The 2022 Banff Meeting Lung Report.

The Lung Session of the 2022 16th Banff Foundation for Allograft Pathology Conference-held in Banff, Alberta-focused on non-rejection lung allograft pathology and novel technologies for the detection of allograft injury. A multidisciplinary panel reviewed the state-of-the-art of current histopathologic entities, serologic studies, and molecular practices, as well as novel applications of digital pathology with artificial intelligence, gene expression analysis, and quantitative image analysis of chest computerized tomography. Current states of need as well as prospective integration of the aforementioned tools and technologies for complete assessment of allograft injury and its impact on lung transplant outcomes were discussed. Key conclusions from the discussion were: (1) recognition of limitations in current standard of care assessment of lung allograft dysfunction; (2) agreement on the need for a consensus regarding the standardized approach to the collection and assessment of pathologic data, inclusive of all lesions associated with graft outcome (eg, non-rejection pathology); and (3) optimism regarding promising novel diagnostic modalities, especially minimally invasive, which should be integrated into large, prospective multicenter studies to further evaluate their utility in clinical practice for directing personalized therapies to improve graft outcomes.

Assessing the role of phosphorylated S6 ribosomal protein in the pathological diagnosis of pulmonary antibody-mediated rejection.

BACKGROUND: Pulmonary antibody-mediated rejection is still a challenging diagnosis as C4d immunostaining has poor sensitivity. Previous studies have indicated that the phosphorylated S6 ribosomal protein, a component of the mammalian target of rapamycin (mTOR) pathway, is correlated with de novo donor-specific antibodies in lung transplantation. The objective of this study was to evaluate the phosphorylation of S6 ribosomal protein as a surrogate for antibody-mediated rejection diagnosis in lung transplant patients. METHODS: This multicentre retrospective study analyzed transbronchial biopsies from 216 lung transplanted patients, 114 with antibody-mediated rejection and 102 without (19 with acute cellular rejection, 17 with ischemia/reperfusion injury, 18 with infection, and 48 without post-transplant complications). Immunohistochemistry was used to quantify phosphorylated S6 ribosomal protein expression in macrophages, endothelium, epithelium, and inter-pathologist agreement was assessed. RESULTS: Median phosphorylated S6 ribosomal protein expression values were higher in antibody-mediated rejection cases than in controls for all cell components, with the highest sensitivity in macrophages (0.9) and the highest specificity in endothelial expression (0.8). The difference was mainly significant in macrophages compared to other post-lung transplantation complications. Inter-pathologist agreement was moderate for macrophages and endothelium, with higher agreement when phosphorylated S6 ribosomal protein expression was dichotomized into positive/negative. The inclusion of phosphorylated S6 ribosomal protein in the diagnostic algorithm could have increased antibody-mediated rejection certainty levels by 25%. CONCLUSIONS: The study supports the role of the mTOR pathway in antibody-mediated rejection-related graft injury and suggests that tissue phosphorylation of S6 ribosomal protein could be a useful surrogate for a more accurate pathological diagnosis of lung antibody-mediated rejection.

Report of the 2022 Banff Heart Concurrent: Focus on non-human leukocyte antigen antibodies in rejection and the pathology of "mixed" rejection.

The Banff Heart Concurrent Session, held as part of the 16th Banff Foundation for Allograft Pathology Conference at Banff, Alberta, Canada, on September 21, 2022, focused on 2 major topics: non-human leukocyte antigen (HLA) antibodies and mixed rejection. Each topic was addressed in a multidisciplinary fashion with clinical, immunological, and pathology perspectives and future developments and prospectives. Following the Banff organization model and principles, the collective aim of the speakers on each topic was to • Determine current knowledge gaps in heart transplant pathology • Identify limitations of current pathology classification systems • Discuss next steps in addressing gaps and refining classification system.

Selenoprotein deficiency disorder predisposes to aortic aneurysm formation.

Aortic aneurysms, which may dissect or rupture acutely and be lethal, can be a part of multisystem disorders that have a heritable basis. We report four patients with deficiency of selenocysteine-containing proteins due to selenocysteine Insertion Sequence Binding Protein 2 (SECISBP2) mutations who show early-onset, progressive, aneurysmal dilatation of the ascending aorta due to cystic medial necrosis. Zebrafish and male mice with global or vascular smooth muscle cell (VSMC)-targeted disruption of Secisbp2 respectively show similar aortopathy. Aortas from patients and animal models exhibit raised cellular reactive oxygen species, oxidative DNA damage and VSMC apoptosis. Antioxidant exposure or chelation of iron prevents oxidative damage in patient's cells and aortopathy in the zebrafish model. Our observations suggest a key role for oxidative stress and cell death, including via ferroptosis, in mediating aortic degeneration.

Graft function and incidence of cardiac allograft vasculopathy in donation after circulatory-determined death heart transplant recipients.

Experience in donation after circulatory-determined death (DCD) heart transplantation (HTx) is expanding. There is limited information on the functional outcomes of DCD HTx recipients. We sought to evaluate functional outcomes in our cohort of DCD recipients. We performed a single-center, retrospective, observational cohort study comparing outcomes in consecutive DCD and donation after brain death (DBD) HTx recipients between 2015 and 2019. Primary outcome was allograft function by echocardiography at 12 and 24 months. Secondary outcomes included incidence of cardiac allograft vasculopathy, treated rejection, renal function, and survival. Seventy-seven DCD and 153 DBD recipients were included. There was no difference in left ventricular ejection fraction at 12 months (59% vs 59%, P = .57) and 24 months (58% vs 58%, P = .87). There was no significant difference in right ventricular function at 12 and 24 months. Unadjusted survival between DCD and DBD recipients at 5 years (85.7% DCD and 81% DBD recipients; P = .45) was similar. There were no significant differences in incidence of cardiac allograft vasculopathy (odds ratio 1.59, P = .21, 95% confidence interval 0.77-3.3) or treated rejection (odds ratio 0.60, P = .12, 95% confidence interval 0.32-1.15) between DBD and DCD recipients. Post-transplant renal function was similar at 1 and 2 years. In conclusion, cardiac allografts from DCD donors perform similarly to a contemporary population of DBD allografts in the medium term.

Lung allograft standardized histological analysis (LASHA) template: A research consensus proposal.

BACKGROUND: Routine monitoring of lung-transplanted patients is crucial for the identification of immunological and non-immunological complications. Determining the etiology of acute allograft dysfunction, particularly in alloimmune-mediated disorders, relies heavily on the lung biopsy with histopathologic analysis. Standardization of the pathologic diagnosis of rejection (e.g., cellular and antibody-mediated) is based on consensus statements and guidelines, indicating the importance of a multidisciplinary approach to achieve a definitive etiological diagnosis. In addition to these statements and guidelines, refinements and standardizations are feasible through systematic analysis morphological, immunophenotypic and molecular alterations observed in transbronchial biopsies. This study is to identify key morphologic features to be assessed, select consistent and reproducible terminology for each histological feature, and provide standardized definitions for pathological assessment and grading. METHODS: A template was created by experts in lung transplantation including pathologists, pulmonologists, immunologists. An initial draft was circulated, followed by discussions and multiple revisions by email and conference calls. RESULTS: The "lung allograft standardized histological analysis - LASHA" template was created and structured as multiple-choice questions with number of fields to be filled in to allow for standardization of results and easy transfer into a future electronic spreadsheet. CONCLUSION: This template will help facilitate multicenter studies through a uniform protocol and correlations with new diagnostic modalities. After validation in large-scale studies, an optimized template could be included in routine clinical practice to enhance graft assessment and medical decision-making.

In Situ Pre-Treatment of Vascularized Composite Allografts With a Targeted Complement Inhibitor Protects Against Brain Death and Ischemia Reperfusion Induced Injuries.

Introduction: Donor brain death (BD) is an unavoidable component of vascularized composite allograft (VCA) transplantation and a key contributor to ischemia-reperfusion injury (IRI). Complement is activated and deposited within solid organ grafts as a consequence of BD and has been shown to exacerbate IRI, although the role of BD and complement in VCA and the role it plays in IRI and VCA rejection has not been studied. Methods: BD was induced in Balb/c donors, and the VCA perfused prior to graft procurement with UW solution supplemented with or without CR2-Crry, a C3 convertase complement inhibitor that binds at sites of complement activation, such as that induced on the endothelium by induction of BD. Following perfusion, donor VCAs were cold stored for 6 hours before transplantation into C57BL/6 recipients. Donor VCAs from living donors (LD) were also procured and stored. Analyses included CR2-Crry graft binding, complement activation, toxicity, injury/inflammation, graft gene expression and survival. Results: Compared to LD VCAs, BD donor VCAs had exacerbated IRI and rejected earlier. Following pretransplant in-situ perfusion of the donor graft, CR2-Crry bound within the graft and was retained post-transplantation. CR2-Crry treatment significantly reduced complement deposition, inflammation and IRI as compared to vehicle-treated BD donors. Treatment of BD donor VCAs with CR2-Crry led to an injury profile not dissimilar to that seen in recipients of LD VCAs. Conclusion: Pre-coating a VCA with CR2-Crry in a clinically relevant treatment paradigm provides localized, and therefore minimally immunosuppressive, protection from the complement-mediated effects of BD induced exacerbated IRI.

A novel injury site-natural antibody targeted complement inhibitor protects against lung transplant injury.

Complement is known to play a role in ischemia and reperfusion injury (IRI). A general paradigm is that complement is activated by self-reactive natural IgM antibodies (nAbs), after they engage postischemic neoepitopes. However, a role for nAbs in lung transplantation (LTx) has not been explored. Using mouse models of LTx, we investigated the role of two postischemic neoepitopes, modified annexin IV (B4) and a subset of phospholipids (C2), in LTx. Antibody deficient Rag1-/- recipient mice were protected from LTx IRI. Reconstitution with either B4 or C2nAb restored IRI, with C2 significantly more effective than B4 nAb. Based on these information, we developed/characterized a novel complement inhibitor composed of single-chain antibody (scFv) derived from the C2 nAb linked to Crry (C2scFv-Crry), a murine inhibitor of C3 activation. Using an allogeneic LTx, in which recipients contain a full nAb repertoire, C2scFv-Crry targeted to the LTx, inhibited IRI, and delayed acute rejection. Finally, we demonstrate the expression of the C2 neoepitope in human donor lungs, highlighting the translational potential of this approach.

A 5-year single-center early experience of heart transplantation from donation after circulatory-determined death donors.

BACKGROUND: In an effort to address the increasing demand for heart transplantation within the United Kingdom (UK), we established a clinical program of heart transplantation from donation after circulatory-determined death (DCD) donors in 2015. After 5 years, we report the clinical early outcomes and impact of the program. METHODS: This is a single-center, retrospective, matched, observational cohort study comparing outcomes of hearts transplanted from DCD donors from March 1, 2015 to February 29, 2020 with those from matched donation after brain death (DBD) donors at Royal Papworth Hospital (RPH) (Cambridge, UK). DCD hearts were either retrieved using thoracoabdominal normothermic regional perfusion or the direct procurement and perfusion technique. All DBD hearts were procured using standard cold static storage. The primary outcomes were recipient 30-day and 1-year survival. RESULTS: During the 5-year study, DCD heart donation increased overall heart transplant activity by 48% (79 for DCD and 164 for DBD). There was no difference in survival at 30 days (97% for DCD vs 99% for DBD, p = 1.00) or 1 year (91% for DCD vs 89% for DBD, p = 0.72). There was no difference in the length of stay in the intensive care unit (7 for DCD vs 6 for DBD days, p = 0.24) or in the hospital (24 for DCD vs 25 for DBD days, p = 0.84). CONCLUSIONS: DCD heart donation increased overall heart transplant activity at RPH by 48%, with no difference in 30-day or 1-year survival in comparison with conventional DBD heart transplantations. DCD heart donation is set to make a dramatic difference in the number of patients who can benefit from heart transplantation.

Severe acute respiratory distress syndrome requiring extracorporeal membrane oxygenation support: a consequence of vaping.

In the past decade, vaping has become more prevalent globally. Since mid-2019, reports have linked the use of vaping devices to lung injury (EVALI). This is the first reported adult case outside the USA to require ECMO for a severe vaping complication. https://bit.ly/39hf2ZY.

Genomic evidence supports a clonal diaspora model for metastases of esophageal adenocarcinoma.

The poor outcomes in esophageal adenocarcinoma (EAC) prompted us to interrogate the pattern and timing of metastatic spread. Whole-genome sequencing and phylogenetic analysis of 388 samples across 18 individuals with EAC showed, in 90% of patients, that multiple subclones from the primary tumor spread very rapidly from the primary site to form multiple metastases, including lymph nodes and distant tissues-a mode of dissemination that we term 'clonal diaspora'. Metastatic subclones at autopsy were present in tissue and blood samples from earlier time points. These findings have implications for our understanding and clinical evaluation of EAC.

The landscape of somatic mutation in normal colorectal epithelial cells.

The colorectal adenoma-carcinoma sequence has provided a paradigmatic framework for understanding the successive somatic genetic changes and consequent clonal expansions that lead to cancer1. However, our understanding of the earliest phases of colorectal neoplastic changes-which may occur in morphologically normal tissue-is comparatively limited, as for most cancer types. Here we use whole-genome sequencing to analyse hundreds of normal crypts from 42 individuals. Signatures of multiple mutational processes were revealed; some of these were ubiquitous and continuous, whereas others were only found in some individuals, in some crypts or during certain periods of life. Probable driver mutations were present in around 1% of normal colorectal crypts in middle-aged individuals, indicating that adenomas and carcinomas are rare outcomes of a pervasive process of neoplastic change across morphologically normal colorectal epithelium. Colorectal cancers exhibit substantially increased mutational burdens relative to normal cells. Sequencing normal colorectal cells provides quantitative insights into the genomic and clonal evolution of cancer.

Heterotopic Ossification following Suprapatellar Intramedullary Nailing.

INTRODUCTION: Tibial shaft fractures are common fractures seen in trauma and orthopedic practice today. The majority of these fractures are treated with intramedullary nailing (IMN) which is rapidly becoming a gold standard. The procedure itself is performed by either a suprapatellar approach or infrapatellar approach. Suprapatellar approach is gaining popularity due to relative ease of insertion, decreased associated risk of anterior knee pain, and more accurate reduction. We report a case of heterotopic ossification noted in the knee following IMN of tibia performed using a suprapatellar approach. CASE REPORT: A 27-year-old male, having sustained a left, Gustilo IIIB tibial shaft fracture following a motor vehicle accident, underwent a reamed intramedullary nail fixation performed through a suprapatellar approach. Two months later, he presented with intra-articular heterotopic ossification which was limiting his knee movement. He then underwent arthroscopic removal of the bony fragments which resolved his symptoms. CONCLUSION: An extensive search of literature did not yield any reported incidence of heterotopic ossification associated with IMN performed through a suprapatellar approach. We present this case report to raise awareness that although IMN through a suprapatellar approach is a safe approach, it does have associated risk of heterotopic ossification which needs to be included in the consent process.

Catastrophic sickling crisis in patient undergoing cardiac transplantation with sickle cell trait.

There is debate in the literature regarding management of patients with sickle cell trait (SCT) undergoing cardiac surgery, since it is recognized that cardiopulmonary bypass presents many precipitating risk factors for a sickling crisis. Despite this, many report successful outcomes without any modification to perioperative management. A 49-year-old woman with SCT (HbS 38%) with postpartum cardiomyopathy underwent cardiac transplantation. The patient was cooled to 34.0°C and retrograde cold blood cardioplegia was infused continuously. The cold ischemic time was 219 minutes and warm ischemic time 46 minutes. After weaning from bypass, she developed global cardiac dysfunction requiring veno-arterial extracorporeal membrane oxygenation. The circuit suddenly stopped, requiring emergency reinstitution of bypass; the circuit had clotted. Transesophageal-echocardiogram revealed thrombus within the left atrium and ventricle. There was no recovery of cardiac function and the patient developed multiorgan failure. At postmortem there was extensive myocardial infarction with evidence of widespread catastrophic intravascular red-cell sickling. This case highlights the danger of complacency in patients with SCT, offering a learning opportunity for the cardiothoracic community to highlight the most serious complication that can occur in this group of patients. We have learned that SCT and cardiac surgery is not a benign combination.

Alveolar Septal Widening as an "Alert" Signal to Look Into Lung Antibody-mediated Rejection: A Multicenter Pilot Study.

BACKGROUND: Antibody-mediated rejection (AMR) plays an important role in allograft dysfunction. Acute lung injury (ALI), endotheliitis, capillary inflammation, and C4d positivity have been described as morphological features conventionally associated with lung AMR. A multidisciplinary, international task force reviewed AMR cases in the context of four face-to-face meetings. Septal widening was a frequent, striking histological feature recognized first and easily at low-power magnification. This study aimed to evaluate whether septal widening could represent an "alert" signal for AMR. METHODS: Following the face-to-face meetings that enabled the classification of cases as AMR or non-AMR, morphometry was performed on biopsies from 48 recipients with definite, probable or possible AMR, 31 controls (negative for any posttransplant injury) and 10 patients with nonimmune-related ALI. RESULTS: Mean alveolar septal thickness was greater in AMR patients than in controls (P < 0.001). Septal thickness was not significantly different between AMR-ALI and non-AMR-ALI. Unexpectedly septal widening was the only histological change detected in some cases with probable or possible AMR that lacked the histological lesions conventionally associated with AMR. The thickness in these cases was similar to that observed in AMR cases with more severe histological injury such as ALI or neutrophilic capillaritis. CONCLUSIONS: Our data suggest that, even if unspecific as the other lesions conventionally associated with AMR, septal widening may represent an "alert" signal to look into lung AMR. A larger prospective study is mandatory to confirm the potential value of septal widening in the multidisciplinary approach of AMR.

Isolated atrial amyloid: a potential contributor to morbidity and mortality following cardiac surgery.

OBJECTIVES: Diffuse cardiac amyloidosis is a significant diagnosis with a poor prognosis. Isolated atrial amyloidosis (IAA) is the most common form of cardiac amyloidosis caused by accumulation of alpha-atrial natriuretic peptide. IAA has been associated with dysrhythmia, but otherwise remains a poorly characterized condition. The impact of incidental IAA on postoperative outcome following cardiac surgery has not previously been reported. The purpose of this study was to examine the impact of isolated atrial amyloid on patient outcomes following cardiac surgery. METHODS: A retrospective analysis was performed of all patients having excision of the left atrial appendage during cardiac surgery at our centre over a 5-year period. Patients with histological evidence of IAA were compared to patients without this diagnosis. IAA was diagnosed by immunohistochemistry for atrial natriuretic peptide. RESULTS: A total of 167 patients underwent left atrial appendage excision and of these 26 (15.6%) were found to have IAA. Preoperative characteristics were similar between the 2 groups. A significantly greater proportion of patients with IAA experienced dysrhythmia requiring implantation of a permanent pacemaker (23.1% vs 7.8%, P = 0.03). There was also a significantly elevated incidence of perioperative death in the IAA group (11.5% vs 1.4%, P = 0.03) and inferior 1-year survival (84.6% vs 96.5%, P = 0.02). CONCLUSIONS: The presence of IAA may be associated with inferior outcomes following cardiac surgery, with increased morbidity in the early postoperative period and inferior long-term survival. Knowledge of the diagnosis preoperatively may facilitate management of patients.

The inhibitory subunit of cardiac troponin (cTnI) is modified by arginine methylation in the human heart.

BACKGROUND: The inhibitory subunit of cardiac troponin (cTnI) is a gold standard cardiac biomarker and also an essential protein in cardiomyocyte excitation-contraction coupling. The interactions of cTnI with other proteins are fine-tuned by post-translational modification of cTnI. Mutations in cTnI can lead to hypertrophic cardiomyopathy. METHODS AND RESULTS: Here we report, for the first time, that cTnI is modified by arginine methylation in human myocardium. Using Western blot, we observed reduced levels of cTnI arginine methylation in human hypertrophic cardiomyopathy compared to dilated cardiomyopathy biopsies. Similarly, using a rat model of cardiac hypertrophy we observed reduced levels of cTnI arginine methylation compared to sham controls. Using mass spectrometry, we identified cTnI methylation sites at R74/R79 and R146/R148 in human cardiac samples. R146 and R148 lie at the boundary between the critical cTnI inhibitory and switch peptides; PRMT1 methylated an extended inhibitory peptide at R146 and R148 in vitro. Mutations at R145 that have been associated with hypertrophic cardiomyopathy hampered R146/R148 methylation by PRMT1 in vitro. H9c2 cardiac-like cells transfected with plasmids encoding for a methylation-deficient R146A/R148A cTnI protein developed cell hypertrophy, with a 32% increase in cell size after 72 h, compared to control cells. DISCUSSION: Our results provide evidence for a novel and significant cTnI post-translational modification. Our work opens the door to translational investigations of cTnI arginine methylation as a biomarker of disease, which can include e.g. cardiomyopathies, myocardial infarction and heart failure, and offers a novel way to investigate the effect of cTnI mutations in the inhibitory/switch peptides.

Data regarding transplant induced germinal center humoral autoimmunity.

This data is related to the research article entitled "Germinal center humoral autoimmunity independently mediates progression of allograft vasculopathy" (Harper et al., 2016) [2]. The data presented here focuses on the humoral autoimmune response triggered by transferred allogeneic CD4 T cells and includes details on: (a) the recipient splenic germinal center (GC) response; (b) augmentation of humoral autoimmunity and accelerated heart allograft rejection following transplantation from donors primed against recipient; (c) flow cytometric analysis of donor and recipient CD4 T cells for signature markers of T follicular helper cell differentiation; (d) in vitro donor endothelial cell migration in response to column purified autoantibody from recipient sera; (e) analysis of development of humoral responses in recipients following adoptive transfer of donor CD4 T cells and; (f) the development of humoral autoimmunity in mixed haematopoietic chimeric mice.

Germinal center humoral autoimmunity independently mediates progression of allograft vasculopathy.

The development of humoral autoimmunity following organ transplantation is increasingly recognised, but of uncertain significance. We examine whether autoimmunity contributes independently to allograft rejection. In a MHC class II-mismatched murine model of chronic humoral rejection, we report that effector antinuclear autoantibody responses were initiated upon graft-versus-host allorecognition of recipient B cells by donor CD4 T-cells transferred within heart allografts. Consequently, grafts were rejected more rapidly, and with markedly augmented autoantibody responses, upon transplantation of hearts from donors previously primed against recipient. Nevertheless, rejection was dependent upon recipient T follicular helper (TFH) cell differentiation and provision of cognate (peptide-specific) help for maintenance as long-lived GC reactions, which diversified to encompass responses against vimentin autoantigen. Heart grafts transplanted into stable donor/recipient mixed haematopoietic chimeras, or from parental strain donors into F1 recipients (neither of which can trigger host adaptive alloimmune responses), nevertheless provoked GC autoimmunity and were rejected chronically, with rejection similarly dependent upon host TFH cell differentiation. Thus, autoantibody responses contribute independently of host adaptive alloimmunity to graft rejection, but require host TFH cell differentiation to maintain long-lived GC responses. The demonstration that one population of helper CD4 T-cells initiates humoral autoimmunity, but that a second population of TFH cells is required for its maintenance as a GC reaction, has important implications for how autoimmune-related phenomena manifest.

Emphysema-associated Autoreactive Antibodies Exacerbate Post-Lung Transplant Ischemia-Reperfusion Injury.

Chronic obstructive pulmonary disease-associated chronic inflammation has been shown to lead to an autoimmune phenotype characterized in part by the presence of lung autoreactive antibodies. We hypothesized that ischemia-reperfusion injury (IRI) liberates epitopes that would facilitate preexisting autoantibody binding, thereby exacerbating lung injury after transplant. We induced emphysema in C57BL/6 mice through 6 months of cigarette smoke (CS) exposure. Mice with CS exposure had significantly elevated serum autoantibodies compared with non-smoke-exposed age-matched (NS) mice. To determine the impact of a full preexisting autoantibody repertoire on IRI, we transplanted BALB/c donor lungs into NS or CS recipients and analyzed grafts 48 hours after transplant. CS recipients had significantly increased lung injury and immune cell infiltration after transplant. Immunofluorescence staining revealed increased IgM, IgG, and C3d deposition in CS recipients. To exclude confounding alloreactivity and confirm the role of preexisting autoantibodies in IRI, syngeneic Rag1-/- (recombination-activating protein 1-knockout) transplants were performed in which recipients were reconstituted with pooled serum from CS or NS mice. Serum from CS-exposed mice significantly increased IRI compared with control mice, with trends in antibody and C3d deposition similar to those seen in allografts. These data demonstrate that pretransplant CS exposure is associated with increased IgM/IgG autoantibodies, which, upon transplant, bind to the donor lung, activate complement, and exacerbate post-transplant IRI.