RESUMO
A standardized, sensitive and universal method for minimal residual disease (MRD) detection in acute myeloid leukemia (AML) is still pending. Although hyperexpression of Wilms' tumor (WT1) gene transcript has been frequently proposed as an MRD marker in AML, wide comparability of the various methods used for evaluating WT1 expression has not been given. We established and standardized a multicenter approach for quantifying WT1 expression by quantitative reverse transcriptase PCR (qRT-PCR), on the basis of a primer/probe set combination at exons 6 and 7. In a series of quality-control rounds, we analyzed 69 childhood AML samples and 47 normal bone marrow (BM) samples from 4 participating centers. Differences in the individual WT1 expressions levels ranged within <0.5 log of the mean in 82% of the cases. In AML samples, the median WT1/1E+04 Abelson (ABL) expression was 3.5E+03 compared with that of 2.3E+01 in healthy BM samples. As 11.5% of childhood AML samples in this cohort harbored WT1 mutations in exon 7, the effect of mutations on WT1 expression has been investigated, showing that mutated cases expressed significantly higher WT1 levels than wild-type cases. Hence, our approach showed high reproducibility and applicability, even in patients with WT1 mutations; therefore, it can be widely used for the quantitation of WT1 expression in future clinical trials.
Assuntos
Exame de Medula Óssea/normas , Genes do Tumor de Wilms , Leucemia Mieloide/patologia , RNA Mensageiro/análise , RNA Neoplásico/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas , Doença Aguda , Adolescente , Adulto , Exame de Medula Óssea/métodos , Criança , Pré-Escolar , Estudos de Coortes , Primers do DNA , Éxons/genética , Feminino , Regulação Leucêmica da Expressão Gênica , Humanos , Lactente , Leucemia Mieloide/diagnóstico , Leucemia Mieloide/genética , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Neoplasia Residual , RNA Mensageiro/genética , RNA Neoplásico/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Sensibilidade e Especificidade , Proteínas WT1/biossíntese , Adulto JovemRESUMO
Assessment of minimal residual disease (MRD) by flow cytometry is considered to be based on the reproducibility of the leukemic immunophenotype detected at diagnosis. However, we previously noticed modulation of surface antigen expression in acute lymphoblastic leukemia (ALL) during the early treatment. Hence, we investigated this in 30 children with B-cell precursor ALL consecutively enrolled in the AIEOP-BFM ALL 2000 protocol. Quantitative expression of seven antigens useful in MRD monitoring was studied at diagnosis and compared to that measured at different time points of remission induction therapy. Downmodulation in the expression of CD10 and CD34 occurred at follow-up. By contrast, upmodulation of CD19, CD20, CD45RA, and CD11a was observed, while the expression of CD58 remained stable. Despite this, we could unambiguously discriminate leukemic cells from normal residual B cells. This holds true when bone marrow (BM) samples from similarly treated T-ALL patients, but not from healthy donors, were used as reference. Our results indicate that immunophenotypic modulation occurs in ALL during the early phases of BFM-type protocols. However, the accuracy of MRD detection by flow cytometry seems not negatively affected if adequate analysis protocols are employed. Investigators should take this phenomenon into account in order to avoid pitfalls in flow cytometric MRD studies.
