RESUMO
In a separate study (F. Courjal et al., Cancer Res., 57: 4360-4367, 1997), we have analyzed by Southern blotting the relationship between DNA amplification and clinicopathological features of breast cancer. Six regions of recurrent amplifications were tested (8p12, 8q24, 11q13, 12q13, 17q12, and 20q13), and the results suggested that there was a relationship between DNA amplification profiles and breast tumor phenotype. We had delineated three subgroups of tumors showing distinct DNA amplification profiles and clinicopathological characteristics: group A, tumors showing amplification at 11q13 and/or 8p12 and/or 20q13; group B, tumors amplified at ERBB2 and/or MYC and/or MDM2/SAS; and group C, tumors with no detectable amplification. The aim of the present work was to characterize extensively the amplification profiles in the different subgroups of tumors. Sixty-one breast tumors distributed in all three subgroups were studied by comparative genomic hybridization (CGH). There was an overall good agreement between Southern blotting results and CGH data. As expected, CGH revealed gains undetected by Southern blotting. Most of these gains occurred in regions for which no adapted probes were available but also revealed nondetected amplifications at 8q24 or 20q13. Tumors showed multiple aberrations with a medium number of 5.6 copy number variations/tumor, whereas, according to Southern blotting results, 38% of the tumors analyzed were devoid of any amplification. This proportion fell to 6.5% after CGH analysis. Recurrent gains were observed in tumors from all three subgroups, albeit at varying incidences, and involved 1q, 8q, 17q23-q24, and 20q13. Gains covered large regions of DNA and could possibly include several cores of amplification. Some events, such as gains at 16p11-p12 and 14q or losses at 22q, showed more restricted distributions, suggesting the existence of additional sets of preferential coamplifications. The complexity of genetic profiles revealed by CGH indicates that breast cancer development depends on a large (yet undetermined) number of genetic events. The description of molecular phenotypes in breast cancer may therefore prove to be complex, and it should be interesting to see how many breast tumor subtypes will be defined in the end.
Assuntos
Neoplasias da Mama/genética , Cromossomos Humanos/genética , DNA de Neoplasias/genética , Amplificação de Genes , Hibridização de Ácido Nucleico , Adulto , Idoso , Southern Blotting , Neoplasias da Mama/classificação , Neoplasias da Mama/patologia , Cromossomos Humanos/ultraestrutura , Feminino , Humanos , Processamento de Imagem Assistida por Computador , Pessoa de Meia-Idade , Fenótipo , Receptor ErbB-2/genéticaRESUMO
DNA amplification is frequent in breast cancer and has been associated with specific clinicopathological parameters and/or worsened course of the disease. In the present work, we were interested in further defining the association linking the occurrence of DNA amplification to the emergence of specific breast tumor phenotype. To this aim, we studied by Southern blotting a total of 1875 breast tumor DNAs with 26 probes mapping at 15 distinct chromosomal localizations. Of the 26 loci tested, 11 loci showed elevated levels of amplification, 9 loci showed occasional and/or low level of DNA copy number increase, and 6 loci showed very rare or no variation. This allowed us to define six amplified domains mapping at 8p12, 8q24, 11q13, 12q13, 17q12, and 20q13.2, respectively. Over 60% of the tumors analyzed presented at least one amplification at one of these localizations. Amplifications often covered large regions of DNA and bore complex patterns involving coamplification of several colocalized markers. Statistical analysis revealed correlations associating DNA amplification with breast tumor phenotype, as well as sets of preferential coamplifications. Based on these correlations, we defined three subsets of breast cancer according to their patterns of DNA amplification. The first subset (group A) was organized around the amplifications at 11q13 and/or 8p12 and was predominantly composed of estrogen receptor-positive tumors and presented a large proportion of lobular cancers. The second subset (group B) was organized around the amplifications of ERBB2 and/or MYC. These tumors were mostly estrogen receptor-negative and of the ductal invasive type. The third subset (group C) corresponded to tumors in which no amplification was detected in the present screen. Tumors in this group were largely diploid and of low histopathological grading.
Assuntos
Neoplasias da Mama/genética , Cromossomos Humanos/genética , DNA de Neoplasias/genética , Amplificação de Genes , Adulto , Idoso , Southern Blotting , Neoplasias da Mama/classificação , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/patologia , Carcinoma Lobular/genética , Carcinoma Lobular/patologia , Mapeamento Cromossômico , Cromossomos Humanos/ultraestrutura , Sondas de DNA , Estrogênios , Feminino , Genes myc , Marcadores Genéticos , Humanos , Pessoa de Meia-Idade , Invasividade Neoplásica , Neoplasias Hormônio-Dependentes/genética , Neoplasias Hormônio-Dependentes/patologia , Fenótipo , Receptor ErbB-2/genéticaRESUMO
Breast cancer heterogeneity can be related directly to its variability at the genetic level. Thus, tumor genotyping could be a valuable approach to define breast tumor subtypes. It has been shown that it is possible to delineate subgroups of breast tumors according to specific sets of DNA amplifications. The aim of the present work was to study the prognostic significance of these DNA amplifications. We studied DNA amplification at eight genes or loci (AIB1, CCND1, EMS1, ERBB2, FGFR1, MDM2, MYC, and RMC20C001) as well as p53 mutations in a series of 640 breast cancer patients who had not received presurgical therapy and analyzed the correlations with survival DNA amplification was assessed by Southern blotting and was scored positive when exceeding three to five copies. Mutations in the p53 gene were searched by four-color fluorescent single. strand conformational polymorphism, using an automated sequencer. Of the nine genetic alterations tested, four (CCND1, EMS1, FGFR1, and p53 mutations) showed a significant association with reduced disease-free (DFS) and/or overall survival (OVS) in the unselected set of patients by univariate test. Correlations for p53 were found only when selecting mutations in exons 5 or 7. Analysis of node-negative and -positive subgroups of patients showed that MDM2 amplification and p53 mutations bore prognostic significance in node-negative patients, whereas amplification of CCND1, EMS1, and FGFR1 correlated with poor outcome in node-positive patients. Multivariate analysis on an unselected set of patients retained significance for the amplification of EMS1, FGFR1, and MDM2 with DFS, of CCND1 with OVS, and of RMC20C001 with both DFS and OVS. Interestingly, stratified analysis according to nodal status confirmed results obtained in the univariate tests: significance of MDM2 amplification and p53 mutations in node-negative and that of CCND1, EMS1, and FGFR1 in node-positive patients. We also observed an association between the number of genetic alterations observed in a tumor and poor prognosis. Patients with two or more amplified loci had a worsened outcome. Strongly correlating coamplifications such as CCND1 and FGFR1, as well as ERBB2 and MYC, were associated with a significant reduction of patient survival, thus indicating cooperative effects. Our data support the idea that genetic alterations in breast cancer are not only helpful for phenotyping purposes, but can also represent powerful prognostic indicators in the clinical practice.
Assuntos
Neoplasias da Mama/genética , Amplificação de Genes , Genes p53 , Mutação , Proteínas Nucleares , Neoplasias da Mama/mortalidade , Mapeamento Cromossômico , Ciclina D1/genética , Feminino , Genes erbB-2 , Genes myc , Genótipo , Humanos , Análise Multivariada , Fenótipo , Prognóstico , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-mdm2RESUMO
Chromosome 17q is frequently rearranged in breast cancer. Allelotyping studies have proposed the existence of at least four regions of allelic imbalance (AI). Here we present a study combining allelotyping using 19 CA repeat markers mapping in the 17q21-25 region and molecular cytogenetics (CGH and FISH). Allelotyping was undertaken on 178 pairs of cognate tumor and normal DNA in order to determine the number of regions of AI and define the shortest overlaps. AI ranged from 34-54% of the informative cases according to the marker and, overall, 66% of the tumors presented AI at one of the markers tested. Analysis of the patterns of imbalances revealed at least five common regions of imbalance respectively defined by markers: D17S855, which is intragenic of BRCA1 (SRO 1), D17S1607 (SRO 2), D17S1855 (SRO 3), between D17S789 and D17S785 (SRO 4) and D17S784 (SRO 5). In order to characterize the nature of the genetic events revealed by allelotyping we performed CGH analysis on a subset of 43 tumors presenting variable patterns of imbalance. CGH showed that AI at 17q could represent four different types of genetic events: loss of chromosome 17, gain of 17q, gain of 17q22-q24, loss of 17q11-q21 and/or 17q25-qter. Some of these anomalies could occur concomitantly within the same tumor. Since 35% of the tumors analysed by CGH presented gains, these data indicated that AI at 17q were not solely indicative of losses of genetic material and could also represent DNA amplification. Gains were most commonly observed in the 17q23-q24 regions. This suggested that AI in SRO 2 and SRO 3 corresponded to DNA amplification. To assess this, we isolated BAC clones by PCR screening for markers D17S1607 and D17S1855 and used these in FISH experiments on six breast tumor cell lines and 14 breast cancer specimens. FISH results showed that both D17S1607 and D17S1855 were frequently involved in DNA amplification (8-30 copies). Altogether, our data show that allelotyping can be efficiently used in amplicon mapping. Clinico-pathological correlations indicated that imbalance at 17q preferentially occurred in high grade, PR- and ERBB2 amplified tumors.
Assuntos
Neoplasias da Mama/genética , Carcinoma Intraductal não Infiltrante/genética , Carcinoma Lobular/genética , Aberrações Cromossômicas , DNA de Neoplasias/genética , Adulto , Idoso , Alelos , Carcinoma/genética , Feminino , Humanos , Hibridização in Situ Fluorescente , Pessoa de Meia-Idade , Monossomia , Hibridização de Ácido NucleicoRESUMO
The EWS/TEC gene fusion generated by the t(9;22) chromosomal translocation found in extraskeletal myxoid chondrosarcomas encodes a fusion protein containing the amino-terminal domain of the EWS protein fused to the whole coding sequence of the orphan nuclear receptor TEC. We have compared the DNA-binding and transcriptional activation properties of various TEC isoforms and the corresponding EWS/TEC fusion proteins. Band-shift experiments show that the full-length TEC receptor can efficiently bind the NGFI-B Response Element (NBRE), whereas an isoform lacking the entire carboxyl-terminal domain of the receptor binds much less efficiently the NBRE. Addition of the amino-terminal domain of EWS to either isoforms does not alter significantly their DNA-binding properties to the NBRE. Co-transfection experiments of COS cells and human chondrocytes indicate that whereas TEC moderately activates transcription from a NBRE-containing promoter, the corresponding EWS/TEC fusion protein is a highly potent transcriptional activator of the same promoter, being approximately 270-fold more active than the native receptor. EWS/TEC may thus exert its oncogenic potential in chrondrosarcomas by activating the transcription of target genes involved in cell proliferation.
Assuntos
Condrossarcoma/metabolismo , Cromossomos Humanos Par 22 , Cromossomos Humanos Par 9 , Proteínas de Ligação a DNA/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Elementos de Resposta/genética , Sarcoma de Ewing/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Translocação Genética , Animais , Fusão Gênica Artificial , Células COS , Condrossarcoma/genética , Humanos , Proteínas de Neoplasias/genética , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares , Proteínas de Fusão Oncogênica/genética , Isoformas de Proteínas , Receptores Citoplasmáticos e Nucleares/genética , Receptores de Esteroides , Transativadores/genéticaRESUMO
Deletions and rearrangements involving chromosome 6q have been reported in a number of human cancers such as ovarian and breast tumors as well as melanoma and hemopoietic malignancies. To gain insight into the regions undergoing deletions on the long arm of chromosome 6, we performed a survey of loss of heterozygosity (LOH) at 11 CA repeat markers, mapping from 6q13 to 6q27 in 83 matched sets of tumor and blood DNAs from breast tumor patients. LOH was observed at all tested markers with frequencies ranging from 11.4 to 39.8% of the informative cases, whereas D2S123, a marker linked to the HMSH2 gene mapping in a region rarely presenting allele losses, showed 3.2%. LOH patterns were often complex, with a number of tumors presenting multiple interstitial losses, thus indicating that chromosome 6q can be severely rearranged in breast cancer. Patterns of losses and correlation between LOH occurring at adjacent markers suggested the existence of three (possibly four) distinct regions of allele losses. These were defined by: D6S251-D6S434 (6q13), D6S292-D6S310-D6S314-D6S311 (6q24-q25), and D6S441-D6S281 (6q27). The fourth and more hypothetical region was in the 6q21 region and defined by D6S287-D6S407. Interestingly, the region at 6q24-q25 defined by D6S292-D6S310-D6S314-D6S311 was predominantly observed in evolved and aggressive breast tumors. LOH at D6S314 was correlated with PR- tumors. All together, our data suggest the possible presence of several genes on 6q whose alteration may play a role in breast cancer formation and development.
Assuntos
Neoplasias da Mama/genética , Cromossomos Humanos Par 6/genética , Perda de Heterozigosidade , Alelos , Neoplasias da Mama/patologia , Deleção Cromossômica , DNA de Neoplasias/análise , DNA de Neoplasias/genética , Repetições de Dinucleotídeos , Feminino , Marcadores Genéticos , Humanos , Pessoa de Meia-IdadeRESUMO
The CCND2 and FGF6 genes are both located at band p13 of human chromosome 12. The two genes are coamplified in some cases (3.6%) of breast tumors. They are physically linked within a 500 kb genomic segment. Thus, both 11q13 and 12p13 chromosomal regions host related linked cyclin and FGF genes, and can be amplified in human tumors. This strengthens the hypothesis of genome duplication involving chromosomes 11 and 12.
RESUMO
Members of the Rho/Rac/Cdc42Hs family of GTPases have been shown to participate in many aspects of the signaling of cell growth and differentiation. Although the biochemical properties of these GTPases have been extensively studied, very little is known about the structure of the corresponding genes. To gain insight on the evolution of the Rho family, we were interested in studying the genomic structure of several members. We report here the structure and the localization to 22q12 of the human Rac2 gene, as well as the localization to 17qter of Rac3, a new member closely related to Rac1 and Rac2. Unlike the structure of its closest relative ARH-G gene, which contains a single intron, Rac2 is made of at least 7 exons, spanning over 18 kb of DNA. Comparison of gene structure and exonic borders suggests that the emergence of the whole superfamily took place early during evolution.
Assuntos
Cromossomos Humanos Par 17/genética , Cromossomos Humanos Par 22/genética , GTP Fosfo-Hidrolases , Proteínas de Ligação ao GTP/genética , Sequência de Aminoácidos , Proteínas de Ciclo Celular/genética , Mapeamento Cromossômico , Cromossomos Humanos Par 17/ultraestrutura , Cromossomos Humanos Par 22/ultraestrutura , Evolução Molecular , Éxons , Humanos , Hibridização in Situ Fluorescente , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/genética , Proteína cdc42 de Ligação ao GTP , Proteínas rac de Ligação ao GTP , Proteínas rho de Ligação ao GTPRESUMO
Evidence of the involvement of cyclin genes in genetic alterations in human cancer is growing. In the present study, we investigated the amplification, in human breast and ovarian cancer, of 5 cyclin genes; cyclin A, cyclin D1, cyclin D2, cyclin D3 and cyclin E. For this purpose, a series of 1,171 breast and 237 ovarian tumors tested for DNA amplification by Southern blotting and a subset of 132 breast and 22 ovarian cancers were analyzed for RNA expression levels by slot-blot and Northern blotting. In breast tumors, only cyclin D1 was found to be activated in a sizeable fraction of the tumors (amplification 12.6%, overexpression 19%). Cyclin A, D2, D3, and E genes never, or only on rare occasions, showed increased DNA copy numbers and were never found overexpressed at the RNA level. Amplification of cyclin D1 correlated with ER+ breast cancer and the presence of lymph-node metastasis. Interestingly, we were also able to determine an association with invasive lobular carcinoma. Our data suggest that cyclin D1 activation determines the evolution of a particular subset of estrogen-responsive tumors. Data obtained in ovarian tumors contrasted with observations in breast cancer. Cyclin D1 DNA amplification was much less frequent in ovarian than in breast tumors (3.3% vs. 12.6%), whereas cyclin E amplification and overexpression were observed in a significant number of cases (12.5% and 18.0% respectively). Cyclin A, cyclin D2 and D3 rarely showed anomalies at the DNA level and were never overexpressed. No clear correlation could be observed between amplification of the cyclin E gene and tumor type, stage or grade in ovarian cancer. Data presented here suggest distinct pathways of cyclin activation in human breast and ovarian cancer.
Assuntos
Neoplasias da Mama/genética , Ciclinas/genética , Amplificação de Genes , Expressão Gênica , Proteínas Oncogênicas/genética , Neoplasias Ovarianas/genética , Proteínas Proto-Oncogênicas , Northern Blotting , Southern Blotting , Ciclina D1 , DNA de Neoplasias/análise , Feminino , Humanos , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas c-akt , RNA Mensageiro/metabolismo , Receptores de Estrogênio/análise , Células Tumorais CultivadasRESUMO
The spontaneous expression of HLA class I and class II molecules in two human breast carcinoma cell lines (MCF7, T47D) and their modulation during epidermal growth factor treatment are reported. Transcription was analysed by Northern blot and hybridisation with HLA class II and class I cDNA specific probes. The expression of cell surface determinants was examined by internal protein labelling with 35s-methionine, immunoprecipitation with monoclonal antibodies specific for HLA class I or class II, followed by isolation of the immune complex on protein A-Sepharose; at least a quantification of glycoprotein was performed by chromatofocusing. Glycoprotein quantification showed a significant increase of HLA class I and class II (DR) antigen expression after stimulation by epidermal growth factor (0.02 microgram ml-1) in the two cell lines, when compared with untreated cell controls. However, with epidermal growth factor treatment of MCF7 and T47D cells, low increases in the amounts of HLA class I and class II RNA were obtained. These differences between expressed antigens and correspondent RNA amounts would be explained by the fact that EGF in these two cell lines acts more in post-transcription for HLA class I and class II antigens.
Assuntos
Fator de Crescimento Epidérmico/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes MHC da Classe II , Genes MHC Classe I , Adenocarcinoma/genética , Adenocarcinoma/imunologia , Northern Blotting , Neoplasias da Mama/genética , Neoplasias da Mama/imunologia , Linhagem Celular , Sondas de DNA , Feminino , Antígenos HLA-D/genética , Antígenos de Histocompatibilidade Classe I/genética , Humanos , RNA Neoplásico/genética , RNA Neoplásico/isolamento & purificaçãoRESUMO
The authors report the immunogenotype of two cases of Richter's syndrome. The immunoglobulin gene rearrangement pattern obtained on Southern Blot analysis was found in both cases to be the same in leukemic blood cells and in the tissue involved by the lymphoma. The beta chain and gamma chain T-cell receptor gene rearrangement pattern exhibited a germ-line configuration in the peripheral blood cells and in the lymph node in Case 2, whereas in Case 1 the lymph node had a gene rearrangement in the beta chain, as well as in the gamma chain T-cell receptor, and the leukemic cells from bone marrow were found to be in a germ-line configuration for T-cell receptors (beta and gamma chains).
Assuntos
DNA de Neoplasias/análise , Leucemia Linfocítica Crônica de Células B/complicações , Linfoma Difuso de Grandes Células B/genética , Idoso , Southern Blotting , Células Clonais , Genótipo , Humanos , Linfoma Difuso de Grandes Células B/etiologia , Masculino , Hibridização de Ácido Nucleico , SíndromeRESUMO
Nineteen French breast and breast-ovarian cancer families were tested for linkage with five chromosome 17q markers. The five breast-ovarian cancer families as a group give positive evidence for linkage, whereas the 14 breast cancer-only families do not. Heterogeneity of linkage of breast and breast-ovarian cancers is significant in France and supports the existence of more than one susceptibility gene.
Assuntos
Neoplasias da Mama/genética , Mapeamento Cromossômico/métodos , Cromossomos Humanos Par 17 , Proto-Oncogenes , Adulto , Idoso , Distribuição de Qui-Quadrado , Saúde da Família , Feminino , França , Ligação Genética , Marcadores Genéticos , Predisposição Genética para Doença , Variação Genética , Humanos , Escore Lod , Masculino , Pessoa de Meia-Idade , Síndromes Neoplásicas Hereditárias/genética , Neoplasias Ovarianas/genética , Linhagem , Polimorfismo de Fragmento de RestriçãoRESUMO
DNA amplification seems to be particularly frequent in human breast tumours and has been associated with cancer evolution and aggressiveness. Recent data indicate that new events should be added to the list, such as the amplifications at chromosome 20q13 or the MDM2 gene. The present work aimed at determining the incidence and clinicopathological signification of these amplifications in a large series of breast and ovarian tumours. We tested 1371 breast and 179 ovarian tumours by Southern blotting and observed amplification of 20q13 in 5.4% breast and 2.8% ovarian carcinomas, whereas MDM2 was found amplified in 5.3% and 3.8% of breast and ovarian tumours respectively. MDM2 RNA expression levels were analysed in a subset of 57 breast tumours and overexpression was observed in 4/57 (7%) of the tumours. Elevated expression levels coincided with amplification of the gene. In breast cancer, 20q13 and MDM2 amplifications seem to define subsets of aggressive tumours. Indeed, 20q13 was correlated to axillary nodal involvement and occurred preferentially in younger patients (< 50 years). Furthermore, 20q13 correlated, as did MDM2 amplification, to aneuploidy. In parallel, we had also tested our tumour DNAs for amplification of CCND1, ERBB-2 and MYC, which made it possible to test for correlations with 20q13 or MDM2 amplifications. Whereas 20q13 showed a very strong correlation to CCND1 amplification, that of MDM2 was prevalent in MYC-amplified tumours. Interestingly, 20q13 and MDM2 amplifications showed some degree of correlation to each other, which may possibly be owing to the fact that both events occurred preferentially in aneuploid tumours. In ovarian cancer, no statistically significant correlation was observed. However, 20q13 amplification occurred preferentially in stage 3 tumours and MDM2 was correlated to ERBB-2 amplification. This may suggest that in ovarian tumours also, 20q13 and MDM2 amplifications occur in late or aggressive cancers.