RESUMEN
Several novel therapeutics are poised to change the natural history of chronic lymphocytic leukaemia (CLL) and the increasing use of these therapies has highlighted limitations of traditional disease monitoring methods. Here we demonstrate that circulating tumour DNA (ctDNA) is readily detectable in patients with CLL. Importantly, ctDNA does not simply mirror the genomic information contained within circulating malignant lymphocytes but instead parallels changes across different disease compartments following treatment with novel therapies. Serial ctDNA analysis allows clonal dynamics to be monitored over time and identifies the emergence of genomic changes associated with Richter's syndrome (RS). In addition to conventional disease monitoring, ctDNA provides a unique opportunity for non-invasive serial analysis of CLL for molecular disease monitoring.
Asunto(s)
ADN Tumoral Circulante/genética , Evolución Clonal/genética , Leucemia Linfocítica Crónica de Células B/genética , Adenina/análogos & derivados , Anciano , Anciano de 80 o más Años , Antineoplásicos/uso terapéutico , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteína 3 que Contiene Repeticiones IAP de Baculovirus/genética , Compuestos Bicíclicos Heterocíclicos con Puentes/uso terapéutico , ADN Tumoral Circulante/sangre , Progresión de la Enfermedad , Femenino , Humanos , Leucemia Linfocítica Crónica de Células B/sangre , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Masculino , Persona de Mediana Edad , Factor 88 de Diferenciación Mieloide/genética , Fosfoproteínas/genética , Piperidinas , Proteínas Proto-Oncogénicas p21(ras)/genética , Pirazoles/uso terapéutico , Pirimidinas/uso terapéutico , Factores de Empalme de ARN/genética , Receptor Notch1/genética , Sulfonamidas/uso terapéutico , Resultado del Tratamiento , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Despite aggressive chemotherapy, approximately one-third of children with acute myeloid leukaemia (AML) relapse. More effective treatments are urgently needed. Survivin is an inhibitor-of-apoptosis protein with key roles in regulating cell division, proliferation and apoptosis. Furthermore, high expression of Survivin has been associated with poor clinical outcome in AML. The survivin suppressant YM155 (Sepantronium Bromide) has pre-clinical activity against a range of solid cancers and leukemias, although data in AML is limited. Therefore, we undertook a comprehensive pre-clinical evaluation of YM155 in paediatric AML. YM155 potently inhibited cell viability in a diverse panel of AML cell lines. All paediatric cell lines were particularly sensitive, with a median IC50 of 0.038 µM. Cell cycle analyses demonstrated concentration-dependent increases in a sub-G1 population with YM155 treatment, suggestive of apoptosis that was subsequently confirmed by an increase in annexin-V positivity. YM155-mediated apoptosis was confirmed across a panel of 8 diagnostic bone marrow samples from children with AML. Consistent with the proposed mechanism of action, YM155 treatment was associated with down-regulation of survivin mRNA and protein expression and induction of DNA damage. These data suggest that YM155-mediated inhibition of survivin is a potentially beneficial therapeutic strategy for AML, particularly paediatric disease, and warrants further evaluation.
Asunto(s)
Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Imidazoles/farmacología , Proteínas Inhibidoras de la Apoptosis/antagonistas & inhibidores , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/patología , Naftoquinonas/farmacología , Western Blotting , Niño , Citometría de Flujo , Humanos , Proteínas Inhibidoras de la Apoptosis/genética , Proteínas Inhibidoras de la Apoptosis/metabolismo , Leucemia Mieloide Aguda/metabolismo , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Survivin , Células Tumorales CultivadasRESUMEN
Huntington's disease (HD) is an inherited neurodegenerative disorder caused by the amplification of a polyglutamine stretch at the N terminus of the huntingtin protein. N-terminal fragments of the mutant huntingtin (mHtt) aggregate and form intracellular inclusions in brain and peripheral tissues. Aggregates are an important hallmark of the disease, translating into a high need to quantify them in vitro and in vivo. We developed a one-step TR-FRET-based immunoassay to quantify soluble and aggregated mHtt in cell and tissue homogenates. Strikingly, quantification revealed a decrease of soluble mHtt correlating with an increase of aggregated protein in primary neuronal cell cultures, transgenic R6/2, and HdhQ150 knock-in HD mice. These results emphasize the assay's efficiency for highly sensitive and quantitative detection of soluble and aggregated mHtt and its application in high-throughput screening and characterization of HD models.
Asunto(s)
Enfermedad de Huntington/metabolismo , Inmunoensayo , Proteínas del Tejido Nervioso/análisis , Proteínas Nucleares/análisis , Animales , Células Cultivadas , Transferencia Resonante de Energía de Fluorescencia , Técnicas de Sustitución del Gen , Proteína Huntingtina , Enfermedad de Huntington/patología , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/citología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismoRESUMEN
The antibody-mediated targeted delivery of cytokines, growth factors and immunomodulators offers great potential for the therapy of cancer and other serious conditions. Interferon-alpha has long been used in the clinic for the treatment of patients with certain malignancies or with viral disease. Promising anticancer activity has recently been reported for two fusion proteins consisting of immunoglobulins bearing the interferon-alpha polypeptide at the C-terminal end of the molecule. Here we describe the design, production and characterization of a novel immunocytokine, in which murine interferon-alpha2 was sequentially fused with the tumor-targeting antibody fragment scFv(F8), specific to the alternatively-spliced EDA domain of fibronectin. The resulting fusion protein (F8-IFNa) could be produced to homogeneity and was shown to retain both antigen binding activity and interferon-alpha activity. Biodistribution studies in tumor-bearing mice with radioiodinated protein preparations confirmed the ability of F8-IFNa to selectively localize at the tumor site. However, using two different murine models of cancer (F9 teratocarcinomas and Cloudman S91 melanomas in immunocompetent mice), we could not detect a striking superiority for the therapeutic performance of F8-IFNa as compared to KSF-IFNa, a fusion protein of irrelevant specificity in the mouse which was used as negative control. In the paper, we present hypotheses why the antibody-based pharmacodelivery of interferon-alpha fails to eradicate tumors, in contrast to the situation observed by our group for other immunocytokines, which benefit from a selective localization at the tumor site.
