Living Donor Liver Transplantation for Hepatocellular Carcinoma Within and Outside Traditional Selection Criteria: A Multicentric North American Experience.

OBJECTIVE: To evaluate long-term oncologic outcomes of patients post-living donor liver transplantation (LDLT) within and outside standard transplantation selection criteria and the added value of the incorporation of the New York-California (NYCA) score. BACKGROUND: LDLT offers an opportunity to decrease the liver transplantation waitlist, reduce waitlist mortality, and expand selection criteria for patients with hepatocellular carcinoma (HCC). METHODS: Primary adult LDLT recipients between October 1999 and August 2019 were identified from a multicenter cohort of 12 North American centers. Posttransplantation and recurrence-free survival were evaluated using the Kaplan-Meier method. RESULTS: Three hundred sixty LDLTs were identified. Patients within Milan criteria (MC) at transplantation had a 1, 5, and 10-year posttransplantation survival of 90.9%, 78.5%, and 64.1% versus outside MC 90.4%, 68.6%, and 57.7% ( P = 0.20), respectively. For patients within the University of California San Francisco (UCSF) criteria, respective posttransplantation survival was 90.6%, 77.8%, and 65.0%, versus outside UCSF 92.1%, 63.8%, and 45.8% ( P = 0.08). Fifty-three (83%) patients classified as outside MC at transplantation would have been classified as either low or acceptable risk with the NYCA score. These patients had a 5-year overall survival of 72.2%. Similarly, 28(80%) patients classified as outside UCSF at transplantation would have been classified as a low or acceptable risk with a 5-year overall survival of 65.3%. CONCLUSIONS: Long-term survival is excellent for patients with HCC undergoing LDLT within and outside selection criteria, exceeding the minimum recommended 5-year rate of 60% proposed by consensus guidelines. The NYCA categorization offers insight into identifying a substantial proportion of patients with HCC outside the MC and the UCSF criteria who still achieve similar post-LDLT outcomes as patients within the criteria.

Impact of gut microbiota on liver transplantation.

The gut microbiota has been gaining attention due to its interactions with the human body and its role in pathophysiological processes. One of the main interactions is the "gut-liver axis," in which disruption of the gut mucosal barrier seen in portal hypertension and liver disease can influence liver allograft function over time. For example, in patients who are undergoing liver transplantation, preexisting dysbiosis, perioperative antibiotic use, surgical stress, and immunosuppressive use have each been associated with alterations in gut microbiota, potentially impacting overall morbidity and mortality. In this review, studies exploring gut microbiota changes in patients undergoing liver transplantation are reviewed, including both human and experimental animal studies. Common themes include an increase in Enterobacteriaceae and Enterococcaceae species and a decrease in Faecalibacterium prausnitzii and Bacteriodes, while a decrease in the overall diversity of gut microbiota after liver transplantation.

Review of pediatric combined heart-liver transplantation: A roadmap to success.

BACKGROUND: Combined heart-liver transplantation (CHLT) is a promising technique to address end stage organ failure in patients with concomitant heart failure and chronic liver disease. While most experience with CHLT has involved adult patients, the expanding population of children born with univentricular congenital heart disease who underwent the Fontan procedure and develop Fontan-associated liver disease (FALD) has emerged as a growing indication for pediatric CHLT. METHODS: Currently, CHLT is performed at a select subset of experienced transplant centers, especially in the pediatric population. RESULTS: While technically demanding, CHLT may offer survival benefit when compared to heart transplant alone with decreased rejection of both synchronous allografts and equivalent outcomes with respect to waitlist time and post-operative complications. Limitations in the technique can be attributed to need for an appropriate multidisciplinary care center, challenges with donor organ availability and allocation, and the complexity associated with patient selection and peri-operative management. CONCLUSION: In this review, we summarize the history of CHLT, discuss patient selection, and highlight key facets of peri-operative care in the pediatric population.

Potential association between public medical insurance, waitlist mortality, and utilization of living donor liver transplantation: An analysis of the Scientific Registry of Transplant Recipients.

BACKGROUND: The Affordable Care Act (ACA) and subsequent Medicaid expansion has increased utilization of public health insurance. Living donor liver transplantation (LDLT) increases access to transplant and is associated with improved survival but consistently represents < 5% of LT in the United States. STUDY DESIGN: National registry data were analyzed to evaluate the impact of insurance payor on waitlist mortality and LDLT rates at LDLT centers since implementation of the ACA. RESULTS: Public insurance [Medicare RR 1.18 (1.13-1.22) P < .001, Medicaid RR 1.22 (1.18-1.27) P < .001], Latino ethnicity (P < .001), and lower education level (P = .02) were associated with increased waitlist mortality at LDLT centers. LDLT recipients were more likely to have private insurance (70.4% vs. 59.4% DDLT, P < .001), be Caucasian (92.1% vs. 83% DDLT, P < .001), and have post-secondary education (66.8% vs. 54.1% DDLT, P < .001). Despite 78% of LDLT centers being located in states with Medicaid expansion, there was no change in LDLT utilization among recipients with Medicaid (P = .196) or Medicare (P = .273). CONCLUSION: Despite Medicaid expansion, registry data suggests that patients with public medical insurance may experience higher waitlist mortality and underutilize LDLT at centers offering LDLT. It is possible that Medicaid expansion has not increased access to LDLT.

Case series and systematic review of acquired diaphragmatic hernia after liver transplantation.

BACKGROUND: ADH is a rare and potentially fatal complication following LT. In this study, a systematic review was completed to identify risk factors which may contribute to ADH. METHODS: Transplant databases at three LT programs were reviewed. Four pediatric and zero adult cases were identified. Next, a systematic review was completed. Fourteen studies describing 41 patients with ADH were identified. Patient demographics, transplant characteristics, and features of ADH diagnosis were examined. RESULTS: The majority (90.2%) of ADH were in children. In pediatric LT, 95.1% received a segmental allograft. ADH occurred in the right P diaphragm 92.7% of the time, and 87.8% were repaired primarily. Patient demographics, post-transplant complications, and immunosuppression regimens were broad and failed to predict ADH. Most patients presented with either respiratory or gastrointestinal symptoms. There were two pediatric deaths related to undiagnosed ADH. The combined worldwide incidence of ADH in pediatric LT is 1.5% (34/2319 patients). CONCLUSION: ADH is a rare complication post-LT that primarily occurs in pediatric recipients. When diagnosed early, ADH can be repaired primarily with good outcomes.

Technical optimization of spatially resolved single-cell transcriptomic datasets to study clinical liver disease.

Single cell and spatially resolved 'omic' techniques have enabled deep characterization of clinical pathologies that remain poorly understood, providing unprecedented insights into molecular mechanisms of disease. However, transcriptomic platforms are costly, limiting sample size, which increases the possibility of pre-analytical variables such as tissue processing and storage procedures impacting RNA quality and downstream analyses. Furthermore, spatial transcriptomics have not yet reached single cell resolution, leading to the development of multiple deconvolution methods to predict individual cell types within each transcriptome 'spot' on tissue sections. In this study, we performed spatial transcriptomics and single nucleus RNA sequencing (snRNAseq) on matched specimens from patients with either histologically normal or advanced fibrosis to establish important aspects of tissue handling, data processing, and downstream analyses of biobanked liver samples. We observed that tissue preservation technique impacts transcriptomic data, especially in fibrotic liver. Single cell mapping of the spatial transcriptome using paired snRNAseq data generated a spatially resolved, single cell dataset with 24 unique liver cell phenotypes. We determined that cell-cell interactions predicted using ligand-receptor analysis of snRNAseq data poorly correlated with cellular relationships identified using spatial transcriptomics. Our study provides a framework for generating spatially resolved, single cell datasets to study gene expression and cell-cell interactions in biobanked clinical samples with advanced liver disease.

Technical optimization of spatially resolved single-cell transcriptomic datasets to study clinical liver disease.

Single cell and spatially resolved 'omic' techniques have enabled deep characterization of clinical pathologies that remain poorly understood, providing unprecedented insights into molecular mechanisms of disease. However, transcriptomic platforms are costly, limiting sample size, which increases the possibility of pre-analytical variables such as tissue processing and storage procedures impacting RNA quality and downstream analyses. Furthermore, spatial transcriptomics have not yet reached single cell resolution, leading to the development of multiple deconvolution methods to predict individual cell types within each transcriptome 'spot' on tissue sections. In this study, we performed spatial transcriptomics and single nucleus RNA sequencing (snRNASeq) on matched specimens from patients with either histologically normal or advanced fibrosis to establish important aspects of tissue handling, data processing, and downstream analyses of biobanked liver samples. We observed that tissue preservation technique impacts transcriptomic data, especially in fibrotic liver. Deconvolution of the spatial transcriptome using paired snRNASeq data generated a spatially resolved, single cell dataset with 24 unique liver cell phenotypes. We determined that cell-cell interactions predicted using ligand-receptor analysis of snRNASeq data poorly correlated with celullar relationships identified using spatial transcriptomics. Our study provides a framework for generating spatially resolved, single cell datasets to study gene expression and cell-cell interactions in biobanked clinical samples with advanced liver disease.

Vascular endothelial growth factor expression in hepatic epithelioid hemangioendothelioma: Implications for treatment and surgical management.

Epithelioid hemangioendothelioma (EHE) is a low-grade, malignant vascular tumor that most commonly presents within the liver. Patients with hepatic EHE are often candidates for liver transplantation as the disease is usually multifocal at diagnosis. Although these patients achieve excellent early outcomes post-transplant, there are very few data regarding tumor markers that can further direct chemotherapy in hepatic EHE to prevent recurrent disease. The purpose of this study was to analyze the expression of the angiogenic factor vascular endothelial growth factor (VEGF) and its receptors in hepatic EHE. Six patients with hepatic EHE were assessed for liver transplantation at our center. Pathology specimens of primary and recurrent EHE were analyzed by hematoxylin and eosin staining and by immunofluorescence for VEGF, fetal liver kinase 1 (Flk-1), and fms-related tyrosine kinase 1 (Flt-1) expression. Five patients underwent liver transplantation, and 1 patient underwent liver resection. Biopsy-proven recurrent EHE occurred in 3 patients. VEGF expression was present in 100% of the EHE specimens examined, whereas Flt-1 expression was present in only 1 sample, and Flk-1 was not observed in any of the specimens. In 1 patient with recurrent hepatic EHE post-liver transplantation, a progressive increase in the VEGF fluorescence intensity and distribution was observed. In conclusion, in this series, VEGF expression was observed in all hepatic EHE specimens analyzed. These data suggest that anti-VEGF chemotherapeutic agents will be of use in patients with hepatic EHE, particularly as a means of reducing the tumor volume prior to resection, as a means of treating unresectable or metastatic disease, or as an adjuvant therapy in the setting of liver transplantation.

HCV Eradication with Direct-Acting Antivirals Does Not Impact HCC Progression on the Waiting List or HCC Recurrence after Liver Transplantation.

Background: The introduction of direct-acting antivirals (DAA) for HCV has led to high rates of HCV eradication. Treatment of patients awaiting liver transplantation (LT) has been controversial. Recent data suggests that DAA treatment may accelerate recurrent HCC. The impact of DAA on delisting for HCC progression or recurrent HCC post-LT has not been well characterized. Methods: A retrospective review of both waitlist patients and LT recipients at a single institution was performed. Patient demographics, HCV treatment, HCC features and treatments, biopsy results, and graft and patient survival were evaluated. Patients on the LT waitlist or who were transplanted between January 2014 and December 2015 were included. Data was collected through December 2017 to have a minimum of two years of follow-up. Results: In the study period, 128 adult LT were performed. 44 patients were HCV+, and 68.2% (N=30) also had HCC. 38.6% (N=17) of HCV+ patients received DAA pre-LT, and 94.1% (N=16/17) achieved sustained virologic response (SVR) pre-LT. Among untreated HCV+ patients who underwent LT, 81.5% (N=22/27) received DAA post-LT, with 82.6% achieving SVR post-LT (N=18/22). 82.1% (N=23/28) of untreated post-LT patients underwent liver biopsy prior to therapy, and 52.2% had at least F1 METAVIR fibrosis. 87.5% (N=14/16) of active waitlist patients received DAA and achieved SVR. HCV eradication did not result in higher rates of delisting for HCC progression. Due to local HCC listing criteria of total tumor volume and AFP, 60% (N=18/30) of HCV+/HCC patients were beyond Milan criteria at the time of LT. Despite this, there was no difference in HCC recurrence rates post-LT, whether patients achieved SVR pre- or post-LT. Conclusions: These data suggest that HCV eradication pre-LT does not significantly impact waitlist time for HCV+ patients with HCC. HCV eradication does not impact rates of delisting for HCC progression or rates of HCC recurrence post-LT.

Belatacept and basiliximab diminish human antiporcine xenoreactivity and synergize to inhibit alloimmunity.

BACKGROUND: Maintenance immunosuppression with calcineurin inhibitor therapy has improved survival rates in solid organ transplantation over the past decade. However, these drugs are associated with negative side effects, including nephrotoxicity and new-onset diabetes. Selective immunomodulatory agents such as belatacept and basiliximab have shown great promise in promoting allograft survival. In the present study, in vitro experiments were conducted to determine if these agents could synergize to inhibit immune responses. METHODS: Porcine and human lymphocytes were incubated with each drug and analyzed using flow cytometry to measure binding capability. The inhibitory effects of each drug were evaluated using mixed lymphocyte reactions with drug doses comparable to the trough levels observed in treated human patients. RESULTS: Our data demonstrates that belatacept and basiliximab bind to porcine peripheral blood mononuclear cells. Mixed lymphocyte reactions revealed that both belatacept and basiliximab monotherapy potently inhibited allogeneic immune responses and human antipig xenoreactivity. These data also demonstrate that combination of belatacept and basiliximab produces a synergistic inhibition of allogeneic immune responses. CONCLUSIONS: These studies suggest that the combination of belatacept and basiliximab will potently inhibit alloreactivity in vivo when used as maintenance immunosuppression. We have further shown that belatacept and basiliximab are significantly reduce xenoreactivity of human lymphocytes in vitro.

Hepatic Epithelioid Hemangioendothelioma Presenting as an Enlarging Vascular Lesion within the Spleen.

Epithelioid hemangioendothelioma (EHE) is a rare vascular neoplasm with variable malignant potential that most often presents within the liver. Many patients present with bilobar or extrahepatic disease, and the current treatment paradigm involves liver transplantation, with favorable long term results. Up to 25% of patients are diagnosed incidentally following imaging for other indications, and confirmation of diagnosis requires histologic analysis, as there are no classical imaging features to distinguish hepatic EHE (HEHE) from other solid hepatic lesions. Here we describe a case of microscopic HEHE that was diagnosed following splenectomy for an enlarging vascular tumor within the spleen. Due to the unexpected diagnosis of EHE within the spleen and coexisting but stable appearing liver hemangiomata, a left hepatic lobectomy was performed. Explant histology revealed benign hemangiomata and diffuse, microscopic HEHE. The patient ultimately underwent liver transplantation. HEHE can be a challenging diagnosis, and this case emphasizes that any enlarging vascular lesion, even within the spleen, should prompt a high index of suspicion for HEHE in the setting of known hemangiomata.

Interventional strategies to prevent beta-cell apoptosis in islet transplantation.

A substantial proportion of the transplanted islet mass fails to engraft due to death by apoptosis, and a number of strategies have been explored to inhibit beta-cell loss. Inhibition of extrinsic signals of apoptosis (i.e., cFLIP or A20) have been explored in experimental islet transplantation but have only shown limited impact. Similarly, strategies targeted at intrinsic signal inhibition (i.e., BCL-2) have not yet provided substantial improvement in islet engraftment. Recently, investigation of downstream apoptosis inhibitors that block the final common pathway (i.e., X-linked inhibitor of apoptosis protein [XIAP]) have demonstrated promise in both human and rodent models of engraftment. In addition, XIAP has enhanced long-term murine islet allograft survival. The complexities of both intrinsic and extrinsic apoptotic pathway inhibition are discussed in depth.

Factors influencing the loss of beta-cell mass in islet transplantation.

Recent advances in clinical islet transplantation have clearly demonstrated that this procedure can provide excellent glycemic control and often insulin independence in a population of patients with type 1 diabetes. A key limitation in the widespread application of clinical islet transplantation is the requirement of 10,000 islet equivalents/kg in most recipients, generally derived from two or more cadaveric donors. It has been determined that a majority of the transplanted islets fail to engraft and become fully functional. In this review article, the factors that contribute to this early loss of islets following transplantation are discussed in depth.

XIAP overexpression in human islets prevents early posttransplant apoptosis and reduces the islet mass needed to treat diabetes.

The Edmonton Protocol for treatment of type 1 diabetes requires islets from two or more donors to achieve euglycemia in a single recipient, primarily because soon after portal infusion, the majority of the transplanted cells undergo apoptosis due to hypoxia and hypoxia reperfusion injury. X-linked inhibitor of apoptosis protein (XIAP) is a potent endogenous inhibitor of apoptosis that is capable of blocking the activation of multiple downstream caspases, and XIAP overexpression has previously been shown to enhance engraftment of a murine beta-cell line. In this study, human islets transduced with a XIAP-expressing recombinant adenovirus were resistant to apoptosis and functionally recovered following in vitro stresses of hypoxia and hypoxia with reoxygenation (models reperfusion injury). Furthermore Ad-XIAP transduction dramatically reduced the number of human islets required to reverse hyperglycemia in chemically diabetic immunodeficient mice. These results suggest that by transiently overexpressing XIAP in the immediate posttransplant period, human islets from a single donor might be used to effectively treat two diabetic recipients.

Neonatal porcine islets exhibit natural resistance to hypoxia-induced apoptosis.

BACKGROUND: Despite the success of the Edmonton protocol for human islet transplantation, an alternate source of islet tissue must be developed if beta-cell replacement therapy is to see widespread application. Neonatal porcine islets (NPI) represent one potential source of tissue. When human or rodent islets are transplanted, the majority of cells undergo hypoxia-induce apoptosis soon after the grafts are placed in the recipient. In the present study, we investigated whether NPI were similarly sensitive to hypoxia. METHODS: NPI were exposed to hypoxia and hypoxia/reoxygenation using an in vitro hypoxic chamber. Afterwards, viability, frequency of apoptosis, and beta-cell function were evaluated. NPI and adult porcine islets were transplanted into chemically diabetic, immunodeficient mice and graft apoptosis was assessed 24 hours and seven days posttransplant. RESULTS: NPI demonstrated a remarkable capacity to resist apoptosis and maintain insulin secretion despite severe stresses such as hypoxia/reoxygenation. One day after transplantation, NPI grafts showed limited apoptosis, confined to rare strongly insulin positive cells. In contrast, adult porcine islet grafts underwent widespread apoptosis. Western blotting revealed that NPI express high levels of at least one potent endogenous antiapoptotic protein (XIAP). CONCLUSIONS: The majority of cells within transplanted human islets undergo apoptosis soon after portal infusion. In contrast, NPI have the capacity to resist this early posttransplant apoptosis, with likely reduced antigen release and diminished immune stimulation. NPI appear to contain a population of insulin-low to insulin-negative pre-beta-cells, which are resistant to hypoxia-induced apoptosis and still capable of differentiating into mature beta-cells.

AEB071 (sotrastaurin) does not exhibit toxic effects on human islets in vitro, nor after transplantation into immunodeficient mice.

AEB071 (AEB, sotrastaurin), a specific inhibitor of protein kinase C, reduces T-lymphocyte activation and cytokine release. AEB delays islet allograft rejection in rats and prevents rejection when combined with cyclosporine. Since many immunosuppressive agents have toxic effects on the function of transplanted islets, we investigated whether this was also the case with AEB. Human islets were transplanted into Rag-knockout mice randomly assigned to vehicle control, AEB or sirolimus treatment groups. Non-fasting blood glucose levels, body weight and glucose tolerance was measured in recipients. In a separate experiment, human islets were cultured in the presence of AEB and assayed for glucose dependent insulin secretion and level of ß-cell apoptosis. Eighty-six percent of the AEB-treated recipients achieved normoglycemia following transplant (compared with none in sirolimus-treated group, p < 0.05). AEB-treated recipients exhibited similar glucose homeostasis as vehicle-treated controls, which was better than in sirolimus-treated recipients. Human islets cultured with AEB showed similar rates of ß-cell apoptosis (p = 0.98 by one-way ANOVA) and glucose stimulated insulin secretion (p = 0.15) as those cultured with vehicle. These results suggest that AEB is not associated with toxic effects on islet engraftment or function. AEB appears to be an appropriate immunosuppressive candidate for clinical trials in islet transplantation.