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The presence of the translocation t(9;22)(q34;q11), leading to the BCR::ABL1 fusion transcript, is the hallmark of chronic myeloid leukemia (CML). Nevertheless, atypical presentation at diagnosis can be challenging. However, although most patients with CML are diagnosed with the e13a2 or e14a2 BCR::ABL1 fusion transcripts, about 5% of them carry rare BCR::ABL1 fusion transcripts, such as e19a2, e8a2, e13a3, e14a3, e1a3, and e6a2. In particular, the e6a2 fusion transcript has been associated with clinically aggressive disease frequently presenting in accelerated or blast crisis phases. To date, there is limited evidence on the efficacy of front-line second-generation tyrosine kinase inhibitors for this genotype. Here, we report two patients, in whom the diagnosis of CML was challenging. The use of primers recognizing more distant exons from the common BCR::ABL1 breakpoint region correctly identified the atypical BCR::ABL1 e6a2 fusion transcript. Treatment with the second-generation tyrosine kinase inhibitor nilotinib was effective in our patient expressing the atypical e6a2 BCR::ABL1 fusion transcript.
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OBJECTIVES: Our aim was to establish and characterize a novel pancreatic ductal adenocarcinoma cell line from a patient in whom the origin of the invasive carcinoma could be traced back to the intraductal papillary mucinous neoplasm (IPMN) precursor lesion. METHODS: The primary patient-derived tumor was propagated in immunocompromised mice for 2 generations and used to establish a continuous in vitro culture termed ASAN-PaCa. Transplantation to fertilized chicken eggs confirmed the tumorigenic potential in vivo. Molecular analyses included karyotyping, next-generation genomic sequencing, expression analysis of marker proteins, and mucin-profiling. RESULTS: The analysis of marker proteins confirmed the epithelial nature of the established cell line, and revealed that the expression of the mucin MUC1 was higher than that of MUC2 and MUC5AC. ASAN-PaCa cells showed rapid in vitro and in vivo growth and multiple chromosomal aberrations. They harbored mutations in KRAS (Q61H), TP53 (Y220C), and RNF43 (I47V and L418M) but lacked either IPMN-specific GNAS or presumed pancreatic ductal adenocarcinoma-driving mutations in KRAS (codons 12/13), SMAD, and CDKN2A genes. CONCLUSIONS: ASAN-PaCa cell line represents a novel preclinical model of pancreatic adenocarcinoma arising in the background of IPMN, and offers an opportunity to study how further introduction of known driver mutations might contribute to pancreatic carcinogenesis.
Assuntos
Adenocarcinoma , Neoplasias Pancreáticas , Animais , Linhagem Celular , Humanos , Camundongos , Mucina-2RESUMO
To circumvent donor-to-donor heterogeneity which may lead to inconsistent results after treatment of acute graft-versus-host disease with mesenchymal stromal cells generated from single donors we developed a novel approach by generating these cells from pooled bone marrow mononuclear cells of 8 healthy "3(rd)-party" donors. Generated cells were frozen in 209 vials and designated as mesenchymal stromal cell bank. These vials served as a source for generation of clinical grade mesenchymal stromal cell end-products, which exhibited typical mesenchymal stromal cell phenotype, trilineage differentiation potential and at later passages expressed replicative senescence-related markers (p21 and p16). Genetic analysis demonstrated their genomic stability (normal karyotype and a diploid pattern). Importantly, clinical end-products exerted a significantly higher allosuppressive potential than the mean allosuppressive potential of mesenchymal stromal cells generated from the same donors individually. Administration of 81 mesenchymal stromal cell end-products to 26 patients with severe steroid-resistant acute graft-versus-host disease in 7 stem cell transplant centers who were refractory to many lines of treatment, induced a 77% overall response at the primary end point (day 28). Remarkably, although the cohort of patients was highly challenging (96% grade III/IV and only 4% grade II graft-versus-host disease), after treatment with mesenchymal stromal cell end-products the overall survival rate at two years follow up was 71±11% for the entire patient cohort, compared to 51.4±9.0% in graft-versus-host disease clinical studies, in which mesenchymal stromal cells were derived from single donors. Mesenchymal stromal cell end-products may, therefore, provide a novel therapeutic tool for the effective treatment of severe acute graft-versus-host disease.
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Doença Enxerto-Hospedeiro/diagnóstico , Doença Enxerto-Hospedeiro/terapia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais , Doadores de Tecidos , Adolescente , Adulto , Células da Medula Óssea , Técnicas de Cultura de Células , Diferenciação Celular , Proliferação de Células , Criança , Pré-Escolar , Resistência a Medicamentos , Feminino , Doença Enxerto-Hospedeiro/etiologia , Doença Enxerto-Hospedeiro/mortalidade , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Humanos , Terapia de Imunossupressão , Lactente , Masculino , Células-Tronco Mesenquimais/citologia , Pessoa de Meia-Idade , Prognóstico , Índice de Gravidade de Doença , Esteroides/uso terapêutico , Análise de Sobrevida , Resultado do Tratamento , Adulto JovemRESUMO
We describe a boy with developmental delay, speech delay, and minor dysmorphic features with a heterozygous de novo â¼460 kb deletion at 2p13.2 involving only parts of EXOC6B present in about 50% of lymphocytes. This widely expressed gene encodes the exocyst component 6B, which is part of a multiprotein complex required for targeted exocytosis. Little is known about the effect of EXOC6B haploinsufficiency. In 2008, a patient with a complex syndromic phenotype, including left renal agenesis, neutropenia, recurrent pulmonary infections, long bone diaphysis broadening, growth retardation, and developmental delay (DD) was found to carry a de novo translocation t(2;7) involving TSN3 and EXOC6B. Further characterization of the translocation indicated that disruption of TSN3 may be responsible for the skeletal phenotype. Recently, a heterozygous deletion of EXOC6B along with a deletion of the CYP26B1 gene has been reported in a boy with intellectual disability, speech delay, hyperactivity, facial asymmetry, a dysplastic ear, brachycephaly, and mild joint contractures. Additionally, disruption of EXOC6B by a de novo balanced translocation t(2;8) has been described in a patient with developmental delay, epilepsy, autistic and aggressive behavior. This is the first report of a de novo deletion affecting only EXOC6B in an individual with developmental delay. In conclusion, based on our findings and recent data from the literature, there is evidence that EXOC6B and the exocyst complex might play an important role in the molecular pathogenesis of intellectual disability.
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Anormalidades Múltiplas/genética , Cromossomos Humanos Par 2/genética , Proteínas de Ligação ao GTP/genética , Deleção de Genes , Haploinsuficiência/genética , Deficiência Intelectual/genética , Deficiência Intelectual/fisiopatologia , Humanos , Hibridização in Situ Fluorescente , Masculino , TurquiaRESUMO
PURPOSE: In acute myeloid leukemia (AML), studies based on whole-genome sequencing have shown genomic diversity within leukemic clones. The aim of this study was to address clonal heterogeneity in AML based on metaphase cytogenetics. PATIENTS AND METHODS: This analysis included all patients enrolled onto two consecutive, prospective, randomized multicenter trials of the Study Alliance Leukemia. Patients were newly diagnosed with non-M3 AML and were fit for intensive chemotherapy. RESULTS: Cytogenetic subclones were detected in 418 (15.8%) of 2,639 patients from the whole study population and in 418 (32.8%) of 1,274 patients with aberrant karyotypes. Among those, 252 karyotypes (60.3%) displayed a defined number of distinct subclones, and 166 (39.7%) were classified as composite karyotypes. Subclone formation was particularly frequent in the cytogenetically adverse group, with subclone formation in 69.0%, 67.1%, and 64.8% of patients with complex aberrant, monosomal, and abnl(17p) karyotypes (P < .001 each). Two-subclone patterns typically followed a mother-daughter evolution, whereas for ≥ three subclones, a branched pattern prevailed. In non-core binding factor AML, subclone formation was associated with inferior event-free and overall survival and was confirmed as an independent predictor of poor prognosis in multivariate analysis. Subgroup analysis showed that subclone formation adds prognostic information particularly in the cytogenetic adverse-risk group. Allogeneic stem-cell transplantation improved the prognosis of patients with subclone karyotypes as shown in landmark analyses. CONCLUSION: Cytogenetic subclones are frequent in AML and permit tracing of clonal evolution and architecture. They bear prognostic significance with clonal heterogeneity as an independent adverse prognostic marker in cytogenetically adverse-risk AML.
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Cariotipagem/métodos , Leucemia Mieloide Aguda/genética , Metáfase/genética , Adolescente , Adulto , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Aberrações Cromossômicas , Feminino , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/mortalidade , Masculino , Pessoa de Meia-Idade , Estudos Multicêntricos como Assunto , Prognóstico , Ensaios Clínicos Controlados Aleatórios como Assunto , Resultado do Tratamento , Adulto JovemRESUMO
Transient myeloproliferative disorder (TMD) of the newborn and acute megakaryoblastic leukaemia (AMKL) in children with Down syndrome (DS) represent paradigmatic models of leukaemogenesis. Chromosome 21 gene dosage effects and truncating mutations of the X-chromosomal transcription factor GATA1 synergize to trigger TMD and AMKL in most patients. Here, we report the occurrence of TMD, which spontaneously remitted and later progressed to AMKL in a patient without DS but with a distinct dysmorphic syndrome. Genetic analysis of the leukaemic clone revealed somatic trisomy 21 and a truncating GATA1 mutation. The analysis of the patient's normal blood cell DNA on a genomic single nucleotide polymorphism (SNP) array revealed a de novo germ line 2·58 Mb 15q24 microdeletion including 41 known genes encompassing the tumour suppressor PML. Genomic context analysis of proteins encoded by genes that are included in the microdeletion, chromosome 21-encoded proteins and GATA1 suggests that the microdeletion may trigger leukaemogenesis by disturbing the balance of a hypothetical regulatory network of normal megakaryopoiesis involving PML, SUMO3 and GATA1. The 15q24 microdeletion may thus represent the first genetic hit to initiate leukaemogenesis and implicates PML and SUMO3 as novel components of the leukaemogenic network in TMD/AMKL.
Assuntos
Cromossomos Humanos Par 15/genética , Síndrome de Down/genética , Leucemia Megacarioblástica Aguda/genética , Transtornos Mieloproliferativos/genética , Proteínas Nucleares/genética , Deleção de Sequência , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética , Ubiquitinas/genética , Criança , Pré-Escolar , Síndrome de Down/patologia , Fator de Transcrição GATA1/genética , Humanos , Lactente , Leucemia Megacarioblástica Aguda/tratamento farmacológico , Leucemia Megacarioblástica Aguda/patologia , Masculino , Transtornos Mieloproliferativos/tratamento farmacológico , Transtornos Mieloproliferativos/patologia , Proteína da Leucemia PromielocíticaRESUMO
BACKGROUND: Exosomes are small membrane vesicles with a size of 40-100 nm that are released by different cell types from a late endosomal cellular compartment. They can be found in various body fluids including plasma, malignant ascites, urine, amniotic fluid and saliva. Exosomes contain proteins, miRNAs and mRNAs (exosome shuttle RNA, esRNA) that could serve as novel platform for diagnosis. METHOD: We isolated exosomes from amniotic fluid, saliva and urine by differential centrifugation on sucrose gradients. Marker proteins were identified by Western blot and FACS analysis after adsorption of exosomes to latex beads. We extracted esRNA from exosomes, carried out RT-PCR, and analyzed amplified products by restriction length polymorphism. RESULTS: Exosomes were positive for the marker proteins CD24, CD9, Annexin-1 and Hsp70 and displayed the correct buoyant density and orientation of antigens. In sucrose gradients the exosomal fractions contained esRNA that could be isolated with sufficient quantity for further analysis. EsRNAs were protected in exosomes from enzymatic degradation. Amniotic fluid esRNA served as template for the typing of the CD24 single nucleotide polymorphism (rs52812045). It also allowed sex determination of the fetus based on the detection of the male specific ZFY gene product. CONCLUSIONS: Our data demonstrate that exosomes from body fluids carry esRNAs which can be analyzed and offers access to the transcriptome of the host organism. The exosomal lipid bilayer protects the genetic information from degradation. As the isolation of exosomes is a minimally invasive procedure, this technique opens new possibilities for diagnostics.
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Líquidos Corporais/metabolismo , Técnicas e Procedimentos Diagnósticos , Exossomos/metabolismo , Líquido Amniótico/metabolismo , Antígeno CD24/genética , Micropartículas Derivadas de Células/metabolismo , Centrifugação com Gradiente de Concentração , Feminino , Feto/metabolismo , Humanos , Masculino , Polimorfismo de Nucleotídeo Único/genética , Estabilidade de RNA , Saliva/metabolismo , Análise para Determinação do Sexo , UrinaRESUMO
Chromosomal instability (CIN), defined by an elevated frequency of the occurrence of novel chromosomal aberrations, is strongly implicated in the generation of aneuploidy, one of the hallmarks of human cancers. As for aneuploidy itself, the role of CIN in the evolution and progression of malignancy is a matter still open to debate. We investigated numerical as well as structural CIN in primary CD34-positive cells by determining the cell-to-cell variability of the chromosome content using fluorescence-in situ-hybridization (FISH). Thereby, CIN was measured in 65 patients with myelodysplastic syndromes (MDS), acute myeloid leukaemia (AML) and control subjects. Among MDS patients, a subgroup with elevated levels of CIN was identified. At a median follow-up of 17.2 months, all patients within this 'high CIN' subgroup had died or progressed to AML, while 80% of MDS patients with normal CIN levels had stable disease (P < 0.001). Notably, there was no statistically significant difference between 'normal CIN' and 'high CIN' MDS patients regarding established risk factors. Hence, elevated CIN levels were associated with poor outcome, and our method provided additional prognostic information beyond conventional cytogenetics. Furthermore, in all three MDS patients for whom serial measurements were available, development of AML was preceded by increasing CIN levels. In conclusion, elevated CIN levels may be valuable as an early indicator of poor prognosis in MDS, hence corroborating the concept of CIN as a driving force in tumour progression.
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Instabilidade Cromossômica/genética , Análise Citogenética , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/terapia , Adulto , Idoso , Progressão da Doença , Feminino , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Masculino , Pessoa de Meia-Idade , Fatores de Risco , Resultado do Tratamento , Células Tumorais CultivadasRESUMO
By means of the tumorigenicity assay applying DNA from a patient with a gastric carcinoma (MA) we have already reported the identification of the putative oncogene myeov. In addition we have shown its involvement in t(11;14)-positive multiple myelomas and amplifications of breast tumours and esophageal carcinomas. The failure of myeov cDNA to induce tumour formation in NIH/3T3 cells prompted us to analyze the human sequences present in our MA-T1a1 tertiary transfectants. Sequence analysis revealed the presence of the human oncogene hst (fgf4) at a distance of approximately 9kb from the myeov gene in our MA-T1a1 tertiary transfectants. Both myeov and hst (fgf4) are normally situated approximately 475-kb apart at band 11q13, a region that is frequently amplified and overexpressed in various tumours. Southern and Northern blot analyses confirmed our sequence data and showed rearrangement of hst sequences during the transfection process and its expression in our MA-T1a1 tertiary transfectants.
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Transformação Celular Neoplásica/genética , Fator 4 de Crescimento de Fibroblastos/genética , Proteínas Proto-Oncogênicas/genética , Neoplasias Gástricas/genética , Transfecção/métodos , Animais , Sequência de Bases , DNA de Neoplasias/genética , Fator 4 de Crescimento de Fibroblastos/biossíntese , Rearranjo Gênico , Humanos , Camundongos , Camundongos Nus , Células NIH 3T3 , Proteínas Proto-Oncogênicas/biossínteseRESUMO
The myeov gene has been isolated by the tumorigenicity assay and is localized at chromosome 11q13, a frequent site for chromosomal rearrangements in various carcinomas and B-cell neoplasms. In addition, myeov is coamplified with cyclin D1 and overexpressed in carcinomas of various organs. The mechanisms of myeov regulation remain enigmatic. The 5'-untranslated region (5'-UTR) of the myeov gene is long, encompasses several upstream AUGs, and is predicted to fold in a strong secondary structure, suggesting that its translation might be regulated by an internal ribosomal entry site. Here we show that initial experiments using monocistronic and dicistronic reporter constructs supported this assumption. However, the application of in vitro transcription/translation assays, Northern blot analysis, and promoterless dicistronic constructs revealed promoter activity of the myeov 5'-UTR. DNA transfection of dicistronic DNA constructs, normal and mutated forms of myeov cDNA fragments cloned in a eukaryotic expression vector, and direct RNA transfection analysis revealed that upstream AUG triplets in the 5'-UTR of the myeov transcript abrogate translation. Alternative splicing mechanisms in specific cell types and/or developmental stage may evade this translation control. Control experiments suggest that the 5'-UTR from encephalomyocarditis virus, when inserted at the midpoint of a dicistronic vector, is also able to function as a cryptic promoter.
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Regulação Neoplásica da Expressão Gênica , Proteínas Oncogênicas/biossíntese , Proteínas Oncogênicas/genética , Fases de Leitura Aberta , Regiões 5' não Traduzidas , Sequência de Bases , Northern Blotting , Linhagem Celular , Cromossomos Humanos Par 11 , Ciclina D1/metabolismo , DNA/metabolismo , DNA Complementar/metabolismo , Vírus da Encefalomiocardite/genética , Genes Reporter , Vetores Genéticos , Humanos , Immunoblotting , Luciferases/metabolismo , Modelos Genéticos , Dados de Sequência Molecular , Mutação , Regiões Promotoras Genéticas , Biossíntese de Proteínas , Estrutura Secundária de Proteína , Proteínas Proto-Oncogênicas , RNA/metabolismo , Transcrição Gênica , TransfecçãoRESUMO
A large variety of biological processes is mediated by stimulation of the receptor tyrosine kinase MET. Screening a mouse embryo cDNA library, we were able to identify several novel, putative intracellular TPR/MET-substrates: SNAPIN, DCOHM, VAV-1, Sorting nexin 2, Death associated protein kinase 3, SMC-1, Centromeric protein C, and hTID-1. Interactions as identified by yeast two-hybrid analysis were validated in vitro and in vivo by mammalian two-hybrid studies, a far-western assay and coimmunoprecipitation. Participation in apoptosis-regulating mechanisms through interaction with DAPK-3 and cell cycle control via binding to nuclear proteins such as CENPC and SMC-1 are possible new aspects of intracellular MET signaling.
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Mapeamento de Interação de Proteínas/métodos , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/metabolismo , Receptores de Fatores de Crescimento/química , Receptores de Fatores de Crescimento/metabolismo , Animais , Western Blotting/métodos , Embrião de Mamíferos/química , Embrião de Mamíferos/metabolismo , Biblioteca Gênica , Humanos , Rim/citologia , Rim/embriologia , Camundongos , Mutagênese Sítio-Dirigida/genética , Proteínas de Fusão Oncogênica/química , Proteínas de Fusão Oncogênica/metabolismo , Peptídeos , Ligação Proteica , Proteínas Proto-Oncogênicas c-met , Ratos , Análise de Sequência de Proteína/métodos , Especificidade por Substrato , Técnicas do Sistema de Duplo-HíbridoRESUMO
Several oncogenes isolated by the NIH/3T3 transformation assay, i.e., dbl, dbs, lbc, lfc, lsc, net, ost and tim, contain a Dbl homology (DH) and a pleckstrin-homology (PH) domain and act as GEFs (guanine nucleotide exchange factors) for Rho-like GTPases. In a search for genes with oncogenic potential in DNA from the monocytic leukaemia cell line U937, we identified an amino-terminal truncated form of gef-h1, a gene encoding a GEF for RhoA. These data support the idea that a systematic search for mutations and/or deletions of GEFs in human cancer is promising.
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DNA/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Células 3T3 , Animais , Sequência de Bases , DNA/administração & dosagem , Fatores de Troca do Nucleotídeo Guanina/genética , Células HeLa , Humanos , Camundongos , Dados de Sequência Molecular , Fatores de Troca de Nucleotídeo Guanina Rho , Homologia de Sequência do Ácido Nucleico , Transfecção , Células U937RESUMO
Cytogenetic analysis of a pediatric T-cell acute lymphoblastic leukemia (ALL) cell line (HPB-ALL) revealed the cryptic t(5;14)(q35;q32.2), recently found in 15-20% pediatric T-ALL patients, with 5q35 and 14q32.2 breakpoints at 5'-HOX11L2 and 3'-BCL11B, respectively. Expression of both BCL11B, which is hematologically restricted to T cells, and HOX11L2 was detected, whereas adjacent genes at 5q35 (RANBP17) and 14q32 (VRK1, HSU88895) were not dysregulated. Apparently, t(5;14)(q35;q32.2) serves to activate HOX11L2 by juxtaposition with elements downstream of BCL11B, implying a novel dysregulatory mechanism comparable to TCRA/D or IGH juxtaposition. As well as providing molecular cytogenetic documentation of t(5;14) ALL, this report validates HPB-ALL cells for investigating this important new disease entity in vitro.
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Regulação Neoplásica da Expressão Gênica/genética , Proteínas de Homeodomínio/metabolismo , Leucemia-Linfoma de Células T do Adulto/genética , Proteínas de Neoplasias/genética , Proteínas Oncogênicas/metabolismo , Translocação Genética/genética , Quebra Cromossômica/genética , Deleção Cromossômica , Cromossomos Humanos Par 14/genética , Cromossomos Humanos Par 16/genética , Cromossomos Humanos Par 5/genética , Análise Citogenética/métodos , Rearranjo Gênico/genética , Genes Homeobox/genética , Proteínas de Homeodomínio/biossíntese , Proteínas de Homeodomínio/genética , Humanos , Leucemia-Linfoma de Células T do Adulto/patologia , Proteínas de Neoplasias/biossíntese , Proteínas Oncogênicas/biossíntese , Proteínas Oncogênicas/genética , Proteínas Proto-Oncogênicas , Transcrição Gênica/genética , Células Tumorais CultivadasRESUMO
Rearrangements of chromosome 11q13 are frequently observed in human cancer. The 11q13 region harbors several chromosomal breakpoint clusters found in hematologic malignancies and exhibits frequent DNA amplification in carcinomas. DNA amplification patterns in breast tumors are consistent with the existence of at least 4 individual amplification units, suggesting the activation of more than 1 gene in this region. Two candidate oncogenes have been identified, CCND1 and EMS1/CORTACTIN, representing centrally localized amplification units. Genes involved in the proximal and distal amplicons remain to be identified. Recently we reported on a putative transforming gene, MYEOV, mapping 360 kb centromeric to CCND1. This gene was found to be rearranged and activated concomitantly with CCND1 in a subset of t(11;14)(q13;q32)-positive multiple myeloma (MM) cell lines. To evaluate the role of the MYEOV gene in the proximal amplification core, we tested 946 breast tumors for copy number increase of MYEOV relative to neighboring genes or markers. RNA expression levels were studied in a subset of 72 tumors for which both RNA and DNA were available. Data presented here show that the MYEOV gene is amplified in 9.5% (90/946) and abnormally expressed in 16.6% (12/72) of breast tumors. Amplification patterns showed that MYEOV was most frequently coamplified with CCND1 (74/90), although independent amplification of MYEOV could also be detected (16/90). Abnormal expression levels correlated only partially with DNA amplification. MYEOV DNA amplification correlated with estrogen and progesterone receptor-positive cancer, invasive lobular carcinoma type and axillary nodal involvement. In contrast to CCND1 amplification, no association with disease outcome could be found. Our data suggest that MYEOV is a candidate oncogene activated in the amplification core located proximal to CCND1.
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Neoplasias da Mama/genética , Centrômero/genética , Ciclina D1/genética , Amplificação de Genes/genética , Proteínas Oncogênicas/genética , Oncogenes , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Cromossomos Humanos Par 11 , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Prognóstico , Proteínas Proto-Oncogênicas , RNA Neoplásico/análise , Células Tumorais CultivadasRESUMO
DNA amplifications at 11q13 are frequently observed in esophageal squamous cell carcinoma (ESC) and correlate with a malignant phenotype. Although this amplicon spans a region of several megabases and harbors numerous genes, CCND1 and EMS1 are thought to be the relevant candidates in ESC. We investigated whether the putative transforming gene MYEOV, mapping 360 kb centromeric to CCND1 and activated concomitantly with CCND1 in a subset of t(11;14)(q13;q32) positive multiple myeloma cell lines, represents a target of 11q13 amplification in ESC. To evaluate the role of MYEOV in ESC, we tested 31 ESC cell lines and 48 primary tumors for copy number levels of MYEOVand demonstrated that MYEOV was always coamplified with CCND1. However, MYEOV expression levels correlated only inconsistently with DNA amplification data. Treatment with the demethylating agent 5-aza-2'-deoxycytidine restored MYEOV expression in a subset of cell lines exhibiting DNA amplification without high MYEOV expression, suggesting that MYEOV is transcriptionally silenced by a DNA methylation mechanism in most of the latter cell lines. Our results indicate that MYEOV is a coamplified gene with CCND1 at 11q13, but its activation is sometimes inhibited by an epigenetic mechanism.
Assuntos
Azacitidina/análogos & derivados , Carcinoma de Células Escamosas/genética , Cromossomos Humanos Par 11/genética , Ciclina D1/genética , Neoplasias Esofágicas/genética , Amplificação de Genes/genética , Proteínas de Neoplasias/genética , Proteínas Oncogênicas/genética , Células Tumorais Cultivadas/efeitos dos fármacos , Azacitidina/farmacologia , Northern Blotting , Southern Blotting , Carcinoma de Células Escamosas/patologia , Ciclina D1/metabolismo , Metilação de DNA/efeitos dos fármacos , Decitabina , Neoplasias Esofágicas/patologia , Inativação Gênica/efeitos dos fármacos , Humanos , Proteínas Oncogênicas/metabolismo , Proteínas Proto-OncogênicasRESUMO
The BCR-ABL fusion, the molecular equivalent of the Philadelphia translocation, gains importance for treatment stratification in adult acute lymphoblastic leukemia (ALL). In this prospective study, samples from 478 patients with CD10(+) B-cell precursor ALL (c-ALL and pre-B ALL) underwent BCR-ABL reverse transcription-polymerase chain reaction (RT-PCR) analysis with double testing of positive samples. Patients were stratified according to the PCR result and treated in 2 German Multicenter Trials of Adult ALL. The outcome was followed and the prognostic impact of BCR-ABL was compared to clinical risk features. Of the 478 samples, 432 had an evaluable BCR-ABL result. Thirty-seven percent of the c-ALL and pre-B ALL patients were BCR-ABL(+) (p190, 77%; p210, 20%; simultaneous p190/p210, 3%). BCR-ABL positivity was associated with the high-risk features of older age (45 years versus 30 years median age; P =.0001) and higher white blood cell counts (23 500/microL versus 11 550/microL; P =.0001). Univariate and multivariate analyses revealed BCR-ABL as the leading factor for a poor prognosis (P =.0001) in comparison to clinical risk criteria. Irrespective of the breakpoint, presence of any BCR-ABL transcript predicted a lower chance of initial treatment response (68.4% versus 84.6%; P =.001) and a lower probability of disease-free survival at 3 years (0.13 versus 0.47; P =.0001). This bad outcome was not influenced by postinduction high-dose treatment stratifications. The results show a high prevalence of BCR-ABL fusion transcripts with predominance of p190. BCR-ABL RT-PCR is confirmed as a sensitive, rapid method to diagnose t(9;22), and p190 and p210 are unequivocally demonstrated as the most important predictors of poor long-term survival despite intensified chemotherapy.