RESUMO
BACKGROUND: Gene expression profiling has the potential to offer consistent, objective diagnostic test results once a standardized protocol has been established. We investigated the robustness, precision, and reproducibility of microarray technology. METHODS: One hundred sixty individual patient samples representing 11 subtypes of acute and chronic leukemias, myelodysplastic syndromes, and nonleukemia as a control group were centrally collected and diagnosed as part of the daily routine in the Munich Leukemia Laboratory. The custom AmpliChip Leukemia research microarray was used for technical analyses of quadruplicate mononuclear cell lysates in 4 different laboratories in Germany (D), Austria (A), and Switzerland (CH) (the DACH study). RESULTS: Total-RNA preparations were successfully performed in 637 (99.5%) of 640 cases. Mean differences between pairs of laboratories in the total-RNA yield from the same sample ranged from 0.02 mug to 1.03 mug. Further processing produced 622 successful in vitro transcription reactions (97.6%); the mean differences between laboratories in the cRNA yield from the same sample ranged from 0.40 mug to 6.18 mug. After hybridization to microarrays, a mean of 47.6%, 46.5%, 46.2%, and 46.4% of probe sets were detected as present for the 4 laboratories, with mean signal-intensity scaling factors of 3.1, 3.7, 4.0, and 4.2, respectively. In unsupervised hierarchical cluster and principal component analyses, replicates from the same patient always clustered closely together, with no indications of any association between gene expression profiles due to different operators or laboratories. CONCLUSIONS: Microarray analysis can be performed with high interlaboratory reproducibility and with comparable quality and high technical precision across laboratories.
Assuntos
Perfilação da Expressão Gênica , Laboratórios/normas , Leucemia/genética , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Reprodutibilidade dos TestesRESUMO
Gene expression profiling has the potential to enhance current methods for the diagnosis of haematological malignancies. Here, we present data on 204 analyses from an international standardization programme that was conducted in 11 laboratories as a prephase to the Microarray Innovations in LEukemia (MILE) study. Each laboratory prepared two cell line samples, together with three replicate leukaemia patient lysates in two distinct stages: (i) a 5-d course of protocol training, and (ii) independent proficiency testing. Unsupervised, supervised, and r(2) correlation analyses demonstrated that microarray analysis can be performed with remarkably high intra-laboratory reproducibility and with comparable quality and reliability.
Assuntos
Perfilação da Expressão Gênica/normas , Leucemia/genética , Análise de Sequência com Séries de Oligonucleotídeos/normas , Adenocarcinoma/genética , Neoplasias da Mama/genética , Carcinoma/genética , Europa (Continente) , Feminino , Perfilação da Expressão Gênica/métodos , Humanos , Leucemia/diagnóstico , Neoplasias Hepáticas/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Projetos Piloto , RNA , Padrões de Referência , Reprodutibilidade dos Testes , Singapura , Estados UnidosAssuntos
Elementos de DNA Transponíveis , Leucemia Linfocítica Crônica de Células B/genética , Proteínas de Neoplasias/genética , Polimorfismo Genético , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-bcl-2/genética , Estudos de Casos e Controles , Clonagem Molecular , Frequência do Gene , Genótipo , Humanos , Proteína de Sequência 1 de Leucemia de Células Mieloides , Reação em Cadeia da Polimerase , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Valor Preditivo dos Testes , Prognóstico , Análise de Sequência de DNARESUMO
Microarray technology has been proposed as an addition to the methods in current use for diagnosing leukemia. Before a new technology can be used in a diagnostic setting, the method has to be shown to produce robust results. It is known that, given the technical aspects of specimen sampling and target preparation, global gene expression patterns can change dramatically. Various parameters such as RNA degradation, shipment time, sample purity, and patient age can principally influence measured gene expression. However, thus far, no information has been available on the robustness of a diagnostic gene expression signature. We demonstrate here that for a subset of acute leukemia, expression profiling is applicable in a diagnostic setting, considering various influencing parameters. With the use of a set of differentially expressed genes, that is, a diagnostic gene expression signature, four genetically defined acute myeloid leukemia subtypes with recurrent chromosomal aberrations can clearly be identified. In addition, we show that preparation by different operators and using different sample-handling procedures did not impair the robustness of diagnostic expression signatures. In conclusion, our results provide additional support for the applicability of microarrays in a diagnostic setting, and we have been encouraged to enroll patients in a prospective study in which microarrays will be tested as an additional routine diagnostic method in parallel with standard diagnostic procedures.
