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BACKGROUND: Adjuvant immune checkpoint blockade (ICB) following chemoradiotherapy and adding ICB to chemotherapy have been key advances for stages III-IV non-small cell lung cancer (NSCLC) treatment. However, known biomarkers like PD-L1 are not consistently indicative of ICB response. Other markers within the tumor immune microenvironment (TIME) may better reflect ICB response and/or resistance mechanisms, but an understanding of how TIMEs differ between stage III and IV NSCLC has not been explored. METHODS: Real-world data from unresectable, stage III-IV, non-squamous, pretreatment NSCLCs (stage III n = 106, stage IV n = 285) were retrospectively analyzed. PD-L1 immunohistochemistry (IHC) was compared to CD274 gene expression. Then, differential gene expression levels, pathway enrichment, and immune infiltrate between stages were calculated from whole-transcriptome RNA-seq. Analyses were stratified by EGFR status. RESULTS: PD-L1 IHC and CD274 expression in tumor cells were highly correlated (n = 295, P < 2.2e-16, â´ = 0.74). CTLA4 expression was significantly increased in stage III tumors (P = 1.32e-04), while no differences were observed for other ICB-related genes. Metabolic pathway activity was significantly enriched in stage IV tumors (P = 0.004), whereas several immune-related KEGG pathways were enriched in stage III. Stage IV tumors had significantly increased macrophage infiltration (P = 0.0214), and stage III tumors had a significantly higher proportion of CD4 + T cells (P = 0.017). CD4 + T cells were also relatively more abundant in EGFR-mutant tumors vs. wild-type (P = 0.0081). CONCLUSION: Directly comparing the TIMEs of stage III and IV NSCLC, these results carry implications for further studies of ICB response in non-resectable stage III NSCLC and guide further research of prognostic biomarkers and therapeutic targets.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Humanos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Antígeno B7-H1/metabolismo , Estudios Retrospectivos , Biomarcadores , Microambiente Tumoral , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Receptores ErbB , Biomarcadores de TumorRESUMEN
Nur77 (Nr4a1) belongs to a small family of orphan nuclear receptors that are rapidly induced by BCR stimulation, yet little is known about its function in B cells. We have previously characterized a reporter of Nr4a1 transcription, Nur77-eGFP, in which GFP expression faithfully detects Ag encounter by B cells in vitro and in vivo. In this study, we report that Nur77 expression correlates with the degree of self-reactivity, counterselection, and anergy among individual B cell clones from two distinct BCR transgenic mouse models but is dispensable for all of these tolerance mechanisms. However, we identify a role for Nur77 in restraining survival of self-reactive B cells in the periphery under conditions of competition for a limited supply of the survival factor BAFF. We find that Nur77 deficiency results in the progressive accumulation of self-reactive B cells in the mature repertoire with age and is sufficient to break B cell tolerance in VH3H9 H chain transgenic mice. We thus propose that Nur77 is upregulated in self-reactive B cells in response to chronic Ag stimulation and selectively restricts the survival of these cells, gradually pruning self-reactivity from the mature repertoire to impose a novel layer of peripheral B cell tolerance.
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Antígenos/farmacología , Linfocitos B/inmunología , Tolerancia Inmunológica/efectos de los fármacos , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/inmunología , Receptores de Antígenos de Linfocitos B/inmunología , Animales , Antígenos/inmunología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/inmunología , Ratones , Ratones Noqueados , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genética , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/inmunología , Receptores de Antígenos de Linfocitos B/genéticaRESUMEN
RATIONALE: Ventricular arrhythmias remain the leading cause of death in patients suffering myocardial ischemia. Myeloperoxidase, a heme enzyme released by polymorphonuclear neutrophils, accumulates within ischemic myocardium and has been linked to adverse left ventricular remodeling. OBJECTIVE: To reveal the role of myeloperoxidase for the development of ventricular arrhythmias. METHODS AND RESULTS: In different murine models of myocardial ischemia, myeloperoxidase deficiency profoundly decreased vulnerability for ventricular tachycardia on programmed right ventricular and burst stimulation and spontaneously as assessed by ECG telemetry after isoproterenol injection. Experiments using CD11b/CD18 integrin-deficient (CD11b-/-) mice and intravenous myeloperoxidase infusion revealed that neutrophil infiltration is a prerequisite for myocardial myeloperoxidase accumulation. Ventricles from myeloperoxidase-deficient (Mpo-/-) mice showed less pronounced slowing and decreased heterogeneity of electric conduction in the peri-infarct zone than wild-type mice. Expression of the redox-sensitive gap junctional protein Cx43 (Connexin 43) was reduced in the peri-infarct area of wild-type compared with Mpo-/- mice. In isolated wild-type cardiomyocytes, Cx43 protein content decreased on myeloperoxidase/H2O2 incubation. Mapping of induced pluripotent stem cell-derived cardiomyocyte networks and in vivo investigations linked Cx43 breakdown to myeloperoxidase-dependent activation of matrix metalloproteinase 7. Moreover, Mpo-/- mice showed decreased ventricular postischemic fibrosis reflecting reduced accumulation of myofibroblasts. Ex vivo, myeloperoxidase was demonstrated to induce fibroblast-to-myofibroblast transdifferentiation by activation of p38 mitogen-activated protein kinases resulting in upregulated collagen generation. In support of our experimental findings, baseline myeloperoxidase plasma levels were independently associated with a history of ventricular arrhythmias, sudden cardiac death, or implantable cardioverter-defibrillator implantation in a cohort of 2622 stable patients with an ejection fraction >35% undergoing elective diagnostic cardiac evaluation. CONCLUSIONS: Myeloperoxidase emerges as a crucial mediator of postischemic myocardial remodeling and may evolve as a novel pharmacological target for secondary disease prevention after myocardial ischemia.
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Arritmias Cardíacas/metabolismo , Isquemia Miocárdica/metabolismo , Miocitos Cardíacos/metabolismo , Peroxidasa/deficiencia , Remodelación Ventricular/fisiología , Animales , Arritmias Cardíacas/patología , Células Cultivadas , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Isquemia Miocárdica/patología , Miocitos Cardíacos/patología , Técnicas de Cultivo de ÓrganosRESUMEN
Neutrophil recruitment and activation are principal events in inflammation. Upon activation neutrophils release myeloperoxidase (MPO), a heme enzyme, which binds to and transcytoses endothelial cells. Whereas the significance of the subendothelial deposition of MPO has evolved as a critical prerequisite for the enzyme's suppression of nitric oxide (NOâ ) bioavailability, the functional consequences of MPO binding to and interaction with endothelial and smooth muscle cells remain poorly understood. Cultured human endothelial cells (HUVECs) were exposed to MPO. Gene expression of the endothelin receptor type B (ETRB), which is critically involved not only in endothelin-1 clearance, but also in endothelin-mediated vasoconstriction, was significantly increased. Real time PCR, Western blotting and immunofluorescence confirmed up-regulation of ETRB in MPO-treated endothelial cells. Inhibition of MPO's enzymatic activity blunted the increase in ETRB protein expression. Treatment of the cells with the MAP kinase inhibitors PD98059 or SB203580 indicates that MPO activates ETRB expression via MAP kinase pathways. On human smooth muscle cells (HAoSMCs), which not only express the endothelin receptor type B (ETRB) but also express the endothelin receptor type A (ETRA), MPO also stimulated ETRB expression as opposed to ETRA expression, which remained unchanged. Functional ex vivo organ bath chamber studies with MPO-incubated rat femoral artery sections revealed increased ETRB agonist dependent constriction. Binding of MPO to endothelial and vascular smooth muscle cells increases expression of the endothelin receptor type B (ETRB) via classical MAP kinase pathways. This suggests that MPO not only affects vasomotion by reducing the bioavailability of vasodilating molecules but also by increasing responsiveness to vasoconstrictors, further advocating for MPO as a central, leukocyte-derived regulator of vascular tone.
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Aorta/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Músculo Liso Vascular/metabolismo , Peroxidasa/metabolismo , Receptor de Endotelina B/metabolismo , Vasoconstricción/fisiología , Animales , Aorta/citología , Biomarcadores/metabolismo , Western Blotting , Células Cultivadas , Perfilación de la Expresión Génica , Células Endoteliales de la Vena Umbilical Humana/citología , Humanos , Técnicas para Inmunoenzimas , Masculino , Músculo Liso Vascular/citología , Análisis de Secuencia por Matrices de Oligonucleótidos , Peroxidasa/genética , ARN Mensajero/genética , Ratas , Ratas Wistar , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptor de Endotelina A/genética , Receptor de Endotelina A/metabolismo , Receptor de Endotelina B/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Regulación hacia ArribaRESUMEN
Myeloperoxidase (MPO) is a heme enzyme abundantly expressed in polymorphonuclear neutrophils. MPO is enzymatically capable of catalyzing the generation of reactive oxygen species (ROS) and the consumption of nitric oxide (NO). Thus MPO has both potent microbicidal and, upon binding to the vessel wall, pro-inflammatory properties. Interestingly, MPO - a highly cationic protein - has been shown to bind to both endothelial cells and leukocyte membranes. Given the anionic surface charge of red blood cells, we investigated binding of MPO to erythrocytes. Red blood cells (RBCs) derived from patients with elevated MPO plasma levels showed significantly higher amounts of MPO by flow cytometry and ELISA than healthy controls. Heparin-induced MPO-release from patient-derived RBCs was significantly increased compared to controls. Ex vivo experiments revealed dose and time dependency for MPO-RBC binding, and immunofluorescence staining as well as confocal microscopy localized MPO-RBC interaction to the erythrocyte plasma membrane. NO-consumption by RBC-membrane fragments (erythrocyte "ghosts") increased with incrementally greater concentrations of MPO during incubation, indicating preserved catalytic MPO activity. In vivo infusion of MPO-loaded RBCs into C57BL/6J mice increased local MPO tissue concentrations in liver, spleen, lung, and heart tissue as well as within the cardiac vasculature. Further, NO-dependent relaxation of aortic rings was altered by RBC bound-MPO and systemic vascular resistance significantly increased after infusion of MPO-loaded RBCs into mice. In summary, we find that MPO binds to RBC membranes in vitro and in vivo, is transported by RBCs to remote sites in mice, and affects endothelial function as well as systemic vascular resistance. RBCs may avidly bind circulating MPO, and act as carriers of this leukocyte-derived enzyme.
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Síndrome Coronario Agudo/sangre , Eritrocitos/metabolismo , Insuficiencia Cardíaca/sangre , Peroxidasa/sangre , Síndrome Coronario Agudo/patología , Animales , Aorta/efectos de los fármacos , Transporte Biológico , Células Cultivadas , Endotelio Vascular/efectos de los fármacos , Eritrocitos/patología , Corazón/efectos de los fármacos , Insuficiencia Cardíaca/patología , Heparina/química , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Óxido Nítrico/metabolismo , Óxido Nítrico/farmacología , Técnicas de Cultivo de Órganos , Peroxidasa/farmacología , Unión Proteica , Técnicas de Cultivo de Tejidos , Resistencia Vascular/efectos de los fármacosRESUMEN
BACKGROUND: Myeloperoxidase (MPO) is an abundant hemoprotein expressed by neutrophil granulocytes that is recognized to play an important role in the development of vascular diseases. Upon degranulation from circulating neutrophil granulocytes, MPO binds to the surface of endothelial cells in an electrostatic-dependent manner and undergoes transcytotic migration to the underlying extracellular matrix (ECM). However, the mechanisms governing the binding of MPO to subendothelial ECM proteins, and whether this binding modulates its enzymatic functions are not well understood. METHODS: We investigated MPO binding to ECM derived from aortic endothelial cells, aortic smooth muscle cells, and fibroblasts, and to purified ECM proteins, and the modulation of these associations by glycosaminoglycans. The oxidizing and chlorinating potential of MPO upon binding to ECM proteins was tested. RESULTS: MPO binds to the ECM proteins collagen IV and fibronectin, and this association is enhanced by the pre-incubation of these proteins with glycosaminoglycans. Correspondingly, an excess of glycosaminoglycans in solution during incubation inhibits the binding of MPO to collagen IV and fibronectin. These observations were confirmed with cell-derived ECM. The oxidizing and chlorinating potential of MPO was preserved upon binding to collagen IV and fibronectin; even the potentiation of MPO activity in the presence of collagen IV and fibronectin was observed. CONCLUSIONS: Collectively, the data reveal that MPO binds to ECM proteins on the basis of electrostatic interactions, and MPO chlorinating and oxidizing activity is potentiated upon association with these proteins. GENERAL SIGNIFICANCE: Our findings provide new insights into the molecular mechanisms underlying the interaction of MPO with ECM proteins.
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Proteínas de la Matriz Extracelular/metabolismo , Glicosaminoglicanos/metabolismo , Peroxidasa/metabolismo , Células Cultivadas , Colágeno Tipo IV/metabolismo , Dimerización , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Proteínas de la Matriz Extracelular/química , Fibronectinas/metabolismo , Humanos , Nitratos/metabolismo , Estrés Oxidativo , Unión Proteica , Tirosina/metabolismoRESUMEN
Recruitment of polymorphonuclear neutrophils (PMNs) remains a paramount prerequisite in innate immune defense and a critical cofounder in inflammatory vascular disease. Neutrophil recruitment comprises a cascade of concerted events allowing for capture, adhesion and extravasation of the leukocyte. Whereas PMN rolling, binding, and diapedesis are well characterized, receptor-mediated processes, mechanisms attenuating the electrostatic repulsion between the negatively charged glycocalyx of leukocyte and endothelium remain poorly understood. We provide evidence for myeloperoxidase (MPO), an abundant PMN-derived heme protein, facilitating PMN recruitment by its positive surface charge. In vitro, MPO evoked highly directed PMN motility, which was solely dependent on electrostatic interactions with the leukocyte's surface. In vivo, PMN recruitment was shown to be MPO-dependent in a model of hepatic ischemia and reperfusion, upon intraportal delivery of MPO and in the cremaster muscle exposed to local inflammation or to intraarterial MPO application. Given MPO's affinity to both the endothelial and the leukocyte's surface, MPO evolves as a mediator of PMN recruitment because of its positive surface charge. This electrostatic MPO effect not only displays a so far unrecognized, catalysis-independent function of the enzyme, but also highlights a principal mechanism of PMN attraction driven by physical forces.
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Infiltración Neutrófila , Neutrófilos/fisiología , Peroxidasa/fisiología , Fenómenos Físicos , Animales , Células Cultivadas , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Infiltración Neutrófila/inmunología , Neutrófilos/metabolismo , Peroxidasa/química , Peroxidasa/genética , Peroxidasa/metabolismo , Unión Proteica/fisiología , Electricidad Estática , Propiedades de SuperficieRESUMEN
AIMS: Observational studies have suggested a mechanistic link between the leucocyte-derived enzyme myeloperoxidase (MPO) and vasomotor function. Here, we tested whether MPO is systemically affecting vascular tone in humans. METHODS AND RESULTS: A total of 12 135 patients were screened for leucocyte peroxidase activity. We identified 15 individuals with low MPO expression and activity (MPO(low)), who were matched with 30 participants exhibiting normal MPO protein content and activity (control). Nicotine-dependent activation of leucocytes caused attenuation of endothelial nitric oxide (NO) bioavailability in the control group (P < 0.01), but not in MPO(low) individuals (P = 0.12); here the MPO burden of leucocytes correlated with the degree of vasomotor dysfunction (P = 0.008). To directly test the vasoactive properties of free circulating MPO, the enzyme was injected into the left atrium of anaesthetized, open-chest pigs. Myeloperoxidase plasma levels peaked within minutes and rapidly declined thereafter, reflecting vascular binding of MPO. Blood flow in the left anterior descending artery and the internal mammary artery (IMA) as well as myocardial perfusion decreased following MPO injection when compared with albumin-treated animals (P < 0.001). Isolated IMA-rings from animals subjected to MPO revealed markedly diminished relaxation in response to acetylcholine (P < 0.01) and nitroglycerine as opposed to controls (P < 0.001). CONCLUSION: Myeloperoxidase elicits profound effects on vascular tone of conductance and resistance vessels in vivo. These findings not only call for revisiting the biological functions of leucocytes as systemic and mobile effectors of vascular tone, but also identify MPO as a critical systemic regulator of vasomotion in humans and thus a potential therapeutic target.
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Neutrófilos/enzimología , Peroxidasa/deficiencia , Sistema Vasomotor/enzimología , Adulto , Anciano , Animales , Velocidad del Flujo Sanguíneo , Circulación Coronaria/fisiología , Endotelio Vascular/enzimología , Hemodinámica/fisiología , Humanos , Masculino , Arterias Mamarias/fisiología , Metaloproteinasa 9 de la Matriz/metabolismo , Persona de Mediana Edad , Activación Neutrófila , Nicotina/farmacología , Óxido Nítrico/metabolismo , Peroxidasa/metabolismo , Peroxidasa/farmacología , Sus scrofa , Vasodilatación/fisiología , Adulto JovenRESUMEN
BACKGROUND: The nitric oxide synthase inhibitor asymmetrical dimethylarginine (ADMA) and the leukocyte-derived hemoprotein myeloperoxidase (MPO) are associated with cardiovascular diseases. Activation of monocytes and polymorphonuclear neutrophils (PMNs) with concomitant release of MPO is regulated in a nitric oxide-dependent fashion. The aim of the study was to investigate a potential 2-way interaction between ADMA and MPO. METHODS AND RESULTS: Ex vivo, ADMA uptake by isolated human PMNs, the principal source of MPO in humans, significantly impaired nitric oxide synthase activity determined by gas chromatography-mass spectrometry. In humans, short-term ADMA infusion (0.0125 mg · kg(-1) · min(-1)) significantly increased MPO plasma concentrations. Functionally, PMN exposure to ADMA enhanced leukocyte adhesion to endothelial cells, augmented NADPH oxidase activity, and stimulated PMN degranulation, resulting in release of MPO. In vivo, a 28-day ADMA infusion (250 µmol · kg(-1) · d(-1)) in C57Bl/6 mice significantly increased plasma MPO concentrations, whereas this ADMA effect on MPO was attenuated by human dimethylarginine dimethylaminohydrolase1 (hDDAH1) overexpression. Moreover, the MPO-derived reactive molecule hypochlorous acid impaired recombinant hDDAH1 activity in vitro. In MPO(-/-) mice, the lipopolysaccharide-induced increase in systemic ADMA concentrations was abrogated. CONCLUSIONS: ADMA profoundly impairs nitric oxide synthesis of PMNs, resulting in increased PMN adhesion to endothelial cells, superoxide generation, and release of MPO. In addition, MPO impairs DDAH1 activity. Our data reveal an ADMA-induced cycle of PMN activation, enhanced MPO release, and subsequent impairment of DDAH1 activity. These findings not only highlight so far unrecognized cytokine-like properties of ADMA but also identify MPO as a regulatory switch for ADMA bioavailability under inflammatory conditions.
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Arginina/análogos & derivados , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Óxido Nítrico Sintasa de Tipo III/antagonistas & inhibidores , Peroxidasa/metabolismo , Amidohidrolasas/genética , Amidohidrolasas/metabolismo , Animales , Arginina/farmacología , Enfermedades Cardiovasculares/metabolismo , Enfermedades Cardiovasculares/fisiopatología , Relación Dosis-Respuesta a Droga , Femenino , Células HL-60 , Humanos , Técnicas In Vitro , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Modelos Animales , Neutrófilos/citología , Óxido Nítrico/metabolismo , Peroxidasa/deficiencia , Peroxidasa/genética , Transducción de Señal/fisiología , Superóxidos/metabolismoRESUMEN
The efficacy of immune checkpoint blockade (ICB) varies greatly among metastatic non-small cell lung cancer (NSCLC) patients. Loss of heterozygosity at the HLA-I locus (HLA-LOH) has been identified as an important immune escape mechanism. However, despite HLA-I disruptions in their tumor, many patients have durable ICB responses. Here we seek to identify HLA-I-independent features associated with ICB response in NSCLC. We use single-cell profiling to identify tumor-infiltrating, clonally expanded CD4+ T cells that express a canonical cytotoxic gene program and NSCLC cells with elevated HLA-II expression. We postulate cytotoxic CD4+ T cells mediate anti-tumor activity via HLA-II on tumor cells and augment HLA-I-dependent cytotoxic CD8+ T cell interactions to drive ICB response in NSCLC. We show that integrating tumor extrinsic cytotoxic gene expression with tumor mutational burden is associated with longer time to progression in a real-world cohort of 123 NSCLC patients treated with ICB regimens, including those with HLA-LOH.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Biomarcadores de Tumor/genética , Linfocitos T CD8-positivos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Humanos , Inmunoterapia , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genéticaRESUMEN
PURPOSE: Biliary tract cancers (BTCs) are aggressive cancers that carry a poor prognosis. An enhanced understanding of the immune landscape of anatomically and molecularly defined subsets of BTC may improve patient selection for immunotherapy and inform immune-based combination treatment strategies. METHODS: We analyzed deidentified clinical, genomic, and transcriptomic data from the Tempus database to determine the mutational frequency and mutational clustering across the three major BTC subtypes (intrahepatic cholangiocarcinoma [IHC], extrahepatic cholangiocarcinoma, and gallbladder cancer). We subsequently determined the relationship between specific molecular alterations and anatomical subsets and features of the BTC immune microenvironment. RESULTS: We analyzed 454 samples of BTC, of which the most commonly detected alterations were TP53 (42.5%), CDKN2A (23.4%), ARID1A (19.6%), BAP1 (15.5%), KRAS (15%), CDKN2B (14.2%), PBRM1 (11.7%), IDH1 (11.7%), TERT (8.4%), KMT2C (10.4%) and LRP1B (8.4%), and FGFR2 fusions (8.7%). Potentially actionable molecular alterations were identified in 30.5% of BTCs including 39.1% of IHC. Integrative cluster analysis revealed four distinct molecular clusters, with cluster 4 predominately associated with FGFR2 rearrangements and BAP1 mutations in IHC. Immune-related biomarkers indicative of an inflamed tumor-immune microenvironment were elevated in gallbladder cancers and in cluster 1, which was enriched for TP53, KRAS, and ATM mutations. Multiple common driver genes, including TP53, FGFR2, IDH1, TERT, BRAF, and BAP1, were individually associated with unique BTC immune microenvironments. CONCLUSION: BTC subtypes exhibit diverse DNA alterations, RNA inflammatory signatures, and immune biomarkers. The association between specific BTC anatomical subsets, molecular alterations, and immunophenotypes highlights new opportunities for therapeutic development.
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Neoplasias de los Conductos Biliares , Neoplasias del Sistema Biliar , Colangiocarcinoma , Neoplasias de la Vesícula Biliar , Neoplasias de los Conductos Biliares/genética , Conductos Biliares Intrahepáticos/patología , Neoplasias del Sistema Biliar/genética , Colangiocarcinoma/genética , Neoplasias de la Vesícula Biliar/genética , Genómica , Humanos , Proteínas Proto-Oncogénicas p21(ras)/genética , Transcriptoma/genética , Microambiente Tumoral/genéticaRESUMEN
Inflammation and angiogenesis are intimately linked, and their dysregulation leads to pathological angiogenesis in human diseases. 15-lipoxygenase (15-LOX) and lipoxin A(4) receptors (ALX) constitute a LXA(4) circuit that is a key feature of inflammatory resolution. LXA(4) analogs have been shown to regulate vascular endothelial growth factor (VEGF)-A-induced angiogenic response in vitro. 15-LOX and ALX are highly expressed in the avascular and immune-privileged cornea. However, the role of this endogenous LXA(4) circuit in pathological neovascularization has not been determined. We report that suture-induced chronic injury in the cornea triggered polymorphonuclear leukocytes (PMN) infiltration, pathological neovascularization, and up-regulation of mediators of inflammatory angiogenesis, namely VEGF-A and the VEGF-3 receptor (FLT4). Up-regulation of the VEGF circuit and neovascularization correlated with selective changes in both 15-LOX (Alox15) and ALX (Fpr-rs2) expression and a temporally defined increase in basal 15-LOX activity. More importantly, genetic deletion of 15-LOX or 5-LOX, key and obligatory enzymes in the formation of LXA(4), respectively, led to exacerbated inflammatory neovascularization coincident with increased VEGF-A and FLT4 expression. Direct topical treatment with LXA(4), but not its metabolic precursor 15-hydroxyeicosatetraenoic acid, reduced expression of VEGF-A and FLT4 and inflammatory angiogenesis and rescued 15-LOX knockout mice from exacerbated angiogenesis. In summary, our findings and the prominent expression of 15-LOX and ALX in epithelial cells and macrophages place the LXA(4) circuit as an endogenous regulator of pathological angiogenesis.
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Enfermedades de la Córnea/metabolismo , Lipoxinas/metabolismo , Neovascularización Patológica/metabolismo , Administración Tópica , Animales , Araquidonato 15-Lipooxigenasa/metabolismo , Araquidonato 5-Lipooxigenasa/metabolismo , Autacoides/metabolismo , Córnea/irrigación sanguínea , Córnea/efectos de los fármacos , Córnea/patología , Enfermedades de la Córnea/complicaciones , Enfermedades de la Córnea/enzimología , Regulación hacia Abajo/efectos de los fármacos , Eliminación de Gen , Inflamación/complicaciones , Inflamación/enzimología , Lipoxinas/administración & dosificación , Lipoxinas/farmacología , Redes y Vías Metabólicas/efectos de los fármacos , Ratones , Neovascularización Patológica/complicaciones , Neovascularización Patológica/enzimología , FenotipoRESUMEN
RNA sequencing (RNA-seq) provides an efficient high-throughput technique to robustly characterize the tumor immune microenvironment (TME). The increasing use of RNA-seq in clinical and basic science settings provides a powerful opportunity to access novel therapeutic biomarkers in the TME. Advanced computational methods are making it possible to resolve the composition of the tumor immune infiltrate, infer the immunological phenotypes of those cells, and assess the immune receptor repertoire in RNA-seq data. These immunological characterizations have increasingly important implications for guiding immunotherapy use. Here, we highlight recent studies that demonstrate the potential utility of RNA-seq in clinical settings, review key computational methods used for characterizing the TME for precision cancer immunotherapy, and discuss important considerations in data interpretation and current technological limitations.
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Biomarcadores de Tumor , Neoplasias/genética , Neoplasias/patología , Microambiente Tumoral/genética , Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Inmunoterapia/métodos , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Linfocitos Infiltrantes de Tumor/patología , Neoplasias/inmunología , Medicina de Precisión/métodos , Análisis de Secuencia de ARNRESUMEN
Patient responses to cancer immunotherapy are shaped by their unique genomic landscape and tumor microenvironment. Clinical advances in immunotherapy are changing the treatment landscape by enhancing a patient's immune response to eliminate cancer cells. While this provides potentially beneficial treatment options for many patients, only a minority of these patients respond to immunotherapy. In this work, we examined RNA-seq data and digital pathology images from individual patient tumors to more accurately characterize the tumor-immune microenvironment. Several studies implicate an inflamed microenvironment and increased percentage of tumor infiltrating immune cells with better response to specific immunotherapies in certain cancer types. We developed NEXT (Neural-based models for integrating gene EXpression and visual Texture features) to more accurately model immune infiltration in solid tumors. To demonstrate the utility of the NEXT framework, we predicted immune infiltrates across four different cancer types and evaluated our predictions against expert pathology review. Our analyses demonstrate that integration of imaging features improves prediction of the immune infiltrate. Of note, this effect was preferentially observed for B cells and CD8 T cells. In sum, our work effectively integrates both RNA-seq and imaging data in a clinical setting and provides a more reliable and accurate prediction of the immune composition in individual patient tumors.
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Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/patología , Microambiente Tumoral/genética , Microambiente Tumoral/inmunología , Biología Computacional , Femenino , Expresión Génica , Humanos , Inmunoterapia , Masculino , Modelos Biológicos , Neoplasias/genética , Neoplasias/inmunología , Neoplasias/terapia , Redes Neurales de la Computación , ARN/genéticaRESUMEN
We developed and clinically validated a hybrid capture next generation sequencing assay to detect somatic alterations and microsatellite instability in solid tumors and hematologic malignancies. This targeted oncology assay utilizes tumor-normal matched samples for highly accurate somatic alteration calling and whole transcriptome RNA sequencing for unbiased identification of gene fusion events. The assay was validated with a combination of clinical specimens and cell lines, and recorded a sensitivity of 99.1% for single nucleotide variants, 98.1% for indels, 99.9% for gene rearrangements, 98.4% for copy number variations, and 99.9% for microsatellite instability detection. This assay presents a wide array of data for clinical management and clinical trial enrollment while conserving limited tissue.
RESUMEN
Genomic analysis of paired tumor-normal samples and clinical data can be used to match patients to cancer therapies or clinical trials. We analyzed 500 patient samples across diverse tumor types using the Tempus xT platform by DNA-seq, RNA-seq and immunological biomarkers. The use of a tumor and germline dataset led to substantial improvements in mutation identification and a reduction in false-positive rates. RNA-seq enhanced gene fusion detection and cancer type classifications. With DNA-seq alone, 29.6% of patients matched to precision therapies supported by high levels of evidence or by well-powered studies. This proportion increased to 43.4% with the addition of RNA-seq and immunotherapy biomarker results. Combining these data with clinical criteria, 76.8% of patients were matched to at least one relevant clinical trial on the basis of biomarkers measured by the xT assay. These results indicate that extensive molecular profiling combined with clinical data identifies personalized therapies and clinical trials for a large proportion of patients with cancer and that paired tumor-normal plus transcriptome sequencing outperforms tumor-only DNA panel testing.
Asunto(s)
Genómica/métodos , Neoplasias/genética , Análisis de Secuencia de ADN/métodos , Análisis de Secuencia de ARN/métodos , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/inmunología , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Terapia Molecular Dirigida , Neoplasias/tratamiento farmacológico , Neoplasias/inmunología , Medicina de PrecisiónRESUMEN
Bivalirudin, a direct thrombin inhibitor, has emerged as an important alternative to heparin in patients undergoing percutaneous coronary intervention. However, it remains elusive if potentially adverse extracoagulant properties are responsible for the fact that its favorable effects in clinical studies are mainly driven by a reduction in bleeding events. The aim of the current study was to determine the effects and mechanisms of acute treatment with bivalirudin in comparison to heparin on NO bioavailability, an important factor for the pathogenesis of ischemic events. In particular, we studied the interaction between bivalirudin and myeloperoxidase (MPO), a leukocyte-derived enzyme that consumes endothelial-derived nitric oxide (NO), modifies a variety of biological targets, and thus affects the integrity of the vessel wall. In patients undergoing elective percutaneous coronary intervention, bivalirudin, in contrast to heparin, exhibited a significant decrease in plasma MPO levels (p = 0.03) accompanied by a deterioration of flow-mediated dilation (p = 0.02), a surrogate for endothelial NO bioavailability. In vitro experiments revealed avid binding of bivalirudin to both bovine aortic endothelial cells (BAEC) and MPO. Methylation of bivalirudin carboxyl groups at the carboxyl-terminal end revealed the specific binding site of bivalirudin to MPO. Bivalirudin-facilitated binding of MPO to BAEC resulted also in functional changes in terms of increased NO consumption as well as enhanced MPO-mediated redox modifications. These results illustrate dichotomous extracoagulant properties of heparins and thrombin inhibitors and suggest that bivalirudin acutely impairs endothelial NO bioavailability, thereby underscoring the potentially critical role of MPO as a mediator of vascular function.
Asunto(s)
Antitrombinas/farmacología , Hirudinas/farmacología , Óxido Nítrico/metabolismo , Fragmentos de Péptidos/farmacología , Peroxidasa/metabolismo , Anciano , Animales , Disponibilidad Biológica , Bovinos , Femenino , Técnica del Anticuerpo Fluorescente , Hirudinas/metabolismo , Humanos , Masculino , Metilación , Persona de Mediana Edad , Fragmentos de Péptidos/metabolismo , Peroxidasa/sangre , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Espectrometría de Masa por Ionización de Electrospray , Tirosina/análogos & derivados , Tirosina/metabolismo , VasodilataciónAsunto(s)
Competencia Clínica , Evaluación Educacional , Humanos , Lugar de Trabajo , Condiciones de TrabajoRESUMEN
We have developed a clinically validated NGS assay that includes tumor, germline and RNA sequencing. We apply this assay to clinical specimens and cell lines, and we demonstrate a clinical sensitivity of 98.4% and positive predictive value of 100% for the clinically actionable variants measured by the assay. We also demonstrate highly accurate copy number measurements and gene rearrangement identification.