RESUMEN
Eltrombopag has been previously shown to be effective in reversing azacitidine-mediated thrombocytopenia. This was further investigated in the SUPPORT trial, a phase III study assessing the efficacy/safety of eltrombopag plus azacitidine in patients with intermediate- to high-risk myelodysplastic syndromes and thrombocytopenia. The results did not support a clinical benefit for the addition of eltrombopag to azacitidine. We investigated if the somatic mutational profiles in the patient cohort were associated with treatment outcomes. Based on the available data, we observed no imbalance in the mutational profiles between treatment arms or a clear association between identified somatic mutations and clinical outcomes.
RESUMEN
Langerhans cell histiocytosis (LCH) is a rare, heterogenous, neoplastic disorder primarily affecting children. BRAF mutations have been reported in >50% of patients with LCH. The selective BRAF inhibitor, dabrafenib, in combination with the MEK1/2 inhibitor, trametinib, has been approved in select BRAF V600-mutant solid tumors. Two open-label phase 1/2 studies were conducted in pediatric patients with BRAF V600-mutant, recurrent/refractory malignancies treated with dabrafenib monotherapy (CDRB436A2102; NCT01677741) or dabrafenib plus trametinib (CTMT212X2101; NCT02124772). The primary objectives of both studies were to determine safe and tolerable doses that achieve similar exposure to the approved doses for adults. Secondary objectives included safety, tolerability, and preliminary antitumor activity. Thirteen and 12 patients with BRAF V600-mutant LCH received dabrafenib monotherapy and in combination with trametinib, respectively. Investigator-assessed objective response rates per Histiocyte Society criteria were 76.9% (95% confidence interval [CI], 46.2-95.0) and 58.3% (95% CI, 27.7-84.8) in the monotherapy and combination studies, respectively. More than 90% of responses were ongoing at study completion. The most common treatment-related adverse events (AEs) were vomiting and increased blood creatinine with monotherapy and pyrexia, diarrhea, dry skin, decreased neutrophil count, and vomiting with combination therapy. Two patients each discontinued treatment with monotherapy and combination therapy because of AEs. Overall, dabrafenib monotherapy or in combination with trametinib demonstrated clinical efficacy and manageable toxicity in relapsed/refractory BRAF V600-mutant pediatric LCH, with most responses ongoing. Safety was consistent with that reported in other pediatric and adult conditions treated with dabrafenib plus trametinib.
Asunto(s)
Histiocitosis de Células de Langerhans , Adulto , Niño , Humanos , Proteínas Proto-Oncogénicas B-raf/genéticaRESUMEN
PURPOSE: BRAF V600 mutations occur in many childhood cancers, including approximately 20% of low-grade gliomas (LGGs). Here, we describe a phase I/II study establishing pediatric dosing and pharmacokinetics of trametinib with or without dabrafenib, as well as efficacy and safety in a disease-specific cohort with BRAF V600-mutant LGG; other cohorts will be reported elsewhere. METHODS: This is a four-part, phase I/II study (ClinicalTrials.gov identifier: NCT02124772) in patients age < 18 years with relapsed/refractory malignancies: trametinib monotherapy dose finding (part A) and disease-specific expansion (part B), and dabrafenib + trametinib dose finding (part C) and disease-specific expansion (part D). The primary objective assessed in all patients in parts A and C was to determine pediatric dosing on the basis of steady-state pharmacokinetics. Disease-specific efficacy and safety (across parts A-D) were secondary objectives. RESULTS: Overall, 139 patients received trametinib (n = 91) or dabrafenib + trametinib (n = 48). Trametinib dose-limiting toxicities in > 1 patient (part A) included mucosal inflammation (n = 3) and hyponatremia (n = 2). There were no dose-limiting toxicities with combination therapy (part C). The recommended phase II dose of trametinib, with or without dabrafenib, was 0.032 mg/kg once daily for patients age < 6 years and 0.025 mg/kg once daily for patients age ≥ 6 years; dabrafenib dosing in the combination was as previously identified for monotherapy. In 49 patients with BRAF V600-mutant glioma (LGG, n = 47) across all four study parts, independently assessed objective response rates were 15% (95% CI, 1.9 to 45.4) for monotherapy (n = 13) and 25% (95% CI, 12.1 to 42.2) for combination (n = 36). Adverse event-related treatment discontinuations were more common with monotherapy (54% v 22%). CONCLUSION: The trial design provided efficient evaluation of pediatric dosing, safety, and efficacy of single-agent and combination targeted therapy. Age-based and weight-based dosing of trametinib with or without dabrafenib achieved target concentrations with manageable safety and demonstrated clinical efficacy and tolerability in BRAF V600-mutant LGG.
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Glioma , Melanoma , Neoplasias Cutáneas , Humanos , Niño , Adolescente , Melanoma/tratamiento farmacológico , Melanoma/genética , Proteínas Proto-Oncogénicas B-raf/genética , Recurrencia Local de Neoplasia/tratamiento farmacológico , Oximas/efectos adversos , Piridonas , Glioma/tratamiento farmacológico , Glioma/genética , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Mutación , Neoplasias Cutáneas/tratamiento farmacológicoRESUMEN
The transcription factor NFATC2 induces ß cell proliferation in mouse and human islets. However, the genomic targets that mediate these effects have not been identified. We expressed active forms of Nfatc2 and Nfatc1 in human islets. By integrating changes in gene expression with genomic binding sites for NFATC2, we identified approximately 2200 transcriptional targets of NFATC2. Genes induced by NFATC2 were enriched for transcripts that regulate the cell cycle and for DNA motifs associated with the transcription factor FOXP. Islets from an endocrine-specific Foxp1, Foxp2, and Foxp4 triple-knockout mouse were less responsive to NFATC2-induced ß cell proliferation, suggesting the FOXP family works to regulate ß cell proliferation in concert with NFATC2. NFATC2 induced ß cell proliferation in both mouse and human islets, whereas NFATC1 did so only in human islets. Exploiting this species difference, we identified approximately 250 direct transcriptional targets of NFAT in human islets. This gene set enriches for cell cycle-associated transcripts and includes Nr4a1. Deletion of Nr4a1 reduced the capacity of NFATC2 to induce ß cell proliferation, suggesting that much of the effect of NFATC2 occurs through its induction of Nr4a1. Integration of noncoding RNA expression, chromatin accessibility, and NFATC2 binding sites enabled us to identify NFATC2-dependent enhancer loci that mediate ß cell proliferation.
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Proliferación Celular , Regulación de la Expresión Génica , Células Secretoras de Insulina/metabolismo , Factores de Transcripción NFATC/metabolismo , Elementos de Respuesta , Transcripción Genética , Animales , Humanos , Ratones Noqueados , Factores de Transcripción NFATC/genéticaRESUMEN
Astrocytes display diverse morphologies in different regions of the central nervous system. Whether astrocyte diversity is attributable to developmental processes and bears functional consequences, especially in humans, is unknown. RNA-seq of human pluripotent stem cell-derived regional astrocytes revealed distinct transcript profiles, suggesting differential functional properties. This was confirmed by differential calcium signaling as well as effects on neurite growth and blood-brain barrier formation. Distinct transcriptional profiles and functional properties of human astrocytes generated from regionally specified neural progenitors under the same conditions strongly implicate the developmental impact on astrocyte diversity. These findings provide a rationale for renewed examination of regional astrocytes and their role in the pathogenesis of psychiatric and neurological disorders.
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Astrocitos/fisiología , Diferenciación Celular/genética , Neurogénesis/genética , Células Madre Pluripotentes/fisiología , Transcriptoma , Secuencia de Bases , Biomarcadores/análisis , Biomarcadores/metabolismo , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Células Madre Pluripotentes Inducidas/fisiología , Células-Madre Neurales/fisiología , Especificidad de Órganos/genética , Prosencéfalo/citología , Prosencéfalo/metabolismo , Análisis de Secuencia de ARNRESUMEN
How mutations in glial fibrillary acidic protein (GFAP) cause Alexander disease (AxD) remains elusive. We generated iPSCs from two AxD patients and corrected the GFAP mutations to examine the effects of mutant GFAP on human astrocytes. AxD astrocytes displayed GFAP aggregates, recapitulating the pathological hallmark of AxD. RNA sequencing implicated the endoplasmic reticulum, vesicle regulation, and cellular metabolism. Corroborating this analysis, we observed enlarged and heterogeneous morphology coupled with perinuclear localization of endoplasmic reticulum and lysosomes in AxD astrocytes. Functionally, AxD astrocytes showed impaired extracellular ATP release, which is responsible for attenuated calcium wave propagation. These results reveal that AxD-causing mutations in GFAP disrupt intracellular vesicle regulation and impair astrocyte secretion, resulting in astrocyte dysfunction and AxD pathogenesis.
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Astrocitos/metabolismo , Proteína Ácida Fibrilar de la Glía/genética , Mutación/genética , Orgánulos/metabolismo , Adenosina Trifosfato/metabolismo , Enfermedad de Alexander/metabolismo , Enfermedad de Alexander/patología , Animales , Astrocitos/citología , Señalización del Calcio , Diferenciación Celular , Retículo Endoplásmico/metabolismo , Humanos , Lisosomas/metabolismo , Ratones , Agregado de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Azacitidine treatment of myelodysplastic syndromes (MDSs) generally exacerbates thrombocytopenia during the first treatment cycles. A Study of Eltrombopag in Myelodysplastic Syndromes Receiving Azacitidine (SUPPORT), a phase 3, randomized, double-blind, placebo-controlled study, investigated the platelet supportive effects of eltrombopag given concomitantly with azacitidine. International Prognostic Scoring System intermediate-1, intermediate-2, or high-risk MDS patients with baseline platelets <75 × 109/L were randomized 1:1 to eltrombopag (start, 200 mg/d [East Asians, 100 mg/d], maximum, 300 mg/d [East Asians, 150 mg/d]) or placebo, plus azacitidine (75 mg/m2 subcutaneously once daily for 7 days every 28 days). The primary end point was the proportion of patients platelet transfusion-free during cycles 1 through 4 of azacitidine therapy. Based on planned interim analyses, an independent data monitoring committee recommended stopping the study prematurely because efficacy outcomes crossed the predefined futility threshold and for safety reasons. At termination, 28/179 (16%) eltrombopag and 55/177 (31%) placebo patients met the primary end point. Overall response (International Working Group criteria; complete, marrow, or partial response) occurred in 20% and 35% of eltrombopag and placebo patients, respectively, by investigator assessment. There was no difference in hematologic improvement in any cell lineage between the 2 arms. There was no improvement in overall or progression-free survival. Adverse events with ≥10% occurrence in the eltrombopag vs placebo arm were febrile neutropenia and diarrhea. Compared with azacitidine alone, eltrombopag plus azacitidine worsened platelet recovery, with lower response rates and a trend toward increased progression to acute myeloid leukemia. This trial was registered at www.clinicaltrials.gov as #NCT02158936.
Asunto(s)
Azacitidina/administración & dosificación , Benzoatos/administración & dosificación , Hidrazinas/administración & dosificación , Síndromes Mielodisplásicos/terapia , Transfusión de Plaquetas , Pirazoles/administración & dosificación , Trombocitopenia/terapia , Adulto , Anciano , Anciano de 80 o más Años , Azacitidina/efectos adversos , Benzoatos/efectos adversos , Método Doble Ciego , Femenino , Humanos , Hidrazinas/efectos adversos , Masculino , Persona de Mediana Edad , Síndromes Mielodisplásicos/complicaciones , Síndromes Mielodisplásicos/mortalidad , Pirazoles/efectos adversos , Factores de Riesgo , Trombocitopenia/complicaciones , Trombocitopenia/mortalidadRESUMEN
BACKGROUND: Human pluripotent stem cells offer the best available model to study the underlying cellular and molecular mechanisms of human embryonic lineage specification. However, it is not fully understood how individual stem cells exit the pluripotent state and transition towards their respective progenitor states. RESULTS: Here, we analyze the transcriptomes of human embryonic stem cell-derived lineage-specific progenitors by single-cell RNA-sequencing (scRNA-seq). We identify a definitive endoderm (DE) transcriptomic signature that leads us to pinpoint a critical time window when DE differentiation is enhanced by hypoxia. The molecular mechanisms governing the emergence of DE are further examined by time course scRNA-seq experiments, employing two new statistical tools to identify stage-specific genes over time (SCPattern) and to reconstruct the differentiation trajectory from the pluripotent state through mesendoderm to DE (Wave-Crest). Importantly, presumptive DE cells can be detected during the transitory phase from Brachyury (T) (+) mesendoderm toward a CXCR4 (+) DE state. Novel regulators are identified within this time window and are functionally validated on a screening platform with a T-2A-EGFP knock-in reporter engineered by CRISPR/Cas9. Through loss-of-function and gain-of-function experiments, we demonstrate that KLF8 plays a pivotal role modulating mesendoderm to DE differentiation. CONCLUSIONS: We report the analysis of 1776 cells by scRNA-seq covering distinct human embryonic stem cell-derived progenitor states. By reconstructing a differentiation trajectory at single-cell resolution, novel regulators of the mesendoderm transition to DE are elucidated and validated. Our strategy of combining single-cell analysis and genetic approaches can be applied to uncover novel regulators governing cell fate decisions in a variety of systems.
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Diferenciación Celular/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Células Madre Embrionarias Humanas/citología , ARN/genética , Técnicas de Cultivo de Célula , Endodermo/crecimiento & desarrollo , Endodermo/metabolismo , Regulación del Desarrollo de la Expresión Génica , Hepatocitos/citología , Humanos , Células Madre Pluripotentes/citología , Análisis de la Célula Individual/métodosRESUMEN
UNLABELLED: A recent article identified an artifact in multiple single-cell RNA-seq (scRNA-seq) datasets generated by the Fluidigm C1 platform. Specifically, Leng et al. showed significantly increased gene expression in cells captured from sites with small or large plate output IDs. We refer to this artifact as an ordering effect (OE). Including OE genes in downstream analyses could lead to biased results. To address this problem, we developed a statistical method and software called OEFinder to identify a sorted list of OE genes. OEFinder is available as an R package along with user-friendly graphical interface implementations which allows users to check for potential artifacts in scRNA-seq data generated by the Fluidigm C1 platform. AVAILABILITY AND IMPLEMENTATION: OEFinder is freely available at https://github.com/lengning/OEFinder CONTACT: rstewart@morgridge.org or lengning1@gmail.com SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
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Análisis de Secuencia de ARN , Análisis de la Célula Individual , Programas Informáticos , Animales , ARNRESUMEN
Oscillatory gene expression is fundamental to development, but technologies for monitoring expression oscillations are limited. We have developed a statistical approach called Oscope to identify and characterize the transcriptional dynamics of oscillating genes in single-cell RNA-seq data from an unsynchronized cell population. Applying Oscope to a number of data sets, we demonstrated its utility and also identified a potential artifact in the Fluidigm C1 platform.
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Interpretación Estadística de Datos , Modelos Genéticos , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodos , Algoritmos , Análisis de Varianza , Células Madre Embrionarias/fisiología , Perfilación de la Expresión Génica/métodos , Perfilación de la Expresión Génica/estadística & datos numéricos , Humanos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Análisis de Secuencia de ARN/estadística & datos numéricos , Análisis de la Célula Individual/estadística & datos numéricos , Programas InformáticosRESUMEN
BACKGROUND: Mass spectrometry (MS) data are often generated from various biological or chemical experiments and there may exist outlying observations, which are extreme due to technical reasons. The determination of outlying observations is important in the analysis of replicated MS data because elaborate pre-processing is essential for successful analysis with reliable results and manual outlier detection as one of pre-processing steps is time-consuming. The heterogeneity of variability and low replication are often obstacles to successful analysis, including outlier detection. Existing approaches, which assume constant variability, can generate many false positives (outliers) and/or false negatives (non-outliers). Thus, a more powerful and accurate approach is needed to account for the heterogeneity of variability and low replication. FINDINGS: We proposed an outlier detection algorithm using projection and quantile regression in MS data from multiple experiments. The performance of the algorithm and program was demonstrated by using both simulated and real-life data. The projection approach with linear, nonlinear, or nonparametric quantile regression was appropriate in heterogeneous high-throughput data with low replication. CONCLUSION: Various quantile regression approaches combined with projection were proposed for detecting outliers. The choice among linear, nonlinear, and nonparametric regressions is dependent on the degree of heterogeneity of the data. The proposed approach was illustrated with MS data with two or more replicates.