RESUMO
BACKGROUND: Idiopathic interstitial pneumonias (IIPs) are a group of heterogeneous, somewhat unpredictable diseases characterized by progressive scarring of the interstitium. Since lung function is a key determinant of survival, we reasoned that the transcriptional profile in IIP lung tissue would be associated with measures of lung function, and could enhance prognostic approaches to IIPs. RESULTS: Using gene expression profiling of 167 lung tissue specimens with IIP diagnosis and 50 control lungs, we identified genes whose expression is associated with changes in lung function (% predicted FVC and % predicted DLCO) modeled as categorical (severe vs mild disease) or continuous variables while adjusting for smoking status and IIP subtype; false discovery rate (FDR) approach was used to correct for multiple comparisons. This analysis identified 58 transcripts that are associated with mild vs severe disease (categorical analysis), including those with established role in fibrosis (ADAMTS4, ADAMTS9, AGER, HIF-1α, SERPINA3, SERPINE2, and SELE) as well as novel IIP candidate genes such as rhotekin 2 (RTKN2) and peptidase inhibitor 15 (PI15). Protein-protein interactome analysis of 553 genes whose expression is significantly associated with lung function when modeled as continuous variables demonstrates that more severe presentation of IIPs is characterized by an increase in cell cycle progression and apoptosis, increased hypoxia, and dampened innate immune response. Our findings were validated in an independent cohort of 131 IIPs and 40 controls at the mRNA level and for one gene (RTKN2) at the protein level by immunohistochemistry in a subset of samples. CONCLUSIONS: We identified commonalities and differences in gene expression among different subtypes of IIPs. Disease progression, as characterized by lower measures of FVC and DLCO, results in marked changes in expression of novel and established genes and pathways involved in IIPs. These genes and pathways represent strong candidates for biomarker studies and potential therapeutic targets for IIP severity.
Assuntos
Regulação da Expressão Gênica , Pneumonias Intersticiais Idiopáticas/genética , Pneumonias Intersticiais Idiopáticas/fisiopatologia , Pulmão/fisiopatologia , Proteínas/genética , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Proteína ADAMTS4 , Proteína ADAMTS9 , Adulto , Idoso , Selectina E/genética , Selectina E/metabolismo , Feminino , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Pessoa de Meia-Idade , Pró-Colágeno N-Endopeptidase/genética , Pró-Colágeno N-Endopeptidase/metabolismo , Receptor para Produtos Finais de Glicação Avançada/genética , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Serpina E2/genética , Serpina E2/metabolismo , Serpinas/genética , Serpinas/metabolismoRESUMO
Although most cases of chronic obstructive pulmonary disease (COPD) occur in smokers, only a fraction of smokers develop the disease. We hypothesized distinct molecular signatures for COPD and emphysema in the peripheral blood mononuclear cells (PBMCs) of current and former smokers. To test this hypothesis, we identified and validated PBMC gene expression profiles in smokers with and without COPD. We generated expression data on 136 subjects from the COPDGene study, using Affymetrix U133 2.0 microarrays (Affymetrix, Santa Clara, CA). Multiple linear regression with adjustment for covariates (gender, age, body mass index, family history, smoking status, and pack-years) was used to identify candidate genes, and ingenuity pathway analysis was used to identify candidate pathways. Candidate genes were validated in 149 subjects according to multiplex quantitative real-time polymerase chain reaction, which included 75 subjects not previously profiled. Pathways that were differentially expressed in subjects with COPD and emphysema included those that play a role in the immune system, inflammatory responses, and sphingolipid (ceramide) metabolism. Twenty-six of the 46 candidate genes (e.g., FOXP1, TCF7, and ASAH1) were validated in the independent cohort. Plasma metabolomics was used to identify a novel glycoceramide (galabiosylceramide) as a biomarker of emphysema, supporting the genomic association between acid ceramidase (ASAH1) and emphysema. COPD is a systemic disease whose gene expression signatures in PBMCs could serve as novel diagnostic or therapeutic targets.
Assuntos
Gangliosídeos/sangue , Regulação da Expressão Gênica , Leucócitos Mononucleares/metabolismo , Doença Pulmonar Obstrutiva Crônica/sangue , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , Feminino , Humanos , Masculino , Metabolômica/métodos , Pessoa de Meia-Idade , Doença Pulmonar Obstrutiva Crônica/diagnóstico , Enfisema Pulmonar/sangue , Enfisema Pulmonar/diagnóstico , Reação em Cadeia da Polimerase em Tempo RealRESUMO
BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is an untreatable lung disease with a median survival of only 3-5 years that is diagnosed using a combination of clinical, radiographic and pathologic criteria. Histologically, IPF is characterised by usual interstitial pneumonia (UIP), a fibrosing interstitial pneumonia with a pattern of heterogeneous, subpleural regions of fibrotic and remodelled lung. We hypothesised that gene expression profiles of lung tissue may identify molecular subtypes of disease that could classify subtypes of IPF/UIP that have clinical implications. METHODS AND FINDINGS: We collected transcriptional profiles on lung tissue from 119 patients with IPF/UIP and 50 non-diseased controls. Differential expression of individual transcripts was identified using an analysis of covariance (ANCOVA) model incorporating the clinical diagnosis of each patient as well as age, gender and smoking status. Validation was performed in an independent cohort of 111 IPF/UIP and 39 non-diseased controls. Our analysis identified two subtypes of IPF/UIP based on a strong molecular signature associated with expression of genes previously associated with fibrosis (matrix metalloproteinases, osteopontin, keratins), cilium genes and genes with unknown function. We demonstrate that elevated expression of cilium genes is associated with more extensive microscopic honeycombing and higher expression of both the airway mucin gene MUC5B and the metalloproteinase MMP7, a gene recently implicated in attenuating ciliated cell differentiation during wound repair. CONCLUSIONS: Expression of cilium genes appears to identify two unique molecular phenotypes of IPF/UIP. The different molecular profiles may be relevant to therapeutic responsiveness in patients with IPF/UIP.
Assuntos
Cílios/genética , Fibrose Pulmonar Idiopática/genética , Fibrose Pulmonar Idiopática/patologia , Metaloproteinase 7 da Matriz/genética , Mucina-5B/genética , Adulto , Idoso , Estudos de Casos e Controles , Perfilação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , RNA/análise , TranscriptomaRESUMO
Idiopathic pulmonary fibrosis (IPF) is associated with the accumulation of collagen-secreting fibroblasts and myofibroblasts in the lung parenchyma. Many mechanisms contribute to their accumulation, including resistance to apoptosis. In previous work, we showed that exposure to the proinflammatory cytokines TNF-α and IFN-γ reverses the resistance of lung fibroblasts to apoptosis. In this study, we investigate the underlying mechanisms. Based on an interrogation of the transcriptomes of unstimulated and TNF-α- and IFN-γ-stimulated primary lung fibroblasts and the lung fibroblast cell line MRC5, we show that among Fas-signaling pathway molecules, Fas expression was increased â¼6-fold in an NF-κB- and p38(mapk)-dependent fashion. Prevention of the increase in Fas expression using Fas small interfering RNAs blocked the ability of TNF-α and IFN-γ to sensitize fibroblasts to Fas ligation-induced apoptosis, whereas enforced adenovirus-mediated Fas overexpression was sufficient to overcome basal resistance to Fas-induced apoptosis. Examination of lung tissues from IPF patients revealed low to absent staining of Fas in fibroblastic cells of fibroblast foci. Collectively, these findings suggest that increased expression of Fas is necessary and sufficient to overcome the resistance of lung fibroblasts to Fas-induced apoptosis. Our findings also suggest that approaches aimed at increasing Fas expression by lung fibroblasts and myofibroblasts may be therapeutically relevant in IPF.
Assuntos
Apoptose/imunologia , Fibroblastos/imunologia , Pulmão/imunologia , Pulmão/patologia , Fibrose Pulmonar/imunologia , Regulação para Cima/imunologia , Receptor fas/biossíntese , Animais , Apoptose/genética , Linhagem Celular , Linhagem Celular Transformada , Células Cultivadas , Proteína Ligante Fas/biossíntese , Proteína Ligante Fas/genética , Fibroblastos/metabolismo , Fibroblastos/patologia , Perfilação da Expressão Gênica , Humanos , Estudos Longitudinais , Pulmão/metabolismo , Camundongos , Camundongos Knockout , Estudos Prospectivos , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/patologia , Regulação para Cima/genética , Receptor fas/deficiência , Receptor fas/genéticaRESUMO
RATIONALE: Vascular remodeling in pulmonary arterial hypertension (PAH) involves proliferation and migration of endothelial and smooth muscle cells, leading to obliterative vascular lesions. Previous studies have indicated that the endothelial cell proliferation is quasineoplastic, with evidence of monoclonality and instability of short DNA microsatellite sequences. OBJECTIVES: To assess whether there is larger-scale genomic instability. METHODS: We performed genome-wide microarray copy number analysis on pulmonary artery endothelial cells and smooth muscle cells isolated from the lungs of patients with PAH. MEASUREMENTS AND MAIN RESULTS: Mosaic chromosomal abnormalities were detected in PAEC cultures from five of nine PAH lungs but not in normal (n = 8) or disease control subjects (n = 5). Fluorescent in situ hybridization analysis confirmed the presence of these abnormalities in vivo in two of three cases. One patient harbored a germline mutation of BMPR2, the primary genetic cause of PAH, and somatic loss of chromosome-13, which constitutes a second hit in the same pathway by deleting Smad-8. In two female subjects with mosaic loss of the X chromosome, methylation analysis showed that the active X was deleted. One subject also showed completely skewed X-inactivation in the nondeleted cells, suggesting the pulmonary artery endothelial cell population was clonal before the acquisition of the chromosome abnormality. CONCLUSIONS: Our data indicate a high frequency of genetically abnormal subclones within PAH lung vessels and provide the first definitive evidence of a second genetic hit in a patient with a germline BMPR2 mutation. We propose that these chromosome abnormalities may confer a growth advantage and thus contribute to the progression of PAH.
Assuntos
Remodelação das Vias Aéreas/fisiologia , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/genética , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/patologia , Adulto , Proliferação de Células , Criança , Aberrações Cromossômicas , Deleção Cromossômica , Variações do Número de Cópias de DNA , Células Endoteliais/patologia , Células Endoteliais/fisiologia , Feminino , Rearranjo Gênico , Estudo de Associação Genômica Ampla , Instabilidade Genômica , Mutação em Linhagem Germinativa , Humanos , Hibridização in Situ Fluorescente , Pulmão/citologia , Análise em Microsséries , Pessoa de Meia-Idade , Miócitos de Músculo Liso/patologia , Polimorfismo de Nucleotídeo Único , Artéria Pulmonar/citologia , Artéria Pulmonar/fisiologia , Doença Pulmonar Obstrutiva Crônica/genética , Doença Pulmonar Obstrutiva Crônica/patologia , Inativação do Cromossomo XRESUMO
Despite widespread expression of epidermal growth factor (EGF) receptors (EGFRs) and EGF family ligands in non-small-cell lung cancer (NSCLC), EGFR-specific tyrosine kinase inhibitors (TKIs) such as gefitinib exhibit limited activity in this cancer. We propose that autocrine growth signaling pathways distinct from EGFR are active in NSCLC cells. To this end, gene expression profiling revealed frequent coexpression of specific fibroblast growth factors (FGFs) and FGF receptors (FGFRs) in NSCLC cell lines. It is noteworthy that FGF2 and FGF9 as well as FGFR1 IIIc and/or FGFR2 IIIc mRNA and protein are frequently coexpressed in NSCLC cell lines, especially those that are insensitive to gefitinib. Specific silencing of FGF2 reduced anchorage-independent growth of two independent NSCLC cell lines that secrete FGF2 and coexpress FGFR1 IIIc and/or FGFR2 IIIc. Moreover, a TKI [(+/-)-1-(anti-3-hydroxy-cyclopentyl)-3-(4-methoxy-phenyl)-7-phenylamino-3,4-dihydro-1H-pyrimido-[4,5-d]pyrimidin-2-one (RO4383596)] that targets FGFRs inhibited basal FRS2 and extracellular signal-regulated kinase phosphorylation, two measures of FGFR activity, as well as proliferation and anchorage-independent growth of NSCLC cell lines that coexpress FGF2 or FGF9 and FGFRs. By contrast, RO4383596 influenced neither signal transduction nor growth of NSCLC cell lines lacking FGF2, FGF9, FGFR1, or FGFR2 expression. Thus, FGF2, FGF9 and their respective high-affinity FGFRs comprise a growth factor autocrine loop that is active in a subset of gefitinib-insensitive NSCLC cell lines.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Neoplasias Pulmonares/genética , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais/genética , Linhagem Celular Tumoral , Fatores de Crescimento de Fibroblastos/genética , Humanos , RNA Interferente Pequeno/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/genéticaRESUMO
RATIONALE: Circulating leukocyte RNA transcripts are systemic markers of inflammation, which have not been studied in cystic fibrosis (CF) lung disease. Although the standard assessment of pulmonary treatment response is FEV(1), a measure of airflow limitation, the lack of systemic markers to reflect changes in lung inflammation critically limits the testing of proposed therapeutics. OBJECTIVES: We sought to prospectively identify and validate peripheral blood leukocyte genes that could mark resolution of pulmonary infection and inflammation using a model by which RNA transcripts could increase the predictive value of spirometry. METHODS: Peripheral blood mononuclear cells were isolated from 10 patients with CF and acute pulmonary exacerbations before and after therapy. RNA expression profiling revealed that 10 genes significantly changed with treatment when compared with matched non-CF and control subjects with stable CF to establish baseline transcript abundance. Peripheral blood mononuclear cell RNA transcripts were prospectively validated, using real-time polymerase chain reaction amplification, in an independent cohort of acutely ill patients with CF (n = 14). Patients who responded to therapy were analyzed using general estimating equations and multiple logistic regression, such that changes in FEV(1)% predicted were regressed with transcript changes. MEASUREMENTS AND MAIN RESULTS: Three genes, CD64, ADAM9, and CD36, were significant and independent predictors of a therapeutic response beyond that of FEV(1) alone (P < 0.05). In both cohorts, receiver operating characteristic analysis revealed greater accuracy when genes were combined with FEV(1). CONCLUSIONS: Circulating mononuclear cell transcripts characterize a response to the treatment of pulmonary exacerbations. Even in small patient cohorts, changes in gene expression in conjunction with FEV(1) may enhance current outcomes measures for treatment response.
Assuntos
Antibacterianos/uso terapêutico , Fibrose Cística/sangue , Precursores de RNA/sangue , Doença Aguda , Adulto , Biomarcadores/sangue , Estudos de Coortes , Fibrose Cística/tratamento farmacológico , Fibrose Cística/genética , Citocinas/sangue , Feminino , Volume Expiratório Forçado , Perfilação da Expressão Gênica/métodos , Humanos , Leucócitos/metabolismo , Masculino , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Valor Preditivo dos Testes , Estudos Prospectivos , Precursores de RNA/genética , Curva ROC , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Espirometria/métodos , Adulto JovemRESUMO
Human herpesvirus-8 (HHV-8) is the causative agent of Kaposi's sarcoma and is associated with the angioproliferative disorders primary effusion lymphoma and multicentric Castleman's disease. Evidence of HHV-8 infection within the pulmonary vasculature of patients with idiopathic pulmonary arterial hypertension (IPAH) has been described. We hypothesize that HHV-8 infection of pulmonary microvascular endothelial cells results in an apoptotic-resistant phenotype characteristic of severe pulmonary arterial hypertension. Our objective was to investigate the ability of HHV-8 to infect human pulmonary microvascular endothelial cells in vitro and characterize the phenotypic effect of this infection. Human pulmonary microvascular endothelial cells were exposed to HHV-8 using two methods (direct virus and co-culture technique). The presence of lytic and latent infection was confirmed. Changes in endothelial cell gene and protein expression and effects on cellular apoptosis were measured. HHV-8 can both lytically and latently infect primary human pulmonary microvascular endothelial cells in vitro. HHV-8 infection results in significant changes in gene expression, including alterations of pathways important to cellular apoptosis. HHV-8 infection also alters expression of genes integral to the bone morphogenic protein pathway, including down-regulation of bone morphogenic protein-4. Other genes previously implicated in the development of PAH are affected by HHV-8 infection, and cells infected with HHV-8 are resistant to apoptosis.
Assuntos
Vasos Sanguíneos/citologia , Vasos Sanguíneos/virologia , Células Endoteliais/virologia , Infecções por Herpesviridae/metabolismo , Apoptose/efeitos dos fármacos , Vasos Sanguíneos/enzimologia , Proteína Morfogenética Óssea 4/farmacologia , Proteínas de Transporte/farmacologia , Caspase 3/metabolismo , Caspase 7/metabolismo , Células Cultivadas , Meios de Cultivo Condicionados , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/enzimologia , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Regulação da Expressão Gênica/efeitos dos fármacos , Herpesvirus Humano 8 , Marcação In Situ das Extremidades Cortadas , Interleucina-6/farmacologia , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Reação em Cadeia da Polimerase , Reprodutibilidade dos TestesRESUMO
The epidermal growth factor receptor (EGFR) is overexpressed in the majority of non-small cell lung cancers (NSCLC). EGFR tyrosine kinase inhibitors, such as gefitinib and erlotinib, produce 9% to 27% response rates in NSCLC patients. E-Cadherin, a calcium-dependent adhesion molecule, plays an important role in NSCLC prognosis and progression, and interacts with EGFR. The zinc finger transcriptional repressor, ZEB1, inhibits E-cadherin expression by recruiting histone deacetylases (HDAC). We identified a significant correlation between sensitivity to gefitinib and expression of E-cadherin, and ZEB1, suggesting their predictive value for responsiveness to EGFR-tyrosine kinase inhibitors. E-Cadherin transfection into a gefitinib-resistant line increased its sensitivity to gefitinib. Pretreating resistant cell lines with the HDAC inhibitor, MS-275, induced E-cadherin along with EGFR and led to a growth-inhibitory and apoptotic effect of gefitinib similar to that in gefitinib-sensitive NSCLC cell lines including those harboring EGFR mutations. Thus, combined HDAC inhibitor and gefitinib treatment represents a novel pharmacologic strategy for overcoming resistance to EGFR inhibitors in patients with lung cancer.
Assuntos
Antineoplásicos/farmacologia , Caderinas/biossíntese , Receptores ErbB/antagonistas & inibidores , Neoplasias Pulmonares/patologia , Quinazolinas/farmacologia , Biomarcadores Tumorais/análise , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/fisiologia , Gefitinibe , Inibidores de Histona Desacetilases , Proteínas de Homeodomínio/biossíntese , Humanos , Valor Preditivo dos Testes , Prognóstico , Fatores de Transcrição/biossíntese , Transfecção , Homeobox 1 de Ligação a E-box em Dedo de ZincoRESUMO
The modest response of patients with head and neck squamous cell carcinoma (HNSCC) and non-small cell lung carcinoma (NSCLC) to epithelial growth factor receptor tyrosine kinase inhibitors such as gefitinib and erlotinib indicates the need for the development of biomarkers to predict response. We determined gefitinib sensitivity in a panel of HNSCC cell lines by a 5-day 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and confirmed these responses with analysis of downstream signaling by immunoblotting and cell cycle arrest. Basal gene expression profiles were then determined by microarray analysis and correlated with gefitinib response. These data were combined with previously reported NSCLC microarray results to generate a broader predictive index. Common markers of resistance between the two tumor types included genes associated with the epithelial to mesenchymal transition. We confirmed that increased protein expression of vimentin combined with the loss of E-cadherin, claudin 4, and claudin 7 by immunoblotting was associated with gefitinib resistance in both HNSCC and NSCLC cell lines. In addition, the loss of the Ca(2+)-independent cell-cell adhesion molecules EpCAM and TROP2 in resistant lines was confirmed by immunofluorescence. Tumor xenografts derived from the gefitinib-sensitive UM-SCC-2 were growth-delayed by gefitinib, whereas the gefitinib-resistant 1483 xenografts were unaffected. These data support a role for epithelial to mesenchymal transition in establishing gefitinib resistance for both HNSCC and NSCLC, and indicate that clinical trials should address whether these biomarkers will be useful for patient selection.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma de Células Escamosas/patologia , Resistencia a Medicamentos Antineoplásicos , Células Epiteliais/patologia , Neoplasias de Cabeça e Pescoço/patologia , Neoplasias Pulmonares/patologia , Mesoderma/patologia , Quinazolinas/farmacologia , Animais , Antineoplásicos/farmacologia , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Feminino , Gefitinibe , Camundongos , Camundongos Nus , Análise de Sequência com Séries de OligonucleotídeosRESUMO
Bioinformatics pipelines are an integral component of next-generation sequencing (NGS). Processing raw sequence data to detect genomic alterations has significant impact on disease management and patient care. Because of the lack of published guidance, there is currently a high degree of variability in how members of the global molecular genetics and pathology community establish and validate bioinformatics pipelines. Improperly developed, validated, and/or monitored pipelines may generate inaccurate results that may have negative consequences for patient care. To address this unmet need, the Association of Molecular Pathology, with organizational representation from the College of American Pathologists and the American Medical Informatics Association, has developed a set of 17 best practice consensus recommendations for the validation of clinical NGS bioinformatics pipelines. Recommendations include practical guidance for laboratories regarding NGS bioinformatics pipeline design, development, and operation, with additional emphasis on the role of a properly trained and qualified molecular professional to achieve optimal NGS testing quality.
Assuntos
Biologia Computacional/normas , Guias como Assunto , Sequenciamento de Nucleotídeos em Larga Escala/normas , Patologia Molecular/normas , Humanos , Laboratórios , Reprodutibilidade dos Testes , Estados UnidosRESUMO
Persistent bronchial dysplasia is associated with increased risk of developing invasive squamous cell carcinoma (SCC) of the lung. In this study, we hypothesized that differences in gene expression profiles between persistent and regressive bronchial dysplasia would identify cellular processes that underlie progression to SCC. RNA expression arrays comparing baseline biopsies from 32 bronchial sites that persisted/progressed to 31 regressive sites showed 395 differentially expressed genes [ANOVA, FDR ≤ 0.05). Thirty-one pathways showed significantly altered activity between the two groups, many of which were associated with cell-cycle control and proliferation, inflammation, or epithelial differentiation/cell-cell adhesion. Cultured persistent bronchial dysplasia cells exhibited increased expression of Polo-like kinase 1 (PLK1), which was associated with multiple cell-cycle pathways. Treatment with PLK1 inhibitor induced apoptosis and G2-M arrest and decreased proliferation compared with untreated cells; these effects were not seen in normal or regressive bronchial dysplasia cultures. Inflammatory pathway activity was decreased in persistent bronchial dysplasia, and the presence of an inflammatory infiltrate was more common in regressive bronchial dysplasia. Regressive bronchial dysplasia was also associated with trends toward overall increases in macrophages and T lymphocytes and altered polarization of these inflammatory cell subsets. Increased desmoglein 3 and plakoglobin expression was associated with higher grade and persistence of bronchial dysplasia. These results identify alterations in the persistent subset of bronchial dysplasia that are associated with high risk for progression to invasive SCC. These alterations may serve as strong markers of risk and as effective targets for lung cancer prevention.Significance: Gene expression profiling of high-risk persistent bronchial dysplasia reveals changes in cell-cycle control, inflammatory activity, and epithelial differentiation/cell-cell adhesion that may underlie progression to invasive SCC. Cancer Res; 78(17); 4971-83. ©2018 AACR.
Assuntos
Carcinoma de Células Escamosas/genética , Inflamação/genética , Neoplasias Pulmonares/genética , Lesões Pré-Cancerosas/genética , Adulto , Idoso , Biópsia , Brônquios/metabolismo , Brônquios/patologia , Broncopatias/genética , Broncopatias/patologia , Carcinoma de Células Escamosas/patologia , Pontos de Checagem do Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Proliferação de Células/genética , Desmogleína 3/genética , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Inflamação/patologia , Neoplasias Pulmonares/patologia , Masculino , Metaplasia , Pessoa de Meia-Idade , Lesões Pré-Cancerosas/patologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , gama Catenina/genética , Quinase 1 Polo-LikeRESUMO
Tyrosine kinase inhibitors (TKI) of the epidermal growth factor receptor (EGFR) produce objective responses in a minority of patients with advanced-stage non-small cell lung cancer (NSCLC), and about half of all treated patients progress within 6 weeks of instituting therapy. Because the target of these agents is known, it should be possible to develop biological predictors of response, but EGFR protein levels have not been proven useful as a predictor of TKI response in patients and the mechanism of primary resistance is unclear. We used microarray gene expression profiling to uncover a pattern of gene expression associated with sensitivity to EGFR-TKIs by comparing NSCLC cell lines that were either highly sensitive or highly resistant to gefitinib. This sensitivity-associated expression profile was used to predict gefitinib sensitivity in a panel of NSCLC cell lines with known gene expression profiles but unknown gefitinib sensitivity. Gefitinib sensitivity was then determined for members of this test panel, and the microarray-based sensitivity prediction was correct in eight of nine NSCLC cell lines. Gene and protein expression differences were confirmed with a combination of quantitative reverse transcription-PCR, flow cytometry, and immunohistochemistry. This gene expression pattern related to gefitinib sensitivity was independent from sensitivity associated with EGFR mutations. Several genes associated with sensitivity encode proteins involved in HER pathway signaling or pathways that interrelate to the HER signaling pathway. Some of these genes could be targets of pharmacologic interventions to overcome primary resistance.
Assuntos
Antineoplásicos/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Quinazolinas/uso terapêutico , Caderinas/metabolismo , Análise por Conglomerados , Ensaios de Seleção de Medicamentos Antitumorais , Receptores ErbB/genética , Citometria de Fluxo/métodos , Gefitinibe , Expressão Gênica , Perfilação da Expressão Gênica/classificação , Humanos , Concentração Inibidora 50 , Família Multigênica , Mutação/efeitos dos fármacos , Reação em Cadeia da Polimerase/métodos , Inibidores de Proteínas Quinases , Proteoma/efeitos dos fármacos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas p21(ras) , Resultado do Tratamento , Células Tumorais Cultivadas , Proteínas rasRESUMO
OBJECTIVES: Circulating levels of the proinflammatory mediator High Mobility Group Box Protein 1 (HMGB1) are increased in septic patients and may contribute to sepsis-induced organ dysfunction. Although HMGB1 has been shown to activate neutrophils from healthy volunteers, the responses of neutrophils from septic patients to HMGB1 have not been reported. In the present study we evaluated gene expression and activation of major intracellular signaling pathways in peripheral blood neutrophils obtained from patients with sepsis-induced acute lung injury after culture with HMGB1 or LPS. DESIGN: Ex-vivo study performed in neutrophils from patients with sepsis-induced acute lung injury. SETTING: Immunology and genetics laboratory at an academic medical center. PATIENTS AND PARTICIPANTS: Twenty-two adult patients with sepsis-induced acute lung injury. MEASUREMENTS AND RESULTS: Using gene arrays, distinct patterns of gene expression were found in neutrophils from septic patients after stimulation with HMGB1 or LPS. While more than three-quarters of the genes upregulated by HMGB1 in neutrophils from septic patients also demonstrated increased expression after culture with LPS, the majority of genes affected by LPS did not show altered expression in neutrophils stimulated with HMGB1. Culture of neutrophils with HMGB1 induced downregulation of its own expression, a finding not present after exposure to LPS. Although HMGB1 and LPS both increased nuclear translocation of NF-kappaB, the magnitude of this effect was greater in LPS stimulated neutrophils from patients with sepsis-induced acute lung injury. CONCLUSION: These findings demonstrate that the patterns of gene expression differ between neutrophils from septic patients stimulated with HMGB1 or LPS, and also that neutrophils from septic patients are not anergic but instead demonstrate intact activation of NF-kappaB after exposure to LPS or HMGB1.
Assuntos
Perfilação da Expressão Gênica , Proteína HMGB1/fisiologia , Lipopolissacarídeos/farmacologia , Neutrófilos/efeitos dos fármacos , Síndrome do Desconforto Respiratório/metabolismo , Sepse/complicações , Adulto , Idoso , Idoso de 80 Anos ou mais , Células Cultivadas , Feminino , Regulação da Expressão Gênica , Proteína HMGB1/farmacologia , Humanos , Masculino , Pessoa de Meia-Idade , NF-kappa B/metabolismo , Neutrófilos/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Síndrome do Desconforto Respiratório/sangue , Síndrome do Desconforto Respiratório/etiologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Comprehensive genetic profiling is increasingly important for the clinical workup of hematologic tumors, as specific alterations are now linked to diagnostic characterization, prognostic stratification and therapy selection. To characterize relevant genetic and genomic alterations in myeloid malignancies maximally, we utilized a comprehensive strategy spanning fluorescence in situ hybridization (FISH), classical karyotyping, Chromosomal Microarray (CMA) for detection of copy number variants (CNVs) and Next generation Sequencing (NGS) analysis. In our cohort of 569 patients spanning the myeloid spectrum, NGS and CMA testing frequently identified mutations and copy number changes in the majority of genes with important clinical associations, such as TP53, TET2, RUNX1, SRSF2, APC and ATM. Most importantly, NGS and CMA uncovered medically actionable aberrations in 75.6% of cases normal by FISH/cytogenetics testing. NGS identified mutations in 65.5% of samples normal by CMA, cytogenetics and FISH, whereas CNVs were detected in 10.1% cases that were normal by all other methodologies. Finally, FISH or cytogenetics, or both, were abnormal in 14.1% of cases where NGS or CMA failed to detect any changes. Multiple mutations and CNVs were found to coexist, with potential implications for patient stratification. Thus, high throughput genomic tumor profiling through targeted DNA sequencing and CNV analysis complements conventional methods and leads to more frequent detection of actionable alterations.
Assuntos
Cromossomos Humanos/genética , Citogenética/métodos , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Hibridização in Situ Fluorescente/métodos , Transtornos Mieloproliferativos/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Estudos de Coortes , Variações do Número de Cópias de DNA/genética , Humanos , Mutação/genética , Transtornos Mieloproliferativos/diagnóstico , Carga TumoralRESUMO
BACKGROUND: Hypoplastic left heart syndrome (HLHS) is a heterogeneous, lethal combination of congenital malformations characterized by severe underdevelopment of left heart structures, resulting in a univentricular circulation. The genetic determinants of this disorder are largely unknown. Evidence of copy number variants (CNVs) contributing to the genetic etiology of HLHS and other congenital heart defects has been mounting. However, the functional effects of such CNVs have not been examined, particularly in cases where the variant of interest is found in only a single patient. METHODS AND RESULTS: Whole-genome SNP microarrays were employed to detect CNVs in two patient cohorts (N = 70 total) predominantly diagnosed with some form of nonsyndromic HLHS. We discovered 16 rare or private variants adjacent to or overlapping 20 genes associated with cardiovascular or premature lethality phenotypes in mouse knockout models. We evaluated the impact of selected variants on the expression of nine of these genes through quantitative PCR on cDNA derived from patient heart tissue. Four genes displayed significantly altered expression in patients with an overlapping or proximal CNV verses patients without such CNVs. CONCLUSION: Rare and private genomic imbalances perturb transcription of genes that potentially affect cardiogenesis in a subset of nonsyndromic HLHS patients.
Assuntos
Variações do Número de Cópias de DNA , Cardiopatias Congênitas/genética , Síndrome do Coração Esquerdo Hipoplásico/genética , Transcrição Gênica , Animais , Estudos de Coortes , DNA Complementar/metabolismo , Éxons , Feminino , Genótipo , Humanos , Masculino , Cadeias de Markov , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Chromosomal instability is central to the process of carcinogenesis. The genome-wide detection of somatic chromosomal alterations (SCA) in small premalignant lesions remains challenging because sample heterogeneity dilutes the aberrant cell information. To overcome this hurdle, we focused on the B allele frequency data from single-nucleotide polymorphism microarrays (SNP arrays). The difference of allelic fractions between paired tumor and normal samples from the same patient (delta-θ) provides a simple but sensitive detection of SCA in the affected tissue. We applied the delta-θ approach to small, heterogeneous clinical specimens, including endobronchial biopsies and brushings. Regions identified by delta-θ were validated by FISH and quantitative PCR in heterogeneous samples. Distinctive genomic variations were successfully detected across the whole genome in all invasive cancer cases (6 of 6), carcinoma in situ (3 of 3), and high-grade dysplasia (severe or moderate; 3 of 11). Not only well-described SCAs in lung squamous cell carcinoma, but also several novel chromosomal alterations were frequently found across the preinvasive dysplastic cases. Within these novel regions, losses of putative tumor suppressors (RNF20 and SSBP2) and an amplification of RASGRP3 gene with oncogenic activity were observed. Widespread sampling of the airway during bronchoscopy demonstrated that field cancerization reflected by SCAs at multiple sites was detectable. SNP arrays combined with delta-θ analysis can detect SCAs in heterogeneous clinical sample and expand our ability to assess genomic instability in the airway epithelium as a biomarker of lung cancer risk.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma de Células Escamosas/genética , Instabilidade Cromossômica/genética , Estudo de Associação Genômica Ampla/métodos , Neoplasias Pulmonares/genética , Análise em Microsséries , Polimorfismo de Nucleotídeo Único , Lesões Pré-Cancerosas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma de Células Escamosas/patologia , Linhagem Celular , Aberrações Cromossômicas , Humanos , Perda de Heterozigosidade , Neoplasias Pulmonares/patologia , Lesões Pré-Cancerosas/patologiaRESUMO
Interleukin-16 (IL-16) is a multifunctional cytokine that has been associated with autoimmune and allergic diseases. To investigate comprehensively whether IL-16 is also associated with chronic obstructive pulmonary disease (COPD) and emphysema, we performed an integrated analysis of multiple "omics" data. Over 500 subjects participating in the COPDGene® study donated blood and were clinically characterized and genetically profiled. IL-16 mRNA levels were measured in peripheral blood mononuclear cells (PBMC), and protein levels were measured in fresh frozen plasma. A multivariate analysis found plasma IL-16 positively associated with age and body mass index, and negatively associated with current smoking and emphysema in the upper lobes. PBMC IL-16 expression was positively associated with gender and a composite score for airflow obstruction, emphysema, and gas trapping. Whole-genome expression quantitative trait locus (eQTL) analysis identified a novel IL-16 missense SNP (rs11556218) associated with lower IL-16 in plasma. In summary, an integrated "omics" analysis in a very large cohort identified an association between decreased IL-16 and emphysema and discovered a novel IL-16 cis-eQTL. Thus IL-16 plasma levels and IL-16 genotyping may be useful in a personalized medicine approach for lung disease.
Assuntos
Enfisema/sangue , Interleucina-16/sangue , Doença Pulmonar Obstrutiva Crônica/sangue , Corticosteroides/farmacologia , Corticosteroides/uso terapêutico , Idoso , Biomarcadores/sangue , Enfisema/tratamento farmacológico , Enfisema/genética , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Leucócitos Mononucleares/metabolismo , Masculino , Pessoa de Meia-Idade , Fenótipo , Polimorfismo de Nucleotídeo Único , Proteômica , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Doença Pulmonar Obstrutiva Crônica/genética , Locos de Características Quantitativas , Fumar/efeitos adversos , Fumar/sangueRESUMO
Genomics and proteomics have emerged as key technologies in biomedical research, resulting in a surge of interest in training by investigators keen to incorporate these technologies into their research. At least two types of training can be envisioned in order to produce meaningful results, quality publications and successful grant applications: (1) immediate short-term training workshops and (2) long-term graduate education or visiting scientist programs. We aimed to fill the former need by providing a comprehensive hands-on training course in genomics, proteomics and informatics in a coherent, experimentally-based framework. This was accomplished through a National Heart, Lung, and Blood Institute (NHLBI)-sponsored 10-day Genomics and Proteomics Hands-on Workshop held at National Jewish Health (NJH) and the University of Colorado School of Medicine (UCD). The course content included comprehensive lectures and laboratories in mass spectrometry and genomics technologies, extensive hands-on experience with instrumentation and software, video demonstrations, optional workshops, online sessions, invited keynote speakers, and local and national guest faculty. Here we describe the detailed curriculum and present the results of short- and long-term evaluations from course attendees. Our educational program consistently received positive reviews from participants and had a substantial impact on grant writing and review, manuscript submissions and publications.