RESUMEN
Overcoming apoptosis resistance is one major issue in glioblastoma (GB) therapies. Accumulating evidence indicates that resistance to apoptosis in GB is mediated via upregulation of pro-survival BCL2-family members. The synthetic BH3-mimetic ABT-737 effectively targets BCL2, BCL2 like 1 and BCL2 like 2 but still barely affects cell survival which is presumably due to its inability to inhibit myeloid cell leukemia 1 (MCL1). The constitutively active serine/threonine kinase proviral integration site for moloney murine leukemia virus 1 (PIM1) was recently found to be overexpressed in GB patient samples and to maintain cell survival in these tumors. For different GB cell lines, Western Blot, mitochondrial fractionation, fluorescence microscopy, effector caspase assays, flow cytometry, and an adult organotypic brain slice transplantation model were used to investigate the putative PIM1/MCL1 signaling axis regarding potential synergistic effects with ABT-737. We demonstrate that combination of the PIM1 inhibitor SGI-1776 or the pan-PIM kinase inhibitor AZD1208 with ABT-737 strongly sensitizes GB cells to apoptosis. Unexpectedly, this effect was found to be MCL1-independent, but could be partially blocked by caspase 8 (CASP8) inhibition. Remarkably, the analysis of autophagy markers in combination with the observation of massive accumulation and hampered degradation of autophagosomes suggests a completely novel function of PIM1 as a late stage autophagy regulator, maintaining the autophagic flux at the level of autophagosome/lysosome fusion. Our data indicate that PIM1 inhibition and ABT-737 synergistically induce apoptosis in an MCL1-independent but CASP8-dependent manner in GB. They also identify PIM1 as a suitable target for overcoming apoptosis resistance in GB.
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Apoptosis/efectos de los fármacos , Glioblastoma/metabolismo , Proteínas Proto-Oncogénicas c-pim-1/metabolismo , Animales , Antineoplásicos/farmacología , Apoptosis/fisiología , Autofagia/fisiología , Compuestos de Bifenilo/farmacología , Línea Celular Tumoral , Supervivencia Celular , Glioma/patología , Humanos , Ratones , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Nitrofenoles/farmacología , Fragmentos de Péptidos/metabolismo , Piperazinas/farmacología , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas c-pim-1/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-pim-1/fisiología , Sulfonamidas/farmacología , TiazolidinasRESUMEN
We describe the case of a boy with acute myeloid leukemia with translocation t(6;11)(p22.2;q23) and insertion ins(11;9)(q23;p21.3p21.3). Translocation t(6;11)(p22.2;q23) involving the short arm of chromosome 6 has not been previously described. The LDI-PCR showed the presence of KMT2A-MLLT3 fusion and identified the BTN3A1 (butyrophilin subfamily 3 member A1) gene on 6p22.2 as the other KMT2A translocation partner. The BTN3A1 gene has never been described in the context of acute leukemia. Although this fusion is out of frame, as the antisense strand of BTN3A1 is fused to the sense strand of KMT2A, the loss of heterozygosity of the BTN3A1 gene might contribute to the malignancy of leukemic cells.
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Leucemia Mieloide Aguda/genética , Translocación Genética , Antígenos CD/genética , Butirofilinas/genética , Cromosomas Humanos Par 11/genética , Cromosomas Humanos Par 6/genética , Cromosomas Humanos Par 9/genética , N-Metiltransferasa de Histona-Lisina/genética , Humanos , Masculino , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteínas Nucleares/genéticaRESUMEN
UNLABELLED: Essentials Von Willebrand disease IIC Miami features high von Willebrand factor (VWF) with reduced function. We aimed to identify and characterize the elusive underlying mutation in the original family. An inframe duplication of VWF exons 9-10 was identified and characterized. The mutation causes a defect in VWF multimerization and decreased VWF clearance from the circulation. SUMMARY: Background A variant of von Willebrand disease (VWD) type 2A, phenotype IIC (VWD2AIIC), is characterized by recessive inheritance, low von Willebrand factor antigen (VWF:Ag), lack of VWF high-molecular-weight multimers, absence of VWF proteolytic fragments and mutations in the VWF propeptide. A family with dominantly inherited VWD2AIIC but markedly elevated VWF:Ag of > 2 U L(-1) was described as VWD type IIC Miami (VWD2AIIC-Miami) in 1993; however, the molecular defect remained elusive. Objectives To identify the molecular mechanism underlying the phenotype of the original VWD2AIIC-Miami. Patients and Methods We studied the original family with VWD2AIIC-Miami phenotypically and by genotyping. The identified mutation was recombinantly expressed and characterized by standard techniques, confocal imaging and in a mouse model, respectively. Results By Multiplex ligation-dependent probe amplification we identified an in-frame duplication of VWF exons 9-10 (c.998_1156dup; p.Glu333_385dup) in all patients. Recombinant mutant (rm)VWF only presented as a dimer. Co-expressed with wild-type VWF, the multimer pattern was indistinguishable from patients' plasma VWF. Immunofluorescence studies indicated retention of rmVWF in unusually large intracellular granules in the endoplasmic reticulum. ADAMTS-13 proteolysis of rmVWF under denaturing conditions was normal; however, an aberrant proteolytic fragment was apparent. A decreased ratio of VWF propeptide to VWF:Ag and a 1-desamino-8-d-arginine vasopressin (DDAVP) test in one patient indicated delayed VWF clearance, which was supported by clearance data after infusion of rmVWF into VWF(-/-) mice. Conclusion The unique phenotype of VWD2 type IIC-Miami results from dominant impairment of multimer assembly, an aberrant structure of mutant mature VWF and reduced clearance in vivo.
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Mutación , Enfermedad de von Willebrand Tipo 2/genética , Factor de von Willebrand/genética , Adulto , Anciano , Animales , Desamino Arginina Vasopresina/química , Retículo Endoplásmico/metabolismo , Femenino , Genes Dominantes , Genes Recesivos , Genotipo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Fenotipo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Enfermedad de von Willebrand Tipo 2/metabolismo , Factor de von Willebrand/metabolismoRESUMEN
The ALOX5 gene encodes 5-lipoxygenase (5-LO), a key enzyme of inflammatory reactions, which is transcriptionally activated by trichostatin A (TSA). Physiologically, 5-LO expression is induced by calcitriol and/or transforming growth factor-ß. Regulation of 5-LO mRNA involves promoter activation and elongation control within the 3'-portion of the ALOX5 gene. Here we focused on the ALOX5 promoter region. Transcriptional initiation was associated with an increase in histone H3 lysine 4 trimethylation in a TSA-inducible manner. Therefore, we investigated the effects of the MLL (mixed lineage leukemia) protein and its derivatives, MLL-AF4 and AF4-MLL, respectively. MLL-AF4 was able to enhance ALOX5 promoter activity by 47-fold, which was further stimulated when either vitamin D receptor and retinoid X receptor or SMAD3/SMAD4 were co-transfected. In addition, we investigated several histone deacetylase inhibitors (HDACi) in combination with gene knockdown experiments (HDAC1-3, MLL). We were able to demonstrate that a combined inhibition of HDAC1-3 induces ALOX5 promoter activity in an MLL-dependent manner. Surprisingly, a constitutive activation of ALOX5 by MLL-AF4 was inhibited by class I HDAC inhibitors, by relieving inhibitory functions deriving from MLL.Conversely, a knockdown of MLL increased the effects mediated by MLL-AF4. Thus, HDACi treatment seems to switch 'inactive MLL' into 'active MLL' and overwrites the dominant functions deriving from MLL-AF4.
RESUMEN
Our focus is the identification, characterisation and functional analysis of different MLL fusions. In general, MLL fusion proteins are encoded by large cDNA cassettes that are difficult to transduce into haematopoietic stem cells. This is due to the size limitations of the packaging process of those vector-encoded RNAs into retro- or lentiviral particles. Here, we present our efforts in establishing a universal vector system to analyse different MLL fusions. The universal cloning system was embedded into the backbone of the Sleeping Beauty transposable element. This transposon has no size limitation and displays no integration preference, thereby avoiding the integration into active genes or their promoter regions. We utilised this novel system to test different MLL fusion alleles (MLL-NEBL, NEBL-MLL, MLL-LASP1, LASP1-MLL, MLL-MAML2, MAML2-MLL, MLL-SMAP1 and SMAP1-MLL) in appropriate cell lines. Stable cell lines were analysed for their growth behaviour, focus formation and colony formation capacity and ectopic Hoxa gene transcription. Our results show that only 1/4 tested direct MLL fusions, but 3/4 tested reciprocal MLL fusions exhibit oncogenic functions. From these pilot experiments, we conclude that a systematic analysis of more MLL fusions will result in a more differentiated picture about the oncogenic capacity of distinct MLL fusions.
Asunto(s)
Elementos Transponibles de ADN , Vectores Genéticos , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Transfección/métodos , Transposasas/metabolismo , Animales , Línea Celular , Proliferación Celular , Supervivencia Celular , Clonación Molecular , Elementos Transponibles de ADN/genética , Fibroblastos/metabolismo , N-Metiltransferasa de Histona-Lisina , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Ratones , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteínas de Fusión Oncogénica/genética , Factores de Tiempo , Transcripción Genética , Translocación Genética , Transposasas/genéticaRESUMEN
MLL-AF4 fusion is hallmark in high-risk infant pro-B-acute lymphoblastic leukemia (pro-B-ALL). Our limited understanding of MLL-AF4-mediated transformation reflects the absence of human models reproducing this leukemia. Hematopoietic stem/progenitor cells (HSPCs) constitute likely targets for transformation. We previously reported that MLL-AF4 enhanced hematopoietic engraftment and clonogenic potential in cord blood (CB)-derived CD34+ HSPCs but was not sufficient for leukemogenesis, suggesting that additional oncogenic lesions are required for MLL-AF4-mediated transformation. MLL-AF4+ pro-B-ALL display enormous levels of FLT3, and occasionally FLT3-activating mutations, thus representing a candidate cooperating event in MLL-AF4+ pro-B-ALL. We have explored whether FLT3.TKD (tyrosine kinase domain) mutation or increased expression of FLT3.WT (wild type) cooperates with MLL-AF4 to immortalize/transform CB-CD34+ HSPCs. In vivo, FLT3.TKD/FLT3.WT alone, or in combination with MLL-AF4, enhances hematopoietic repopulating function of CB-CD34+ HSPCs without impairing migration or hematopoietic differentiation. None of the animals transplanted with MLL-AF4+FLT3.TKD/WT-CD34+ HSPCs showed any sign of disease after 16 weeks. In vitro, enforced expression of FLT3.TKD/FLT3.WT conveys a transient overexpansion of MLL-AF4-expressing CD34+ HSPCs associated to higher proportion of cycling cells coupled to lower apoptotic levels, but does not augment clonogenic potential nor confer stable replating. Together, FLT3 activation does not suffice to immortalize/transform MLL-AF4-expressing CB-CD34+ HSPCs, suggesting the need of alternative (epi)-genetic cooperating oncogenic lesions.
Asunto(s)
Antígenos CD34/inmunología , Proteínas de Unión al ADN/metabolismo , Sangre Fetal/inmunología , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Proteínas Nucleares/metabolismo , Tirosina Quinasa 3 Similar a fms/metabolismo , Animales , Transformación Celular Neoplásica , Técnicas de Cocultivo , N-Metiltransferasa de Histona-Lisina , Humanos , Ligandos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Factores de Elongación TranscripcionalRESUMEN
The biological process of differentiation - from a fertilized egg to a human being - is a consecutive mechanism that leads to the establishment of tissue-specific gene expression, but also to a coordinated shut-down of all those genes that are not necessary for a given cell type. This process is accompanied by posttranslational modifications of the chromatin (DNA methylation and covalent histone modifications), also termed the "epigenetic layer". All epigenetic processes are mediated by protein complexes that either mediate specific DNA methylation patterns, or modify nucleosomal proteins in a covalent fashion (acetylation, methylation, phosphorylation and ubiquitinylation). One important player involved in epigenetics is the MLL protein which represents a histone H3 methyltransferase. The MLL gene gained much attention because of its frequent genetic rearrangements, thereby creating oncogenic MLL fusion genes that cause acute leukemia in pediatric and adult patients. This article is summarizing certain functional aspects about MLL, but is mainly emphasizing on an alternative splice event within the PHD domain. This changes the biological properties of the MLL protein, thereby influencing its ability of being either a transcriptional activator or repressor.
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Empalme Alternativo/genética , Proteína de la Leucemia Mieloide-Linfoide/genética , Activación Transcripcional/genética , Secuencia de Bases , Células Cultivadas , Exones/genética , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , ARN/biosíntesis , ARN/aislamiento & purificación , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Levaduras/genéticaRESUMEN
Rearrangements affecting the MLL gene in hematological malignancies are associated with poor prognosis. Most often they are reciprocal translocations and more rarely complex forms involving at least 3 chromosomes. We describe an unusual case with cutaneous leukemic infiltrates that waxed and waned until progression to acute myeloid leukemia, AML-M5. The leukemic cells harbored a novel apparent 3-way translocation t(6;19;11)(p22.2;p13.1;q23.3). We utilized advanced molecular cytogenetic methods including 24-color karyotyping, high-resolution array comparative genomic hybridization (aCGH) and DNA sequencing to characterize the genomic complement in the leukemic cells from aspirated bone marrow cells at AML diagnosis. Karyotyping showed 47,XY,t(6;19;11)(p22;p13;q23),+der(6)t(6;11)(p22;q23)[17]/48,sl,+8[3]/48,sl,+8,der(12)t(1;12)(q11;p13)[3]/ 48,sdl,der(Y)t(Y;1)(q12;q11),+8[7] conferring MLL-ELL fusion. Oligo-aCGH analysis confirmed gains of 6p22qter and 11q23.3qter involving the CMAHP and MLL genes, respectively. DNA sequencing disclosed an additional breakpoint at 6p24.3 (at RREB1 gene). Retrospective fluorescence in situ hybridization revealed presence of the MLL-involving rearrangement in the initial stages of disease before clear morphological signs of bone marrow involvement. The patient responded well to therapy and remains in remission>6 years from diagnosis. This apparent 3-way translocation is remarkable because of its rarity and presentation with myeloid sarcoma, and may, as more cases are characterized, further our understanding onto how such complex translocations contribute to promote leukemogenesis and respond to therapy.
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Leucemia Mieloide Aguda/genética , Oxigenasas de Función Mixta/genética , Proteína de la Leucemia Mieloide-Linfoide/genética , Factores de Elongación Transcripcional/genética , Translocación Genética , Secuencia de Bases , Células de la Médula Ósea/patología , Cromosomas Humanos Par 11 , Cromosomas Humanos Par 19 , Cromosomas Humanos Par 6 , Hibridación Genómica Comparativa , Proteínas de Unión al ADN/genética , Progresión de la Enfermedad , Fusión Génica , N-Metiltransferasa de Histona-Lisina , Humanos , Lactante , Masculino , Datos de Secuencia Molecular , Factores de Transcripción/genética , TrisomíaAsunto(s)
Cromosomas Humanos Par 11/genética , Cromosomas Humanos Par 2/genética , Leucemia Mielomonocítica Aguda/genética , Proteínas de Fusión Oncogénica/genética , Translocación Genética , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Secuencia de Bases , Células de la Médula Ósea/química , Células de la Médula Ósea/ultraestructura , Bandeo Cromosómico , Cromosomas Humanos Par 11/ultraestructura , Cromosomas Humanos Par 2/ultraestructura , Citarabina/administración & dosificación , Etopósido/administración & dosificación , Humanos , Idarrubicina/administración & dosificación , Lactante , Recién Nacido , Leucemia Mielomonocítica Aguda/tratamiento farmacológico , Leucemia Mielomonocítica Aguda/patología , Masculino , Datos de Secuencia Molecular , Tamizaje Neonatal , Proteínas de Fusión Oncogénica/análisis , Inducción de Remisión , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The chromosomal translocation t(4;11)(q21;q23) is a frequent genetic aberration of the mixed lineage leukemia (MLL) gene, predominantly associated with high-risk acute lymphoblastic leukemia (ALL) in pediatric patients. Previous studies demonstrated that mice transplanted with hematopoietic cells expressing the AF4-MLL fusion protein develop proB ALL. The AF4-MLL oncoprotein becomes activated by Taspase1-mediated hydrolysis, which subsequently leads to a heterodimer of the cleavage products AF4-MLL·N and MLL·C. This protein-protein interaction is due to the FYRN and FYRC interaction domains present in both protein fragments. Heterodimerization subsequently induces high-molecular-weight protein complex formation that is protected against SIAH1/2-mediated polyubiquitinylation. Here, we attempted to selectively block this initial heterodimerization step, aiming to prevent the oncogenic activation of the AF4-MLL multiprotein complex. The minimal interaction interface was experimentally defined first in a bacterial two-hybrid system, and then in mammalian cells by using a biosensor assay. Expression of the FYRC domain, or smaller portions thereof, resulted in the inhibition of heterodimer formation, and blocked AF4-MLL multiprotein complex formation with subsequent destruction of the AF4-MLL oncoprotein. Thus, it is in principle possible to specifically target the AF4-MLL protein. This knowledge can now be exploited to design inhibitory decoys in order to destroy the AF4-MLL oncoprotein.
Asunto(s)
Cromosomas Humanos Par 11/genética , Cromosomas Humanos Par 4/genética , Proteínas de Unión al ADN/metabolismo , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Translocación Genética , Proteínas de Unión al ADN/genética , Células HeLa , N-Metiltransferasa de Histona-Lisina , Humanos , Immunoblotting , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteínas Nucleares/genética , Proteínas de Fusión Oncogénica/antagonistas & inhibidores , Proteínas de Fusión Oncogénica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Multimerización de Proteína , Estructura Terciaria de Proteína , Factores de Elongación TranscripcionalRESUMEN
Expression of the AF4-MLL fusion protein in murine hematopoietic progenitor/stem cells results in the development of proB acute lymphoblastic leukemia. In this study, we affinity purified the AF4-MLL and AF4 protein complexes to elucidate their function. We observed that the AF4 complex consists of 11 binding partners and exhibits positive transcription elongation factor b (P-TEFb)-mediated activation of promoter-arrested RNA polymerase (pol) II in conjunction with several chromatin-modifying activities. In contrast, the AF4-MLL complex consists of at least 16 constituents including P-TEFb kinase, H3K4(me3) and H3K79(me3) histone methyltransferases (HMT), a protein arginine N-methyltransferase and a histone acetyltransferase. These findings suggest that the AF4-MLL protein disturbs the fine-tuned activation cycle of promoter-arrested RNA Pol II and causes altered histone methylation signatures. Thus, we propose that these two processes are key to trigger cellular reprogramming that leads to the onset of acute leukemia.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Epigénesis Genética , Leucemia/etiología , Proteína de la Leucemia Mieloide-Linfoide/fisiología , Proteínas Nucleares/fisiología , Proteínas de Fusión Oncogénica/fisiología , Factor B de Elongación Transcripcional Positiva/metabolismo , Cromatografía en Gel , Proteínas de Unión al ADN/aislamiento & purificación , Activación Enzimática , Histona Metiltransferasas , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Humanos , Metilación , Proteína de la Leucemia Mieloide-Linfoide/aislamiento & purificación , Proteínas Nucleares/aislamiento & purificación , Proteínas de Fusión Oncogénica/aislamiento & purificación , Fosforilación , Factores de Elongación TranscripcionalRESUMEN
Translocations involving the mixed lineage leukemia (MLL) gene, localized at 11q23, frequently occur in pediatric acute myeloid leukemia (AML). We recently reported differences in prognosis between the different translocation partners, suggesting differences in biological background. To unravel the latter, we used microarrays to generate gene expression profiles of 245 pediatric AML cases, including 53 MLL-rearranged cases. Thereby, we identified a specific gene expression signature for t(9;11)(p22;q23), and identified BRE (brain and reproductive organ expressed) to be discriminative for t(9;11)(p22;q23) (P<0.001) when compared with other MLL subtypes. Patients with high BRE expression showed a significantly better 3-year relapse-free survival (pRFS) (80±13 vs 30±10%, P=0.02) within MLL-rearranged AML cases. Moreover, multivariate analysis identified high BRE expression as an independent favorable prognostic factor within pediatric AML for RFS (HR=0.2, P=0.04). No significant differences were identified for 3-year event-free survival or for 3-year overall survival. Forced expression of BRE did not result in altered cell proliferation, apoptosis or drug sensitivity, which could explain the favorable outcome. In conclusion, overexpression of the BRE gene is predominantly found in MLL-rearranged AML with t(9;11)(p22;q23). Although further investigation for the role of BRE in leukemogenesis and outcome is warranted, high BRE expression is an independent prognostic factor for pRFS in pediatric AML.
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Reordenamiento Génico , Leucemia Mieloide Aguda/genética , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteínas del Tejido Nervioso/genética , Adolescente , Niño , Preescolar , Cromosomas Humanos Par 11 , Cromosomas Humanos Par 9 , Femenino , N-Metiltransferasa de Histona-Lisina , Humanos , Lactante , Recién Nacido , Leucemia Mieloide Aguda/mortalidad , Masculino , Translocación GenéticaRESUMEN
Acute lymphoblastic leukemia (ALL) in infants younger than 1 year is a rare but relatively homogeneous disease ( approximately 80% MLL gene rearranged, approximately 70% CD10-negative) when compared with childhood and adult ALL. Several studies in children and adults with ALL have shown that minimal residual disease (MRD) status is a strong and independent prognostic factor. We therefore evaluated the prognostic significance of MRD in infant ALL. Ninety-nine infant patients treated according to the Interfant-99 protocol were included in this study. MRD was analyzed by real-time quantitative PCR analysis of rearranged immunoglobulin genes, T-cell receptor genes and MLL genes at various time points (TP) during therapy. Higher MRD levels at the end of induction (TP2) and consolidation (TP3) were significantly associated with lower disease-free survival. Combined MRD information at TP2 and TP3 allowed recognition of three patients groups that significantly differed in outcome. All MRD-high-risk patients (MRD levels > or =10(-4) at TP3; 26% of patients) relapsed. MRD-low-risk patients (MRD level <10(-4) at both TP2 and TP3) constituted 44% of patients and showed a relapse-rate of only 13%, whereas remaining patients (MRD-medium-risk patients; 30% of patients) had a relapse rate of 31%. Comparison between the current Interfant-06 stratification at diagnosis and the here presented MRD-based stratification showed that both stratifications recognized different subgroups of patients. These data indicate that MRD diagnostics has added value for recognition of risk groups in infant ALL and that MRD diagnostics can be used for treatment intervention in infant ALL as well.
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Neoplasia Residual/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Estudios de Seguimiento , Reordenamiento Génico , Genes de Inmunoglobulinas , N-Metiltransferasa de Histona-Lisina , Humanos , Lactante , Recién Nacido , Proteína de la Leucemia Mieloide-Linfoide/genética , Neoplasia Residual/tratamiento farmacológico , Neoplasia Residual/genética , Reacción en Cadena de la Polimerasa , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Pronóstico , Receptores de Antígenos de Linfocitos T/genética , Recurrencia , Resultado del TratamientoRESUMEN
BACKGROUND: We studied 24 Hungarian patients from 23 unrelated families to identify the genetic background of the entire type 3 von Willebrand disease (VWD) population in this country. The current report focuses on the molecular characterization of a novel large deletion. RESULTS: A large partial deletion (delExon1-3) of the 5'-region of the von Willebrand factor gene (VWF) was detected in 12/48 alleles (25% of all type 3 alleles). The 5'-deletion breakpoint is located in the untranslated region between VWF and CD9, whereas the 3' breakpoint is in intron 3 of VWF. Analysis of the breakpoints showed Alu Y and Alu SP repetitive sequences at the ends of the deletion, suggesting that a recombination event caused the subsequent loss of the 35-kb fragment. DelExon1-3 was not found in any of the other screened populations. CONCLUSION: We report a large novel deletion including exons 1, 2 and 3 of VWF commonly causing type 3 VWD in the Hungarian population. This mutation, probably caused by an Alu-mediated recombination event, and subsequently distributed in Hungary by a founder effect, seems to be unique to Hungarian patients with a high allele frequency. Together, delExon1-3 and 2435delC make up 37.5% of the genetic defects in Hungarian patients with VWD type 3. This offers a rational approach to molecular testing of relevant families in Hungary.
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Eliminación de Gen , Enfermedades de von Willebrand/genética , Factor de von Willebrand/genética , Región de Flanqueo 5' , Elementos Alu , Exones , Familia , Efecto Fundador , Frecuencia de los Genes , Humanos , Hungría , Intrones , Enfermedades de von Willebrand/clasificación , Enfermedades de von Willebrand/epidemiologíaAsunto(s)
Inversión Cromosómica , Proteínas de Unión al ADN/genética , Leucemia-Linfoma de Células T del Adulto/genética , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteínas Nucleares/genética , Factores de Transcripción/genética , Adolescente , Cromosomas Humanos Par 11 , Supervivencia sin Enfermedad , Trasplante de Células Madre Hematopoyéticas , Humanos , Masculino , Neoplasias Primarias Secundarias/genética , Proteínas de Fusión Oncogénica , TransactivadoresRESUMEN
Data on secondary acute lymphoblastic leukaemia (sALL) following ALL treatment are very rare. However, the incidence might be underestimated as sALLs without a significant lineage shift might automatically be diagnosed as relapses. Examination of immunoglobulin and T-cell receptor gene rearrangements brought a new tool that can help in discrimination between relapse and sALL. We focused on the recurrences of childhood ALL to discover the real frequency of the sALL after ALL treatment. We compared clonal markers in matched presentation and recurrence samples of 366 patients treated according to the Berlin-Frankfurt-Munster (BFM)-based protocols. We found two cases of sALL and another three, where the recurrence is suspicious of being sALL rather than relapse. Our proposal for the 'secondary ALL after ALL' diagnostic criteria is as follows: (A) No clonal relationship between diagnosis and recurrence; (B) significant immunophenotypic shift--significant cytogenetic shift--gain/loss of a fusion gene. For the sALL (A) plus at least one (B) criterion should be fulfilled. With these criteria, the estimated frequency of the sALL after ALL is according to our data 0.5-1.5% of ALL recurrences on BFM-based protocols. Finally, we propose a treatment strategy for the patients with secondary disease.
Asunto(s)
Técnicas de Diagnóstico Molecular/métodos , Neoplasias Primarias Secundarias/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Antineoplásicos/efectos adversos , Preescolar , Diagnóstico Diferencial , Femenino , Reordenamiento Génico de Linfocito T , Genes de Inmunoglobulinas , Humanos , Inmunofenotipificación , Incidencia , Masculino , Neoplasias Primarias Secundarias/inducido químicamente , Leucemia-Linfoma Linfoblástico de Células Precursoras/inducido químicamente , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , RecurrenciaRESUMEN
The human mixed lineage leukemia (MLL) gene is frequently involved in genetic rearrangements with more than 55 different translocation partner genes, all associated with acute leukemia. Reciprocal chromosomal translocations generate two MLL fusion alleles, where 5'- and 3'-portions of MLL are fused to gene segments of given fusion partners. In case of t(4;11) patients, about 80% of all patients exhibit both reciprocal fusion alleles, MLL.AF4 and AF4.MLL, respectively. By contrast, 20% of all t(4;11) patients seem to encode only the MLL.AF4 fusion allele. Here, we analyzed these 'MLL.AF4(+)/AF4.MLL(-)' patients at the genomic DNA level to unravel their genetic situation. Cryptic translocations and three-way translocations were found in this group of t(4;11) patients. Reciprocal MLL fusions with novel translocation partner genes, for example NF-KB1 and RABGAP1L, were identified and actively transcribed in leukemic cells. In other patients, the reciprocal 3'-MLL gene segment was fused out-of-frame to PBX1, ELF2, DSCAML1 and FXYD6. The latter rearrangements caused haploinsufficiency of genes that are normally expressed in hematopoietic cells. Finally, patients were identified that encode only solitary 3'-MLL gene segments on the reciprocal allele. Based on these data, we propose that all t(4;11) patients exhibit reciprocal MLL alleles, but due to the individual recombination events, provide different pathological disease mechanisms.