Estudo comparativo da detecção de Doença Residual Mínima pelas técnicas de imunofenotipagem por citometria de fluxo e reação em cadeia da polimerase, em crianças e adolescentes com diagnóstico de Leucemia Linfoide Aguda

Abstract

Introduction: Current treatment strategies of childhood acute lymphoblastic leukemia (ALL) result in a long-term remission rate of about 90%, however the chance of cure is less among patients who relapse. Stratification schemes using clinical and biological factors have been proposed to classify patients for the relapse risk, with the ultimate goal of offering high-risk patients an effective treatment. The detection of minimal residual disease (MRD) leukemic residual cells in small proportions, verified during the treatment currently represents the main prognostic factor of ALL in children and can suggest a change in the previously started chemotherapeutic treatment. The methods currently recommended to detect MRD are multiparameter flow cytometry (FC) and quantitative real time polymerase chain reaction (RQPCR) of the clonal rearrangements regions of T-cell receptor (TCR) and/or immunoglobulin (Ig) genes. Both methods show good performance in detecting leukemic residual cells, but they are expensive, require qualified staff and are frequently restricted to reference centers, especially the RQ-PCR. Thus, the aim of this study was to investigate whether FC and conventional PCR have good concordance in the detection of MRD at the end of the remission induction phase (D35) and at the end of the remission consolidation phase (D78) of childhood ALL treatment. We also evaluated associations between the DRM results and clinical data of ALL patients. Methods: Between September 2013 and December 2016, 42 children admitted to the Hospital das Clínicas/UFMG with recent ALL diagnosis (35 with B-ALL and 7 with T-ALL) were prospectively studied. The patients were treated according to the protocol of the Brazilian Group for the Treatment of Childhood Acute Leukemia (GBTLI LLA-2009). The bone marrow (BM) samples collected at diagnosis were evaluated by FC, for the immunophenotypic characterization of leukemic cells, and by PCR, for the detection of clonal rearrangements of Ig and TCR genes. The MRD was monitored by FC in the BM samples collected on days 15, 35 and 78 of the treatment and, by PCR, on days 35 and 78. Results: MRD measurements were performed in 107 samples by FC and in 63 samples by conventional PCR. Using 0.01% as a cutoff point, MRD was detected by FC in 87.8%, 17.1% and 28% of the samples taken at D15, D35 and D78, respectively. Otherwise, by PCR, MRD was detected in 13.2% and 12% of the samples analyzed at D35 and D78, respectively. MRD was evaluated by both assays in 61/68 (89.7%) samples collected at D35 and D78. The overall concordance between the two methods was 88.5%, being greater at D35 (91.9%; Kappa: 0.62) in comparison with the D78 analysis (83.3%; Kappa: 0.52). The discordant results were elucidated by the RQ-PCR. When prognostic factors were analyzed, the affected cellular lineage was significantly associated with PCR in both time points (D35: p = 0.037; D78: p = 0.032), being greater the MRD detection frequency in T-ALL subjects. The clinical follow-up period varied from 6 to 46 months (median: 27 months). The overall survival rate (OS) at 3.8 years was 75.1% (± 9.9%) and the event-free survival rate (EFS) was 50.5% (± 21.4%). Higher OS and EFS were observed in B-ALL patients than in T-ALL patients (p = 0,001 and p = 0,001, respectively) and also in patients with white blood cell (WBC) less than 50.000 cells/µL when compared to those with WBC greater than or equal to 50.000 cells/µL (p = 0.0001 and p = 0,00009, respectively). Conclusion: MRD detection by FC and conventional PCR showed comparable results in childhood ALL, when samples were collected at medullar regeneration phases. Both methods are accessible to regions with restricted financial and technological resources and, when concurrently used, may be an effective tool to monitor the chemotherapeutic treatment of children with ALL. Further studies including additional time and a larger number of cases are needed to obtain a more consistent survival analysis.