Abstract
Simple SummaryBy supporting the selection of the most suitable treatment protocol, the advancement of diagnostic methods contributes to achieving the best possible outcome for pediatric cases of acute lymphoblastic leukemia (ALL). In this study, we focused on a novel possibility in the flow cytometric (FC) analysis, as this method is the initial, crucial step in the diagnostic algorithm of ALL and can determine further diagnostic and therapeutic strategies. After the retrospective, multidimensional dot-plot-based FC analysis of 72 bone marrow samples of children with ALL, we found that the integrated appearance of immunophenotype resulted in a simple, quick, and accurate method. Furthermore, associations between immunophenotype and cytogenetic alterations were detected, which enabled the identification of cases with potential adverse outcome by completing the conventional FC analysis with multidimensional dot-plots. Standardized multi-center studies would be required to validate our results.Multicolor flow cytometry (FC) evaluation has a key role in the diagnosis and prognostic stratification of ALL. Our aim was to create new analyzing protocols using multidimensional dot-plots. Seventy-two pediatric patients with ALL were included in this single-center study. Data of a normal BM sample and three BM samples of patients with BCP-ALL were merged, then all B cell populations of the four samples were presented in a single radar dot-plot, and those parameters and locations were selected in which the normal and pathological cell populations differed from each other the most. The integrated profile of immunophenotype resulted in a simple, rapid, and accurate method. There were no significant differences between the percentages of lymphoblasts in the detection of minimal residual disease (MRD) by multidimensional or conventional FC method (p = 0.903 at Day 15 and p = 0.155 at Day 33). Furthermore, we found associations between the position and the number of clusters of blast cells in the radar plots and cytogenetic properties (p = 0.002 and p < 0.0001 by the position and p = 0.02 by the number of subclones). FC analysis based on multidimensional dot-plots is not only a rapid, easy-to-use method, but can also provide additional information to screen cases which require detailed genetic examination.
Highlights
Acute lymphoblastic leukemia (ALL) is the most common pediatric malignant disease with an annual incidence of approximately 3.5/100,000 children worldwide; the overall 3-year survival rate can be as high as 87% if proper treatment is applied [1,2]
flow cytometry (FC) investigation, to some extent, is able to predict the presence of certain cytogenetic aberrations, as there are evidences of associations between the immunophenotype of pathological cells and cytogenetic aberrations, e.g., the absence of CD10 marker on abnormal lymphoblasts is associated with KMT2A gene rearrangement, and CD66c and CD25 positivity is associated with the presence of Philadelphia chromosome in ALL cases [9,10]
The expression of intracellular A subunit of coagulation factor XIII (FXIII-A) needs to be mentioned here; though it is present in pathological B-cell lymphoblasts, the absence of this marker refers to the presence of a genetic subgroup belonging to the new provisional category of “B-other” genetic entity introduced by the World Health Organization (WHO) in 2016
Summary
Acute lymphoblastic leukemia (ALL) is the most common pediatric malignant disease with an annual incidence of approximately 3.5/100,000 children worldwide; the overall 3-year survival rate can be as high as 87% if proper treatment is applied [1,2]. FC investigation, to some extent, is able to predict the presence of certain cytogenetic aberrations, as there are evidences of associations between the immunophenotype of pathological cells and cytogenetic aberrations, e.g., the absence of CD10 marker on abnormal lymphoblasts is associated with KMT2A gene rearrangement, and CD66c and CD25 positivity is associated with the presence of Philadelphia chromosome in ALL cases [9,10]. The standardized FC method is based on well-defined antibody clones labelled with the recommended fluorochromes in 8 or 10-color settings, followed by conventional analysis, which uses bivariate dot-plots. In this case, we gain information about the expression of CD markers one by one, whether each antigen is expressed or not, and even if its presence is weak or bright positive [6]. In case of MRD detection, it is a challenge to identify the abnormal cells in the regenerating bone marrow (BM), where all the stages of normal maturation are present during therapy; an immunophenotype shift of abnormal cells can occur [23,24,25]
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