RESUMEN
Molecular monitoring of chronic myeloid leukemia patients using robust BCR-ABL1 tests standardized to the International Scale (IS) is key to proper disease management, especially when treatment cessation is considered. Most laboratories currently use a time-consuming sample exchange process with reference laboratories for IS calibration. A World Health Organization (WHO) BCR-ABL1 reference panel was developed (MR(1)-MR(4)), but access to the material is limited. In this study, we describe the development of the first cell-based secondary reference panel that is traceable to and faithfully replicates the WHO panel, with an additional MR(4.5) level. The secondary panel was calibrated to IS using digital PCR with ABL1, BCR and GUSB as reference genes and evaluated by 44 laboratories worldwide. Interestingly, we found that >40% of BCR-ABL1 assays showed signs of inadequate optimization such as poor linearity and suboptimal PCR efficiency. Nonetheless, when optimized sample inputs were used, >60% demonstrated satisfactory IS accuracy, precision and/or MR(4.5) sensitivity, and 58% obtained IS conversion factors from the secondary reference concordant with their current values. Correlation analysis indicated no significant alterations in %BCR-ABL1 results caused by different assay configurations. More assays achieved good precision and/or sensitivity than IS accuracy, indicating the need for better IS calibration mechanisms.
Asunto(s)
Proteínas de Fusión bcr-abl/análisis , Calibración , Proteínas de Fusión bcr-abl/normas , Genes abl , Humanos , Reacción en Cadena de la Polimerasa , Proteínas Proto-Oncogénicas c-bcr/genética , Estándares de Referencia , Organización Mundial de la SaludRESUMEN
Treatment of chronic myeloid leukemia (CML) with tyrosine kinase inhibitors has advanced to a stage where many patients achieve very low or undetectable levels of disease. Remarkably, some of these patients remain in sustained remission when treatment is withdrawn, suggesting that they may be at least operationally cured of their disease. Accurate definition of deep molecular responses (MRs) is therefore increasingly important for optimal patient management and comparison of independent data sets. We previously published proposals for broad standardized definitions of MR at different levels of sensitivity. Here we present detailed laboratory recommendations, developed as part of the European Treatment and Outcome Study for CML (EUTOS), to enable testing laboratories to score MR in a reproducible manner for CML patients expressing the most common BCR-ABL1 variants.
Asunto(s)
Regulación Leucémica de la Expresión Génica , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Calibración , Europa (Continente) , Proteínas de Fusión bcr-abl/genética , Perfilación de la Expresión Génica , Variación Genética , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Límite de Detección , Reacción en Cadena de la Polimerasa , Reproducibilidad de los Resultados , Resultado del TratamientoRESUMEN
Serial quantification of BCR-ABL1 mRNA is an important therapeutic indicator in chronic myeloid leukaemia, but there is a substantial variation in results reported by different laboratories. To improve comparability, an internationally accepted plasmid certified reference material (CRM) was developed according to ISO Guide 34:2009. Fragments of BCR-ABL1 (e14a2 mRNA fusion), BCR and GUSB transcripts were amplified and cloned into pUC18 to yield plasmid pIRMM0099. Six different linearised plasmid solutions were produced with the following copy number concentrations, assigned by digital PCR, and expanded uncertainties: 1.08±0.13 × 10(6), 1.08±0.11 × 10(5), 1.03±0.10 × 10(4), 1.02±0.09 × 10(3), 1.04±0.10 × 10(2) and 10.0±1.5 copies/µl. The certification of the material for the number of specific DNA fragments per plasmid, copy number concentration of the plasmid solutions and the assessment of inter-unit heterogeneity and stability were performed according to ISO Guide 35:2006. Two suitability studies performed by 63 BCR-ABL1 testing laboratories demonstrated that this set of 6 plasmid CRMs can help to standardise a number of measured transcripts of e14a2 BCR-ABL1 and three control genes (ABL1, BCR and GUSB). The set of six plasmid CRMs is distributed worldwide by the Institute for Reference Materials and Measurements (Belgium) and its authorised distributors (https://ec.europa.eu/jrc/en/reference-materials/catalogue/; CRM code ERM-AD623a-f).
Asunto(s)
Proteínas de Fusión bcr-abl/genética , Proteínas de Fusión bcr-abl/metabolismo , Plásmidos/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/normas , Calibración , Clonación Molecular , ADN , Proteínas de Escherichia coli/genética , Dosificación de Gen , Humanos , Proteínas de Transporte de Membrana/genética , Proteínas Proto-Oncogénicas c-bcr/genética , ARN Mensajero/metabolismo , Estándares de ReferenciaRESUMEN
The blood serum concentrations of progesterone and estradiol-17beta were measured at various stages of the oestrous cycle of seven female mules, and their reproductive tracts were examined ultrasonographically at the same stages.
Asunto(s)
Equidae/fisiología , Estradiol/sangre , Estro/fisiología , Ovario/diagnóstico por imagen , Progesterona/sangre , Útero/diagnóstico por imagen , Animales , Cuello del Útero/diagnóstico por imagen , Equidae/anatomía & histología , Equidae/sangre , Estro/sangre , Detección del Estro , Femenino , Ovulación , Detección de la Ovulación/veterinaria , UltrasonografíaRESUMEN
Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL) has a dismal prognosis. We prospectively evaluated minimal residual disease (MRD) by measuring BCR/ ABL levels with a quantitative real-time PCR procedure after induction and after consolidation in 45 adults with Ph+ ALL who obtained complete hematological remission after a high-dose daunorubicin induction schedule. At diagnosis, the mean BCR-ABL/GUS ratio was 1.55 +/- 1.78. A total of 42 patients evaluable for outcome analysis were operationally divided into two MRD groups: good molecular responders (GMRs; n = 28) with > 2 log reduction of residual disease after induction and > 3 log reduction after consolidation therapy, and poor molecular responders (PMRs; n = 14) who, despite complete hematological remission, had a higher MRD at both time points. In GMR, the actuarial probability of relapse-free, disease-free and overall survival at two years was 38, 27 and 48%, respectively, as compared to 0, 0 and 0% in PMR (P = 0.0035, 0.0076 and 0.0026, respectively). Salvage therapy induced a second sustained complete hematological remission in three GMR patients, but in no PMR patient. Our data indicate that, as already shown in children, adult Ph+ ALL patients have a heterogeneous sensitivity to treatment, and that early quantification of residual disease is a prognostic parameter in this disease.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Proteínas de Fusión bcr-abl/genética , Cromosoma Filadelfia , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Adolescente , Adulto , Antibióticos Antineoplásicos/uso terapéutico , Antimetabolitos Antineoplásicos/uso terapéutico , Antineoplásicos Fitogénicos/uso terapéutico , Asparaginasa/uso terapéutico , Citarabina/uso terapéutico , Daunorrubicina/uso terapéutico , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Neoplasia Residual/tratamiento farmacológico , Neoplasia Residual/genética , Neoplasia Residual/mortalidad , Leucemia-Linfoma Linfoblástico de Células Precursoras/mortalidad , Valor Predictivo de las Pruebas , Pronóstico , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Vincristina/uso terapéuticoRESUMEN
Two novel IL2-dependent cell lines, DERL-2 and DERL-7, were established from a patient with hepatosplenic gammadelta T cell lymphoma. This patient presented, at diagnosis, two discrete populations of CD56+ cells, one TCRgammadelta+, the second lacking T cell-restricted antigens. The cell lines derived displayed features corresponding to the two cellular components of the disease: DERL-2 was CD56+/CD3+/TcRgammadelta+ while DERL-7 was CD56+/CD3-/TcRgammadelta-. Along with CD56, the two cell lines shared the expression of CD7, CD2, CD158b and CD117. Karyotype analysis showed that both cell lines were near-diploid, with iso-7q and loss of one chromosome 10. In addition, DERL-2 showed 5q+ in all metaphases analyzed, while DERL-7 revealed loss of one chromosome 4. Genotypically, both cell lines shared the same STR pattern at nine loci and demonstrated an identical rearranged pattern of the T cell receptor genes beta, gamma and delta, with respect to the original tumor cells. These data indicated that both cell lines and the original neoplastic populations were T cell-derived and arose from a common ancestor. Among a large panel of cytokines tested, only SCF was able to substitute IL2 in supporting cell proliferation. Moreover, SCF and IL2 acted synergistically, dramatically enhancing cell growth. These cell lines may represent a model to further analyze the overlap area between T and NK cell malignancies, and may provide new information about the synergistic action of IL2 and SCF on normal and neoplastic T/NK cells.
Asunto(s)
Linfoma no Hodgkin/inmunología , Linfoma no Hodgkin/patología , Células Tumorales Cultivadas/citología , Adulto , Complejo CD3/análisis , Antígeno CD56/análisis , División Celular/efectos de los fármacos , Análisis Citogenético , Sinergismo Farmacológico , Reordenamiento Génico de la Cadena beta de los Receptores de Antígenos de los Linfocitos T/genética , Genotipo , Humanos , Inmunofenotipificación , Interleucina-2/farmacología , Masculino , Receptores de Antígenos de Linfocitos T gamma-delta/análisis , Receptores de Antígenos de Linfocitos T gamma-delta/genética , Factor de Células Madre/farmacologíaRESUMEN
A new intrauterine device for contraception was tested on nine bitches. After it had been implanted, the bitches were mated but none of them became pregnant. Over a two-year period no side effects were observed, except in a bulldog bitch in which signs of oestrus persisted until the device had been removed.
Asunto(s)
Anticoncepción/veterinaria , Perros/fisiología , Dispositivos Intrauterinos/veterinaria , Animales , Anticoncepción/métodos , Estro/fisiología , FemeninoRESUMEN
BACKGROUND AND OBJECTIVE: Large granular lymphocytes derive from two major lineages: one expressing the CD3 surface antigen (T-lymphocytes), and the other lacking this marker (NK-cells). Although developmental overlaps between natural killer cells and T-cells have been described, malignancies derived from these two cell types are considered as distinct lymphoid disorders. DESIGN AND METHODS: We report the case of a 30-year old man affected by a lymphoma/leukemia syndrome presenting with hepatosplenic lymphoma which rapidly transformed into aggressive NK-leukemia. Extensive flow cytometry studies and molecular analysis were repeated during the course of the disease, and showed an unexpected changing pattern. RESULTS: At diagnosis, flow cytometry analysis showed the co-existence of two cell populations, one CD56(+), CD3(+), TcRgd(+), and the other CD56(+), CD3(-) and TcRgd(-). Molecular analysis showed that the TcR genes had the same clonally rearranged pattern involving b, g and d genes in both populations. At disease relapse and during the terminal refractory phase, only CD3(-) cells were present. INTERPRETATION AND CONCLUSIONS: This is an unusual case of CD56(+) aggressive lymphoma/leukemia characterized by the clonal expansion of two phenotypically different cell populations, variably balanced during the course of the disease. The presence of the same TcR genomic rearrangement suggests the origin from a common progenitor able to differentiate along both T- and NK-pathways.