RESUMEN
Histone methyltransferases (HMTs) are important epigenetic regulators of gene transcription and are disrupted at the genomic level in a spectrum of human tumours including haematological malignancies. Using high-resolution single nucleotide polymorphism (SNP) arrays, we identified recurrent deletions of the SETD2 locus in 3% (8/261) of chronic lymphocytic leukaemia (CLL) patients. Further validation in two independent cohorts showed that SETD2 deletions were associated with loss of TP53, genomic complexity and chromothripsis. With next-generation sequencing we detected mutations of SETD2 in an additional 3.8% of patients (23/602). In most cases, SETD2 deletions or mutations were often observed as a clonal event and always as a mono-allelic lesion, leading to reduced mRNA expression in SETD2-disrupted cases. Patients with SETD2 abnormalities and wild-type TP53 and ATM from five clinical trials employing chemotherapy or chemo-immunotherapy had reduced progression-free and overall survival compared with cases wild type for all three genes. Consistent with its postulated role as a tumour suppressor, our data highlight SETD2 aberration as a recurrent, early loss-of-function event in CLL pathobiology linked to aggressive disease.
Asunto(s)
Genómica , N-Metiltransferasa de Histona-Lisina/genética , Leucemia Linfocítica Crónica de Células B/genética , Mutación , Proteínas de la Ataxia Telangiectasia Mutada/genética , Supervivencia sin Enfermedad , Femenino , Genes Supresores de Tumor , Histona Metiltransferasas , Humanos , Leucemia Linfocítica Crónica de Células B/enzimología , Leucemia Linfocítica Crónica de Células B/mortalidad , Masculino , Pronóstico , Tasa de Supervivencia , Proteína p53 Supresora de Tumor/genéticaRESUMEN
The biological features of IGHV-M chronic lymphocytic leukemia responsible for disease progression are still poorly understood. We undertook a longitudinal study close to diagnosis, pre-treatment and post relapse in 13 patients presenting with cMBL or Stage A disease and good-risk biomarkers (IGHV-M genes, no del(17p) or del(11q) and low CD38 expression) who nevertheless developed progressive disease, of whom 10 have required therapy. Using cytogenetics, fluorescence in situ hybridisation, genome-wide DNA methylation and copy number analysis together with whole exome, targeted deep- and Sanger sequencing at diagnosis, we identified mutations in established chronic lymphocytic leukemia driver genes in nine patients (69%), non-coding mutations (PAX5 enhancer region) in three patients and genomic complexity in two patients. Branching evolutionary trajectories predominated (n=9/13), revealing intra-tumoural epi- and genetic heterogeneity and sub-clonal competition before therapy. Of the patients subsequently requiring treatment, two had sub-clonal TP53 mutations that would not be detected by standard methodologies, three qualified for the very-low-risk category defined by integrated mutational and cytogenetic analysis and yet had established or putative driver mutations and one patient developed progressive, therapy-refractory disease associated with the emergence of an IGHV-U clone. These data suggest that extended genomic and immunogenetic screening may have clinical utility in patients with apparent good-risk disease.
Asunto(s)
Exoma/genética , Dosificación de Gen , Cadenas Pesadas de Inmunoglobulina/genética , Leucemia Linfocítica Crónica de Células B/genética , Mutación , Adolescente , Adulto , Anciano , Células Clonales , Análisis Citogenético , Progresión de la Enfermedad , Heterogeneidad Genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Estudios Longitudinales , Persona de Mediana Edad , Riesgo , Adulto JovenRESUMEN
Genome-wide array approaches and sequencing analyses are powerful tools for identifying genetic aberrations in cancers, including leukemias and lymphomas. However, the clinical and biological significance of such aberrations and their subclonal distribution are poorly understood. Here, we present the first genome-wide array based study of pre-treatment and relapse samples from patients with B-cell chronic lymphocytic leukemia (B-CLL) that uses the computational statistical tool OncoSNP. We show that quantification of the proportion of copy number alterations (CNAs) and copy neutral loss of heterozygosity regions (cnLOHs) in each sample is feasible. Furthermore, we (i) reveal complex changes in the subclonal architecture of paired samples at relapse compared with pre-treatment, (ii) provide evidence supporting an association between increased genomic complexity and poor clinical outcome (iii) report previously undefined, recurrent CNA/cnLOH regions that expand or newly occur at relapse and therefore might harbor candidate driver genes of relapse and/or chemotherapy resistance. Our findings are likely to impact on future therapeutic strategies aimed towards selecting effective and individually tailored targeted therapies.
Asunto(s)
Biomarcadores de Tumor/genética , Aberraciones Cromosómicas , Células Clonales/patología , Leucemia Linfocítica Crónica de Células B/genética , Recurrencia Local de Neoplasia/genética , Polimorfismo de Nucleótido Simple/genética , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Dosificación de Gen , Genoma Humano , Genómica , Humanos , Leucemia Linfocítica Crónica de Células B/terapia , Pérdida de Heterocigocidad , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia/terapia , Análisis de Secuencia por Matrices de Oligonucleótidos , PronósticoAsunto(s)
Proteínas de Ciclo Celular/genética , Deleción Cromosómica , Cromosomas Humanos Par 11 , Proteínas de Unión al ADN/genética , Leucemia Linfocítica Crónica de Células B/genética , Mutación , Proteínas Serina-Treonina Quinasas/genética , Homeostasis del Telómero , Proteínas Supresoras de Tumor/genética , Proteínas de la Ataxia Telangiectasia Mutada , Roturas del ADN de Doble Cadena , Humanos , Leucemia Linfocítica Crónica de Células B/patología , Estadificación de NeoplasiasRESUMEN
Historically, genes targeted by recurrent chromosomal deletions have been identified within the smallest genomic region shared in all patients, the minimally deleted region (MDR). However, deletions this small do not occur in all patients and are a simplification of the impact larger heterogeneous deletions have during carcinogenesis. We use the example of 13q14 deletions in chronic lymphocytic leukemia to show that genes outside MDRs are associated with disease progression. Genomic profiling of 224 patients identified 205 copy number alterations on chromosome 13 in 132 cases. Deletions including DLEU2 were heterogeneous (845 Kb-96.2 Mb) and identified two breakpoint cluster regions within short interspersed nuclear elements proximal to DLEU2 and within long interspersed nuclear elements/L1 repeats distal to GUCY1B2. After defining a deletion class on the basis of size and location, we show that (a) at diagnosis, larger deletions (class II) were associated with a significantly increased risk of disease progression (odds ratio=12.3; P=0.005), (b) in progressive patients, class II deletions were enriched (P=0.02) and (c) this association was independent of IgVH mutational status, ZAP70 expression and ATM/TP53 deletion. Deletion of a 1 Mb gene cluster (48.2-49.2 Mb), including SETDB2, PHF11 and RCBTB1, was significantly associated (P<0.01) with disease progression. Here, we show that the deletion of genes outside MDRs can influence clinical outcome.
Asunto(s)
Leucemia Linfocítica Crónica de Células B/genética , Deleción Cromosómica , Trastornos de los Cromosomas/genética , Cromosomas Humanos Par 13/genética , Progresión de la Enfermedad , Dosificación de Gen , Humanos , Familia de Multigenes , Polimorfismo de Nucleótido Simple , PronósticoAsunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Proteínas de Ciclo Celular/genética , Proteínas de Unión al ADN/genética , Etopósido/administración & dosificación , Genes p53 , Imidazoles/administración & dosificación , Leucemia Linfocítica Crónica de Células B/genética , Mutación , Piperazinas/administración & dosificación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Supresoras de Tumor/genética , Proteínas de la Ataxia Telangiectasia Mutada , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Humanos , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológicoAsunto(s)
Reordenamiento Génico de Cadena Pesada de Linfocito B , Genes de Inmunoglobulinas , Cadenas Pesadas de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Leucemia Linfocítica Crónica de Células B/epidemiología , Factores Sexuales , Linfocitos B/metabolismo , Estudios de Cohortes , ADN de Neoplasias/genética , Bases de Datos Genéticas , Femenino , Humanos , Leucemia Linfocítica Crónica de Células B/genética , Masculino , ARN Mensajero/genética , ARN Neoplásico/genéticaRESUMEN
BACKGROUND: The clinical course of chronic lymphocytic leukaemia (CLL) is variable. ZAP-70 expression is believed to provide prognostic information. The flow cytometric detection of ZAP-70 is difficult because it is an intracellular antigen with weak expression in CLL. Consensus has not been reached as to the best method for measurement. METHODS: We analyzed 72 CLL patient samples for ZAP-70 expression and IgVH mutational status. Sensitivity and specificity of ZAP-70 expression against IgVH mutational status were assessed for two clones (2F3.2 and 1E7.2) and for four methods of analysis: percentage positivity (PP), comparing test to isotype control, ratio of geometric means of test and isotype control, and percentage and ratiometric methods comparing test and T/NK cell populations. The effects of anticoagulant, collection times, and time to analysis were also evaluated. RESULTS: Sensitivity and specificity were 85 and 88%, respectively, for Upstate PP; 70 and 88% for Caltag PP; 89 and 91% for Upstate ratio; 89 and 88% for Caltag ratio. Intraobserver variability was smaller when ZAP-70 expression was assessed using a ratiometric approach rather than the percentage method. By 48 h, we observed an average decrease of 13% in the Caltag ratio in the heparin preserved samples compared to an increase of 3% in those collected in EDTA. Within the first 24-h period, a greater percent variability was observed in those samples collected into EDTA compared with heparin. CONCLUSION: Our data support a rapid method for ZAP-70 measurement using commercially available fixation/permeabilization reagents, a conjugated antibody, and a ratiometric method of analysis that minimizes subjective interpretation of the results. This is a method of ZAP-70 assessment that could be included in a routine diagnostic CLL panel; however, the choice of anticoagulant and time of analysis after collection are critical factors in accurate assessment of ZAP-70 expression.
Asunto(s)
Anticuerpos/inmunología , Anticoagulantes/farmacología , Citometría de Flujo/métodos , Leucemia Linfocítica Crónica de Células B/diagnóstico , Proteína Tirosina Quinasa ZAP-70/análisis , Reacciones Antígeno-Anticuerpo , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/química , Biomarcadores de Tumor/inmunología , Progresión de la Enfermedad , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/metabolismo , Persona de Mediana Edad , Mutación , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Coloración y Etiquetado , Factores de Tiempo , Proteína Tirosina Quinasa ZAP-70/efectos de los fármacos , Proteína Tirosina Quinasa ZAP-70/inmunologíaAsunto(s)
Macroglobulinemia de Waldenström/terapia , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Monoclonales de Origen Murino , Cladribina/uso terapéutico , Quimioterapia Combinada , Inhibidores Enzimáticos/uso terapéutico , Humanos , Factores Inmunológicos/uso terapéutico , Inmunosupresores/uso terapéutico , Intercambio Plasmático , Pronóstico , Rituximab , Trasplante de Células Madre/métodos , Talidomida/uso terapéutico , Vidarabina/análogos & derivados , Vidarabina/uso terapéutico , Macroglobulinemia de Waldenström/tratamiento farmacológico , Macroglobulinemia de Waldenström/inmunologíaRESUMEN
Molecular cloning of immunoglobulin heavy chain (IGH) translocation breakpoints identifies genes of biological importance in the development of normal and malignant B cells. Long-distance inverse PCR (LDI-PCR) was first applied to amplification of IGH gene translocations targeted to the joining (IGHJ) regions. We report here successful amplification of the breakpoint of IGH translocations targeted to switch (IGHS) regions by LDI-PCR. To detect IGHS translocations, Southern blot assays using 5' and 3' switch probes were performed. Illegitimate Smu rearrangements were amplified from the 5' end (5'Smu LDI-PCR) from the alternative derivative chromosome, and those of Sgamma or Salpha were amplified from the 3' end (3'Sgamma or 3'alpha LDI-PCR) from the derivative chromosome 14. Using a combination of these methods, we have succeeded in amplifying IGHS translocation breakpoints involving FGFR3/MMSET on 4p16, BCL6 on 3q27, MYC on 8q24, IRTA1 on 1q21 and PAX5 on 9p13 as well as BCL11A on 2p13 and CCND3 on 6p21. The combination of LDI-PCR for IGHJ and IGHS allows rapid molecular cloning of almost all IGH gene translocation breakpoints.
Asunto(s)
Rotura Cromosómica/genética , Reordenamiento Génico , Genes de Cambio/genética , Cadenas Pesadas de Inmunoglobulina/genética , Región de Cambio de la Inmunoglobulina/genética , Linfoma/genética , Translocación Genética , Secuencia de Bases , Southern Blotting , Proteínas Portadoras/genética , Cromosomas Humanos Par 14 , Clonación Molecular , Proteínas de Unión al ADN/genética , Humanos , Cadenas mu de Inmunoglobulina/genética , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Factor de Transcripción PAX5 , Reacción en Cadena de la Polimerasa , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-bcl-6 , Proteínas Proto-Oncogénicas c-myc/genética , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos , Receptores de Factores de Crecimiento de Fibroblastos/genética , Proteínas Represoras , Factores de Transcripción/genética , Células Tumorales CultivadasAsunto(s)
Genes de Inmunoglobulinas/genética , Leucemia Linfocítica Crónica de Células B/diagnóstico , Regiones Determinantes de Complementariedad/genética , ADN de Neoplasias/análisis , Humanos , Región Variable de Inmunoglobulina/genética , Leucemia Linfocítica Crónica de Células B/genética , Técnicas de Diagnóstico Molecular , Eliminación de SecuenciaRESUMEN
The P2X7 receptor, a plasma membrane ATP-gated ion channel that plays a role in lymphocyte apoptosis, has been suggested as an important contributory factor to the pathogenesis of chronic lymphocytic leukaemia (CLL). The P2X7 gene resides on chromosome 12 and is polymorphic in the population at large (1513A/C) with the A and C alleles encoding fully active and nonfunctional proteins, respectively. We have evaluated the significance of this polymorphism by genotyping 144 patients with CLL and 348 healthy controls using a tetraprimer ARMS assay. We found no significant difference in allele frequency between patients and controls. Although patients with the C allele (A/C heterozygotes or C/C homozygotes) had a marginally shorter survival than those who were homozygous for the A allele, this difference was not significant for either the patient group considered as a whole or for IgVH-mutated/unmutated subsets. Finally, no association was found between trisomy 12 and P2X7 genotype. We conclude that the influence, if any, of P2X7 genotype on susceptibility to CLL or clinical outcome is small.
Asunto(s)
Cromosomas Humanos Par 12 , Leucemia Linfocítica Crónica de Células B/genética , Polimorfismo Genético , Receptores Purinérgicos P2/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Aberraciones Cromosómicas , Cartilla de ADN , Genotipo , Humanos , Leucemia Linfocítica Crónica de Células B/epidemiología , Leucemia Linfocítica Crónica de Células B/mortalidad , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Receptores Purinérgicos P2X7 , Valores de Referencia , Análisis de SupervivenciaRESUMEN
B-cell-specific plasma-membrane proteins are potential targets for either small molecule or antibody-based therapies. We have sought to annotate proteins expressed at the cell surface membrane in patients with chronic lymphocytic leukemia (CLL) using plasma-membrane-based proteomic analysis to identify previously uncharacterized and potentially B-cell-specific proteins. Proteins from plasma-membrane fractions were separated on one-dimensional gels and trypsinized fractions subjected to high-throughput MALDI-TOF mass spectrometry. Using this method, many known B-cell surface antigens were detected, but also known proteins not previously described in this disease or in this cellular compartment, including cell surface receptors, membrane-associated enzymes and secreted proteins, and completely unknown proteins. To validate the method, we show that BLK, a B-cell-specific kinase, is located in the CLL-plasma-membrane fraction. We also describe two novel proteins (MIG2B and B-cell novel protein #1, BCNP1), which are expressed preferentially in B cells. MIG2B is in a highly conserved and defined gene family containing two plasma-membrane-binding ezrin/radixin/moesin domains and a pleckstrin homology domain; the Caenorhabditis elegans homolog (UNC-112) is a membrane-associated protein that colocalizes with integrin at cell-matrix adhesion complexes. BCNP1 is a completely unknown protein with three predicted transmembrane domains, with three alternatively spliced final exons. Proteomic analysis may thus define new potential therapeutic targets.
Asunto(s)
Linfocitos B/química , Leucemia Linfocítica Crónica de Células B/patología , Proteínas de la Membrana/aislamiento & purificación , Proteínas de Neoplasias/aislamiento & purificación , Proteómica , Proteínas Reguladoras de la Apoptosis , Linfocitos B/patología , Secuencia de Bases , Western Blotting , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Sistemas de Lectura Abierta , Isoformas de Proteínas , Estructura Terciaria de Proteína , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Waldenström's macroglobulinaemia (WM) is most usefully defined as a distinct chronic lymphoproliferative disorder with characteristic marrow morphology and phenotype; although nodal morphology if available will reveal a lymphoplasmacytoid lymphoma, the presence of a significant IgM paraprotein defines the clinical features of the disease. The clonal cell is a B-cell expressing IgM, CD19, and CD20 but not IgD, CD5, CD10 or CD23 and has somatic hypermutation of immunoglobulin heavy chain variable regions consistent with a post-germinal centre origin. Treatment of WM has been dependent on alkylating agents with or without coriticosteroids for many years, supplemented by the use of therapeutic plasmapheresis in the initial stages for patients at risk from the clinical consequences of hyperviscosity. This approach to treatment results in response rates of approximately 60% with a median survival of about 60 months. There is increasing evidence to show that the purine analogues fludarabine and cladribine which are active in the treatment of patients who are resistant to alkylating agents such as chlorambucil may be able to achieve higher response rates when used as initial therapy. A prospective trial is being undertaken to compare fludarabine and chlorambucil as initial treatment; because of the effect of subsequent active treatment on patients who do not respond to the first treatment choice, the long-term outcome may be similar for both groups. Recent advances in therapy include the use of therapeutic monoclonal antibodies such as rituximab and the use of autologous or allogeneic transplant procedures for selected patients.
Asunto(s)
Macroglobulinemia de Waldenström/diagnóstico , Macroglobulinemia de Waldenström/tratamiento farmacológico , Antineoplásicos/administración & dosificación , Antineoplásicos/uso terapéutico , Diagnóstico Diferencial , Humanos , Investigación , Resultado del TratamientoRESUMEN
B cell chronic lymphocytic leukemia (CLL) lacks a consistent genetic abnormality. However, immunoglobulin V(H) gene segment mutation analysis has provided insights into the pathogenesis of these diseases and allowed the development of powerful prognostic markers. Immunoglobulin gene chromosomal translocations are rare in CLL and involve a distinct subset of genes including BCL3, BCL11A and CCND2. BCL2 translocations in CLL appear to arise via a different mechanism from comparable translocations seen in B cell non-Hodgkin lymphoma.
Asunto(s)
Genes de Inmunoglobulinas , Leucemia Linfocítica Crónica de Células B/genética , Animales , Secuencia de Bases , Reordenamiento Génico de Linfocito B , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Ratones , Datos de Secuencia Molecular , Mutación , Alineación de Secuencia , Hipermutación Somática de Inmunoglobulina , Translocación GenéticaRESUMEN
The t(2;14)(p13;q32.3) involving the BCL11A and IGH genes is a rare but recurrent chromosomal aberration in B-cell malignancies. Hitherto, juxtaposition of BCL11A and IGH has only been described in B-cell chronic lymphocytic leukemia (B-CLL) and immunocytoma. As subgroups of B-CLL can be distinguished by the pattern of somatic mutation of immunoglobulin variable (V) genes we investigated four lymphomas with IGH/BCL11A involvement for IGH hypermutation. Clonal V(H) gene rearrangements were amplified; in all four cases, sequencing of the amplificates revealed the rearranged V(H) genes to lack somatic mutations. These results suggest that t(2;14)(p13;q32.3) is associated with a subset of B-CLL/immunocytoma characterized by non-mutated IG genes deriving from pre-germinal center B cells. As the translocations in both informative cases are targeted to the switch regions of the IGG2 gene, which is mainly used in T cell-independent immune responses, these translocations presumably occurred in activated B cells in the course of T cell-independent immune responses outside the germinal center.