Can multi-color flowcytometric immunophenotyping replace PCR-based detection of minimal residual disease in childhood acute lymphoblastic leukemia?
01 / 2005 - 12 / 2009
- BACKGROUND. Large-scale clinical minimal residual disease (MRD) studies in childhood acute lymphoblastic leukemia (ALL) have clearly shown the prognostic value of MRD information for predicting relapse. Based on the kinetics of tumor reduction during the first three months of follow-up, using MRD techniques with sensitivities of at least 0.01%, three risk groups with highly significant differences in relapse rates could be discriminated. So far, most MRD studies in childhood ALL have used PCR techniques employing immunoglobulin (Ig) and/or T cell receptor (TCR) gene rearrangements as patient-specific targets. Over the last five years, we have developed and standardized our molecular techniques that are needed for implementation of real-time quantitative (RQ)-PCR-based MRD diagnostics for treatment stratification. In the new DCOG-ALL10 treatment protocol (starting in the second half of 2004), our standardized PCR-based MRD diagnostics will be used for treatment stratification, with treatment reduction for low-risk patients (risk of relapse <5%) and treatment intensification for intermediate-risk patients (risk of relapse 25%) as well as for high-risk patients (risk of relapse >75%). PCR-based risk group stratification is, however, labor-intensive, time-consuming, highly complex, and expensive. This is particularly caused by the complexity of the Ig/TCR gene rearrangement patterns in ALL, which need to be identified in the ALL cells at diagnosis, and the high frequency of subclone formation, which requires the usage of preferably two MRD-PCR targets per patient. Identification of the RQ-PCR targets takes 4 to 6 weeks and measuring the MRD levels in a follow-up sample takes 1 to 2 weeks, implying that the first PCR-based results can not be obtained earlier than 6 to 8 weeks after diagnosis. In contrast, flow cytometric MRD analysis in childhood ALL is much faster, relatively simple, and approximately 2 to 3-fold cheaper, but so far has been hampered by lower sensitivity and, to a lesser extent, applicability. In addition, flow cytometric immunophenotyping has the advantage of analysis at the single-cell level, allowing recognition and characterization of small subpopulations, including the presumed CD34-positive leukemic stem cells. - AIMS. The aims of this translational research project are: 1. To investigate whether flow cytometric MRD detection in childhood ALL can be improved in order to detect an aberrant immunophenotype in over 95% of patients with sensitivities that reach consistently at least 0.01%, preferably 0.001%. The challenge is to simplify the MRD method, while maintaining or increasing the reliability. 2. To evaluate the clinical value of flow cytometric MRD detection in childhood ALL at predefined sampling points and to analyze whether flow cytometric MRD data are at least as good for risk group stratification as RQ-PCR-based MRD data. - PLAN OF INVESTIGATION. Improvement of sensitivity and applicability of flow cytometric MRD analysis should particularly be possible using recently introduced multi-color flow cytometers (applying 6-color labelings) and by inclusion of new markers/antibodies that have become available, e.g. through gene expression profiling studies of normal and leukemic cell samples. MRD will be analyzed using the optimized flow cytometric method in pediatric ALL patients enrolled in the Dutch DCOG-ALL10 treatment protocol. To evaluate the clinical relevance of flow cytometric MRD analysis, (a) flow cytometric MRD data will be compared with the routinely obtained PCR-based MRD data from the same sample; (b) for each patient, the flow cytometry-based MRD risk group will be compared with the PCR-based MRD risk group; and (c) flow cytometry-based MRD risk groups will be compared with PCR-based MRD risk groups in relation to clinical outcome. Furthermore, we aim to identify and characterize leukemic cell subsets, particularly presumed leukemic stem cells, at diagnosis and to monitor these subpopulations by flow cytometry during treatment, as monitoring of these cells may give more insight into true treatment efficacy. - PERSPECTIVE. This study will show whether MRD analysis in childhood ALL using improved flow cytometric immunophenotyping results in comparable risk group stratification as the currently used labor-intensive, highly complex, and expensive PCR-based MRD analysis. If so, future MRD information can be obtained much faster, cheaper, and potentially more accurate, allowing MRD-based risk group classification and subsequent therapy stratification also during earlier phases of therapy.