RESUMO
BACKGROUND: Minimal residual disease is an important independent prognostic factor in childhood acute lymphoblastic leukemia. The classical detection methods such as multiparameter flow cytometry and real-time quantitative polymerase chain reaction analysis are expensive, time-consuming and complex, and require considerable technical expertise. DESIGN AND METHODS: We analyzed 229 consecutive children with acute lymphoblastic leukemia treated according to the GBTLI-99 protocol at three different Brazilian centers. Minimal residual disease was analyzed in bone marrow samples at diagnosis and on days 14 and 28 by conventional homo/heteroduplex polymerase chain reaction using a simplified approach with consensus primers for IG and TCR gene rearrangements. RESULTS: At least one marker was detected by polymerase chain reaction in 96.4% of the patients. By combining the minimal residual disease results obtained on days 14 and 28, three different prognostic groups were identified: minimal residual disease negative on days 14 and 28, positive on day 14/negative on day 28, and positive on both. Five-year event-free survival rates were 85%, 75.6%, and 27.8%, respectively (p<0.0001). The same pattern of stratification held true for the group of intensively treated children. When analyzed in other subgroups of patients such as those at standard and high risk at diagnosis, those with positive B-derived CD10, patients positive for the TEL/AML1 transcript, and patients in morphological remission on a day 28 marrow, the event-free survival rate was found to be significantly lower in patients with positive minimal residual disease on day 28. Multivariate analysis demonstrated that the detection of minimal residual disease on day 28 is the most significant prognostic factor. CONCLUSIONS: This simplified strategy for detection of minimal residual disease was feasible, reproducible, cheaper and simpler when compared with other methods, and allowed powerful discrimination between children with acute lymphoblastic leukemia with a good and poor outcome.
Assuntos
Neoplasia Residual/diagnóstico , Neoplasia Residual/genética , Reação em Cadeia da Polimerase/métodos , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Adolescente , Antígenos CD/análise , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Criança , Pré-Escolar , Feminino , Citometria de Fluxo/métodos , Citometria de Fluxo/estatística & dados numéricos , Rearranjo Gênico , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias kappa de Imunoglobulina/genética , Lactente , Estimativa de Kaplan-Meier , Masculino , Análise Multivariada , Neoplasia Residual/metabolismo , Reação em Cadeia da Polimerase/estatística & dados numéricos , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Prognóstico , Modelos de Riscos Proporcionais , Receptores de Antígenos de Linfócitos T/genética , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Resultado do TratamentoRESUMO
The leptomeningeal involvement of central nervous system is defined in the most centers by the presence of blast cells in the CSF or the presence of cranial-nerve palsies. Sometimes, cytology does not allow clear distinction between lymphoblasts and normal cells, and auxiliary methods to the precise identification of leukemic cells in cerebrospinal fluid is necessary. We analyzed CSF from 11 consecutive patients, in whom a differential diagnosis of leptomeningeal involvement was made, including 4 patients at diagnosis and 7 patients during the treatment by cytomorphological analysis and PCR and automatic sequencing. Six patients were considered with leptomeningeal involvement by conventional analysis: unequivocal cytomorphological involvement was considered in 5 patients, and in one it was assumed to be due to cranial-nerve palsy, with no blast cells detected in cerebrospinal fluid. In 2 it was considered suspicious and in 3 negative. PCR and sequencing analysis showed involvement in 6 patients; 5 of the 6 patients were considered to have leptomeningeal involvement based on clinical and cytomorphological criteria, and, in one of the patients, it was suspicious. Our data suggest that the use of PCR and sequencing can be useful in confirming CNS leukemia and eliminating other conditions when used together with the cytomorphological analysis.