RESUMO
ETV6/CBFA2 (TEL/AML1) is the most frequent genetic abnormality associated with acute lymphoblastic leukemias in children, and is associated with a favorable prognosis. To investigate the influence of ETV6/CBFA2 on cellular transformation, the fusion gene was cloned into a murine ecotropic retroviral vector and transduced into IL-3-dependent Ba/F3 and 32Dcl.3 and IL-7-dependent IxN/2b murine hematopoietic cell lines. Different variants of ETV6/CBFA2, corresponding to CBFA2 alternatively spliced variants, and the reciprocal product CBFA2/ETV6, were stably expressed in each of these cell lines. However, although Western blot analysis demonstrated expression of each variant, none of the stable cell lines expressing CBFA2/ETV6 or the variants conferred factor-independent growth. We further investigated the effect of ETV6/CBFA2 expression in vivo by generating transgenic mice in which expression of the fusion was directed to lymphoid cells using the immunoglobulin heavy chain enhancer/promoter. Four founder mice were identified showing transmission and expression of the chimeric product. The mice were bred for five generations and followed for more than 24 months. The mice did not develop a malignant hematologic disorder, nor did they display histopathologic, morphologic, or immunophenotypic abnormalities, although ETV6/CBFA2 expression was confirmed in each line. We conclude that the expression of ETV6/CBFA2 alone is not sufficient for induction of growth factor independence in hematopoietic cell lines or hematologic disease in transgenic mice.
Assuntos
Transformação Celular Neoplásica , Neoplasias Hematológicas/etiologia , Neoplasias Hematológicas/genética , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Proteínas de Fusão Oncogênica/genética , Animais , Western Blotting , Diferenciação Celular , Linhagem Celular , Subunidade alfa 2 de Fator de Ligação ao Core , Eletroporação , Elementos Facilitadores Genéticos , Citometria de Fluxo , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Leucemia/etiologia , Leucemia/genética , Camundongos , Camundongos Transgênicos , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Retroviridae/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Ativação Transcricional , Transdução GenéticaRESUMO
The molecular cloning of the t(5;10)(q33;q22) associated with atypical chronic myeloid leukemia (CML) is reported. Fluorescence in situ hybridization (FISH), Southern blot, and reverse transcriptase- polymerase chain reaction analysis demonstrated that the translocation resulted in an H4/platelet-derived growth factor receptor betaR (PDGFbetaR) fusion transcript that incorporated 5' sequences from H4 fused in frame to 3' PDGFbetaR sequences encoding the transmembrane, WW-like, and tyrosine kinase domains. FISH combined with immunophenotype analysis showed that t(5;10)(q33;q22) was present in CD13(+) and CD14(+) cells but was not observed in CD3(+) or CD19(+) cells. H4 has previously been implicated in pathogenesis of papillary thyroid carcinoma as a fusion partner of RET. The H4/RET fusion incorporates 101 amino acids of H4, predicted to encode a leucine zipper dimerization domain, whereas the H4/PDGFbetaR fusion incorporated an additional 267 amino acids of H4. Retroviral transduction of H4/PDGFbetaR, but not a kinase-inactive mutant, conferred factor-independent growth to Ba/F3 cells and caused a T-cell lymphoblastic lymphoma in a murine bone marrow transplantation assay of transformation. Mutational analysis showed that the amino-terminal H4 leucine zipper domain (amino acids 55-93), as well as H4 amino acids 101 to 386, was required for efficient induction of factor-independent growth of Ba/F3 cells. Tryptophan-to-alanine substitutions in the PDGFbetaR WW-like domain at positions 566/593, or tyrosine-to-phenylalanine substitutions at PDGFbetaR positions 579/581 impaired factor-independent growth of Ba/F3 cells. H4/PDGFbetaR is an oncoprotein expressed in t(5;10)(q33;q22) atypical CML and requires dimerization motifs in the H4 moiety, as well as residues implicated in signal transduction by PDGFbetaR, for efficient induction of factor-independent growth of Ba/F3 cells. (Blood. 2001;97:3910-3918)
Assuntos
Carcinoma Papilar/genética , Cromossomos Humanos Par 10 , Cromossomos Humanos Par 5 , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Proteínas/genética , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Neoplasias da Glândula Tireoide/genética , Translocação Genética , Transformação Celular Neoplásica/genética , Cromossomos Humanos Par 10/genética , Cromossomos Humanos Par 5/genética , Clonagem Molecular , Análise Citogenética , Proteínas do Citoesqueleto , DNA de Neoplasias/genética , DNA de Neoplasias/isolamento & purificação , Rearranjo Gênico , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/etiologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Mutagênese , Células Mieloides/metabolismo , Células Mieloides/patologia , Proteínas de Fusão Oncogênica , Estrutura Terciária de Proteína , Proteínas/metabolismo , TransfecçãoRESUMO
Between 1977 and 1996, cytogenetic investigations were performed on 182 childhood (< or = 16 yr) acute lymphoblastic leukemias (ALL), constituting 94% (182 of 194) of all ALL patients diagnosed and treated at the Departments of Pediatrics, Lund and Malmo University Hospitals, Sweden, during these two decades. The cytogenetic analyses were successful in 152 cases (84%). The failure rate was higher for the ALL investigated before 1987 (30% vs. 4%, p < 0.0001), and also the incidence of cytogenetically normal cases was higher during 1977-86 (43% vs. 25%, p < 0.05). Clonal chromosomal abnormalities were found in 103 (68%) ALL. Structural rearrangements were detected, by chromosome banding alone, in 76 cases (50%). Fluorescence in situ hybridization (FISH) was used to identify cases with t(12;21), 11q23 rearrangements, and 9p deletions, using probes for ETV6/CBFA2, MLL, and CDKN2A/B, in 72 cases from which cells in fixative and/or unstained metaphase preparations were available. In total, the most common structural rearrangements were del(9p) (17%), t(12;21) (15%), del(6q) (8%), and MLL rearrangements (4%). Six (32%) of nineteen cytogenetically normal ALL analyzed by FISH harbored cryptic abnormalities; three displayed t(12;21) and four had del(9p), one of which also carried a t(12;21). Five (45%) of the t(12;21)-positive ALL showed +der(21)t(12;21) or ider(21)(q10)t(12;21), resulting in the formation of double fusion genes. Among the more rare aberrations, eight structural rearrangements were identified as novel recurrent ALL-associated abnormalities, and nine cases harbored rearrangements previously not reported. Sixteen cases displayed karyotypically unrelated clones at different investigations. Seven ALL (5%) showed simple chromosomal changes, unrelated to the aberrations detected at diagnosis, during morphologic and clinical remission, and in all but one instance the patients remained in remission, with the abnormal clone disappearing in subsequent investigations. This indicates that the emergence of novel clonal chromosomal aberrations during remission in childhood ALL is rather common and does not by necessity predict a forthcoming relapse.
Assuntos
Hibridização in Situ Fluorescente , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Adolescente , Criança , Pré-Escolar , Deleção Cromossômica , Cromossomos Humanos Par 12/genética , Cromossomos Humanos Par 12/ultraestrutura , Cromossomos Humanos Par 21/genética , Cromossomos Humanos Par 21/ultraestrutura , Cromossomos Humanos Par 9/ultraestrutura , Células Clonais/ultraestrutura , Sondas de DNA , Feminino , Humanos , Imunofenotipagem , Lactente , Contagem de Leucócitos , Masculino , Células-Tronco Neoplásicas/ultraestrutura , Oncogenes , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/mortalidade , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/mortalidade , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Suécia/epidemiologia , Translocação Genética , Resultado do TratamentoRESUMO
Sclerosing epithelioid fibrosarcoma (SEF) is a recently described entity. It is a low-grade sarcoma that occurs primarily in the deep soft tissues of the extremities of adults. It may histologically simulate benign lesions such as fibroma and myxoma or malignancies such as sclerosing carcinoma and lymphoma, extraskeletal myxoid chondrosarcoma, clear cell sarcoma of tendons and aponeuroses, and synovial sarcoma, depending on the lesion's cellularity, degree of fibrosis, and amount of myxoid matrix. There are no previously published cytogenetic studies of this tumor. We found the karyotype 40-45,XY,add(9)(p13),add(10)(p11),-12,-13,-18,add(18)(q11),add(20)(q11) in a SEF of a 14-year-old boy, by using chromosome banding. Fluorescence in situ hybridization showed that both the add(10) and the add(18) contained amplified sequences from 12q13 and 12q15, including the HMGIC gene. Chromosome 18 material was present in the add(9) and terminally in the add(10). The karyotype of this case indicates that SEF is unrelated to extraskeletal myxoid chondrosarcoma, clear cell sarcoma, and synovial sarcoma. When compared with the findings in other soft tissue tumors such as well-differentiated liposarcoma and low-grade malignant fibrous histiocytoma, the chromosome banding and in situ hybridization data add support to the notion that SEF is a relatively low grade variant of fibrosarcoma.
Assuntos
Cromossomos Humanos Par 10/genética , Cromossomos Humanos Par 12/genética , Fibrossarcoma/genética , Doenças do Pé/genética , Translocação Genética/genética , Adolescente , Fibrossarcoma/patologia , Doenças do Pé/patologia , Humanos , Hibridização in Situ Fluorescente , Cariotipagem , MasculinoRESUMO
Jumping translocations (JT) are characterized by the relocalization of the same part of a donor to several recipient chromosomes. Although JT occasionally are constitutional, most are associated with hematologic malignancies. In such cases, JT usually arise during disease progression and are associated with poor prognosis. Despite its clinical importance, this cytogenetic phenomenon has not been characterized at the molecular level. We have analyzed JT in a juvenile chronic myelomonocytic leukemia that subsequently transformed to an acute myeloid leukemia. Detailed fluorescence in situ hybridization (FISH) analyses showed that the cytogenetically identical donor breakpoint at 3q21 was highly heterogeneous. In fact, more than 10 distinct breakpoints, four of which mapped within YACs, were identified. Analyses of samples during disease progression showed that the breakpoint complexity decreased, indicating clonal selection. Hence, the 3q21 breakpoints displayed a spatial as well as a temporal heterogeneity, revealing that JT are highly unstable, showing great variation in the size of donor segment. The breaks at the recipient chromosomes were mapped within the subtelomeric regions. The general telomere length was not affected and an underlying replication error resulting in microsatellite instability was excluded. We conclude that the emergence of JT is unlikely to cause fusion genes or to affect the expression of genes located in the breakpoint regions. The identification of YACs spanning the breakpoints, ie, YACs 913c7, 937g5, 948c2 and 955g1, may facilitate the isolation of DNA sequences leading to a genetic instability associated with the origin of multiple translocations.
Assuntos
Cromossomos Humanos Par 3/genética , Leucemia Mielomonocítica Crônica/genética , Translocação Genética/genética , Pré-Escolar , Cromossomos Humanos Par 11/genética , Cromossomos Humanos Par 15/genética , Cromossomos Humanos Par 22/genética , Cromossomos Humanos Par 7/genética , Evolução Fatal , Humanos , Hibridização in Situ Fluorescente , Cariotipagem , Masculino , Repetições de Microssatélites/genéticaAssuntos
Fusão Gênica Artificial , Proteínas de Ligação a DNA/genética , Genes abl , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mieloide Crônica Atípica BCR-ABL Negativa/genética , Proteínas Repressoras , Fatores de Transcrição/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Feminino , Humanos , Hibridização in Situ Fluorescente , Lactente , Masculino , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas c-ets , Variante 6 da Proteína do Fator de Translocação ETSRESUMO
Cyclin D2, encoded by CCND2 at 12p13, takes part in the regulation of the cell cycle and has been suggested as a candidate for gene amplification in lymphoid malignancies. CCND2 is often overexpressed in chronic B-cell disorders, and we recently detected genomic amplification of the chromosomal region containing CCND2 in two of three investigated non-Hodgkin lymphomas (NHLs) with cytogenetic abnormalities involving 12p. In the present study, 58 NHLs without karyotypic evidence of 12p aberrations were analyzed by fluorescence in situ hybridization with probes for CCND2. No genomic amplification was found, strongly suggesting that this abnormality is rare in such NHLs.
Assuntos
Ciclinas/genética , Linfoma não Hodgkin/genética , Aberrações Cromossômicas , Cromossomos Humanos Par 12 , Ciclina D2 , Amplificação de Genes , Humanos , Hibridização in Situ FluorescenteRESUMO
Thirty-two hematologic malignancies--nine with cytogenetically identified 12p abnormalities and 23 with whole or partial losses of chromosome 12--were selected for fluorescence in situ hybridization (FISH) investigations of 12p. These analyses revealed structural 12p changes, such as translocations, deletions, insertions, inversions and amplification, in 20 cases. ETV6 rearrangements were detected in three acute leukemias. One acute undifferentiated leukemia had t(4;12)(q12;p13) as the sole anomaly. The second case, an acute myeloid leukemia (AML), displayed complex abnormalities involving, among others, chromosomes 9 and 12. The third case, also an AML, had an insertion of the distal part of ETV6 into chromosome arm 11q and into multiple ring chromosomes, which also contained chromosome 11 material, resulting in an amplification of a possible fusion gene. The fusion partners in these cases remain to be identified. Thirty-one additional breakpoints on 12p could be characterized in detail. The majority of these breaks were shown to result in interchromosomal rearrangements, possibly indicating the location of hitherto unrecognized genes of importance in the pathogenesis of hematologic malignancies. The FISH analyses disclosed terminal or interstitial 12p deletions in 18 cases. Seven myeloid malignancies showed deletions restricted to a region, including ETV6 and CDKN1B, which has been reported to be frequently lost in leukemias. In four cases, the deletions involved both these genes, whereas two AML displayed loss of CDKN1B but not ETV6, supporting previously reported findings indicating a region of deletion not including this gene. However, one myelodysplastic syndrome lacked one copy of ETV6 but not CDKN1B. Hence, we suggest a minimal region of deletion on 12p located between the ETV6 and CDKN1B genes.
Assuntos
Aberrações Cromossômicas , Cromossomos Humanos Par 12 , Neoplasias Hematológicas/genética , Leucemia/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Mapeamento Cromossômico , Feminino , Deleção de Genes , Rearranjo Gênico , Marcadores Genéticos , Humanos , Hibridização in Situ Fluorescente , Cariotipagem , Linfoma não Hodgkin/genética , Masculino , Pessoa de Meia-Idade , Policitemia Vera/genéticaRESUMO
A BCR/ABL-negative chronic myeloid leukemia (CML) with t(12;14) (p12;q11-13) as the sole chromosomal abnormality was investigated by fluorescence in situ hybridization (FISH), which disclosed a cryptic insertion of ETV6 (previously called TEL), located at 12p12, into ABL at chromosome band 9q34. ETV6/ABL fusion was confirmed by RT-PCR, revealing that the first five exons of ETV6 were fused in frame with ABL at exon 2. Wild-type ETV6 was expressed, in accordance with the FISH results showing no deletion of the second ETV6 allele. ETV6/ABL chimeric transcripts have previously been reported in acute leukemias, but never before in CML. The present case suggests that ETV6/ABL positivity may constitute a new genetic subgroup of BCR-negative CML.
Assuntos
Proteínas de Ligação a DNA/genética , Genes abl/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Proteínas de Fusão Oncogênica/genética , Proteínas Repressoras , Fatores de Transcrição/genética , Adulto , Southern Blotting , Células da Medula Óssea/química , Bandeamento Cromossômico , Mapeamento Cromossômico , Cromossomos Humanos Par 12/genética , Cromossomos Humanos Par 14/genética , DNA de Neoplasias/isolamento & purificação , Proteínas de Fusão bcr-abl/genética , Humanos , Hibridização in Situ Fluorescente , Masculino , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas c-ets , Translocação Genética/genética , Variante 6 da Proteína do Fator de Translocação ETSRESUMO
The cytogenetically unidentifiable t(12;21)(p12:q22), resulting in ETV6/CBFA2 fusion, is the most frequent chromosomal aberration in childhood acute lymphoblastic leukaemia ALL). We report a variant, ider(21)(q10)t(12:21)(p12;q22), which was shown to contain double ETV6/CBFA2 fusions by fluorescence in situ hybridization. This is the second case of such an ider(21) in childhood ALL, suggesting that it is a new recurrent abnormality. Since the ider(21) is cytogenetically indistinguishable from i(21)(q10) and idic(21)(p11), changes associated with similar clinical features as the t(12;21), i.e. pre-B-cell ALL and age 1-10 years, we suggest that all ALL displaying these changes should be tested for ETV6/CBFA2 fusion transcript.
Assuntos
Cromossomos Humanos Par 12/genética , Cromossomos Humanos Par 21/genética , Proteínas de Ligação a DNA/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteínas Repressoras , Fatores de Transcrição/genética , Translocação Genética , Criança , Humanos , Hibridização in Situ Fluorescente , Masculino , Proteínas de Fusão Oncogênica/genética , Proteínas Proto-Oncogênicas c-ets , Variante 6 da Proteína do Fator de Translocação ETSRESUMO
Seventy-nine acute myeloid leukemias (AML) and myelodysplastic syndromes without cytogenetic evidence of 12p aberrations were investigated by fluorescence in situ hybridization with probes for ETV6 and CDKN1B (previously called TEL and KIP1, respectively) to ascertain whether abnormalities of these genes are frequently undetected by standard chromosome banding analyses and, if so, whether they are associated with specific karyotypic patterns and morphologic features. One of sixty cytogenetically aberrant myeloid malignancies, an AML with a complex karyotype including del(5q) and del(20q), showed a hemizygous interstitial deletion of the ETV6 and CDKN1B loci. No concomitant rearrangement of the other ETV6 allele was detected. Two of nineteen cytogenetically normal AML displayed a hemizygous interstitial deletion involving CDKN1B, but not ETV6. Thus, cryptic deletions of these genes seem to be rare in cytogenetically abnormal myeloid malignancies without 12p aberrations (2%), whereas they may be more frequent in karyotypically normal AML (10%). Furthermore, the present findings show that the deletions may be narrow, not including the ETV6 gene, and indirectly suggest that CDKN1B, or a closely located genomic segment, is the target of 12p deletions.
Assuntos
Proteínas de Ciclo Celular , Deleção Cromossômica , Cromossomos Humanos Par 12 , Proteínas de Ligação a DNA/genética , Leucemia Mieloide/genética , Proteínas Associadas aos Microtúbulos/genética , Síndromes Mielodisplásicas/genética , Proteínas Repressoras , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor , Doença Aguda , Idoso , Aberrações Cromossômicas , Inibidor de Quinase Dependente de Ciclina p27 , Humanos , Masculino , Proteínas Proto-Oncogênicas c-ets , Variante 6 da Proteína do Fator de Translocação ETSRESUMO
Homology searches in the Expressed Sequence Tag Database were performed using SPYGQ-rich regions as query sequences to find genes encoding protein regions similar to the N-terminal parts of the sarcoma-associated EWS and FUS proteins. Clone 22911 (T74973), encoding a SPYGQ-rich region in its 5' end, and several other clones that overlapped 22911 were selected. The combined data made it possible to assemble a full-length cDNA sequence. This cDNA sequence is 1677 bp, containing an initiation codon ATG, an open reading frame of 400 amino acids, a poly(A) signal, and a poly(A) tail. We found 100% identity between the 5' part of the consensus sequence and the 598-bp-long sequence named TFG. The TFG sequence is fused to the 3' end of NTRK1, generating the TRK-T3 fusion transcript found in papillary thyroid carcinoma. The cDNA therefore represents the full-length transcript of the TFG gene. TFG was localized to 3q11-q12 by fluorescence in situ hybridization. The 3' and the 5' ends of the TFG cDNA probe hybridized to a 2.2-kb band on Northern blot filters in all tissues examined.
Assuntos
Proteínas/genética , Neoplasias da Glândula Tireoide/genética , Sequência de Aminoácidos , Sequência de Bases , Mapeamento Cromossômico , Cromossomos Humanos Par 3/genética , Clonagem Molecular , DNA Complementar/genética , Expressão Gênica , Humanos , Hibridização in Situ Fluorescente , Dados de Sequência MolecularAssuntos
Medula Óssea/patologia , Aberrações Cromossômicas , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Pré-Escolar , Humanos , Hibridização in Situ Fluorescente , Cariotipagem , Masculino , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Indução de RemissãoRESUMO
A total of 70 human immunodeficiency virus type 1 (HIV-1) and 42 HIV-2 antibody-positive serum samples, collected from groups of individuals in which only one type of HIV prevails, were tested for cross-reactivity against HIV-2 and HIV-1 proteins by Western blot (WB) (immunoblot), radioimmunoprecipitation assay (RIPA), neutralization analysis, and enzyme-linked immunosorbent assay with as antigen synthetic peptides representing selected parts of the envelope (env) glycoproteins. Cross-reactions against the env glycoproteins were observed by WB in 10% (7 of 70) and by RIPA in 40% (28 of 70) of the HIV-1 antibody-positive serum samples and by WB in 29% (12 of 42) and by RIPA in 48% (20 of 42) of the HIV-2 antibody-positive serum samples. Testing by enzyme-linked immunosorbent assay against a 36-amino-acid peptide (Cys-301-Cys-336) of the external glycoprotein of strain HTLV-IIIB of HIV-1 (HIV-1HTLV-IIIB) (known to represent a dominating, linear neutralizing site) showed type-specific reactions in 67% (38 of 57) of HIV-1 antibody-positive serum samples. Type-specific reactions against a homologous 35-amino-acid peptide of strain SBL-6669 of HIV-2 (HIV-2SBL-6669) were found in 75% (30 of 40) of HIV-2 antibody-positive serum samples, and these reactions were correlated to neutralization against HIV-2SBL-6669. Cross-reactions against these peptides were seen in 23% (13 of 57) and 33% (13 of 40) of the HIV-1 and HIV-2 antibody-positive serum samples, respectively. These cross-reactions were correlated to cross-neutralization against HIV-1HTLV-IIIB and HIV-2SBL-6669. Cross-neutralization against one heterotypic virus strain was demonstrated in 16% (9 of 57) of HIV-1 antibody-positive serum samples and in 22% (5 of 22) of HIV-2 antibody-positive serum samples, but no correlation was found between cross-neutralization and env cross-reactivity in WB or RIPA.