RESUMO
PURPOSE: After an initial response to androgen ablation, most prostate tumors recur, ultimately progressing to highly aggressive androgen-independent cancer. The molecular mechanisms underlying progression are not well known in part due to the rarity of androgen-independent samples from primary and metastatic sites. EXPERIMENTAL DESIGN: We compared the gene expression profiles of 10 androgen-independent primary prostate tumor biopsies with 10 primary, untreated androgen-dependent tumors. Samples were laser capture microdissected, the RNA was amplified, and gene expression was assessed using Affymetrix Human Genome U133A GeneChip. Differential expression was examined with principal component analysis, hierarchical clustering, and Student's t testing. Analysis of gene ontology was done with Expression Analysis Systematic Explorer and gene expression data were integrated with genomic alterations with Differential Gene Locus Mapping. RESULTS: Unsupervised principal component analysis showed that the androgen-dependent and androgen-independent tumors segregated from one another. After filtering the data, 239 differentially expressed genes were identified. Two main gene ontologies were found discordant between androgen-independent and androgen-dependent tumors: macromolecule biosynthesis was down-regulated and cell adhesion was up-regulated in androgen-independent tumors. Other differentially expressed genes were related to interleukin-6 signaling as well as angiogenesis, cell adhesion, apoptosis, oxidative stress, and hormone response. The Differential Gene Locus Mapping analysis identified nine regions of potential chromosomal deletion in the androgen-independent tumors, including 1p36, 3p21, 6p21, 8p21, 11p15, 11q12, 12q23, 16q12, and 16q21. CONCLUSIONS: Taken together, these data identify several unique characteristics of androgen-independent prostate cancer that may hold potential for the development of targeted therapeutic intervention.
Assuntos
Androgênios/metabolismo , Antineoplásicos Hormonais/farmacologia , Regulação Neoplásica da Expressão Gênica , Regulação da Expressão Gênica , Neoplasias da Próstata/genética , Neoplasias da Próstata/terapia , Idoso , Antagonistas de Androgênios/metabolismo , Biópsia , Adesão Celular , Deleção Cromossômica , Mapeamento Cromossômico , Cromossomos/ultraestrutura , Análise por Conglomerados , Progressão da Doença , Regulação para Baixo , Deleção de Genes , Genoma , Humanos , Interleucina-6/metabolismo , Lasers , Masculino , Pessoa de Meia-Idade , Modelos Estatísticos , Metástase Neoplásica , Análise de Sequência com Séries de Oligonucleotídeos , Estresse Oxidativo , Análise de Componente Principal , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , RNA/metabolismo , Transdução de Sinais , Regulação para CimaRESUMO
Frozen tissue specimens are the gold standard for molecular analysis. However, snap freezing presents several challenges regarding collection and storage of tissue, and preservation of histological detail. We evaluate an alternative preservation method, ethanol fixation followed by paraffin embedding, by analyzing expression profiles of microdissected cells on Affymetrix oligonucleotide arrays of three matched benign prostatic hyperplasia (BPH) and tumor samples processed with each preservation method. Frozen samples generated an average present call of 26% of the probe sets, compared to 4.5% in ethanol-paraffin samples. Eighty-eight percent of the probe sets called present in the ethanol-paraffin samples were also present in the frozen specimens. Comparing ethanol-paraffin BPH to tumor, 52 probe sets showed a twofold differential expression or higher in at least two cases, 23 of which were also differentially expressed in at least one frozen case. Despite a significant drop in the number of transcripts detectable, the data suggests that the obtainable information in ethanol-fixed samples may be useful for molecular profiling where frozen tissue is not available. However, ethanol fixation and paraffin embedding of tissue specimens is not optimal for high-throughput mRNA expression analysis. Improved methods for transcript profiling of archival samples, and/or tissue processing are still required.
Assuntos
Etanol/farmacologia , Regulação da Expressão Gênica , Próstata/patologia , Hiperplasia Prostática/patologia , Manejo de Espécimes/métodos , Fixação de Tecidos/métodos , Fixadores , Congelamento , Perfilação da Expressão Gênica , Genoma Humano , Humanos , Lasers , Masculino , Hibridização de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos , Parafina/química , Inclusão em Parafina/métodos , RNA/metabolismo , RNA Mensageiro/metabolismo , RNA NeoplásicoRESUMO
Using a general strategy for evaluating clinical tissue specimens, we found that 70% ethanol fixation and paraffin embedding is a useful method for molecular profiling studies. Human prostate and kidney were used as test tissues. The protein content of the samples was analyzed by one-dimensional gel electrophoresis, immunoblot, two-dimensional gel electrophoresis, and layered expression scanning. In each case, the fixed and embedded tissues produced results similar to that obtained from snap-frozen specimens, although the protein quantity was somewhat decreased. Recovery of mRNA was reduced in both quantity and quality in the ethanol-fixed samples, but was superior to that obtained from formalin-fixed samples and sufficient to perform reverse transcription polymerase chain reactions. Recovery of DNA from ethanol-fixed specimens was superior to formalin-fixed samples as determined by one-dimensional gel electrophoresis and polymerase chain reaction. In conclusion, specimens fixed in 70% ethanol and embedded in paraffin produce good histology and permit recovery of DNA, mRNA, and proteins sufficient for several downstream molecular analyses. Complete protocols and additional discussion of relevant issues are available on an accompanying website (http://cgap-mf.nih.gov/).