RESUMO
This study presents a case of lymphocytic ganglioneuritis in a dog secondary to intervertebral disc extrusion that mimicked a peripheral nerve sheath tumour on magnetic resonance imaging. A four-year-old spayed female dachshund with lumbar pain was imaged via magnetic resonance. A tubular, space-occupying, contrast-enhancing lesion was noted in the right intervertebral foramen at L6 to L7. This was presumed to represent focal enlargement of the right sixth lumbar spinal nerve. A right-sided haemilaminectomy was performed at L6 to L7 and material that grossly resembled extruded nucleus pulposus was removed. The right L6 dorsal root ganglion, dorsal nerve root and proximal spinal nerve were severely enlarged and a partial thickness biopsy was collected from the dorsal root ganglion. Results of histopathological examination of the submitted tissue samples were consistent with extruded disc material and lymphocytic ganglioneuritis. To the author's knowledge, this is the first published report of lymphocytic ganglioneuritis secondary to intervertebral disc disease in a dog.
Assuntos
Doenças do Cão/patologia , Deslocamento do Disco Intervertebral/veterinária , Vértebras Lombares , Neurite (Inflamação)/veterinária , Animais , Diagnóstico Diferencial , Doenças do Cão/cirurgia , Cães , Feminino , Deslocamento do Disco Intervertebral/patologia , Deslocamento do Disco Intervertebral/cirurgia , Dor Lombar/veterinária , Imageamento por Ressonância Magnética/veterinária , Neoplasias de Bainha Neural/patologia , Neurite (Inflamação)/patologia , Neurite (Inflamação)/cirurgiaRESUMO
BACKGROUND: The reliability and validity of magnetic resonance imaging (MRI) for detecting neoplastic, inflammatory, and cerebrovascular brain lesions in dogs are unknown. OBJECTIVES: To estimate sensitivity, specificity, and inter-rater agreement of MRI for classifying histologically confirmed neoplastic, inflammatory, and cerebrovascular brain disease in dogs. ANIMALS: One hundred and twenty-one client-owned dogs diagnosed with brain disease (n = 77) or idiopathic epilepsy (n = 44). METHODS: Retrospective, multi-institutional case series; 3 investigators analyzed MR images for the presence of a brain lesion with and without knowledge of case clinical data. Investigators recorded most likely etiologic category (neoplastic, inflammatory, cerebrovascular) and most likely specific disease for all brain lesions. Sensitivity, specificity, and inter-rater agreement were calculated to estimate diagnostic performance. RESULTS: MRI was 94.4% sensitive (95% confidence interval [CI] = 88.7, 97.4) and 95.5% specific (95% CI = 89.9, 98.1) for detecting a brain lesion with similarly high performance for classifying neoplastic and inflammatory disease, but was only 38.9% sensitive for classifying cerebrovascular disease (95% CI = 16.1, 67.0). In general, high specificity but not sensitivity was retained for MR diagnosis of specific brain diseases. Inter-rater agreement was very good for overall detection of structural brain lesions (κ = 0.895, 95% CI = 0.792, 0.998, P < .001) and neoplastic lesions, but was only fair for cerebrovascular lesions (κ = 0.299, 95% CI = 0, 0.761, P = .21). CONCLUSIONS AND CLINICAL IMPORTANCE: MRI is sensitive and specific for identifying brain lesions and classifying disease as inflammatory or neoplastic in dogs. Cerebrovascular disease in general and specific inflammatory, neoplastic, and cerebrovascular brain diseases were frequently misclassified.
Assuntos
Encefalopatias/veterinária , Doenças do Cão/diagnóstico , Imageamento por Ressonância Magnética/veterinária , Animais , Encefalopatias/diagnóstico , Encefalopatias/patologia , Diagnóstico Diferencial , Doenças do Cão/patologia , Cães , Feminino , Imuno-Histoquímica , Imageamento por Ressonância Magnética/métodos , Masculino , Variações Dependentes do Observador , Reprodutibilidade dos Testes , Estudos Retrospectivos , Sensibilidade e EspecificidadeRESUMO
The t(8;21) translocation fuses the DNA-binding domain of the hematopoietic master regulator RUNX1 to the ETO protein. The resultant RUNX1/ETO fusion protein is a leukemia-initiating transcription factor that interferes with RUNX1 function. The result of this interference is a block in differentiation and, finally, the development of acute myeloid leukemia (AML). To obtain insights into RUNX1/ETO-dependant alterations of the epigenetic landscape, we measured genome-wide RUNX1- and RUNX1/ETO-bound regions in t(8;21) cells and assessed to what extent the effects of RUNX1/ETO on the epigenome depend on its continued expression in established leukemic cells. To this end, we determined dynamic alterations of histone acetylation, RNA Polymerase II binding and RUNX1 occupancy in the presence or absence of RUNX1/ETO using a knockdown approach. Combined global assessments of chromatin accessibility and kinetic gene expression data show that RUNX1/ETO controls the expression of important regulators of hematopoietic differentiation and self-renewal. We show that selective removal of RUNX1/ETO leads to a widespread reversal of epigenetic reprogramming and a genome-wide redistribution of RUNX1 binding, resulting in the inhibition of leukemic proliferation and self-renewal, and the induction of differentiation. This demonstrates that RUNX1/ETO represents a pivotal therapeutic target in AML.
Assuntos
Cromatina/genética , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Proteínas Proto-Oncogênicas/genética , Fatores de Transcrição/metabolismo , Translocação Genética , Acetilação , Sítios de Ligação , Fator de Ligação a CCAAT/genética , Fator de Ligação a CCAAT/metabolismo , Diferenciação Celular/genética , Linhagem Celular Tumoral , Cromatina/metabolismo , Cromossomos Humanos Par 21 , Cromossomos Humanos Par 8 , Análise por Conglomerados , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Perfilação da Expressão Gênica , Inativação Gênica , Histonas/metabolismo , Humanos , Mutação , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas/metabolismo , RNA Polimerase II/metabolismo , Proteína 1 Parceira de Translocação de RUNX1 , Fatores de Transcrição/genética , Ativação TranscricionalRESUMO
Early genetic events in the development of high-grade serous ovarian cancer (HGSOC) may define the molecular basis of the profound structural and numerical instability of chromosomes in this disease. To discover candidate genetic changes we sequentially passaged cells from a karyotypically normal hTERT immortalised human ovarian surface epithelial line (IOSE25) resulting in the spontaneous formation of colonies in soft agar. Cell lines transformed ovarian surface epithelium 1 and 4 (TOSE 1 and 4) established from these colonies had an abnormal karyotype and altered morphology, but were not tumourigenic in immunodeficient mice. TOSE cells showed loss of heterozygosity (LOH) at TP53, increased nuclear p53 immunoreactivity and altered expression profile of p53 target genes. The parental IOSE25 cells contained a missense, heterozygous R175H mutation in TP53, whereas TOSE cells had LOH at the TP53 locus with a new R273H mutation at the previous wild-type TP53 allele. Cytogenetic and array CGH analysis of TOSE cells also revealed a focal genomic amplification of CXCR4, a chemokine receptor commonly expressed by HGSOC cells. TOSE cells had increased functional CXCR4 protein and its abrogation reduced epidermal growth factor receptor (EGFR) expression, as well as colony size and number. The CXCR4 ligand, CXCL12, was epigenetically silenced in TOSE cells and its forced expression increased TOSE colony size. TOSE cells had other cytogenetic changes typical of those seen in HGSOC ovarian cancer cell lines and biopsies. In addition, enrichment of CXCR4 pathway in expression profiles from HGSOC correlated with enrichment of a mutated TP53 gene expression signature and of EGFR pathway genes. Our data suggest that mutations in TP53 and amplification of the CXCR4 gene locus may be early events in the development of HGSOC, and associated with chromosomal instability.
Assuntos
Transformação Celular Neoplásica/genética , Ovário/citologia , Receptores CXCR4/genética , Proteína Supressora de Tumor p53/genética , Animais , Células Epiteliais/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Cariotipagem , Perda de Heterozigosidade , Camundongos , Ovário/metabolismo , RNA MensageiroAssuntos
Linfócitos B/metabolismo , Cromossomos Humanos Par 21 , Cromossomos Humanos Par 8 , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Expressão Gênica , Leucemia Mieloide Aguda/genética , Proteínas de Fusão Oncogênica/genética , Fator de Transcrição Brn-3A/genética , Translocação Genética , Humanos , Proteína 1 Parceira de Translocação de RUNX1RESUMO
Massively parallel pyrosequencing allows sensitive deep sequencing to detect molecular aberrations. Thus far, data are limited on the technical performance in a clinical diagnostic setting. Here, we investigated as an international consortium the robustness, precision and reproducibility of amplicon next-generation deep sequencing across 10 laboratories in eight countries. In a cohort of 18 chronic myelomonocytic leukemia patients, mutational analyses were performed on TET2, a frequently mutated gene in myeloproliferative neoplasms. Additionally, hotspot regions of CBL and KRAS were investigated. The study was executed using GS FLX sequencing instruments and the small volume 454 Life Sciences Titanium emulsion PCR setup. We report a high concordance in mutation detection across all laboratories, including a robust detection of novel variants, which were undetected by standard Sanger sequencing. The sensitivity to detect low-level variants present with as low as 1-2% frequency, compared with the 20% threshold for Sanger-based sequencing is increased. Together with the output of high-quality long reads and fast run time, we demonstrate the utility of deep sequencing in clinical applications. In conclusion, this multicenter analysis demonstrated that amplicon-based deep sequencing is technically feasible, achieves high concordance across multiple laboratories and allows a broad and in-depth molecular characterization of cancer specimens with high diagnostic sensitivity.
Assuntos
Proteínas de Ligação a DNA/genética , Sequenciamento de Nucleotídeos em Larga Escala , Leucemia Mielomonocítica Crônica/genética , Mutação/genética , Proteínas Proto-Oncogênicas c-cbl/genética , Proteínas Proto-Oncogênicas/genética , Proteínas ras/genética , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Análise Mutacional de DNA , Dioxigenases , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Prognóstico , Proteínas Proto-Oncogênicas p21(ras)RESUMO
Historically, genes targeted by recurrent chromosomal deletions have been identified within the smallest genomic region shared in all patients, the minimally deleted region (MDR). However, deletions this small do not occur in all patients and are a simplification of the impact larger heterogeneous deletions have during carcinogenesis. We use the example of 13q14 deletions in chronic lymphocytic leukemia to show that genes outside MDRs are associated with disease progression. Genomic profiling of 224 patients identified 205 copy number alterations on chromosome 13 in 132 cases. Deletions including DLEU2 were heterogeneous (845 Kb-96.2 Mb) and identified two breakpoint cluster regions within short interspersed nuclear elements proximal to DLEU2 and within long interspersed nuclear elements/L1 repeats distal to GUCY1B2. After defining a deletion class on the basis of size and location, we show that (a) at diagnosis, larger deletions (class II) were associated with a significantly increased risk of disease progression (odds ratio=12.3; P=0.005), (b) in progressive patients, class II deletions were enriched (P=0.02) and (c) this association was independent of IgVH mutational status, ZAP70 expression and ATM/TP53 deletion. Deletion of a 1 Mb gene cluster (48.2-49.2 Mb), including SETDB2, PHF11 and RCBTB1, was significantly associated (P<0.01) with disease progression. Here, we show that the deletion of genes outside MDRs can influence clinical outcome.
Assuntos
Leucemia Linfocítica Crônica de Células B/genética , Deleção Cromossômica , Transtornos Cromossômicos/genética , Cromossomos Humanos Par 13/genética , Progressão da Doença , Dosagem de Genes , Humanos , Família Multigênica , Polimorfismo de Nucleotídeo Único , PrognósticoRESUMO
BACKGROUND: The incidence of human papillomavirus-associated vulval neoplasia is increasing worldwide; yet the associated genetic changes remain poorly understood. METHODS: We have used single-nucleotide polymorphism microarray analysis to perform the first high-resolution investigation of genome-wide allelic imbalance in vulval neoplasia. Our sample series comprised 21 high-grade vulval intraepithelial neoplasia and 6 vulval squamous cell carcinomas, with paired non-lesional samples used to adjust for normal copy number variation. RESULTS: Overall the most common recurrent aberrations were gains at 1p and 20, with the most frequent deletions observed at 2q, 3p and 10. Copy-neutral loss of heterozygosity at 6p was a recurrent event in vulval intraepithelial neoplasia. The pattern of genetic alterations differed from the characteristic changes we previously identified in cutaneous squamous cell carcinomas. Vulval neoplasia samples did not exhibit gain at 5p, a frequent recurrent aberration in a series of cervical tumours analysed elsewhere using an identical protocol. CONCLUSION: This series of 27 vulval samples comprises the largest systematic genome-wide analysis of vulval neoplasia performed to date. Despite shared papillomavirus status and regional proximity, our data suggest that the frequency of certain genetic alterations may differ in vulval and cervical tumours.
Assuntos
Alphapapillomavirus/fisiologia , Carcinoma in Situ/genética , Carcinoma de Células Escamosas/genética , Perfilação da Expressão Gênica/métodos , Neoplasias Vulvares/genética , Carcinoma in Situ/etiologia , Carcinoma in Situ/virologia , Carcinoma de Células Escamosas/etiologia , Carcinoma de Células Escamosas/virologia , Aberrações Cromossômicas , DNA Viral/análise , Feminino , Regulação Neoplásica da Expressão Gênica , Genômica/métodos , Papillomavirus Humano 16/fisiologia , Humanos , Perda de Heterozigosidade , Análise de Sequência com Séries de Oligonucleotídeos , Polimorfismo de Nucleotídeo Único , Neoplasias do Colo do Útero/genética , Neoplasias Vulvares/etiologia , Neoplasias Vulvares/virologia , Displasia do Colo do Útero/genéticaRESUMO
Although childhood high hyperdiploid acute lymphoblastic leukemia is associated with a favorable outcome, 20% of patients still relapse. It is important to identify these patients already at diagnosis to ensure proper risk stratification. We have investigated 11 paired diagnostic and relapse samples with single nucleotide polymorphism array and mutation analyses of FLT3, KRAS, NRAS and PTPN11 in order to identify changes associated with relapse and to ascertain the genetic evolution patterns. Structural changes, mainly cryptic hemizygous deletions, were significantly more common at relapse (P<0.05). No single aberration was linked to relapse, but four deletions, involving IKZF1, PAX5, CDKN2A/B or AK3, were recurrent. On the basis of the genetic relationship between the paired samples, three groups were delineated: (1) identical genetic changes at diagnosis and relapse (2 of 11 cases), (2) clonal evolution with all changes at diagnosis being present at relapse (2 of 11) and (3) clonal evolution with some changes conserved, lost or gained (7 of 11), suggesting the presence of a preleukemic clone. This ancestral clone was characterized by numerical changes only, with structural changes and RTK-RAS mutations being secondary to the high hyperdiploid pattern.
Assuntos
Deleção Cromossômica , Diploide , Genes ras/genética , Mutação/genética , Recidiva Local de Neoplasia/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Receptores Proteína Tirosina Quinases/genética , Adolescente , Evolução Biológica , Biomarcadores Tumorais/genética , Criança , Pré-Escolar , Células Clonais , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Recidiva Local de Neoplasia/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Polimorfismo de Nucleotídeo Único/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologiaRESUMO
Our previous work identified a chromosomal translocation t(4;6) in prostate cancer cell lines and primary tumors. Using probes located on 4q22 and 6q15, the breakpoints identified in LNCaP cells, we performed fluorescence in situ hybridization analysis to detect this translocation in a large series of clinical localized prostate cancer samples treated conservatively. We found that t(4;6)(q22;q15) occurred in 78 of 667 cases (11.7%). The t(4;6)(q22;q15) was not independently associated with patient outcome. However, it occurs more frequently in high clinical T stage, high tumor volume specimens and in those with high baseline PSA (P=0.001, 0.001 and 0.01, respectively). The t(4;6)(q22;q15) occurred more frequently in samples with two or more TMPRSS2:ERG fusion genes caused by internal deletion than in samples without these genomic alterations, but this correlation is not statistically significant (P=0.0628). The potential role of this translocation in the development of human prostate cancer is discussed.
Assuntos
Cromossomos Humanos Par 4/genética , Cromossomos Humanos Par 6/genética , Neoplasias da Próstata/genética , Translocação Genética , Instabilidade Genômica , Humanos , Hibridização in Situ Fluorescente , Masculino , Proteínas de Fusão Oncogênica/genética , Prognóstico , Neoplasias da Próstata/patologiaRESUMO
BACKGROUND: Oral submucous fibrosis (OSF) is a high-risk pre-cancerous condition where 7-13% of these patients develop head and neck squamous cell carcinoma (HNSCC). To date there is no cancer predictive markers for OSF patients. Genomic instability hallmarks early genetic events during malignant transformation causing loss of heterozygosity (LOH) and chromosomal copy number abnormality. However, to date there is no study on genomic instability in OSF. Although this condition is known as a high-risk pre-cancerous condition, there is no data regarding the genomic status of this disease in terms of genetic susceptibility to malignant transformation. METHODS: In this study, we investigated the existence of genetic signatures for carcinogenesis in OSF. We employed the high-resolution genome-wide Affymetrix Mapping single nucleotide polymorphism microarray technique to 'fingerprint' global genomic instability in the form of LOH in 15 patient-matched OSF-blood genomic DNA samples. RESULTS: This rapid high-resolution mapping technique has revealed for the first time that a small number of discrete hot-spot LOH loci appeared in 47-53% of the OSF tissues studied. Many of these LOH loci were previously identified regions of genomic instability associated with carcinogenesis of the HNSCC. CONCLUSION: To our knowledge, this is the first evidence that genomic instability in the form of LOH is present in OSF. We hypothesize that the genomic instability detected in OSF may play an important role in malignant transformation. Further functional association studies on these putative genes may reveal potential predictive oral cancer markers for OSF patients.
Assuntos
Transformação Celular Neoplásica/genética , Impressões Digitais de DNA , Perda de Heterozigosidade/genética , Fibrose Oral Submucosa/genética , Lesões Pré-Cancerosas/genética , Adulto , Idoso , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Feminino , Marcadores Genéticos , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Masculino , Análise em Microsséries , Pessoa de Meia-Idade , Fibrose Oral Submucosa/patologia , Polimorfismo de Nucleotídeo Único/genética , Lesões Pré-Cancerosas/patologiaRESUMO
To identify genomic abnormalities characteristic of pancreatic ductal adenocarcinoma (PDAC) in vivo, a panel of 27 microdissected PDAC specimens were analysed using high-density microarrays representing approximately 116 000 single nucleotide polymorphism (SNP) loci. We detected frequent gains of 1q, 2, 3, 5, 7p, 8q, 11, 14q and 17q (> or =78% of cases), and losses of 1p, 3p, 6, 9p, 13q, 14q, 17p and 18q (> or =44%). Although the results were comparable with those from array CGH, regions of those genetic changes were defined more accurately by SNP arrays. Integrating the Ensembl public data, we have generated 'gene' copy number indices that facilitate the search for novel candidates involved in pancreatic carcinogenesis. Copy numbers in a subset of the genes were validated using quantitative real-time PCR. The SKAP2/SCAP2 gene (7p15.2), which belongs to the src family kinases, was most frequently (63%) amplified in our sample set and its recurrent overexpression (67%) was confirmed by reverse transcription-PCR. Furthermore, fluorescence in situ hybridization and in situ RNA hybridization analyses for this gene have demonstrated a significant correlation between DNA copy number and mRNA expression level in an independent sample set (P<0.001). These findings indicate that the dysregulation of SKAP2/SCAP2, which is mostly caused by its increased gene copy number, is likely to be associated with the development of PDAC.
Assuntos
Cromossomos Humanos/genética , DNA de Neoplasias/genética , Dosagem de Genes , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Neoplasias Pancreáticas/genética , Polimorfismo de Nucleotídeo Único , Adenocarcinoma/genética , Adenocarcinoma/patologia , Adulto , Idoso , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Aberrações Cromossômicas , DNA de Neoplasias/metabolismo , Feminino , Humanos , Hibridização in Situ Fluorescente , Peptídeos e Proteínas de Sinalização Intracelular/genética , Perda de Heterozigosidade , Masculino , Microdissecção , Pessoa de Meia-Idade , Hibridização de Ácido Nucleico , Neoplasias Pancreáticas/patologia , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Single-nucleotide polymorphism (SNP) array analysis was performed using the 10K GeneChip array on a series of 26 paired follicular lymphoma (FL) and transformed-FL (t-FL) biopsies and the lymphoma cell lines SCI-1, DoHH2 and RL2261. Regions of acquired homozygosity were detected in 43/52 (83%) primary specimens with a mean of 1.7 and 3.0 aberrations in the FL and t-FL, respectively. A notable feature was the occurrence of recurring sites of acquired uniparental disomy (aUDP) on 6p, 9p, 12q and 17p in cell lines and primary samples. Homozygosity of 9p and 17p arose predominantly in t-FL and in three cases rendered the cell homozygous for a pre-existing mutation of either CDKN2A or TP53. These data suggest that mutation precedes mitotic recombination, which leads to the removal of the remaining wild-type allele. In all, 18 cases exhibited abnormalities in both FL and t-FL samples. In 10 cases blocks of homozygosity were detected in FL that were absent in the subsequent t-FL sample. These differences support the notion that FL and t-FL may arise in a proportion of patients by divergence from a common malignant ancestor cell rather than by clonal evolution from an antecedent FL.
Assuntos
Genoma Humano/genética , Linfoma Folicular/genética , Dissomia Uniparental , Adulto , Idoso , Linhagem Celular Transformada , Cromossomos , Homozigoto , Humanos , Pessoa de Meia-Idade , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Polimorfismo de Nucleotídeo Único , Recombinação GenéticaRESUMO
MicroRNAs (miRNAs) are short single-stranded RNAs that have a potentially important role in gene regulation. Using a quantitative real-time polymerase chain reaction assay specific to the mature miRNA, the expression level of a selected group of haematopoietic tissue-specific miRNAs was measured across a set of 30 primary adult acute myeloid leukaemia (AML) with a normal karyotype. The expression levels of each miRNA were correlated with the genome-wide mRNA expression profiles in the same leukaemias. This revealed that miR-181a correlated strongly with the AML morphological sub-type and with the expression of genes previously identified through sequence analysis as potential interaction targets. Three other miRNAs, miR-10a, miR-10b and miR-196a-1, showed a clear correlation with HOX gene expression.
Assuntos
Perfilação da Expressão Gênica , Leucemia Mieloide Aguda/genética , MicroRNAs/análise , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Genes Homeobox , Humanos , Leucemia Mieloide Aguda/classificação , Leucemia Mieloide Aguda/patologia , Masculino , Pessoa de Meia-IdadeAssuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/genética , Genes do Tumor de Wilms , Leucemia Mieloide/genética , Proteínas Mutantes/genética , Tirosina Quinase 3 Semelhante a fms/genética , Doença Aguda , Adulto , Idoso , Análise Mutacional de DNA , Primers do DNA , Éxons/genética , Reações Falso-Negativas , Feminino , Genótipo , Humanos , Cariotipagem , Leucemia Mieloide/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , Proteínas Mutantes/fisiologia , Reação em Cadeia da Polimerase , Estrutura Terciária de Proteína/genética , Sequências de Repetição em Tandem , Falha de TratamentoRESUMO
The chromosomal translocation t(8;21) is associated with 10-15% of all cases of acute myeloid leukaemia (AML). The resultant fusion protein AML1/MTG8 interferes with haematopoietic gene expression and is an important regulator of leukaemogenesis. We studied the effects of small interfering RNA (siRNA)-mediated AML1/MTG8 depletion on global gene expression in t(8;21)-positive leukaemic cell lines and in primary AML blasts using cDNA arrays, oligonucleotide arrays and real-time reverse transcription-polymerase chain reaction (RT-PCR). Suppression of AML1/MTG8 results in the increased expression of genes associated with myeloid differentiation, such as AZU1, BPI, CTSG, LYZ and RNASE2 as well as of antiproliferative genes such as IGFBP7, MS4A3 and SLA both in blasts and in cell lines. Furthermore, expression levels of several genes affiliated with drug resistance or indicative of poor prognosis AML (BAALC, CD34, PRG2, TSPAN7) are affected by AML1/MTG8 depletion. In conclusion, siRNA-mediated suppression of AML1/MTG8 cause very similar changes in gene expression pattern in t(8;21)-positive cell lines and in primary AML blasts. Furthermore, the results suggest that the specific targeting of AML1/MTG8 function may be a promising approach for complementing existing treatment strategies.
Assuntos
Diferenciação Celular/genética , Proliferação de Células , Cromossomos Humanos Par 21 , Cromossomos Humanos Par 8 , Subunidade alfa 2 de Fator de Ligação ao Core/fisiologia , Proteínas de Ligação a DNA/fisiologia , Regulação Neoplásica da Expressão Gênica/fisiologia , Leucemia Mieloide/genética , Proteínas Proto-Oncogênicas/fisiologia , RNA Interferente Pequeno/fisiologia , Fatores de Transcrição/fisiologia , Translocação Genética , Doença Aguda , Sequência de Bases , Linhagem Celular Tumoral , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Primers do DNA , Proteínas de Ligação a DNA/genética , Perfilação da Expressão Gênica , Humanos , Leucemia Mieloide/patologia , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Proto-Oncogênicas/genética , Proteína 1 Parceira de Translocação de RUNX1 , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/genéticaRESUMO
Although chromosome translocations are well-documented recurrent events in hematological malignancies and soft tissue sarcomas, their significance in carcinomas is less clear. We report here the molecular characterization of the reciprocal translocation t(1;15)(p22;q22) in the prostate carcinoma cell line, LNCaP. The chromosome 1 breakpoint was localized to a single BAC clone, RP11-290M5, by sequential FISH analysis of clones selected from the NCBI chromosome 1 map. This was further refined to a 580-bp region by Southern blot analysis. A 2.85-kb fragment spanning the der(1) breakpoint was amplified by long-range inverse PCR. The breakpoint on chromosome 1 was shown to lie between the CYR61 and the DDAH1 genes with the der(1) junctional sequence linking the CYR61 gene to the TSPAN3 (TM4SF8) gene on chromosome 15. Confirmatory PCR and FISH mapping of the der(15) showed loss of chromosome material proximal to the breakpoint on chromosome 15, containing the PSTPIP1 and RCN2 genes. On the available evidence we conclude that this translocation does not result in an in-frame gene fusion. Comparative expressed sequence hybridization (CESH) and comparative genomic hybridization (CGH) analysis, showed relative down-regulation of gene expression surrounding the breakpoint, but no gross change in genomic copy number. Real-time quantitative RT-PCR for genes around the breakpoint supported the CESH data. Therefore, here we may have revealed a gene down-regulation mechanism associated with a chromosome translocation, either through small deletion at the breakpoint or through another means of chromosome domain related gene regulation.
Assuntos
Cromossomos Humanos Par 15 , Cromossomos Humanos Par 1 , Neoplasias da Próstata/genética , Translocação Genética , Sequência de Bases , Southern Blotting , Proteínas de Ligação ao Cálcio/genética , Linhagem Celular Tumoral , Bandeamento Cromossômico , Mapeamento Cromossômico , Proteína Rica em Cisteína 61 , Primers do DNA , Humanos , Proteínas Imediatamente Precoces/genética , Hibridização in Situ Fluorescente , Peptídeos e Proteínas de Sinalização Intercelular/genética , Masculino , Dados de Sequência Molecular , Hibridização de Ácido Nucleico/métodos , Reação em Cadeia da Polimerase/métodosRESUMO
BACKGROUND: BCL2 is upregulated in nodal and extranodal B-cell non-Hodgkin's lymphomas, with a consequent antiapoptotic effect. However, loss of BCL2 has also been noted in some malignancies, suggesting a different molecular pathogenesis. OBJECTIVES: To investigate genomic and protein expression status of BCL2 and to compare the results with that of JUNB in primary cutaneous lymphomas (PCLs). METHODS: We analysed gene copy number of BCL2 and JUNB in 88 DNA samples from 80 patients with PCL consisting of Sézary syndrome/mycosis fungoides (SS/MF), primary cutaneous B-cell lymphoma (PCBCL) and primary cutaneous CD30+ anaplastic large cell lymphoma (C-ALCL) by the use of real-time polymerase chain reaction (PCR) and immunohistochemistry (IHC). Real-time PCR and IHC findings were subsequently compared with the results of additional fluorescent in situ hybridization (FISH) analysis of 23 cases of SS and Affymetrix cDNA expression microarray study of two primary cutaneous T-cell lymphoma (CTCL) cell lines. RESULTS: Real-time PCR analysis showed loss of BCL2 gene copy number in 22 of 80 PCL cases (28%), including 17 of 42 SS/MF, three of 13 C-ALCL and two of 33 PCBCL samples, and gain of BCL2 in four PCBCL samples. Gain of JUNB was identified in 18 of 71 PCL cases (25%), including nine of 35 SS/MF, seven of 13 C-ALCL and two of 31 PCBCL samples. IHC analysis revealed absent nuclear expression of BCL2 protein in 47 of 73 PCL cases, comprising 28 of 36 SS/MF, eight of eight C-ALCL and 11 of 29 PCBCL cases. In contrast, BCL2 protein expression was detected in 26 of 73 PCL cases, consisting of 18 of 29 PCBCL and eight of 36 SS/MF cases. JUNB protein expression was present in tumour cells from 30 of 33 of SS/MF and eight of eight C-ALCL, and was absent in tumour cells from 18 of 27 PCBCL cases. A comparison between BCL2 and JUNB revealed loss of BCL2 and gain of JUNB in five of 35 SS/MF samples, and expression of JUNB protein and absent BCL2 expression in 25 SS/MF and eight of eight C-ALCL cases. In contrast, expression of BCL2 and absent JUNB expression were detected in 67% of PCBCL cases. Additional FISH analysis revealed deletion of BCL2 in 19 of 23 SS cases (83%), including eight cases with BCL2 loss shown by real-time PCR. Furthermore, Affymetrix expression microarray demonstrated decreased expression of proapoptotic and antiapoptotic genes involved in BCL2 signalling pathways such as BOK, BIM, HRK, RASA1 and STAT2 in two CTCL cell lines with BCL2 loss and absent BCL2 expression. Increased expression of JUNB was also identified in the MF cell line. CONCLUSIONS: These findings provide a comprehensive assessment of BCL2 and JUNB status in PCL, and suggest that there is a selection pressure in a subset of CTCL cases for tumour cells showing BCL2 loss and upregulation of JUNB primarily through chromosomal deletion and amplification, respectively.
Assuntos
Genes bcl-2 , Genes jun , Linfoma/genética , Neoplasias Cutâneas/genética , Humanos , Técnicas Imunoenzimáticas , Hibridização in Situ Fluorescente , Linfoma/metabolismo , Linfoma de Células B/genética , Linfoma de Células B/metabolismo , Linfoma Anaplásico de Células Grandes/genética , Linfoma Anaplásico de Células Grandes/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Reação em Cadeia da Polimerase/métodos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Síndrome de Sézary/genética , Síndrome de Sézary/metabolismo , Neoplasias Cutâneas/metabolismo , Células Tumorais CultivadasRESUMO
The Mixed Lineage Leukemia (MLL) gene has been identified in 11q23 translocations. The aim of the present study is the investigation of the frequency of MLL gene rearrangements in cases of de novo myelodysplastic syndromes (MDS). Sixty-two patients with de novo MDS were included in the analysis. The detection of MLL gene rearrangements was performed by Southern blot. Clonal karyotypic abnormalities were found in 15/50 (30%) cases. 11q23 abnormalities were not detected. One case with RAEB and a complex karyotype presented a del (11)(q13); further analysis by FISH revealed loss of one copy of MLL gene in all metaphases. Southern blot revealed germline bands in all cases using Eco RI and in 61/62 cases with Bam HI. The case with RAEB and a del (11)(q13) revealed a rearranged band following only Bam HI digestion, but not Eco RI. Rearrangements of MLL gene within exons 5-9 were not identified in this series of adult de novo MDS, indicating that this molecular abnormality is not involved in the pathogenesis of this group of hemopoietic disorders.
Assuntos
Proteínas de Ligação a DNA/genética , Síndromes Mielodisplásicas/genética , Proto-Oncogenes , Fatores de Transcrição , Southern Blotting , Cromossomos Humanos Par 11/genética , Rearranjo Gênico/genética , Histona-Lisina N-Metiltransferase , Humanos , Hibridização in Situ Fluorescente , Cariotipagem , Síndromes Mielodisplásicas/patologia , Proteína de Leucina Linfoide-Mieloide , Proteínas de Neoplasias/genéticaRESUMO
Histone deacetylase (HDAC) and histone acetyltransferase (HAT) functions are associated with various cancers, and the inhibition of HDAC has been found to arrest disease progression. Here, we have investigated the gene expression profiles of leukaemic cells in response to the HDAC inhibitor trichostatin A (TSA) using oligonucleotide microarrays. Nucleosomal histone acetylation was monitored in parallel and the expression profiles of selected genes were confirmed by quantitative polymerase chain reaction (PCR). A large number of genes (9% of the genome) were found to be similarly regulated in CCRF-CEM and HL-60 cells in response to TSA, and genes showing primary and secondary responses could be distinguished by temporal analysis of gene expression. A small fraction of genes were highly sensitive to histone hyper-acetylation, including XRCC1, HOXB6, CDK10, MYC, MYB, NMI and CBFA2T3 and many were trans-acting factors relevant to cancer. The most rapidly repressed gene was MKRN3, an imprinted gene involved in the Prader-Willi syndrome.