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1.
J Hum Genet ; 65(2): 165-174, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31772335

RESUMO

Somatic mutational mosaicism is a common feature of monogenic genetic disorders, particularly in diseases such as retinoblastoma, with high rates of de novo mutations. The detection and quantification of mosaicism is particularly relevant in these diseases, since it has important implications for genetic counseling, patient management, and probably also on disease onset and progression. In order to assess the rate of somatic mosaicism (high- and low-level mosaicism) in sporadic retinoblastoma patients, we analyzed a cohort of 153 patients with sporadic retinoblastoma using ultra deep next-generation sequencing. High-level mosaicism was detected in 14 out of 100 (14%) bilateral patients and in 11 out of 29 (38%) unilateral patients in whom conventional Sanger sequencing identified a pathogenic mutation in blood DNA. In addition, low-level mosaicism was detected in 3 out of 16 (19%) unilateral patients in whom conventional screening was negative in blood DNA. Our results also reveal that mosaicism was associated to delayed retinoblastoma onset particularly in unilateral patients. Finally we compared the level of mosaicism in different tissues to identify the best DNA source to identify mosaicism in retinoblastoma patients. In light of these results we recommended analyzing the mosaic status in all retinoblastoma patients using accurate techniques such as next-generation sequencing, even in those cases in which conventional Sanger sequencing identified a pathogenic mutation in blood DNA. Our results suggest that a significant proportion of those cases are truly mosaics that could have been overlooked. This information should be taking into consideration in the management and genetic counseling of retinoblastoma patients and families.

2.
Am J Hematol ; 92(9): E534-E541, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28612357

RESUMO

The International Prognostic Scoring System and its revised form (IPSS-R) are the most widely used indices for prognostic assessment of patients with myelodysplastic syndromes (MDS), but can only partially account for the observed variation in patient outcomes. This study aimed to evaluate the relative contribution of patient condition and mutational status in peripheral blood when added to the IPSS-R, for estimating overall survival and the risk of leukemic transformation in patients with MDS. A prospective cohort (2006-2015) of 200 consecutive patients with MDS were included in the study series and categorized according to the IPSS-R. Patients were further stratified according to patient condition (assessed using the multidimensional Lee index for older adults) and genetic mutations (peripheral blood samples screened using next-generation sequencing). The change in likelihood-ratio was tested in Cox models after adding individual covariates. The addition of the Lee index to the IPSS-R significantly improved prediction of overall survival [hazard ratio (HR) 3.02, 95% confidence interval (CI) 1.96-4.66, P < 0.001), and mutational analysis significantly improved prediction of leukemic evolution (HR 2.64, 1.56-4.46, P < 0.001). Non-leukemic death was strongly linked to patient condition (HR 2.71, 1.72-4.25, P < 0.001), but not to IPSS-R score (P = 0.35) or mutational status (P = 0.75). Adjustment for exposure to disease-modifying therapy, evaluated as a time-dependent covariate, had no effect on the proposed model's predictive ability. In conclusion, patient condition, assessed by the multidimensional Lee index and patient mutational status can improve the prediction of clinical outcomes of patients with MDS already stratified by IPSS-R.


Assuntos
Síndromes Mielodisplásicas/sangue , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/mortalidade , Taxa de Sobrevida , Intervalo Livre de Doença , Feminino , Seguimentos , Humanos , Masculino , Estudos Prospectivos , Espanha/epidemiologia
3.
Br J Cancer ; 117(2): 256-265, 2017 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-28557976

RESUMO

BACKGROUND: In B-cell precursor acute lymphoblastic leukaemia (B-ALL), the identification of additional genetic alterations associated with poor prognosis is still of importance. We determined the frequency and prognostic impact of somatic mutations in children and adult cases with B-ALL treated with Spanish PETHEMA and SEHOP protocols. METHODS: Mutational status of hotspot regions of TP53, JAK2, PAX5, LEF1, CRLF2 and IL7R genes was determined by next-generation deep sequencing in 340 B-ALL patients (211 children and 129 adults). The associations between mutation status and clinicopathological features at the time of diagnosis, treatment outcome and survival were assessed. Univariate and multivariate survival analyses were performed to identify independent prognostic factors associated with overall survival (OS), event-free survival (EFS) and relapse rate (RR). RESULTS: A mutation rate of 12.4% was identified. The frequency of adult mutations was higher (20.2% vs 7.6%, P=0.001). TP53 was the most frequently mutated gene (4.1%), followed by JAK2 (3.8%), CRLF2 (2.9%), PAX5 (2.4%), LEF1 (0.6%) and IL7R (0.3%). All mutations were observed in B-ALL without ETV6-RUNX1 (P=0.047) or BCR-ABL1 fusions (P<0.0001). In children, TP53mut was associated with lower OS (5-year OS: 50% vs 86%, P=0.002) and EFS rates (5-year EFS: 50% vs 78.3%, P=0.009) and higher RR (5-year RR: 33.3% vs 18.6% P=0.037), and was independently associated with higher RR (hazard ratio (HR)=4.5; P=0.04). In adults, TP53mut was associated with a lower OS (5-year OS: 0% vs 43.3%, P=0.019) and a higher RR (5-year RR: 100% vs 61.4%, P=0.029), whereas JAK2mut was associated with a lower EFS (5-year EFS: 0% vs 30.6%, P=0.035) and a higher RR (5-year RR: 100% vs 60.4%, P=0.002). TP53mut was an independent risk factor for shorter OS (HR=2.3; P=0.035) and, together with JAK2mut, also were independent markers of poor prognosis for RR (TP53mut: HR=5.9; P=0.027 and JAK2mut: HR=5.6; P=0.036). CONCLUSIONS: TP53mut and JAK2mut are potential biomarkers associated with poor prognosis in B-ALL patients.


Assuntos
Biomarcadores Tumorais/genética , Janus Quinase 2/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Proteína Supressora de Tumor p53/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Linfócitos B/patologia , Criança , Pré-Escolar , Intervalo Livre de Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Mutação , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Prognóstico , Receptores de Citocinas/biossíntese , Resultado do Tratamento
4.
J Hematol Oncol ; 10(1): 83, 2017 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-28399885

RESUMO

BACKGROUND: Chronic lymphocytic leukemia (CLL) is a highly genetically heterogeneous disease. Although CLL has been traditionally considered as a mature B cell leukemia, few independent studies have shown that the genetic alterations may appear in CD34+ hematopoietic progenitors. However, the presence of both chromosomal aberrations and gene mutations in CD34+ cells from the same patients has not been explored. METHODS: Amplicon-based deep next-generation sequencing (NGS) studies were carried out in magnetically activated-cell-sorting separated CD19+ mature B lymphocytes and CD34+ hematopoietic progenitors (n = 56) to study the mutational status of TP53, NOTCH1, SF3B1, FBXW7, MYD88, and XPO1 genes. In addition, ultra-deep NGS was performed in a subset of seven patients to determine the presence of mutations in flow-sorted CD34+CD19- early hematopoietic progenitors. Fluorescence in situ hybridization (FISH) studies were performed in the CD34+ cells from nine patients of the cohort to examine the presence of cytogenetic abnormalities. RESULTS: NGS studies revealed a total of 28 mutations in 24 CLL patients. Interestingly, 15 of them also showed the same mutations in their corresponding whole population of CD34+ progenitors. The majority of NOTCH1 (7/9) and XPO1 (4/4) mutations presented a similar mutational burden in both cell fractions; by contrast, mutations of TP53 (2/2), FBXW7 (2/2), and SF3B1 (3/4) showed lower mutational allele frequencies, or even none, in the CD34+ cells compared with the CD19+ population. Ultra-deep NGS confirmed the presence of FBXW7, MYD88, NOTCH1, and XPO1 mutations in the subpopulation of CD34+CD19- early hematopoietic progenitors (6/7). Furthermore, FISH studies showed the presence of 11q and 13q deletions (2/2 and 3/5, respectively) in CD34+ progenitors but the absence of IGH cytogenetic alterations (0/2) in the CD34+ cells. Combining all the results from NGS and FISH, a model of the appearance and expansion of genetic alterations in CLL was derived, suggesting that most of the genetic events appear on the hematopoietic progenitors, although these mutations could induce the beginning of tumoral cell expansion at different stage of B cell differentiation. CONCLUSIONS: Our study showed the presence of both gene mutations and chromosomal abnormalities in early hematopoietic progenitor cells from CLL patients.


Assuntos
Aberrações Cromossômicas , Células-Tronco Hematopoéticas/patologia , Leucemia Linfocítica Crônica de Células B/patologia , Mutação , Antígenos CD19 , Antígenos CD34 , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Hibridização in Situ Fluorescente/métodos , Leucemia Linfocítica Crônica de Células B/genética
5.
Leuk Res ; 56: 82-87, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28222336

RESUMO

Our study aimed to analyze the presence of mutations in SF3B1 and other spliceosome-related genes in myelodysplastic syndromes with ringed sideroblasts (MDS-RS) by combining conventional Sanger and next-generation sequencing (NGS) methods, and to determine the feasibility of this approach in a clinical setting. 122 bone marrow samples from MDS-RS patients were studied. Initially, exons 14 and 15 of the SF3B1 gene were analyzed by Sanger sequencing. Secondly, they were studied by NGS covering besides SF3B1, SRSF2, U2AF1 and ZRSR2 genes. An 86% of all patients showed mutations in the SF3B1 gene. Six of them, which were not identifiable by conventional sequencing in the first diagnostic step, were revealed by NGS. In addition, 19.5% of cases showed mutations in other splicing genes: SRSF2, U2AF1, and ZRSR2. Furthermore, 8.7% of patients had two mutations in SF3B1, SF3B1 and SRSF2, and SF3B1 and U2AF1, while 5.7% showed no mutations in the four spliceosome-related genes analyzed. The combined use of conventional Sanger and NGS allows the identification of mutations in spliceosome-related genes in almost all MDS patients with RS. This two-step approach is affordable and could be useful as a complementary technique in cases with an unclear diagnosis.


Assuntos
Anemia Sideroblástica/genética , Síndromes Mielodisplásicas/diagnóstico , Síndromes Mielodisplásicas/genética , Spliceossomos/genética , Anemia Sideroblástica/diagnóstico , Medula Óssea , Humanos , Métodos , Mutação , Fosfoproteínas/genética , Fatores de Processamento de RNA/genética , Análise de Sequência de DNA , Fatores de Processamento de Serina-Arginina/genética , Fator de Processamento U2AF/genética
6.
Eur J Haematol ; 98(2): 142-148, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27717146

RESUMO

The presence of chromosomal gains other than trisomy 12 in chronic lymphocytic leukaemia (CLL) is unusual. However, some patients may show gains on several chromosomes simultaneously suggesting a hyperdiploid karyotype. OBJECTIVE: The objective of this study was to analyse by FISH the frequency and prognostic impact of hyperdiploidy in CLL. METHOD: A review of 1359 consecutive cases diagnosed with CLL referred for FISH analysis to a unique institution was carried out. Hyperdiploidy was considered when a gain of at least three of the five FISH probes used was observed. RESULTS: Seven cases (0.51%) with hyperdiploidy were found, confirming that it is a rare event in this disease. Although most patients presented with early Binet stages at diagnosis, six of seven (86%) shortly progressed. The median of time to the first therapy (TTFT) and overall survival (OS) for the patients with hyperdiploidy were short (1.4 months and 20 months, respectively). Moreover, comparing them with a control group of patients (non-hyperdiploid) with completed follow-up data, TTFT and OS of the patients with hyperdiploidy were significantly shorter than the control group. CONCLUSION: The presence of hyperdiploidy is uncommon and probably associated with poor prognostic markers in CLL.


Assuntos
Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/mortalidade , Poliploidia , Idoso , Biomarcadores , Estudos de Casos e Controles , Aberrações Cromossômicas , Feminino , Humanos , Imunofenotipagem , Hibridização in Situ Fluorescente , Estimativa de Kaplan-Meier , Leucemia Linfocítica Crônica de Células B/diagnóstico , Leucemia Linfocítica Crônica de Células B/terapia , Masculino , Pessoa de Meia-Idade , Mutação , Estadiamento de Neoplasias , Prognóstico , Estudos Retrospectivos , Tempo para o Tratamento , Resultado do Tratamento , Proteína Supressora de Tumor p53/genética
8.
PLoS One ; 11(10): e0164370, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27741277

RESUMO

To explore novel genetic abnormalities occurring in myelodysplastic syndromes (MDS) through an integrative study combining array-based comparative genomic hybridization (aCGH) and next-generation sequencing (NGS) in a series of MDS and MDS/myeloproliferative neoplasms (MPN) patients. 301 patients diagnosed with MDS (n = 240) or MDS/MPN (n = 61) were studied at the time of diagnosis. A genome-wide analysis of DNA copy number abnormalities was performed. In addition, a mutational analysis of DNMT3A, TET2, RUNX1, TP53 and BCOR genes was performed by NGS in selected cases. 285 abnormalities were identified in 71 patients (23.6%). Three high-risk MDS cases (1.2%) displayed chromothripsis involving exclusively chromosome 13 and affecting some cancer genes: FLT3, BRCA2 and RB1. All three cases carried TP53 mutations as revealed by NGS. Moreover, in the whole series, the integrative analysis of aCGH and NGS enabled the identification of cryptic recurrent deletions in 2p23.3 (DNMT3A; n = 2.8%), 4q24 (TET2; n = 10%) 17p13 (TP53; n = 8.5%), 21q22 (RUNX1; n = 7%), and Xp11.4 (BCOR; n = 2.8%), while mutations in the non-deleted allele where found only in DNMT3A (n = 1), TET2 (n = 3), and TP53 (n = 4). These cryptic abnormalities were detected mainly in patients with normal (45%) or non-informative (15%) karyotype by conventional cytogenetics, except for those with TP53 deletion and mutation (15%), which had a complex karyotype. In addition to well-known copy number defects, the presence of chromothripsis involving chromosome 13 was a novel recurrent change in high-risk MDS patients. Array CGH analysis revealed the presence of cryptic abnormalities in genomic regions where MDS-related genes, such as TET2, DNMT3A, RUNX1 and BCOR, are located.


Assuntos
Aberrações Cromossômicas , Síndromes Mielodisplásicas/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Cromossomos Humanos Par 13 , Hibridização Genômica Comparativa , Subunidade alfa 2 de Fator de Ligação ao Core/genética , DNA/química , DNA/isolamento & purificação , DNA/metabolismo , DNA (Citosina-5-)-Metiltransferases/genética , Variações do Número de Cópias de DNA , Análise Mutacional de DNA , Proteínas de Ligação a DNA/genética , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Cariótipo , Masculino , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/diagnóstico , Síndromes Mielodisplásicas/patologia , Proteínas Proto-Oncogênicas/genética , Recidiva , Risco , Proteína Supressora de Tumor p53/genética , Adulto Jovem
9.
Oncotarget ; 7(21): 30492-503, 2016 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-27127180

RESUMO

The biological and molecular events that underlie bone marrow fibrosis in patients with myelodysplastic syndromes are poorly understood, and its prognostic role in the era of the Revised International Prognostic Scoring System (IPSS-R) is not yet fully determined. We have evaluated the clinical and biological events that underlie bone marrow fibrotic changes, as well as its prognostic role, in a well-characterized prospective patient cohort (n=77) of primary MDS patients. The degree of marrow fibrosis was linked to parameters of erythropoietic failure, marrow cellularity, p53 protein accumulation, WT1 gene expression, and serum levels of CXCL9 and CXCL10, but not to other covariates including the IPSS-R score. The presence of bone marrow fibrosis grade 2 or higher was associated with the presence of mutations in cohesin complex genes (31.5% vs. 5.4%, p=0.006). By contrast, mutations in CALR, JAK2, PDGFRA, PDGFRB,and TP53 were very rare. Survival analysis showed that marrow fibrosis grade 2 or higher was a relevant significant predictor for of overall survival, and independent of age, performance status, and IPSS-R score in multivariate analysis.


Assuntos
Medula Óssea/metabolismo , Análise Mutacional de DNA/métodos , Mutação , Síndromes Mielodisplásicas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Medula Óssea/patologia , Quimiocina CXCL10/sangue , Quimiocina CXCL9/sangue , Feminino , Fibrose , Humanos , Masculino , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/metabolismo , Síndromes Mielodisplásicas/patologia , Prognóstico , Estudos Prospectivos , Análise de Sobrevida , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas WT1/genética , Proteínas WT1/metabolismo
10.
Leuk Res ; 46: 30-6, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27111859

RESUMO

Deletion 13q (13q-) is the most common cytogenetic aberration in chronic lymphocytic leukemia (CLL) and is associated with the most favorable prognosis as the sole cytogenetic abnormality. However, it is heterogeneous whereby CLL patients with higher percentages of 13q- cells (13q-H) have a more aggressive clinical course and a distinct gene expression profile. The microRNA (miRNA) expression profile of CLL gives additional biological and prognostic information, but its expression in 13q- CLL has not been examined in detail. The miRNA expression of clonal B cell lymphocytes (CD19+ cells) of 38 CLL patients and normal B cells of six healthy donors was analyzed. CLL patients with higher percentages of 13q- cells (≥80%) showed a different level of miRNA expression from patients with lower percentages (<80%). Interestingly, miR-143 was downregulated and miR-155 was overexpressed in 13q-H. This deregulation affected important validated target genes involved in apoptosis (BCL2, MDM2, TP53INP1) and proliferation (KRAS, PI3K-AKT signaling), that could lead to decreased apoptosis and increased proliferation in 13q-H patients. This study provides new evidence about the heterogeneity of the 13q deletion in CLL patients, showing that miRNA regulation could be involved in several significant pathways deregulated in CLL patients with a high number of losses in 13q.


Assuntos
Transtornos Cromossômicos/genética , Perfilação da Expressão Gênica , Leucemia Linfocítica Crônica de Células B/genética , MicroRNAs/genética , Apoptose/genética , Linfócitos B/metabolismo , Linfócitos B/patologia , Proliferação de Células/genética , Aberrações Cromossômicas , Deleção Cromossômica , Cromossomos Humanos Par 13/genética , Variação Genética , Humanos , Leucemia Linfocítica Crônica de Células B/mortalidade , Prognóstico
11.
PLoS One ; 11(2): e0148972, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26872047

RESUMO

UNLABELLED: Identifying additional genetic alterations associated with poor prognosis in acute lymphoblastic leukemia (ALL) is still a challenge. AIMS: To characterize the presence of additional DNA copy number alterations (CNAs) in children and adults with ALL by whole-genome oligonucleotide array (aCGH) analysis, and to identify their associations with clinical features and outcome. Array-CGH was carried out in 265 newly diagnosed ALLs (142 children and 123 adults). The NimbleGen CGH 12x135K array (Roche) was used to analyze genetic gains and losses. CNAs were analyzed with GISTIC and aCGHweb software. Clinical and biological variables were analyzed. Three of the patients showed chromothripsis (cth6, cth14q and cth15q). CNAs were associated with age, phenotype, genetic subtype and overall survival (OS). In the whole cohort of children, the losses on 14q32.33 (p = 0.019) and 15q13.2 (p = 0.04) were related to shorter OS. In the group of children without good- or poor-risk cytogenetics, the gain on 1p36.11 was a prognostic marker independently associated with shorter OS. In adults, the gains on 19q13.2 (p = 0.001) and Xp21.1 (p = 0.029), and the loss of 17p (p = 0.014) were independent markers of poor prognosis with respect to OS. In summary, CNAs are frequent in ALL and are associated with clinical parameters and survival. Genome-wide DNA copy number analysis allows the identification of genetic markers that predict clinical outcome, suggesting that detection of these genetic lesions will be useful in the management of patients newly diagnosed with ALL.


Assuntos
Biomarcadores Tumorais/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Hibridização Genômica Comparativa , Variações do Número de Cópias de DNA , Feminino , Dosagem de Genes , Frequência do Gene , Marcadores Genéticos , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Humanos , Lactente , Recém-Nascido , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Leucemia-Linfoma Linfoblástico de Células Precursoras/mortalidade , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Modelos de Riscos Proporcionais , Resultado do Tratamento , Adulto Jovem
12.
Br J Haematol ; 172(3): 428-38, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26567765

RESUMO

The introduction of Rituximab has improved the outcome and survival rates of Burkitt lymphoma (BL). However, early relapse and refractoriness are current limitations of BL treatment and new biological factors affecting the outcome of these patients have not been explored. This study aimed to identify the presence of genomic changes that could predict the response to new therapies in BL. Forty adolescent and adult BL patients treated with the Dose-Intensive Chemotherapy Including Rituximab (Burkimab) protocol (Spanish Programme for the Study and Treatment of Haematological Malignancies; PETHEMA) were analysed using array-based comparative genomic hybridization (CGH). In addition, the presence of TP53, TCF3 (E2A), ID3 and GNA13 mutations was assessed by next-generation sequencing (NGS). Ninety-seven per cent of the patients harboured genomic imbalances. Losses on 11q, 13q, 15q or 17p were associated with a poor response to Burkimab therapy (P = 0·038), shorter progression-free survival (PFS; P = 0·007) and overall survival (OS; P = 0·009). The integrative analysis of array-CGH and NGS showed that 26·3% (5/19) and 36·8% (7/19) of patients carried alterations in the TP53 and TCF3 genes, respectively. TP53 alterations were associated with shorter PFS (P = 0·011) while TCF3 alterations were associated with shorter OS (P = 0·032). Genetic studies could be used for risk stratification of BL patients treated with the Burkimab protocol.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linfoma de Burkitt/tratamento farmacológico , Linfoma de Burkitt/genética , Aberrações Cromossômicas , Adolescente , Adulto , Idoso , Hibridização Genômica Comparativa/métodos , Feminino , Genoma , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Prognóstico , Rituximab/administração & dosagem , Resultado do Tratamento , Adulto Jovem
13.
PLoS One ; 10(11): e0143073, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26630574

RESUMO

To analyze the impact of the 11q deleted (11q-) cells in CLL patients on the time to first therapy (TFT) and overall survival (OS), 2,493 patients with CLL were studied. 242 patients (9.7%) had 11q-. Fluorescence in situ hybridization (FISH) studies showed a threshold of 40% of deleted cells to be optimal for showing that clinical differences in terms of TFT and OS within 11q- CLLs. In patients with ≥40% of losses in 11q (11q-H) (74%), the median TFT was 19 months compared with 44 months in CLL patients with <40% del(11q) (11q-L) (P<0.0001). In the multivariate analysis, only the presence of 11q-L, mutated IGHV status, early Binet stage and absence of extended lymphadenopathy were associated with longer TFT. Patients with 11q-H had an OS of 90 months, while in the 11q-L group the OS was not reached (P = 0.008). The absence of splenomegaly (P = 0.02), low LDH (P = 0.018) or ß2M (P = 0.006), and the presence of 11q-L (P = 0.003) were associated with a longer OS. In addition, to detect the presence of mutations in the ATM, TP53, NOTCH1, SF3B1, MYD88, FBXW7, XPO1 and BIRC3 genes, a select cohort of CLL patients with losses in 11q was sequenced by next-generation sequencing of amplicons. Eighty % of CLLs with 11q- showed mutations and fewer patients with low frequencies of 11q- had mutations among genes examined (50% vs 94.1%, P = 0.023). In summary, CLL patients with <40% of 11q- had a long TFT and OS that could be associated with the presence of fewer mutated genes.


Assuntos
Deleção Cromossômica , Cromossomos Humanos Par 11 , Leucemia Linfocítica Crônica de Células B/diagnóstico , Leucemia Linfocítica Crônica de Células B/genética , Mutação , Proteínas de Neoplasias/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Hibridização in Situ Fluorescente , Cariótipo , Leucemia Linfocítica Crônica de Células B/mortalidade , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/imunologia , Prognóstico , Estudos Retrospectivos , Análise de Sobrevida
14.
Eur J Haematol ; 93(5): 422-8, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24813417

RESUMO

To assess the presence of genetic imbalances in patients with myeloproliferative neoplasms (MPNs), 38 patients with chronic eosinophilia were studied by array comparative genomic hybridization (aCGH): seven had chronic myelogenous leukaemia (CML), BCR-ABL1 positive, nine patients had myeloproliferative neoplasia Ph- (MPN-Ph-), three had a myeloid neoplasm associated with a PDGFRA rearrangement, and the remaining two cases were Lymphoproliferative T neoplasms associated with eosinophilia. In addition, 17 patients had a secondary eosinophilia and were used as controls. Eosinophilic enrichment was carried out in all cases. Genomic imbalances were found in 76% of all MPN patients. Losses on 20q were the most frequent genetic abnormality in MPNs (32%), affected the three types of MPN studied. This study also found losses at 11q13.3 in 26% of patients with MPN-Ph- and in 19p13.11 in two of the three patients with an MPN associated with a PDGFRA rearrangement. In addition, 29% of patients with CML had losses on 8q24. In summary, aCGH revealed clonality in eosinophils in most MPNs, suggesting that it could be a useful technique for defining clonality in these diseases. The presence of genetic losses in new regions could provide new insights into the knowledge of these MPN associated with eosinophilia.


Assuntos
Aberrações Cromossômicas , Eosinofilia/genética , Eosinófilos/metabolismo , Genoma , Neoplasias Hematológicas/genética , Transtornos Mieloproliferativos/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Cromossomos Humanos Par 11/química , Cromossomos Humanos Par 19/química , Cromossomos Humanos Par 20/química , Cromossomos Humanos Par 8/química , Doença Crônica , Células Clonais , Hibridização Genômica Comparativa , Eosinofilia/diagnóstico , Eosinofilia/patologia , Eosinófilos/patologia , Feminino , Proteínas de Fusão bcr-abl/genética , Instabilidade Genômica , Neoplasias Hematológicas/diagnóstico , Neoplasias Hematológicas/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Transtornos Mieloproliferativos/diagnóstico , Transtornos Mieloproliferativos/patologia , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética
15.
Methods Mol Biol ; 973: 121-45, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23412787

RESUMO

BAC array-CGH is a powerful method to identify DNA copy number changes (gains, amplifications and deletions) on a genome-wide scale, and to map these changes to genomic sequence. It is based on the analysis of genomic DNA isolated from test and reference cell populations, the differential labelling with fluorescent dyes and the co-hybridization with a genomic array. BAC array-CGH has proven to be a specific, sensitive, and reliable technique, with considerable advantages compared to other methods used for the analysis of DNA copy number changes. The application of genome scanning technologies and the recent advances in bioinformatics tools that enable us to perform a robust and highly sensitive analysis of array-CGH data, useful not only for genome scanning of tumor cells but also in the identification of novel cancer related genes, oncogenes and suppressor genes. Cytogenetic analysis provides essential information for diagnosis and prognosis in patients with hematologic malignancies such as lymphomas. However, the chromosomal interpretation in non-Hodgkin lymphoma (NHL) is sometimes inconclusive. Copy number aberrations identified by BAC array-CGH analyses could be a complementary methodology to chromosomal analysis. In NHL the genomic imbalances might have a prognostic rather than a diagnostic value. In fact, the diagnosis of NHL is based on pathological and molecular cytogenetics data. Furthermore genetic variations and their association with specific types of lymphoma development, and elucidation of the variable genetic pathways leading to lymphoma development, are important directions for future cancer research. Array-CGH, along with FISH and PCR, will be used for routine diagnostic purposes in near future.


Assuntos
Hibridização Genômica Comparativa/métodos , Linfoma Difuso de Grandes Células B/genética , Adulto , Idoso , Aberrações Cromossômicas , Cromossomos Artificiais Bacterianos/genética , DNA/genética , DNA/isolamento & purificação , Feminino , Dosagem de Genes , Humanos , Linfoma Difuso de Grandes Células B/diagnóstico , Masculino , Pessoa de Meia-Idade , Análise de Sobrevida , Adulto Jovem
16.
J Neurooncol ; 110(1): 69-77, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22864683

RESUMO

Even though much progress has been made towards understanding the molecular nature of glioma, the survival rates of patients affected by this tumour have not changed significantly over recent years. Better knowledge of this malignancy is still needed in order to predict its outcome and improve patient treatment. VAV1 is an GDP/GTP exchange factor for Rho/Rac proteins with oncogenic potential that is involved in the regulation of cytoskeletal dynamics and cell migration. Here we report its overexpression in 59 patients diagnosed with high-grade glioma, and the associated upregulation of a number of genes coding for proteins also involved in cell invasion- and migration-related processes. Unexpectedly, immunohistochemical experiments revealed that VAV1 is not expressed in glioma cells. Instead, VAV1 is found in non-tumoural astrocyte-like cells that are located either peritumouraly or perivascularly. We propose that the expression of VAV1 is linked to synergistic signalling cross-talk between cancer and infiltrating cells. Interestingly, we show that the pattern of expression of VAV1 could have a role in the neoplastic process in glioblastoma tumours.


Assuntos
Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Proteínas Proto-Oncogênicas c-vav/biossíntese , Microambiente Tumoral/fisiologia , Adulto , Idoso , Neoplasias Encefálicas/mortalidade , Neoplasias Encefálicas/patologia , Análise por Conglomerados , Hibridização Genômica Comparativa , Intervalo Livre de Doença , Feminino , Glioblastoma/mortalidade , Glioblastoma/patologia , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Proto-Oncogênicas c-vav/análise , Receptor Cross-Talk/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa
17.
Pharmacogenet Genomics ; 22(5): 381-8, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22388797

RESUMO

BACKGROUND: Chronic myeloid leukemia (CML) is a malignant clonal disorder of the hematopoietic system caused by the expression of the BCR/ABL fusion oncogene. It is well known that CML cells are genetically unstable. However, the mechanisms by which these cells acquire genetic alterations are poorly understood. Imatinib mesylate is the standard therapy for newly diagnosed CML patients. Imatinib mesylate targets the oncogenic kinase activity of BCR-ABL. OBJECTIVE: To study the gene expression profile of bone marrow hematopoietic cells in the same patients with CML before and 1 month after imatinib therapy. METHODS: Samples from patients with CML were analyzed using Affymetrix GeneChip Expression Arrays. RESULTS: A total of 594 differentially expressed genes, most of which (393 genes) were downregulated, as a result of imatinib therapy were observed. CONCLUSION: The blockade of oncoprotein Bcr-Abl by imatinib could cause a decrease in the expression of key DNA repair genes and substantially modify the expression profile of the bone marrow cells in the first days of therapy.


Assuntos
Proteínas de Fusão bcr-abl/metabolismo , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Piperazinas/uso terapêutico , Pirimidinas/uso terapêutico , Adolescente , Adulto , Benzamidas , Células da Medula Óssea/metabolismo , Proteínas de Ciclo Celular/genética , Dano ao DNA/genética , Reparo do DNA/genética , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Proteínas de Fusão bcr-abl/genética , Perfilação da Expressão Gênica , Humanos , Mesilato de Imatinib , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Masculino , Pessoa de Meia-Idade
18.
Ann Surg Oncol ; 19(7): 2367-79, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22395973

RESUMO

BACKGROUND: Glioblastoma multiforme (GBM) is the most common primary tumor of the central nervous system in adults. Patients with GBM have few treatment options, and their disease is invariably fatal. Molecularly targeted agents offer the potential to improve patient treatment; however, the use of these will require a fuller understanding of the genetic changes in this complex tumor. METHODS: We analyzed a series of 32 patients with GBM with array comparative genomic hybridization in combination with gene expression analysis. We focused on the recurrent breakpoints found by spectral karyotyping (SKY). RESULTS: By SKY we identified 23 recurrent breakpoints of the 202 translocations found in GBM cases. Gains and losses were identified in chromosomal regions close to the breakpoints by array comparative genomic hybridization. We evaluated the genes located in the regions involved in the breakpoints in depth. A list of 406 genes that showed a level of expression significantly different between patients and control subjects was selected to determine their effect on survival. Genes CACNA2D3, PPP2R2B, SIK, MAST3, PROM1, and PPP6C were significantly associated with shorter survival (median 200 days vs. 450 days, P≤0.03). CONCLUSIONS: We present a list of genes located in regions of breakpoints that could be grounds for future studies to determine whether they are crucial in the pathogenesis of this type of tumor, and we provide a list of six genes associated with the clinical outcome of patients with GBM.


Assuntos
Biomarcadores Tumorais/genética , Aberrações Cromossômicas , Hibridização Genômica Comparativa , Perfilação da Expressão Gênica , Glioblastoma/genética , Glioblastoma/patologia , Cariotipagem Espectral , Adulto , Idoso , Estudos de Casos e Controles , Feminino , Glioblastoma/mortalidade , Humanos , Cariotipagem , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Análise de Sequência com Séries de Oligonucleotídeos , Prognóstico , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Risco , Taxa de Sobrevida
19.
PLoS One ; 6(9): e24939, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21957467

RESUMO

Splenic marginal zone lymphomas (SMZL) are an uncommon type of B-cell non-Hodgkin's lymphoma (NHL-B) in which no specific chromosomal translocations have been described. In contrast, the most frequent cytogenetic abnormality is the loss of the long arm of chromosome 7 (7q). Previous reports have located this loss in the 7q32 region. In order to better characterize the genomic imbalances in SMZL, molecular studies were carried out in 73 patients with SMZL. To gain insight into the mapping at 7q a tiling array was also used. The results confirmed the loss of 7q as the most frequent change. In addition, several abnormalities, including 4q22.1, 1q21.3-q22, 6q25.3, 20q13.33, 3q28, 2q23.3-q24.1 and 17p13, were also present. A loss of 7q22.1 at 99925039-101348479 bp was observed in half of the cases. The region of 7q22.1 has not previously been characterised in SMZL. Our results confirmed the presence of a new region of loss on chromosome 7 in these NHL.


Assuntos
Cromossomos Humanos Par 7/genética , Linfoma de Zona Marginal Tipo Células B/genética , Neoplasias Esplênicas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Deleção Cromossômica , Cromossomos Artificiais Bacterianos/genética , Hibridização Genômica Comparativa , Feminino , Humanos , Hibridização in Situ Fluorescente , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Reprodutibilidade dos Testes
20.
J Am Acad Dermatol ; 64(3): 573-8, 578.e1, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20691500

RESUMO

BACKGROUND: Onychomatricoma (OM) is a rare benign tumor of the nail matrix in which genome-wide analyses have never been performed. It is clinically characterized by an increased transversal curvature of the nail plate, a longitudinal yellowish discoloration, and splinter hemorrhages. Once the nail plate has been removed, fingerlike fibrokeratogenous projections appear through the proximal nailfold. Histologically, it is a fibroepithelial tumor with well-established features. In this article, a comprehensive review of this tumor is made. OBJECTIVE: We performed a genome-wide analysis of an OM, in an attempt to shed light on the mechanisms underlying its development. METHODS: We report a 36-year-old man who was given a diagnosis of OM involving his fourth right toenail. To investigate molecular genetic alterations, we carried out two approaches, fluorescent in situ hybridization and array-based comparative genomic hybridization, in our patient. RESULTS: Genomic testing of OM showed 34 genomic alterations, with most of the genomic losses being on chromosome 11. Array-based comparative genomic hybridization showed the deletion of 11p15.4, which harbors STIM-1, 11q14.2 (RP-11 292E14), which harbors the Cathepsin C gene, 11q14 (RP11-281F10-RP11-265F24), and 11q21 (RP11-203F8 and RP11 183A22). LIMITATIONS: This work is an initial approach to a genome-wide study of this tumor. Further studies (with more cases) must be conducted to pinpoint possible candidate genes for the development of OM. CONCLUSIONS: Array-based comparative genomic hybridization showed important genomic alterations in OM, especially genomic losses. Most genomic losses affected the chromosome 11 in our patient. The STIM-1 and the Cathepsin C genes might play a role in the development of OM.


Assuntos
Doenças da Unha/patologia , Unhas/patologia , Neoplasias Cutâneas/patologia , Adulto , Cromossomos Humanos Par 11/genética , Hibridização Genômica Comparativa , Estudo de Associação Genômica Ampla , Humanos , Hibridização in Situ Fluorescente , Masculino , Doenças da Unha/genética , Neoplasias Cutâneas/genética
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