RESUMEN
Treatment of CML with the tyrosine kinase inhibitor (TKI) imatinib mesylate results in the emergence of point mutations within the kinase domain (KD) of the BCR-ABL1 fusion transcript. The introduction of next-generation TKIs that can overcome the effects of some BCR-ABL1 KD mutations requires quantitative mutation profiling methods to assess responses. We report the design and validation of such quantitative assays, using pyrosequencing and mutation-specific RT-PCR techniques, to allow sequential monitoring and illustrate their use in tracking specific KD mutations (e.g. G250E, T315I, and M351T) following changes in therapy. Pyrosequencing and mutation-specific RT-PCR allows sequential monitoring of specific mutations and identification of rapid clonal shifts in response to kinase inhibitor therapy in CML. Rapid reselection of TKI-resistant clones occurs following therapy switch in CML.
Asunto(s)
Análisis Mutacional de ADN/métodos , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Inhibidores de Proteínas Quinasas/uso terapéutico , Sustitución de Aminoácidos , Benzamidas , Células Clonales/efectos de los fármacos , Células Clonales/metabolismo , Resistencia a Antineoplásicos/genética , Proteínas de Fusión bcr-abl/genética , Humanos , Mesilato de Imatinib , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Mutación/efectos de los fármacos , Piperazinas/uso terapéutico , Pirimidinas/uso terapéutico , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND: BCR-ABL kinase domain (KD) mutations are detected in approximately 45% of patients with imatinib-resistant chronic myeloid leukemia. Patterns of KD mutations in Philadelphia chromosome (Ph)-positive acute lympho- blastic leukemia (ALL) are less well studied. METHODS: The authors assessed KD mutations in patients with recurrent Ph-positive ALL after treatments that included 1 or more kinase inhibitors (n = 24 patients) or no prior kinase inhibitor (KI) therapy (n = 12 patients). RESULTS: ABL KD mutations were detected by direct sequencing in 15 of 17 patients (88%) who had recurrent Ph-positive ALL and received prior imatinib (n = 16) or dasatinib (n = 1) treatment and in 6 of 7 patients (86%) who had resistant/recurrent tumors treated with >or=2 KIs compared with 0 of 12 patients with recurrent Ph-positive ALL who never received KIs. A restricted set of mutations was observed, mostly Y253H and T315I, and were detected on average 10 months after KI initiation, and mutations were not detected in the initial tumor samples before KI therapy in 12 patients who were assessed. Using a more sensitive pyrosequencing method, mutations were not detected at codons 315 and 253 in the diagnostic samples from those 12 patients or in 30 patients with Ph-positive ALL who never developed recurrent disease. CONCLUSIONS: ABL KD mutations, especially at codons 315 and 253, emerged at the time of disease recurrence in the vast majority of patients who had Ph-positive ALL and received maintenance KI therapy. Thus, the authors concluded that ongoing KI exposure may alter the patterns of recurrence and favor the outgrowth of clones with KI-resistant mutations.
Asunto(s)
Genes abl , Mutación Puntual , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Benzamidas , Niño , Preescolar , Resistencia a Antineoplásicos , Femenino , Proteínas de Fusión bcr-abl , Humanos , Mesilato de Imatinib , Masculino , Persona de Mediana Edad , Modelos Moleculares , Piperazinas/uso terapéutico , Inhibidores de Proteínas Quinasas/uso terapéutico , Pirimidinas/uso terapéutico , Recurrencia , Terapia RecuperativaRESUMEN
To further characterize the genotypic features of splenic (S) and nodal (N) marginal zone lymphomas (MZL) we compared eight SMZL and five NMZL by array-based comparative genomic hybridization (aCGH). Arbitrarily, aberrations were divided into major imbalances, defined as gains or losses involving five or more contiguous genetic loci, and minor imbalances, defined as those involving four or fewer loci. SMZL, but not NMZL, demonstrated major imbalances. These included deletions involving various lengths of 7q (three cases), and 14q23q24 (one case) and gains of 9p13p21 (one case), 13q21q33 (one case) and 16p13.1 (one case). Common minor imbalances in SMZL were: loss of sonic hedgehog gene (SHH) at 7q36.2 (four cases), loss of protection of telomere 1 gene (POT1) at 7q31.32 (three cases), and gain of glioma associated oncogene 1 (GLI1) at 12q13.2 (three cases). Common minor alterations in NMZL were: loss of the fas-associated via death domain gene (FADD) at 11q13.2 (three cases) and gain of GLI1 (five cases). In conclusion, SMZL, but not NMZL, demonstrates large genomic imbalances and frequent loss of the 7q31.32 and 7q36.2 regions involving POT1 and SHH, respectively. In NMZL, loss of FADD and gain of GLI1 are frequent events.
Asunto(s)
Cromosomas Humanos Par 7/genética , Enfermedades Linfáticas/genética , Linfoma de Células B de la Zona Marginal/genética , Neoplasias del Bazo/genética , Anciano , Hibridación Genómica Comparativa/métodos , Análisis Mutacional de ADN , ADN de Neoplasias/genética , ADN de Neoplasias/aislamiento & purificación , Femenino , Proteínas Hedgehog/genética , Proteínas Hedgehog/fisiología , Humanos , Hibridación Fluorescente in Situ , Enfermedades Linfáticas/fisiopatología , Linfoma de Células B de la Zona Marginal/fisiopatología , Masculino , Persona de Mediana Edad , Hibridación de Ácido Nucleico/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Complejo Shelterina , Neoplasias del Bazo/fisiopatología , Telómero/genética , Proteínas de Unión a Telómeros/genética , Proteínas de Unión a Telómeros/fisiologíaRESUMEN
Chronic myeloid leukemia (CML) with T315I mutation has been reported to have poor prognosis. We analyzed 27 patients with T315I, including 20 who developed T315I after imatinib failure (representing 11% of 186 patients with imatinib failure), and 7 of 23 who developed new mutations after second tyrosine kinase inhibitor (TKI). Median follow-up from mutation detection was 18 months. At the time of T315I detection, 10 were in chronic phase (CP), 9 in accelerated phase, and 8 in blast phase. Except for the lack of response to second TKIs (P = .002), there was no difference in patient characteristics and outcome between patients with T315I and those with other or no mutations. Patients in CP had a 2-year survival rate of 87%. Although the T315I mutation is resistant to currently available TKIs, survival of patients with T315I remains mostly dependent on the stage of the disease, with many CP patients having an indolent course.
Asunto(s)
Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Piperazinas/uso terapéutico , Mutación Puntual , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/genética , Pirimidinas/uso terapéutico , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Sustitución de Aminoácidos , Benzamidas , Niño , Resistencia a Antineoplásicos/genética , Femenino , Proteínas de Fusión bcr-abl , Humanos , Mesilato de Imatinib , Leucemia Mielógena Crónica BCR-ABL Positiva/enzimología , Masculino , Persona de Mediana Edad , Resultado del TratamientoRESUMEN
Dasatinib and nilotinib are potent tyrosine kinase inhibitors (TKIs) with activity against many imatinib-resistant chronic myeloid leukemia (CML) clones with BCR-ABL kinase domain (KD) mutations, except T315I. We assessed for changes in the BCR-ABL KD mutation status in 112 patients with persistent CML who received a second-generation TKI after imatinib failure. Sixty-seven different KD mutations were detected before the start of therapy with a second TKI, with T315I seen in 15%. Equal numbers of patients received nilotinib or dasatinib following imatinib, and 18 received 3 TKIs. Response rates were similar for patients with and without mutations, regardless of mutation site except for T315I. Overall, 29 patients (26%) developed new KD mutations after therapy with a second (n = 24) or third (n = 5) TKI, but only 4 (4%) developed T315I. In 73% of cases, the KD mutations that persisted or developed following switch to new TKI were at sites also found in prior in vitro TKI mutagenesis assays. Although there is only a mild increase in mutation frequency with sequential TKI treatment, novel mutations do occur and mutation regression/acquisition/persistence generally reflects the in vitro differential sensitivity predicted for each TKI. In this study, there was no marked increase in development of T315I.
Asunto(s)
Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Mutación , Inhibidores de Proteínas Quinasas/administración & dosificación , Proteínas Tirosina Quinasas/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Estudios de Seguimiento , Proteínas de Fusión bcr-abl , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/enzimología , Masculino , Persona de Mediana Edad , Estructura Terciaria de Proteína , Proteínas Tirosina Quinasas/antagonistas & inhibidoresRESUMEN
Molecular recognition of small molecule ligands by the nucleic acid aptamers for tobramycin, ATP, and FMN has been examined using electrospray ionization mass spectrometry (ESI-MS). Mass spectrometric data for binding stoichiometry and relative binding affinity correlated well with solution data for tobramycin aptamer complexes, in which aptamer/ligand interactions are mediated by hydrogen bonds. For the ATP and FMN aptamers, where ligand interactions involve both hydrogen bonding and significant pi-stacking, the relative binding affinities determined by MS did not fully correlate with results obtained from solution experiments. Some high-affinity aptamer/ligand complexes appeared to be destabilized in the gas phase by internal Coulombic repulsion. In CAD experiments, complexes with a greater number of intermolecular hydrogen bonds exhibited greater gas-phase stability even in cases when solution binding affinities were equivalent. These results indicate that in at least some cases, mass spectrometric data on aptamer/ligand binding affinities should be used in conjunction with complementary techniques to fully assess aptamer molecular recognition properties.
Asunto(s)
Aptámeros de Nucleótidos/química , Conformación de Ácido Nucleico , Espectrometría de Masa por Ionización de Electrospray/métodos , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/química , Sitios de Unión , Mononucleótido de Flavina/química , Ligandos , Tobramicina/análogos & derivados , Tobramicina/químicaRESUMEN
Most biopolymer drugs to date have been proteins. However, the ability to select nucleic acid binding species (aptamers) has led to the development of protein inhibitors and modulators that are small, readily synthesized nucleic acids. The techniques for optimizing, stabilizing, and delivering nucleic acid therapies are just beginning to be developed, but the same engineering flexibility that has so far allowed the generation of multiple, high affinity and specificity binding species appears to also apply to the methods for adapting nucleic acids to clinical applications. We review the selection and characterization of various aptamers and their applications to a variety of disease states, and then focus on the hurdles that must be overcome for the use of aptamers as both exogenously delivered drugs and as gene therapies.