RESUMEN
CONTEXT: Correct diagnosis of the tissue origin of a metastatic cancer is the first step in disease management, but it is frequently difficult using standard pathologic methods. Microarray-based gene expression profiling has shown great promise as a new tool to address this challenge. OBJECTIVE: Adoption of microarray technologies in the clinic remains limited. We aimed to bridge this technological gap by developing a real-time quantitative polymerase chain reaction (RT-PCR) assay. DESIGN: We constructed a microarray database of 466 frozen and 112 formalin-fixed, paraffin-embedded (FFPE) samples of both primary and metastatic tumors, measuring expression of 22,000 genes. From the microarray database, we used a genetic algorithm to search for gene combinations optimal for multitumor classification. A 92-gene RT-PCR assay was then designed and used to generate a database for 481 frozen and 119 FFPE tumor samples. RESULTS: The microarray-based K-nearest neighbor classifier demonstrated 84% accuracy in classifying 39 tumor types via cross-validation and 82% accuracy in predicting 112 independent FFPE samples. We successfully translated the microarray database to the RT-PCR platform, which allowed an overall success rate of 87% in classifying 32 different tumor classes in the validation set of 119 FFPE tumor samples. CONCLUSIONS: The RT-PCR-based expression assay involving 92 genes represents a powerful tool for accurately and objectively identifying the site of origin for metastatic tumors, especially in the cases of cancer of unknown primary. The assay uses RT-PCR and routine FFPE samples, making it suitable for rapid clinical adoption.
Asunto(s)
Perfilación de la Expresión Génica , Metástasis de la Neoplasia/diagnóstico , Metástasis de la Neoplasia/genética , Neoplasias , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Algoritmos , Bases de Datos Factuales , Femenino , Humanos , Masculino , Neoplasias/clasificación , Neoplasias/diagnóstico , Neoplasias/genéticaRESUMEN
Human embryonic stem (hES) cells hold promise for generating an unlimited supply of cells for replacement therapies. To characterize hES cells at the molecular level, we obtained 148,453 expressed sequence tags (ESTs) from undifferentiated hES cells and three differentiated derivative subpopulations. Over 32,000 different transcripts expressed in hES cells were identified, of which more than 16,000 do not match closely any gene in the UniGene public database. Queries to this EST database revealed 532 significantly upregulated and 140 significantly downregulated genes in undifferentiated hES cells. These data highlight changes in the transcriptional network that occur when hES cells differentiate. Among the differentially regulated genes are several components of signaling pathways and transcriptional regulators that likely play key roles in hES cell growth and differentiation. The genomic data presented here may facilitate the derivation of clinically useful cell types from hES cells.
Asunto(s)
Diferenciación Celular/genética , Etiquetas de Secuencia Expresada , Perfilación de la Expresión Génica , Transducción de Señal/genética , Células Madre/metabolismo , Antígenos CD/genética , Antígenos CD/fisiología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , División Celular/efectos de los fármacos , División Celular/genética , División Celular/fisiología , Línea Celular , Receptor gp130 de Citocinas , Dimetilsulfóxido/farmacología , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Embrión de Mamíferos/citología , Factores de Crecimiento de Fibroblastos/genética , Factores de Crecimiento de Fibroblastos/fisiología , Expresión Génica/efectos de los fármacos , Biblioteca de Genes , Sustancias de Crecimiento/farmacología , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/fisiología , Interleucina-6 , Factor Inhibidor de Leucemia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/fisiología , Proteína Nodal , Proteínas/genética , Proteínas/fisiología , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/fisiología , ARN/genética , ARN/aislamiento & purificación , Receptores de Factores de Crecimiento de Fibroblastos/genética , Receptores de Factores de Crecimiento de Fibroblastos/fisiología , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Transducción de Señal/fisiología , Células Madre/citología , Factores de Transcripción/genética , Factores de Transcripción/fisiología , Transcripción Genética/genética , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/fisiología , Tretinoina/farmacología , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genética , Proteínas WntRESUMEN
Cone arrestin (CAR) is a novel member of the arrestin superfamily expressed in retinal cone photoreceptors and the pineal gland. To understand the regulatory mechanisms controlling its cone- and pineal-specific expression, and to facilitate further functional studies using gene knockout approaches, we characterized the genomic organization and the 5'-flanking region of the mouse CAR (mCAR) gene. The mCAR gene is comprised of 17 exons and 16 introns, encoding five alternatively spliced transcripts. A 215-bp proximal promoter fragment containing a TATA box, an Sp1 site and four cone-rod homeobox-binding sites is sufficient to direct expression in cultured retinoblastoma cells and in cone photoreceptors and the pineal gland in transgenic Xenopus laevis.