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Abstract Background The incidence of hepatocellular carcinoma (HCC) is increasing in the western world over the past decades. As liver resection (LR) represents one of the most efficient treatment options, advantages of anatomic (ALR) versus non‐anatomic liver resection (NALR) show a lack of consistent evidence. Therefore, the aim of this study was to investigate complications and survival rates after both resection types. Methods This is a multicentre cohort study using retrospectively and prospectively collected data. We included all patients undergoing LR for HCC between 2009 and 2020 from three specialised centres in Switzerland and Germany. Complication and survival rates after ALR versus NALR were analysed using uni‐ and multivariate Cox regression models. Results Two hundred and ninety‐eight patients were included. Median follow‐up time was 52.76 months. 164/298 patients (55%) underwent ALR. Significantly more patients with cirrhosis received NALR (n = 94/134; p

Background & Aims: AXL and MERTK expression on circulating monocytes modulated immune responses in patients with cirrhosis (CD14+HLA-DR+AXL+) and acute-on-chronic liver failure (CD14+MERTK+). AXL expression involved enhanced efferocytosis, sustained phagocytosis, but reduced tumor necrosis factor-α/interleukin-6 production and T-cell activation, suggesting a homeostatic function. Axl was expressed on murine airway in tissues contacting the external environment, but not interstitial lung- and tissue-resident synovial lining macrophages. Here, we assessed AXL expression on tissue macrophages in patients with cirrhosis. Methods: Using multiplexed immunofluorescence we compared AXL expression in liver biopsies in cirrhosis (n = 22), chronic liver disease (n = 8), non-cirrhotic portal hypertension (n = 4), and healthy controls (n = 4). Phenotype and function of isolated primary human liver macrophages were characterized by flow cytometry (cirrhosis, n = 11; control, n = 14) ex vivo. Also, AXL expression was assessed on peritoneal (n = 29) and gut macrophages (n = 16) from cirrhotic patients. Regulation of AXL expression was analyzed in vitro and ex vivo using primary hepatic stellate cells (HSCs), LX-2 cells, and GAS6 in co-culture experiments. Results: AXL was expressed on resident (CD68+) but not tissue-infiltrating (MAC387+) liver macrophages, hepatocytes, HSCs, or sinusoidal endothelial cells. Prevalence of hepatic CD68+AXL+ cells significantly decreased with cirrhosis progression: (healthy, 90.2%; Child-Pugh A, 76.1%; Child-Pugh B, 64.5%; and Child-Pugh C, 18.7%; all P < .05) and negatively correlated with Model for End-Stage Liver Disease and C-reactive protein (all P < .05). AXL-expressing hepatic macrophages were CD68highHLA-DRhighCD16highCD206high. AXL expression also decreased on gut and peritoneal macrophages from cirrhotic patients but increased in regional lymph nodes. GAS6, enriched in the cirrhotic liver, appeared to be secreted by HSCs and down-regulate AXL in vitro. Conclusions: Decreased AXL expression on resident liver macrophages in advanced cirrhosis, potentially in response to activated HSC-secreted GAS6, suggests a role for AXL in the regulation of hepatic immune homeostasis.

Meier, Nuciforo, Coto-Llerena et al. report a rare case of a patient with hepatocellular carcinoma with neuroendocrine differentiation. The authors establish organoids that recapitulate the genetic features of the patient’s tumor and are used for ex vivo drug testing to guide therapy selection in the patient.

Hepatocellular carcinomas (HCCs) usually arise from chronic liver disease (CLD). Precancerous cells in chronically inflamed environments may be ‘epigenetically primed’, sensitising them to oncogenic transformation. We investigated whether epigenetic priming in CLD may affect HCC outcomes by influencing the genomic and transcriptomic landscapes of HCC. Analysis of DNA methylation arrays from 10 paired CLD‐HCC identified 339 shared dysregulated CpG sites and 18 shared differentially methylated regions compared with healthy livers. These regions were associated with dysregulated expression of genes with relevance in HCC, including ubiquitin D (UBD), cytochrome P450 family 2 subfamily C member 19 (CYP2C19) and O‐6‐methylguanine‐DNA methyltransferase (MGMT). Methylation changes were recapitulated in an independent cohort of nine paired CLD‐HCC. High CLD methylation score, defined using the 124 dysregulated CpGs in CLD and HCC in both cohorts, was associated with poor survival, increased somatic genetic alterations and TP53 mutations in two independent HCC cohorts. Oncogenic transcriptional and methylation dysregulation is evident in CLD and compounded in HCC. Epigenetic priming in CLD sculpts the transcriptional landscape of HCC and creates an environment favouring the acquisition of genetic alterations, suggesting that the extent of epigenetic priming in CLD could influence disease outcome.

Plain language summary Focal nodular hyperplasia (FNH) is a lesion resulting from the abnormal growth of liver cells. It is typically considered a benign tumor that does not become malignant. In rare cases, FNH may occur alongside malignant hepatocellular carcinoma (HCC). In these cases, it is not known whether the malignant HCC may derive from the benign FNH. In this study, we reported on the analysis of a 74-year-old female patient with co-occurring FNH and HCC. We found that the FNH and HCC lesions were in fact genetically related, suggesting that the FNH gave rise to the HCC lesions. Furthermore, we found multiple cell populations within the FHN lesion that may be precursors to the HCC lesions suggesting that, in rare cases, FNH may be capable of progressing to malignant HCC. These findings may help to refine the surveillance strategy for these lesions.

ObjectiveThe novel picture archiving and communication system (PACS), compatible with virtual reality (VR) software, displays cross-sectional images in VR. VR magnetic resonance cholangiopancreatography (MRCP) was tested to improve the anatomical understanding and intraoperative performance of minimally invasive cholecystectomy (CHE) in surgical trainees.DesignWe used an immersive VR environment to display volumetric MRCP data (Specto VRTM). First, we evaluated the tolerability and comprehensibility of anatomy with a validated simulator sickness questionnaire (SSQ) and examined anatomical landmarks. Second, we compared conventional MRCP and VR MRCP by matching three-dimensional (3D) printed models and identifying and measuring common bile duct stones (CBDS) using VR MRCP. Third, surgical trainees prepared for CHE with either conventional MRCP or VR MRCP, and we measured perioperative parameters and surgical performance (validated GOALS score).SettingThe study was conducted out at Clarunis, University Center for Gastrointestinal and Liver Disease, Basel, Switzerland.ParticipantsFor the first and second study step, doctors from all specialties and years of experience could participate. In the third study step, exclusively surgical trainees were included. Of 74 participating clinicians, 34, 27, and 13 contributed data to the first, second, and third study phases, respectively.ResultsAll participants determined the relevant biliary structures with VR MRCP. The median SSQ score was 0.75 (IQR: 0, 3.5), indicating good tolerability. Participants selected the corresponding 3D printed model faster and more reliably when previously studying VR MRCP compared to conventional MRCP: We obtained a median of 90 s (IQR: 55, 150) and 72.7% correct answers with VR MRCP versus 150 s (IQR: 100, 208) and 49.6% correct answers with conventional MRCP, respectively (p

Hepatocellular carcinoma (HCC) typically develops from a background of cirrhosis resulting from chronic inflammation. This inflammation is frequently associated with chronic liver diseases (CLD). The advent of next generation sequencing has enabled extensive analyses of molecular aberrations in HCC. However, less attention has been directed to the chronically inflamed background of the liver, prior to HCC emergence and during recurrence following surgery. Hepatocytes within chronically inflamed liver tissues present highly activated inflammatory signaling pathways and accumulation of a complex mutational landscape. In this altered environment, cells may transform in a stepwise manner toward tumorigenesis. Similarly, the chronically inflamed environment which persists after resection may impact the timing of HCC recurrence. Advances in research are allowing an extensive epigenomic, transcriptomic and proteomic characterization of CLD which define the emergence of HCC or its recurrence. The amount of data generated will enable the understanding of oncogenic mechanisms in HCC from the CLD perspective and provide the possibility to identify robust biomarkers or novel therapeutic targets for the treatment of primary and recurrent HCC. Importantly, biomarkers defined by the analysis of CLD tissue may permit the early detection or prevention of HCC emergence and recurrence. In this review, we compile the current omics based evidence of the contribution of CLD tissues to the emergence and recurrence of HCC.

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 HSPA1A. 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.

Abstract Background The physical oral health and dental behaviour of patients after solid organ transplantation (SOT) has repeatedly been reported as insufficient. The objective of this systematic review was to detect whether the oral health-related quality of life (OHRQoL) of patients after SOT is reduced compared to that of healthy individuals. Methods A systematic literature search was performed by two independent individuals based on the PubMed, Web of Science and Scopus databases by using the following search terms: “transplantation” AND “oral health-related quality of life”. The findings were checked to determine eligibility, whereby publication prior to 31 October 2020, examination of adult patients (age at least 18 years) with SOT, reporting of an OHRQoL outcome and full text in English language were the prerequisites for inclusion in the qualitative analysis. Quality appraisal of the included studies was performed using the Agency for Healthcare Research and Quality methodology checklist. Results Seven of 25 studies that examined patients after kidney (3), heart (2), liver (1) and lung transplantation (1) were included. Four studies included healthy controls, and five studies included a cohort of patients before transplantation for comparison. Clinical oral health examinations were heterogeneous between groups. The majority of studies (5/7) applied the short form of the “Oral Health Impact Profile” (OHIP 14) to assess OHRQoL. The OHIP 14 values ranged between 1.7 and 8.9 across studies, indicating an unaffected or just slightly reduced OHRQoL. Only one study found better OHRQoL in patients after SOT compared to a group before SOT, and one study confirmed worse OHRQoL of SOT recipients compared to a healthy control. Only two studies revealed an association between OHRQoL and oral health parameters. Furthermore, two studies each found a relationship between OHRQoL and general health-related quality of life or disease-related parameters. Conclusions Patients after SOT show an unaffected or only slightly reduced OHRQoL, which was mainly independent of the insufficient oral status. This might indicate a shift in the perception threshold for oral diseases and conditions caused by the general health burden related to the SOT.

The use of patient-derived organoids (PDO) as a valuable alternative to in vivo models significantly increased over the last years in cancer research. The ability of PDOs to genetically resemble tumor heterogeneity makes them a powerful tool for personalized drug screening. Despite the extensive optimization of protocols for the generation of PDOs from colorectal tissue, there is still a lack of standardization of tissue handling prior to processing, leading to microbial contamination of the organoid culture. Here, using a cohort of 16 patients diagnosed with colorectal carcinoma (CRC), we aimed to test the efficacy of phosphate-buffered saline (PBS), penicillin/streptomycin (P/S), and Primocin, alone or in combination, in preventing organoid cultures contamination when used in washing steps prior to tissue processing. Each CRC tissue was divided into 5 tissue pieces, and treated with each different washing solution, or none. After the washing steps, all samples were processed for organoid generation following the same standard protocol. We detected contamination in 62.5% of the non-washed samples, while the use of PBS or P/S-containing PBS reduced the contamination rate to 50% and 25%, respectively. Notably, none of the organoid cultures washed with PBS/Primocin-containing solution were contaminated. Interestingly, addition of P/S to the washing solution reduced the percentage of living cells compared to Primocin. Taken together, our results demonstrate that, prior to tissue processing, adding Primocin to the tissue washing solution is able to eliminate the risk of microbial contamination in PDO cultures, and that the use of P/S negatively impacts organoids growth. We believe that our easy-to-apply protocol might help increase the success rate of organoid generation from CRC patients.

Abstract Background Intra-arterial therapy with embolics is established for the treatment of malignancies of the liver. However, there are no studies comparing the different effects of various embolics used in clinical practice. Herein, we analyzed the effect of 3 different embolics on tumor growth in a rat model of colorectal liver metastases. Methods Eight days after subcapsular implantation of 5 × 105 colorectal cancer cells (CC531) in the left liver lobe of WAG/Rij rats were randomized into 4 groups (n = 8) and underwent intra-arterial hepatic therapy. Animals received either EmboCept S®, DC Bead® or Lipiodol® Ultra-Fluid. Animals of the control group received a comparable amount of saline. Tumor growth was measured on day 8 and 11 using a three-dimensional 40 MHz ultrasound device. On day 11 tumor and liver tissue were removed for histological and immunohistochemical analyses. Results On day 11 animals of the control group showed a tumor growth of ~ 60% compared to day 8. Application of Lipiodol Ultra-Fluid® did not significantly influence tumor growth (~ 40%). In contrast, treatment with EmboCept S® or DC Bead® completely inhibited tumor growth. Of interest, application of EmboCept S® did not only completely inhibit tumor growth but even decreased tumor size. Immunohistochemical analysis showed a significant increase of necrotic areas within the tumors after application of EmboCept S® and DC Bead® compared to Lipiodol® Ultra-Fluid. Conclusion The present study demonstrates that an intra-arterial therapy with EmboCept S® and DC Bead®, but not Lipiodol® Ultra-Fluid, results in a complete inhibition of rat colorectal liver metastatic growth.

Background. Neuroendocrine tumors (NETs) are a rare entity and are most commonly found in the gastroenteropancreatic tract. The clinical outcome depends on the potential resectability, grade, and stage. Here, we report a case of a tumor debulking in a metastatic NET of the pancreas. A 25-year-old woman with stable metastatic NET of the pancreas G2 T4N1M1 (hepatic, extrahepatic) already underwent several therapies. Case Presentation. A 25-year-old woman with stable metastatic NET of the pancreas G2 T4N1M1 (hepatic, extrahepatic) already underwent several pharmaceutical therapies. Due to the young age, the G2 characteristic, and the stable liver disease, the decision for debulking was made. Based on a 3D CT scan, an embolization was successfully performed directly prior to a pylorus-preserving pancreatic head resection, advanced interaortocaval lymph node dissection, and an atypical liver resection within segment VI. Histological workup revealed a stage pT3, G2, pN1 (29/34), pM1c (hepatic and extrahepatic), L1, V0, Pn0 with complete surgical resection of the primary tumor (180 mm). The excision of the liver segment V showed a completely resected metastasis. Conclusions. In this patient, extensive surgery of a pancreatic NET with the aim of a prolonged progression-free survival was performed. Close cooperation between different disciplines is absolutely mandatory. Modern imaging allowed a precise therapy plan to be worked out.

Background. Arterial ex situ back-table perfusion (BP) reportedly reduces ischemic-type biliary lesion after liver transplantation. We aimed to verify these findings in a prospective investigation. Methods. Our prospective, randomized, controlled, multicenter study involved livers retrieved from patients in 2 German regions, and compared the outcomes of standard aortic perfusion to those of aortic perfusion combined with arterial ex situ BP. The primary endpoint was the incidence of ischemic-type biliary lesions over a follow-up of 2 years after liver transplantation, whereas secondary endpoints included 2-year graft survival, initial graft damage as reflected by transaminase levels, and functional biliary parameters at 6 months after transplantation. Results. A total of 75 livers preserved via standard aortic perfusion and 75 preserved via standard aortic perfusion plus arterial BP were treated using a standardized protocol. The incidence of clinically apparent biliary lesions after liver transplantation (n = 9 for both groups; P = 0.947), the 2-year graft survival rate (standard aortic perfusion, 74%; standard aortic perfusion plus arterial BP, 68%; P = 0.34), and incidence of initial graft injury did not differ between the 2 perfusion modes. Although 33 of the 77 patients with cholangiography workups exhibited injured bile ducts, only 10 had clinical symptoms. Conclusions. Contrary to previous findings, the present study indicated that additional ex situ BP did not prevent ischemic-type biliary lesions or ischemia-reperfusion injury after liver transplantation. Moreover, there was considerable discrepancy between cholangiography findings regarding bile duct changes and clinically apparent cholangiopathy after transplantation, which should be considered when assessing ischemic-type biliary lesions.

BackgroundGraft-derived cell-free DNA (GcfDNA), which is released into the blood stream by necrotic and apoptotic cells, is a promising noninvasive organ integrity biomarker. In liver transplantation (LTx), neither conventional liver function tests (LTFs) nor immunosuppressive drug monitoring are very effective for rejection monitoring. We therefore hypothesized that the quantitative measurement of donor-derived cell-free DNA (cfDNA) would have independent value for the assessment of graft integrity, including damage from acute rejection.Methods and findingsTraditional LFTs were performed and plasma GcfDNA was monitored in 115 adults post-LTx at three German transplant centers as part of a prospective, observational, multicenter cohort trial. GcfDNA percentage (graft cfDNA/total cfDNA) was measured using droplet digital PCR (ddPCR), based on a limited number of predefined single nucleotide polymorphisms, enabling same-day turn-around. The same method was used to quantify blood microchimerism. GcfDNA was increased >50% on day 1 post-LTx, presumably from ischemia/reperfusion damage, but rapidly declined in patients without graft injury within 7 to 10 d to a median ConclusionsIn this study, determination of GcfDNA in plasma by ddPCR allowed for earlier and more sensitive discrimination of acute rejection in LTx patients as compared with conventional LFTs. Potential blood microchimerism was quantitatively low and had no significant influence on GcfDNA value. Further research, which should ideally include protocol biopsies, will be needed to establish the practical value of GcfDNA measurements in the management of LTx patients.

Highly promising preclinical data obtained in cultured cells and in nude mice bearing xenografts contrast with the rather modest clinical efficacy of Polo-like kinase 1 (Plk1) inhibitors. In the present study, we investigated if Plk1 might be a suitable target in hepatocellular carcinoma (HCC) and if a genetically engineered mouse tumor model that well reflects the tumor cell and micro-environmental features of naturally occurring cancers might be suitable to study anti-Plk1 therapy. Analysis of Plk1 expression in human HCC samples confirmed that HCC express much higher Plk1 levels than the adjacent normal liver tissue. Inhibition of Plk1 by an adenovirus encoding for a short hairpin RNA against Plk1 or by the small-molecule inhibitor BI 2536 reduced the viability of HCC cell lines and inhibited HCC xenograft progression in nude mice. Treatment of transforming growth factor (TGF) α/c-myc bitransgenic mice with BI 2536 during hepatocarcinogenesis reduced the number of dysplastic foci and of Ki-67-positive cells within the foci, indicating diminished tumorigenesis. In contrast, BI 2536 had no significant effect on HCC progression in the transgenic mouse HCC model as revealed by magnetic resonance imaging. Measurement of BI 2536 by mass spectrometry revealed considerably lower BI 2536 levels in HCC compared with the adjacent normal liver tissue. In conclusion, low intratumoral levels are a novel mechanism of resistance to the Plk1 inhibitor BI 2536. Plk1 inhibitors achieving sufficient intratumoral levels are highly promising in HCC treatment.

In a mouse model of established extrahepatic colorectal metastasis, we analyzed whether stromal cellderived factor (SDF) 1 stimulates tumor cell migration in vitro, angiogenesis, tumor growth in vivo. METHODS: Using chemotaxis chambers, CT26.WT colorectal tumor cell migration was studied under stimulation with different concentrations of SDF-1. To evaluate angiogenesis, tumor growth in vivo, green fluorescent protein-transfected CT26.WT cells were implanted in dorsal skinfold chambers of syngeneic BALB/c mice. After 5 days, tumors were locally exposed to SDF-1. Cell proliferation, tumor microvascularization, growth were studied during a further 9-day period using intravital fluorescence microscopy, histology, immunohistochemistry. Tumors exposed to PBS only served as controls. RESULTS:In vitro, > 30% of unstimulated CT26.WT cells showed expression of the SDF-1 receptor CXCR4. On chemotaxis assay, SDF-1 provoked a dose-dependent increase in cell migration. In vivo, SDF-1 accelerated neovascularization, induced a significant increase in tumor growth. Capillaries of SDF-1-treated tumors showed significant dilation. Of interest, SDF-1 treatment was associated with a significantly increased expression of proliferating cell nuclear antigen, a downregulation of cleaved caspase-3. CONCLUSION: Our study indicates that the CXC chemokine SDF-1 promotes tumor cell migration in vitro, tumor growth of established extrahepatic metastasis in vivo due to angiogenesis-dependent induction of tumor cell proliferation, inhibition of apoptotic cell death.

Portal hyperperfusion after extended hepatectomy or small-for-size liver transplantation may induce organ dysfunction and failure. The underlying mechanisms, however, are still not completely understood. Herein, we analysed whether hepatectomy-associated portal hyperperfusion induces a hepatic arterial buffer response, i.e., an adaptive hepatic arterial constriction, which may cause hepatocellular hypoxia and organ dysfunction.Sprague-Dawley rats underwent 30%, 70% and 90% hepatectomy. Baseline measurements before hepatectomy served as controls. Hepatic arterial and portal venous flows were analysed by ultrasonic flow measurement. Microvascular blood flow and mitochondrial redox state were determined by intravital fluorescence microscopy. Hepatic tissue pO2 was analysed by polarographic techniques. Hepatic function and integrity were studied by bromosulfophthalein bile excretion and liver histology.Portal blood flow was 2- to 4-fold increased after 70% and 90% hepatectomy. This, however, did not provoke a hepatic arterial buffer response. Nonetheless, portal hyperperfusion and constant hepatic arterial blood flow were associated with a reduced mitochondrial redox state and a decreased hepatic tissue pO2 after 70% and 90% hepatectomy. Microvascular blood flow increased significantly after hepatectomy and functional sinusoidal density was found only slightly reduced. Major hepatectomy further induced a 2- to 3-fold increase of bile flow. This was associated with a 2-fold increase of bromosulfophthalein excretion.Portal hyperperfusion after extended hepatectomy does not induce a hepatic arterial buffer response but reduces mitochondrial redox state and hepatocellular oxygenation. This is not due to a deterioration of microvascular perfusion, but rather due to a relative hypermetabolism of the remnant liver after major resection.