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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., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Introduction: WNT1-inducible signalling pathway protein 1 (WISP1) promotes progression of several tumor entities often correlating with worse prognosis. Here its expression regulation and role in the progression of chronic liver diseases (CLD) was investigated., Methods: WISP1 expression was analyzed in human HCC datasets, in biopsies and serum samples and an HCC patient tissue microarray (TMA) including correlation to clinicopathological parameters. Spatial distribution of WISP1 expression was determined using RNAscope analysis. Regulation of WISP1 expression was investigated in cytokine-stimulated primary mouse hepatocytes (PMH) by array analysis and qRT-PCR. Outcome of WISP1 stimulation was analyzed by IncuCyte S3-live cell imaging, qRT-PCR, and immunoblotting in murine AML12 cells., Results: In a TMA, high WISP1 expression was positively correlated with early HCC stages and male sex. Highest WISP1 expression levels were detected in patients with cirrhosis as compared to healthy individuals, patients with early fibrosis, and non-cirrhotic HCC in liver biopsies, expression datasets and serum samples. WISP1 transcripts were predominantly detected in hepatocytes of cirrhotic rather than tumorous liver tissue. High WISP1 expression was associated with better survival. In PMH, AML12 and HepaRG, WISP1 was identified as a specific TGF-β1 target gene. Accordingly, expression levels of both cytokines positively correlated in human HCC patient samples. WISP1-stimulation induced the expression of Bcl-xL, PCNA and p21 in AML12 cells., Conclusions: WISP1 expression is induced by TGF-β1 in hepatocytes and is associated with cirrhotic liver disease. We propose a crucial role of WISP1 in balancing pro- and anti-tumorigenic effects during premalignant stages of CLD., Competing Interests: Declaration of competing interest NMB, AD, SD, TF, MPE, RL, KB, JGH, DG, SR, MT, AP, II, SH, SA, KS, CT, TC, OW, EB, NR, ER, TSW and PG have no conflicts of interest to declare. FF has received travel support from Ipsen, Abbvie, Astrazeneca and speaker's fees from AbbVie, MSD, Ipsen, Astra. MSM has served as a consultant or received speaker's honorary from ThermoFisher, Merck, GlaxoSmithKline, Roche, Incyte and Novartis. LQ was an employee of Thermo Fisher Scientific at the time of submission of the manuscript, but Thermo Fisher Scientific was not involved in and had no influence on the actual study., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background & Aims: Metabolic and transcriptional programs respond to extracellular matrix-derived cues in complex environments, such as the tumor microenvironment. Here, we demonstrate how lysyl oxidase (LOX), a known factor in collagen crosslinking, contributes to the development and progression of cholangiocarcinoma (CCA)., Methods: Transcriptomes of 209 human CCA tumors, 143 surrounding tissues, and single-cell data from 30 patients were analyzed. The recombinant protein and a small molecule inhibitor of the LOX activity were used on primary patient-derived CCA cultures to establish the role of LOX in migration, proliferation, colony formation, metabolic fitness, and the LOX interactome. The oncogenic role of LOX was further investigated by RNAscope and in vivo using the AKT/NICD genetically engineered murine CCA model., Results: We traced LOX expression to hepatic stellate cells and specifically hepatic stellate cell-derived inflammatory cancer-associated fibroblasts and found that cancer-associated fibroblast-driven LOX increases oxidative phosphorylation and metabolic fitness of CCA, and regulates mitochondrial function through transcription factor A, mitochondrial. Inhibiting LOX activity in vivo impedes CCA development and progression. Our work highlights that LOX alters tumor microenvironment-directed transcriptional reprogramming of CCA cells by facilitating the expression of the oxidative phosphorylation pathway and by increasing stemness and mobility., Conclusions: Increased LOX is driven by stromal inflammatory cancer-associated fibroblasts and correlates with diminished survival of patients with CCA. Modulating the LOX activity can serve as a novel tumor microenvironment-directed therapeutic strategy in bile duct pathologies., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background & Aims: Liver injury after COVID-19 vaccination is very rare and shows clinical and histomorphological similarities with autoimmune hepatitis (AIH). Little is known about the pathophysiology of COVID-19 vaccine-induced liver injury (VILI) and its relationship to AIH. Therefore, we compared VILI with AIH., Methods: Formalin-fixed and paraffin-embedded liver biopsy samples from patients with VILI (n = 6) and from patients with an initial diagnosis of AIH (n = 9) were included. Both cohorts were compared by histomorphological evaluation, whole-transcriptome and spatial transcriptome sequencing, multiplex immunofluorescence, and immune repertoire sequencing., Results: Histomorphology was similar in both cohorts but showed more pronounced centrilobular necrosis in VILI. Gene expression profiling showed that mitochondrial metabolism and oxidative stress-related pathways were more and interferon response pathways were less enriched in VILI. Multiplex analysis revealed that inflammation in VILI was dominated by CD8 + effector T cells, similar to drug-induced autoimmune-like hepatitis. In contrast, AIH showed a dominance of CD4 + effector T cells and CD79a + B and plasma cells. T-cell receptor (TCR) and B-cell receptor sequencing showed that T and B cell clones were more dominant in VILI than in AIH. In addition, many T cell clones detected in the liver were also found in the blood. Interestingly, analysis of TCR beta chain and Ig heavy chain variable-joining gene usage further showed that TRBV6-1, TRBV5-1, TRBV7-6, and IgHV1-24 genes are used differently in VILI than in AIH., Conclusions: Our analyses support that SARS-CoV-2 VILI is related to AIH but also shows distinct differences from AIH in histomorphology, pathway activation, cellular immune infiltrates, and TCR usage. Therefore, VILI may be a separate entity, which is distinct from AIH and more closely related to drug-induced autoimmune-like hepatitis., Impact and Implications: Little is known about the pathophysiology of COVID-19 vaccine-induced liver injury (VILI). Our analysis shows that COVID-19 VILI shares some similarities with autoimmune hepatitis, but also has distinct differences such as increased activation of metabolic pathways, a more prominent CD8+ T cell infiltrate, and an oligoclonal T and B cell response. Our findings suggest that VILI is a distinct disease entity. Therefore, there is a good chance that many patients with COVID-19 VILI will recover completely and will not develop long-term autoimmune hepatitis., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: Hepatocellular adenoma (HCA) is a rare liver tumour, which can have atypical morphological features such as cytological atypia, pseudoglandular architecture, and altered reticulin framework. Little is known about the genetic and epigenetic alterations of such HCAs and whether they show the alterations classically found in hepatocellular carcinoma (HCC) or in HCA without atypical morphology., Methods: We analysed five HCAs with atypical morphological features and one HCA with transition to HCC. Every tumour was subtyped by immunohistochemistry, sequenced by a targeted NGS panel, and analysed on a DNA methylation microarray., Results: Subtyping of the five HCAs with atypical features revealed two β-catenin mutated HCA (b-HCA), two β-catenin mutated inflammatory HCA (b-IHCA), and one sonic hedgehog activated HCA (shHCA). None of them showed mutations typically found in HCC, such as, e.g. TERT or TP53 mutations. The epigenomic pattern of HCAs with atypical morphological features clustered with reference data for HCAs without atypical morphological features but not with HCC. Similarly, phyloepigenetic trees using the DNA methylation data reproducibly showed that HCAs with morphological atypia are much more similar to nonmalignant samples than to malignant samples. Finally, atypical HCAs showed no relevant copy number variations (CNV)., Conclusion: In our series, mutational, DNA methylation, as well as CNV analyses, supported a relationship of atypical HCAs with nonatypical HCAs rather than with HCC. Therefore, in cases with difficult differential diagnosis between HCC and HCA, it might be advisable to perform targeted sequencing and/or combined methylation/copy number profiling., (© 2022 The Authors. Histopathology published by John Wiley & Sons Ltd.)

The ability to align individual cellular information from multiple experimental sources is fundamental for a systems-level understanding of biological processes. However, currently available tools are mainly designed for single-cell transcriptomics matching and integration, and generally rely on a large number of shared features across datasets for cell matching. This approach underperforms when applied to single-cell proteomic datasets due to the limited number of parameters simultaneously accessed and lack of shared markers across these experiments. Here, we introduce a cell-matching algorithm, matching with partial overlap (MARIO) that accounts for both shared and distinct features, while consisting of vital filtering steps to avoid suboptimal matching. MARIO accurately matches and integrates data from different single-cell proteomic and multimodal methods, including spatial techniques and has cross-species capabilities. MARIO robustly matched tissue macrophages identified from COVID-19 lung autopsies via codetection by indexing imaging to macrophages recovered from COVID-19 bronchoalveolar lavage fluid by cellular indexing of transcriptomes and epitopes by sequencing, revealing unique immune responses within the lung microenvironment of patients with COVID., (© 2023. The Author(s).)

Background & Aims: Cholangiocarcinoma (CCA) is a heterogeneous and lethal malignancy, the molecular origins of which remain poorly understood. MicroRNAs (miRs) target diverse signalling pathways, functioning as potent epigenetic regulators of transcriptional output. We aimed to characterise miRNome dysregulation in CCA, including its impact on transcriptome homeostasis and cell behaviour., Methods: Small RNA sequencing was performed on 119 resected CCAs, 63 surrounding liver tissues, and 22 normal livers. High-throughput miR mimic screens were performed in three primary human cholangiocyte cultures. Integration of patient transcriptomes and miRseq together with miR screening data identified an oncogenic miR for characterization. MiR-mRNA interactions were investigated by a luciferase assay. MiR-CRISPR knockout cells were generated and phenotypically characterized in vitro (proliferation, migration, colony, mitochondrial function, glycolysis) and in vivo using subcutaneous xenografts., Results: In total, 13% (140/1,049) of detected miRs were differentially expressed between CCA and surrounding liver tissues, including 135 that were upregulated in tumours. CCA tissues were characterised by higher miRNome heterogeneity and miR biogenesis pathway expression. Unsupervised hierarchical clustering of tumour miRNomes identified three subgroups, including distal CCA-enriched and IDH1 mutant-enriched subgroups. High-throughput screening of miR mimics uncovered 71 miRs that consistently increased proliferation of three primary cholangiocyte models and were upregulated in CCA tissues regardless of anatomical location, among which only miR-27a-3p had consistently increased expression and activity in several cohorts. FoxO signalling was predominantly downregulated by miR-27a-3p in CCA, partially through targeting of FOXO1. MiR-27a knockout increased FOXO1 levels in vitro and in vivo, impeding tumour behaviour and growth., Conclusions: The miRNomes of CCA tissues are highly remodelled, impacting transcriptome homeostasis in part through regulation of transcription factors like FOXO1. MiR-27a-3p arises as an oncogenic vulnerability in CCA., Impact and Implications: Cholangiocarcinogenesis entails extensive cellular reprogramming driven by genetic and non-genetic alterations, but the functional roles of these non-genetic events remain poorly understood. By unveiling global miRNA upregulation in patient tumours and their functional ability to increase proliferation of cholangiocytes, these small non-coding RNAs are implicated as critical non-genetic alterations promoting biliary tumour initiation. These findings identify possible mechanisms for transcriptome rewiring during transformation, with potential implications for patient stratification., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

Rationale: Coronavirus disease 2019 (COVID-19) can lead to acute respiratory distress syndrome with fatal outcomes. Evidence suggests that dysregulated immune responses, including autoimmunity, are key pathogenic factors. Objectives: To assess whether IgA autoantibodies target lung-specific proteins and contribute to disease severity. Methods: We collected 147 blood, 9 lung tissue, and 36 BAL fluid samples from three tertiary hospitals in Switzerland and one in Germany. Severe COVID-19 was defined by the need to administer oxygen. We investigated the presence of IgA autoantibodies and their effects on pulmonary surfactant in COVID-19 using the following methods: immunofluorescence on tissue samples, immunoprecipitations followed by mass spectrometry on BAL fluid samples, enzyme-linked immunosorbent assays on blood samples, and surface tension measurements with medical surfactant. Measurements and Main Results: IgA autoantibodies targeting pulmonary surfactant proteins B and C were elevated in patients with severe COVID-19 but not in patients with influenza or bacterial pneumonia. Notably, pulmonary surfactant failed to reduce surface tension after incubation with either plasma or purified IgA from patients with severe COVID-19. Conclusions: Our data suggest that patients with severe COVID-19 harbor IgA autoantibodies against pulmonary surfactant proteins B and C and that these autoantibodies block the function of lung surfactant, potentially contributing to alveolar collapse and poor oxygenation.

Introduction: Colorectal carcinoma (CRC) is among the most common carcinomas in women and men. In the advanced stage, patients are treated based on the RAS status. Recent studies indicate that in the future, in addition to KRAS and NRAS, alterations in other genes, such as PIK3CA or TP53, will be considered for therapy. Therefore, it is important to know the mutational landscape of routinely diagnosed CRC., Method: We report the molecular profile of 512 Swiss CRC patients analyzed by targeted next-generation sequencing as part of routine diagnostics at our institute., Results: Pathogenic and likely pathogenic variants were found in 462 (90%) CRC patients. Variants were detected in TP53 (54.3%), KRAS (48.2%), PIK3CA (15.6%), BRAF (13.5%), SMAD4 (10.5%), FBXW7 (7.8%), NRAS (3.5%), PTEN (2.7%), ERBB2 (1.6%), AKT1 (1.5%), and CTNNB1 (0.9%). The remaining pathogenic alterations were found in the genes ATM(n= 1), MAP2K1(n= 1), and IDH2(n= 1)., Discussion/conclusions: Our analysis revealed the prevalence of potential predictive markers in a large cohort of CRC patients obtained during routine diagnostic analysis. Furthermore, our study is the first of this size to uncover the molecular landscape of CRC in Switzerland., (© 2022 The Author(s). Published by S. Karger AG, Basel.)

Obesity, characterized by chronic low-grade inflammation of the adipose tissue, is associated with adverse coronavirus disease 2019 (COVID-19) outcomes, yet the underlying mechanism is unknown. To explore whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection of adipose tissue contributes to pathogenesis, we evaluated COVID-19 autopsy cases and deeply profiled the response of adipose tissue to SARS-CoV-2 infection in vitro. In COVID-19 autopsy cases, we identified SARS-CoV-2 RNA in adipocytes with an associated inflammatory infiltrate. We identified two distinct cellular targets of infection: adipocytes and a subset of inflammatory adipose tissue-resident macrophages. Mature adipocytes were permissive to SARS-CoV-2 infection; although macrophages were abortively infected, SARS-CoV-2 initiated inflammatory responses within both the infected macrophages and bystander preadipocytes. These data suggest that SARS-CoV-2 infection of adipose tissue could contribute to COVID-19 severity through replication of virus within adipocytes and through induction of local and systemic inflammation driven by infection of adipose tissue-resident macrophages.

Growing evidence links COVID-19 with acute and long-term neurological dysfunction. However, the pathophysiological mechanisms resulting in central nervous system involvement remain unclear, posing both diagnostic and therapeutic challenges. Here we show outcomes of a cross-sectional clinical study (NCT04472013) including clinical and imaging data and corresponding multidimensional characterization of immune mediators in the cerebrospinal fluid (CSF) and plasma of patients belonging to different Neuro-COVID severity classes. The most prominent signs of severe Neuro-COVID are blood-brain barrier (BBB) impairment, elevated microglia activation markers and a polyclonal B cell response targeting self-antigens and non-self-antigens. COVID-19 patients show decreased regional brain volumes associating with specific CSF parameters, however, COVID-19 patients characterized by plasma cytokine storm are presenting with a non-inflammatory CSF profile. Post-acute COVID-19 syndrome strongly associates with a distinctive set of CSF and plasma mediators. Collectively, we identify several potentially actionable targets to prevent or intervene with the neurological consequences of SARS-CoV-2 infection., (© 2022. The Author(s).)

Next-generation sequencing (NGS) of tumor cell-derived DNA/RNA to screen for targetable genomic alterations is now widely available and has become part of routine practice in oncology. NGS testing strategies depend on cancer type, disease stage and the impact of results on treatment selection. The European Society for Medical Oncology (ESMO) has recently published recommendations for the use of NGS in patients with advanced cancer. We complement the ESMO recommendations with a practical review of how oncologists should read and interpret NGS reports. A concise and straightforward NGS report contains details of the tumor sample, the technology used and highlights not only the most important and potentially actionable results, but also other pathogenic alterations detected. Variants of unknown significance should also be listed. Interpretation of NGS reports should be a joint effort between molecular pathologists, tumor biologists and clinicians. Rather than relying and acting on the information provided by the NGS report, oncologists need to obtain a basic level of understanding to read and interpret NGS results. Comprehensive annotated databases are available for clinicians to review the information detailed in the NGS report. Molecular tumor boards do not only stimulate debate and exchange, but may also help to interpret challenging reports and to ensure continuing medical education., Competing Interests: Disclosure SS: grants: Swiss National Cancer Foundation (fellowship), Fill the Gap (Career development grant), Von Tobel Foundation (research funding), Bristol Myers Squibb (BMS) (research funding), AstraZeneca (research funding), Janssen (research funding); consulting fees/advisory boards (all institutional): BMS, Merck Sharp & Dohme (MSD), Boehringer Ingelheim, AstraZeneca. Travel support: Takeda, MSD, Boehringer-Ingelheim. WJ: grants: AstraZeneca; honoraria: Bayer, Janssen; consulting fees: GlaxoSmithKline (GSK); advisory board: GSK, Janssen, MSD. CB: advisory board: AstraZeneca, Pfizer, Roche, Takeda, Boehringer Ingelheim, Janssen; travel support: AstraZeneca, Takeda. MM: Swiss National Science Foundation grant; consulting fees, payment or honoraria for lectures etc. (all institutional): Merck, GlaxoSmithKline, Roche, Novartis, ThermoFisher. SR: grants (all research funding): AstraZeneca, Merck, Roche, Amgen; consulting fees/payment or honoraria for lectures, expert testimony etc. (all institutional): Amgen, AstraZeneca, Bayer, BMS, Boehringer-Ingelheim, Eli Lilly, Janssen, Merck, MSD, Novartis, Otsuka, Pfizer, PharmaMar, Roche Pharma and Roche Diagnostics, Sanofi, Takeda; travel support (institutional): Roche, Eli Lilly, BMS, Amgen, AstraZeneca, MSD; participation on data safety monitoring board or Advisory board; Roche (institutional); other: vice president of SAKK; elected member of the Swiss Federal Drug Commission. AO: advisory role (compensated, institutional): AstraZeneca, Astellas, Bayer, Janssen, Molecular Partners, MSD, Pfizer, Roche, Sanofi Aventis (compensated, institutional) Novartis, Janssen, Bayer, MSD, AstraZeneca (compensated); research support (institutional): TEVA, Janssen; travel support: Astellas, Bayer, Janssen, Sanofi Aventis; speakers bureau (compensated, institutional): Astellas, Bayer, Janssen. MJ: advisory role (institutional): Novartis, AstraZeneca, Basilea Pharmaceutica, Bayer, BMS, Debiopharm, MSD, Roche, Sanofi; research funding: Swiss Cancer Research; travel grants: Roche, Sanofi, Takeda. All other authors have declared no conflicts of interest., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: Hepatocellular carcinoma with neuroendocrine differentiation (HCC-NED) is a very rare subtype of primary liver cancer. Treatment allocation in these patients therefore remains a challenge., Methods: We report the case of a 74-year-old man with a HCC-NED. The tumor was surgically removed in curative intent. Histopathological work-up revealed poorly differentiated hepatocellular carcinoma (Edmondson-Steiner grade IV) with diffuse expression of neuroendocrine markers synaptophysin and chromogranin. Three months after resection, multifocal recurrence of the HCC-NED was observed. In the meantime, tumor organoids have been generated from the resected HCC-NED and extensively characterized. Sensitivity to a number of drugs approved for the treatment of HCC or neuroendocrine carcinomas was tested in vitro., Results: Based on the results of the in vitro drug screening, etoposide and carboplatin are used as first line palliative combination treatment. With genomic analysis revealing a NTRK1 -mutation of unknown significance (kinase domain) and tumor organoids found to be sensitive to entrectinib, a pan-TRK inhibitor, the patient was treated with entrectinib as second line therapy. After only two weeks, treatment is discontinued due to deterioration of the patient's general condition., Conclusion: The rapid establishment of patient-derived tumor organoids allows in vitro drug testing and thereby personalized treatment choices, however clinical translation remains a challenge. To the best of our knowledge, this report provides a first proof-of-principle for using organoids for personalized medicine in this rare subtype of primary liver cancer., Competing Interests: Competing interestsThe authors declare no competing interest., (© The Author(s) 2022.)

Understanding the mechanisms of HIV tissue persistence necessitates the ability to visualize tissue microenvironments where infected cells reside; however, technological barriers limit our ability to dissect the cellular components of these HIV reservoirs. Here, we developed protein and nucleic acid in situ imaging (PANINI) to simultaneously quantify DNA, RNA, and protein levels within these tissue compartments. By coupling PANINI with multiplexed ion beam imaging (MIBI), we measured over 30 parameters simultaneously across archival lymphoid tissues from healthy or simian immunodeficiency virus (SIV)-infected nonhuman primates. PANINI enabled the spatial dissection of cellular phenotypes, functional markers, and viral events resulting from infection. SIV infection induced IL-10 expression in lymphoid B cells, which correlated with local macrophage M2 polarization. This highlights a potential viral mechanism for conditioning an immunosuppressive tissue environment for virion production. The spatial multimodal framework here can be extended to decipher tissue responses in other infectious diseases and tumor biology., Competing Interests: Declaration of interests G.P.N. and M.A. are cofounders and stockholders of Ionpath Inc., which manufactures the instrument used in this manuscript. G.P.N. and Y.G. are cofounders and stockholders of Akoya Biosciences, Inc. and inventors on patent US9909167. S.J.R. is a Scientific Advisory Board member for KITE/Gilead and Immunitas Therapeutics. S.J., X.R.-C., Y.B., and G.P.N. are inventors on pending patent US17433542 related in part to this work. The other authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Chronic liver inflammation causes continuous liver damage with progressive liver fibrosis and cirrhosis, which may eventually lead to hepatocellular carcinoma (HCC). Whereas the 10-year incidence for HCC in patients with cirrhosis is approximately 20%, many of these patients remain tumor free for their entire lives. Clarifying the mechanisms that define the various outcomes of chronic liver inflammation is a key aspect in HCC research. In addition to a wide variety of contributing factors, microRNAs (miRNAs) have also been shown to be engaged in promoting liver cancer. Therefore, we wanted to characterize miRNAs that are involved in the development of HCC, and we designed a longitudinal study with formalin-fixed and paraffin-embedded liver biopsy samples from several pathology institutes from Switzerland. We examined the miRNA expression by nCounterNanostring technology in matched nontumoral liver tissue from patients developing HCC (n = 23) before and after HCC formation in the same patient. Patients with cirrhosis (n = 26) remaining tumor free within a similar time frame served as a control cohort. Comparison of the two cohorts revealed that liver tissue from patients developing HCC displayed a down-regulation of miR-579-3p as an early step in HCC development, which was further confirmed in a validation cohort. Correlation with messenger RNA expression profiles further revealed that miR-579-3p directly attenuated phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) expression and consequently protein kinase B (AKT) and phosphorylated AKT. In vitro experiments and the use of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology confirmed that miR-579-3p controlled cell proliferation and cell migration of liver cancer cell lines. Conclusion: Liver tissues from patients developing HCC revealed changes in miRNA expression. miR-579-3p was identified as a novel tumor suppressor regulating phosphoinositide 3-kinase-AKT signaling at the early stages of HCC development., (© 2022 The Authors. Hepatology Communications published by Wiley Periodicals LLC on behalf of American Association for the Study of Liver Diseases.)

Proteogenomic analyses of hepatocellular carcinomas (HCC) have focused on early-stage, HBV-associated HCCs. Here we present an integrated proteogenomic analysis of HCCs across clinical stages and etiologies. Pathways related to cell cycle, transcriptional and translational control, signaling transduction, and metabolism are dysregulated and differentially regulated on the genomic, transcriptomic, proteomic and phosphoproteomic levels. We describe candidate copy number-driven driver genes involved in epithelial-to-mesenchymal transition, the Wnt-β-catenin, AKT/mTOR and Notch pathways, cell cycle and DNA damage regulation. The targetable aurora kinase A and CDKs are upregulated. CTNNB1 and TP53 mutations are associated with altered protein phosphorylation related to actin filament organization and lipid metabolism, respectively. Integrative proteogenomic clusters show that HCC constitutes heterogeneous subgroups with distinct regulation of biological processes, metabolic reprogramming and kinase activation. Our study provides a comprehensive overview of the proteomic and phophoproteomic landscapes of HCCs, revealing the major pathways altered in the (phospho)proteome., (© 2022. The Author(s).)

Background: NUT carcinoma is a highly aggressive and rare subset of squamous cell carcinoma with grim prognosis. It is under-recognized by both pathologists and oncologists. Recognition is challenging due to its rareness and the fact that its clinical and laboratory features as well as morphological and immunohistochemical characteristics may mimic other malignancies. Case presentation: An interesting case of NUT carcinoma in a 47-year-old male with a large tumor mass in the inferior part of the mediastinum and left lung and increased levels of serum alpha fetoprotein (AFP) is described. Immunohistochemical analysis of both the primary tumor in a bronchoscopy specimen and an excisional biopsy of a subcutaneous metastasis showed positivity for AFP and leukocyte common antigen (LCA) that were misleading and resulted in diagnostic pitfalls of mediastinal germ cell tumor (clinically) and hematolymphoid neoplasm (pathologic report). Immunohistochemical demonstration of NUT protein expression revealed the proper diagnosis, which was further confirmed by RNA sequencing revealing a BRD4- NUTM1 gene fusion. Conclusions: Since NUT carcinoma can show a wide spectrum of histological and immunophenotypic features and can clinically mimic other tumors, use of RNA sequencing with identification of specific NUTM1 fusion partner could be crucial when there are discrepant clinical and histopathological findings. As well, since the category of so-called NUTM1 -rearranged neoplasms is rapidly expanding, identification of NUTM1 fusion partner may be essential for the appropriate clinical management.

Background: Focal nodular hyperplasia (FNH) is typically considered a benign tumor of the liver without malignant potential. The co-occurrence of FNH and hepatocellular carcinoma (HCC) has been reported in rare cases. In this study we sought to investigate the clonal relationship between these lesions in a patient with FNH-HCC co-occurrence., Methods: A 74-year-old female patient underwent liver tumor resection. The resected nodule was subjected to histologic analyses using hematoxylin and eosin stain and immunohistochemistry. DNA extracted from microdissected FNH and HCC regions was subjected to whole exome sequencing. Clonality analysis were performed using PyClone., Results: Histologic analysis reveals that the nodule consists of an FNH and two adjoining HCC components with distinct histopathological features. Immunophenotypic characterization and genomic analyses suggest that the FNH is clonally related to the HCC components, and is composed of multiple clones at diagnosis, that are likely to have progressed to HCC through clonal selection and/or the acquisition of additional genetic events., Conclusion: To the best of our knowledge, our work is the first study showing a clonal relationship between FNH and HCC. We show that FNH may possess the capability to undergo malignant transformation and to progress to HCC in very rare cases., Competing Interests: Competing interestsM.S.M. has received speaker’s honoraria from Thermo Fisher and honoraria as an advisory board member from Novartis. The other authors declare no competing interests., (© The Author(s) 2022.)

Although initial immunophenotypical studies on peripheral blood and bronchoalveolar lavage samples have provided a glimpse into the immunopathology of COVID-19, analyses of pulmonary draining lymph nodes are currently scarce. 22 lethal COVID-19 cases and 28 controls were enrolled in this study. Pulmonary draining lymph nodes (mediastinal, tracheal, peribronchial) were collected at autopsy. Control lymph nodes were selected from a range of histomorphological sequelae [unremarkable histology, infectious mononucleosis, follicular hyperplasia, non-SARS related HLH, extrafollicular plasmablast activation, non-SARS related diffuse alveolar damage (DAD), pneumonia]. Samples were mounted on a tissue microarray and underwent immunohistochemical staining for a selection of immunological markers and in-situ hybridization for Epstein Barr Virus (EBV) and SARS-CoV-2. Gene expression profiling was performed using the HTG EdgeSeq Immune Response Panel. Characteristic patterns of a dysregulated immune response were detected in COVID-19: 1. An accumulation of extrafollicular plasmablasts with a relative paucity or depletion of germinal centers. 2. Evidence of T-cell dysregulation demonstrated by immunohistochemical paucity of FOXP3+, Tbet+ and LEF1+ positive T-cells and a downregulation of key genes responsible for T-cell crosstalk, maturation and migration as well as a reactivation of herpes viruses in 6 COVID-19 lymph nodes (EBV, HSV). 3. Macrophage activation by a M2-polarized, CD163+ phenotype and increased incidence of hemophagocytic activity. 4. Microvascular dysfunction, evidenced by an upregulation of hemostatic (CD36, PROCR, VWF) and proangiogenic (FLT1, TEK) genes and an increase of fibrin microthrombi and CD105+ microvessels. Taken together, these findings imply widespread dysregulation of both innate and adoptive pathways with concordant microvascular dysfunction in severe COVID-19., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Haslbauer, Zinner, Stalder, Schneeberger, Menter, Bassetti, Mertz, Went, Matter and Tzankov.)

Background: A dysregulated immune response is considered one of the major factors leading to severe COVID-19. Previously described mechanisms include the development of a cytokine storm, missing immunoglobulin class switch, antibody-mediated enhancement, and aberrant antigen presentation., Objectives: To understand the heterogeneity of immune response in COVID-19, a thorough investigation of histomorphological patterns in regional lymph nodes was performed., Materials and Methods: Lymph nodes from the cervical, mediastinal, and hilar regions were extracted from autopsies of patients with lethal COVID-19 (n = 20). Histomorphological characteristics, SARS-CoV‑2 qRT-PCR, and gene expression profiling on common genes involved in immunologic response were analyzed., Results: Lymph nodes displayed moderate to severe capillary stasis and edema, an increased presence of extrafollicular plasmablasts, mild to moderate plasmacytosis, a dominant population of CD8 + T‑cells, and CD11c/CD68 + histiocytosis with hemophagocytic activity. Out of 20 cases, 18 presented with hypoplastic or missing germinal centers with a decrease of follicular dendritic cells and follicular T‑helper cells. A positive viral load was detected by qRT-PCR in 14 of 20 cases, yet immunohistochemistry for SARS-CoV-2 N-antigen revealed positivity in sinus histiocytes of only one case. Gene expression analysis revealed an increased expression of STAT1, CD163, granzyme B, CD8A, MZB1, and PAK1, as well as CXCL9., Conclusions: Taken together, our findings imply a dysregulated immune response in lethal COVID-19. The absence/hypoplasia of germinal centers and increased presence of plasmablasts implies a transient B‑cell response, implying an impaired development of long-term immunity against SARS-CoV‑2 in such occasions., (© 2021. The Author(s).)

Understanding viral tropism is an essential step toward reducing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission, decreasing mortality from coronavirus disease 2019 (COVID-19) and limiting opportunities for mutant strains to arise. Currently, little is known about the extent to which distinct tissue sites in the human head and neck region and proximal respiratory tract selectively permit SARS-CoV-2 infection and replication. In this translational study, we discover key variabilities in expression of angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2), essential SARS-CoV-2 entry factors, among the mucosal tissues of the human proximal airways. We show that SARS-CoV-2 infection is present in all examined head and neck tissues, with a notable tropism for the nasal cavity and tracheal mucosa. Finally, we uncover an association between smoking and higher SARS-CoV-2 viral infection in the human proximal airway, which may explain the increased susceptibility of smokers to developing severe COVID-19. This is at least partially explained by differences in interferon (IFN)-β1 levels between smokers and non-smokers., Competing Interests: I.T.L. is currently an employee and shareholder of Moderna, although this work was conducted prior to/independent of his employment. I.T.L. had also received research support unrelated to this study from Genentech (Roche). Moderna did not fund or participate in this study in any form., (© 2021.)

Background: multisystem inflammatory syndrome in children (MIS-C) is a new disease associated with a recent infection with severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2). Affected children can present predominantly with abdominal symptoms, fever and high inflammatory parameters that might lead to a consult by the pediatric surgeon and an indication for surgery., Methods: clinical data of three patients with MIS-C that underwent surgery were collected. Histopathological analysis of the appendix was performed., Results: we present the clinical course of three children with fever, abdominal pain and vomiting for several days. Clinical examination and highly elevated inflammation markers led to indication for laparoscopy; appendectomy was performed in two patients. Because of intraoperative findings or due to lack of postoperative improvement, all patients were reevaluated and tested positive for MIS-C associated laboratory parameters and were subsequently treated with corticosteroids, intravenous immunoglobulins, acetyl salicylic acid and/or light molecular weight heparin., Conclusions: we discuss the implications of MIS-C as a new differential diagnosis and stress the importance of assessing the previous medical history, identifying patterns of symptoms and critically surveilling the clinical course. We implemented an algorithm for pediatric surgeons to consider MIS-C as a differential diagnosis for acute abdomen that can be integrated into the surgical workflow.

Background: Genetic aberrations in hepatocellular carcinoma (HCC) are well known, but the functional consequences of such aberrations remain poorly understood., Results: Here, we explored the effect of defined genetic changes on the transcriptome, proteome and phosphoproteome in twelve tumors from an mTOR-driven hepatocellular carcinoma mouse model. Using Network-based Integration of multi-omiCS data (NetICS), we detected 74 'mediators' that relay via molecular interactions the effects of genetic and miRNA expression changes. The detected mediators account for the effects of oncogenic mTOR signaling on the transcriptome, proteome and phosphoproteome. We confirmed the dysregulation of the mediators YAP1, GRB2, SIRT1, HDAC4 and LIS1 in human HCC., Conclusions: This study suggests that targeting pathways such as YAP1 or GRB2 signaling and pathways regulating global histone acetylation could be beneficial in treating HCC with hyperactive mTOR signaling., (© 2021. The Author(s).)

Emerging evidence points toward an intricate relationship between the pandemic of coronavirus disease 2019 (COVID-19) and diabetes. While preexisting diabetes is associated with severe COVID-19, it is unclear whether COVID-19 severity is a cause or consequence of diabetes. To mechanistically link COVID-19 to diabetes, we tested whether insulin-producing pancreatic β cells can be infected by SARS-CoV-2 and cause β cell depletion. We found that the SARS-CoV-2 receptor, ACE2, and related entry factors (TMPRSS2, NRP1, and TRFC) are expressed in β cells, with selectively high expression of NRP1. We discovered that SARS-CoV-2 infects human pancreatic β cells in patients who succumbed to COVID-19 and selectively infects human islet β cells in vitro. We demonstrated that SARS-CoV-2 infection attenuates pancreatic insulin levels and secretion and induces β cell apoptosis, each rescued by NRP1 inhibition. Phosphoproteomic pathway analysis of infected islets indicates apoptotic β cell signaling, similar to that observed in type 1 diabetes (T1D). In summary, our study shows SARS-CoV-2 can directly induce β cell killing., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

An excess formation of neutrophil extracellular traps (NETs), previously shown to be strongly associated with cytokine storm and acute respiratory distress syndrome (ARDS) with prevalent endothelial dysfunction and thrombosis, has been postulated to be a central factor influencing the pathophysiology and clinical presentation of severe COVID-19. A growing number of serological and morphological evidence has added to this assumption, also in regard to potential treatment options. In this study, we used immunohistochemistry and histochemistry to trace NETs and their molecular markers in autopsy lung tissue from seven COVID-19 patients. Quantification of key immunomorphological features enabled comparison with non-COVID-19 diffuse alveolar damage. Our results strengthen and extend recent findings, confirming that NETs are abundantly present in seriously damaged COVID-19 lung tissue, especially in association with microthrombi of the alveolar capillaries. In addition, we provide evidence that low-density neutrophils (LDNs), which are especially prone to NETosis, contribute substantially to COVID-19-associated lung damage in general and vascular blockages in particular., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this article., (Copyright © 2021 Astrid Obermayer et al.)

Rationale: Adenylosuccinate lyase (ADSL) is an essential enzyme for de novo purine biosynthesis. Here we sought to investigate the putative role of ADSL in colorectal carcinoma (CRC) carcinogenesis and response to antimetabolites. Methods: ADSL expression levels were assessed by immunohistochemistry or retrieved from The Cancer Genome Atlas (TCGA) dataset. The effects of ADSL silencing or overexpression were evaluated on CRC cell proliferation, cell migration and cell-cycle. In vivo tumor growth was assessed by the chicken chorioallantoic membrane (CAM). Transfected cell lines or patient-derived organoids (PDO) were treated with 5-fluorouracil (5-FU) and 6-mercaptopurine (6-MP) and drug response was correlated with ADSL expression levels. Metabolomic and transcriptomic profiling were performed to identify dysregulated pathways and ADSL downstream effectors. Mitochondrial respiration and glycolytic capacity were measured using Seahorse; mitochondrial membrane potential and the accumulation of ROS were measured by FACS using MitoTracker Red and MitoSOX staining, respectively. Activation of canonical pathways was assessed by immunohistochemistry and immunoblotting. Results: ADSL expression is significantly increased in CRC tumors compared to non-tumor tissue. ADSL-high CRCs show upregulation of genes involved in DNA synthesis, DNA repair and cell cycle. Accordingly, ADSL overexpression accelerated progression through the cell cycle and significantly increased proliferation and migration in CRC cell lines. Additionally, ADSL expression increased tumor growth in vivo and sensitized CRCs to 6-MP in vitro , ex vivo (PDOs) and in vivo (CAM model). ADSL exerts its oncogenic function by affecting mitochondrial function via alteration of the TCA cycle and impairment of mitochondrial respiration. The KEAP1-NRF2 and mTORC1-cMyc axis are independently activated upon ADSL overexpression and may favor the survival and proliferation of ROS-accumulating cells, favoring DNA damage and tumorigenesis. Conclusions: Our results suggest that ADSL is a novel oncogene in CRC, modulating mitochondrial function, metabolism and oxidative stress, thus promoting cell cycle progression, proliferation and migration. Our results also suggest that ADSL is a predictive biomarker of response to 6-mercaptopurine in the pre-clinical setting., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Transcriptional enhancer factor domain family member 4 (TEAD4) is a downstream effector of the conserved Hippo signaling pathway, regulating the expression of genes involved in cell proliferation and differentiation. It is up-regulated in several cancer types and is associated with metastasis and poor prognosis. However, its role in hepatocellular carcinoma (HCC) remains largely unexplored. Using data from The Cancer Genome Atlas, we found that TEAD4 was overexpressed in HCC and was associated with aggressive HCC features and worse outcome. Overexpression of TEAD4 significantly increased proliferation and migration rates in HCC cells in vitro as well as tumor growth in vivo . Additionally, RNA sequencing analysis of TEAD4 -overexpressing HCC cells demonstrated that TEAD4 overexpression was associated with the up-regulation of genes involved in epithelial-to-mesenchymal transition, proliferation, and protein-folding pathways. Among the most up-regulated genes following TEAD4 overexpression were the 70-kDa heat shock protein (HSP70) family members HSPA6 and HSPA1 A. Chromatin immunoprecipitation-quantitative real-time polymerase chain reaction experiments demonstrated that TEAD4 regulates HSPA6 and HSPA1A expression by directly binding to their promoter and enhancer regions. The pharmacologic inhibition of HSP70 expression in TEAD4 -overexpressing cells reduced the effect of TEAD4 on cell proliferation. Finally, by overexpressing TEAD4 in yes-associated protein ( YAP )/transcriptional coactivator with PDZ binding motif ( TAZ )-knockdown HCC cells, we showed that the effect of TEAD4 on cell proliferation and its regulation of HSP70 expression does not require YAP and TAZ, the main effectors of the Hippo signaling pathway. Conclusion: A novel Hippo-independent mechanism for TEAD4 promotes cell proliferation and tumor growth in HCC by directly regulating HSP70 family members., (© 2021 The Authors. Hepatology Communications published by Wiley Periodicals LLC on behalf of the American Association for the Study of Liver Diseases.)

Introduction: In December 2019, the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic broke out. The virus rapidly spread globally, resulting in a major world public-health crisis. The major disease manifestation occurs in the respiratory tract. However, further studies documented other systemic involvement. This study investigates histopathologic eye changes in postmortem material of coronavirus disease 2019 (COVID-19) patients., Methods: Sections of formalin-fixed, paraffin-embedded eyes from 5 patients (10 eyes) who died of COVID-19 at the University Hospital in Basel were included. Gross examination and histological evaluation were performed by 3 independent ophthalmopathologists. Immunohistochemical staining was performed using antibodies against fibrin, cleaved caspase 3, and ACE-2. Five enucleated eyes of patients not infected with SARS-CoV-2 served as control group. All cases have been studied for presence of SARS-CoV-2 RNA by means of reverse transcription PCR and RNA in situ hybridization (ISH). The choroidal vessels of one case were analyzed with electron microscope., Results: Ophthalmopathologically, 8 eyes from 4 patients displayed swollen endothelial cells in congested choroidal vessels. No further evidence of specific eye involvement of SARS-CoV-2 was found in any of the patients. In the 8 eyes with evidence of changes due to SARS-CoV-2, immunohistochemical staining demonstrated fibrin microthrombi, apoptotic changes of endothelial and inflammatory cells. In control eyes, ACE-2 was detectable in the conjunctiva, cornea, retina, and choroidea and displayed significantly lower amounts of stained cells as in COVID-19 eyes. SARS-CoV-2 RNA was detectable in both bulbi of 2/5 patients, yet ISH failed to visualize viruses. Electron microscopy showed no significant results due to the artifacts., Discussion/conclusion: As already described in other organs of COVID-19 patients, the ophthalmological examination revealed-microthrombi, that is, hypercoagulation and vasculopathy most probably due to endothelial damage. A possible viral spread to the endothelial cells via ACE-2 provides one pathophysiological explanation. The expression of ACE-2 receptors in the conjunctiva hints toward its susceptibility to infection. To what extend eyes, function is disrupted by SARS-CoV-2 is subject to further studies, especially in the clinic., (© 2021 The Author(s). Published by S. Karger AG, Basel.)

The discovery of actionable oncogenic driver alterations has significantly improved treatment options for patients with advanced non-small cell lung cancer (NSCLC). In lung adenocarcinoma (LUAD), approved drugs or drugs in clinical development can target more than half of these altered oncogenic driver genes. In particular, several gene fusions have been discovered in LUAD, including ALK , ROS1 , NTRK , RET , NRG1 and FGFR . All these fusions involve tyrosine kinases (TK), which are activated due to structural rearrangements on the DNA level. Although the overall prevalence of these fusions in LUAD is rare, their detection is extremely important, as they are linked to an excellent response to TK inhibitors. Therefore, reliable screening methods applicable to small tumor samples (biopsies and cytology specimens) are required in the diagnostic workup of advanced NSCLC. Several methods are at disposal in a routine laboratory to demonstrate, directly or indirectly, the presence of a gene fusion. These methods include immunohistochemistry (IHC), fluorescence in-situ hybridization (FISH), reverse transcriptase-polymerase chain reaction (RT-PCR), multiplex digital color-coded barcode technology or next-generation sequencing (NGS) either on DNA or RNA level. In our review, we will summarize the increasing number of relevant fusion genes in NSCLC, point out their underlining molecular mechanisms and discuss different methods for the detection of fusion genes., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/tlcr-20-676). The series “Looking for Chimeras in NSCLC: Widen Therapeutic Options Targeting Oncogenic Fusions” was commissioned by the editorial office without any funding or sponsorship. Dr. MSM reports personal fees from Thermo Fisher, outside the submitted work. Dr. SS reports personal fees from MSD, personal fees from Astra Zeneca, personal fees from Boehringer Ingelheim, personal fees from Roche, personal fees from Pfizer, personal fees from Thermo Fisher Scientific, outside the submitted work. Dr. LB reports grants from Sanofi, grants and personal fees from Roche, grants and personal fees from MSD, personal fees from Astra Zeneca, personal fees from Bayer, personal fees from BMS, personal fees from Boehringer Ingelheim, personal fees from Pfizer, personal fees from Takeda, outside the submitted work. The other author has no other conflicts of interest to declare., (2020 Translational Lung Cancer Research. All rights reserved.)

Objectives: Tumor mutational burden (TMB) has emerged as a promising predictive biomarker for immune checkpoint inhibitor therapy. While the feasibility of TMB analysis on formalin-fixed paraffin-embedded (FFPE) samples has been thoroughly evaluated, only limited analyses have been performed on cytological samples, and no dedicated study has investigated concordance of TMB between different sample types. Here, we assessed TMB on matched histological and cytological samples from lung cancer patients and evaluated the accuracy of TMB estimation in these sample types., Materials and Methods: We analyzed mutations and resulting TMB in FFPE samples and matched ethanol-fixed cytological smears (n = 12 matched pairs) by using a targeted next-generation sequencing assay (Oncomine™ Tumor Mutational Load). Two different variant allele frequency (VAF) thresholds were used to estimate TMB (VAF = 5% or 10%)., Results: At 5% VAF threshold, 73% (107/147) of mutations were concordantly detected in matched histological and cytological samples. Discordant variants were mainly unique to FFPE samples (34/40 discordant variants) and mostly C:G > T:A transitions with low allelic frequency, likely indicating formalin fixation artifacts. Increasing the VAF threshold to 10% clearly increased the number of concordantly detected mutations in matched histological and cytological samples to 96% (100/106 mutations), and drastically reduced the number of FFPE-only mutations (from 34 to 4 mutations). In contrast, cytological samples showed consistent mutation count and TMB values at both VAF thresholds. Using FFPE samples, 2 out of 12 patients were classified as TMB-high at VAF cutoff of 5% but TMB-low at 10%, whereas cytological specimens allowed consistent patient classification independently from VAF cutoff., Conclusion: Our results show that cytological smears provide more consistent TMB values due to high DNA quality and lack of formalin-fixation induced artifacts. Therefore, cytological samples should be the preferred sample type for robust TMB estimation., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

The coronavirus SARS-CoV-2 is the causative agent of the ongoing severe acute respiratory disease pandemic COVID-19. Tissue and cellular tropism is one key to understanding the pathogenesis of SARS-CoV-2. We investigate the expression and subcellular localization of the SARS-CoV-2 receptor, angiotensin-converting enzyme 2 (ACE2), within the upper (nasal) and lower (pulmonary) respiratory tracts of human donors using a diverse panel of banked tissues. Here, we report our discovery that the ACE2 receptor protein robustly localizes within the motile cilia of airway epithelial cells, which likely represents the initial or early subcellular site of SARS-CoV-2 viral entry during host respiratory transmission. We further determine whether ciliary ACE2 expression in the upper airway is influenced by patient demographics, clinical characteristics, comorbidities, or medication use, and show the first mechanistic evidence that the use of angiotensin-converting enzyme inhibitors (ACEI) or angiotensin II receptor blockers (ARBs) does not increase susceptibility to SARS-CoV-2 infection through enhancing the expression of ciliary ACE2 receptor. These findings are crucial to our understanding of the transmission of SARS-CoV-2 for prevention and control of this virulent pathogen.

Cholangiocarcinoma (CCA) mortality rates are increasing as a result of rising incidence and limited curative treatment(s) for patients with advanced disease. NOTCH pathway reactivation has been reported in biliary malignancies to conflicting degrees, hindering prioritization of key therapeutic targets within the network and identification of candidate responder patients for NOTCH-directed therapies. We analyzed genomic data from 341 patients with CCA and identified NOTCH1 significantly increased in a subgroup characterized by distinct stromal infiltration. Network-wide imbalance of the NOTCH pathway was seen in CCA, including correlation of NOTCH1 with NOTCH3 and NOTCH ligands. Given the diversity of observed NOTCH receptor engagement, γ-secretase modulation was rationalized as a therapeutic option. Indeed, subcutaneous transplantation of sensitive and resistant CCA cell lines pretreated with a γ-secretase inhibitor (GSi) cocktail demonstrated the antineoplastic effects of GSi in a subset of CCA and led to the development of a 225-gene responder signature. This signature was validated in an independent cohort of 119 patients. Further, this signature was enriched in liver tumors initiated by hydrodynamic injections of activated-NOTCH as compared with the AKT-RAS-driven tumors. Candidate GSi-responder patients were characterized by distinct transcriptomes overlapping with previous hepatobiliary metastasis and stemness, unique stromal properties, and dysfunctional intratumoral immune infiltration. Pan-cancer analysis identified 41.9% of cancer types to harbor prospective GSi-responder patients, which was adapted into a 20-gene GSi-sensitivity score metric capable of discriminating nanomolar versus micromolar sensitivity to a cell-permeable GSi (Z-LLNle-CHO) across 60 diverse tumor lines (area under the curve = 1). Conclusion: We have established a GSi-responder signature with evidence across several patient cohorts, as well as in vitro and in vivo models, to enable precision medicine application of NOTCH-directed therapy in CCA as well as prospectively across diverse malignancies., (© 2019 by the American Association for the Study of Liver Diseases.)

Introduction: Gastrointestinal stromal tumor (GIST) is the most common mesenchymal neoplasm of the gastrointestinal tract. It has distinct molecular features and primarily affects the KIT and PDGFRA genes., Objective: We wanted to assess the molecular profile of 68 GIST patients who were sequenced consecutively between 2014 and 2019 at our institute of pathology., Methods: Our cohort comprised 60 primary and 8 metastatic GIST patients; 43 and 57% of the cases, respectively, were analyzed by Sanger sequencing or next-generation sequencing (NGS)., Results: Of the 60 primary GIST patients, 47 (78%) showed a KIT mutation; 2 cases showed a double KIT mutation, and 1 of these was a therapy-naive GIST. Nine (15%) patients harbored a PDGFRA mutation, 2 (3%) had a BRAF mutation, 1 (2%) had a PIK3CA mutation, and 1 (2%) did not show any mutation. One BRAF and the PIK3CA mutation have not been described in GIST before. All metastatic GIST harbored exclusively KIT mutations., Conclusion: A retrospective analysis of GIST sequenced at our institute revealed incidences of KIT and PDGFRA mutations comparable to those in other cohorts from Europe. Interestingly, we found 2 previously undescribed mutations in the BRAF and PIK3CA genes as well as 1 treatment-naive case with a double KIT mutation in exon 11., (© 2020 S. Karger AG, Basel.)

Background & Aims: Liver macrophages can be involved in both pathogen clearance and/or pathogenesis. To get further insight on their role during chronic hepatitis B virus (HBV) infections, our aim was to phenotypically and functionally characterize in vivo and ex vivo the interplay between HBV, primary human liver macrophages (PLMs) and primary blood monocytes differentiated into pro-inflammatory or anti-inflammatory macrophages (M1-MDMs or M2-MDMs, respectively)., Methods: PLMs or primary blood monocytes, either ex vivo differentiated into M1-MDMs or M2-MDMs, were exposed to HBV and their activation followed by ELISA or quantitative reverse transcription PCR (RT-qPCR). Liver biopsies from HBV-infected patients were analysed by RT-qPCR or immunohistochemistry. Viral parameters in HBV-infected primary human hepatocytes and differentiated HepaRG cells were followed by ELISA, qPCR and RT-qPCR analyses., Results: HBc protein was present within the macrophages of liver biopsies taken from HBV-infected patients. Macrophages from HBV-infected patients also expressed higher levels of anti-inflammatory macrophage markers than those from non-infected patients. Ex vivo exposure of naive PLMs to HBV led to reduced secretion of pro-inflammatory cytokines. Upon exposure to HBV or HBV-producing cells during differentiation and activation, M1-MDMs secreted less IL-6 and IL-1β, whereas M2-MDMs secreted more IL-10 when exposed to HBV during activation. Finally, cytokines produced by M1-MDMs, but not those produced by HBV-exposed M1-MDMs, decreased HBV infection of hepatocytes., Conclusions: Altogether, our data strongly suggest that HBV modulates liver macrophage functions to favour the establishment of infection., Lay Summary: Hepatitis B virus modulates liver macrophage function in order to favour the establishment and likely maintenance of infection. It impairs the production of the antiviral cytokine IL-1β, while promoting that of IL-10 in the microenvironment. This phenotype can be recapitulated in naive liver macrophages or monocyte-derived-macrophages ex vivo by short exposure to the virus or cells replicating the virus, thus suggesting an "easy to implement" mechanism of inhibition., (Copyright © 2019 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Strawberry gingivitis is a rare oral manifestation of granulomatosis with polyangiitis (GPA, formerly known as Wegener's granulomatosis). It manifests as a red-purple hyperplastic gingivitis that frequently goes unrecognized as a disease-specific symptom, especially if it is the primary and only manifestation of the disease. GPA is a systemic necrotizing granulomatous vasculitis that takes a fatal course without treatment. Oral health care providers, who are among the first to examine the oral cavity, should be familiar with its typical appearance, clinical course, diagnostic parameters, and management. This article highlights the challenges to early-stage diagnosis of initial multiple gingival enlargements because histologic biopsies are often nonspecific and histology alone may not be sufficient to make a correct diagnosis of GPA from gingival specimens. Because strawberry gingivitis may be the first manifestation of GPA, awareness of it should be increased so that it can be diagnosed by its unique clinical appearance and additional related diagnostic parameters even if the histologic gingival findings are nonspecific., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Golgi phosphoprotein 2 (GOLPH2) has been shown to be involved in chronic inflammatory processes and carcinogenesis. GOLPH2 is prominently overexpressed in hepatocellular carcinoma, melanoma, glioblastoma, prostate, lung, and colorectal cancer. With a low and tightly regulated expression in non-malignant tissues, GOLPH2 has been proposed as an attractive target for cancer therapy. However, GOLPH2 is predominantly located intracellularly and when situated outside of the cell it is proteolytically cleaved and shed from the cell surface. Until now, GOLPH2 has been regarded as an "undruggable" target., Objective: We sought to create antibodies that specifically bind to GOLPH2 overexpressing tumor cells., Patients and Methods: Antibodies binding to membranous GOLPH2 despite shedding of the protein were generated from a scFV library screening. These antibodies target the part of GOLPH2 that remains at the cell surface after proteolytic cleavage. These antibodies were then tested in vitro and in vivo., Results: Two candidates (G2-1 and G2-2) showed target specific binding in vitro. Utilizing a tumor array (n = 128 tumors) with G2-2 and a reference antibody, a GOLPH2 expression scoring system was established. Rapid internalization of the antibodies was noted so this was exploited to deliver a toxic payload of pyrrolobenzodiazepine (PBD). In two patient-derived xenograft (PDX)-models, colorectal and lung cancer, the G2-2 antibody drug conjugate (ADC) displayed high efficacy with significant tumor responses (P = 0.001; P = 0.013) and improved survival (P = 0.0001; P = 0.0011) compared with controls., Conclusions: Treatment with GOLPH2-directed antibodies induces durable responses in colorectal and lung cancer models. With a robust companion assay for GOLPH2 positivity at hand our findings prepare for the translation into a clinical trial.

Current methods in molecular pathology Abstract. Macroscopy and microscopy were the cornerstones of tumor analysis in pathology for many years. Recently, we have witnessed an enormous increase in knowledge of genetic and epigenetic alterations occurring in tumors. The detection of these alterations is becoming increasingly important during pathological work-up because they have an important impact on the diagnosis, prognosis, therapy, and prevention of tumors. It is therefore crucial to have appropriate methods available to detect these genetic alterations, such as mutations, translocations or changes in the methylation profile. In the following review article, we will present current methods that are being applied in molecular pathology.

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related deaths worldwide. Treatment options for patients with advanced-stage disease are limited. A major obstacle in drug development is the lack of an in vivo model that accurately reflects the broad spectrum of human HCC. Patient-derived xenograft (PDX) tumor mouse models could overcome the limitations of cancer cell lines. PDX tumors maintain the genetic and histologic heterogeneity of the originating tumors and are used for preclinical drug development in various cancers. Controversy exists about their genetic and molecular stability through serial passaging in mice. We aimed to establish PDX models from human HCC biopsies and to characterize their histologic and molecular stability during serial passaging. A total of 54 human HCC needle biopsies that were derived from patients with various underlying liver diseases and tumor stages were transplanted subcutaneously into immunodeficient, nonobese, diabetic/severe combined immunodeficiency gamma-c mice; 11 successfully engrafted. All successfully transplanted HCCs were Edmondson grade III or IV. HCC PDX tumors retained the histopathologic, transcriptomic, and genomic characteristics of the original HCC biopsies over 6 generations of retransplantation. These characteristics included Edmondson grade, expression of tumor markers, tumor gene signature, tumor-associated mutations, and copy number alterations. Conclusion: PDX mouse models can be established from undifferentiated HCCs, with an overall success rate of approximately 20%. The transplanted tumors represent the entire spectrum of the molecular landscape of HCCs and preserve the characteristics of the originating tumors through serial passaging. HCC PDX models are a promising tool for preclinical personalized drug development.

Commercially available targeted panels miss genomic regions frequently altered in hepatocellular carcinoma (HCC). We sought to design and benchmark a sequencing assay for genomic screening of HCC. We designed an AmpliSeq custom panel targeting all exons of 33 protein-coding and two long noncoding RNA genes frequently mutated in HCC, TERT promoter, and nine genes with frequent copy number alterations. By using this panel, the profiling of DNA from fresh-frozen (n = 10, 1495×) and/or formalin-fixed, paraffin-embedded (FFPE) tumors with low-input DNA (n = 36, 530×) from 39 HCCs identified at least one somatic mutation in 90% of the cases. Median of 2.5 (range, 0 to 74) and 3 (range, 0 to 76) mutations were identified in fresh-frozen and FFPE tumors, respectively. Benchmarked against the mutations identified from Illumina whole-exome sequencing (WES) of the corresponding fresh-frozen tumors (105×), 98% (61 of 62) and 100% (104 of 104) of the mutations from WES were detected in the 10 fresh-frozen tumors and the 36 FFPE tumors, respectively, using the HCC panel. In addition, 18 and 70 somatic mutations in coding and noncoding genes, respectively, not found by WES were identified by using our HCC panel. Copy number alterations between WES and our HCC panel showed an overall concordance of 86%. In conclusion, we established a cost-effective assay for the detection of genomic alterations in HCC., (Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

Macrophages have been recognized as key players in non-alcoholic fatty liver disease (NAFLD). Our aim was to assess whether pharmacological attenuation of macrophages can be achieved by imatinib, an anti-leukemia drug with known anti-inflammatory and anti-diabetic properties, and how this impacts on NAFLD. We analyzed the pro- and anti-inflammatory gene expression of murine macrophages and human monocytes in vitro in the presence or absence of imatinib. In a time-resolved study, we characterized metabolic disease manifestations such as hepatic steatosis, systemic and adipose tissue inflammation as well as lipid and glucose metabolism in obese mice at one and three months of imatinib treatment. Our results showed that imatinib lowered pro-inflammatory markers in murine macrophages and human monocytes in vitro. In obese mice, imatinib reduced TNFα-gene expression in peritoneal and liver macrophages and systemic lipid levels at one month. This was followed by decreased hepatic steatosis, systemic and adipose tissue inflammation and increased insulin sensitivity after three months. As the transcription factor sterol regulatory element-binding protein (SREBP) links lipid metabolism to the innate immune response, we assessed the gene expression of SREBPs and their target genes, which was indeed downregulated in the liver and partially in peritoneal macrophages. In conclusion, targeting both inflammatory and lipogenic pathways in macrophages and liver as shown by imatinib could represent an attractive novel therapeutic strategy for patients with NAFLD.

Hepatocellular carcinoma (HCC) is the most common primary liver cancer and the second most frequent cause of cancer-related mortality worldwide. The multikinase inhibitor sorafenib is the only treatment option for advanced HCC. Due to tumor heterogeneity, its efficacy greatly varies between patients and is limited due to adverse effects and drug resistance. Current in vitro models fail to recapitulate key features of HCCs. We report the generation of long-term organoid cultures from tumor needle biopsies of HCC patients with various etiologies and tumor stages. HCC organoids retain the morphology as well as the expression pattern of HCC tumor markers and preserve the genetic heterogeneity of the originating tumors. In a proof-of-principle study, we show that liver cancer organoids can be used to test sensitivity to sorafenib. In conclusion, organoid models can be derived from needle biopsies of liver cancers and provide a tool for developing tailored therapies., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Hepatocellular carcinomas (HCCs) are not routinely biopsied, resulting in a lack of tumor materials for molecular profiling. Here we sought to determine whether plasma-derived cell-free DNA (cfDNA) captures the genetic alterations of HCC in patients who have not undergone systemic therapy., Patients and Methods: Frozen biopsies from the primary tumor and plasma were synchronously collected from 30 prospectively recruited, systemic treatment-naïve HCC patients. Deep sequencing of the DNA from the biopsies, plasma-derived cfDNA and matched germline was carried out using a panel targeting 46 coding and non-coding genes frequently altered in HCCs., Results: In 26/30 patients, at least one somatic mutation was detected in biopsy and/or cfDNA. Somatic mutations in HCC-associated genes were present in the cfDNA of 63% (19/30) of the patients and could be detected 'de novo' without prior knowledge of the mutations present in the biopsy in 27% (8/30) of the patients. Mutational load and the variant allele fraction of the mutations detected in the cfDNA positively correlated with tumor size and Edmondson grade. Crucially, among the seven patients in whom the largest tumor was ≥5 cm or was associated with metastasis, at least one mutation was detected 'de novo' in the cfDNA of 86% (6/7) of the cases. In these patients, cfDNA and tumor DNA captured 87% (80/92) and 95% (87/92) of the mutations, suggesting that cfDNA and tumor DNA captured similar proportions of somatic mutations., Conclusion: In patients with high disease burden, the use of cfDNA for genetic profiling when biopsy is unavailable may be feasible. Our results support further investigations into the clinical utility of cfDNA in a larger cohort of patients.

Histidine phosphorylation, the so-called hidden phosphoproteome, is a poorly characterized post-translational modification of proteins. Here we describe a role of histidine phosphorylation in tumorigenesis. Proteomic analysis of 12 tumours from an mTOR-driven hepatocellular carcinoma mouse model revealed that NME1 and NME2, the only known mammalian histidine kinases, were upregulated. Conversely, expression of the putative histidine phosphatase LHPP was downregulated specifically in the tumours. We demonstrate that LHPP is indeed a protein histidine phosphatase. Consistent with these observations, global histidine phosphorylation was significantly upregulated in the liver tumours. Sustained, hepatic expression of LHPP in the hepatocellular carcinoma mouse model reduced tumour burden and prevented the loss of liver function. Finally, in patients with hepatocellular carcinoma, low expression of LHPP correlated with increased tumour severity and reduced overall survival. Thus, LHPP is a protein histidine phosphatase and tumour suppressor, suggesting that deregulated histidine phosphorylation is oncogenic.

Steatosis is a frequent histological feature of hepatitis C virus (HCV) infection. Cohort studies of patients with chronic hepatitis C identified HCV genotype 3 (HCV GT3) as the prevalent steatotic genotype. Moreover, Huh-7 cells over-expressing HCV GT3 core protein accumulate more triglyceride in larger lipid droplets than cells expressing core proteins of other HCV genotypes. However, little is known about the relationship of steatosis and HCV infection at the cellular level in vivo. In this study, we used highly sensitive multiplex in situ hybridization methodology together with lipid staining to investigate HCV-induced lipid droplet accumulation at the cellular level in liver biopsies. Consistent with previous reports, histological steatosis grades were significantly higher in GT3 compared to GT1 infected livers, but independent of viral load. Using nile red lipid stainings, we observed that the frequency of lipid droplet containing cells was similar in HCV GT1- and HCV GT3-infected livers. Lipid droplet formation preferentially occurred in HCV-infected cells irrespective of the genotype, but was also observed in noninfected cells. These findings demonstrate that the main difference between GT1- and GT3-induced steatosis is the size of lipid droplets, but not the number or relative distribution of lipid droplets in infected vs uninfected hepatocytes., (© 2017 John Wiley & Sons Ltd.)

The TP53 gene is the most commonly mutated gene in human cancers and mutations in TP53 have been shown to have either gain-of-function or loss-of-function effects. Using the data generated by The Cancer Genome Atlas, we sought to define the spectrum of TP53 mutations in hepatocellular carcinomas (HCCs) and their association with clinicopathologic features, and to determine the oncogenic and mutational signatures in TP53 -mutant HCCs. Compared to other cancer types, HCCs harbored distinctive mutation hotspots at V157 and R249, whereas common mutation hotspots in other cancer types, R175 and R273, were extremely rare in HCCs. In terms of clinicopathologic features, in addition to the associations with chronic viral infection and high Edmondson grade, we found that TP53 somatic mutations were less frequent in HCCs with cholestasis or tumor infiltrating lymphocytes, but were more frequent in HCCs displaying necrotic areas. An analysis of the oncogenic signatures based on the genetic alterations found in genes recurrently altered in HCCs identified four distinct TP53 -mutant subsets, three of which were defined by CTNNB1 mutations, 1q amplifications or 8q24 amplifications, respectively, that co-occurred with TP53 mutations. We also found that mutational signature 12, a liver cancer-specific signature characterized by T>C substitutions, was prevalent in HCCs with wild-type TP53 or with missense TP53 mutations, but not in HCCs with deleterious TP53 mutations. Finally, whereas patients with HCCs harboring deleterious TP53 mutations had worse overall and disease-free survival than patients with TP53 -wild-type HCCs, patients with HCCs harboring missense TP53 mutations did not have worse prognosis. In conclusion, our results highlight the importance to consider the genetic heterogeneity among TP53 -mutant HCCs in studies of biomarkers and molecular characterization of HCCs.