RESUMEN
Sustained undetectable molecular residual disease (UMRD) is obtained in a minority of patients with chronic myeloid leukemia treated with tyrosine kinase inhibitors. It remains unclear whether these patients are definitively cured of their leukemia or whether leukemic stem cells (LSCs) persist in their BM. We have evaluated the presence of BCR-ABL-expressing marrow LSCs in 6 patients with chronic myeloid leukemia with sustained UMRD induced by IFN-α (n = 3), imatinib mesylate after IFN-α failure (n = 2), and dasatinib after imatinib intolerance (n = 1). Purified CD34(+) cells were used for clonogenic and long-term culture-initiating cell assays performed on classic or HOXB4-expressing MS-5 feeders. Using this strategy, we identified BCR-ABL-expressing LSCs in all patients. Interestingly, long-term culture-initiating cell assays with MS-5/HOXB4 stromal feeders increased detected numbers of LSCs in 3 patients. The relation between LSC persistency and a potential risk of disease relapse for patients with durable UMRD (on or off tyrosine kinase inhibitor therapy) warrants further investigation.
Asunto(s)
Leucemia Mielógena Crónica BCR-ABL Positiva/diagnóstico , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Células Madre Neoplásicas/fisiología , Adulto , Anciano , Algoritmos , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Supervivencia Celular , Femenino , Humanos , Límite de Detección , Masculino , Persona de Mediana Edad , Modelos Biológicos , Técnicas de Diagnóstico Molecular/normas , Técnicas de Diagnóstico Molecular/estadística & datos numéricos , Neoplasia Residual , Células Madre Neoplásicas/patología , Inducción de Remisión , Células Tumorales CultivadasRESUMEN
Recent technological advances in cell reprogramming by generation of induced pluripotent stem cells (iPSC) offer major perspectives in disease modelling and future hopes for providing novel stem cells sources in regenerative medicine. However, research on iPSC still requires refining the criteria of the pluripotency stage of these cells and exploration of their equivalent functionality to human embryonic stem cells (ESC). We report here on the use of infrared microspectroscopy to follow the spectral modification of somatic cells during the reprogramming process. We show that induced pluripotent stem cells (iPSC) adopt a chemical composition leading to a spectral signature indistinguishable from that of embryonic stem cells (ESC) and entirely different from that of the original somatic cells. Similarly, this technique allows a distinction to be made between partially and fully reprogrammed cells. We conclude that infrared microspectroscopy signature is a novel methodology to evaluate induced pluripotency and can be added to the tests currently used for this purpose.
Asunto(s)
Reprogramación Celular/fisiología , Animales , Reprogramación Celular/genética , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
Tyrosine kinase inhibitors (TKIs) have revolutionized the treatment of chronic myeloid leukemia (CML). However, resistance is occasionally observed, mainly due to mutations within the BCR-ABL kinase domain. The T315I substitution confers complete resistance to all TKIs commonly used in clinical practice. To date, the hierarchical level of stem cells in which this mutation initially appears has not been studied. The aim of this study was to evaluate the behavior of T315I mutated cells and to study the presence of potential additional mutations in progenitors and stem cells from a patient with CML. A comprehensive analysis of BCR-ABL(315I) mRNA expressing cells in mononuclear cells, in CD34+âcell populations, and in their primitive long-term culture-initiating cell (LTC-IC) derived progenitors was performed. We show that the T315I substitution arises in primitive Ph1 stem cells without altering their myeloid and erythroid terminal differentiation potential. Leukemic cells expressing a T315I mutated BCR-ABL display a progressive decline in LTC-IC assays as described for non-mutated CML cells at diagnosis. Finally, in our experiments, additional non-315 ABL mutations were identified in hematopoietic stem cell colonies. This observation is suggestive of genetic instability affecting CML progenitors.
Asunto(s)
Resistencia a Antineoplásicos/genética , Genes abl , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Mutación Missense , Células Progenitoras Mieloides/metabolismo , Anciano , Sustitución de Aminoácidos/genética , Sustitución de Aminoácidos/fisiología , Antineoplásicos/uso terapéutico , Análisis Mutacional de ADN/métodos , Humanos , Isoleucina/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Masculino , Células Progenitoras Mieloides/patología , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Polimorfismo de Nucleótido Simple , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Treonina/genéticaRESUMEN
Tyrosine kinase inhibitors (TKIs) have dramatically improved the treatment of chronic myeloid leukemia (CML). However, resistances are occasionally observed, mainly due to mutations within the BCR-ABL kinase domain. The T315I substitution confers complete resistance to TKIs commonly used in clinical practice. In the present study, we used an allele-specific quantitative-RT-PCR to perform a molecular follow-up of BCR-ABL transcripts harboring the T315I mutation. We retrospectively quantified BCR-ABL315I mRNA in five patients who acquired the T315I mutation. Our results highlight the relevance of allele-specific Q-RT-PCR experiments for the monitoring of mutated BCR-ABL transcripts and suggest that the kinetics of emergence of T315I mutant mRNA is influenced by the stage of the disease and the presence of previous BCR-ABL kinase domain mutations.