Banff 2022 Liver Group Meeting report: Monitoring long-term allograft health.

The Banff Working Group on Liver Allograft Pathology met in September 2022. Participants included hepatologists, surgeons, pathologists, immunologists, and histocompatibility specialists. Presentations and discussions focused on the evaluation of long-term allograft health, including noninvasive and tissue monitoring, immunosuppression optimization, and long-term structural changes. Potential revision of the rejection classification scheme to better accommodate and communicate late T cell-mediated rejection patterns and related structural changes, such as nodular regenerative hyperplasia, were discussed. Improved stratification of long-term maintenance immunosuppression to match the heterogeneity of patient settings will be central to improving long-term patient survival. Such personalized therapeutics are in turn contingent on a better understanding and monitoring of allograft status within a rational decision-making approach, likely to be facilitated in implementation with emerging decision-support tools. Proposed revisions to rejection classification emerging from the meeting include the incorporation of interface hepatitis and fibrosis staging. These will be opened to online testing, modified accordingly, and subject to consensus discussion leading up to the next Banff conference.

Obliteration of portal venules contributes to portal hypertension in biliary cirrhosis.

The effects of the obliteration of portal venules (OPV) in cirrhotic portal hypertension are poorly understood. To investigate its contribution to portal hypertension in biliary cirrhosis and its underlying mechanism, we evaluated OPV using two-dimensional (2D) histopathology in liver explants from patients with biliary atresia (BA, n = 63), primary biliary cholangitis (PBC, n = 18), and hepatitis B-related cirrhosis (Hep-B-cirrhosis, n = 35). Then, three-dimensional (3D) OPV was measured by X-ray phase-contrast CT in two parallel models in rats following bile duct ligation (BDL) or carbon tetrachloride (CCl4) administration, representing biliary cirrhosis and post-necrotic cirrhosis, respectively. The portal pressure was also measured in the two models. Finally, the effects of proliferative bile ducts on OPV were investigated. We found that OPV was significantly more frequent in patients with biliary cirrhosis, including BA (78.57 ± 16.45%) and PBC (60.00 ± 17.15%), than that in Hep-B-cirrhotic patients (29.43 ± 14.94%, p < 0.001). OPV occurred earlier, evidenced by the paired liver biopsy at a Kasai procedure (KP), and was irreversible even after a successful KP in the patients with BA. OPV was also significantly more frequent in the BDL models than in the CCl4 models, as shown by 2D and 3D quantitative analysis. Portal pressure was significantly higher in the BDL model than that in the CCl4 model. With the proliferation of bile ducts, portal venules were compressed and irreversibly occluded, contributing to the earlier and higher portal pressure in biliary cirrhosis. OPV, as a pre-sinusoidal component, plays a key role in the pathogenesis of portal hypertension in biliary cirrhosis. The proliferated bile ducts and ductules gradually take up the 'territory' originally attributed to portal venules and compress the portal venules, which may lead to OPV in biliary cirrhosis. © 2024 The Pathological Society of Great Britain and Ireland.

Multiple infusions of ex vivo-expanded regulatory T cells promote CD163+ myeloid cells and kidney allograft survival in non-lymphodepleted non-human primates.

Clinical verification of adoptively transferred regulatory T cell (Treg) efficacy in transplantation remains challenging. Here, we examined the influence of autologous ex vivo-expanded polyclonal Tregs on kidney graft survival in a clinically relevant non-human primate model. Peripheral blood Tregs were isolated and expanded using artificial antigen presenting cells. Immunosuppression was comprised of tapered tacrolimus and CTLA4 immunoglobulin, in five animals each without or with Treg infusions. Escalating Treg doses were administered 6, 10, 13, 16, 20, 23, 27 and 30 days after transplant. Infused Tregs were monitored for Treg signature, anti-apoptotic (Bcl-2) and proliferation (Ki67) marker expression. Treg infusions prolonged median graft survival time significantly from 35 to 70 days. Treg marker (Ki67 and Bcl-2) expression by infused Tregs diminished after their infusion but remained comparable to that of circulating native Tregs. No major changes in circulating donor-reactive T cell responses or total Treg percentages, or in graft-infiltrating T cell subsets were observed with Treg infusion. However, Treg infusion was associated with significant increases in CD163 expression by circulating HLA-DR+ myeloid cells and elevated levels of circulating soluble CD163. Further, graft-infiltrating CD163+ cells were increased with Treg infusion. Thus, multiple Treg infusions were associated with M2-like myeloid cell enhancement that may mediate immunomodulatory, anti-inflammatory and graft reparative effects.

Next-generation pathology detection of T cell-antigen-presenting cell immune synapses in human liver allografts.

BACKGROUND AND AIMS: In otherwise near-normal appearing biopsies by routine light microscopy, next-generation pathology (NGP) detected close pairings (immune pairs; iPAIRs) between lymphocytes and antigen-presenting cells (APCs) that predicted immunosuppression weaning failure in pediatric liver transplant (LTx) recipients (Immunosuppression Withdrawal for Stable Pediatric Liver Transplant Recipients [iWITH], NCT01638559). We hypothesized that NGP-detected iPAIRs enrich for true immune synapses, as determined by nuclear shape metrics, intercellular distances, and supramolecular activation complex (SMAC) formation. APPROACH AND RESULTS: Intralobular iPAIRs (CD45 high lymphocyte-major histocompatibility complex II + APC pairs; n = 1167, training set) were identified at low resolution from multiplex immunohistochemistry-stained liver biopsy slides from several multicenter LTx immunosuppression titration clinical trials (iWITH; NCT02474199 (Donor Alloantigen Reactive Tregs (darTregs) for Calcineurin Inhibitor (CNI) Reduction (ARTEMIS); Prospective Longitudinal Study of iWITH Screen Failures Secondary to Histopathology). After excluding complex multicellular aggregates, high-resolution imaging was used to examine immune synapse formation ( n = 998). By enriching for close intranuclear lymphocyte-APC distance (mean: 0.713 µm) and lymphocyte nuclear flattening (mean ferret diameter: 2.1), SMAC formation was detected in 29% of iPAIR-engaged versus 9.5% of unpaired lymphocytes. Integration of these morphometrics enhanced NGP detection of immune synapses (ai-iSYN). Using iWITH preweaning biopsies from eligible patients ( n = 53; 18 tolerant, 35 nontolerant; testing set), ai-iSYN accurately predicted (87.3% accuracy vs. 81.4% for iPAIRs; 100% sensitivity, 75% specificity) immunosuppression weaning failure. This confirmed the presence and importance of intralobular immune synapse formation in liver allografts. Stratification of biopsy mRNA expression data by immune synapse quantity yielded the top 20 genes involved in T cell activation and immune synapse formation and stability. CONCLUSIONS: NGP-detected immune synapses (subpathological rejection) in LTx patients prior to immunosuppression reduction suggests that NGP-detected (allo)immune activity usefulness for titration of immunosuppressive therapy in various settings.

Transcriptome and Exome Analyses of Hepatocellular Carcinoma Reveal Patterns to Predict Cancer Recurrence in Liver Transplant Patients.

Hepatocellular carcinoma (HCC) is one of the most lethal human cancers. Liver transplantation has been an effective approach to treat liver cancer. However, significant numbers of patients with HCC experience cancer recurrence, and the selection of suitable candidates for liver transplant remains a challenge. We developed a model to predict the likelihood of HCC recurrence after liver transplantation based on transcriptome and whole-exome sequencing analyses. We used a training cohort and a subsequent testing cohort based on liver transplantation performed before or after the first half of 2012. We found that the combination of transcriptome and mutation pathway analyses using a random forest machine learning correctly predicted HCC recurrence in 86.8% of the training set. The same algorithm yielded a correct prediction of HCC recurrence of 76.9% in the testing set. When the cohorts were combined, the prediction rate reached 84.4% in the leave-one-out cross-validation analysis. When the transcriptome analysis was combined with Milan criteria using the k-top scoring pairs (k-TSP) method, the testing cohort prediction rate improved to 80.8%, whereas the training cohort and the combined cohort prediction rates were 79% and 84.4%, respectively. Application of the transcriptome/mutation pathways RF model on eight tumor nodules from 3 patients with HCC yielded 8/8 consistency, suggesting a robust prediction despite the heterogeneity of HCC. Conclusion: The genome prediction model may hold promise as an alternative in selecting patients with HCC for liver transplant.

Graft IL-33 regulates infiltrating macrophages to protect against chronic rejection.

Alarmins, sequestered self-molecules containing damage-associated molecular patterns, are released during tissue injury to drive innate immune cell proinflammatory responses. Whether endogenous negative regulators controlling early immune responses are also released at the site of injury is poorly understood. Herein, we establish that the stromal cell-derived alarmin interleukin 33 (IL-33) is a local factor that directly restricts the proinflammatory capacity of graft-infiltrating macrophages early after transplantation. By assessing heart transplant recipient samples and using a mouse heart transplant model, we establish that IL-33 is upregulated in allografts to limit chronic rejection. Mouse cardiac transplants lacking IL-33 displayed dramatically accelerated vascular occlusion and subsequent fibrosis, which was not due to altered systemic immune responses. Instead, a lack of graft IL-33 caused local augmentation of proinflammatory iNOS+ macrophages that accelerated graft loss. IL-33 facilitated a metabolic program in macrophages associated with reparative and regulatory functions, and local delivery of IL-33 prevented the chronic rejection of IL-33-deficient cardiac transplants. Therefore, IL-33 represents what we believe is a novel regulatory alarmin in transplantation that limits chronic rejection by restraining the local activation of proinflammatory macrophages. The local delivery of IL-33 in extracellular matrix-based materials may be a promising biologic for chronic rejection prophylaxis.

Enhancing the Value of Histopathological Assessment of Allograft Biopsy Monitoring.

Traditional histopathological allograft biopsy evaluation provides, within hours, diagnoses, prognostic information, and mechanistic insights into disease processes. However, proponents of an array of alternative monitoring platforms, broadly classified as "invasive" or "noninvasive" depending on whether allograft tissue is needed, question the value proposition of tissue histopathology. The authors explore the pros and cons of current analytical methods relative to the value of traditional and illustrate advancements of next-generation histopathological evaluation of tissue biopsies. We describe the continuing value of traditional histopathological tissue assessment and "next-generation pathology (NGP)," broadly defined as staining/labeling techniques coupled with digital imaging and automated image analysis. Noninvasive imaging and fluid (blood and urine) analyses promote low-risk, global organ assessment, and "molecular" data output, respectively; invasive alternatives promote objective, "mechanistic" insights by creating gene lists with variably increased/decreased expression compared with steady state/baseline. Proponents of alternative approaches contrast their preferred methods with traditional histopathology and: (1) fail to cite the main value of traditional and NGP-retention of spatial and inferred temporal context available for innumerable objective analyses and (2) belie an unfamiliarity with the impact of advances in imaging and software-guided analytics on emerging histopathology practices. Illustrative NGP examples demonstrate the value of multidimensional data that preserve tissue-based spatial and temporal contexts. We outline a path forward for clinical NGP implementation where "software-assisted sign-out" will enable pathologists to conduct objective analyses that can be incorporated into their final reports and improve patient care.

Late graft dysfunction after pediatric heart transplantation is associated with fibrosis and microvasculopathy by automated, digital whole-slide analysis.

BACKGROUND: Histopathologic features of late graft dysfunction (LGD) in endomyocardial biopsies (EMBs) after pediatric heart transplantation (HT) have been incompletely described and rarely quantified. We employed automated, morphometric analysis of whole-slide EMB images to objectively quantify fibrosis and microvasculopathy after pediatric HT. METHODS: Nine recipients with clinical LGD were matched with controls on age, listing diagnosis, crossmatch and time since HT. Fibrosis was quantified as percent tissue area with fibrosis and capillary density as capillaries per unit area, number of capillary "neighbors" within 30 µm of each myocyte and myocyte-to-nearest-capillary diffusion distance. Clinical data, including all EMB reports, were also reviewed. RESULTS: The groups were well matched for age at HT (median 4.0 vs 3.1 years), listing diagnosis (50% congenital heart disease for each), positive crossmatch (11% each) and days post-HT (2,628 vs 2,894, p = 0.69). Despite a similar number of previous EMBs (median 23 each, p = 0.43), areas occupied by fibrosis were greater in LGD cases (44.5% vs 23.2%, p = 0.012). Capillary number/area data were not statistically different between LGD cases and controls (378/mm2 vs 559/mm2, p = 0.57), but LGD cases more commonly had zero capillary neighbors (35% vs 20%, p = 0.02) and greater myocyte-to-nearest-capillary distances (27.1 µm vs 18.7 µm, p = 0.005). Cumulative rejection history correlated with fibrosis (r = 0.49, p = 0.039) and myocyte-to-nearest-capillary distance (r = 0.5, p = 0.036). CONCLUSIONS: LGD after pediatric HT is associated with previous rejection and characterized histologically by fibrosis and microvasculopathy, which are not readily appreciated by traditional semi-quantitative EMB analysis. Software-assisted EMB analysis may enable greater pathophysiologic understanding of LGD and identification of targets for future study and intervention.

Highly heterogeneous genomic landscape of uterine leiomyomas by whole exome sequencing and genome-wide arrays.

OBJECTIVE: To determine the genomic signatures of human uterine leiomyomas and prevalence of MED12 mutations in human uterine leiomyosarcomas. DESIGN: Retrospective cohort study. SETTING: Not applicable. PATIENT(S): This study included a set of 16 fresh frozen leiomyoma and corresponding unaffected myometrium specimens as well as 153 leiomyosarcomas collected from women diagnosed with uterine leiomyomas or leiomyosarcomas who underwent clinically indicated abdominal hysterectomy. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Whole exome sequencing and high-resolution X-chromosome and whole genome single nucleotide polymorphism microarray analyses were performed on leiomyoma samples negative for the known MED12 mutations and compared with their corresponding myometrium. Leiomyosarcoma specimens were examined for exon 2 MED12 mutations to evaluate the frequency of MED12 mutated leiomyosarcomas. RESULT(S): Our results indicate remarkable genomic heterogeneity of leiomyoma lesions. MED12-negative leiomyomas contain copy number alterations involving the Mediator complex subunits such as MED8, MED18, CDK8, and long intergenic nonprotein coding RNA340 (CASC15), which may affect the Mediator architecture and/or its transcriptional activity. We also identified mutations in a number of genes that were implicated in leiomyomagenesis such as COL4A6, DCN, and AHR, as well as novel genes: NRG1, ADAM18, HUWE1, FBXW4, FBXL13, and CAPRIN1. CONCLUSION(S): Mutations in genes implicated in cell-to-cell interactions and remodeling of the extracellular matrix and genomic aberrations involving genes coding for the Mediator complex subunits were identified in uterine leiomyomas. Additionally, we discovered that ∼4.6% of leiomyosarcomas harbored MED12 exon 2 mutations, but the relevance of this association with molecular pathogenesis of leiomyosarcoma remains unknown.