Análisis mutacional de p53 en neoplasias hematológicas

Descripció del recurs: 5 juny 2003Consultable des del TDXTítol obtingut de la portada digitalitzadaLas mutaciones puntuales de p53 representan la lesión molecular más común en neoplasias humanas y se han asociado con mala evolución clínica. Los objetivos de este trabajo han sido: - Establecer la aplicabilidad del análisis mutacional de p53 en las neoplasias hematológicas. - Conocer la contribución de las mutaciones de p53 en las transformaciones agresivas de los síndromes mieloproliferativos crónicos bcr/abl-. - Determinar la frecuencia de inactivación de p53 en leucemias mieloides con reordenamientos MLL y con translocaciones cromosómicas que afectan a 17p. - Analizar la frecuencia de mutaciones de p53 en leucemias agudas linfoblásticas de línea T y B. - Estudiar el rendimiento del análisis mutacional de p53 en casos con diagnóstico morfológico de Burkitt. - Estudiar la presencia de mutaciones en procesos linfoproliferativos de baja frecuencia: neoplasias NK y reordenamientos NPM/ALK. - Conocer la frecuencia de mutaciones de p53 en linfoma folicular. - Comparar el espectro mutacional encontrado en nuestros casos con el descrito en las bases de datos de p53. - Aplicar los métodos de análisis mutacional de p53 a las muestras obtenidas de dos hermanos pertenecientes a una familia con síndrome de Li-Fraumeni. De los resultados originados en forma de artículos y anexos se puede concluir que: - Las mutaciones de p53 son eventos moleculares comunes en las transformaciones agresivas de síndromes mieloproliferativos crónicos bcr/abl -. - Las neoplasias hematológicas con reordenamientos de 17p presentan a menudo mutaciones del gen p53. - Las neoplasias mieloides con lesiones estructurales del brazo largo del cromosoma 3, a pesar de su mal pronóstico, no muestran mutaciones de p53. - Las leucemias agudas mieloides del adulto con reordenamientos del gen MLL pueden presentar mutaciones de p53. Esta asociación es particularmente relevante en casos con cariotipo complejo, deleciones del brazo largo del cromosoma 5 o en casos de LAM secundaria. - Las mutaciones de p53 son frecuentes en dos subgrupos moleculares de leucemias infantiles: c-myc y MLL+. - Las mutaciones de p53 rara vez se asocian a LAL-T en el momento del diagnóstico - En el 11% de los casos de LAL de línea B del niño en recaída se encuentran mutaciones de p53. - Las LAL-B con morfología L3 y los linfomas de Burkitt presentan con frecuencia mutaciones de p53. - Las mutaciones de p53 a menudo son responsables de la fase final agresiva del linfoma folicular y del manto. - Los linfomas foliculares en estadios avanzados pueden presentar mutaciones de p53 en ausencia de transformación histológica evidente. - En linfomas con reordenamientos NPM/ALK no se detectaron mutaciones de p53 . - No se han detectado mutaciones de p53 en linfomas/leucemias NK. - La distribución de las mutaciones recogidas en el presente trabajo coincide con los hallazgos acumulados en las bases de datos internacionales.P53 point mutations are the most common molecular lesions described in human neoplasia. The aims of this work were as follows: - To establish the applicability of the mutational analysis of p53 in hematologic malignancies. - To gain some insight into the mechanisms associated with the acute transformations of bcr/abl- chronic myeloproliferative disorders. - To determine the frequency of p53 mutations in acute myeloid leukemias with MLL rearrangements and 17p translocations. - To study the presence of p53 mutations in T-cell and B-cell lineage acute lymphoblastic leukemias (T-ALL and B-ALL). - To perform the same analysis in cases with Burkitt's lymphoma. - To investigate the p53 status in rare hematologic malignancies i.e. NK leukemias and NPM/ALK lymphomas. - To analyze the p53 mutations in cases with follicular lymphoma. - To establish the mutational spectrum in our cases. - To investigate the p53 gene in one Li-Fraumeni pedigree. Based on the results obtained in this thesis, it may be concluded that: - p53 mutations are commonly found in the acute transfromations of bcr/abl- chronic myeloproliferative disorders. - Myeloid malignancies with 17p cytogenetic lesions are frequently associated with p53 mutations. - Myeloid malignancies with cytogenetic involvement of the long arm of chromosome 3 did not show p53 mutations. - In some instances, acute myeloid leukemias (AML) with MLL rearrangements had p53 mutations. This association merits investigation in cases with a complex karyotype, deletions at the long arm of chromosome 5 and in secondary AML. - P53 mutations are common in c-myc and MLL+ B-ALL. - P53 are rarely detected in T-ALL at diagnosis. - P53 mutations are present in a significant percentage (11%) of B-ALL at relapse. - Burkitt's lymphoma cases frequently displayed p53 mutations. - p53 mutations play a role in the acute transformations arising from follicular and mantle lymphomas. - Follicular lymphomas may harbor p53 mutations even in the absence of documented histologic transformation. - P53 mutations are not detected in NK leukemias or in NPM/ALK lymphomas. - Mutational spectrum obtained from the mutants described in this work is consistent with that reported in international databases

TET2 missense variants in human neoplasia. A proposal of structural and functional classification

The human TET2 gene plays a pivotal role in the epigenetic regulation of normal and malignant hematopoiesis. Somatic TET2 mutations have been repeatedly identified in age-related clonal hematopoiesis and in myeloid neoplasms ranging from acute myeloid leukemia (AML) to myeloproliferative neoplasms. However, there have been no attempts to systematically explore the structural and functional consequences of the hundreds of TET2 missense variants reported to date. We have sequenced the TET2 gene in 189 Spanish AML patients using Sanger sequencing and NGS protocols. Next, we performed a thorough bioinformatics analysis of TET2 protein and of the expected impact of all reported TET2 missense variants on protein structure and function, exploiting available structure-and-function information as well as 3D structure prediction tools. We have identified 38 TET2 allelic variants in the studied patients, including two frequent SNPs: p.G355D (10 cases) and p.I1762V (28 cases). Four of the detected mutations are reported here for the first time: c.122C>T (p.P41L), c.4535C>G (p.A1512G), c.4760A>G (p.D1587G), and c.5087A>T (p.Y1696F). We predict a complex multidomain architecture for the noncatalytic regions of TET2, and in particular the presence of well-conserved α+β globular domains immediately preceding and following the actual catalytic unit. Further, we provide a rigorous interpretation of over 430 missense SNVs that affect the TET2 catalytic domain, and we hypothesize explanations for ~700 additional variants found within the regulatory regions of the protein. Finally, we propose a systematic classification of all missense mutants and SNPs reported to date into three major categories (severe, moderate, and mild), based on their predicted structural and functional impact. The proposed classification of missense TET2 variants would help to assess their clinical impact on human neoplasia and may guide future structure-and-function investigations of TET family members

Estudis dels principals arbres ornamentals, no autòctons, disponibles a Catalunya

Després de l'estudi realitzat per la ICEA sobre els arbres autòctons, ha estat convenient tractar dels arbres naturalitzats i adaptats. Aquests són molt més nombrosos a Catalunya, de manera que per cada arbre autòcton en corresponen deu dels altres. Per motius que expliquem, l'estudi se centra en un col?lectiu de 119 arbres no autòctons. D'acord amb una enquesta, la meitat dels arbres indicats són fàcils de produir i es comercialitzen regularment. Els restants, per problemes de propagació, de cultiu o de rendibilitat, tenen una comercialització més variable. Les produccions anuals per arbre estan dins l'interval de 1.000 a 5.000 unitats per any, per a 36 espècies, o sigui, el 30 % del total. Les restants es reparteixen entre els intervals superiors a 5.000 i inferiors a 1.000. L'aplicació determinada per a cada arbre, en relació a les 41 comarques catalanes, permet conèixer quins són els arbres adients per a cadas cuna. Els quadres de característiques i d'exigències arrodoneixen l'estudi, proporcionant una informació completa per a cada un dels 119 arbres. Dins el col?lectiu, hi ha 37 arbres de flor, dels quals hem realitzat l'estudi fenològic. L'Àrea Metropolitana de Barcelona reuneix excel?lents condicions per a la plantació d'aquests arbres de flor. De les 37 espècies, n'hi van bé 33, o sigui, un 90 %.After the publication of the research on the catalan native tree by the ICEA, it has become convenient to study the naturalized and adapted tree. In Catalonia, there are ten naturalized and adapted trees for each native one. As we will explain later, this research focuses on a group of 119 non-native trees. Following a survey, half of these 119 trees are easy to produce and to marked. The remainders, which have some problems to propagate, cultivate and become rendible, present a more variable marketing. The annual production is between 1.000 and 5.000 trees for each specie; that means a 30 % of the total production. Remainders are between more them 5.000 and less them 1.000. The chapter «Application» indicate the species of non-native trees wich are more convenient to each of the different 41 catalan region. To provide better documentation, the study is completed with information about the «Characteristics» and the «Requirements» of these 119 trees. Because of their flowering, 37 species are thoroughly studied, Barcelona?s Metropolitan Area has an ideal climate for these trees. In fact, 33 out of 37 species, that is a 90 %, can be perfectly adapted to this area.Después del estudio realizado por la ICEA sobre el árbol autóctono, ha sido conveniente tratar el árbol naturalizado y adaptado. Éste es mucho más numeroso en Cataluña, de forma que por cada árbol autóctono, corresponden diez de los otros. Por los motivos que se explican, el estudio se centra en un colectivo de 119 árboles no autóctonos. Según una encuesta, la mitad de los árboles indicados son fáciles de producir y se comercializan regularmente. Los restantes, por problemas de propagación, cultivo o rentabilidad, presentan una comercialización más variable. Las producciones anuales por árbol resultan estar en el intervalo de 1.000 a 5.000 unidades por año, para 36 especies, o sea, el 30 % del total. Las restantes se reparten entre los intervalos superiores a 5.000 e inferiores a 1.000. La aplicación determinada para cada árbol, con relación a las 41 comarcas catalanas, permite conocer cuales son los árboles convenientes para cada comarca. Los cuadros de características y de exigencias redondean el estudio, proporcionando una información completa para cada uno de los 119 árboles. En el colectivo hay 37 árboles de flor, de los que se ha realizado el estudio fenológico. El Área Metropolitana de Barcelona reúne excelentes condiciones para la plantación de estos árboles de flor. De las 37 especies, 33, o sea, el 90 %, se adaptan perfectamente a la zona

Many signs, one mutation : Early onset of de novo GATA2 deficiency syndrome. A case report

We report a case with a broad spectrum of symptoms, related to GATA2 deficiency syndrome, which emerged as early as at 6 months of age. They ranged from lymphedema, deafness to myelodysplastic syndrome (MDS). Non-hematologic symptoms may long precede myelodysplastic syndrome diagnosis in patients with GATA2 mutations

Bone marrow fibrosis, sequence variant of asxl1, and Sjögren syndrome : A case report

Only proven pathogenic mutations associated with myeloid neoplasms are key to establish the clonal nature of the bone marrow fibrosis. In cases with genetic variants of uncertain meaning, the clinical picture may be required to rule out secondary causes. Only proven pathogenic mutations associated with myeloid neoplasms are key to establish the clonal nature of the bone marrow fibrosis. In cases with genetic variants of uncertain meaning, the clinical picture may be required to rule out secondary causes

Treatment with G-CSF reduces acute myeloid leukemia blast viability in the presence of bone marrow stroma

BACKGROUND: The resulting clinical impact of the combined use of G-CSF with chemotherapy as a chemosensitizing strategy for treatment of acute myeloid leukemia (AML) patients is still controversial. In this study, the effect of ex vivo treatment with G-CSF on AML primary blasts was studied. METHODS: Peripheral blood mononuclear cells from AML patients were treated with G-CSF at increasing doses, alone or in co-culture with HS-5 stromal cells. Cell viability and surface phenotype was determined by flow cytometry 72 h after treatment. For clonogenicity assays, AML primary samples were treated for 18 h with G-CSF at increasing concentrations and cultured in methyl-cellulose for 14 days. Colonies were counted based on cellularity and morphology criteria. RESULTS: The presence of G-CSF reduced the overall viability of AML cells co-cultured with bone marrow stroma; whereas, in absence of stroma, a negligible effect was observed. Moreover, clonogenic capacity of AML cells was significantly reduced upon treatment with G-CSF. Interestingly, reduction in the AML clonogenic capacity correlated with the sensitivity to chemotherapy observed in vivo. CONCLUSIONS: These ex vivo results would provide a biological basis to data available from studies showing a clinical benefit with the use of G-CSF as a priming agent in patients with a chemosensitive AML and would support implementation of further studies exploring new strategies of chemotherapy priming in AML

Ultrastructural, cytogenetic, and molecular findings in mast cell leukemia : Case report

We report a de novo aleukemic form of MCL with a complex monosomic karyotype with LOH for multiple chromosomes and TP53 mutation. Additionally, whereas D816V KIT was not found, the c-Kit transmembrane domain p.M541L variant was detected which is the most common SNP of KIT gene in humans with controversial pathogenic role. In these cases, it is crucial to perform a rapid broad molecular study for an accurate diagnosis which could help to initiate targeted therapy

The expression level of BAALC-associated microRNA miR-3151 is an independent prognostic factor in younger patients with cytogenetic intermediate-risk acute myeloid leukemia

Acute myeloid leukemia (AML) is a heterogeneous disease whose prognosis is mainly related to the biological risk conferred by cytogenetics and molecular profiling. In elderly patients (>= 60 years) with normal karyotype AML miR-3151 have been identified as a prognostic factor. However, miR-3151 prognostic value has not been examined in younger AML patients. In the present work, we have studied miR-3151 alone and in combination with BAALC, its host gene, in a cohort of 181 younger intermediate-risk AML (IR-AML) patients. Patients with higher expression of miR-3151 had shorter overall survival (P = 0.0025), shorter leukemia-free survival (P = 0.026) and higher cumulative incidence of relapse (P = 0.082). Moreover, in the multivariate analysis miR-3151 emerged as independent prognostic marker in both the overall series and within the unfavorable molecular prognostic category. Interestingly, the combined determination of both miR-3151 and BAALC improved this prognostic stratification, with patients with low levels of both parameters showing a better outcome compared with those patients harboring increased levels of one or both markers (P = 0.003). In addition, we studied the microRNA expression profile associated with miR-3151 identifying a six-microRNA signature. In conclusion, the analysis of miR-3151 and BAALC expression may well contribute to an improved prognostic stratification of younger patients with IR-AML

A novel deep targeted sequencing method for minimal residual disease monitoring in acute myeloid leukemia

A high proportion of patients with acute myeloid leukemia who achieve minimal residual disease negative status ultimately relapse because a fraction of pathological clones remains undetected by standard methods. We designed and validated a high-throughput sequencing method for minimal residual disease assessment of cell clonotypes with mutations of NPM1, IDH1/2 and/or FLT3-single nucleotide variants. For clinical validation, 106 follow-up samples from 63 patients in complete remission were studied by sequencing, evaluating the level of mutations detected at diagnosis. The predictive value of minimal residual disease status by sequencing, multiparameter flow cytometry, or quantitative polymerase chain reaction analysis was determined by survival analysis. The sequencing method achieved a sensitivity of 10-4 for single nucleotide variants and 10-5 for insertions/deletions and could be used in acute myeloid leukemia patients who carry any mutation (86% in our diagnostic data set). Sequencing-determined minimal residual disease positive status was associated with lower disease-free survival (hazard ratio 3.4, P=0.005) and lower overall survival (hazard ratio 4.2, P<0.001). Multivariate analysis showed that minimal residual disease positive status determined by sequencing was an independent factor associated with risk of death (hazard ratio 4.54, P=0.005) and the only independent factor conferring risk of relapse (hazard ratio 3.76, P=0.012). This sequencing-based method simplifies and standardizes minimal residual disease evaluation, with high applicability in acute myeloid leukemia. It is also an improvement upon flow cytometry- and quantitative polymerase chain reaction-based prediction of outcomes of patients with acute myeloid leukemia and could be incorporated in clinical settings and clinical trials.This study was supported by the Subdirección General de Investigación Sanitaria (Instituto de Salud Carlos III, Spain) grants PI13/02387 and PI16/01530, and the CRIS against Cancer foundation grant 2014/0120. ML holds a postdoctoral fellowship of the Spanish Ministry of Economy and Competitiveness (FPDI-2013- 16409). PRP holds a postdoctoral fellowship of the Spanish Instituto de Salud Carlos III: Contrato Predoctoral de Formación en Investigación en Salud i-PFIS (IFI 14/00008).S