Ester Mejstříková

Detection of residual B precursor lymphoblastic leukemia by uniform gating flow cytometry

Residual disease (RD) is an important prognostic factor in acute lymphoblastic leukemia (ALL). Flow cytometry (FC)‐based RD detection is easy to perform, but interpretation requires expert analysis due to individual differences among patients.

Detection and monitoring of normal and leukemic cell populations with hierarchical clustering of flow cytometry data

Flow cytometry is a valuable tool in research and diagnostics including minimal residual disease (MRD) monitoring of hematologic malignancies. However, its gradual advancement toward increasing numbers of fluorescent parameters leads to information rich datasets, which are challenging to analyze by standard gating and do not reflect the multidimensionality of the data.

Loss of B cells and their precursors is the most constant feature of GATA-2 deficiency in childhood myelodysplastic syndrome

GATA-2 deficiency was recently described as common cause of overlapping syndromes of immunodeficiency, lymphedema, familiar myelodysplastic syndrome or acute myeloid leukemia. The aim of our study was to analyze bone marrow and peripheral blood samples of children with myelodysplastic syndrome or aplastic anemia to define prevalence of the GATA2 mutation and to assess whether mutations in GATA-2 transcription factor exhibit specific immunophenotypic features. The prevalence of a GATA2 mutation in a consecutively diagnosed cohort of children was 14% in advanced forms of myelodysplastic syndrome (refractory anemia with excess blasts, refractory anemia with excess blasts in transformation, and myelodysplasia-related acute myeloid leukemia), 17% in refractory cytopenia of childhood, and 0% in aplastic anemia. In GATA-2-deficient cases, we found the most profound B-cell lymphopenia, including its progenitors in blood and bone marrow, which correlated with significantly diminished intronRSS-Kde recombination excision circles in comparison to other myelodysplastic syndrome/aplastic anemia cases. The other typical features of GATA-2 deficiency (monocytopenia and natural killer cell lymphopenia) were less discriminative. In conclusion, we suggest screening for GATA2 mutations in pediatric myelodysplastic syndrome, preferentially in patients with impaired B-cell homeostasis in bone marrow and peripheral blood (low number of progenitors, intronRSS-Kde recombination excision circles and naïve cells).

Childhood near-tetraploid acute lymphoblastic leukemia: an EGIL study on 36 cases

Objectives:  Patients with near‐tetraploid (karyotype: 81 – 103 chromosomes) acute lymphoblastic leukemia (NT‐ALL) constitute about 1% of childhood ALL and data reported on them are limited and controversial. The aim of the study was to enlarge the knowledge on these rarely occurring ALL.

The TREC/KREC assay for the diagnosis and monitoring of patients with DiGeorge syndrome.

DiGeorge syndrome (DGS) presents with a wide spectrum of thymic pathologies. Nationwide neonatal screening programs of lymphocyte production using T-cell recombination excision circles (TREC) have repeatedly identified patients with DGS. We tested what proportion of DGS patients could be identified at birth by combined TREC and kappa-deleting element recombination circle (KREC) screening. Furthermore, we followed TREC/KREC levels in peripheral blood (PB) to monitor postnatal changes in lymphocyte production. Methods TREC/KREC copies were assessed by quantitative PCR (qPCR) and were related to the albumin control gene in dry blood spots (DBSs) from control (n = 56), severe immunodeficiency syndrome (SCID, n = 10) and DGS (n = 13) newborns. PB was evaluated in DGS children (n = 32), in diagnostic samples from SCID babies (n = 5) and in 91 controls. Results All but one DGS patient had TREC levels in the normal range at birth, albeit quantitative TREC values were significantly lower in the DGS cohort. One patient had slightly reduced KREC at birth. Postnatal DGS samples revealed reduced TREC numbers in 5 of 32 (16%) patients, whereas KREC copy numbers were similar to controls. Both TREC and KREC levels showed a more pronounced decrease with age in DGS patients than in controls (p<0.0001 for both in a linear model). DGS patients had higher percentages of NK cells at the expense of T cells (p<0.0001). The patients with reduced TREC levels had repeated infections in infancy and developed allergy and/or autoimmunity, but they were not strikingly different from other patients. In 12 DGS patients with paired DBS and blood samples, the TREC/KREC levels were mostly stable or increased and showed similar kinetics in respective patients. Conclusions The combined TREC/KREC approach with correction via control gene identified 1 of 13 (8%) of DiGeorge syndrome patients at birth in our cohort. The majority of patients had TREC/KREC levels in the normal range.

Flow Diagnostics Essential Code: A Simple and Brief Format for the Summary of Leukemia Phenotyping

Flow cytometry is a valuable part in the routine diagnostics of acute leukemia (AL). Although internationally recognized definitions of main AL subsets are available, there is currently no consensus format for the short summary of clinical flow cytometry reports. Since clinical reports are too long for most database purposes, there is a need for a standardized format of their short summaries.

Low marginal zone-like B lymphocytes and natural antibodies characterize skewed B-lymphocyte subpopulations in del22q11 DiGeorge patients.

PURPOSE Patients with DiGeorge syndrome suffer from T-lymphopenia. T-cells are important for the maturation and regulation of B-cell function. Our aim was to characterize the B-cell compartment in DiGeorge syndrome patients. METHODS B-cell subset phenotypization using flow cytometry. Serum BAFF (B-cell activating factor) and serum anti-alpha-galactosyl IgM measurement using ELISA. Serum IgG measurement using nephelometry. RESULTS We observed a significantly increased number of naïve B-cells and decreased number of switched memory B-cells in DiGeorge patients. Furthermore, we observed increased BAFF levels and a trend toward hypergammaglobulinemia later in life. Surprisingly, we detected a decrease in marginal zone-like (MZ-like) B-cells and natural antibodies in DiGeorge patients. CONCLUSION The maturation of B-cells is impaired in DiGeorge patients, with high naïve and low switched memory B-cell numbers being observed. There is a clear trend toward hypergammaglobulinemia later in life, coupled with increased serum BAFF levels. Surprisingly, the T-independent humoral response is also impaired, with low numbers of MZ-like B-cell and low levels of anti-alpha-galactosyl IgM natural antibodies being detected.

Reprogramming of B cell acute lymphoblastic leukemia cells: Do we need to shoot a moving target?

In a recent paper, McClellan et al. (1) report that blasts from some precursor B cell acute lymphoblastic leukemia (B-ALL) cases transdifferentiate (reprogram) into nonmalignant cells. Although showing induced reprogramming in both Philadelphia chromosome-positive (Ph+) and Ph− cases, the authors concentrate on Ph+ leukemias, proposing reprogramming to be a possible therapeutic modality for this high-risk group of B-ALLs. Although the findings are highly interesting and relevant, we would like to raise several points of concern.

Cytokines, growth, and environment factors in bone marrow plasma of acute lymphoblastic leukemia pediatric patients

Acute lymphoblastic leukemia (ALL) cells depend on the microenvironment of the host in vivo and do not survive in in vitro culture. Conversely, the suppression of non-malignant tissues is one of the leading characteristics of the course of ALL. Both the non-malignant suppression and malignant cell survival may be partly affected by soluble factors within the bone marrow (BM) environment. Here, we aimed to identify proteins in BM plasma of children with ALL that may contribute to ALL aggressiveness and/or the microenvironment-mediated survival of ALL cells. LBMp (leukemic bone marrow plasma) at the time of ALL diagnosis was compared to control plasma of bone marrow (CBMp) or peripheral blood (CPBp) using a cytokine antibody array. The cytokine antibody array enabled simultaneous detection of 79 proteins per sample. Candidate proteins exhibiting significantly different profiles were further analyzed and confirmed by ELISA.mRNAexpression of one of the candidate proteins (TIMP1) was studied using quantitative reverse transcriptase polymerase chain reaction (qRTPCR). The cytokine antibody array experiments identified 23 proteins that differed significantly (p<0.05); of these, two proteins (TIMP1 and LIF) withstood the Bonferroni correction. In contrast, little difference was observed between CBMp and CPBp. At the diagnosis of ALL, changes in the soluble microenvironment are detectable in BM plasma. These changes probably participate in the pathogenesis and/or result from the changes in the cell composition.

How to make usage of the standardized EuroFlow 8-color protocols possible for instruments of different manufacturers

A critical component of the EuroFlow standardization of leukemia/lymphoma immunophenotyping is instrument setup. Initially, the EuroFlow consortium developed a step-by-step standard operating protocol for instrument setup of ≥8-color flow cytometers that were available in 2006, when the EuroFlow activities started. Currently, there are 14 instruments from 9 manufacturers capable of 3-laser excitation and ≥8 color measurements. The specific adaptations required in the instrument set-up to enable them to acquire the standardized 8-color EuroFlow protocols are described here. Overall, all 14 instruments can be fitted with similar violet, blue and red lasers for simultaneous measurements of ≥8 fluorescent dyes. Since individual instruments differ both on their dynamic range (scale) and emission filters, it is not accurate to simply recalculate the target values to different scale, but adjustment of PMT voltages to a given emission filter and fluorochrome, is essential. For this purpose, EuroFlow has developed an approach using Type IIB (spectrally matching) particles to set-up standardized and fully comparable fluorescence measurements, in instruments from different manufacturers, as demonstrated here for the FACSCanto II, and Navios and MACSQuant flow cytometers. Data acquired after such adjustment on any of the tested cytometry platforms could be fully superimposed and therefore analyzed together. The proposed approach can be used to derive target values for any combination of spectrally distinct fluorochromes and any distinct emission filter of any new flow cytometry platform, which enables the measurement of the 8-color EuroFlow panels in a standardized way, by creating superimposable datafiles.