Assuntos
Metilação de DNA , DNA de Neoplasias/metabolismo , Regulação Leucêmica da Expressão Gênica , Leucemia Linfocítica Crônica de Células B/metabolismo , Linfoma de Célula do Manto/metabolismo , Proteínas de Neoplasias/biossíntese , Proteínas do Tecido Nervoso/biossíntese , Receptores Imunológicos/biossíntese , Feminino , Humanos , Leucemia Linfocítica Crônica de Células B/patologia , Linfoma de Célula do Manto/patologia , Masculino , Proteínas RoundaboutRESUMO
Quantitative PCR (qPCR) for detection of fusion transcripts and overexpressed genes is a promising tool for following minimal residual disease (MRD) in patients with hematological malignancies. Its widespread clinical use has to some extent been hampered by differences in data analysis and presentation that complicate multicenter clinical trials. To address these issues, we designed a highly flexible MRD-reporting software program, in which data from various qPCR platforms can be imported, processed, and presented in a uniform manner to generate intuitively understandable reports. The software was tested in a two-step quality control (QC) study; the first step involved eight centers, whose previous experience with the software ranged from none to extensive. The participants received cDNA from consecutive samples from a BCR-ABL+ chronic myeloid leukemia (CML) patient and an acute myeloid leukemia (AML) patient with both CBFß-MYH11 and WT1 target genes, they conducted qPCR on their respective hardware platforms and generated a series of reports with pre-defined features. In step two, five centers used the software to report BCR-ABL+ MRD in a harmonized manner, applying their recently obtained CML international scale conversion factors. The QC study demonstrated that this MRD-reporting software is suitable for efficient handling of qPCR data, generation of MRD reports and harmonization of MRD data.
Assuntos
Sistemas de Gerenciamento de Base de Dados , Bases de Dados Genéticas , Neoplasia Residual/genética , Relatório de Pesquisa/normas , Reação em Cadeia da Polimerase Via Transcriptase Reversa/estatística & dados numéricos , DNA Complementar/genética , DNA de Neoplasias/genética , Europa (Continente)/epidemiologia , Genes do Tumor de Wilms , Humanos , Serviços de Informação , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mieloide Aguda/genética , Neoplasia Residual/epidemiologia , Proteínas de Fusão Oncogênica/genética , Controle de Qualidade , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Pesquisa Translacional Biomédica/métodosRESUMO
Individualized PCR strategies hamper comparability of molecular results between different laboratories in several fields of medicine. To harmonize BCR-ABL mRNA quantification an international multicenter trial involving 37 laboratories in 14 countries was initiated using 10 samples, each containing various dilutions (10, 2, 1 and 0.1%) of b3a2 or b2a2 BCR-ABL positive in normal leukocytes and negative controls. A novel control plasmid (pME-2) was designed for external calibration containing BCR-ABL and glucuronidase-beta (GUS) sequences. Median BCR-ABL/ABL ratios were 9.1, 1.8, 0.85 and 0.11% in b3a2 samples and 9.5, 1.6, 0.84 and 0.11% in b2a2 samples. Median BCR-ABL/GUS ratios were 3.4, 0.77, 0.37 and 0.042% in b3a2 samples and 2.8, 0.48, 0.29 and 0.031% in b2a2 samples. The coefficients of variation were 0.62 for ratios BCR-ABL/ABL and 1.03 for ratios BCR-ABL/GUS. Five of 37 evaluable participating laboratories (13%) detected low BCR-ABL copy numbers in negative control samples; one laboratory failed to detect BCR-ABL in a low-level sample. We conclude that the use of a common control plasmid does indeed improve comparability of BCR-ABL mRNA quantification results. However, further standardizing efforts like introducing a calibrator and regular control rounds are needed.
