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Chromothripsis-associated chromosome 21 amplification orchestrates transformation to blast-phase MPN through targetable overexpression of DYRK1A
Abstract Chromothripsis, the chaotic shattering and repair of chromosomes, is common in cancer. Whether chromothripsis generates actionable therapeutic targets remains an open question. In a cohort of 64 patients in blast phase of a myeloproliferative neoplasm (BP-MPN), we describe recurrent amplification of a region of chromosome 21q (‘chr. 21amp’) in 25%, driven by chromothripsis in a third of these cases. We report that chr. 21amp BP-MPN has a particularly aggressive and treatment-resistant phenotype. DYRK1A, a serine threonine kinase, is the only gene in the 2.7-megabase minimally amplified region that showed both increased expression and chromatin accessibility compared with non-chr. 21amp BP-MPN controls. DYRK1A is a central node at the nexus of multiple cellular functions critical for BP-MPN development and is essential for BP-MPN cell proliferation in vitro and in vivo, and represents a druggable axis. Collectively, these findings define chr. 21amp as a prognostic biomarker in BP-MPN, and link chromothripsis to a therapeutic target.
Measure selection for an electronic patient-reported outcome (ePRO) system for CAR T-cell therapy patients: a modified Delphi consensus study
Background: Chimeric Antigen Receptor (CAR) T-cell therapies are effective for treating haematological cancers but carry risks of toxicity and substantial symptom burden. Patient-reported outcomes (PROs) could significantly enhance clinical management for patients undergoing these treatments. However, guidance on selection of PRO measures for monitoring adverse event and quality of life after CAR T-cell therapy is limited. This study aimed to achieve consensus among patients and healthcare professionals on the selection of PRO measures for an electronic PRO (ePRO) system for CAR T-cell therapy clinical settings. Methods: Two-round modified Delphi study (online survey and consensus meeting) conducted from December 2023 to January 2024 to select PRO measures for the ePRO system, guided by a conceptual framework with four measurement domains: symptom burden, impacts of cancer and CAR T-cell therapy, treatment tolerability, and health-related quality of life (HRQoL). Database searches (PubMed, ePROVIDE, COSMIN, and COMET) and licensing websites of cancer-specific PRO measures identified 113 PRO measures. Measures were pre-specified for treatment tolerability and HRQoL domains and concept mapping established conceptual coverage for the remaining domains. Seven PRO measures were shortlisted and prespecified inclusion thresholds and stopping criteria guided Delphi panel selection. Registration: ISRCTN11232653. Findings: Nineteen participants (5 CAR T-cell patients, 14 healthcare professionals/researchers) recruited from a UK National Health Service (NHS) cellular therapy centre and professional networks took part in Round One (Delphi online survey). Shortlisted measures were rated for relevance, comprehensiveness, and ease of understanding for the symptom burden and impacts of cancer and CAR T-cell treatment domains. Consensus was achieved after Round One, precluding the requirement for Round 2 (consensus meeting). The Symptom Burden Questionnaire™ (SBQ™) and the Quality of Life in Adult Cancer Survivors (QLACS) were selected to represent the Symptom Burden and Impacts domains, respectively. These measures, EQ5D-5L, measuring HRQoL, and Functional Assessment of Chronic Illness Therapy–Item GP5 (FACT-GP5), single-item global indicator of cancer treatment tolerability, will be included in the ePRO system. Interpretation: In the absence of guidance on PRO measure selection for CAR T-cell therapies, consensus-based methods represent an important step towards use of PROs with this clinical population. Modest sample size and representativeness of the patient subgroup are limitations of this study. Funding: This study is funded by the National Institute for Health and Care Research (NIHR) Blood and Transplant Research Unit in Precision Cellular Therapeutics (NIHR203339). The views expressed are those of the authors and not necessarily those of the NIHR, NHS Blood and Transplant, or the Department of Health and Social Care.
Genome-Wide Association Studies of Down Syndrome Associated Congenital Heart Defects Suggests a Genetically Heterogeneous Risk for CHD in DS.
Congenital heart defects (CHDs) are the most common structural birth defect and are present in 40%-50% of children born with Down syndrome (DS). To characterize the genetic architecture of DS-associated CHD, we sequenced genomes of a multiethnic group of children with DS and a CHD (n = 886: atrioventricular septal defects (AVSD), n = 438; atrial septal defects (ASD), n = 122; ventricular septal defects (VSD), n = 170; other types of CHD, n = 156) and DS with a structurally normal heart (DS + NH, n = 572). We performed four genome-wide association study (GWAS) for common variants (MAF > 0.05) comparing DS with CHD, stratified by CHD-subtype, to DS + NH controls. Although no SNP achieved genome-wide significance, multiple loci in each analysis achieved suggestive significance (p
Deconvoluting clonal and cellular architecture in IDH-mutant acute myeloid leukemia.
Isocitrate dehydrogenase 1/2 (IDH) mutations are early initiating events in acute myeloid leukemia (AML). The complex clonal architecture and cellular heterogeneity in IDH-mutant AML underlies the heterogeneous clinical presentation and outcomes. Integrating single-cell genotyping and transcriptomics, we demonstrate a stem-like and inflammatory phenotype of IDH-mutant AML and identify clone-specific programs associated with NPM1, NRAS, and SRSF2 co-mutations. Furthermore, these clones had distinct responses to treatment with combination IDH inhibitors and chemotherapy, including elimination, reconstitution of myeloid differentiation, or retention within progenitor populations. At relapse after IDH inhibitor monotherapy, we identify upregulated stemness, inflammation, mitochondrial metabolism, and anti-apoptotic factors, as well as downregulated major histocompatibility complex (MHC) class II antigen presentation. At the pre-leukemic stage, we observe upregulation of IDH2-associated pathways, including inflammation. We deliver a detailed phenotyping of IDH-mutant AML and a framework for dissecting contributions of recurrently mutated genes in AML at diagnosis and following therapy, with implications for precision medicine.
Tumor-Infiltrating Clonal Hematopoiesis.
BACKGROUND: Clonal hematopoiesis of indeterminate potential (CHIP) is an age-related condition associated with increased mortality among patients with cancer. CHIP mutations with high variant-allele frequencies can be detected in tumors, a phenomenon we term tumor-infiltrating clonal hematopoiesis (TI-CH). The frequency of TI-CH and its effect on tumor evolution are unclear. METHODS: We characterized CHIP and TI-CH in 421 patients with early-stage non-small-cell lung cancer (NSCLC) from the TRACERx study and in 49,351 patients from the MSK-IMPACT pan-cancer cohort. We studied the association of TI-CH with survival and disease recurrence and evaluated the functional effect of TET2-mutant CHIP on the biologic features of lung tumors. RESULTS: Among patients with NSCLC, 42% of those with CHIP had TI-CH. TI-CH independently predicted an increased risk of death or recurrence, with an adjusted hazard ratio of 1.80 (95% confidence interval [CI], 1.23 to 2.63) as compared with the absence of CHIP and an adjusted hazard ratio of 1.62 (95% CI, 1.02 to 2.56) as compared with CHIP in the absence of TI-CH. Among patients with solid tumors, 26% of those with CHIP had TI-CH. TI-CH conferred a risk of death from any cause that was 1.17 times (95% CI, 1.06 to 1.29) as high as the risk with CHIP in the absence of TI-CH. TET2 mutations were the strongest genetic predictor of TI-CH; such mutations enhanced monocyte migration to lung tumor cells, fueled a myeloid-rich tumor microenvironment in mice, and resulted in the promotion of tumor organoid growth. CONCLUSIONS: TI-CH increased the risk of disease recurrence or death among patients with NSCLC and the risk of death from any cause among patients with solid tumors. TI-CH remodeled the tumor immune microenvironment and accelerated tumor organoid growth, findings that support a role for an aging-related hematologic clonal proliferation in cancer evolution. (Funded by the Royal Society and others.).
Lymphatic vessels in bone support regeneration after injury.
Blood and lymphatic vessels form a versatile transport network and provide inductive signals to regulate tissue-specific functions. Blood vessels in bone regulate osteogenesis and hematopoiesis, but current dogma suggests that bone lacks lymphatic vessels. Here, by combining high-resolution light-sheet imaging and cell-specific mouse genetics, we demonstrate presence of lymphatic vessels in mouse and human bones. We find that lymphatic vessels in bone expand during genotoxic stress. VEGF-C/VEGFR-3 signaling and genotoxic stress-induced IL6 drive lymphangiogenesis in bones. During lymphangiogenesis, secretion of CXCL12 from proliferating lymphatic endothelial cells is critical for hematopoietic and bone regeneration. Moreover, lymphangiocrine CXCL12 triggers expansion of mature Myh11+ CXCR4+ pericytes, which differentiate into bone cells and contribute to bone and hematopoietic regeneration. In aged animals, such expansion of lymphatic vessels and Myh11-positive cells in response to genotoxic stress is impaired. These data suggest lymphangiogenesis as a therapeutic avenue to stimulate hematopoietic and bone regeneration.
SUMIC: A Simple Ultrafast Multicolor Immunolabelling and Clearing Approach for Whole-Organ and Large Tissue 3D Imaging
High-resolution whole-organ imaging of cleared tissues captures cellular and molecular insights within the intact tissue and tumour microenvironments. However, current immunolabelling and clearing methods are complicated and time-consuming; extending to several weeks. Here, we developed S imple U ltrafast M ulticolor I mmunolabelling and C learing or SUMIC , a method that enables multicolor immunolabelling and clearing of whole murine organs and human tissues within 2 to 2.5 days. Moreover, SUMIC is simple, robust, non-hazardous and versatile comprising antigen retrieval, permeabilization, collagenase-based digestion, immunolabelling, dehydration, and clearing. SUMIC permits quantitative and singlecell resolution analysis and detection of rare cells in whole organs, for example, round αSMA positive cells in the thymus. Upon volumetric imaging, SUMIC-processed samples retain normal tissue architecture and can be used for paraffin-embedding and histology. We employed the SUMIC method for whole-organ mapping of lymphatic vessels across different ages and organs. This analysis revealed the expansion of lymphatic vessels in endocrine tissues but not in any other organs with aging. Hence, SUMIC will accelerate discoveries compared to other whole organ imaging pipelines.
Vitamin D Attenuates Oxidative Damage and Inflammation in Retinal Pigment Epithelial Cells.
Age-related macular degeneration (AMD), the most common visual disorder in elderly people, is characterized by the formation of deposits beneath the retinal pigment epithelium (RPE) and by dysfunction of RPE and photoreceptor cells. The biologically active form of vitamin D, 1,25-(OH)2D3 (VITD), is categorized as a multifunctional steroid hormone that modulates many transcriptional processes of different genes and is involved in a broad range of cellular functions. Epidemiological and genetic association studies demonstrate that VITD may have a protective role in AMD, while single nucleotide polymorphisms in the vitamin D metabolism gene (CYP24A1) increase the risk of AMD. However, the functional mechanisms of VITD in AMD are not fully understood. In the current study, we investigated the impact of VITD on H2O2-induced oxidative stress and inflammation in human RPE cells. We demonstrate that exposure to H2O2 caused significantly reduced cell viability, increased production of reactive oxygen species (ROS), lowered expression of antioxidant enzymes and enhanced inflammation. VITD exposure notably counteracted the above H2O2-induced effects. Our data suggest that VITD protects the RPE from oxidative damage and elucidate molecular mechanisms of VITD deficiency in the development of AMD.
Deletion of TSPO Resulted in Change of Metabolomic Profile in Retinal Pigment Epithelial Cells.
Age-related macular degeneration is the main cause of vision loss in the aged population worldwide. Drusen, extracellular lesions formed underneath the retinal pigment epithelial (RPE) cells, are a clinical feature of AMD and associated with AMD progression. RPE cells support photoreceptor function by providing nutrition, phagocytosing outer segments and removing metabolic waste. Dysfunction and death of RPE cells are early features of AMD. The translocator protein, TSPO, plays an important role in RPE cholesterol efflux and loss of TSPO results in increased intracellular lipid accumulation and reactive oxygen species (ROS) production. This study aimed to investigate the impact of TSPO knockout on RPE cellular metabolism by identifying the metabolic differences between wildtype and knockout RPE cells, with or without treatment with oxidized low density lipoprotein (oxLDL). Using liquid chromatography mass spectrometry (LC/MS), we differentiated several metabolic pathways among wildtype and knockout cells. Lipids amongst other intracellular metabolites were the most influenced by loss of TSPO and/or oxLDL treatment. Glucose, amino acid and nucleotide metabolism was also affected. TSPO deletion led to up-regulation of fatty acids and glycerophospholipids, which in turn possibly affected the cell membrane fluidity and stability. Higher levels of glutathione disulphide (GSSG) were found in TSPO knockout RPE cells, suggesting TSPO regulates mitochondrial-mediated oxidative stress. These data provide biochemical insights into TSPO-associated function in RPE cells and may shed light on disease mechanisms in AMD.
TSPO Ligands Promote Cholesterol Efflux and Suppress Oxidative Stress and Inflammation in Choroidal Endothelial Cells.
Choroidal endothelial cells supply oxygen and nutrients to retinal pigment epithelial (RPE) cells and photoreceptors, recycle metabolites, and dispose of metabolic waste through the choroidal blood circulation. Death of the endothelial cells of the choroid may cause abnormal deposits including unesterified and esterified cholesterol beneath RPE cells and within Bruch's membrane that contribute to the progression of age-related macular degeneration (AMD), the most prevalent cause of blindness in older people. Translocator protein (TSPO) is a cholesterol-binding protein that is involved in mitochondrial cholesterol transport and other cellular functions. We have investigated the role of TSPO in choroidal endothelial cells. Immunocytochemistry showed that TSPO was localized to the mitochondria of choroidal endothelial cells. Choroidal endothelial cells exposed to TSPO ligands (Etifoxine or XBD-173) had significantly increased cholesterol efflux, higher expression of cholesterol homeostasis genes (LXRα, CYP27A1, CYP46A1, ABCA1 and ABCG1), and reduced biosynthesis of cholesterol and phospholipids from [14C]acetate, when compared to untreated controls. Treatment with TSPO ligands also resulted in reduced production of reactive oxygen species (ROS), increased antioxidant capacity, and reduced release of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α and VEGF) induced by oxidized LDL. These data suggest TSPO ligands may offer promise for the treatment of AMD.
Risk Stratification in Older Intensively Treated Patients With AML.
PURPOSE: AML is a genetically heterogeneous disease, particularly in older patients. In patients older than 60 years, survival rates are variable after the most important curative approach, intensive chemotherapy followed by allogeneic hematopoietic cell transplantation (allo-HCT). Thus, there is an urgent need in clinical practice for a prognostic model to identify older patients with AML who benefit from curative treatment. METHODS: We studied 1,910 intensively treated patients older than 60 years with AML and high-risk myelodysplastic syndrome (HR-MDS) from two cohorts (NCRI-AML18 and HOVON-SAKK). The median patient age was 67 years. Using a random survival forest, clinical, molecular, and cytogenetic variables were evaluated in an AML development cohort (n = 1,204) for association with overall survival (OS). Relative weights of selected variables determined the prognostic model, which was validated in AML (n = 491) and HR-MDS cohorts (n = 215). RESULTS: The complete cohort had a high frequency of poor-risk features, including 2022 European LeukemiaNet adverse-risk (57.3%), mutated TP53 (14.4%), and myelodysplasia-related genetic features (65.1%). Nine variables were used to construct four groups with highly distinct 4-year OS in the (1) AML development, (2) AML validation, and (3) HR-MDS test cohorts ([1] favorable: 54% ± 4%, intermediate: 38% ± 2%, poor: 21% ± 2%, very poor: 4% ± 1%; [2] 54% ± 9%, 43% ± 4%, 27% ± 4%, 4% ± 3%; and [3] 54% ± 10%, 33% ± 6%, 14% ± 5%, 0% ± 3%, respectively). This new AML60+ classification improves current prognostic classifications. Importantly, patients within the AML60+ intermediate- and very poor-risk group significantly benefited from allo-HCT, whereas the poor-risk patients showed an indication, albeit nonsignificant, for improved outcome after allo-HCT. CONCLUSION: The new AML60+ classification provides prognostic information for intensively treated patients 60 years and older with AML and HR-MDS and identifies patients who benefit from intensive chemotherapy and allo-HCT.
Tracking in situ checkpoint inhibitor-bound target T cells in patients with checkpoint-induced colitis.
The success of checkpoint inhibitors (CPIs) for cancer has been tempered by immune-related adverse effects including colitis. CPI-induced colitis is hallmarked by expansion of resident mucosal IFNγ cytotoxic CD8+ T cells, but how these arise is unclear. Here, we track CPI-bound T cells in intestinal tissue using multimodal single-cell and subcellular spatial transcriptomics (ST). Target occupancy was increased in inflamed tissue, with drug-bound T cells located in distinct microdomains distinguished by specific intercellular signaling and transcriptional gradients. CPI-bound cells were largely CD4+ T cells, including enrichment in CPI-bound peripheral helper, follicular helper, and regulatory T cells. IFNγ CD8+ T cells emerged from both tissue-resident memory (TRM) and peripheral populations, displayed more restricted target occupancy profiles, and co-localized with damaged epithelial microdomains lacking effective regulatory cues. Our multimodal analysis identifies causal pathways and constitutes a resource to inform novel preventive strategies.
HLA-dependent variation in SARS-CoV-2 CD8 + T cell cross-reactivity with human coronaviruses.
The conditions and extent of cross-protective immunity between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and common-cold human coronaviruses (HCoVs) remain open despite several reports of pre-existing T cell immunity to SARS-CoV-2 in individuals without prior exposure. Using a pool of functionally evaluated SARS-CoV-2 peptides, we report a map of 126 immunogenic peptides with high similarity to 285 MHC-presented peptides from at least one HCoV. Employing this map of SARS-CoV-2-non-homologous and homologous immunogenic peptides, we observe several immunogenic peptides with high similarity to human proteins, some of which have been reported to have elevated expression in severe COVID-19 patients. After combining our map with SARS-CoV-2-specific TCR repertoire data from COVID-19 patients and healthy controls, we show that public repertoires for the majority of convalescent patients are dominated by TCRs cognate to non-homologous SARS-CoV-2 peptides. We find that for a subset of patients, >50% of their public SARS-CoV-2-specific repertoires consist of TCRs cognate to homologous SARS-CoV-2-HCoV peptides. Further analysis suggests that this skewed distribution of TCRs cognate to homologous or non-homologous peptides in COVID-19 patients is likely to be HLA-dependent. Finally, we provide 10 SARS-CoV-2 peptides with known cognate TCRs that are conserved across multiple coronaviruses and are predicted to be recognized by a high proportion of the global population. These findings may have important implications for COVID-19 heterogeneity, vaccine-induced immune responses, and robustness of immunity to SARS-CoV-2 and its variants.
Evaluating performance of existing computational models in predicting CD8+ T cell pathogenic epitopes and cancer neoantigens.
T cell recognition of a cognate peptide-major histocompatibility complex (pMHC) presented on the surface of infected or malignant cells is of the utmost importance for mediating robust and long-term immune responses. Accurate predictions of cognate pMHC targets for T cell receptors would greatly facilitate identification of vaccine targets for both pathogenic diseases and personalized cancer immunotherapies. Predicting immunogenic peptides therefore has been at the center of intensive research for the past decades but has proven challenging. Although numerous models have been proposed, performance of these models has not been systematically evaluated and their success rate in predicting epitopes in the context of human pathology has not been measured and compared. In this study, we evaluated the performance of several publicly available models, in identifying immunogenic CD8+ T cell targets in the context of pathogens and cancers. We found that for predicting immunogenic peptides from an emerging virus such as severe acute respiratory syndrome coronavirus 2, none of the models perform substantially better than random or offer considerable improvement beyond HLA ligand prediction. We also observed suboptimal performance for predicting cancer neoantigens. Through investigation of potential factors associated with ill performance of models, we highlight several data- and model-associated issues. In particular, we observed that cross-HLA variation in the distribution of immunogenic and non-immunogenic peptides in the training data of the models seems to substantially confound the predictions. We additionally compared key parameters associated with immunogenicity between pathogenic peptides and cancer neoantigens and observed evidence for differences in the thresholds of binding affinity and stability, which suggested the need to modulate different features in identifying immunogenic pathogen versus cancer peptides. Overall, we demonstrate that accurate and reliable predictions of immunogenic CD8+ T cell targets remain unsolved; thus, we hope our work will guide users and model developers regarding potential pitfalls and unsettled questions in existing immunogenicity predictors.