RESUMEN
The aggregation of hypertrophic macrophages constitutes the basis of all granulomatous diseases, such as tuberculosis or sarcoidosis, and is decisive for disease pathogenesis. However, macrophage-intrinsic pathways driving granuloma initiation and maintenance remain elusive. We found that activation of the metabolic checkpoint kinase mTORC1 in macrophages by deletion of the gene encoding tuberous sclerosis 2 (Tsc2) was sufficient to induce hypertrophy and proliferation, resulting in excessive granuloma formation in vivo. TSC2-deficient macrophages formed mTORC1-dependent granulomatous structures in vitro and showed constitutive proliferation that was mediated by the neo-expression of cyclin-dependent kinase 4 (CDK4). Moreover, mTORC1 promoted metabolic reprogramming via CDK4 toward increased glycolysis while simultaneously inhibiting NF-κB signaling and apoptosis. Inhibition of mTORC1 induced apoptosis and completely resolved granulomas in myeloid TSC2-deficient mice. In human sarcoidosis patients, mTORC1 activation, macrophage proliferation and glycolysis were identified as hallmarks that correlated with clinical disease progression. Collectively, TSC2 maintains macrophage quiescence and prevents mTORC1-dependent granulomatous disease with clinical implications for sarcoidosis.
Asunto(s)
Granuloma/inmunología , Macrófagos/inmunología , Complejos Multiproteicos/metabolismo , Sarcoidosis/inmunología , Serina-Treonina Quinasas TOR/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Línea Celular , Quinasa 4 Dependiente de la Ciclina/metabolismo , Progresión de la Enfermedad , Granuloma/tratamiento farmacológico , Humanos , Macrófagos/efectos de los fármacos , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , ARN Interferente Pequeño/genética , Sarcoidosis/tratamiento farmacológico , Transducción de Señal , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor/genéticaRESUMEN
BACKGROUND: Langerhans cell histiocytosis (LCH) is a histiocytic disorder driven by a constitutive activation of the MAPK signaling pathway in myeloid cells. In 50-60% of cases, it is caused by the BRAFV600E mutation. There is evidence that levels of BRAFV600E in the peripheral blood of patients with LCH correlate with disease burden and could be used as marker for disease extent and response to therapy. However, there is currently no consensus on how testing for minimal disseminated disease should be performed. METHODS: Different approaches to determine the mutation load in patients with LCH were assessed and longitudinal evaluation of patient DNA during treatment with chemotherapy and/or the RAF inhibitor vemurafenib was performed. DNA was isolated from whole blood, different leukocyte subsets, and circulating cell-free DNA (ccf-DNA). RESULTS: We show that determining BRAF levels from whole blood is superior to using ccfDNA. Furthermore, it is important to identify the clinically relevant BRAF-mutated cellular subpopulations such as CD14+ monocytes or CD1c+ DCs, since other blood cells can also harbor the mutation and therefore confound whole blood or ccfDNA measurements. CONCLUSION: Our data support the view that single-agent treatment with an RAF inhibitor reduces disease activity but does not cure LCH.
Asunto(s)
Histiocitosis de Células de Langerhans/sangre , Histiocitosis de Células de Langerhans/genética , Proteínas Mutantes/sangre , Proteínas Mutantes/genética , Proteínas Proto-Oncogénicas B-raf/sangre , Proteínas Proto-Oncogénicas B-raf/genética , Sustitución de Aminoácidos , Biomarcadores/sangre , Ácidos Nucleicos Libres de Células/sangre , Ácidos Nucleicos Libres de Células/genética , Preescolar , ADN/sangre , ADN/genética , Análisis Mutacional de ADN , Femenino , Marcadores Genéticos , Histiocitosis de Células de Langerhans/tratamiento farmacológico , Humanos , Lactante , Estudios Longitudinales , Masculino , Proteínas Mutantes/antagonistas & inhibidores , Mutación Missense , Reacción en Cadena de la Polimerasa , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Vemurafenib/uso terapéuticoRESUMEN
Langerhans cell histiocytosis (LCH) is a MAPK pathway-driven disease characterized by the accumulation of CD1a+ langerin+ cells of unknown origin. We have previously reported that the Notch signaling pathway is active in LCH lesions and that the Notch ligand Jagged2 (JAG2) induces CD1a and langerin expression in monocytes in vitro. Here we show that Notch signaling induces monocytes to acquire an LCH gene signature and that Notch inhibition suppresses the LCH phenotype. In contrast, while also CD1c+ dendritic cells or IL-4-stimulated CD14+ monocytes acquire CD1a and langerin positivity in culture, their gene expression profiles and surface phenotypes are more different from primary LCH cells. We propose a model where CD14+ monocytes serve as LCH cell precursor and JAG2-mediated activation of the Notch signaling pathway initiates a differentiation of monocytes toward LCH cells in selected niches and thereby contributes to LCH pathogenesis.
Asunto(s)
Diferenciación Celular , Histiocitosis de Células de Langerhans/metabolismo , Proteína Jagged-2/metabolismo , Receptores de Lipopolisacáridos/metabolismo , Monocitos/metabolismo , Transducción de Señal , Antígenos CD/metabolismo , Proliferación Celular , Histiocitosis de Células de Langerhans/patología , Humanos , Lectinas Tipo C/metabolismo , Lectinas de Unión a Manosa/metabolismo , Fenotipo , Receptores Notch/metabolismo , Transcripción GenéticaRESUMEN
Langerhans cell histiocytosis (LCH) is a rare neoplasm predominantly affecting children. It occupies a hybrid position between cancers and inflammatory diseases, which makes it an attractive model for studying cancer development. To explore the molecular mechanisms underlying the pathophysiology of LCH and its characteristic clinical heterogeneity, we investigated the transcriptomic and epigenomic diversity in primary LCH lesions. Using single-cell RNA sequencing, we identified multiple recurrent types of LCH cells within these biopsies, including putative LCH progenitor cells and several subsets of differentiated LCH cells. We confirmed the presence of proliferative LCH cells in all analyzed biopsies using IHC, and we defined an epigenomic and gene-regulatory basis of the different LCH-cell subsets by chromatin-accessibility profiling. In summary, our single-cell analysis of LCH uncovered an unexpected degree of cellular, transcriptomic, and epigenomic heterogeneity among LCH cells, indicative of complex developmental hierarchies in LCH lesions. SIGNIFICANCE: This study sketches a molecular portrait of LCH lesions by combining single-cell transcriptomics with epigenome profiling. We uncovered extensive cellular heterogeneity, explained in part by an intrinsic developmental hierarchy of LCH cells. Our findings provide new insights and hypotheses for advancing LCH research and a starting point for personalizing therapy.See related commentary by Gruber et al., p. 1343.This article is highlighted in the In This Issue feature, p. 1325.
Asunto(s)
Epigénesis Genética , Epigenómica , Histiocitosis de Células de Langerhans/genética , Biomarcadores , Biopsia , Susceptibilidad a Enfermedades , Epigenómica/métodos , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Histiocitosis de Células de Langerhans/diagnóstico , Histiocitosis de Células de Langerhans/metabolismo , Humanos , Inmunohistoquímica , Radiografía , Análisis de la Célula IndividualRESUMEN
BACKGROUND: The extent of lymph node dissection (LND) in bladder cancer (BCa) patients at the time of radical cystectomy may affect oncologic outcome. OBJECTIVE: To evaluate whether extended versus limited LND prolongs recurrence-free survival (RFS). DESIGN, SETTING, AND PARTICIPANTS: Prospective, multicenter, phase-III trial patients with locally resectable T1G3 or muscle-invasive urothelial BCa (T2-T4aM0). INTERVENTION: Randomization to limited (obturator, and internal and external iliac nodes) versus extended LND (in addition, deep obturator, common iliac, presacral, paracaval, interaortocaval, and para-aortal nodes up to the inferior mesenteric artery). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The primary endpoint was RFS. Secondary endpoints included cancer-specific survival (CSS), overall survival (OS), and complications. The trial was designed to show 15% advantage of 5-yr RFS by extended LND. RESULTS AND LIMITATIONS: In total, 401 patients were randomized from February 2006 to August 2010 (203 limited, 198 extended). The median number of dissected nodes was 19 in the limited and 31 in the extended arm. Extended LND failed to show superiority over limited LND with regard to RFS (5-yr RFS 65% vs 59%; hazard ratio [HR]=0.84 [95% confidence interval 0.58-1.22]; p=0.36), CSS (5-yr CSS 76% vs 65%; HR=0.70; p=0.10), and OS (5-yr OS 59% vs 50%; HR=0.78; p=0.12). Clavien grade ≥3 lymphoceles were more frequently reported in the extended LND group within 90d after surgery. Inclusion of T1G3 tumors may have contributed to the negative study result. CONCLUSIONS: Extended LND failed to show a significant advantage over limited LND in RFS, CSS, and OS. A larger trial is required to determine whether extended compared with limited LND leads to a small, but clinically relevant, survival difference (ClinicalTrials.gov NCT01215071). PATIENT SUMMARY: In this study, we investigated the outcome in bladder cancer patients undergoing cystectomy based on the anatomic extent of lymph node resection. We found that extended removal of lymph nodes did not reduce the rate of tumor recurrence in the expected range.
Asunto(s)
Cistectomía/métodos , Escisión del Ganglio Linfático/métodos , Neoplasias de la Vejiga Urinaria/cirugía , Anciano , Quimioterapia Adyuvante , Cistectomía/efectos adversos , Cistectomía/mortalidad , Progresión de la Enfermedad , Femenino , Alemania , Humanos , Escisión del Ganglio Linfático/efectos adversos , Escisión del Ganglio Linfático/mortalidad , Metástasis Linfática , Masculino , Persona de Mediana Edad , Invasividad Neoplásica , Estadificación de Neoplasias , Supervivencia sin Progresión , Estudios Prospectivos , Medición de Riesgo , Factores de Riesgo , Factores de Tiempo , Neoplasias de la Vejiga Urinaria/mortalidad , Neoplasias de la Vejiga Urinaria/patologíaRESUMEN
OBJECTIVE: To evaluate the mRNA expression of lymphangiogenesis and proliferation markers and to examine its association with histopathological characteristics and clinical outcome in patients with urothelial carcinoma of the bladder (UCB) after radical cystectomy (RC). PATIENTS AND METHODS: Gene expression analysis of the vascular endothelial growth -C and -D (VEGF-C/-D), its receptor VEGF receptor-3 (VEGFR-3), MKI67, and RACGAP1 was performed in 108 patients after radical cystectomy and their correlation with clinical-pathological parameters was investigated. Uni- and multivariate regression analyses were used to identify predictors for cancer-specific survival (CSS), recurrence-free survival (RFS) and overall survival (OS) after RC. RESULTS: The expression of RACGAP1 and VEGFR-3 showed an association with a higher pT stage (P = 0.049; Pâ¯=â¯0.009). MKI67 showed an association with a high-grade urothelial carcinoma of the bladder (Pâ¯=â¯0.021). VEGFR-3 expression was significantly associated with the presence of lymphovascular invasion (LVI) (P = 0.016) and lymph node metastases (pN+) (Pâ¯=â¯0.028). With the univariate analysis, overexpression of VEGFR-3 (Pâ¯=â¯0.029) and the clinical-pathological parameters pT stage (P < 0.0001), pN+ (Pâ¯=â¯0.0004), LVI (P < 0.0001) and female gender (Pâ¯=â¯0.021) were significantly associated with a reduced CSS. Multivariate analysis identified a higher pT stage (Pâ¯=â¯0.017) and LVI (Pâ¯=â¯0.008) as independent predictors for reduced CSS. Independent predictors for reduced OS were a higher pT stage (Pâ¯=â¯0.0007) and LVI (Pâ¯=â¯0.0021), while overexpression of VEGF-D was associated with better OS (P < 0.0001). CONCLUSIONS: The mRNA expression of the investigated markers showed associations with common histopathological parameters. Increased expression of VEGF-D is independently associated with better overall survival.