RESUMO
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.
Assuntos
DNA Tumoral Circulante/genética , Evolução Clonal/genética , Leucemia Linfocítica Crônica de Células B/genética , Adenina/análogos & derivados , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/uso terapêutico , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteína 3 com Repetições IAP de Baculovírus/genética , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , DNA Tumoral Circulante/sangue , Progressão da Doença , Feminino , Humanos , Leucemia Linfocítica Crônica de Células B/sangue , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , Fator 88 de Diferenciação Mieloide/genética , Fosfoproteínas/genética , Piperidinas , Proteínas Proto-Oncogênicas p21(ras)/genética , Pirazóis/uso terapêutico , Pirimidinas/uso terapêutico , Fatores de Processamento de RNA/genética , Receptor Notch1/genética , Sulfonamidas/uso terapêutico , Resultado do Tratamento , Proteína Supressora de Tumor p53/genéticaRESUMO
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.
Assuntos
Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Imidazóis/farmacologia , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/patologia , Naftoquinonas/farmacologia , Western Blotting , Criança , Citometria de Fluxo , Humanos , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Leucemia Mieloide Aguda/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Survivina , Células Tumorais CultivadasRESUMO
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.
Assuntos
Doença de Huntington/metabolismo , Imunoensaio , Proteínas do Tecido Nervoso/análise , Proteínas Nucleares/análise , Animais , Células Cultivadas , Transferência Ressonante de Energia de Fluorescência , Técnicas de Introdução de Genes , Proteína Huntingtina , Doença de Huntington/patologia , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismoRESUMO
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.
