Impact of waiting time on post-transplant survival for recipients with hepatocellular carcinoma: A natural experiment randomized by blood group.

Background & Aims: When listing for liver transplantation, one can transplant as soon as possible or introduce a test-of-time to better select patients, as the tumor's biological behavior is observed. Knowing the degree of harm caused by time itself is essential to advise patients and decide on the maximum duration of the test-of-time. Therefore, we investigated the causal effect of waiting time on post-transplant survival for patients with hepatocellular carcinoma. Methods: We analyzed the UNOS-OPTN dataset and exploited a natural experiment created by blood groups. Relations between variables and assumptions were described in a causal graph. Selection bias was addressed by inverse probability weighting. Confounding was avoided using instrumental variable analysis, with an additive hazards model in the second stage. The causal effect was evaluated by estimating the difference in 5-year overall survival if all patients waited 2 months instead of 12 months. Upper bounds of the test-of-time were evaluated for probable scenarios by means of simulation. Results: The F-statistic of the first stage was 86.3. The effect of waiting 12 months vs. 2 months corresponded with a drop in overall survival rate of 5.07% (95% CI 0.277-9.69) and 8.33% (95% CI 0.47-15.60) at 5- and 10-years post-transplant, respectively. The median survival dropped by 3.41 years from 16.21 years (95% CI 15.98-16.60) for those waiting 2 months to 12.80 years (95% CI 10.72-15.90) for those waiting 12 months. Conclusions: From a patient's perspective, the choice between ablate-and-wait vs. immediate transplantation is in favor of immediate transplantation. From a policy perspective, the extra waiting time can be used to increase the utility of scarce donor livers. However, the duration of the test-of-time is bounded, and it likely should not exceed 8 months. Impact and implications: When listing patients with liver cancer for transplantation, it is unclear whether a test-of-time or immediate transplantation offer better outcomes at the population level. In this study, we found that increased liver transplant waiting times are detrimental in patients with liver cancer. Furthermore, our simulation showed that a pre-operative observational period can be useful to ensure good donor liver allocation, but that its duration should not exceed 8 months.

Cholangiocarcinoma landscape in Europe: Diagnostic, prognostic and therapeutic insights from the ENSCCA Registry.

BACKGROUND & AIMS: Cholangiocarcinoma (CCA) is a rare and heterogeneous biliary cancer, whose incidence and related mortality is increasing. This study investigates the clinical course of CCA and subtypes (intrahepatic [iCCA], perihilar [pCCA], and distal [dCCA]) in a pan-European cohort. METHODS: The ENSCCA Registry is a multicenter observational study. Patients were included if they had a histologically proven diagnosis of CCA between 2010-2019. Demographic, histomorphological, biochemical, and clinical studies were performed. RESULTS: Overall, 2,234 patients were enrolled (male/female=1.29). iCCA (n = 1,243) was associated with overweight/obesity and chronic liver diseases involving cirrhosis and/or viral hepatitis; pCCA (n = 592) with primary sclerosing cholangitis; and dCCA (n = 399) with choledocholithiasis. At diagnosis, 42.2% of patients had local disease, 29.4% locally advanced disease (LAD), and 28.4% metastatic disease (MD). Serum CEA and CA19-9 showed low diagnostic sensitivity, but their concomitant elevation was associated with increased risk of presenting with LAD (odds ratio 2.16; 95% CI 1.43-3.27) or MD (odds ratio 5.88; 95% CI 3.69-9.25). Patients undergoing resection (50.3%) had the best outcomes, particularly with negative-resection margin (R0) (median overall survival [mOS] = 45.1 months); however, margin involvement (R1) (hazard ratio 1.92; 95% CI 1.53-2.41; mOS = 24.7 months) and lymph node invasion (hazard ratio 2.13; 95% CI 1.55-2.94; mOS = 23.3 months) compromised prognosis. Among patients with unresectable disease (49.6%), the mOS was 10.6 months for those receiving active palliative therapies, mostly chemotherapy (26.2%), and 4.0 months for those receiving best supportive care (20.6%). iCCAs were associated with worse outcomes than p/dCCAs. ECOG performance status, MD and CA19-9 were independent prognostic factors. CONCLUSION: CCA is frequently diagnosed at an advanced stage, a proportion of patients fail to receive cancer-specific therapies, and prognosis remains dismal. Identification of preventable risk factors and implementation of surveillance in high-risk populations are required to decrease cancer-related mortality. LAY SUMMARY: This is, to date, the largest international (pan-European: 26 hospitals and 11 countries) observational study, in which the course of cholangiocarcinoma has been investigated, comparing the 3 subtypes based on the latest International Classification of Diseases 11th Edition (ICD-11) (i.e., intrahepatic [2C12], perihilar [2C18], or distal [2C15] affected bile ducts), which come into effect in 2022. General and tumor-type specific features at diagnosis, risk factors, biomarker accuracy, as well as patient management and outcomes, are presented and compared, outlining the current clinical state of cholangiocarcinoma in Europe.

Application of human liver organoids as a patient-derived primary model for HBV infection and related hepatocellular carcinoma.

The molecular events that drive hepatitis B virus (HBV)-mediated transformation and tumorigenesis have remained largely unclear, due to the absence of a relevant primary model system. Here we propose the use of human liver organoids as a platform for modeling HBV infection and related tumorigenesis. We first describe a primary ex vivo HBV-infection model derived from healthy donor liver organoids after challenge with recombinant virus or HBV-infected patient serum. HBV-infected organoids produced covalently closed circular DNA (cccDNA) and HBV early antigen (HBeAg), expressed intracellular HBV RNA and proteins, and produced infectious HBV. This ex vivo HBV-infected primary differentiated hepatocyte organoid platform was amenable to drug screening for both anti-HBV activity and drug-induced toxicity. We also studied HBV replication in transgenically modified organoids; liver organoids exogenously overexpressing the HBV receptor sodium taurocholate co-transporting polypeptide (NTCP) after lentiviral transduction were not more susceptible to HBV, suggesting the necessity for additional host factors for efficient infection. We also generated transgenic organoids harboring integrated HBV, representing a long-term culture system also suitable for viral production and the study of HBV transcription. Finally, we generated HBV-infected patient-derived liver organoids from non-tumor cirrhotic tissue of explants from liver transplant patients. Interestingly, transcriptomic analysis of patient-derived liver organoids indicated the presence of an aberrant early cancer gene signature, which clustered with the hepatocellular carcinoma (HCC) cohort on The Cancer Genome Atlas Liver Hepatocellular Carcinoma dataset and away from healthy liver tissue, and may provide invaluable novel biomarkers for the development of HCC and surveillance in HBV-infected patients.

Machine perfusion in abdominal organ transplantation: Current use in the Netherlands.

Scarcity of donor organs and the increment in patients awaiting a transplant increased the use of organs from expanded criteria donors or donation after circulatory death. Due to the suboptimal outcomes of these donor organs, there is an increased interest in better preservation methods, such as ex vivo machine perfusion or abdominal regional perfusion to improve outcomes. This state-of-the-art review aims to discuss the available types of perfusion techniques, its potential benefits and the available evidence in kidney, liver and pancreas transplantation. Additionally, translational steps from animal models towards clinical studies will be described, as well as its application to clinical practice, with the focus on the Netherlands. Despite the lack of evidence from randomized controlled trials, currently available data suggest especially beneficial effects of normothermic regional perfusion on biliary complications and ischemic cholangiopathy after liver transplantation. For ex vivo machine perfusion in kidney transplantation, hypothermic machine perfusion has proven to be beneficial over static cold storage in a randomized controlled trial, while normothermic machine perfusion is currently under investigation. For ex vivo machine perfusion in liver transplantation, normothermic machine perfusion has proven to reduce discard rates and early allograft dysfunction. In response to clinical studies, hypothermic machine perfusion for deceased donor kidneys has already been implemented as standard of care in the Netherlands.

Low skeletal muscle mass as a risk factor for postoperative delirium in elderly patients undergoing colorectal cancer surgery.

Background: Both low skeletal muscle mass (LSMM) and delirium are frequently seen in elderly patients. This study aimed to investigate the association between preoperative LSMM and postoperative delirium (POD) in elderly patients undergoing colorectal cancer (CRC) surgery and to design a model to predict POD. Patients and methods: This is a retrospective observational cohort study. Patients aged 70 years or older undergoing CRC surgery from January 2013 to October 2015 were included in this study. The cross-sectional skeletal muscle area at the level of the third lumbar vertebra using computed tomography was adjusted for patients' height, resulting in the skeletal muscle index. The lowest quartile per sex was defined as LSMM. Short Nutritional Assessment Questionnaire for Residential Care and KATZ-Activities of Daily Living were used to define malnourishment and physical dependency, respectively. POD was diagnosed using the Delirium Observational Screening Scale and geriatricians' notes. Results: Median age of the 251 included patients was 76 years (IQR, 73-80 years), of whom 56% of patients were males, 24% malnourished, and 15% physically impaired. LSMM and POD were diagnosed in 65 and 33 (13%) patients, respectively. POD occurred significantly more in patients with LSMM (25%) compared with patients without LSMM (10%), P=0.006. In the multivariate analysis, age, history of delirium, and LSMM were significantly associated with POD. In addition, this effect increased in patients with LSMM and malnourishment (P=0.019) or physical dependency (P=0.017). Conclusion: Age, history of delirium, LSMM, and malnourishment or physical dependency were independently associated with POD. Our nomogram could be used to identify patients at an increased risk for delirium. These patients may benefit from intensive monitoring to prevent POD.

Interaction of immunosuppressants with HCV antivirals daclatasvir and asunaprevir: combined effects with mycophenolic acid.

AIM: To investigate the specific effects of immunosuppressants on the antiviral action of daclatasvir and asunaprevir. METHODS: The antiviral activity of daclatasvir (DCV) and asunaprevir (ASV) combined with immunosuppressants was tested using two in vitro models for hepatitis C virus (HCV) infection. RESULTS: Tacrolimus, rapamycin and cyclosporine did not negatively affect the antiviral action of DCV or ASV. Mycophenolic acid (MPA) showed additive antiviral effects combined with these direct acting antivirals (DAAs). MPA induces interferon-stimulated genes (ISGs) and is a potent GTP synthesis inhibitor. DCV or ASV did not induce ISGs expression nor affected ISG induction by MPA. Rather, the combined antiviral effect of MPA with DCV and ASV was partly mediated via inhibition of GTP synthesis. CONCLUSION: Immunosuppressants do not negatively affect the antiviral activity of DAAs. MPA has additive effect on the antiviral action of DCV and ASV. This combined benefit needs to be confirmed in prospective clinical trials.

Management and outcome of hepatocellular adenoma with massive bleeding at presentation.

AIM: To evaluate outcome of acute management and risk of rebleeding in patients with massive hemorrhage due to hepatocellular adenoma (HCA). METHODS: This retrospective cohort study included all consecutive patients who presented to our hospital with massive hemorrhage (grade II or III) due to ruptured HCA and were admitted for observation and/or intervention between 1999-2016. The diagnosis of HCA was based on radiological findings from contrast-enhanced magnetic resonance imaging (MRI) or pathological findings from biopsy or resection of the HCA. Hemorrhage was diagnosed based on findings from computed tomography or MRI. Medical records were reviewed for demographic features, clinical presentation, tumor features, initial and subsequent management, short- and long-term complications and patient and lesion follow-up. RESULTS: All patients were female (n = 23). Treatment in the acute phase consisted of embolization (n = 9, 39.1%), conservative therapy (n = 13, 56.5%), and other intervention (n = 1, 4.3%). Median hemoglobin level decreased significantly more on days 0-3 in the intervention group than in the patients initially treated conservatively (0.9 mmol/L vs 2.4 mmol/L respectively, P = 0.006). In total, 4 patients suffered severe short-term complications, which included hypovolemic shock, acute liver failure and abscess formation. After a median follow-up of 36 mo, tumor regression in non-surgically treated patients occurred with a median reduction of 76 mm down to 25 mm. Four patients underwent secondary (elective) treatment (i.e., tumor resection) to address HCA size of > 5 cm and/or desire for future pregnancy. One case of rebleeding was documented (4.3%). None of the patients experienced long-term complication (mean follow-up time: 36 mo). CONCLUSION: With a 4.3% risk of rebleeding, secondary (elective) treatment of HCA after massive hemorrhage may only be considered in patients with persistent HCA > 5 cm.

Intrahepatic cholangiocarcinoma: current perspectives.

Intrahepatic cholangiocarcinoma (ICC) is the second most common malignancy arising from the liver. ICC makes up about 10% of all cholangiocarcinomas. It arises from the peripheral bile ducts within the liver parenchyma, proximal to the secondary biliary radicals. Histologically, the majority of ICCs are adenocarcinomas. Only a minority of patients (15%) present with resectable disease, with a median survival of less than 3 years. Multidisciplinary management of ICC is complicated by large differences in disease course for individual patients both across and within tumor stages. Risk models and nomograms have been developed to more accurately predict survival of individual patients based on clinical parameters. Predictive risk factors are necessary to improve patient selection for systemic treatments. Molecular differences between tumors, such as in the epidermal growth factor receptor status, are promising, but their clinical applicability should be validated. For patients with locally advanced disease, several treatment strategies are being evaluated. Both hepatic arterial infusion chemotherapy with floxuridine and yttrium-90 embolization aim to downstage locally advanced ICC. Selected patients have resectable disease after downstaging, and other patients might benefit because of postponing widespread dissemination and biliary obstruction.

Hydroxylated collagen peptide in urine as biomarker for detecting colorectal liver metastases.

The clinical efficacy of carcinoembryonic antigen (CEA) as a marker of colorectal liver metastasis is limited, motivating a search for new biomarkers. Recently, urine proteomic analysis revealed AGPP(-OH)GEAGKP(-OH)GEQGVP(-OH)GDLGAP(-OH)GP (AGP), a promising peptide for this application. This study aimed to determine whether combining urine AGP testing with serum CEA analyses improves the sensitivity of detecting colorectal liver metastases. Urine samples from 100 patients with CRLM were collected prospectively and compared to three control groups: healthy kidney donors, patients who were relapse-free for 24 months after curative CRLM surgery, and primary colorectal cancer patients. A stable isotope labeled peptide standard was used to quantify the abundance of AGP in urine samples by selective reaction monitoring. Combined testing of urine AGP levels and serum CEA levels revealed a significantly increased sensitivity compared to CEA alone (85% vs. 68%, P<0.001; specificity 84% and 91%, respectively). No correlation was found between CEA and AGP-positive test results within individual patients (r(2) = 0.08). Urine AGP testing was negative in the three control groups. These results indicate that collagen-derived urine AGP peptide with a specific hydroxylation pattern combined with serum CEA levels may significantly improve the detection of colorectal liver metastases in patients at risk.

GITR engagement in combination with CTLA-4 blockade completely abrogates immunosuppression mediated by human liver tumor-derived regulatory T cells ex vivo.

In liver cancer tumor-infiltrating regulatory T cells (Ti-Treg) are potent suppressors of tumor-specific T-cell responses and express high levels of the Treg-associated molecules cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and glucocorticoid-induced tumor necrosis factor receptor (GITR). In this study, we have evaluated the capacity of GITR-ligation, CTLA-4-blockade and a combination of both treatments to alleviate immunosuppression mediated by Ti-Treg. Using ex vivo isolated cells from individuals with hepatocellular carcinoma (HCC) or liver metastases from colorectal cancer (LM-CRC) we show that treatment with a soluble form of the natural ligand of GITR (GITRL), or with blocking antibodies to CTLA-4, reduces the suppression mediated by human liver tumor-infiltrating CD4+Foxp3+ Treg, thereby restoring proliferation and cytokine production by effector T cells. Importantly, combined treatment with low doses of both molecules exhibited stronger recovery of T cell function compared with either treatment alone. Our data suggest that in patients with primary and secondary liver cancer both GITR-ligation and anti-CTLA-4 mAb can improve the antitumor immunity by abrogating Ti-Treg mediated suppression.

Post-Transplantation Immunoadsorption Can Be Withheld in ABO-Incompatible Kidney Transplant Recipients.

After ABO-incompatible kidney transplantation, postoperative plasma exchange (PE) or immunoadsorption (IA) is performed per protocol or depending on postoperative A/B-titers to prevent acute rejection. However, the need for postoperative PE or IA is not known. Since 2006, 30 consecutive patients received three standard postoperative IAs. Starting from 2009, the last 46 patients received only preoperative IA. Preoperative desensitization consisted of rituximab, tacrolimus, mycophenolate mofetil, prednisone and intravenous immunoglobulins. Antigen-specific IA was performed pre-operatively with the Glycosorb device. Biopsy-proven acute rejections either antibody-mediated (AMR) or mixed cellular and antibody-mediated (MAR) within 3 months were recorded. The postoperative titer in patients with postoperative IA did not exceed 1:16 (IgG 1:4 [<2-16] median and range). The postoperative IgG titer was not significantly different after abandoning postoperative IA, although three patients had titers of 1:32 and one patient even 1:128. Rejections tended to be more frequent in the group with postoperative IA: 6 AMR and 3 MAR were recorded in 30 patients, vs. 4 AMR and 1 MAR in the 46 patients without postoperative IA (30 vs. 11%, P = 0.067). Baseline characteristics differed however: in the group with postoperative IA the vast majority had blood group O (87 vs. 52%, P = 0.003). Also, the IgG titer on the day of transplantation was higher (1:4 [<2-16] vs. 1:2 [<2-32], P = 0.007). All 14 patients with AMR and MAR rejections had postoperative IgG titers ≤1:16. Postoperative removal of A/B-antibodies can be safely removed from the ABOi transplantation protocol using strict preoperative criteria for antibody lowering.

Tumor-infiltrating plasmacytoid dendritic cells promote immunosuppression by Tr1 cells in human liver tumors.

CD4+ type 1 T regulatory (Tr1) cells have a crucial role in inducing tolerance. Immune regulation by these cells is mainly mediated through the secretion of high amounts of IL-10. Several studies have suggested that this regulatory population may be involved in tumor-mediated immune-suppression. However, direct evidence of a role for Tr1 cells in human solid tumors is lacking. Using ex vivo isolated cells from individuals with hepatocellular carcinoma (HCC; n = 39) or liver metastases from colorectal cancer (LM-CRC; n = 60) we identify a CD4+FoxP3-IL-13-IL-10+ T cell population in tumors of individuals with primary or secondary liver cancer that is characterized as Tr1 cells by the expression of CD49b and the lymphocyte activation gene 3 (LAG-3) and strong suppression activity of T cell responses in an IL-10 dependent manner. Importantly, the presence of tumor-infiltrating Tr1 cells is correlated with tumor infiltration of plasmacytoid dendritic cells (pDCs). pDCs exposed to tumor-derived factors enhance IL-10 production by Tr1 cells through up-regulation of the inducible co-stimulatory ligand (ICOS-L). These findings suggest a role for pDCs and ICOS-L in promoting intra-tumoral immunosuppression by Tr1 cells in human liver cancer, which may foster tumor progression and which might interfere with attempts of immunotherapeutic intervention.

Human Allogeneic Bone Marrow and Adipose Tissue Derived Mesenchymal Stromal Cells Induce CD8+ Cytotoxic T Cell Reactivity.

INTRODUCTION: For clinical applications, Mesenchymal Stromal Cells (MSC) can be isolated from bone marrow and adipose tissue of autologous or allogeneic origin. Allogeneic cell usage has advantages but may harbor the risk of sensitization against foreign HLA. Therefore, we evaluated whether bone marrow and adipose tissue-derived MSC are capable of inducing HLA-specific alloreactivity. METHODS: MSC were isolated from healthy human Bone Marrow (BM-MSC) and adipose tissue (ASC) donors. Peripheral Blood Mononuclear Cells (PBMC) were co-cultured with HLA-AB mismatched BM-MSC or ASC precultured with or without IFNy. After isolation via FACS sorting, the educated CD8+ T effector populations were exposed for 4 hours to Europium labeled MSC of the same HLA make up as in the co-cultures or with different HLA. Lysis of MSC was determined by spectrophotometric measurement of Europium release. RESULTS: CD8+ T cells educated with BM-MSC were capable of HLA specific lysis of BM-MSC. The maximum lysis was 24% in an effector:target (E:T) ratio of 40:1. Exposure to IFNγ increased HLA-I expression on BM-MSC and increased lysis to 48%. Co-culturing of PBMC with IFNγ-stimulated BM-MSC further increased lysis to 76%. Surprisingly, lysis induced by ASC was significantly lower. CD8+ T cells educated with ASC induced a maximum lysis of 13% and CD8+ T cells educated with IFNγ-stimulated ASC of only 31%. CONCLUSION: Allogeneic BM-MSC, and to a lesser extend ASC, are capable of inducing HLA specific reactivity. These results should be taken into consideration when using allogeneic MSC for clinical therapy.

Human Bone Marrow- and Adipose Tissue-derived Mesenchymal Stromal Cells are Immunosuppressive In vitro and in a Humanized Allograft Rejection Model.

BACKGROUND: Recent studies with bone marrow (BM)-derived Mesenchymal Stromal Cells (MSC) in transplant recipients demonstrate that treatment with MSC is safe and clinically feasible. While BM is currently the preferred source of MSC, adipose tissue is emerging as an alternative. To develop efficient therapies, there is a need for preclinical efficacy studies in transplantation. We used a unique humanized transplantation model to study the in vivo immunosuppressive effect of human BM-MSC and adipose tissue-derived MSC (ASC). METHODS: Gene expression of BM-MSC and ASC and their capacity to inhibit activated PBMC proliferation was evaluated. The in vivo immunosuppressive effect of BM-MSC and ASC was studied in a humanized mouse model. SCID mice were transplanted with human skin grafts and injected with human allogeneic PBMC with or without administration of BM-MSC or ASC. The effect of MSC on skin graft rejection was studied by immunohistochemistry and PCR. RESULTS: BM-MSC and ASC expressed TGFß, CXCL-10 and IDO. IDO expression and acitivity increased significantly in BM-MSC and ASC upon IFN-γ stimulation. IFN-γ stimulated BM-MSC and ASC inhibited the proliferation of activated PBMC in a significant and dose dependent manner. In our humanized mouse model, alloreactivity was marked by pronounced CD45+ T-cell infiltrates consisting of CD4+ and CD8+ T cells and increased IFN-γ expression in the skin grafts which were all significantly inhibited by both BM-MSC and ASC. CONCLUSION: BM-MSC and ASC are immunosuppressive in vitro and suppress alloreactivity in a preclinical humanized transplantation model.

Management strategy after diagnosis of Abernethy malformation: a case report.

INTRODUCTION: The Abernethy malformation is a rare anomaly with a widely variable clinical presentation. Many diagnostic dilemmas have been reported. Nowadays, with the evolution of medical imaging, diagnosis can be made more easily, but management of patients with an Abernethy malformation is still open for discussion. CASE PRESENTATION: In this case study, we describe a 34-year-old Caucasian man who presented with a large hepatocellular carcinoma in the presence of an Abernethy malformation, which was complicated by the development of pulmonary arterial hypertension. CONCLUSION: This case underlines the importance of regular examination of patients with an Abernethy malformation, even in older patients, to prevent complications and to detect liver lesions at an early stage.