Surgical pulmonary arterioplasty at bidirectional cavopulmonary anastomosis leads to favorable pulmonary hemodynamics at final stage palliation.

Objective: Pulmonary arterioplasty (PA plasty) at bidirectional cavopulmonary anastomosis (BDCA) is associated with increased morbidity, but outcomes to final stage palliation are unknown. We sought to determine the influence of PA plasty on pulmonary artery growth and hemodyamics at Fontan. Methods: We retrospectively reviewed clinical data and outcomes for BDCA patients from 2006 to 2018. PA plasty was categorized by extent (type 1-4), as previously described. Outcomes included pulmonary artery reintervention and mortality before final palliation. Results: Five hundred eighty-eight patients underwent BDCA. One hundred seventy-nine patients (30.0%) underwent concomitant PA plasty. Five hundred seventy (97%) patients (169 [94%] PA plasty) survived to BDCA discharge. One hundred forty out of 570 survivors (25%) required PA/Glenn reintervention before final stage palliation (59 out of 169 [35%]) PA plasty; 81 out of 401 (20%) non-PA plasty; P < .001). Twelve-, 24-, and 36-month freedom from reintervention after BDCA was 80% (95% CI, 74-86%), 75% (95% CI, 69-82%), and 64% (95% CI, 57-73%) for PA plasty, and 95% (95% CI, 93-97%), 91% (95% CI, 88-94%), and 81% (95% CI, 76-85%) for non-PA plasty (P < .001). Prefinal stage mortality was 37 (6.3%) (14 out of 169 PA plasty; 23 out of 401 non-PA plasty; P = .4). Five hundred four (144 PA plasty and 360 non-PA plasty) patients reached final stage palliation (471 Fontan, 26 1.5-ventricle, and 7 2-ventricular repair). Pre-Fontan PA pressure and pulmonary vascular resistance were 10 mm Hg (range, 9-12 mm Hg) and 1.6 mm Hg (range, 1.3-1.9 mm Hg) in PA plasty and 10 mm Hg (range, 8-12 mm Hg) and 1.5 mm Hg (range, 1.3-1.9 mm Hg) in non-PA plasty patients, respectively (P = .29, .6). Fontan hospital mortality, length of stay, and morbidity were similar. Conclusions: PA plasty at BDCA does not confer additional mortality risk leading to final palliation. Despite increased pulmonary artery reintervention, there was reliable pulmonary artery growth and favorable pulmonary hemodynamics at final stage palliation.

Spatially resolved immune exhaustion within the alloreactive microenvironment predicts liver transplant rejection.

Allograft rejection is common following clinical organ transplantation, but defining specific immune subsets mediating alloimmunity has been elusive. Calcineurin inhibitor dose escalation, corticosteroids, and/or lymphocyte depleting antibodies have remained the primary options for treatment of clinical rejection episodes. Here, we developed a highly multiplexed imaging mass cytometry panel to study the immune response in archival biopsies from 79 liver transplant (LT) recipients with either no rejection (NR), acute T cell-mediated rejection (TCMR), or chronic rejection (CR). This approach generated a spatially resolved proteomic atlas of 461,816 cells (42 phenotypes) derived from 96 pathologist-selected regions of interest. Our analysis revealed that regulatory (HLADR+ Treg) and PD1+ T cell phenotypes (CD4+ and CD8+ subsets), combined with variations in M2 macrophage polarization, were a unique signature of active TCMR. These data provide insights into the alloimmune microenvironment in clinical LT, including identification of potential targets for focused immunotherapy during rejection episodes and suggestion of a substantial role for immune exhaustion in TCMR.

Finance, race, ethnicity, and spoken language impact clinical outcomes for children with acute liver failure.

BACKGROUND: Pediatric acute liver failure (PALF) is an emergency, necessitating prompt referral and management at an experienced liver transplant center. Social determinants of health (SDOH) drive healthcare disparities and can affect many aspects of disease presentation, access to care, and ultimately clinical outcomes. Potential associations between SDOH and PALF outcomes, including spontaneous recovery (SR), liver transplant (LT) or death, are unknown. This study aims to investigate how SDOH may affect PALF and therefore identify areas for intervention to mitigate unrecognized disparities. METHODS: A retrospective, single-center cohort was analyzed and then compared and validated with data from the multicenter National Institutes of Health PALF Study Group. The single-center review included 145 patients admitted with PALF using diagnostic codes. Medical records were reviewed to extract patient demographics, family structure, inpatient social worker assessments, and clinical outcomes. Data were stratified by outcome. RESULTS: This analysis determined that level of family support (p = .02), caretaker employment (p = .02), patient age, race, and language (p = .01) may impact clinical outcomes. Specifically, the cohort of children that died had the largest proportion of non-English speaking patients with limited support systems and parents who worked full-time. Conversely, patients who underwent LT more often belonged to English-speaking families with a homemaker and extensive support systems. CONCLUSION: This study suggests that SDOH impact PALF outcomes and highlights patient populations facing additional challenges during an already complex healthcare emergency. These associations may indicate unconscious biases held by transplant teams when evaluating waitlist candidacy, as well as barriers to healthcare access. Strategies to better understand the broader applicability of our findings and, if confirmed, efforts to mitigate social disparities, may improve clinical outcomes in PALF.

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.

CHALF Score: A Novel Tool to Rapidly Risk Stratify Children in Need of Liver Transplant Evaluation During Acute Liver Failure.

BACKGROUND: Pediatric acute liver failure (PALF) can require emergent liver transplantation (LT, >25%) or lead to death (~15%). Existing models cannot predict clinical trajectory or survival with native liver (SNL). We aimed to create a predictive model for PALF clinical outcomes based on admission variables. METHODS: A retrospective, single-center PALF cohort (April 2003 to January 2022) was identified using International Classification of Disease codes, selected using National Institutes of Health PALF Study Group (PALFSG) criteria, and grouped by clinical outcome (SNL, LT, or death). Significant admission variables were advanced for feature selection using least absolute shrinkage and selection operator regression with bootstrapping (5000×). A predictive model of SNL versus LT or death was created using logistic regression and validated using PALFSG data. RESULTS: Our single-center cohort included 147 patients (58% SNL, 32% LT, 10% expired), while the PALFSG validation cohort included 492 patients (50% SNL, 35% LT, 15% expired). Admission variables associated with SNL included albumin (odds ratio [OR], 16; P < 0.01), ammonia (OR, 2.37; P < 0.01), and total bilirubin (OR, 2.25; P < 0.001). A model using these variables predicted SNL versus LT or death with high accuracy (accuracy [0.75 training, 0.70 validation], area under the curve [0.83 training, 0.78 validation]). A scaled score (CHLA-acute liver failure score) was created that predicted SNL versus LT or death with greater accuracy (C statistic 0.83) than Pediatric End-Stage Liver Disease (C statistic 0.76) and admission liver injury unit (C statistic 0.76) scores. CONCLUSIONS: The CHLA-acute liver failure score predicts SNL versus LT or mortality in PALF using admission laboratories with high accuracy. This novel, externally validated model offers an objective guide for urgent referral to a pediatric LT center.

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.

Spatially resolved immune exhaustion within the alloreactive microenvironment predicts liver transplant rejection.

Allograft rejection is a frequent complication following solid organ transplantation, but defining specific immune subsets mediating alloimmunity has been elusive due to the scarcity of tissue in clinical biopsy specimens. Single cell techniques have emerged as valuable tools for studying mechanisms of disease in complex tissue microenvironments. Here, we developed a highly multiplexed imaging mass cytometry panel, single cell analysis pipeline, and semi-supervised immune cell clustering algorithm to study archival biopsy specimens from 79 liver transplant (LT) recipients with histopathological diagnoses of either no rejection (NR), acute T-cell mediated rejection (TCMR), or chronic rejection (CR). This approach generated a spatially resolved proteomic atlas of 461,816 cells derived from 98 pathologist-selected regions of interest relevant to clinical diagnosis of rejection. We identified 41 distinct cell populations (32 immune and 9 parenchymal cell phenotypes) that defined key elements of the alloimmune microenvironment (AME), identified significant cell-cell interactions, and established higher order cellular neighborhoods. Our analysis revealed that both regulatory (HLA-DR+ Treg) and exhausted T-cell phenotypes (PD1+CD4+ and PD1+CD8+ T-cells), combined with variations in M2 macrophage polarization, were a unique signature of TCMR. TCMR was further characterized by alterations in cell-to-cell interactions among both exhausted immune subsets and inflammatory populations, with expansion of a CD8 enriched cellular neighborhood comprised of Treg, exhausted T-cell subsets, proliferating CD8+ T-cells, and cytotoxic T-cells. These data enabled creation of a predictive model of clinical outcomes using a subset of cell types to differentiate TCMR from NR (AUC = 0.96 ± 0.04) and TCMR from CR (AUC = 0.96 ± 0.06) with high sensitivity and specificity. Collectively, these data provide mechanistic insights into the AME in clinical LT, including a substantial role for immune exhaustion in TCMR with identification of novel targets for more focused immunotherapy in allograft rejection. Our study also offers a conceptual framework for applying spatial proteomics to study immunological diseases in archival clinical specimens.

Immunologic benefits of maternal living donor allografts in pediatric liver transplantation: fewer rejection episodes and no evidence of de novo allosensitization.

BACKGROUND: Pediatric liver transplant (LT) recipients of maternal living liver donor (LLD) grafts have been reported to experience fewer rejection episodes. However, it is unclear whether this benefit translates to reduction in developing donor-specific antibody (DSA) among maternal-LLD recipients. The aim of this study was to compare immunologic outcomes among maternal-LLD, non-maternal-LLD, and deceased donor liver transplant (DDLT) recipients. METHODS: Children (≤18 years) who underwent LT between 1/1998 and 12/2019 at two high-volume LT centers in North America were evaluated. Patients were divided into three groups by type of graft received (maternal-LLD, non-maternal LLD, and DDLT). Clinical variables and outcomes were compared according to each graft type. RESULTS: A total of 450 pediatric primary LT were analyzed: 275 (61.1%) DDLT, 73 (16.2%) maternal-LLD, and 102 (22.6%) non-maternal-LLD. Children receiving LLD grafts were less likely to develop rejection when compared to the DDLT group (DDLT 46.9% vs. maternal-LLD 31.5% vs. non-maternal-LLD 28.4%, p = 0.001). There was no difference in rejection rates between maternal and non-maternal-LLD recipients. A higher percentage of maternal-LLD recipients were on immunosuppression monotherapy compared to non-maternal-LLD and DDLT recipients (6.7% vs. 1.2 vs. 2.4%, respectively). A subgroup of 68 patients were tested for DSA post-LT. Maternal-LLD recipients were less likely to develop de novo DSA (maternal-LLD 11.8% vs. non-maternal-LLD 19.3% vs. DDLT 43%, p = 0.018). None of the maternal-LLD recipients developed antibody-mediated rejection. CONCLUSIONS: These data support the concept of immunologic benefit of maternal-LLD in pediatric LT, with lower rates of rejection and allosensitization post-LT when compared to DDLT recipients.

Clinical Value of Surveillance Biopsies in Pediatric Liver Transplantation.

Although pediatric liver transplantation (LT) results in excellent long-term outcomes, a high incidence of early acute cellular rejection and late graft fibrosis persists. Routine measurement of allograft enzymes may not reliably detect rejection episodes, identify candidates for immunosuppression minimization, or indicate allograft fibrosis. Surveillance biopsies (SBs) can provide valuable information in this regard, but their role in pediatric LT is not fully established. A retrospective cohort of 236 pediatric LT recipients from a high-volume center was studied to characterize the risks and benefits of SB versus for-cause biopsies (FCBs). The study population was 47.1% male and 54.7% Hispanic, and 31% received living donor grafts. Our data suggest that patients in the SB group had better transplant outcomes (rejection-free, graft, and patient survival) compared with patients who had FCBs or who never underwent biopsy. Among 817 biopsies obtained from 236 patients, 150 (18.4%) were SBs. Only 6 patients had a biopsy-related complication, and none were observed in the SB subset. Graft biochemical blood tests did not accurately predict rejection severity on biopsy, with aspartate aminotransferase area under the receiver operating characteristic curve (AUROC) 0.66, alanine aminotransferase AUROC 0.65 (very poor predictions), and gamma-glutamyltransferase AUROC 0.58 (no prediction). SBs identified subclinical rejection in 18.6% of biopsies, whereas 63.3% of SBs had evidence of fibrosis. SBs prompted changes in immunosuppression including dose reduction. Our experience suggests that SB in pediatric LT is safe, offers valuable information about subclinical rejection episodes, and can guide management of immunosuppression, including minimization. Improved outcomes with SB were likely multifactorial, potentially relating to a more favorable early posttransplant course and possible effect of management optimization through SB. Further multicenter studies are needed to examine the role of SBs on long-term outcomes in pediatric LT.

Non-invasive biomarkers of Fontan-associated liver disease.

BACKGROUND & AIMS: Fontan-associated liver disease (FALD) has emerged as an important morbidity following surgical palliation of single ventricle congenital heart disease. In this study, non-invasive biomarkers that may be associated with severity of FALD were explored. METHODS: A retrospective cohort of paediatric patients post-Fontan who underwent liver biopsy at a high volume at a paediatric congenital heart disease centre was reviewed. RESULTS: Among 106 patients, 66% were male and 69% were Hispanic. The mean age was 14.4 ± 3.5 years, and biopsy was performed 10.8 ± 3.6 years post-Fontan. The mean BMI was 20.8 ± 5 kg/m2, with 27.4% meeting obesity criteria. Bridging fibrosis was observed in 35% of patients, and 10.4% of all patients had superimposed steatosis. Bridging fibrosis was associated with lower platelet counts (168.3 ± 58.4 vs. 203.9 ± 65.8 K/µl for congestive hepatic fibrosis score [CHFS] 0-2b, p = 0.009), higher bilirubin (1.7 ± 2.2 vs. 0.9 ± 0.7 mg/dl, p = 0.0090), higher aspartate aminotransferase-to-platelet ratio index [APRI] and fibrosis-4 [FIB-4] scores (APRI: 0.5 ± 0.3 vs. 0.4 ± 0.1, p <0.01 [AUC: 0.69] and FIB-4: 0.6 ± 0.4 vs. 0.4 ± 0.2, p <0.01 [AUC: 0.69]), and worse overall survival (median 2 years follow-up post-biopsy, p = 0.027). Regression modelling of temporal changes in platelet counts before and after biopsy correlated with fibrosis severity (p = 0.005). CONCLUSIONS: In this large, relatively homogeneous adolescent population in terms of age, ethnicity, and Fontan duration, bridging fibrosis was observed in 35% of patients within the first decade post-Fontan. Bridging fibrosis was associated with worse survival. Changes in platelet counts, even years before biopsy, and APRI/FIB-4 scores had modest discriminatory power in identifying patients with advanced fibrosis. Steatosis may represent an additional risk factor for disease progression in obese patients. Further prospective studies are necessary to develop strategies to screen for FALD in the adolescent population. LAY SUMMARY: In this study, the prevalence of Fontan-associated liver disease (FALD) in the young adult population and clinical variables that may be predictive of fibrosis severity or adverse outcomes were explored. Several lab-based, non-invasive markers of bridging fibrosis in FALD were identified, suggesting that these values may be followed as a prognostic biomarker for FALD progression in the adolescent population.

Clinicopathologic Characteristics of Late Acute Antibody-mediated Rejection in Pediatric Liver Transplantation.

BACKGROUND: An early and accurate diagnosis of liver antibody-mediated rejection (AMR) followed by timely intervention is important for clinical management but remains challenging. The aim of this study was to assess the clinicopathologic characteristics and outcomes of late acute AMR in pediatric liver transplantation recipients. METHODS: We performed a retrospective review of 739 ABO-identical/compatible allograft liver biopsies from 199 pediatric transplantation recipients. RESULTS: Based on Banff 2016 AMR criteria, 3 recipients fulfilled the criteria for definite for late acute AMR, 2 met the criteria for suspicious for AMR, and 2 were indeterminate for AMR. We further assessed the clinicopathologic characteristics of these 7 patients. All 7 patients had at least 1 biopsy with a histopathologic pattern compatible with acute AMR. Additionally, we observed accompanied moderately to markedly dilated portal/central veins and endothelialitis disproportionate to the degree of bile duct injury in all 7 patients; periportal/perivenular hepatocyte necrosis was seen in 6 of 7 patients; and arteritis was seen in 3 of 7 patients. In each case, microvascular C4d deposition was present in at least 1 biopsy. Posttransplant donor specific anti-HLA antibodies were detected in 5 patients. Two of 7 patients were retransplanted, and 2 died after developing refractory AMR. The remaining 5 patients were alive with stable graft function at a median follow-up of 4.1 years. CONCLUSIONS: Our data suggest that acute AMR in pediatric liver grafts is rare, can develop late, and may be associated with graft loss or patient death. The recurrent histopathologic findings of moderately to markedly dilated portal/central veins and endothelialitis disproportionate to the degree of bile duct injury are features that appear unique to pediatric acute AMR of liver grafts.