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

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

Atezolizumab/bevacizumab or lenvatinib in hepatocellular carcinoma: Multicenter real-world study with focus on bleeding and thromboembolic events.

Background & Aims: Atezolizumab/bevacizumab (atezo/bev) and lenvatinib have demonstrated efficacy as first-line therapies for hepatocellular carcinoma (HCC). However, vascular endothelial growth factor (VEGF) inhibition with these therapies may be associated with the risk of bleeding and thromboembolic events. In this study, we evaluated the efficacy and safety with focus on the bleeding and thromboembolic events of atezo/bev vs. lenvatinib in a large, multicenter real-world population. Methods: This study is based on HCC cohorts from seven centers in Germany and Austria. Incidences of bleeding or thromboembolic events and efficacy outcomes were assessed and compared. Results: In total, 464 patients treated with atezo/bev (n = 325) or lenvatinib (n = 139) were analyzed. Both groups were balanced with respect to demographics, presence of liver cirrhosis, and variceal status. Duration of therapy did not differ between groups. Within 3 months of therapy, bleeding episodes were described in 57 (18%) patients receiving atezo/bev compared with 15 (11%) patients receiving lenvatinib (p = 0.07). Variceal hemorrhage occurred in 11 (3%) patients treated with atezo/bev compared with 4 (3%) patients treated with lenvatinib (p = 0.99). Thromboembolic events were reported in 19 (6%) of patients in the atezo/bev cohort compared with 5 (4%) patients in the lenvatinib cohort (p = 0.37). In addition, incidence of overall bleeding, variceal hemorrhage, and thromboembolic events did not differ significantly in patients who received either atezo/bev or lenvantinib for 6 months. Conclusions: Safety considerations related to bleeding and thromboembolic events may not be helpful in guiding clinical decision-making when choosing between atezo/bev and lenvatinib. Impact and implications: The inhibition of VEGF by current first-line therapies for HCC, such as atezolizumab/bevacizumab or lenvatinib, may be associated with the risk of bleeding and thromboembolic events. Studies comparing the incidence of these side effects between atezolizumab/bevacizumab and lenvatinib, which are preferred treatments over sorafenib for HCC, are needed. Differences in this side effect profile may influence the choice of first-line therapy by treating physicians. Because no significant differences were observed regarding bleeding or thromboembolic events between both therapies in the present study, we conclude that safety considerations related to these events may not be helpful in guiding clinical decision-making when choosing between atezolizumab/bevacizumab and lenvatinib.

Changing treatment landscape associated with improved survival in advanced hepatocellular carcinoma: a nationwide, population-based study.

BACKGROUND AND AIMS: The treatment of hepatocellular carcinoma (HCC) is undergoing a historic transformation with the approval of several new systemic therapies in the last few years. This study aimed to examine the impact of this changing landscape on survival and costs in a Western nationwide, real-world cohort. METHODS: A nationwide representative claims database (InGef) was screened for HCC cases between 2015 and 2020. Survival in an era with only sorafenib (period A, January 2015 to July 2018) and after approval of lenvatinib and other systemic treatments (period B, August 2018 to December 2020) was analysed. Health care costs were assessed. RESULTS: We identified 2876 individuals with HCC in the study period. The proportion of patients receiving systemic therapy increased significantly over time, from 11.8% in 2015 to 15.1% in 2020 (p < 0.0001). The median overall survival in period B was 6.5 months (95% confidence interval [CI]: 4.9-8.9) and in period A was 5.3 months (95% CI: 4.5-6.3; p = 0.046). In period B, the median overall survival with lenvatinib was 9.7 months (95% CI: 6.3-18.4) versus 4.8 months with sorafenib (95% CI: 4.0-7.1, p = 0.008). Costs for prescription drugs per patient increased from €6150 in 2015 to €9049 in 2020 (p < 0.0001), and costs for outpatient care per patient increased from €1646 to €2149 (p = 0.0240). CONCLUSION: The approval of new systemic therapies resulted in a survival benefit in patients with HCC. The magnitude of the effect is modest and associated with a moderate increase in health costs.

Fulminant Liver Failure after Treatment with a Checkpoint Inhibitor for Gastric Cancer: A Case Report and Review of the Literature.

Nivolumab is a promising monoclonal antibody inhibitor of programmed death-1, a protein on the surface of T-cells. As such, it is approved for use in patients with multiple advanced malignancies and can significantly elongate progression-free survival. However, monoclonal antibody inhibitors can lead to adverse hepatic reactions, which in rare cases result in further hepatic damage. Herein, we present a case of a patient with locally advanced gastric carcinoma treated with fluorouracil, oxaliplatin, docetaxel and the checkpoint inhibitor nivolumab. Five months after her first dosage of nivolumab and without a preexisting liver disease, she presented with transaminitis. During the course of her stay, the patient developed status epilepticus, which required mechanical ventilation followed by fulminant hepatic failure. A subsequent liver biopsy revealed severe liver damage with extensive confluent parenchymal necrosis corresponding to checkpoint-inhibitor-induced hepatitis. Alternative reasons for this hepatic failure were ruled out. Despite aggressive therapeutic interventions including corticosteroids and plasma exchange, the patient died due to liver failure. Although hepatic failure is rarely seen in patients with checkpoint inhibitor therapy, it requires early awareness and rapid intervention.

Transjugular intrahepatic portosystemic shunt creation (TIPS) in the angio-CT-a hybrid intervention with image fusion.

OBJECTIVE: For transjugular intrahepatic portosystemic shunt (TIPS) creation, ultrasound guidance for portal vein puncture is strongly recommended. However, outside regular hours of service, a skilled sonographer might be lacking. Hybrid intervention suites combine CT imaging with conventional angiography allowing to project 3D information into the conventional 2D imaging and further CT-fluoroscopic puncture of the portal vein. The purpose of this study was to assess whether TIPS using angio-CT facilitates the procedure for a single interventional radiologist. METHODS: All TIPS procedures from 2021 and 2022 which took place outside regular working hours were included (n = 20). Ten TIPS procedures were performed with just fluoroscopy guidance and ten procedures using angio-CT. For the angio-CT TIPS, a contrast-enhanced CT was performed on the angiography table. From the CT, a 3D volume was created using virtual rendering technique (VRT). The VRT was blended with the conventional angiography image onto the live monitor and used as guidance for the TIPS needle. Fluoroscopy time, area dose product, and interventional time were assessed. RESULTS: Hybrid intervention with angio-CT did lead to a significantly shorter fluoroscopy time and interventional time (p = 0.034 for both). Mean radiation exposure was significantly reduced, too (p = 0.04). Furthermore, the mortality rate was lower in patients who underwent the hybrid TIPS (0% vs 33%). CONCLUSION: TIPS procedure in angio-CT performed by only one interventional radiologist is quicker and reduces radiation exposure for the interventionalist compared to mere fluoroscopy guidance. The results further indicate increased safety using angio-CT. CLINICAL RELEVANCE STATEMENT: This study aimed to evaluate the feasibility of using angio-CT in TIPS procedures during non-standard working hours. Results indicated that the use of angio-CT significantly reduced fluoroscopy time, interventional time, and radiation exposure, while also leading to improved patient outcomes. KEY POINTS: • Image guiding such as ultrasound is recommended for transjugular intrahepatic portosystemic shunt creation but might be not available for emergency cases outside of regular working hours. • Transjugular intrahepatic portosystemic shunt creation using an angio-CT with image fusion is feasible for only one physician under emergency settings and results in lower radiation exposure and faster procedures. • Transjugular intrahepatic portosystemic shunt creation using an angio-CT with image fusion seems to be safer than using mere fluoroscopy guidance.

Regional transplant rates depend more on physician-dependent variables than on proximity to transplant center.

PURPOSE: The objective of this work was to uncover inequalities in access to liver transplantation in Bavaria, Germany. METHODS: For this purpose, the annual transplantation rate per 1 million inhabitants for the respective districts was determined from the aggregated postal codes of the place of residence of transplanted patients. The variables examined were proximity and travel time to the nearest transplant center, as well as the care category of the regional hospital. In addition, we assessed whether the head of gastroenterology at the regional hospital through which liver transplant candidates are referred was trained at a liver transplant center. RESULTS: We could not demonstrate a direct relationship between proximity or travel time to the nearest transplant center and access to liver transplantation. Multivariate regression analysis shows that liver transplant training (p < 0.0001) of the chief physician (gastroenterologist) of the regional hospital was the most decisive independent factor for access to liver transplantation within a district. CONCLUSION: We show that the transplant training experience of the head of gastroenterology at a regional hospital is an independent factor for the regional transplantation rate. Therefore, it appears important to maintain some liver transplant expertise outside the transplant centers in order to properly identify and assign potential transplant candidates for transplantation.

[(Rare) infectious hepatitis as an important differential diagnosis of unclear hepatopathy]. / (Seltene) infektiöse Hepatitiden als wichtige Differenzialdiagnose der unklaren Hepatopathie.

BACKGROUND: Liver biopsies to determine the cause of unclear hepatopathy and acute liver failure represent a diagnostic hallmark and require close cooperation between clinicians and pathologists. The commonly acute presentation of hepatic dysfunction warrants a rapid diagnosis. Infectious causes of hepatitis may be identified by the pathologist, supporting further diagnostic and therapeutic steps. OBJECTIVES: Rare infectious causes of hepatic dysfunction with distinct histomorphological features are presented. MATERIALS AND METHODS: Retrospective cases of liver biopsies for evaluation of hepatic dysfunction with infection confirmed by laboratory tests were selected from the archive of the institute of pathology of TUM and evaluated for morphologic diagnostic criteria. RESULTS AND CONCLUSIONS: Infections with adenovirus, Herpes simplex virus, Hepatitis A virus, and coxiella burnettii are rare findings in liver core biopsies but able to cause hepatic dysfunction that present with distinct histomorphologic alterations that can contribute to the identification of the causative agent. Rare infectious causes should be considered in any patient presenting with hepatic dysfunction of unknown etiology. Knowledge of the histomorphologic criteria by the pathologist is crucial to initiate further diagnostic testing and treatment.

Real-World Data for Lenvatinib in Hepatocellular Carcinoma (ELEVATOR): A Retrospective Multicenter Study.

Background: Lenvatinib is approved as first-line treatment for patients with advanced hepatocellular carcinoma (HCC). The efficacy of lenvatinib in Caucasian real-world patients is insufficiently defined. The purpose of this study was to evaluate the efficacy of lenvatinib in a multi-center cohort (ELEVATOR) from Germany and Austria. Methods: A retrospective data analysis of 205 patients treated with first-line systemic lenvatinib at 14 different sites was conducted. Overall survival, progression free survival, overall response rate and adverse event rates were assessed and analyzed. Results: Patients receiving lenvatinib in the real-world setting reached a median overall survival of 12.8 months, which was comparable to the results reported from the REFLECT study. Median overall survival (mOS) and progression free survival (mPFS) was superior in those patients who met the inclusion criteria of the REFLECT study compared to patients who failed to meet the inclusion criteria (mOS 15.6 vs 10.2 months, HR 0.55, 95% CI 0.38-0.81, p=0.002; mPFS 8.1 vs 4.8 months HR 0.65, 95% CI 0.46-0.91, p=0.0015). For patients with an impaired liver function according to the Albumin-Bilirubin (ALBI) grade, or reduced ECOG performance status ≥2, survival was significantly shorter compared to patients with sustained liver function (ALBI grade 1) and good performance status (ECOG performance status 0), respectively (HR 1.69, 95% CI 1.07-2.66, p=0.023; HR 2.25, 95% CI 1.19-4.23, p=0.012). Additionally, macrovascular invasion (HR 1.55, 95% CI 1.02-2.37, p=0.041) and an AFP ≥200 ng/mL (HR 1.56, 95% CI 1.03-2.34, p=0.034) were confirmed as independent negative prognostic factors in our cohort of patients with advanced HCC. Conclusion: Overall, our data confirm the efficacy of lenvatinib as first-line treatment and did not reveal new or unexpected side effects in a large retrospective Caucasian real-world cohort, supporting the use of lenvatinib as meaningful alternative for patients that cannot be treated with IO-based combinations in first-line HCC.

Sorafenib Versus Lenvatinib-Based Sequential Systemic Therapy for Advanced Hepatocellular Carcinoma: A Real-World Analysis.

The optimal treatment sequence of tyrosine kinase inhibitor (TKI)-based therapy in patients with hepatocellular carcinoma (HCC) remains unclear. Therefore, sequential systemic therapy after first-line therapy with sorafenib or lenvatinib was compared in a retrospective real-world cohort. In total, 164 patients with HCC were included. Child B cirrhosis was present in 26 patients (16.5%), whereas 132 patients (83.5%) had preserved liver function. In total, 72 patients (44%) discontinued systemic therapy after first-line therapy while 51 (31%) and 31 (19%) patients received 2 or more treatment lines. Most notably, median overall survival (mOS) was influenced by liver functional status and patient performance status at the beginning of first-line therapy. Patients receiving a sequential therapy regimen had significantly longer mOS compared to patients that discontinued systemic therapy after omitting first-line treatment. The choice of the initial TKI did not impact mOS. A clear deterioration of liver function could be observed during the course of TKI-based treatment.

CRISPR somatic genome engineering and cancer modeling in the mouse pancreas and liver.

Genetically engineered mouse models (GEMMs) transformed the study of organismal disease phenotypes but are limited by their lengthy generation in embryonic stem cells. Here, we describe methods for rapid and scalable genome engineering in somatic cells of the liver and pancreas through delivery of CRISPR components into living mice. We introduce the spectrum of genetic tools, delineate viral and nonviral CRISPR delivery strategies and describe a series of applications, ranging from gene editing and cancer modeling to chromosome engineering or CRISPR multiplexing and its spatio-temporal control. Beyond experimental design and execution, the protocol describes quantification of genetic and functional editing outcomes, including sequencing approaches, data analysis and interpretation. Compared to traditional knockout mice, somatic GEMMs face an increased risk for mouse-to-mouse variability because of the higher experimental demands of the procedures. The robust protocols described here will help unleash the full potential of somatic genome manipulation. Depending on the delivery method and envisaged application, the protocol takes 3-5 weeks.

Atezolizumab and bevacizumab with transarterial chemoembolization in hepatocellular carcinoma: the DEMAND trial protocol.

The combination of the anti-PD-L1 antibody atezolizumab and the anti-VEGF bevacizumab is the first approved immunotherapeutic regimen for first-line therapy in patients with unresectable hepatocellular carcinoma (HCC), currently approved in more than 80 countries. The efficacy and tolerability of this regimen suggest that the use of atezolizumab + bevacizumab could be extended to the treatment of patients with intermediate-stage HCC in combination with transarterial chemoembolization (TACE). The authors describe the rationale and design of the DEMAND study. This investigator-initiated, multicenter, randomized phase II study is the first trial to evaluate the safety and efficacy of atezolizumab + bevacizumab prior to or in combination with TACE in patients with intermediate-stage HCC. The primary end point is the 24-month survival rate; secondary end points include objective response rate, progression-free survival, safety and quality of life. Clinical Trial Registration: NCT04224636 (ClinicalTrials.gov).

High rate of complete histopathological response in hepatocellular carcinoma patients after combined transarterial chemoembolization and stereotactic body radiation therapy.

BACKGROUND: Liver transplantation (LT) presents a curative treatment option in patients with early stage hepatocellular carcinoma (HCC) who are not eligible for resection or ablation therapy. Due to a risk of up 30% for waitlist drop-out upon tumor progression, bridging therapies are used to halt tumor growth. Transarterial chemoembolization (TACE) and less commonly stereotactic body radiation therapy (SBRT) or a combination of TACE and SBRT, are used as bridging therapies in LT. However, it remains unclear if one of those treatment options is superior. The analysis of explant livers after transplantation provides the unique opportunity to investigate treatment response by histopathology. AIM: To analyze histopathological response to a combination of TACE and SBRT in HCC in comparison to TACE or SBRT alone. METHODS: In this multicenter retrospective study, 27 patients who received liver transplantation for HCC were analyzed. Patients received either TACE or SBRT alone, or a combination of TACE and SBRT as bridging therapy to liver transplantation. Liver explants of all patients who received at least one TACE and/or SBRT were analyzed for the presence of residual vital tumor tissue by histopathology to assess differences in treatment response to bridging therapies. Statistical analysis was performed using Fisher-Freeman-Halton exact test, Kruskal-Wallis and Mann-Whitney-U tests. RESULTS: Fourteen patients received TACE only, four patients SBRT only, and nine patients a combination therapy of TACE and SBRT. There were no significant differences between groups regarding age, sex, etiology of underlying liver disease or number and size of tumor lesions. Strikingly, analysis of liver explants revealed that almost all patients in the TACE and SBRT combination group (8/9, 89%) showed no residual vital tumor tissue by histopathology, whereas TACE or SBRT alone resulted in significantly lower rates of complete histopathological response (0/14, 0% and 1/4, 25%, respectively, P value < 0.001). CONCLUSION: Our data suggests that a combination of TACE and SBRT increases the rate of complete histopathological response compared to TACE or SBRT alone in bridging to liver transplantation.

Liver transplantation versus watchful waiting in hepatocellular carcinoma patients with complete response to bridging therapy - a retrospective observational study.

Bridging therapy to prevent progression on the waiting list can result in a sustained complete response (sCR). In some patients, the liver transplantation (LT) risk might exceed those of tumor recurrence. We thus evaluated whether a watchful waiting (CR-WW) strategy could be a feasible alternative to transplantation (CR-LT). We performed a retrospective analysis of overall survival (OS) and recurrence-free survival (RFS) of patients with a sCR (CR > 6 months). Permitted bridging included thermoablation, resection, and combinations of either with transarterial chemoembolization. Patients were divided into the intended treatment strategies CR-WW and CR-LT. 39 (18.40%) sCR patients from 212 were investigated. 22 patients were treated with a CR-LT and 17 patients a CR-WW strategy. Five-year RFS was lower in the CR-WW than in the CR-LT group [53.3% (22.1%; 77.0%) and 84.0% (57.6%; 94.7%)]. 29.4% (5/17) CR-WW patients received salvage transplantation because of recurrence. OS (5-year) was 83.9% [56.8%; 94.7%] after LT and 75.4% [39.8%; 91.7%] after WW. Our analysis shows that the intuitive decision made by our patients in agreement with their treating physicians for a watchful waiting strategy in sCR can be justified. Applied on a larger scale, this strategy could help to reduce the pressure on the donor pool.

Age independent survival benefit for patients with hepatocellular carcinoma (HCC) without metastases at diagnosis: a population-based study.

OBJECTIVE: Hepatocellular carcinoma (HCC) is a major cause of death worldwide and its incidence is expected to increase globally. Aim of this study was to assess whether the implementation of screening policies and the improvement of treatment options translated into a real-world survival benefit in HCC patients. DESIGN: 4078 patients diagnosed with HCC between 1998 and 2016 from the Munich Cancer Registry were analysed. Tumour characteristics and outcome were analysed by time period and according to age and presence of metastases at diagnosis. Overall survival (OS) was analysed using Kaplan-Meier method and relative survival (RS) was computed for cancer-specific survival. Cox proportional hazard models were conducted to control for prognostic variables. RESULTS: While incidence of HCC remained substantially stable, tumours were diagnosed at increasingly earlier stages, although the median age at diagnosis increased. The 3 years RS in HCC improved from 19.8% in 1998-2002, 22.4% in 2003-2007, 30.6% in 2008-2012 up to 31.0% in 2013-2016. Median OS increased from 6 months in 1998-2002 to 12 months in 2008-2016. However, analysis according to the metastatic status showed that survival improved only in patients without metastases at diagnosis whereas the prognosis of patients with metastatic disease remained unchanged. CONCLUSION: These real-world data show that, in contrast to the current assumptions, the incidence of HCC did not increase in a representative German region. Earlier diagnosis, likely related to the implementation of screening programmes, translated into an increasing employment of effective therapeutic options and a clear survival benefit in patients without metastases at diagnosis, irrespective of age.

Progression after Immunotherapy for Fibrolamellar Carcinoma.

BACKGROUND: Fibrolamellar carcinoma (FLC) is a rare malignancy of the liver that differs from typical hepatocellular carcinoma (HCC) in several aspects such as the absence of underlying liver disease and occurrence in younger patients. Even though the survival rates in FLC are slightly better than in typical HCC, the prognosis of metastatic FLC remains deleterious. Several reports suggest that systemic chemotherapy regimens can successfully be used to halt disease progression in FLC, while targeted tumor therapy with sorafenib seems to be of limited efficiency. However, results from controlled clinical trials investigating systemic therapies in FLC are virtually nonexistent. Therefore, the choice of treatment often relies on case series with limited numbers of patients. Immunotherapy with checkpoint inhibitors is an emerging cancer therapy in several solid malignancies including HCC. Currently, there do not exist any reports on the use of checkpoint inhibitors in FLC. CASE REPORT: Here, we describe a case of advanced FLC in a young man receiving immunotherapy, who progressed after 3 months of treatment - similar to 2 other patients with advanced FLC at our hospital. CONCLUSION: While immunotherapy seems to be a promising treatment with limited side effects in several other tumor entities, there is currently no data supporting tumor response in FLC.

Hippo Signaling in the Liver - A Long and Ever-Expanding Story.

The first description of Hippo signaling in mammals a little more than 10 years ago showed a striking phenotype in the liver, linking the role of this signaling pathway to organ size control and carcinogenesis. Even though Hippo signaling has been extensively studied in the liver and other organs over the recent years, many open questions remain in our understanding of its role in hepatic physiology and disease. The functions of Hippo signaling extend well beyond cancer and organ size determination: components of upstream Hippo signaling and the downstream effectors YAP and TAZ are involved in a multitude of cell and non-cell autonomous functions including cell proliferation, survival, development, differentiation, metabolism, and cross-talk with the immune system. Moreover, regulation and biological functions of Hippo signaling are often organ or even cell type specific - making its role even more complex. Here, we give a concise overview of the role of Hippo signaling in the liver with a focus on cell-type specific functions. We outline open questions and future research directions that will help to improve our understanding of this important pathway in liver disease.

Promotion of growth factor signaling as a critical function of ß-catenin during HCC progression.

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related deaths worldwide. ß-catenin is widely thought to be a major oncogene in HCC based on the frequency of mutations associated with aberrant Wnt signaling in HCC patients. Challenging this model, our data reveal that ß-catenin nuclear accumulation is restricted to the late stage of the disease. Until then, ß-catenin is primarily located at the plasma membrane in complex with multiple cadherin family members where it drives tumor cell survival by enhancing the signaling of growth factor receptors such as EGFR. Therefore, our study reveals the evolving nature of ß-catenin in HCC to establish it as a compound tumor promoter during the progression of the disease.

Constitutive and Inducible Systems for Genetic In Vivo Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein Injection.

In research models of liver cancer, regeneration, inflammation, and fibrosis, flexible systems for in vivo gene expression and silencing are highly useful. Hydrodynamic tail vein injection of transposon-based constructs is an efficient method for genetic manipulation of hepatocytes in adult mice. In addition to constitutive transgene expression, this system can be used for more advanced applications, such as shRNA-mediated gene knock-down, implication of the CRISPR/Cas9 system to induce gene mutations, or inducible systems. Here, the combination of constitutive CreER expression together with inducible expression of a transgene or miR-shRNA of choice is presented as an example of this technique. We cover the multi-step procedure starting from the preparation of sleeping beauty-transposon constructs, to the injection and treatment of mice, and the preparation of liver tissue for analysis by immunostaining. The system presented is a reliable and efficient approach to achieve complex genetic manipulations in hepatocytes. It is specifically useful in combination with Cre/loxP-based mouse strains and can be applied to a variety of models in the research of liver disease.

Human hepatic organoids for the analysis of human genetic diseases.

We developed an in vitro model system where induced pluripotent stem cells (iPSCs) differentiate into 3-dimensional human hepatic organoids (HOs) through stages that resemble human liver during its embryonic development. The HOs consist of hepatocytes, and cholangiocytes, which are organized into epithelia that surround the lumina of bile duct-like structures. The organoids provide a potentially new model for liver regenerative processes, and were used to characterize the effect of different JAG1 mutations that cause: (a) Alagille syndrome (ALGS), a genetic disorder where NOTCH signaling pathway mutations impair bile duct formation, which has substantial variability in its associated clinical features; and (b) Tetralogy of Fallot (TOF), which is the most common form of a complex congenital heart disease, and is associated with several different heritable disorders. Our results demonstrate how an iPSC-based organoid system can be used with genome editing technologies to characterize the pathogenetic effect of human genetic disease-causing mutations.