tumors in a multicenter study using immunohistochemistry and compared our results with previously published 882 cases of PLC, including 570 HCC and 312 iCCA. Most carcinomas showed strong positivity for E-cadherin. Strong N-cadherin positivity was present in HCC and iCCA. However, except for clear cell renal cell carcinoma (23.6% of cases) and thyroid cancer (29.2%), N-cadherin was only in some instances faintly expressed in adenocarcinomas of the gastrointestinal tract (0%-0.5%), lung (7.1%), pancreas (3.9%), gynecological organs (0%-7.4%), breast (2.2%) as well as in urothelial (9.4%) and squamous cell carcinoma (0%-5.6%). As expected, N-cadherin was detected in neuroendocrine tumors (25%-75%), malignant melanoma (46.2%) and malignant mesothelioma (41%). In conclusion, N-cadherin is a useful marker for the distinction of PLC vs liver metastases of extrahepatic carcinomas (P < .01).

ALK1 controls hepatic vessel formation, angiodiversity, and angiocrine functions in hereditary hemorrhagic telangiectasia of the liver.

BACKGROUND AND AIMS: In hereditary hemorrhagic telangiectasia (HHT), severe liver vascular malformations are associated with mutations in the Activin A Receptor-Like Type 1 ( ACVRL1 ) gene encoding ALK1, the receptor for bone morphogenetic protein (BMP) 9/BMP10, which regulates blood vessel development. Here, we established an HHT mouse model with exclusive liver involvement and adequate life expectancy to investigate ALK1 signaling in liver vessel formation and metabolic function. APPROACH AND RESULTS: Liver sinusoidal endothelial cell (LSEC)-selective Cre deleter line, Stab2-iCreF3 , was crossed with Acvrl1 -floxed mice to generate LSEC-specific Acvrl1 -deficient mice ( Alk1HEC-KO ). Alk1HEC-KO mice revealed hepatic vascular malformations and increased posthepatic flow, causing right ventricular volume overload. Transcriptomic analyses demonstrated induction of proangiogenic/tip cell gene sets and arterialization of hepatic vessels at the expense of LSEC and central venous identities. Loss of LSEC angiokines Wnt2 , Wnt9b , and R-spondin-3 ( Rspo3 ) led to disruption of metabolic liver zonation in Alk1HEC-KO mice and in liver specimens of patients with HHT. Furthermore, prion-like protein doppel ( Prnd ) and placental growth factor ( Pgf ) were upregulated in Alk1HEC-KO hepatic endothelial cells, representing candidates driving the organ-specific pathogenesis of HHT. In LSEC in vitro , stimulation or inhibition of ALK1 signaling counter-regulated Inhibitors of DNA binding (ID)1-3, known Alk1 transcriptional targets. Stimulation of ALK1 signaling and inhibition of ID1-3 function confirmed regulation of Wnt2 and Rspo3 by the BMP9/ALK1/ID axis. CONCLUSIONS: Hepatic endothelial ALK1 signaling protects from development of vascular malformations preserving organ-specific endothelial differentiation and angiocrine signaling. The long-term surviving Alk1HEC-KO HHT model offers opportunities to develop targeted therapies for this severe disease.

Etiology-independent activation of the LTß-LTßR-RELB axis drives aggressiveness and predicts poor prognosis in HCC.

BACKGROUND AND AIMS: HCC is the most common primary liver tumor, with an increasing incidence worldwide. HCC is a heterogeneous malignancy and usually develops in a chronically injured liver. The NF-κB signaling network consists of a canonical and a noncanonical branch. Activation of canonical NF-κB in HCC is documented. However, a functional and clinically relevant role of noncanonical NF-κB and its downstream effectors is not established. APPROACH AND RESULTS: Four human HCC cohorts (total n = 1462) and 4 mouse HCC models were assessed for expression and localization of NF-κB signaling components and activating ligands. In vitro , NF-κB signaling, proliferation, and cell death were measured, proving a pro-proliferative role of v-rel avian reticuloendotheliosis viral oncogene homolog B (RELB) activated by means of NF-κB-inducing kinase. In vivo , lymphotoxin beta was identified as the predominant inducer of RELB activation. Importantly, hepatocyte-specific RELB knockout in a murine HCC model led to a lower incidence compared to controls and lower maximal tumor diameters. In silico , RELB activity and RELB-directed transcriptomics were validated on the The Cancer Genome Atlas HCC cohort using inferred protein activity and Gene Set Enrichment Analysis. In RELB-active HCC, pathways mediating proliferation were significantly activated. In contrast to v-rel avian reticuloendotheliosis viral oncogene homolog A, nuclear enrichment of noncanonical RELB expression identified patients with a poor prognosis in an etiology-independent manner. Moreover, RELB activation was associated with malignant features metastasis and recurrence. CONCLUSIONS: This study demonstrates a prognostically relevant, etiology-independent, and cross-species consistent activation of a lymphotoxin beta/LTßR/RELB axis in hepatocarcinogenesis. These observations may harbor broad implications for HCC, including possible clinical exploitation.

Dual roles of B lymphocytes in mouse models of diet-induced nonalcoholic fatty liver disease.

BACKGROUND AND AIMS: Growing evidence suggests an important role of B cells in the development of NAFLD. However, a detailed functional analysis of B cell subsets in NAFLD pathogenesis is lacking. APPROACH AND RESULTS: In wild-type mice, 21 weeks of high fat diet (HFD) feeding resulted in NAFLD with massive macrovesicular steatosis, modest hepatic and adipose tissue inflammation, insulin resistance, and incipient fibrosis. Remarkably, Bnull (JHT) mice were partially protected whereas B cell harboring but antibody-deficient IgMi mice were completely protected from the development of hepatic steatosis, inflammation, and fibrosis. The common feature of JHT and IgMi mice is that they do not secrete antibodies, whereas HFD feeding in wild-type mice led to increased levels of serum IgG2c. Whereas JHT mice have no B cells at all, regulatory B cells were found in the liver of both wild-type and IgMi mice. HFD reduced the number of regulatory B cells and IL-10 production in the liver of wild-type mice, whereas these increased in IgMi mice. Livers of patients with advanced liver fibrosis showed abundant deposition of IgG and stromal B cells and low numbers of IL-10 expressing cells, compatible with our experimental data. CONCLUSIONS: B lymphocytes have both detrimental and protective effects in HFD-induced NAFLD. The lack of secreted pathogenic antibodies protects partially from NAFLD, whereas the presence of certain B cell subsets provides additional protection. IL-10-producing regulatory B cells may represent such a protective B cell subset.

Clinical, histological and molecular profiling of different stages of alcohol-related liver disease.

OBJECTIVE: Alcohol-related liver disease (ALD) ranges from never-decompensated ALD (ndALD) to the life-threatening decompensated phenotype, known as alcohol-related hepatitis (AH). A multidimensional study of the clinical, histological and molecular features of these subtypes is lacking. DESIGN: Two large cohorts of patients were recruited in an international, observational multicentre study: a retrospective cohort of patients with ndALD (n=110) and a prospective cohort of patients with AH (n=225). Clinical, analytical, immunohistochemistry and hepatic RNA microarray analysis of both disease phenotypes were performed. RESULTS: Age and mean alcohol intake were similar in both groups. AH patients had greater aspartate amino transferase/alanine amino transferase ratio and lower gamma-glutamyl transferase levels than in ndALD patients. Patients with AH demonstrated profound liver failure and increased mortality. One-year mortality was 10% in ndALD and 50% in AH. Histologically, steatosis grade, ballooning and pericellular fibrosis were similar in both groups, while advanced fibrosis, Mallory-Denk bodies, bilirubinostasis, severe neutrophil infiltration and ductular reaction were more frequent among AH patients. Transcriptome analysis revealed a profound gene dysregulation within both phenotypes when compare to controls. While ndALD was characterised by deregulated expression of genes involved in matrisome and immune response, the development of AH resulted in a marked deregulation of genes involved in hepatocyte reprogramming and bile acid metabolism. CONCLUSIONS: Despite comparable alcohol intake, AH patients presented with worse liver function compared with ndALD patients. Bilirubinostasis, severe fibrosis and ductular reaction were prominent features of AH. AH patients exhibited a more profound deregulation of gene expression compared with ndALD patients.

Gastric Bypass Resolves Metabolic Dysfunction-Associated Fatty Liver Disease (MAFLD) in Low-BMI Patients: A Prospective Cohort Study.

OBJECTIVE: Metabolic dysfunction-associated fatty liver disease (MAFLD) reflects the multifactorial pathogenesis of fatty liver disease in metabolically sick patients. The effects of metabolic surgery on MAFLD have not been investigated. This study assesses the impact of Roux-en-Y gastric bypass (RYGB) on MAFLD in a prototypical cohort outside the guidelines for obesity surgery. METHODS: Twenty patients were enrolled in this prospective, single-arm trial investigating the effects of RYGB on advanced metabolic disease (DRKS00004605). Inclusion criteria were an insulin-dependent type 2 diabetes, body mass index of 25 to 35 kg/m 2 , glucagon-stimulated C-peptide of >1.5 ng/mL, glycated hemoglobin >7%, and age 18 to 70 years. A RYGB with intraoperative liver biopsies and follow-up liver biopsies 3 years later was performed. Steatohepatitis was assessed by expert liver pathologists. Data were analyzed using the Wilcoxon rank sum test and a P value <0.05 was defined as significant. RESULTS: MAFLD completely resolved in all patients 3 years after RYGB while fibrosis improved as well. Fifty-five percent were off insulin therapy with a significant reduction in glycated hemoglobin (8.45±0.27% to 7.09±0.26%, P =0.0014). RYGB reduced systemic and hepatic nitrotyrosine levels likely through upregulation of NRF1 and its dependent antioxidative and mitochondrial genes. In addition, central metabolic regulators such as SIRT1 and FOXO1 were upregulated while de novo lipogenesis was reduced and ß-oxidation was improved in line with an improvement of insulin resistance. Lastly, gastrointestinal hormones and adipokines secretion were changed favorably. CONCLUSIONS: RYGB is a promising therapy for MAFLD even in low-body mass index patients with insulin-treated type 2 diabetes with complete histologic resolution. RYGB restores the oxidative balance, adipose tissue function, and gastrointestinal hormones.

CD44 Contributes to the Regulation of MDR1 Protein and Doxorubicin Chemoresistance in Osteosarcoma.

Osteosarcoma is the most common type of pediatric bone tumor. Despite great advances in chemotherapy during the past decades, the survival rates of osteosarcoma patients remain unsatisfactory. Drug resistance is one of the main reasons, leading to treatment failure and poor prognosis. Previous reports correlated expression of cluster of differentiation 44 (CD44) with drug resistance and poor survival of osteosarcoma patients, however the underlying mechanisms are poorly defined. Here, we investigated the role of CD44 in the regulation of drug chemoresistance, using osteosarcoma cells isolated from mice carrying a mutation of the tumor suppressor neurofibromatosis type 2 (Nf2) gene. CD44 expression was knocked-down in the cells using CRISPR/Cas9 approach. Subsequently, CD44 isoforms and mutants were re-introduced to investigate CD44-dependent processes. Sensitivity to doxorubicin was analyzed in the osteosarcoma cells with modified CD44 expression by immunoblot, colony formation- and WST-1 assay. To dissect the molecular alterations induced by deletion of Cd44, RNA sequencing was performed on Cd44-positive and Cd44-negative primary osteosarcoma tissues isolated from Nf2-mutant mice. Subsequently, expression of candidate genes was evaluated by quantitative reverse transcription PCR (qRT-PCR). Our results indicate that CD44 increases the resistance of osteosarcoma cells to doxorubicin by up-regulating the levels of multidrug resistance (MDR) 1 protein expression, and suggest the role of proteolytically released CD44 intracellular domain, and hyaluronan interactions in this process. Moreover, high throughput sequencing analysis identified differential regulation of several apoptosis-related genes in Cd44-positive and -negative primary osteosarcomas, including p53 apoptosis effector related to PMP-22 (Perp). Deletion of Cd44 in osteosarcoma cells led to doxorubicin-dependent p53 activation and a profound increase in Perp mRNA expression. Overall, our results suggest that CD44 might be an important regulator of drug resistance and suggest that targeting CD44 can sensitize osteosarcoma to standard chemotherapy.

[Digital teaching in pathology: experiences from Mainz]. / Digitale Lehre in der Pathologie: Erfahrungen aus Mainz.

BACKGROUND: Due to the corona pandemic, digital teaching has become especially important in education and has led to a restructuring of teaching, not only in the subject of surgical pathology. OBJECTIVES: In this article, different forms of e­learning are presented and illustrated using the example of teaching surgical pathology and neuropathology at the University Medical Center Mainz. RESULTS: Before the onset of the corona pandemic in spring 2020, digitization had already assumed great importance for teaching in the technology- and method-oriented subject of surgical pathology. In particular, the possibility of virtual microscopy via scanned slides with a digital slide server has been used in many pathology institutes. Virtual microscopy often partially or completely replaced conventional microscopy of histologic slide collections. Complementary virtual learning offers are becoming more and more important. These include asynchronously provided lectures or macroscopy videos, video conferences, scripts and communication via learning platforms. In addition, electronic exams have become an indispensable part of teaching. Nevertheless, the corona pandemic revealed how important personal contact with students is to achieve optimal learning success; learning forms with a combination of face-to-face teaching and e­learning in the sense of blended learning are of particular importance. CONCLUSIONS: As part of blended learning, digital teaching is an ideal complement to face-to-face teaching and is changing teaching in the longer term, not only in the field of surgical pathology. Digital learning formats will remain in the future and will at least partially replace classroom formats such as lectures.

Perilipin 1 Expression Differentiates Liposarcoma from Other Types of Soft Tissue Sarcoma.

Lipid droplets, a morphologic feature of adipocytic tumors, are strongly regulated by associated proteins of the perilipin/PAT (perilipin, adipophilin, and tail-interacting protein of 47 kD) family. So far, the use of perilipins as markers for differential diagnosis of soft tissue tumors has only been studied in a few cases. The aim of this study was to investigate the expression of perilipins in 478 human soft tissue tumors and 60 respective normal tissues. Perilipin 1 was immunohistochemically positive in all studied cases of well-differentiated liposarcomas, >90% of myxoid round cell liposarcomas, and >70% of pleomorphic liposarcomas, whereas only the differentiated components of dedifferentiated liposarcomas were immunohistochemically positive for perilipin 1. All other types of soft tissue sarcomas were negative for perilipin 1. Perilipin 2 was more prominent in dedifferentiated and pleomorphic liposarcomas and nearly all other high-grade sarcomas. In well-differentiated liposarcomas, lipomas, or normal adipose tissue, perilipin 2 was virtually absent. In addition, long-term stimulation of adipogenesis in the liposarcoma cell line LiSa-2 restored perilipin 1 expression, as exhibited in the source tumor. Furthermore, knockdown of perilipin 2 or perilipin 3 in LiSa-2 cells influenced lipid droplet number and size as well as cell vitality. In summary, perilipin 1 is a promising marker for the differential diagnosis of liposarcomas from other soft tissue sarcomas, whereas perilipin 2 correlates negatively with tumor grade and may be therapeutically useful.

Yes-associated protein (YAP) induces a secretome phenotype and transcriptionally regulates plasminogen activator Inhibitor-1 (PAI-1) expression in hepatocarcinogenesis.

BACKGROUND: Overexpression and nuclear enrichment of the oncogene yes-associated protein (YAP) cause tumor initiation and support tumor progression in human hepatocellular carcinoma (HCC) via cell autonomous mechanisms. However, how YAP expression in tumor cells affects intercellular communication within the tumor microenvironment is not well understood. METHODS: To investigate how tumor cell-derived YAP is changing the paracrine communication network between tumor cells and non-neoplastic cells in hepatocarcinogenesis, the expression and secretion of cytokines, growth factors and chemokines were analyzed in transgenic mice with liver-specific and inducible expression of constitutively active YAP (YAPS127A). Transcriptomic and proteomic analyses were performed using primary isolated hepatocytes and blood plasma. In vitro, RNAinterference (RNAi), expression profiling, functional analyses and chromatin immunoprecipitation (ChIP) analyses of YAP and the transcription factor TEA domain transcription factor 4 (TEAD4) were performed using immortalized cell lines. Findings were confirmed in cohorts of HCC patients at the transcript and protein levels. RESULTS: YAP overexpression induced the expression and secretion of many paracrine-acting factors with potential impact on tumorous or non-neoplastic cells (e.g. plasminogen activator inhibitor-1 (PAI-1), C-X-C motif chemokine ligand 13 (CXCL13), CXCL16). Expression analyses of human HCC patients showed an overexpression of PAI-1 in human HCC tissues and a correlation with poor overall survival as well as early cancer recurrence. PAI-1 statistically correlated with genes typically induced by YAP, such as connective tissue growth factor (CTGF) and cysteine rich angiogenic inducer 61 (CYR61) or YAP-dependent gene signatures (CIN4/25). In vitro, YAP inhibition diminished the expression and secretion of PAI-1 in murine and human liver cancer cell lines. PAI-1 affected the expression of genes involved in cellular senescence and oncogene-induced senescence was confirmed in YAPS127A transgenic mice. Silencing of TEAD4 as well as treatment with the YAP/TEAD interfering substance Verteporfin reduced PAI-1 expression. ChIP analyses confirmed the binding of YAP and TEAD4 to the gene promoter of PAI-1 (SERPINE1). CONCLUSIONS: These results demonstrate that the oncogene YAP changes the secretome response of hepatocytes and hepatocyte-derived tumor cells. In this context, the secreted protein PAI-1 is transcriptionally regulated by YAP in hepatocarcinogenesis. Perturbation of these YAP-dependent communication hubs including PAI-1 may represent a promising pharmacological approach in tumors with YAP overexpression. Video abstract.

A young patient with type 2 diabetes associated non-alcoholic steatohepatitis, liver cirrhosis, and hepatocellular carcinoma.

The rising prevalence of the metabolic syndrome has led to an increase of non-alcoholic fatty liver disease (NAFLD), and its progressive-inflammatory form called non-alcoholic steatohepatitis (NASH). In recent years, NAFLD and NASH have become major risk factors for developing liver cirrhosis and hepatocellular carcinoma (HCC). In this case, we report a 46-year-old patient with type 2 diabetes mellitus and metabolic comorbidities including obesity and arterial hypertension, who was referred because of rising liver enzymes. After clinical and diagnostic evaluation, the patient was diagnosed with NASH-associated liver cirrhosis, Child-Pugh stage B. A normal blood sugar level was difficult to achieve, and the patient presented with consistently elevated HbA1c-levels irresponsive to insulin therapy. Due to the underlying liver cirrhosis, the patient was enrolled in the HCC-surveillance program. Sonography during follow up showed a focal lesion. On magnetic resonance imaging (MRI), the diagnosis of HCC (BCLC stage A) was confirmed based on typical contrast enhancement and portal-venous wash-out. The patient was evaluated for liver transplantation with a labMELD of 17, and an intermittent therapy with TACE was initiated. Only 2 months after liver transplantation, the patient developed severe and lethal complications. Overall, this case highlights the different medical issues of patients with metabolic syndrome developing a chronic liver disease. In this patient, a rapid progression from NASH-associated liver cirrhosis to HCC was seen, and therefore highlights the importance of close surveillance to identify and treat potential risk factors early in the course of the disease.

Intrahepatic cholangiocarcinoma - influence of resection margin and tumor distance to the liver capsule on survival.

BACKGROUND: Intrahepatic cholangiocarcinoma (ICC) is often diagnosed in advanced stage. Aim of this study was to analyse the influence of resection margins and tumor distance to the liver capsule on survival and recurrence in a single center with a high number of extended resections. METHODS: From January 2008 to June 2018 data of all patients with ICC were collected and further analysed with Kaplan Meier Model, Cox regression or Chi2 test for categorical data. RESULTS: Out of 210 included patients 150 underwent curative intended resection (71.4%). Most patients required extended resections (n = 77; 51.3%). R0-resection was achieved in 131 patients (87.3%) with minimal distances to the resection margin > 1 cm in 22, 0.5-1 cm in 11, 0.1-0.5 cm in 49 patients, and <  0.1 cm in 49 patients. Overall survival (OS) for margins > 0.5 cm compared to 0.5-0.1 cm or R1 was better, but without reaching significance. All three groups had significantly better OS compared to the irresectable group. Recurrence-free survival (RFS) was also better in patients with a margin > 0.5 cm than in the < 0.5-0.1 cm or the R1-group, but even without reaching significance. Different distance to the liver capsule significantly affected OS, but not RFS. CONCLUSIONS: Wide resection margins (> 0.5 cm) should be targeted but did not show significantly better OS or RFS in a cohort with a high percentage of extended resections (> 50%). Wide margins, narrow margins and even R1 resections showed a significant benefit over the irresectable group. Therefore, extended resections should be performed, even if only narrow margins can be achieved.

[Variation in morphology or two entities?] / Morphologische Variabilität oder zwei Entitäten?

A 37-year-old patient presented with B symptoms. On examination, hypodense liver lesions, multiple enlarged and partly confluent lymph nodes in the upper abdomen and retroperitoneum, as well as disseminated splenic and pulmonary foci were detected. Biopsies of a tumor in the coecum and the liver led to the diagnosis of an adenocarcinoma of the colon. In molecular pathology, microsatellite instability was detected. The post-neoadjuvant surgical specimen showed an unusual morphology and the question arose whether a second tumor should be considered.

RSPO2 gene rearrangement: a powerful driver of ß-catenin activation in liver tumours.

OBJECTIVE: We aimed at the identification of genetic alterations that may functionally substitute for CTNNB1 mutation in ß-catenin-activated hepatocellular adenomas (HCAs) and hepatocellular carcinoma (HCC). DESIGN: Large cohorts of HCA (n=185) and HCC (n=468) were classified using immunohistochemistry. The mutational status of the CTNNB1 gene was determined in ß-catenin-activated HCA (b-HCA) and HCC with at least moderate nuclear CTNNB1 accumulation. Ultra-deep sequencing was used to characterise CTNNB1wild-type and ß-catenin-activated HCA and HCC. Expression profiling of HCA subtypes was performed. RESULTS: A roof plate-specific spondin 2 (RSPO2) gene rearrangement resulting from a 46.4 kb microdeletion on chromosome 8q23.1 was detected as a new morphomolecular driver of ß-catenin-activated HCA. RSPO2 fusion positive HCA displayed upregulation of RSPO2 protein, nuclear accumulation of ß-catenin and transcriptional activation of ß-catenin-target genes indicating activation of Wingless-Type MMTV Integration Site Family (WNT) signalling. Architectural and cytological atypia as well as interstitial invasion indicated malignant transformation in one of the RSPO2 rearranged b-HCAs. The RSPO2 gene rearrangement was also observed in three ß-catenin-activated HCCs developing in context of chronic liver disease. Mutations of the human telomerase reverse transcriptase promoter-known to drive malignant transformation of CTNNB1-mutated HCA-seem to be dispensable for RSPO2 rearranged HCA and HCC. CONCLUSION: The RSPO2 gene rearrangement leads to oncogenic activation of the WNT signalling pathway in HCA and HCC, represents an alternative mechanism for the development of b-HCA and may drive malignant transformation without additional TERT promoter mutation.

Application of patient-derived liver cancer cells for phenotypic characterization and therapeutic target identification.

Primary liver cancer (PLC) ranks among the most lethal solid cancers worldwide due to lack of effective biomarkers for early detection and limited treatment options in advanced stages. Development of primary culture models that closely recapitulate phenotypic and molecular diversities of PLC is urgently needed to improve the patient outcome. Long-term cultures of 7 primary liver cancer cell lines of hepatocellular and cholangiocellular origin were established using defined culture conditions. Morphological and histological characteristics of obtained cell lines and xenograft tumors were analyzed and compared to original tumors. Time course analyses of transcriptomic and genomic changes were performed using next-generation sequencing (NGS). Key oncogenic alterations were identified by targeted NGS and cell lines carrying potentially actionable mutations were treated with corresponding specific inhibitors. PDCL fully resembled morphological features of the primary cancers in vitro and in vivo over extended period in culture. Genomic alterations as well as transcriptome profiles showed high similarity with primary tumors and remained stable during long-term culturing. Targeted-NGS confirmed that key oncogenic mutations such as TP53, KRAS, CTNNB1 as well as actionable mutations (e.g. MET, cKIT, KDR) were highly conserved in PDCL and amenable for individualized therapeutic approaches. Integrative genomic and transcriptomic approaches further demonstrated that PDCL more closely resemble molecular and prognostic features of PLC than established cell lines and are valuable tool for direct target evaluation. Our integrative analysis demonstrates that PDCL represents refined model for discovery of relevant molecular subgroups and exploration of precision medicine approaches for the treatment of this deadly disease.

Health-related Quality of Life in Nonalcoholic Fatty Liver Disease Associates With Hepatic Inflammation.

BACKGROUND & AIMS: Chronic liver disease has negative effects on health-related quality of life (HRQL). We analyzed data from the European non-alcoholic fatty liver disease (NAFLD) registry to assess the effects of NAFLD on HRQL. METHODS: We collected data from 304 patients (mean age, 52.3 ± 12.9 years) with histologically defined NAFLD enrolled prospectively into the European NAFLD Registry in Germany, the United Kingdom, and Spain. The chronic liver disease questionnaire (CLDQ) was completed within 6 months of liver biopsy collection. RESULTS: The mean CLDQ overall score was 5.0 ± 1.2, with the lowest score in the category fatigue (4.3 ± 1.6) and the highest scores for activity (5.4 ± 1.4). Women had significantly lower CLDQ scores than men (4.6 ± 1.3 vs 5.3 ± 1.1; P < .001). We found negative correlations between CLDQ scores and presence of obesity (P < .001), type 2 diabetes (P < .001), and dyslipidaemia (P < .01). There was a negative correlation between level of aspartate aminotransferase, but not alanine aminotransferase, and HRQL. Higher histological score of steatosis (1 vs 3) resulted in lower mean CLDQ score (5.3 ± 1.1 vs 4.5 ± 1.4; P < .01); higher level of lobular inflammation (0 vs 3) also resulted in lower mean CLDQ score (5.3 ± 1.2 vs 3.9 ± 1.8; P <. 001). In contrast, advanced fibrosis (F3-4) compared to early or intermediate fibrosis (F0-2) had no significant effect on mean CLDQ score (4.9 ± 1.2 vs 5.1 ± 1.3; P = .072). In multivariate analysis, patients sex, age, presence of type 2 diabetes, and inflammation were independently associated with low HRQL. CONCLUSION: In an analysis of data from the European NAFLD registry, we observed a substantial burden of symptoms in patients. In addition to age, sex, and the presence of diabetes, detection of lobular inflammation in biopsies correlated with lower HRQL.

Surgical therapy of primary hepatic angiosarcoma.

BACKGROUND: Primary hepatic angiosarcoma (PHA) is a rare tumor entity. Radical surgical resection is currently considered the best treatment choice. The aim of this analysis is to report our experience with surgery for PHA. METHODS: All resections of PHA from 01/2002 until 06/2017 were identified from our prospective institutional database. All cases were re-confirmed by a second pathologist. We analyzed completeness of resection, overall (OS) and disease-free survival (DFS). RESULTS: Nine patients with PHA underwent hepatic resection. Median follow-up after surgery was 15.5 months (range: 3-144). At last follow-up 4/9 patients were alive, three of them without recurrence 15, 21 and 144 months after surgery. Five patients developed PHA recurrence. Four of these died 3 to 17 months after surgery. One patient with PHA recurrence is alive 15 months after surgery. Another patient without PHA recurrence died 59 months after surgery from pancreatic cancer. Median OS and DFS after resection was 18 months (range: 3-144 months) and 10 months (range: 2-144 months), respectively. After R-0 resection (n = 8), the median OS and DFS was 59 and 11 months. CONCLUSIONS: Resection of PHA is the only approach to achieve complete tumor removal and offers a chance for long-term survival and should be evaluated in cases of PHA.

Role of α1-adrenoceptor subtypes on corneal epithelial thickness and cell proliferation in mice.

Adrenergic stimuli are important for corneal epithelial structure and healing. The purpose of the present study was to examine the hypothesis that the lack of a single α1-adrenoceptor (α1-AR) subtype affects corneal epithelial thickness and cell proliferation. Expression levels of α1-AR mRNA were determined in mouse cornea using real-time PCR. In mice devoid of one of the three α1-AR subtypes (α1A-AR-/-, α1B-AR-/-, α1D-AR-/-) and in wild-type controls, thickness of individual corneal layers, the number of epithelial cell layers, and average epithelial cell size were determined in cryosections. Endothelial cell density and morphology were calculated in corneal explants, and epithelial cell proliferation rate was determined with immunofluorescence microscopy. Moreover, the ultrastructure of the corneal epithelium was examined by transmission electron microscopy. Messenger RNA for all three α1-AR subtypes was expressed in whole cornea and in corneal epithelium from wild-type mice with a rank order of abundance of α1A ≥ α1B > α1D. In contrast, no α1-AR mRNA was detected in the stroma, and only α1B-AR mRNA was found in the Descemet endothelial complex. Remarkably, corneal epithelial thickness and mean epithelial cell size were reduced in α1A-AR-/- mice. Our findings suggest that the α1A-AR exerts growth effects in mouse corneal epithelial cells.

Hepatocyte-specific deletion of IL1-RI attenuates liver injury by blocking IL-1 driven autoinflammation.

BACKGROUND & AIMS: Interleukin (IL)-1-type cytokines including IL-1α, IL-1ß and interleukin-1 receptor antagonist (IL-1Ra) are among the most potent molecules of the innate immune system and exert biological activities through the ubiquitously expressed interleukin-1 receptor type 1 (IL-1R1). The role of IL-1R1 in hepatocytes during acute liver failure (ALF) remains undetermined. METHODS: The role of IL-1R1 during ALF was investigated using a novel transgenic mouse model exhibiting deletion of all signaling-capable IL-1R isoforms in hepatocytes (Il1r1Hep-/-). RESULTS: ALF induced by D-galactosamine (D-GalN) and lipopolysaccharide (LPS) was significantly attenuated in Il1r1Hep-/- mice leading to reduced mortality. Conditional deletion of Il1r1 decreased activation of injurious c-Jun N-terminal kinases (JNK)/c-Jun signaling, activated nuclear factor-kappa B (NF-κB) p65, inhibited extracellular signal-regulated kinase (ERK) and prevented caspase 3-mediated apoptosis. Moreover, Il1r1Hep-/- mice exhibited reduced local and systemic inflammatory cytokine and chemokine levels, especially TNF-α, IL-1α/ß, IL-6, CC-chemokine ligand 2 (CCL2), C-X-C motif ligand 1 (CXCL-1) and CXCL-2, and a reduced neutrophil recruitment into the hepatic tissue in response to injury. NLRP3 inflammasome expression and caspase 1 activation were suppressed in the absence of the hepatocellular IL-1R1. Inhibition of IL-1R1 using IL-1ra (anakinra) attenuated the severity of liver injury, while IL-1α administration exaggerated it. These effects were lost ex vivo and at later time points, supporting a role of IL-1R1 in inflammatory signal amplification during acute liver injury. CONCLUSION: IL-1R1 in hepatocytes plays a pivotal role in an IL-1-driven auto-amplification of cell death and inflammation in the onset of ALF. LAY SUMMARY: Acute liver injury which can cause lethal liver failure is medicated by a class of proteins called cytokines. Among these, interleukin-1 (IL-1) and the corresponding receptor IL-1R1 play a prominent role in the immune system, but their role in the liver is undetermined. In the current study, a novel mouse model with defective IL-1R1 in liver cells was studied. Mice lacking this receptor in liver cells were protected from cell death to a certain extent. This protection occurred only in the presence of other, neighboring cells, arguing for the involvement of proteins derived from these cells. This effect is called paracrine signaling and the current study has for the first time shown that the IL-1R1 receptor on hepatocytes is involved in acute liver failure in this context. The approved drug anakinra - which blocks IL-1R1 - had the same effect, supporting the proposed mechanism of action. The findings of this study suggest new treatment options for patients with acute liver failure by blocking defined signals of the immune system.