RESUMO
Expression profiling studies have suggested that HER2-amplified breast cancers constitute a heterogeneous group that may be subdivided according to their ER status: HER2-amplified ER-positive breast carcinomas that fall into the luminal B cluster; and HER2-amplified ER-negative cancers which form a distinct molecular subgroup, known as the erbB2 or HER2 subgroup. ER-negative breast cancer differs significantly from ER-positive disease in the pattern, type, and complexity of genetic aberrations. Here we have compared the genomic profiles of ER-positive and ER-negative HER2-amplified cancers using tiling path microarray-based comparative genomic hybridization (aCGH). Validation of the differentially amplified regions was performed in an independent series of 70 HER2-amplified breast cancers. Although HER2-amplified cancers had remarkably complex patterns of molecular genetic aberrations, ER-positive and ER-negative HER2-amplified breast carcinomas shared most molecular genetic features as defined by aCGH. Genome-wide Fisher's exact test analysis revealed that less than 1.5% of the genome was significantly differentially gained or lost in ER-positive versus ER-negative HER2-amplified cancers. However, two regions of amplification were significantly associated with ER-positive carcinomas, one of which mapped to 17q21.2 and encompassed GJC1, IGFBP4, TNS4, and TOP2A. Chromogenic in situ hybridization analysis of an independent validation series confirmed the association between ER status and TOP2A amplification. In conclusion, although hormone receptor status does not determine the overall genetic profile of HER2-amplified breast cancers, specific genetic aberrations may be characteristic of subgroups of HER2 breast cancers.
Assuntos
Neoplasias da Mama/genética , Perfilação da Expressão Gênica , Genes erbB-2 , Análise de Sequência com Séries de Oligonucleotídeos , Receptores de Estrogênio/genética , Antígenos de Neoplasias/genética , Neoplasias da Mama/patologia , DNA Topoisomerases Tipo II/genética , Proteínas de Ligação a DNA/genética , Feminino , Amplificação de Genes , Humanos , Hibridização In Situ/métodos , Proteínas de Ligação a Poli-ADP-RiboseRESUMO
AIMS: Acinic cell carcinomas (ACCs) and secretory carcinomas (SCs) of the breast are rare, low-grade malignancies that preferentially affect young female patients. Owing to the morphological and immunohistochemical similarities between these lesions, they have been proposed to be two morphological variants of the same entity. It has been demonstrated that SCs of the breast consistently harbour the t(12;15)ETV6-NTRK3 translocation. The aim was to determine whether ACCs also harbour ETV6 gene rearrangements and are thus variants of SCs. METHODS AND RESULTS: Using the ETV6 fluorescence in situ hybridization DNA Probe Split Signal (Dako), the presence of ETV6 rearrangements in three SCs and six ACCs was investigated. Cases were considered as harbouring an ETV6 gene rearrangement if >10% of nuclei displayed 'split apart signals' (i.e. red and green signals were separated by a distance greater than the size of two hybridization signals). Whereas the three SCs displayed ETV6 split apart signals in >10% of the neoplastic cells, no ACC showed any definite evidence of ETV6 gene rearrangement. CONCLUSIONS: Based on the lack of ETV6 rearrangements in ACCs, our results strongly support the concept that SCs and ACCs are distinct entities and should be recorded separately in breast cancer taxonomy schemes.
Assuntos
Neoplasias da Mama/genética , Carcinoma de Células Acinares/genética , Rearranjo Gênico , Hibridização in Situ Fluorescente , Proteínas Proto-Oncogênicas c-ets/genética , Proteínas Repressoras/genética , Neoplasias da Mama/patologia , Carcinoma de Células Acinares/patologia , DNA de Neoplasias/análise , Feminino , Humanos , Proteínas Proto-Oncogênicas c-ets/metabolismo , Proteínas Repressoras/metabolismo , Variante 6 da Proteína do Fator de Translocação ETSRESUMO
The effect of cigarette smoke and nicotine on duodenal mucosal bicarbonate secretion (DMBS) was studied in rats. Cigarette smoke but not intravenous nicotine administered acutely to anesthetized rats via a tracheostomy tube stimulated DMBS by 47 +/- 6%. The increase was neurally mediated via atropine-sensitive postganglionic cholinergic neurons. Introduction of cigarette smoke after the infusion of vasoactive intestinal peptide and porcine histidine isoleucine (PHI) also caused a delayed increase in DMBS. However, the magnitude of this increase was similar to that seen in control non-peptide-infused rats. The increase in bicarbonate secretion predominantly involved Brunner's glands. Rats exposed to cigarette smoke for 4 and 8 days before direct instillation of smoke via tracheostomy tube did not show any increase in their DMBS. These studies indicate that in the rat, cigarette smoke increases DMBS, most likely secreted by the Brunner's glands. The increase is neurally mediated via atropine-sensitive postganglionic cholinergic neurons. Gastroenteric neuropeptides do not exert any influence on cigarette smoke-mediated DMBS secretion in the rat. Unlike acute exposure to cigarette smoke, chronic exposure (4 and 8 days) of rats to cigarette smoke abolishes increase in DMBS induced by subsequent exposure to cigarette smoke. This last observation may, in part, may explain the tendency of chronic smokers who have duodenal bulb ulcers to show greater propensity to higher rate of recurrence and protracted healing.
Assuntos
Bicarbonatos/metabolismo , Duodeno/metabolismo , Neurotransmissores/metabolismo , Fumar/metabolismo , Animais , Atropina/farmacologia , Glândulas Duodenais/metabolismo , Úlcera Duodenal/etiologia , Duodeno/efeitos dos fármacos , Hormônios Gastrointestinais/farmacologia , Hexametônio/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Masculino , Peptídeos/farmacologia , Ratos , Ratos Sprague-Dawley , Recidiva , Reserpina/farmacologia , Fumar/efeitos adversos , Tetrodotoxina/farmacologia , Vagotomia , Nervo Vago/fisiologia , Peptídeo Intestinal Vasoativo/farmacologia , CicatrizaçãoRESUMO
Poor-prognosis oestrogen receptor-positive breast cancer is characterised by the presence of high-level focal amplifications. We utilised a focused small interfering RNA screen in 14 breast cancer cell lines to define genes that were pathogenic in three genomic regions focally amplified in oestrogen receptor-positive breast cancer, 8p11-12, 11q13 and 20q. Silencing the GNAS locus, that encodes the G protein alpha stimulatory subunit Gαs, specifically reduced the growth of 20q amplified breast cancer cell lines. Examination of a publically available small hairpin RNA data set confirmed GNAS silencing to be selective for 20q amplified cancer cell lines. Cell lines with 20q amplification were found to overexpress specifically the extra long Gαs splice variant (XLαs). Overexpression of XLαs induced cAMP levels to a greater extent than Gαs, suggesting that amplification of the GNAS locus, and overexpression of the XLαs variant in particular, enhanced cAMP signalling. GNAS silencing in amplified cell lines reduced extracellular-signal-regulated kinase 1/2 (ERK1/2) phosphorylation, and conversely overexpression of exogenous XLαs in a non-amplified cell line increased MEK-ERK1/2 phosphorylation, identifying one potential downstream consequence of enhanced cAMP signalling. Our data indicate that amplification of the GNAS locus may contribute to the pathogenesis of breast cancer, and highlight a previously unrecognised role for the GNAS XLαs variant in cancer.
Assuntos
Neoplasias da Mama/genética , Cromossomos Humanos Par 20/genética , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Western Blotting , Linhagem Celular Tumoral , Cromograninas , Feminino , Citometria de Fluxo , Imunofluorescência , Amplificação de Genes , Loci Gênicos , Humanos , Isoformas de Proteínas/genética , RNA Interferente Pequeno , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Overexpression of the receptor tyrosine kinase ERBB2 (also known as HER2) occurs in around 15% of breast cancers and is driven by amplification of the ERBB2 gene. ERBB2 amplification is a marker of poor prognosis, and although anti-ERBB2-targeted therapies have shown significant clinical benefit, de novo and acquired resistance remains an important problem. Genomic profiling has demonstrated that ERBB2+ve breast cancers are distinguished from ER+ve and 'triple-negative' breast cancers by harbouring not only the ERBB2 amplification on 17q12, but also a number of co-amplified genes on 17q12 and amplification events on other chromosomes. Some of these genes may have important roles in influencing clinical outcome, and could represent genetic dependencies in ERBB2+ve cancers and therefore potential therapeutic targets. Here, we describe an integrated genomic, gene expression and functional analysis to determine whether the genes present within amplicons are critical for the survival of ERBB2+ve breast tumour cells. We show that only a fraction of the ERBB2-amplified breast tumour lines are truly addicted to the ERBB2 oncogene at the mRNA level and display a heterogeneous set of additional genetic dependencies. These include an addiction to the transcription factor gene TFAP2C when it is amplified and overexpressed, suggesting that TFAP2C represents a genetic dependency in some ERBB2+ve breast cancer cells.
Assuntos
Neoplasias da Mama/genética , Amplificação de Genes/genética , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Fator de Transcrição AP-2/metabolismo , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Feminino , Perfilação da Expressão Gênica , Humanos , Células MCF-7 , Interferência de RNA , RNA Interferente Pequeno , Receptor ErbB-2/biossíntese , Fator de Transcrição AP-2/biossínteseRESUMO
Triple negative breast cancers (TNBCs) have a relatively poor prognosis and cannot be effectively treated with current targeted therapies. We searched for genes that have the potential to be therapeutic targets by identifying genes consistently overexpressed when amplified. Fifty-six TNBCs were subjected to high-resolution microarray-based comparative genomic hybridization (aCGH), of which 24 were subjected to genome-wide gene expression analysis. TNBCs were genetically heterogeneous; no individual focal amplification was present at high frequency, although 78.6% of TNBCs harboured at least one focal amplification. Integration of aCGH and expression data revealed 40 genes significantly overexpressed when amplified, including the known oncogenes and potential therapeutic targets, FGFR2 (10q26.3), BUB3 (10q26.3), RAB20 (13q34), PKN1 (19p13.12) and NOTCH3 (19p13.12). We identified two TNBC cell lines with FGFR2 amplification, which both had constitutive activation of FGFR2. Amplified cell lines were highly sensitive to FGFR inhibitor PD173074, and to RNAi silencing of FGFR2. Treatment with PD173074 induced apoptosis resulting partly from inhibition of PI3K-AKT signalling. Independent validation using publicly available aCGH data sets revealed FGFR2 gene was amplified in 4% (6/165) of TNBC, but not in other subtypes (0/214, P=0.0065). Our analysis demonstrates that TNBCs are heterogeneous tumours with amplifications of FGFR2 in a subgroup of tumours.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Amplificação de Genes , Regulação Neoplásica da Expressão Gênica , Animais , Apoptose , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Sobrevivência Celular , Hibridização Genômica Comparativa , Dosagem de Genes/genética , Perfilação da Expressão Gênica , Genômica , Humanos , Ligantes , Fosfatidilinositol 3-Quinases/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Reprodutibilidade dos Testes , Transdução de SinaisRESUMO
BACKGROUND: Previous data implicating genetic and epigenetic events on chromosome 9, including the CDKN2A/2B locus, as molecular predictors of Wilms tumour relapse, have been conflicting. AIMS: To clarify this using genome-wide and focused molecular genetic analysis. METHODS: Microarray-based comparative genomic hybridisation (aCGH) using genome-wide coverage was applied to 76 favourable histology Wilms tumours. Additional investigation of the 9p21 locus was carried out using loss of heterozygosity (LOH) and fluorescence in situ hybridisation (FISH), as well as immunohistochemistry for CDKN2A/p16(INK4a) on a paediatric renal tumour tissue microarray. RESULTS: Approximately half of the tumours were found to show chromosome 9 copy number changes. Those cases which harboured alterations comprised at least four distinct patterns: gain of the entire chromosome, loss of 9p, gain of 9q34, or a more complex combination of gains/losses. None of these tumour groups showed any statistically significant correlation with clinicopathological variables. Deletion mapping of 9p by LOH revealed several regions of overlap, including the CDKN2A/2B locus in 4/34 (11.8%) tumours, which was confirmed to represent hemizygous deletions by FISH. CDKN2A/p16(INK4a) protein expression was predominantly negative in Wilms tumours as assessed by immunohistochemistry on a tissue array, reflecting the expression pattern in normal kidney. However, 38/236 (16.1%) non-anaplastic Wilms tumours, 4/9 (44.4%) anaplastic Wilms tumours, 5/7 (71.4%) rhabdoid tumours of the kidney, and 4/10 (40%) clear cell sarcomas of the kidney showed nuclear CDKN2A/p16(INK4a )immunoreactivity. CONCLUSIONS: These data reveal the complex nature of genetic alterations on chromosome 9 in Wilms tumours, but do not provide evidence for their involvement in or association with treatment failure.
Assuntos
Aberrações Cromossômicas , Cromossomos Humanos Par 9/genética , Inibidor p16 de Quinase Dependente de Ciclina , Genes p16 , Neoplasias Renais/genética , Tumor de Wilms/genética , Biomarcadores Tumorais/metabolismo , Pré-Escolar , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Humanos , Hibridização in Situ Fluorescente , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Perda de Heterozigosidade , Estadiamento de Neoplasias , Prognóstico , Análise de Sobrevida , Análise Serial de Tecidos/métodos , Tumor de Wilms/metabolismo , Tumor de Wilms/patologiaRESUMO
Pure invasive micropapillary carcinoma (MPC) is a special histological type that accounts for 0.7-3% of all breast cancers. MPC has a distinctive growth pattern and a more aggressive clinical behaviour than invasive ductal carcinomas of no special type (IDC-NSTs). To define the molecular characteristics of MPCs, we profiled a series of 12 MPCs and 24 grade and oestrogen receptor (ER)-matched IDC-NSTs using high-resolution microarray comparative genomic hybridization (aCGH). In addition, we generated a tissue microarray containing a series of 24 MPCs and performed immunohistochemical analysis with ER, PR, Ki-67, HER2, CK5/6, CK14, CK17, EGFR, topoisomerase-IIalpha, cyclin D1, caveolin-1, E-cadherin, and beta-catenin antibodies. In situ hybridization probes were employed to evaluate the prevalence of amplification of HER2, TOP2A, EGFR, CCND1, MYC, ESR1, and FGFR1 genes. aCGH analysis demonstrated that MPCs significantly differed from IDC-NSTs at the genomic level. Gains of 1q, 2q, 4p, 6p, 6q23.2-q27, 7p, 7q, 8p, 8q, 9p, 10p, 11q, 12p, 12q, 16p, 17p, 17q, 19p, 20p, 20q, and 21q, and losses of 1p, 2p, 6q11.1-q16.3, 6q21-q22.1, 9p, 11p, 15q, and 19q were more prevalent in MPCs. High-level gains/amplifications of 8p12-p11, 8q12, 8q13, 8q21, 8q23, 8q24, 17q21, 17q23, and 20q13 were significantly associated with MPCs. A comparison between 24 MPCs and a series of 48 grade and ER-matched IDC-NSTs revealed that high cyclin D1 expression, high proliferation rates, and MYC (8q24) amplification were significantly associated with MPCs. Our results demonstrate that MPCs have distinct histological features and molecular genetic profiles supporting the contention that they constitute a distinct pathological entity.
Assuntos
Neoplasias da Mama/genética , Carcinoma Ductal de Mama/genética , Perfilação da Expressão Gênica/métodos , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias da Mama/imunologia , Carcinoma Ductal de Mama/imunologia , Ciclina D1/genética , Progressão da Doença , Feminino , Amplificação de Genes , Marcadores Genéticos , Humanos , Imuno-Histoquímica , Imunofenotipagem , Hibridização in Situ Fluorescente , OncogenesRESUMO
Despite aggressive salvage regimens, approximately half of all children who suffer a Wilms' tumour recurrence will die of their disease. Although there are increasing data on molecular genetic prognostic factors present in the tumour at diagnosis, there is little information regarding the molecular events that occur with Wilms' tumour progression and relapse. In the present study, microarray-based comparative genomic hybridization (aCGH) analysis has been carried out on 58 Wilms' tumour samples, which included 38 untreated primary and 20 recurrent tumours. A higher degree of copy number changes was observed in the recurrent tumours (33.0% genomic clones) than in the primary tumour (21.2%). Paired analysis highlighted the acquisition of 15q gain with high levels of IGF1R expression in the tumour recurrence in two cases. The most statistically significant abnormality acquired between diagnosis and relapse was loss of 17p. One case that experienced 17p loss was classified as favourable histology at diagnosis, but exhibited diffuse anaplasia at recurrence and had a homozygous TP53 deletion. Another instructive case with a constitutional 11p13 deletion presented with bilateral tumours and suffered two subsequent recurrences in the left kidney. A somatic WT1 mutation was found only in the right kidney tumour, while the constitutional 11p13 deletion was the only abnormality detected in the initial left kidney tumour by aCGH. The two subsequent relapses in the left kidney contained an accumulation of additional genetic alterations, including an independent WT1 mutation.
Assuntos
Aberrações Cromossômicas , Cromossomos Humanos Par 11 , Perfilação da Expressão Gênica , Recidiva Local de Neoplasia/genética , Análise de Sequência com Séries de Oligonucleotídeos , Tumor de Wilms/genética , Aniridia/complicações , Aniridia/genética , Análise Mutacional de DNA , Progressão da Doença , Feminino , Deleção de Genes , Genes do Tumor de Wilms , Genes p53 , Homozigoto , Humanos , Interpretação de Imagem Assistida por Computador , Lactente , Masculino , Recidiva Local de Neoplasia/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tumor de Wilms/complicações , Tumor de Wilms/patologiaRESUMO
Despite the excellent survival of Wilms tumour patients treated with multimodality therapy, approximately 15% will suffer from tumour relapse, where response rates are markedly reduced. We have carried out microarray-based comparative genomic hybridisation on a series of 76 Wilms tumour samples, enriched for cases which recurred, to identify changes in DNA copy number associated with clinical outcome. Using 1Mb-spaced genome-wide BAC arrays, the most significantly different genomic changes between favourable histology tumours that did (n = 37), and did not (n = 39), subsequently relapse were gains on 1q, and novel deletions at 12q24 and 18q21. Further relapse-associated loci included losses at 1q32.1, 2q36.3-2q37.1, and gain at 13q31. 1q gains correlated strongly with loss of 1p and/or 16q. In 3 of 11 cases with concurrent 1p(-)/1q(+), a breakpoint was identified at 1p13. Multiple low-level sub-megabase gains along the length of 1q were identified using chromosome 1 tiling-path arrays. One such recurrent region at 1q22-q23.1 included candidate genes RAB25, NES, CRABP2, HDGF and NTRK1, which were screened for mRNA expression using quantitative RT-PCR. These data provide a high-resolution catalogue of genomic copy number changes in relapsing favourable histology Wilms tumours.