Minimal residual disease in Brazilian children with acute lymphoid leukemia: comparison of three detection methods by PCR
Introduction
An enormous diversity of heavy chain immunoglobulin (IgH) molecules and T-cell receptors (TCR) is generated during B- and T-lymphocyte differentiation through the rearrangement of the variable (V), diversity (D), junction (J) and constant (C) gene segment. Additional variability may be generated by random insertion of nucleotides not coded by a germinative lineage (N region) between the V and D or D and J elements. Thus, each lymphocyte obtains a specific combination of the V–(D)–J segments [1], [2], [3], [4], [5]. Lymphoid leukemia cells are similar to normal precursor lymphoid cells, presenting, like the latter, rearranged Ig and TCR genes. Since leukemic cells derive from the same clone, they all tend to present the same rearrangement pattern of Ig and TCR genes. These rearrangements can be amplified by the polymerase chain reaction (PCR) at diagnosis, when neoplasic cells are the majority. They also can be sequenced, and primers or complementary DNA probes which hybridize these sequences derived from leukemic clones can be synthesized and used for the detection and quantification of minimal residual disease (MRD) [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15].
Some questions, however, have been raised about the study of MRD: does its detection have advantages and an independent prognostic significance compared to other classical factors such as white blood cells count, age, immunophenotyping or detection by standard light microscopy? [12]. Can relatively simpler techniques such as the study of clonality with consensus primers be useful when compared to other more complex methods for the detection of MRD based on sequencing and on the use of clone-specific markers? [16], [17]. Furthermore, no studies on the behavior and incidence of MRD in children from developing countries have been published thus far. In an attempt to provide some answers to these questions, the goals of the present study were to investigate the detection of MRD by PCR during different phases of treatment in Brazilian children with acute lymphoid leukemia (ALL), with an analysis of T-cell receptor gamma (TCRγ) using consensus primers, clone-specific primers and a semi-nested reaction, to correlate MRD detection to event-free survival (EFS) and to determine its independent prognostic significance by multivariate analysis, testing its association with age, white blood cell count at diagnosis and immunophenotyping.
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Patients
Seventy-four children with ALL were admitted for treatment at the Pediatric Clinic of the University Hospital, Faculty of Medicine of Ribeirão Preto, University of São Paulo, between December 1990 and April 1996, and 43 of them were eligible for the present study. The criterion used for patient inclusion was the availability of at least three bone marrow samples stored during different treatment phases. Thirty-four of these 43 patients (79%) presented TCRγ rearrangements at diagnosis and were
Results
In the present study, the detection frequency of TCRγ gene rearrangement was 76% for lineage B ALL and 100% for T-ALL. Seventeen patients were analyzed in phase I, 14 in phase II, 21 in phase III, and 23 in phase IV. The presence of more than 5% blasts at the end of induction (week 4) was observed in 4/34 patients (11.7%). After reinduction, two of these suffered a relapse, one did not reach remission and the other one is in complete continuous remission (CCR). The 2% agarose gel was not able
Discussion
Several investigators have observed that not only the presence of MRD during treatment, but also its levels may be associated with relapse [11], [12], [13], [14], [15], [23], [24]. Quantitative or semi-quantitative analyses of MRD have shown that relapse occurs more frequently in patients with high detected MRD levels (10−2–10−3) than in patients with lower levels or undetected MRD [11], [12], [13], [14], [15], [23], [24], [25]. This may probably be an explanation for the lack of a significant
Acknowledgements
C.A. Scrideli provided the concept, design, collected and assembled the data, analyzed the data, drafted the manuscript, provided statistical input and gave final approval. S. Kashima provided technical support. R. Cipolloti, R. Defavery, and J.E. Bernardes provided patient materials and logistical support. L.G. Tone contributed significantly to all aspects of this study as well as providing the critical revision and funding.
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