RESUMO
Undifferentiated pleomorphic sarcoma of bone (UPSb) is a rare tumor often difficult to differentiate from fibrosarcoma of bone (FSb), diagnostically. We applied array comparative genomic hybridization (array CGH) to screen for genes with potential importance in the tumor and compared the results with alterations seen in FSb. Twenty-two fresh frozen tissue specimens from 20 patients (18 primary tumors and 4 local recurrences) with UPSb were studied. DNA was isolated and hybridized onto Agilent 244K CGH oligoarrays. The hybridization data were analyzed using Agilent DNA Analytics Software. The number of changes ranged from 2 to 168 (average = 66). Losses were most frequently seen at 8p, 9p, 10, 13q, and 18q, and gains at 4q, 5p, 6p, 7p, 8q, 12p, 14q, 17q, 19p, 20q, 22q, and X. Homozygous deletions of CDKN2A, RB1, TP53, and ING1 were seen in 8/20, 7/20, 3/20, and 2/20 cases, respectively. Hypermethylation of both p16(INK4a) and p14(ARF) was found in two cases with loss at CDKN2A. Inactivation either of CDKN2A, RB1, or TP53 was detected in 18/20 cases. One case showed high level gains of CDK4 and MDM2. Frequent gains were seen at MYC, PDGFRA, KIT, and KDR. Immunohistochemical positivity of KIT, PDGFRA, KDR, and PDGFRB was found in 8/14, 5/14, 4/14, and 4/14 cases, respectively. The regions most significantly discriminating between UPSb and FSb included RB1 and MYC. No homozygous deletions of RB1 were found in FSb. In conclusion, our analysis showed the disruption of G1/S checkpoint regulation to be crucial for the oncogenesis of UPSb.
Assuntos
Neoplasias Ósseas/genética , Hibridização Genômica Comparativa/métodos , Fase G1/genética , Osteossarcoma/genética , Fase S/genética , Adulto , Idoso , Neoplasias Ósseas/patologia , Criança , Variações do Número de Cópias de DNA , DNA de Neoplasias/análise , DNA de Neoplasias/genética , Feminino , Fibrossarcoma/genética , Fibrossarcoma/patologia , Genes do Retinoblastoma/genética , Genes p16 , Genes p53/genética , Humanos , Masculino , Pessoa de Meia-Idade , Osteossarcoma/patologia , Proteínas Proto-Oncogênicas c-kit/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Sobrevida , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Adulto JovemRESUMO
Very little is known about the genetics of fibrosarcoma (FS) of bone. We applied array comparative genomic hybridization (CGH) to identify genes and genomic regions with potential role in the pathogenesis of this tumor. Seventeen patients with FS of bone were included in the study. Array CGH analysis was carried out in 13 fresh frozen tissue specimens from 11 of these patients (nine primary tumors and four local recurrences). DNA was extracted and hybridizations were performed on Agilent 244K CGH oligoarrays. The data were analyzed using Agilent DNA Analytics Software. The number of changes per patient ranged from 0 to 132 (average = 43). Losses were most commonly detected at 6q, 8p, 9p, 10, 13q, and 20p. CDKN2A was homozygously deleted in 7/11 patients. Hypermethylation of both p16(INK4a) and p14(ARF) was found in 1/14 patients. An internal deletion of STARD13 was found in a region with common losses at 13q13.1. The most frequent gains were seen at 1q, 4q, 5p, 8q, 12p, 15q, 16q, 17q, 20q, 22q, and Xp. Single recurrent high level amplification was detected at 4q12, including KIT, PDGFRA, and KDR. No activating mutations were found in any of them. Immunohistochemistry revealed expression of PDGFRA and/or PDGFRB in 12/17 samples. Moreover, small regions of gains pinpointed genes of particular interest, such as IGF1R at 15q26.3 and CHD1L at 1q21.1. In conclusion, our analysis provided novel findings that can be exploited when searching for markers for diagnosis and prognosis, and targets of therapy in this tumor type.
Assuntos
Neoplasias Ósseas/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , Fibrossarcoma/genética , Amplificação de Genes , Deleção de Genes , Proteínas Proto-Oncogênicas c-kit/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Adulto , Idoso , Neoplasias Ósseas/patologia , Mapeamento Cromossômico , Cromossomos Humanos Par 13 , Cromossomos Humanos X , Hibridização Genômica Comparativa , DNA de Neoplasias/genética , DNA de Neoplasias/isolamento & purificação , Feminino , Fibrossarcoma/patologia , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
PURPOSE: Histologic grade is currently the best predictor of clinical course in chondrosarcoma patients. Grading suffers, however, from extensive interobserver variability and new objective markers are needed. Hence, we have investigated DNA copy numbers in chondrosarcomas with the purpose of identifying markers useful for prognosis and subclassification. EXPERIMENTAL DESIGN: The overall pattern of genomic imbalances was assessed in a series of 67 chondrosarcomas using array comparative genomic hybridization. Statistical analyses were applied to evaluate the significance of alterations detected in subgroups based on clinical data, morphology, grade, tumor size, and karyotypic features. Also, the global gene expression profiles were obtained in a subset of the tumors. RESULTS: Genomic imbalances, in most tumors affecting large regions of the genome, were found in 90% of the cases. Several apparently distinctive aberrations affecting conventional central and peripheral tumors, respectively, were identified. Although rare, recurrent amplifications were found at 8q24.21-q24.22 and 11q22.1-q22.3, and homozygous deletions of loci previously implicated in chondrosarcoma development affected the CDKN2A, EXT1, and EXT2 genes. The chromosomal imbalances in two distinct groups of predominantly near-haploid and near-triploid tumors, respectively, support the notion that polyploidization of an initially hyperhaploid/hypodiploid cell population is a common mechanism of chondrosarcoma progression. Increasing patient age as well as tumor grade were associated with adverse outcome, but no copy number imbalance affected metastasis development or tumor-associated death. CONCLUSION: Despite similarities in the overall genomic patterns, the present findings suggest that some regions are specifically altered in conventional central and peripheral tumors, respectively.
Assuntos
Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Condrossarcoma/genética , Condrossarcoma/patologia , Cromossomos Humanos Par 11/genética , Cromossomos Humanos Par 8/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Feminino , Dosagem de Genes/genética , Perfilação da Expressão Gênica , Humanos , Masculino , Metaloproteases/genética , Metaloproteases/metabolismo , Pessoa de Meia-Idade , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , Deleção de Sequência/genéticaRESUMO
BACKGROUND: Ewing sarcoma family of tumors (ESFT), characterized by t(11;22)(q24;q12), is one of the most common tumors of bone in children and young adults. In addition to EWS/FLI1 gene fusion, copy number changes are known to be significant for the underlying neoplastic development of ESFT and for patient outcome. Our genome-wide high-resolution analysis aspired to pinpoint genomic regions of highest interest and possible target genes in these areas. METHODS: Array comparative genomic hybridization (CGH) and expression arrays were used to screen for copy number alterations and expression changes in ESFT patient samples. A total of 31 ESFT samples were analyzed by aCGH and in 16 patients DNA and RNA level data, created by expression arrays, was integrated. Time of the follow-up of these patients was 5-192 months. Clinical outcome was statistically evaluated by Kaplan-Meier/Logrank methods and RT-PCR was applied on 42 patient samples to study the gene of the highest interest. RESULTS: Copy number changes were detected in 87% of the cases. The most recurrent copy number changes were gains at 1q, 2, 8, and 12, and losses at 9p and 16q. Cumulative event free survival (ESFT) and overall survival (OS) were significantly better (P < 0.05) for primary tumors with three or less copy number changes than for tumors with higher number of copy number aberrations. In three samples copy number imbalances were detected in chromosomes 11 and 22 affecting the FLI1 and EWSR1 loci, suggesting that an unbalanced t(11;22) and subsequent duplication of the derivative chromosome harboring fusion gene is a common event in ESFT. Further, amplifications on chromosomes 20 and 22 seen in one patient sample suggest a novel translocation type between EWSR1 and an unidentified fusion partner at 20q. In total 20 novel ESFT associated putative oncogenes and tumor suppressor genes were found in the integration analysis of array CGH and expression data. Quantitative RT-PCR to study the expression levels of the most interesting gene, HDGF, confirmed that its expression was higher than in control samples. However, no association between HDGF expression and patient survival was observed. CONCLUSION: We conclude that array CGH and integration analysis proved to be effective methods to identify chromosome regions and novel target genes involved in the tumorigenesis of ESFT.
Assuntos
Neoplasias Ósseas/genética , Proteínas de Ligação a Calmodulina/genética , Regulação Neoplásica da Expressão Gênica/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteínas de Ligação a RNA/genética , Sarcoma de Ewing/genética , Adolescente , Criança , Cromossomos Humanos Par 11/genética , Cromossomos Humanos Par 20/genética , Cromossomos Humanos Par 21/genética , Cromossomos Humanos Par 22/genética , Intervalo Livre de Doença , Humanos , Estimativa de Kaplan-Meier , Proteína EWS de Ligação a RNA , Proteínas Repressoras/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sarcoma de Ewing/secundário , Taxa de Sobrevida , Adulto JovemRESUMO
Chondrosarcoma is a malignant bone tumor that is often resistant to chemotherapy and radiotherapy. We applied high resolution oligonucleotide array comparative genomic hybridization to 46 tumor specimens from 44 patients with chondrosarcoma and identified several genes with potential importance for the development of chondrosarcoma. Several homozygous deletions were detected. The tumor suppressor genes CDKN2A and MTAP were each homozygously deleted in four of the cases, and the RB1 gene was homozygously deleted in one. Two homozygous deletions of MTAP did not affect CDKN2A. Deletions were also found to affect genes of the cadherin family, including CDH4 and CDH7, each of which had a targeted homozygous loss in one case, and CDH19, which had a targeted homozygous loss in two cases. Loss of the EXT1 and EXT2 genes was uncommon; EXT1 was homozygously deleted in none and EXT2 in two of the cases, and large heterozygous losses including EXT1 and/or EXT2 were seen in three cases. Targeted gains and amplifications affected the MYC, E2F3, CDK6, PDGFRA, KIT, and PDGFD genes in one case each. The data indicate that chondrosarcomas develop through a combination of genomic imbalances that often affect the RB1 signaling pathway. The inactivation of cadherin genes may also be critical in the pathogenesis of the tumor.
Assuntos
Neoplasias Ósseas/genética , Caderinas/genética , Condrossarcoma/genética , Deleção de Genes , Neoplasias Ósseas/sangue , Condrossarcoma/sangue , Aberrações Cromossômicas , Hibridização Genômica Comparativa , DNA de Neoplasias/sangue , DNA de Neoplasias/genética , Feminino , Amplificação de Genes/genética , Humanos , Masculino , Purina-Núcleosídeo Fosforilase/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
BACKGROUND: Xenografts have been shown to provide a suitable source of tumor tissue for molecular analysis in the absence of primary tumor material. We utilized ES xenograft series for integrated microarray analyses to identify novel biomarkers. METHOD: Microarray technology (array comparative genomic hybridization (aCGH) and micro RNA arrays) was used to screen and identify copy number changes and differentially expressed miRNAs of 34 and 14 passages, respectively. Incubated cells used for xenografting (Passage 0) were considered to represent the primary tumor. Four important differentially expressed miRNAs (miR-31, miR-31*, miR-145, miR-106) were selected for further validation by real time polymerase chain reaction (RT-PCR). Integrated analysis of aCGH and miRNA data was performed on 14 xenograft passages by bioinformatic methods. RESULTS: The most frequent losses and gains of DNA copy number were detected at 9p21.3, 16q and at 8, 15, 17q21.32-qter, 1q21.1-qter, respectively. The presence of these alterations was consistent in all tumor passages. aCGH profiles of xenograft passages of each series resembled their corresponding primary tumors (passage 0). MiR-21, miR-31, miR-31*, miR-106b, miR-145, miR-150*, miR-371-5p, miR-557 and miR-598 showed recurrently altered expression. These miRNAS were predicted to regulate many ES-associated genes, such as genes of the IGF1 pathway, EWSR1, FLI1 and their fusion gene (EWS-FLI1). Twenty differentially expressed miRNAs were pinpointed in regions carrying altered copy numbers. CONCLUSION: In the present study, ES xenografts were successfully applied for integrated microarray analyses. Our findings showed expression changes of miRNAs that were predicted to regulate many ES associated genes, such as IGF1 pathway genes, FLI1, EWSR1, and the EWS-FLI1 fusion genes.
Assuntos
Dosagem de Genes , MicroRNAs/genética , Sarcoma de Ewing/genética , Animais , Análise por Conglomerados , Variações do Número de Cópias de DNA , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Nus , Reprodutibilidade dos Testes , Sarcoma de Ewing/metabolismo , Transplante HeterólogoRESUMO
BACKGROUND AND OBJECTIVES: Translocation-associated gene fusions are well recognized in acute myeloid leukemia. Other molecular genetic changes are less well known. The novel cDNA technology has opened the avenue to large-scale gene expression analysis. Our aim was to perform cDNA microarray analysis of acute myeloid leukemia (AML). DESIGN AND METHODS: We performed cDNA microarray analysis using the Clontech hematology filter (containing 406 genes) on 15 patients to study gene expression profiling in AML. As reference, we used whole bone marrow from 5 healthy donors. RESULTS: Our results revealed 50 differentially expressed genes in at least 3 out of 15 patients. Twenty-two genes were upregulated (ratio > or =4), whereas 28 genes were downregulated (ratio < or =0.25). All but one of the 13 genes tested by real-time polymerase chain reaction (PCR) showed the same expression profiles. Among the overexpressed genes, several were those earlier associated with chromosomal translocations and gene fusions. These genes were FGFR1, MYC, NPM1, DEC, and BCL2. The expression of two upregulated genes, HOXA4 and CSF1R, was significantly higher in patients with a white blood cell count higher than 30 x 10(9)/L cells. In patients whose white blood cell count was higher than 100 x 10(9)/L cells, both CLC and GRN were significantly underexpressed, whereas HOXA4 and DAPK1 were overexpressed. FGFR1 and CAMLG were more frequently significantly overexpressed in patients with CD56 immunophenoytpe. INTERPRETATION AND CONCLUSIONS: Clinical and prognostic significance of differential gene expression should be studied with a larger series of patients by using other techniques, such as quantitative real-time PCR.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Cromossômicas não Histona , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular , Leucemia Mieloide/genética , Proteínas de Neoplasias/genética , Oncogenes/fisiologia , Doença Aguda , Adulto , Idoso , Proteínas Reguladoras de Apoptose , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Proteínas Quinases Dependentes de Cálcio-Calmodulina/fisiologia , Proteínas de Transporte/genética , Proteínas de Transporte/fisiologia , Estudos de Casos e Controles , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Proteínas Quinases Associadas com Morte Celular , Feminino , Genes bcl-2/genética , Genes bcl-2/fisiologia , Genes myc/genética , Genes myc/fisiologia , Proteínas de Homeodomínio , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/fisiologia , Proteínas Nucleares/genética , Proteínas Nucleares/fisiologia , Nucleofosmina , Proteínas Oncogênicas/genética , Proteínas de Ligação a Poli-ADP-Ribose , Progranulinas , RNA/metabolismo , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/fisiologia , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos , Receptor de Fator Estimulador de Colônias de Macrófagos/genética , Receptor de Fator Estimulador de Colônias de Macrófagos/fisiologia , Receptores de Fatores de Crescimento de Fibroblastos/genética , Receptores de Fatores de Crescimento de Fibroblastos/fisiologia , Fatores de Transcrição , Translocação Genética , Proteínas Virais/genética , Proteínas Virais/fisiologiaRESUMO
BACKGROUND: As the treatment results of childhood ALL have improved, avoidance of late effects has become increasingly important. Identification of favorable prognostic factors helps to achieve this goal. PROCEDURE: We studied the prognostic value of TEL-AML1 fusion and a risk factor composed of age, white blood cell count (WBC), lymphomatous features, and hemoglobin level (Hb). We also compared outcome between two cohorts; cohort 1 (n=100) diagnosed 1975-1981, and cohort 2 (n=102) 1989-1991. Both cohorts were retrospectively divided in two groups: very-low-risk (WBC <10 x 10(9)/L, age 2 to <10 years, no lymphomatous features, Hb <90 g/L), and non-low-risk (=the remainder). We performed fluorescent in situ hybridization (FISH) of TEL-AML1 fusion of the marrow samples obtained at diagnosis. RESULTS: The median follow-up is 20 years in cohort 1, and 8 years in cohort 2. In both cohorts, the very-low-risk category comprised one-fourth of the children. TEL-AML1 fusion was more frequent in the very-low-risk (35%) than in the non-low-risk group (17%) (P=0.03). The 8-year event-free survival (EFS) of children with the fusion was better than of those without, 74 vs. 54% (P=0.040). The 8-year EFS in the very-low-risk group was 76% in cohort 1, and 79% in cohort 2 (n.s.). In the non-low-risk groups, EFS was 39 vs. 64% (P=0.02), respectively. CONCLUSIONS: Our data support the reported association of TEL-AML1 fusion with a favorable outcome although the risk group had a greater impact. These very-long-term follow-up data also indicate that children with very-low-risk ALL (slow disease) had a favorable outcome already in the late 1970s, and may be over treated with the contemporary ALL protocols.
Assuntos
Antineoplásicos/administração & dosagem , Proteínas de Fusão Oncogênica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Adolescente , Antineoplásicos/uso terapêutico , Criança , Pré-Escolar , Estudos de Coortes , Subunidade alfa 2 de Fator de Ligação ao Core , Feminino , Seguimentos , Humanos , Hibridização in Situ Fluorescente , Lactente , Masculino , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Prognóstico , Indução de Remissão , Estudos Retrospectivos , Fatores de Risco , Taxa de Sobrevida , Resultado do TratamentoRESUMO
The expression of apoptosis-related genes BCL2, BAX, BCL2L1, BCL2A1, MCL1, DAPK1 and MYC was studied by quantitative real-time polymerase chain reaction on total RNA samples from patients with acute lymphoblastic leukaemia (ALL, n = 16), acute myeloid leukaemia (AML, n = 27), chronic myeloid leukaemia (CML, n = 12), mantle cell lymphoma (MCL, n = 19) and chronic lymphoid leukaemia (CLL, n = 32). BCL2, BAX, BCL2A1, MCL1, DAPK1 and MYC were overexpressed in all patient groups. BCL2L1 was underexpressed in CLL and CML, but not in AML, ALL and MCL. MCL1 levels were significantly higher in CD13 and CD33-positive ALL, and in CD56-positive AML samples. BCL2, BCL2L1, BCL2A1 and MCL1 were overexpressed and DAPK1 was underexpressed in CLL samples with a 11q23 deletion. MYC overexpression was significantly associated with shorter overall survival in MCL (P < 0.01). AML patients with a normal karyotype showed a higher frequency of BCL2A1 overexpression (P < 0.001) than those with an abnormal karyotype.