RESUMEN

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.

Asunto(s)

Carcinoma de Células Escamosas/genética , Inflamación/genética , Neoplasias Pulmonares/genética , Lesiones Precancerosas/genética , Adulto , Anciano , Biopsia , Bronquios/metabolismo , Bronquios/patología , Enfermedades Bronquiales/genética , Enfermedades Bronquiales/patología , Carcinoma de Células Escamosas/patología , Puntos de Control del Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Proliferación Celular/genética , Desmogleína 3/genética , Progresión de la Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Inflamación/patología , Neoplasias Pulmonares/patología , Masculino , Metaplasia , Persona de Mediana Edad , Lesiones Precancerosas/patología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , gamma Catenina/genética , Quinasa Tipo Polo 1

RESUMEN

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.

Asunto(s)

Biología Computacional/normas , Guías como Asunto , Secuenciación de Nucleótidos de Alto Rendimiento/normas , Patología Molecular/normas , Humanos , Laboratorios , Reproducibilidad de los Resultados , Estados Unidos

RESUMEN

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.

Asunto(s)

Cromosomas Humanos/genética , Citogenética/métodos , Genómica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Hibridación Fluorescente in Situ/métodos , Trastornos Mieloproliferativos/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Estudios de Cohortes , Variaciones en el Número de Copia de ADN/genética , Humanos , Mutación/genética , Trastornos Mieloproliferativos/diagnóstico , Carga Tumoral

RESUMEN

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.

Asunto(s)

Variaciones en el Número de Copia de ADN , Cardiopatías Congénitas/genética , Síndrome del Corazón Izquierdo Hipoplásico/genética , Transcripción Genética , Animales , Estudios de Cohortes , ADN Complementario/metabolismo , Exones , Femenino , Genotipo , Humanos , Masculino , Cadenas de Markov , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Reacción en Cadena de la Polimerasa , Polimorfismo de Nucleótido Simple

RESUMEN

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.

Asunto(s)

Regulación de la Expresión Génica , Neumonías Intersticiales Idiopáticas/genética , Neumonías Intersticiales Idiopáticas/fisiopatología , Pulmón/fisiopatología , Proteínas/genética , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Proteína ADAMTS4 , Proteína ADAMTS9 , Adulto , Anciano , Selectina E/genética , Selectina E/metabolismo , Femenino , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Masculino , Persona de Mediana Edad , Procolágeno N-Endopeptidasa/genética , Procolágeno N-Endopeptidasa/metabolismo , Receptor para Productos Finales de Glicación Avanzada/genética , Receptor para Productos Finales de Glicación Avanzada/metabolismo , Serpina E2/genética , Serpina E2/metabolismo , Serpinas/genética , Serpinas/metabolismo

RESUMEN

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.

Asunto(s)

Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Células Escamosas/genética , Inestabilidad Cromosómica/genética , Estudio de Asociación del Genoma Completo/métodos , Neoplasias Pulmonares/genética , Análisis por Micromatrices , Polimorfismo de Nucleótido Simple , Lesiones Precancerosas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Células Escamosas/patología , Línea Celular , Aberraciones Cromosómicas , Humanos , Pérdida de Heterocigocidad , Neoplasias Pulmonares/patología , Lesiones Precancerosas/patología

RESUMEN

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.

Asunto(s)

Biología Computacional/educación , Investigación Biomédica/educación , Biología Computacional/instrumentación , Curriculum , Educación , Humanos , Programas Informáticos

RESUMEN

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.

Asunto(s)

Enfisema/sangre , Interleucina-16/sangre , Enfermedad Pulmonar Obstructiva Crónica/sangre , Corticoesteroides/farmacología , Corticoesteroides/uso terapéutico , Anciano , Biomarcadores/sangre , Enfisema/tratamiento farmacológico , Enfisema/genética , Femenino , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Humanos , Leucocitos Mononucleares/metabolismo , Masculino , Persona de Mediana Edad , Fenotipo , Polimorfismo de Nucleótido Simple , Proteómica , Enfermedad Pulmonar Obstructiva Crónica/tratamiento farmacológico , Enfermedad Pulmonar Obstructiva Crónica/genética , Sitios de Carácter Cuantitativo , Fumar/efectos adversos , Fumar/sangre

RESUMEN

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.

Asunto(s)

Cilios/genética , Fibrosis Pulmonar Idiopática/genética , Fibrosis Pulmonar Idiopática/patología , Metaloproteinasa 7 de la Matriz/genética , Mucina 5B/genética , Adulto , Anciano , Estudios de Casos y Controles , Perfilación de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , ARN/análisis , Transcriptoma

RESUMEN

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.

Asunto(s)

Gangliósidos/sangre , Regulación de la Expresión Génica , Leucocitos Mononucleares/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/sangre , Anciano , Anciano de 80 o más Años , Biomarcadores/sangre , Femenino , Humanos , Masculino , Metabolómica/métodos , Persona de Mediana Edad , Enfermedad Pulmonar Obstructiva Crónica/diagnóstico , Enfisema Pulmonar/sangre , Enfisema Pulmonar/diagnóstico , Reacción en Cadena en Tiempo Real de la Polimerasa

RESUMEN

Lung cancers express lower levels of prostacyclin than normal lung tissues. Prostacyclin prevents lung cancer in a variety of mouse models. A randomized phase II trial comparing oral iloprost (a prostacyclin analog) with placebo in high-risk subjects showed improvement in bronchial histology in former, but not current, smokers. This placebo-controlled study offered the opportunity for investigation of other potential intermediate endpoint and predictive biomarkers to incorporate into chemoprevention trials. Matched bronchial biopsies were obtained at baseline and at 6-month follow-up from 125 high-risk individuals who completed the trial: 31/29 and 37/28 current/former smokers in the iloprost and placebo arm, respectively. We analyzed the expression of 14 selected miRNAs by Real Time PCR in 496 biopsies. The expression of seven miRNAs was significantly correlated with histology at baseline. The expression of miR-34c was inversely correlated with histology at baseline (P < 0.0001) and with change in histology at follow-up (P = 0.0003), independent of treatment or smoking status. Several miRNAs were also found to be differentially expressed in current smokers as compared with former smokers. In current smokers, miR-375 was upregulated at baseline (P < 0.0001) and downregulated after treatment with iloprost (P = 0.0023). No miRNA at baseline reliably predicted a response to iloprost. No biomarker predictive of response to iloprost was found. MiR-34c was inversely correlated with baseline histology and with histology changes. Mir-34c changes at follow-up could be used as a quantitative biomarker that parallels histologic response in formalin-fixed bronchial biopsies in future lung cancer chemoprevention studies.

Asunto(s)

Biomarcadores de Tumor , Bronquios/metabolismo , Iloprost/uso terapéutico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/prevención & control , MicroARNs/fisiología , Administración Oral , Antineoplásicos/administración & dosificación , Antineoplásicos/uso terapéutico , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Bronquios/patología , Estudios de Casos y Controles , Quimioprevención , Humanos , Iloprost/administración & dosificación , Neoplasias Pulmonares/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Placebos , Fumar/efectos adversos , Fumar/patología

Asunto(s)

Carcinoma in Situ/genética , Carcinoma de Células Escamosas/genética , Variaciones en el Número de Copia de ADN , Marcadores Genéticos , Neoplasias Pulmonares/genética , Femenino , Humanos , Masculino

RESUMEN

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 ∼6-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.

Asunto(s)

Apoptosis/inmunología , Fibroblastos/inmunología , Pulmón/inmunología , Pulmón/patología , Fibrosis Pulmonar/inmunología , Regulación hacia Arriba/inmunología , Receptor fas/biosíntesis , Animales , Apoptosis/genética , Línea Celular , Línea Celular Transformada , Células Cultivadas , Proteína Ligando Fas/biosíntesis , Proteína Ligando Fas/genética , Fibroblastos/metabolismo , Fibroblastos/patología , Perfilación de la Expresión Génica , Humanos , Estudios Longitudinales , Pulmón/metabolismo , Ratones , Ratones Noqueados , Estudios Prospectivos , Fibrosis Pulmonar/metabolismo , Fibrosis Pulmonar/patología , Regulación hacia Arriba/genética , Receptor fas/deficiencia , Receptor fas/genética

RESUMEN

Pulmonary arterial hypertension is a common and fatal complication of scleroderma that may involve inflammatory and autoimmune mechanisms. Alterations in the gene expression of peripheral blood mononuclear cells have been previously described in patients with pulmonary arterial hypertension. Our goal is to identify differentially expressed genes in peripheral blood mononuclear cells in scleroderma patients with and without pulmonary hypertension as biomarkers of disease. Gene expression analysis was performed on a Microarray Cohort of scleroderma patients with (n = 10) and without (n = 10) pulmonary hypertension. Differentially expressed genes were confirmed in the Microarray Cohort and validated in a Validation Cohort of scleroderma patients with (n = 15) and without (n = 19) pulmonary hypertension by RT-qPCR. We identified inflammatory and immune-related genes including interleukin-7 receptor (IL-7R) and chemokine receptor 7 as differentially expressed in patients with scleroderma-associated pulmonary hypertension. Flow cytometry confirmed decreased expression of IL-7R on circulating CD4+ T-cells from scleroderma patients with pulmonary hypertension. Differences exist in the expression of inflammatory and immune-related genes in peripheral blood cells from patients with scleroderma-related pulmonary hypertension compared to those with normal pulmonary artery pressures. These findings may have implications as biomarkers to screen at-risk populations for early diagnosis and provide insight into mechanisms of scleroderma-related pulmonary hypertension.

Asunto(s)

Células Sanguíneas/inmunología , Esclerodermia Sistémica/sangre , Esclerodermia Sistémica/complicaciones , Linfocitos T CD4-Positivos/inmunología , Análisis por Conglomerados , Estudios de Cohortes , Demografía , Hipertensión Pulmonar Primaria Familiar , Femenino , Citometría de Flujo , Hemodinámica , Humanos , Hipertensión Pulmonar/sangre , Hipertensión Pulmonar/etiología , Hipertensión Pulmonar/genética , Hipertensión Pulmonar/inmunología , Masculino , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Receptores de Interleucina-7/inmunología , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Esclerodermia Sistémica/genética , Esclerodermia Sistémica/inmunología

RESUMEN

BACKGROUND: A plethora of agents is in early stages of development for colorectal cancer (CRC), including those that target the insulin-like growth factor I receptor (IGFIR) pathway. In the current environment of numerous cancer targets, it is imperative that patient selection strategies be developed with the intent of preliminary testing in the latter stages of phase I trials. The goal of this study was to develop and characterize predictive biomarkers for an IGFIR tyrosine kinase inhibitor, OSI-906, that could be applied in CRC-specific studies of this agent. METHODS: Twenty-seven CRC cell lines were exposed to OSI-906 and classified according to IC(50) value as sensitive (5 micromol/L). Cell lines were subjected to immunoblotting and immunohistochemistry for effector proteins, IGFIR copy number by fluorescence in situ hybridization, KRAS/BRAF/phosphoinositide 3-kinase mutation status, and baseline gene array analysis. The most sensitive and resistant cell lines were used for gene array and pathway analyses, along with shRNA knockdown of highly ranked genes. The resulting integrated genomic classifier was then tested against eight human CRC explants in vivo. RESULTS: Baseline gene array data from cell lines and xenografts were used to develop a k-top scoring pair (k-TSP) classifier, which, in combination with IGFIR fluorescence in situ hybridization and KRAS mutational status, was able to predict with 100% accuracy a test set of patient-derived CRC xenografts. CONCLUSIONS: These results indicate that an integrated approach to the development of individualized therapy is feasible and should be applied early in the development of novel agents, ideally in conjunction with late-stage phase I trials.

Asunto(s)

Biomarcadores de Tumor/análisis , Sistemas de Liberación de Medicamentos , Perfilación de la Expresión Génica , Imidazoles/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Pirazinas/farmacología , Receptor IGF Tipo 1/antagonistas & inhibidores , Animales , Línea Celular Tumoral , Resistencia a Antineoplásicos , Ensayos de Selección de Medicamentos Antitumorales , Estudios de Factibilidad , Femenino , Dosificación de Gen , Técnicas de Silenciamiento del Gen , Genes ras , Humanos , Ratones , Ratones Desnudos , Mutación , Proteínas Proto-Oncogénicas B-raf , Receptor IGF Tipo 1/genética , Reproducibilidad de los Resultados , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

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.

Asunto(s)

Remodelación de las Vías Aéreas (Respiratorias)/fisiología , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/genética , Hipertensión Pulmonar/genética , Hipertensión Pulmonar/patología , Adulto , Proliferación Celular , Niño , Aberraciones Cromosómicas , Deleción Cromosómica , Variaciones en el Número de Copia de ADN , Células Endoteliales/patología , Células Endoteliales/fisiología , Femenino , Reordenamiento Génico , Estudio de Asociación del Genoma Completo , Inestabilidad Genómica , Mutación de Línea Germinal , Humanos , Hibridación Fluorescente in Situ , Pulmón/citología , Análisis por Micromatrices , Persona de Mediana Edad , Miocitos del Músculo Liso/patología , Polimorfismo de Nucleótido Simple , Arteria Pulmonar/citología , Arteria Pulmonar/fisiología , Enfermedad Pulmonar Obstructiva Crónica/genética , Enfermedad Pulmonar Obstructiva Crónica/patología , Inactivación del Cromosoma X

RESUMEN

Doxazolidine (Doxaz) is a functionally distinct formaldehyde conjugate of doxorubicin (Dox) that induces cancer cell death in Dox-sensitive and resistant cells. Pentyl PABC-Doxaz (PPD) is a prodrug of Doxaz that is activated by carboxylesterase 2 (CES2), which is expressed by liver, non-small-cell lung, colon, pancreatic, renal, and thyroid cancer cells. Here, we demonstrate that in two murine models, PPD was effective at slowing tumor growth and demonstrated markedly reduced cardiotoxic and nephrotoxic effects, as well as better tolerance, relative to Dox. Hepatotoxicity, consistent with liver expression of the murine CES2 homologue, was induced by PPD. Unlike irinotecan, a clinical CES2-activated prodrug, PPD produced no visible gastrointestinal damage. Finally, we demonstrate that cellular response to PPD may be predicted with good accuracy using CES2 expression and Doxaz sensitivity, suggesting that these metrics may be useful as clinical biomarkers for sensitivity of a specific tumor to PPD treatment.

Asunto(s)

Carbamatos/metabolismo , Carbamatos/farmacología , Carboxilesterasa/metabolismo , Doxorrubicina/análogos & derivados , Oxazoles/metabolismo , Profármacos/metabolismo , Profármacos/farmacología , Animales , Antineoplásicos/metabolismo , Antineoplásicos/farmacología , Western Blotting , Carbamatos/química , Carbamatos/toxicidad , Carboxilesterasa/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Doxorrubicina/química , Doxorrubicina/metabolismo , Doxorrubicina/farmacología , Doxorrubicina/toxicidad , Evaluación Preclínica de Medicamentos , Femenino , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Miocitos Cardíacos/efectos de los fármacos , Neoplasias/enzimología , Neoplasias/patología , Oxazoles/farmacología , Profármacos/química , Profármacos/toxicidad , Ratas , Análisis de Regresión

RESUMEN

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.

Asunto(s)

Carcinoma de Pulmón de Células no Pequeñas/genética , Factores de Crecimiento de Fibroblastos/metabolismo , Neoplasias Pulmonares/genética , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Transducción de Señal/genética , Línea Celular Tumoral , Factores de Crecimiento de Fibroblastos/genética , Humanos , ARN Interferente Pequeño/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/genética

RESUMEN

The quality of gene expression microarray data has improved dramatically since the first arrays were introduced in the late 1990s. However, the reproducibility of data generated at multiple laboratory sites remains a matter of concern, especially for scientists who are attempting to combine and analyze data from public repositories. We have carried out a study in which a common set of RNA samples was assayed five times in four different laboratories using Affymetrix GeneChip arrays. We observed dramatic differences in the results across laboratories and identified batch effects in array processing as one of the primary causes for these differences. When batch processing of samples is confounded with experimental factors of interest it is not possible to separate their effects, and lists of differentially expressed genes may include many artifacts. This study demonstrates the substantial impact of sample processing on microarray analysis results and underscores the need for randomization in the laboratory as a means to avoid confounding of biological factors with procedural effects.

Asunto(s)

Laboratorios/normas , Análisis de Secuencia por Matrices de Oligonucleótidos/normas , Animales , Cromosomas de los Mamíferos/genética , Análisis por Conglomerados , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Masculino , Ratones , Ratones Endogámicos C57BL , Análisis de Componente Principal , Distribución Aleatoria , Reproducibilidad de los Resultados , Caracteres Sexuales

RESUMEN

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.

Asunto(s)

Antibacterianos/uso terapéutico , Fibrosis Quística/sangre , Precursores del ARN/sangre , Enfermedad Aguda , Adulto , Biomarcadores/sangre , Estudios de Cohortes , Fibrosis Quística/tratamiento farmacológico , Fibrosis Quística/genética , Citocinas/sangre , Femenino , Volumen Espiratorio Forzado , Perfilación de la Expresión Génica/métodos , Humanos , Leucocitos/metabolismo , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Valor Predictivo de las Pruebas , Estudios Prospectivos , Precursores del ARN/genética , Curva ROC , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Espirometría/métodos , Adulto Joven