Inhibition of the transmembrane transporter ABCB1 overcomes resistance to doxorubicin in patient-derived organoid models of HCC.

BACKGROUND: Transarterial chemoembolization is the first-line treatment for intermediate-stage HCC. However, the response rate to transarterial chemoembolization varies, and the molecular mechanisms underlying variable responses are poorly understood. Patient-derived hepatocellular carcinoma organoids (HCCOs) offer a novel platform to investigate the molecular mechanisms underlying doxorubicin resistance. METHODS: We evaluated the effects of hypoxia and doxorubicin on cell viability and cell cycle distribution in 20 patient-derived HCCO lines. The determinants of doxorubicin response were identified by comparing the transcriptomes of sensitive to resistant HCCOs. Candidate genes were validated by pharmacological inhibition. RESULTS: Hypoxia reduced the proliferation of HCCOs and increased the number of cells in the G0/G1 phase of the cell cycle, while decreasing the number in the S phase. The IC50s of the doxorubicin response varied widely, from 29nM to >1µM. Doxorubicin and hypoxia did not exhibit synergistic effects but were additive in some HCCOs. Doxorubicin reduced the number of cells in the G0/G1 and S phases and increased the number in the G2 phase under both normoxia and hypoxia. Genes related to drug metabolism and export, most notably ABCB1, were differentially expressed between doxorubicin-resistant and doxorubicin-sensitive HCCOs. Small molecule inhibition of ABCB1 increased intracellular doxorubicin levels and decreased drug tolerance in resistant HCCOs. CONCLUSIONS: The inhibitory effects of doxorubicin treatment and hypoxia on HCCO proliferation are variable, suggesting an important role of tumor-cell intrinsic properties in doxorubicin resistance. ABCB1 is a determinant of doxorubicin response in HCCOs. Combination treatment of doxorubicin and ABCB1 inhibition may increase the response rate to transarterial chemoembolization.

Inflammation-induced TRIM21 represses hepatic steatosis by promoting the ubiquitination of lipogenic regulators.

Nonalcoholic steatohepatitis (NASH) is a leading cause for chronic liver diseases. Current therapeutic options are limited due to an incomplete mechanistic understanding of how steatosis transitions to NASH. Here we show that the TRIM21 E3 ubiquitin ligase is induced by the synergistic actions of proinflammatory TNF-α and fatty acids in livers of humans and mice with NASH. TRIM21 ubiquitinates and degrades ChREBP, SREBP1, ACC1, and FASN, key regulators of de novo lipogenesis, and A1CF, an alternative splicing regulator of the high-activity ketohexokinase-C (KHK-C) isoform and rate-limiting enzyme of fructose metabolism. TRIM21-mediated degradation of these lipogenic activators improved steatosis and hyperglycemia as well as fructose and glucose tolerance. Our study identifies TRIM21 as a negative regulator of liver steatosis in NASH and provides mechanistic insights into an immunometabolic crosstalk that limits fatty acid synthesis and fructose metabolism during metabolic stress. Thus, enhancing this natural counteracting force of steatosis through inhibition of key lipogenic activators via TRIM21-mediated ubiquitination may provide a therapeutic opportunity to treat NASH.

The impact of floating photovoltaic power plants on lake water temperature and stratification.

Floating photovoltaics (FPV) refers to photovoltaic power plants anchored on water bodies with modules mounted on floats. FPV represents a relatively new technology in Europe and is currently showing a rapid growth in deployment. However, effects on thermal characteristics of lakes are largely unknown, yet these are crucial for licensing and approval of such plants. Here, we quantify FPV impacts on lake water temperature, energy budget and thermal stratification of a lake through measurements of near-surface lateral wind flow, irradiance, air and water temperatures at one of the largest commercial German facilities, situated on a 70 m deep dredging lake in the Upper Rhine Valley, South-West Germany. Underneath the FPV facility, a 73% reduction in irradiance on the lake surface and an average 23% reduction in near-surface wind speed at module height are detected. A three month data set is then used to set up the General Lake Model and simulate scenarios of different FPV occupancies and changing climatic conditions. We observe that a lake coverage with FPV result in a more unstable and shorter thermal stratification during summer, which could mitigate the effects of climate change. The reduction of water temperatures follows a non-linear relationship with increased FPV occupancy. A sensitivity analysis showed that an increased wind reduction by FPV can have a considerable impact on the thermal properties of the lake. However, measurements only suggest small deviations with regard to the thermal properties of the investigated lake. These findings can be used in approval procedures and allow for a more accurate assessment of environmental impacts of future installations.

Studying Hepatitis Virus-Host Interactions in Patient Liver Biopsies.

Infectious diseases are a major contributor to human suffering and the associated socioeconomic burden worldwide. A better understanding of human pathogen-host interactions is a prerequisite for the development of treatment strategies aimed at combatting human pathogen-induced diseases. Model systems that faithfully recapitulate the pathogen-host interactions in humans are critical to gain meaningful insight. Unfortunately, such model systems are not yet available for a number of pathogens. The strict tropism of the hepatitis B (HBV) and C (HCV) viruses for the human liver has made it difficult to study their virus-host interactions during the natural history of these infections. In this case, surplus liver biopsy tissue donated by patients provides an opportunity to obtain a snapshot of the phenomenological and molecular aspects of the human liver of chronically HCV or HBV-infected patients. In this review, we will briefly summarize our own efforts over the years to advance our knowledge of the virus-host interactions during the natural history of chronic HCV and HBV infection.

Preclinical and Phase 1 Assessment of Antisense Oligonucleotide Bepirovirsen in Hepatitis B Virus-Transgenic Mice and Healthy Human Volunteers: Support for Clinical Dose Selection and Evaluation of Safety, Tolerability, and Pharmacokinetics of Single and Multiple Doses.

Dose-dependent reductions in hepatitis B virus (HBV) RNA, DNA, and viral proteins following bepirovirsen administration were observed in HepG2.2.15 cells. In HBV-transgenic mice treated at 50 mg/kg/wk, hepatic HBV RNA and DNA were reduced by 90% and 99%, respectively. Subsequently, a phase 1 first-in-human study assessed pharmacokinetics and tolerability of single (75-450 mg) and multiple (150-450 mg on days 1, 4, 8, 11, 15, and 22) subcutaneous bepirovirsen doses in 96 healthy volunteers. Bepirovirsen at all dose levels was rapidly absorbed (maximum plasma concentration 3-8 hours after dosing), rapidly distributed to peripheral tissues, and slowly eliminated (median plasma terminal half-life: 22.5-24.6 days across cohorts). Plasma exposure (dose-proportional at 150-450 mg) and concentration-time profiles were similar following the first and sixth doses, suggesting little to no plasma accumulation (steady state achieved by day 22). Renal elimination of full-length bepirovirsen accounted for <2% of the total dose. Across the single and multiple dose cohorts, 197 treatment-emergent adverse events were reported, with 99% and 65% classified as mild in severity and local injection site reactions, respectively. In conclusion, bepirovirsen showed an acceptable safety profile in humans with observed pharmacokinetics consistent with the chemical class, warranting further evaluation of bepirovirsen in chronic HBV infection.

Integrative proteogenomic characterization of hepatocellular carcinoma across etiologies and stages.

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-ß-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.

Peroxisomal ß-oxidation acts as a sensor for intracellular fatty acids and regulates lipolysis.

To liberate fatty acids (FAs) from intracellular stores, lipolysis is regulated by the activity of the lipases adipose triglyceride lipase (ATGL), hormone-sensitive lipase and monoacylglycerol lipase. Excessive FA release as a result of uncontrolled lipolysis results in lipotoxicity, which can in turn promote the progression of metabolic disorders. However, whether cells can directly sense FAs to maintain cellular lipid homeostasis is unknown. Here we report a sensing mechanism for cellular FAs based on peroxisomal degradation of FAs and coupled with reactive oxygen species (ROS) production, which in turn regulates FA release by modulating lipolysis. Changes in ROS levels are sensed by PEX2, which modulates ATGL levels through post-translational ubiquitination. We demonstrate the importance of this pathway for non-alcoholic fatty liver disease progression using genetic and pharmacological approaches to alter ROS levels in vivo, which can be utilized to increase hepatic ATGL levels and ameliorate hepatic steatosis. The discovery of this peroxisomal ß-oxidation-mediated feedback mechanism, which is conserved in multiple organs, couples the functions of peroxisomes and lipid droplets and might serve as a new way to manipulate lipolysis to treat metabolic disorders.

Supervised learning based on tumor imaging and biopsy transcriptomics predicts response of hepatocellular carcinoma to transarterial chemoembolization.

Although transarterial chemoembolization (TACE) is the most widely used treatment for intermediate-stage, unresectable hepatocellular carcinoma (HCC), it is only effective in a subset of patients. In this study, we combine clinical, radiological, and genomics data in supervised machine-learning models toward the development of a clinically applicable predictive classifier of response to TACE in HCC patients. Our study consists of a discovery cohort of 33 tumors through which we identify predictive biomarkers, which are confirmed in a validation cohort. We find that radiological assessment of tumor area and several transcriptomic signatures, primarily the expression of FAM111B and HPRT1, are most predictive of response to TACE. Logistic regression decision support models consisting of tumor area and RNA-seq gene expression estimates for FAM111B and HPRT1 yield a predictive accuracy of ∼90%. Reverse transcription droplet digital PCR (RT-ddPCR) confirms these genes in combination with tumor area as a predictive classifier for response to TACE.

Interferon lambda 4 impairs hepatitis C viral antigen presentation and attenuates T cell responses.

Genetic variants of the interferon lambda (IFNL) gene locus are strongly associated with spontaneous and IFN treatment-induced clearance of hepatitis C virus (HCV) infections. Individuals with the ancestral IFNL4-dG allele are not able to clear HCV in the acute phase and have more than a 90% probability to develop chronic hepatitis C (CHC). Paradoxically, the IFNL4-dG allele encodes a fully functional IFNλ4 protein with antiviral activity against HCV. Here we describe an effect of IFNλ4 on HCV antigen presentation. Only minor amounts of IFNλ4 are secreted, because the protein is largely retained in the endoplasmic reticulum (ER) where it induces ER stress. Stressed cells are significantly weaker activators of HCV specific CD8+ T cells than unstressed cells. This is not due to reduced MHC I surface presentation or extracellular IFNλ4 effects, since T cell responses are restored by exogenous loading of MHC with HCV antigens. Rather, IFNλ4 induced ER stress impairs HCV antigen processing and/or loading onto the MHC I complex. Our results provide a potential explanation for the IFNλ4-HCV paradox.

Ubiquitous expression of HBsAg from integrated HBV DNA in patients with low viral load.

BACKGROUND & AIMS: Loss of serum HBsAg is a hallmark of spontaneous and therapy induced resolution of HBV infection, since it generally reflects a profound decrease in viral replication. However, integrated HBV DNA can contribute to HBsAg expression independent of viral replication. The relative contributions of these sources of HBsAg are not well understood. Specifically, it is not known whether actively transcribed HBV integration could spread throughout the entire liver. METHODS: The relative distribution of HBsAg and HBV RNA in liver biopsy tissue from HBeAg-negative (HBe-) patients was analyzed by immunohistochemistry and in situ hybridization (ISH), respectively. Frozen biopsy tissue was used for molecular analysis of intrahepatic viral RNA, virus-host chimeric transcripts and viral DNA. RESULTS: Immunohistochemistry and ISH analysis revealed HBsAg and HBV RNA positivity in virtually all hepatocytes in the liver of some HBe- patients despite very low viremia. Reverse transcription quantitative PCR and RNA-sequencing analysis confirmed high expression levels of HBV envelope-encoding RNAs. However, the amount of viral transcriptional template (covalently closed circular (ccc)DNA) was too low to support this ubiquitous HBV RNA expression. In contrast, levels of total cellular HBV DNA were consistent with ubiquitous HBV integration. Finally, RNA-sequencing revealed the presence of many HBV-host chimeric transcripts with the potential for HBsAg expression. CONCLUSIONS: Transcriptionally active HBV integration can extend to the entire liver in some HBe- patients. This can lead to ubiquitous HBsAg expression independent of HBV replication. In such patients, HBsAg is probably not a clinically useful surrogate marker for viral resolution or functional cure. LAY SUMMARY: Loss of serum hepatitis B surface antigen (HBsAg) indicates resolution of HBV infection. However, integrated HBV DNA can contribute to HBsAg production independently of viral replication. We investigated the extent of HBsAg-producing viral integration in the livers of patients with low serum viral loads. Our findings suggest that transcriptionally active HBV integration can extend to the entire liver in some patients, questioning the clinical utility of HBsAg as a surrogate marker for viral replication.

Characterization and Application of Precore/Core-Related Antigens in Animal Models of Hepatitis B Virus Infection.

BACKGROUND AND AIMS: The hepatitis B core-related antigen (HBcrAg), a composite antigen of precore/core gene including classical hepatitis B core protein (HBc) and HBeAg and, additionally, the precore-related antigen PreC, retaining the N-terminal signal peptide, has emerged as a surrogate marker to monitor the intrahepatic HBV covalently closed circular DNA (cccDNA) and to define meaningful treatment endpoints. APPROACH AND RESULTS: Here, we found that the woodchuck hepatitis virus (WHV) precore/core gene products (i.e., WHV core-related antigen [WHcrAg]) include the WHV core protein and WHV e antigen (WHeAg) as well as the WHV PreC protein (WPreC) in infected woodchucks. Unlike in HBV infection, WHeAg and WPreC proteins were N-glycosylated, and no significant amounts of WHV empty virions were detected in WHV-infected woodchuck serum. WHeAg was the predominant form of WHcrAg, and a positive correlation was found between the serum WHeAg and intrahepatic cccDNA. Both WHeAg and WPreC antigens displayed heterogeneous proteolytic processing at their C-termini, resulting in multiple species. Analysis of the kinetics of each component of the precore/core-related antigen, along with serum viral DNA and surface antigens, in HBV-infected chimpanzees and WHV-infected woodchucks revealed multiple distinct phases of viral decline during natural resolution and in response to antiviral treatments. A positive correlation was found between HBc and intrahepatic cccDNA but not between HBeAg or HBcrAg and cccDNA in HBV-infected chimpanzees, suggesting that HBc can be a better marker for intrahepatic cccDNA. CONCLUSIONS: In conclusion, careful monitoring of each component of HBcrAg along with other classical markers will help understand intrahepatic viral activities to elucidate natural resolution mechanisms as well as guide antiviral development.

Response prediction of hepatocellular carcinoma undergoing transcatheter arterial chemoembolization: unlocking the potential of CT texture analysis through nested decision tree models.

OBJECTIVES: To investigate if nested multiparametric decision tree models based on tumor size and CT texture parameters from pre-therapeutic imaging can accurately predict hepatocellular carcinoma (HCC) lesion response to transcatheter arterial chemoembolization (TACE). MATERIALS AND METHODS: This retrospective study (January 2011-September 2017) included consecutive pre- and post-therapeutic dynamic CT scans of 37 patients with 92 biopsy-proven HCC lesions treated with drug-eluting bead TACE. Following manual segmentation of lesions according to modified Response Evaluation Criteria in Solid Tumors criteria on baseline arterial phase CT images, tumor size and quantitative texture parameters were extracted. HCCs were grouped into lesions undergoing primary TACE (VT-lesions) or repeated TACE (RT-lesions). Distinct multiparametric decision tree models to predict complete response (CR) and progressive disease (PD) for the two groups were generated. AUC and model accuracy were assessed. RESULTS: Thirty-eight of 72 VT-lesions (52.8%) and 8 of 20 RT-lesions (40%) achieved CR. Sixteen VT-lesions (22.2%) and 8 RT-lesions (40%) showed PD on follow-up imaging despite TACE treatment. Mean of positive pixels (MPP) was significantly higher in VT-lesions compared to RT-lesions (180.5 vs 92.8, p = 0.001). The highest AUC in ROC curve analysis and accuracy was observed for the prediction of CR in VT-lesions (AUC 0.96, positive predictive value 96.9%, accuracy 88.9%). Prediction of PD in VT-lesions (AUC 0.88, accuracy 80.6%), CR in RT-lesions (AUC 0.83, accuracy 75.0%), and PD in RT-lesions (AUC 0.86, accuracy 80.0%) was slightly inferior. CONCLUSIONS: Nested multiparametric decision tree models based on tumor heterogeneity and size can predict HCC lesion response to TACE treatment with high accuracy. They may be used as an additional criterion in the multidisciplinary treatment decision-making process. KEY POINTS: • HCC lesion response to TACE treatment can be predicted with high accuracy based on baseline tumor heterogeneity and size. • Complete response of HCC lesions undergoing primary TACE was correctly predicted with 88.9% accuracy and a positive predictive value of 96.9%. • Progressive disease was correctly predicted with 80.6% accuracy for lesions undergoing primary TACE and 80.0% accuracy for lesions undergoing repeated TACE.

Transition to HBeAg-negative chronic hepatitis B virus infection is associated with reduced cccDNA transcriptional activity.

BACKGROUND & AIMS: HBeAg seroconversion during the natural history of chronic hepatitis B (CHB) is associated with a strong drop in serum HBV DNA levels and a reduction of intrahepatic covalently closed circular DNA (cccDNA) content. Of particular interest is the transition to HBeAg-negative chronic infection (ENCI). ENCI, previously known as inactive carrier state, is characterized by very low or negative viremia and the absence of liver disease. The molecular mechanisms responsible for the transition to ENCI and for the control of viral replication in ENCI are still poorly understood. METHODS: To identify which step(s) in the viral life cycle are controlled during the transition to ENCI, we quantified cccDNA, pre-genomic RNA (pgRNA), total HBV RNA and DNA replicative intermediates in 68 biopsies from patients in different phases of CHB. RESULTS: HBeAg seroconversion is associated with a reduction of cccDNA amounts as well as transcriptional activity. Silencing of cccDNA is particularly pronounced in ENCI, where there was ~46 times less pgRNA per cccDNA compared to HBeAg-negative CHB. Furthermore, a subgroup of patients with HBeAg-negative CHB can be characterized by reduced replication efficiency downstream of pgRNA. CONCLUSIONS: The reduction in serum viral load during the transition to ENCI seems to primarily result from strong inhibition of the transcriptional activity of cccDNA which can be maintained in the absence of liver disease. LAY SUMMARY: During the natural course of chronic hepatitis B virus infections, the immune response can gain control of viral replication. Quantification of viral DNA and RNA in liver biopsies of patients in different stages of chronic hepatitis B allowed us to identify the steps in the viral life cycle that are affected during the transition from active to inactive disease. Therapeutic targeting of these steps might induce sustained inhibition of viral transcription.

Liver biopsy derived induced pluripotent stem cells provide unlimited supply for the generation of hepatocyte-like cells.

BACKGROUND & AIMS: Hepatocyte-like cells (HLCs) differentiated from induced pluripotent stem cells (iPSCs) have emerged as a promising cell culture model to study metabolism, biotransformation, viral infections and inherited liver diseases. iPSCs provide an unlimited supply for the generation of HLCs, but incomplete HLC differentiation remains a major challenge. iPSC may carry-on a tissue of origin dependent expression memory influencing iPSC differentiation into different cell types. Whether liver derived iPSCs (Li-iPSCs) would allow the generation of more fully differentiated HLCs is not known. METHODS: In the current study, we used primary liver cells (PLCs) expanded from liver needle biopsies and reprogrammed them into Li-iPSCs using a non-integrative Sendai virus-based system. Li-iPSCs were differentiated into HLCs using established differentiation protocols. The HLC phenotype was characterized at the protein, functional and transcriptional level. RNA sequencing data were generated from the originating liver biopsies, the Li-iPSCs, fibroblast derived iPSCs, and differentiated HLCs, and used to characterize and compare their transcriptome profiles. RESULTS: Li-iPSCs indeed retain a liver specific transcriptional footprint. Li-iPSCs can be propagated to provide an unlimited supply of cells for differentiation into Li-HLCs. Similar to HLCs derived from fibroblasts, Li-HLCs could not be fully differentiated into hepatocytes. Relative to the originating liver, Li-HLCs showed lower expression of liver specific transcription factors and increased expression of genes involved in the differentiation of other tissues. CONCLUSIONS: PLCs and Li-iPSCs obtained from small pieces of human needle liver biopsies constitute a novel unlimited source for the production of HLCs. Despite the preservation of a liver specific gene expression footprint in Li-iPSCs, the generation of fully differentiated hepatocytes cannot be achieved with the current differentiation protocols.

Hepatocellular Carcinoma Xenografts Established From Needle Biopsies Preserve the Characteristics of the Originating Tumors.

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.

Mapping the Heterogeneity of Histone Modifications on Hepatitis B Virus DNA Using Liver Needle Biopsies Obtained from Chronically Infected Patients.

Covalently closed circular DNA (cccDNA) forms the basis for replication and persistence of hepatitis B virus (HBV) in the chronically infected liver. We have previously shown that viral transcription is subject to regulation by posttranslational modifications (PTMs) of histone proteins bound to cccDNA through analysis of de novo HBV-infected cell lines. We now report the successful adaptation of this chromatin immunoprecipitation sequencing (ChIPseq) approach for analysis of fine-needle patient liver biopsy specimens to investigate the role of histone PTMs in chronically HBV-infected patients. Using 18 specimens from patients in different stages of chronic HBV infection, our work shows that the profile of histone PTMs in chronic infection is more nuanced than previously observed in in vitro models of acute infection. In line with our previous findings, we find that the majority of HBV-derived sequences are associated with the activating histone PTM H3K4me3. However, we show a striking interpatient variability of its deposition in this patient cohort correlated with viral transcription and patient HBV early antigen (HBeAg) status. Unexpectedly, we detected deposition of the classical inhibitory histone PTM H3K9me3 on HBV-DNA in around half of the patient biopsy specimens, which could not be linked to reduced levels of viral transcripts. Our results show that current in vitro models are unable to fully recapitulate the complex epigenetic landscape of chronic HBV infection observed in vivo and demonstrate that fine-needle liver biopsy specimens can provide sufficient material to further investigate the interaction of viral and host proteins on HBV-DNA.IMPORTANCE Hepatitis B virus (HBV) is a major global health concern, chronically infecting millions of patients and contributing to a rising burden of liver disease. The viral genome forms the basis for chronic infection and has been shown to be subject to regulation by epigenetic mechanisms, such as posttranslational modification of histone proteins. Here, we confirm and expand on previous results by adapting a high-resolution technique for analysis of histone modifications for use with patient-derived fine-needle liver biopsy specimens. Our work highlights that the situation in vivo is more complex than predicted by current in vitro models, for example, by suggesting a novel, noncanonical role of the histone modification H3K9me3 in the HBV life cycle. Importantly, enabling the use of fine-needle liver biopsy specimens for such high-resolution analyses may facilitate further research into the epigenetic regulation of the HBV genome.

The effect of particle size, morphology and support on the formation of palladium hydride in commercial catalysts.

The relative amounts of hydrogen retained by a range of supported palladium catalysts have been investigated by a combination of electron microscopy and spectroscopic techniques, including incoherent inelastic neutron scattering. Contrary to expectation, the hydrogen capacity is not determined solely by the metal particle size, but it is a complex interaction between the particle size and its state of aggregation. The nature of the support is not only integral to the amount of hydrogen held by the catalyst, it also causes a marked difference in the rate of release of stored hydrogen from palladium. It is more difficult to fully dehydrogenate palladium on/in the porous activated carbon than on the non-porous carbon black based catalyst. The type of support also results in differences in the form of the residual hydrogen: whether it is α- or ß-hydride phase, subsurface or in the threefold surface site. Our data on the supported catalysts reinforces what has only been seen previously with palladium black and our computational study provides confirmation of the empirical assignments. We also report the first vibrational spectroscopic study of hydrogen adsorbed at the surface of ß-PdH and have observed for the first time hydrogen in the on-top site. This has enabled the relative proportion of bulk- to surface-H occupation in calculated model and in industrial nanoparticles to be estimated.

Organoid Models of Human Liver Cancers Derived from Tumor Needle Biopsies.

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.

Diagnostic Targeted Sequencing Panel for Hepatocellular Carcinoma Genomic Screening.

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.