Expansion of Pathogenic Cardiac Macrophages in Immune Checkpoint Inhibitor Myocarditis.

BACKGROUND: Immune checkpoint inhibitors (ICIs), antibodies targeting PD-1 (programmed cell death protein 1)/PD-L1 (programmed death-ligand 1) or CTLA4 (cytotoxic T-lymphocyte-associated protein 4), have revolutionized cancer management but are associated with devastating immune-related adverse events including myocarditis. The main risk factor for ICI myocarditis is the use of combination PD-1 and CTLA4 inhibition. ICI myocarditis is often fulminant and is pathologically characterized by myocardial infiltration of T lymphocytes and macrophages. Although much has been learned about the role of T-cells in ICI myocarditis, little is understood about the identity, transcriptional diversity, and functions of infiltrating macrophages. METHODS: We used an established murine ICI myocarditis model (Ctla4+/-Pdcd1-/- mice) to explore the cardiac immune landscape using single-cell RNA-sequencing, immunostaining, flow cytometry, in situ RNA hybridization, molecular imaging, and antibody neutralization studies. RESULTS: We observed marked increases in CCR2 (C-C chemokine receptor type 2)+ monocyte-derived macrophages and CD8+ T-cells in this model. The macrophage compartment was heterogeneous and displayed marked enrichment in an inflammatory CCR2+ subpopulation highly expressing Cxcl9 (chemokine [C-X-C motif] ligand 9), Cxcl10 (chemokine [C-X-C motif] ligand 10), Gbp2b (interferon-induced guanylate-binding protein 2b), and Fcgr4 (Fc receptor, IgG, low affinity IV) that originated from CCR2+ monocytes. It is important that a similar macrophage population expressing CXCL9, CXCL10, and CD16α (human homologue of mouse FcgR4) was expanded in patients with ICI myocarditis. In silico prediction of cell-cell communication suggested interactions between T-cells and Cxcl9+Cxcl10+ macrophages via IFN-γ (interferon gamma) and CXCR3 (CXC chemokine receptor 3) signaling pathways. Depleting CD8+ T-cells or macrophages and blockade of IFN-γ signaling blunted the expansion of Cxcl9+Cxcl10+ macrophages in the heart and attenuated myocarditis, suggesting that this interaction was necessary for disease pathogenesis. CONCLUSIONS: These data demonstrate that ICI myocarditis is associated with the expansion of a specific population of IFN-γ-induced inflammatory macrophages and suggest the possibility that IFN-γ blockade may be considered as a treatment option for this devastating condition.

AI-guided histopathology predicts brain metastasis in lung cancer patients.

Brain metastases can occur in nearly half of patients with early and locally advanced (stage I-III) non-small cell lung cancer (NSCLC). There are no reliable histopathologic or molecular means to identify those who are likely to develop brain metastases. We sought to determine if deep learning (DL) could be applied to routine H&E-stained primary tumor tissue sections from stage I-III NSCLC patients to predict the development of brain metastasis. Diagnostic slides from 158 patients with stage I-III NSCLC followed for at least 5 years for the development of brain metastases (Met+, 65 patients) versus no progression (Met-, 93 patients) were subjected to whole-slide imaging. Three separate iterations were performed by first selecting 118 cases (45 Met+, 73 Met-) to train and validate the DL algorithm, while 40 separate cases (20 Met+, 20 Met-) were used as the test set. The DL algorithm results were compared to a blinded review by four expert pathologists. The DL-based algorithm was able to distinguish the eventual development of brain metastases with an accuracy of 87% (p < 0.0001) compared with an average of 57.3% by the four pathologists and appears to be particularly useful in predicting brain metastases in stage I patients. The DL algorithm appears to focus on a complex set of histologic features. DL-based algorithms using routine H&E-stained slides may identify patients who are likely to develop brain metastases from those who will remain disease free over extended (>5 year) follow-up and may thus be spared systemic therapy. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

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.

Transcriptional and Immune Landscape of Cardiac Sarcoidosis.

BACKGROUND: Cardiac involvement is an important determinant of mortality among sarcoidosis patients. Although granulomatous inflammation is a hallmark finding in cardiac sarcoidosis, the precise immune cell populations that comprise the granuloma remain unresolved. Furthermore, it is unclear how the cellular and transcriptomic landscape of cardiac sarcoidosis differs from other inflammatory heart diseases. METHODS: We leveraged spatial transcriptomics (GeoMx digital spatial profiler) and single-nucleus RNA sequencing to elucidate the cellular and transcriptional landscape of cardiac sarcoidosis. Using GeoMX digital spatial profiler technology, we compared the transcriptomal profile of CD68+ rich immune cell infiltrates in human cardiac sarcoidosis, giant cell myocarditis, and lymphocytic myocarditis. We performed single-nucleus RNA sequencing of human cardiac sarcoidosis to identify immune cell types and examined their transcriptomic landscape and regulation. Using multichannel immunofluorescence staining, we validated immune cell populations identified by single-nucleus RNA sequencing, determined their spatial relationship, and devised an immunostaining approach to distinguish cardiac sarcoidosis from other inflammatory heart diseases. RESULTS: Despite overlapping histological features, spatial transcriptomics identified transcriptional signatures and associated pathways that robustly differentiated cardiac sarcoidosis from giant cell myocarditis and lymphocytic myocarditis. Single-nucleus RNA sequencing revealed the presence of diverse populations of myeloid cells in cardiac sarcoidosis with distinct molecular features. We identified GPNMB (transmembrane glycoprotein NMB) as a novel marker of multinucleated giant cells and predicted that the MITF (microphthalmia-associated transcription factor) family of transcription factors regulated this cell type. We also detected additional macrophage populations in cardiac sarcoidosis including HLA-DR (human leukocyte antigen-DR)+ macrophages, SYTL3 (synaptotagmin-like protein 3)+ macrophages and CD163+ resident macrophages. HLA-DR+ macrophages were found immediately adjacent to GPMMB+ giant cells, a distinct feature compared with other inflammatory cardiac diseases. SYTL3+ macrophages were located scattered throughout the granuloma and CD163+ macrophages, CD1c+ dendritic cells, nonclassical monocytes, and T cells were located at the periphery and outside of the granuloma. Finally, we demonstrate mTOR (mammalian target of rapamycin) pathway activation is associated with proliferation and is selectively found in HLA-DR+ and SYLT3+ macrophages. CONCLUSIONS: In this study, we identified diverse populations of immune cells with distinct molecular signatures that comprise the sarcoid granuloma. These findings provide new insights into the pathology of cardiac sarcoidosis and highlight opportunities to improve diagnostic testing.

Functional assays reveal the pathogenic mechanism of a de novo tropomyosin variant identified in patient with dilated cardiomyopathy.

Dilated cardiomyopathy (DCM) is a leading cause of heart failure and a major indicator for heart transplant. Human genetic studies have identified over a thousand causal mutations for DCM in genes involved in a variety of cellular processes, including sarcomeric contraction. A substantial clinical challenge is determining the pathogenicity of novel variants in disease-associated genes. This challenge of connecting genotype and phenotype has frustrated attempts to develop effective, mechanism-based treatments for patients. Here, we identified a de novo mutation (T237S) in TPM1, the gene that encodes the thin filament protein tropomyosin, in a patient with DCM and conducted in vitro experiments to characterize the pathogenicity of this novel variant. We expressed recombinant mutant protein, reconstituted it into thin filaments, and examined the effects of the mutation on thin filament function. We show that the mutation reduces the calcium sensitivity of thin filament activation, as previously seen for known pathogenic mutations. Mechanistically, this shift is due to mutation-induced changes in tropomyosin positioning along the thin filament. We demonstrate that the thin filament activator omecamtiv mecarbil restores the calcium sensitivity of thin filaments regulated by the mutant tropomyosin, which lays the foundation for additional experiments to explore the therapeutic potential of this drug for patients harboring the T237S mutation. Taken together, our results suggest that the TPM1 T237S mutation is likely pathogenic and demonstrate how functional in vitro characterization of pathogenic protein variants in the lab might guide precision medicine in the clinic.

Donor Macrophages Modulate Rejection After Heart Transplantation.

BACKGROUND: Cellular rejection after heart transplantation imparts significant morbidity and mortality. Current immunosuppressive strategies are imperfect, target recipient T cells, and have adverse effects. The innate immune response plays an essential role in the recruitment and activation of T cells. Targeting the donor innate immune response would represent the earliest interventional opportunity within the immune response cascade. There is limited knowledge about donor immune cell types and functions in the setting of cardiac transplantation, and no current therapeutics exist for targeting these cell populations. METHODS: Using genetic lineage tracing, cell ablation, and conditional gene deletion, we examined donor mononuclear phagocyte diversity and macrophage function during acute cellular rejection of transplanted hearts in mice. We performed single-cell RNA sequencing on donor and recipient macrophages and monocytes at multiple time points after transplantation. On the basis of our imaging and single-cell RNA sequencing data, we evaluated the functional relevance of donor CCR2+ (C-C chemokine receptor 2) and CCR2- macrophages using selective cell ablation strategies in donor grafts before transplant. Last, we performed functional validation that donor macrophages signal through MYD88 (myeloid differentiation primary response protein 88) to facilitate cellular rejection. RESULTS: Donor macrophages persisted in the rejecting transplanted heart and coexisted with recipient monocyte-derived macrophages. Single-cell RNA sequencing identified donor CCR2+ and CCR2- macrophage populations and revealed remarkable diversity among recipient monocytes, macrophages, and dendritic cells. Temporal analysis demonstrated that donor CCR2+ and CCR2- macrophages were transcriptionally distinct, underwent significant morphologic changes, and displayed unique activation signatures after transplantation. Although selective depletion of donor CCR2- macrophages reduced allograft survival, depletion of donor CCR2+ macrophages prolonged allograft survival. Pathway analysis revealed that donor CCR2+ macrophages are activated through MYD88/nuclear factor kappa light chain enhancer of activated B cells signaling. Deletion of MYD88 in donor macrophages resulted in reduced antigen-presenting cell recruitment, reduced ability of antigen-presenting cells to present antigen to T cells, decreased emergence of allograft-reactive T cells, and extended allograft survival. CONCLUSIONS: Distinct populations of donor and recipient macrophages coexist within the transplanted heart. Donor CCR2+ macrophages are key mediators of allograft rejection, and deletion of MYD88 signaling in donor macrophages is sufficient to suppress rejection and extend allograft survival. This highlights the therapeutic potential of donor heart-based interventions.

Progestin Primed Ovarian Stimulation (PPOS) protocol yields lower euploidy rate in older patients undergoing IVF.

BACKGROUND: To explore if exogenous progestin required for progestin primed ovarian stimulation (PPOS) protocol compromises the euploidy rate of patients who underwent preimplantation genetic testing cycles when compared to those who received the conventional gonadotropin-releasing hormone (GnRH) antagonist protocol. METHODS: This retrospective cohort study analyzed 128 preimplantation genetic testing for aneuploidy (PGT-A) cycles performed from January 2018 to December 2021 in a single university hospital-affiliated fertility center. Infertile women aged 27 to 45 years old requiring PGT-A underwent either PPOS protocol or GnRH-antagonist protocol with in-vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) for fertilization. Frozen embryo transfers were performed following each PGT-A cycle. Data regarding the two groups were analyzed using the Statistical Package for Social Sciences (SPSS) version 22.0 (SPSS Inc., Chicago, IL). RESULTS: Patients who underwent PPOS treatment had significantly reduced blastocyst formation rate and euploidy rate compared to those who received the GnRH antagonist protocol. Subgroup-analysis was performed by stratifying patients' age into elder and young subgroups (elder: ≥ 38-year-old, young: < 38-year-old). In the elder sub-population, the blastocyst formation rate of the PPOS group was significantly lower than that of the GnRH-antagonist group (45.8 ± 6.1% vs. 59.9 ± 3.8%, p = 0.036). Moreover, the euploidy rate of the PPOS group was only about 20% of that of  the GnRH-antagonist group (5.4% and 26.7%, p = 0.006). In contrast, no significant differences in blastocyst formation rate (63.5 ± 5.7% vs. 67.1 ± 3.2%, p = 0.45) or euploidy rate (30.1% vs. 38.5%, p = 0.221) were observed in the young sub-population. Secondary outcomes, which included implantation rate, biochemical pregnancy rate, clinical pregnancy rate, live birth rate, and miscarriage rate, were comparable between the two treatment groups, regardless of age. CONCLUSION: When compared to the conventional GnRH-antagonist approach, PPOS protocol could potentially reduce the euploidy rate in aging IVF patients. However, due to the retrospective nature of this study, the results are to be interpreted with caution. Before the PPOS protocol is widely implemented, further studies exploring its efficacy in larger populations are needed to define the optimal patient selection suitable for this method. TRIAL REGISTRATION: Human Investigation and Ethical Committee of Chang Gung Medical Foundation (202200194B0).

CT Approach to Lung Injury.

Diffuse alveolar damage (DAD), which represents the pathologic changes seen after acute lung injury, is caused by damage to all three layers of the alveolar wall and can ultimately result in alveolar collapse with loss of the normal pulmonary architecture. DAD has an acute phase that predominantly manifests as airspace disease at CT owing to filling of the alveoli with cells, plasma fluids, and hyaline membranes. DAD then evolves into a heterogeneous organizing phase, with mixed airspace and interstitial disease characterized by volume loss, architectural distortion, fibrosis, and parenchymal loss. Patients with DAD have a severe clinical course and typically require prolonged mechanical ventilation, which may result in ventilator-induced lung injury. In those patients who survive DAD, the lungs will remodel over time, but most will have residual findings at chest CT. Organizing pneumonia (OP) is a descriptive term for a histologic pattern characterized by intra-alveolar fibroblast plugs. The significance and pathogenesis of OP are controversial. Some authors regard it as part of a spectrum of acute lung injury, while others consider it a marker of acute or subacute lung injury. At CT, OP manifests with various forms of airspace disease that are most commonly bilateral and relatively homogeneous in appearance at individual time points. Patients with OP most often have a mild clinical course, although some may have residual findings at CT. In patients with DAD and OP, imaging findings can be combined with clinical information to suggest the diagnosis in many cases, with biopsy reserved for difficult cases with atypical findings or clinical manifestations. To best participate in the multidisciplinary approach to patients with lung injury, radiologists must not only recognize these entities but also describe them with consistent and meaningful terminology, examples of which are emphasized in the article. © RSNA, 2023 See the invited commentary by Kligerman et al in this issue. Quiz questions for this article are available in the supplemental material.

Sensitivity of Antemortem Imaging for Pericarditis: An Institutional Experience.

ABSTRACT: Pericarditis is a challenging diagnosis with nonspecific manifestations, significant clinical implications, and possible mortality. The advancement of imaging, such as echocardiography, computed tomography (CT), and magnetic resonance imaging (MRI), has improved the sensitivity of diagnosis, although limitations remain. In this study, we investigated the prevalence of pericarditis identified at autopsy and correlated these findings with antemortem imaging studies and clinical information. Thirty-four decedents were identified in our archival autopsy records (prevalence, 1.23%) from 2010 to 2021 with a postmortem diagnosis of pericarditis. Thirty-five antemortem imaging studies were performed on 32 decedents, of which CT was the most common (18/35, 51.4%). The sensitivity of antemortem imaging was poor, with CT showing the highest sensitivity at 16.7% (3/18), while echocardiography studies, transthoracic (TTE) and transesophageal (TEE), each had a sensitivity of 0.0%. Pericarditis was determined as the immediate cause of death by autopsy in 13 decedents, of which 3 were diagnosed antemortem. It was considered contributory to the death in 6 decedents, none of which were diagnosed antemortem. In summary, antemortem imaging has limited utility in the diagnosis of pericarditis. It is imperative to examine the pericardium during autopsy to identify a possible cause of death or contributing factor.

Vascular Smooth Muscle Cell Subpopulations and Neointimal Formation in Mouse Models of Elastin Insufficiency.

OBJECTIVE: Using a mouse model of Eln (elastin) insufficiency that spontaneously develops neointima in the ascending aorta, we sought to understand the origin and phenotypic heterogeneity of smooth muscle cells (SMCs) contributing to intimal hyperplasia. We were also interested in exploring how vascular cells adapt to the absence of Eln. Approach and Results: We used single-cell sequencing together with lineage-specific cell labeling to identify neointimal cell populations in a noninjury, genetic model of neointimal formation. Inactivating Eln production in vascular SMCs results in rapid intimal hyperplasia around breaks in the ascending aorta's internal elastic lamina. Using lineage-specific Cre drivers to both lineage mark and inactivate Eln expression in the secondary heart field and neural crest aortic SMCs, we found that cells with a secondary heart field lineage are significant contributors to neointima formation. We also identified a small population of secondary heart field-derived SMCs underneath and adjacent to the internal elastic lamina. Within the neointima of SMC-Eln knockout mice, 2 unique SMC populations were identified that are transcriptionally different from other SMCs. While these cells had a distinct gene signature, they expressed several genes identified in other studies of neointimal lesions, suggesting that some mechanisms underlying neointima formation in Eln insufficiency are shared with adult vessel injury models. CONCLUSIONS: These results highlight the unique developmental origin and transcriptional signature of cells contributing to neointima in the ascending aorta. Our findings also show that the absence of Eln, or changes in elastic fiber integrity, influences the SMC biological niche in ways that lead to altered cell phenotypes.

Imaging of Genetic Thoracic Aortopathy.

Aortopathy is a term most commonly used to describe a group of genetic diseases that predispose patients to an elevated risk of aortic events including aneurysm and acute aortic syndrome. Types of genetic aortopathy are classified as either heritable or congenital, with heritable thoracic aortic disease (HTAD) further subclassified into syndromic HTAD or nonsyndromic HTAD, the former of which is associated with specific phenotypic features. Radiologists may be the first physicians to encounter features of genetic aortopathy, either incidentally or at the time of an acute aortic event. Identifying patients with genetic aortopathy is of substantial importance to clinicians who manage thoracic aortic disease, because aortic diameter thresholds for surgical intervention are often lower than those for nongenetic aortopathy related to aging and hypertension. In addition, when reparative surgery is performed, the approach and extent of the repair may differ in patients with genetic aortopathy. The radiologist should also be familiar with competing diagnoses that can result in acute aortic events, mainly acquired inflammatory and noninflammatory thoracic aortic disease, because these conditions may be associated with increased risks of similar pathologic endpoints. Because many imaging and phenotypic features of various types of genetic aortopathy overlap, diagnosis and determination of appropriate follow-up recommendations can be challenging. A multidisciplinary approach with the use of imaging is often required and, once the diagnosis is made, imaging has additional importance because of the need for lifelong follow-up. ©RSNA, 2022.

Evolution of pathological findings in surveillance biopsies of lung transplant recipients infected with SARS-CoV-2.

BACKGROUND: Previous reports of coronavirus disease 2019 (COVID-19) following lung transplantation generally described a grim prognosis, but these were anecdotal case series of symptomatic patients. A systematic study of the outcomes and pathology of SARS-CoV-2 infection in a large cohort of lung transplant patients is lacking. METHODS: To determine the histopathologic evolution of COVID-19 in lung transplant recipients, we identified all patients who underwent surveillance transbronchial biopsies at our institution, tested positive for SARS-CoV-2, and had multiple pathology specimens available for evaluation. Histology was reviewed and immunofluorescence for SARS-CoV-2 nucleocapsid protein was performed. RESULTS: Ten patients met inclusion criteria. Half (5/10) had incidental diagnosis on routine respiratory pathogen testing at the time of transbronchial biopsy. Six patients were hospitalized, with three requiring intensive care unit (ICU) admission. One patient died. Two specimens showed new onset International Society for Heart and Lung Transplantation (ISHLT) Grade A2 rejection at or following diagnosis. One patient developed bronchiolitis obliterans 111 days following diagnosis and 1 year post transplant. Two patients had organizing pneumonia at diagnosis and three patients showed evolving lung injury following diagnosis. The SARS-CoV-2 nucleocapsid protein was detected in a subset of samples at diagnosis and up to 111 days following diagnosis. CONCLUSIONS: Overall, the pathology of SARS-CoV-2 infection in lung transplant patients is varied, ranging from no pathologic alterations to organizing pneumonia and lung injury. The pathology findings did not necessarily correlate with clinical acuity, as one patient admitted to the ICU had normal pathology. These findings may be generalizable to non-transplant patients and require more follow-up regarding long-term outcomes.

Heterogeneous Cellular Contributions to Elastic Laminae Formation in Arterial Wall Development.

RATIONALE: Elastin is an important ECM (extracellular matrix) protein in large and small arteries. Vascular smooth muscle cells (SMCs) produce the layered elastic laminae found in elastic arteries but synthesize little elastin in muscular arteries. However, muscular arteries have a well-defined internal elastic lamina (IEL) that separates endothelial cells (ECs) from SMCs. The extent to which ECs contribute elastin to the IEL is unknown. OBJECTIVE: To use targeted elastin (Eln) deletion in mice to explore the relative contributions of SMCs and ECs to elastic laminae formation in different arteries. METHODS AND RESULTS: We used SMC- and EC-specific Cre recombinase transgenes with a novel floxed Eln allele to focus gene inactivation in mice. Inactivation of Eln in SMCs using Sm22aCre resulted in depletion of elastic laminae in the arterial wall with the exception of the IEL and SMC clusters in the outer media near the adventitia. Inactivation of elastin in ECs using Tie2Cre or Cdh5Cre resulted in normal medial elastin and a typical IEL in elastic arteries. In contrast, the IEL was absent or severely disrupted in muscular arteries. Interruptions in the IEL resulted in neointimal formation in the ascending aorta but not in muscular arteries. CONCLUSIONS: Combined with lineage-specific fate mapping systems, our knockout results document an unexpected heterogeneity in vascular cells that produce the elastic laminae. SMCs and ECs can independently form an IEL in most elastic arteries, whereas ECs are the major source of elastin for the IEL in muscular and resistance arteries. Neointimal formation at IEL disruptions in the ascending aorta confirms that the IEL is a critical physical barrier between SMCs and ECs in the large elastic arteries. Our studies provide new information about how SMCs and ECs contribute elastin to the arterial wall and how local elastic laminae defects may contribute to cardiovascular disease.

Inappropriate p53 activation during development induces features of CHARGE syndrome.

CHARGE syndrome is a multiple anomaly disorder in which patients present with a variety of phenotypes, including ocular coloboma, heart defects, choanal atresia, retarded growth and development, genitourinary hypoplasia and ear abnormalities. Despite 70-90% of CHARGE syndrome cases resulting from mutations in the gene CHD7, which encodes an ATP-dependent chromatin remodeller, the pathways underlying the diverse phenotypes remain poorly understood. Surprisingly, our studies of a knock-in mutant mouse strain that expresses a stabilized and transcriptionally dead variant of the tumour-suppressor protein p53 (p53(25,26,53,54)), along with a wild-type allele of p53 (also known as Trp53), revealed late-gestational embryonic lethality associated with a host of phenotypes that are characteristic of CHARGE syndrome, including coloboma, inner and outer ear malformations, heart outflow tract defects and craniofacial defects. We found that the p53(25,26,53,54) mutant protein stabilized and hyperactivated wild-type p53, which then inappropriately induced its target genes and triggered cell-cycle arrest or apoptosis during development. Importantly, these phenotypes were only observed with a wild-type p53 allele, as p53(25,26,53,54)(/-) embryos were fully viable. Furthermore, we found that CHD7 can bind to the p53 promoter, thereby negatively regulating p53 expression, and that CHD7 loss in mouse neural crest cells or samples from patients with CHARGE syndrome results in p53 activation. Strikingly, we found that p53 heterozygosity partially rescued the phenotypes in Chd7-null mouse embryos, demonstrating that p53 contributes to the phenotypes that result from CHD7 loss. Thus, inappropriate p53 activation during development can promote CHARGE phenotypes, supporting the idea that p53 has a critical role in developmental syndromes and providing important insight into the mechanisms underlying CHARGE syndrome.

A long noncoding RNA protects the heart from pathological hypertrophy.

The role of long noncoding RNA (lncRNA) in adult hearts is unknown; also unclear is how lncRNA modulates nucleosome remodelling. An estimated 70% of mouse genes undergo antisense transcription, including myosin heavy chain 7 (Myh7), which encodes molecular motor proteins for heart contraction. Here we identify a cluster of lncRNA transcripts from Myh7 loci and demonstrate a new lncRNA-chromatin mechanism for heart failure. In mice, these transcripts, which we named myosin heavy-chain-associated RNA transcripts (Myheart, or Mhrt), are cardiac-specific and abundant in adult hearts. Pathological stress activates the Brg1-Hdac-Parp chromatin repressor complex to inhibit Mhrt transcription in the heart. Such stress-induced Mhrt repression is essential for cardiomyopathy to develop: restoring Mhrt to the pre-stress level protects the heart from hypertrophy and failure. Mhrt antagonizes the function of Brg1, a chromatin-remodelling factor that is activated by stress to trigger aberrant gene expression and cardiac myopathy. Mhrt prevents Brg1 from recognizing its genomic DNA targets, thus inhibiting chromatin targeting and gene regulation by Brg1. It does so by binding to the helicase domain of Brg1, a domain that is crucial for tethering Brg1 to chromatinized DNA targets. Brg1 helicase has dual nucleic-acid-binding specificities: it is capable of binding lncRNA (Mhrt) and chromatinized--but not naked--DNA. This dual-binding feature of helicase enables a competitive inhibition mechanism by which Mhrt sequesters Brg1 from its genomic DNA targets to prevent chromatin remodelling. A Mhrt-Brg1 feedback circuit is thus crucial for heart function. Human MHRT also originates from MYH7 loci and is repressed in various types of myopathic hearts, suggesting a conserved lncRNA mechanism in human cardiomyopathy. Our studies identify a cardioprotective lncRNA, define a new targeting mechanism for ATP-dependent chromatin-remodelling factors, and establish a new paradigm for lncRNA-chromatin interaction.

Suppression of the Reactive Oxygen Response Alleviates Experimental Autoimmune Uveitis in Mice.

Reactive oxygen species (ROS) are produced by host phagocytes and play an important role in antimicrobial actions against various pathogens. Autoimmune uveitis causes blindness and severe visual impairment in humans at all ages worldwide. However, the role of ROS in autoimmune uveitis remains unclear. We used ROS-deficient (Ncf1-/-) mice to investigate the role of ROS in experimental autoimmune uveitis (EAU). Besides, we also used the antioxidant N-acetylcysteine (NAC) treatment to evaluate the effect of suppression of ROS on EAU in mice. The EAU disease scores of Ncf1-/- mice were significantly lower than those of wild-type mice. EAU induction increased the levels of cytokines (interleukin (IL)-1α, IL-1ß, IL-4, IL-6, IL-12, IL-17, and tumor necrosis factor (TNF)-α) and chemokines (monocyte chemoattractant protein (MCP)-1) in the retinas of wild-type mice but not in those of Ncf1-/- mice. EAU induction enhanced the level of NF-κB activity in wild-type mice. However, the level of NF-κB activity in Ncf1-/- mice with EAU induction was low. Treatment with the antioxidant NAC also decreased the severity of EAU in mice with reduced levels of oxidative stress, inflammatory mediators, and NF-κB activation in the retina. We successfully revealed a novel role of ROS in the pathogenesis of EAU and suggest a potential antioxidant role for the treatment of autoimmune uveitis in the future.

Genomic landscape of ductal carcinoma in situ and association with progression.

PURPOSE: The detection rate of breast ductal carcinoma in situ (DCIS) has increased significantly, raising the concern that DCIS is overdiagnosed and overtreated. Therefore, there is an unmet clinical need to better predict the risk of progression among DCIS patients. Our hypothesis is that by combining molecular signatures with clinicopathologic features, we can elucidate the biology of breast cancer progression, and risk-stratify patients with DCIS. METHODS: Targeted exon sequencing with a custom panel of 223 genes/regions was performed for 125 DCIS cases. Among them, 60 were from cases having concurrent or subsequent invasive breast cancer (IBC) (DCIS + IBC group), and 65 from cases with no IBC development over a median follow-up of 13 years (DCIS-only group). Copy number alterations in chromosome 1q32, 8q24, and 11q13 were analyzed using fluorescence in situ hybridization (FISH). Multivariable logistic regression models were fit to the outcome of DCIS progression to IBC as functions of demographic and clinical features. RESULTS: We observed recurrent variants of known IBC-related mutations, and the most commonly mutated genes in DCIS were PIK3CA (34.4%) and TP53 (18.4%). There was an inverse association between PIK3CA kinase domain mutations and progression (Odds Ratio [OR] 10.2, p < 0.05). Copy number variations in 1q32 and 8q24 were associated with progression (OR 9.3 and 46, respectively; both p < 0.05). CONCLUSIONS: PIK3CA kinase domain mutations and the absence of copy number gains in DCIS are protective against progression to IBC. These results may guide efforts to distinguish low-risk from high-risk DCIS.

Will oncotype DX DCIS testing guide therapy? A single-institution correlation of oncotype DX DCIS results with histopathologic findings and clinical management decisions.

Given the increased detection rates of ductal carcinoma in situ (DCIS) and the limited overall survival benefit from adjuvant breast irradiation after breast-conserving surgery, there is interest in identifying subsets of patients who have low rates of ipsilateral breast tumor recurrence such that they might safely forgo radiation. The Oncotype DCIS score is a reverse transcription-PCR (RT-PCR)-based assay that was validated to predict which DCIS cases are most likely to recur. Clinically, these results may be used to assist in selecting which patients with DCIS might safely forgo radiation therapy after breast-conserving surgery; however, little is currently published on how this test is being used in practice. Our study examines traditional histopathologic features used in predicting DCIS risk with Oncotype DCIS results and how these results affect clinical decision-making at our academic institution. Histopathologic features and management decisions for 37 cases with Oncotype DCIS results over the past 4 years were collected. Necrosis, high nuclear grade, biopsy site change, estrogen receptor and progesterone receptor positivity <90% on immunohistochemistry, and Van Nuys Prognostic Index score of 8 or greater were significant predictors of an intermediate-high recurrence score on multivariate regression analysis (P<0.02). Low Oncotype DCIS scores and low nuclear grade were associated with lower rate of radiation therapy (P<0.008). There were seven cases (19%) with Oncotype DCIS results that we considered unexpected in relation to the histopathologic findings (ie, high nuclear grade with comedonecrosis and a low Oncotype score, or hormone receptor discrepancies). Overall, pathologic features correlate with Oncotype DCIS scores but unexpected results do occur, making individual recommendations sometimes challenging.

Breast cancer stem cells: are we ready to go from bench to bedside?

Since the discovery of breast cancer stem cells (BCSCs) more than 10 years ago, a body of exciting research has developed. The intrinsic properties of BCSCs, including self-renewal and the ability to give rise to heterogeneous progeny, make BCSCs a likely source of tumour initiation, heterogeneity, progression and metastasis. BCSCs are also inherently resistant to conventional therapies and are therefore thought to contribute to disease recurrence. In this review, we will focus on both the challenges and recent advances in the characterization of BCSCs with respect to phenotype, molecular signature and their role in the behaviour of the different molecular subtypes of breast cancer. Of most importance is our ability to translate our growing knowledge base into the development of targeted therapies with the goal of reducing adverse outcomes in breast cancer patients.