Phenotypic profiling of CD34+ cells by advanced flow cytometry improves diagnosis of juvenile myelomonocytic leukemia.

Diagnostic criteria for juvenile myelomonocytic leukemia (JMML) are currently well defined, however in some patients diagnosis still remains a challenge. Flow cytometry is a well established tool for diagnosis and follow-up of hematological malignancies, nevertheless it is not routinely used for JMML diagnosis. Herewith, we characterized the CD34+ hematopoietic precursor cells collected from 31 children with JMML using a combination of standardized EuroFlow antibody panels to assess the ability to discriminate JMML cells from normal/reactive bone marrow cell as controls (n=29) or from cells of children with other hematological diseases mimicking JMML (n=9). CD34+ precursors in JMML showed markedly reduced B-cell and erythroid-committed precursors compared to controls, whereas monocytic and CD7+ lymphoid precursors were significantly expanded. Moreover, aberrant immunophenotypes were consistently present in CD34+ precursors in JMML, while they were virtually absent in controls. Multivariate logistic regression analysis showed that combined assessment of the number of CD34+CD7+ lymphoid precursors and CD34+ aberrant precursors or erythroid precursors had a great potential in discriminating JMMLs versus controls. Importantly our scoring model allowed highly efficient discrimination of truly JMML versus patients with JMML-like diseases. In conclusion, we show for the first time that CD34+ precursors from JMML patients display a unique immunophenotypic profile which might contribute to a fast and accurate diagnosis of JMML worldwide by applying an easy to standardize single eight-color antibody combination.

Circular Dichroism as a Rapid Method for Analyzing the Binding of a Targeting Ligand to the Surface of Albumin Nanoparticles.

Circular dichroism (CD) is an excellent and rapid method for analysis of chiral molecules, whose mechanism is based on the absorption of left- and right-hand circularly polarized light. Albumin nanoparticles are biocompatible and easy to modify due to their structure. Tumor cell membranes are among the molecules that direct nanoparticles into the tumor microenvironment, but methods to study them except molecular biology are not well validated yet. The aim of this study was to use circular dichroism as the tool to qualitatively assess ligand binding on the surface of nanoparticles. Human serum albumin (HSA) nanoparticles with encapsulated 5-fluorouracil (5-FU) were coated on MCF-7 cell membranes and subjected to CD analysis. This study was completed using sample and separate 5-FU release analysis. The amount of encapsulated drug in nanoparticles affects the binding of cell membranes on the nanoparticle surface. In addition, it can be suspected that the alpha structure of HSA was mainly used for the interaction, which confirms the effectiveness of using CD as a rapid technique for analyzing ligand-nanoparticle interactions. The release of 5-FU from the nanoparticles proceeds in an uncontrolled manner, making this study in need of further modification and investigation.

Minimal residual disease assessment in B-cell precursor acute lymphoblastic leukemia by semi-automated identification of normal hematopoietic cells: A EuroFlow study.

Presence of minimal residual disease (MRD), detected by flow cytometry, is an important prognostic biomarker in the management of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). However, data-analysis remains mainly expert-dependent. In this study, we designed and validated an Automated Gating & Identification (AGI) tool for MRD analysis in BCP-ALL patients using the two tubes of the EuroFlow 8-color MRD panel. The accuracy, repeatability, and reproducibility of the AGI tool was validated in a multicenter study using bone marrow follow-up samples from 174 BCP-ALL patients, stained with the EuroFlow BCP-ALL MRD panel. In these patients, MRD was assessed both by manual analysis and by AGI tool supported analysis. Comparison of MRD levels obtained between both approaches showed a concordance rate of 83%, with comparable concordances between MRD tubes (tube 1, 2 or both), treatment received (chemotherapy versus targeted therapy) and flow cytometers (FACSCanto versus FACSLyric). After review of discordant cases by additional experts, the concordance increased to 97%. Furthermore, the AGI tool showed excellent intra-expert concordance (100%) and good inter-expert concordance (90%). In addition to MRD levels, also percentages of normal cell populations showed excellent concordance between manual and AGI tool analysis. We conclude that the AGI tool may facilitate MRD analysis using the EuroFlow BCP-ALL MRD protocol and will contribute to a more standardized and objective MRD assessment. However, appropriate training is required for the correct analysis of MRD data.

The Role of Eosinophil-Derived Neurotoxin and Vascular Endothelial Growth Factor in the Pathogenesis of Eosinophilic Asthma.

Asthma is a chronic complex pulmonary disease characterized by airway inflammation, remodeling, and hyperresponsiveness. Vascular endothelial growth factor (VEGF) and eosinophil-derived neurotoxin (EDN) are two significant mediators involved in the pathophysiology of asthma. In asthma, VEGF and EDN levels are elevated and correlate with disease severity and airway hyperresponsiveness. Diversity in VEGF polymorphisms results in the variability of responses to glucocorticosteroids and leukotriene antagonist treatment. Targeting VEGF and eosinophils is a promising therapeutic approach for asthma. We identified lichochalcone A, bevacizumab, azithromycin (AZT), vitamin D, diosmetin, epigallocatechin gallate, IGFBP-3, Neovastat (AE-941), endostatin, PEDF, and melatonin as putative add-on drugs in asthma with anti-VEGF properties. Further studies and clinical trials are needed to evaluate the efficacy of those drugs. AZT reduces the exacerbation rate and may be considered in adults with persistent symptomatic asthma. However, the long-term effects of AZT on community microbial resistance require further investigation. Vitamin D supplementation may enhance corticosteroid responsiveness. Herein, anti-eosinophil drugs are reviewed. Among them are, e.g., anti-IL-5 (mepolizumab, reslizumab, and benralizumab), anti-IL-13 (lebrikizumab and tralokinumab), anti-IL-4 and anti-IL-13 (dupilumab), and anti-IgE (omalizumab) drugs. EDN over peripheral blood eosinophil count is recommended to monitor the asthma control status and to assess the efficacy of anti-IL-5 therapy in asthma.

The kinetics of blast clearance are associated with copy number alterations in childhood B-cell acute lymphoblastic leukemia.

We analyzed the pattern of whole-genome copy number alterations (CNAs) and their association with the kinetics of blast clearance during the induction treatment among 195 pediatric patients with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) who displayed intermediate or high levels of minimal residual disease (MRD). Using unsupervised hierarchical clustering of CNAs > 5 Mbp, we dissected three clusters of leukemic samples with distinct kinetics of blast clearance [A - early slow responders (n=105), B - patients with persistent leukemia (n=24), C - fast responders with the low but detectable disease at the end of induction (n=66)] that corresponded with the patients' clinical features, the microdeletion profile,the presence of gene fusions and patients survival. Low incidence of large CNAs and chromosomal numerical aberrations occurred in cluster A which included ALL samples showing recurrent microdeletions within the genes encoding transcription factors (i.e., IKZF1, PAX5, ETV6, and ERG), DNA repair genes (XRCC3 and TOX), or harboring chromothriptic pattern of CNAs. Low hyperdiploid karyotype with trisomy 8 or hypodiploidy was predominantly observed in cluster B. Whereas cluster C included almost exclusively high-hyperdiploid ALL samples with concomitant mutations in RAS pathway genes. The pattern of CNAs influences the kinetics of leukemic cell clearance and selected aberrations affecting DNA repair genes may contribute to BCP-ALL chemoresistance.

Small RNA-Seq Reveals Similar miRNA Transcriptome in Children and Young Adults with T-ALL and Indicates miR-143-3p as Novel Candidate Tumor Suppressor in This Leukemia.

We aimed to identify miRNAs and pathways specifically deregulated in adolescent and young adult (AYA) T-ALL patients. Small RNA-seq showed no major differences between AYA and pediatric T-ALL, but it revealed downregulation of miR-143-3p in T-ALL patients. Prediction algorithms identified several known and putative oncogenes targeted by this miRNA, including KRAS, FGF1, and FGF9. Pathway analysis indicated signaling pathways related to cell growth and proliferation, including FGFR signaling and PI3K-AKT signaling, with the majority of genes overrepresented in these pathways being predicted targets of hsa-miR-143-3p. By luciferase reporter assays, we validated direct interactions of this miRNA with KRAS, FGF1 and FGF9. In cell proliferation assays, we showed reduction of cell growth upon miR-143-3p overexpression in two T-ALL cell lines. Our study is the first description of the miRNA transcriptome in AYA T-ALL patients and the first report on tumor suppressor potential of miR-143-3p in T-ALL. Downregulation of this miRNA in T-ALL patients might contribute to enhanced growth and viability of leukemic cells. We also discuss the potential role of miR-143-3p in FGFR signaling. Although this requires more extensive validation, it might be an interesting direction, since FGFR inhibition proved promising in preclinical studies in various cancers.

Commonly Assessed Markers in Childhood BCP-ALL Diagnostic Panels and Their Association with Genetic Aberrations and Outcome Prediction.

Immunophenotypic characterization of leukemic cells with the use of flow cytometry (FC) is a fundamental tool in acute lymphoblastic leukemia (ALL) diagnostics. A variety of genetic aberrations underlie specific B-cell precursor ALL (BCP-ALL) subtypes and their identification is of great importance for risk group stratification. These aberrations include: ETV6::RUNX1 fusion gene, Philadelphia chromosome (BCR::ABL1 fusion gene), rearrangements of the KMT2A, TCF3::PBX1 fusion gene and changes in chromosome number (hyperdiploidy and hypodiploidy). Diagnostic panels for BCP-ALL usually include B-cell lineage specific antigens: CD19, CD10, CD20, maturation stage markers: CD34, CD10, CD38, TdT, IgM and other markers useful for possible genetic subtype indication. Some genetic features of leukemic cells (blasts) are associated with expression of certain antigens. This review comprehensively summarizes all known research data on genotype-immunophenotype correlations in BCP-ALL. In some cases, single molecules are predictive of particular genetic subtypes, i.e., NG2 with KMT2A gene rearrangements or CD123 with hyperdiploidy. However, much more information on possible genotype or prognosis can be obtained with wider (≥8-color) panels. In several studies, a quantitative antigen expression scale and advanced statistical analyses were used to further increase the specificity and sensitivity of genotype/immunophenotype correlation detection. Fast detection of possible genotype/immunophenotype correlations makes multicolor flow cytometry an essential tool for initial leukemia diagnostics and stratification.

Efficacy of ruxolitinib in acute lymphoblastic leukemia: A systematic review.

Philadelphia-like acute lymphoblastic leukemia (Ph-like ALL) is a high-risk molecular subtype with a gene expression profile similar to Philadelphia-positive ALL, but not harboring the BCR-ABL1 gene fusion. We aimed to investigate the efficacy of target therapy with the Janus kinase inhibitor, ruxolitinib, in patients with Ph-like ALL and molecular signature of JAK-STAT signaling pathway. A systematic search of the literature was performed to identify reports concerning administration of ruxolitinib in Ph-like ALL patients. Additionally, Polish Pediatric ALL registries were searched for patients with Ph-like ALL treated with ruxolitinib. Extracted information included epidemiological background, somatic aberrations, treatment response, and patient outcome. After PubMed database search, twelve patients were identified, and one was identified in the Polish Pediatric ALL registry. In nine patients gene fusions affecting JAK2 (n = 7) and EPOR (n = 2) were detected. Surface overexpression of CRLF2 and IKZF1 deletions were observed in two and three patients, respectively. Induction failure occurred in all the patients. Therapy with ruxolitinib led to complete (n = 7) and partial (n = 2) remission, in three individuals no information was found. Based on the limited number of studies describing the efficacy of ruxolitinib as an additional compound administrated with standard ALL therapy, we conclude that this approach can be considered in patients with aberrations activating JAK-STAT pathway.

Case report: Successful allogeneic stem cell transplantation in a child with novel GATA2 defect associated B-cell acute lymphoblastic leukemia.

GATA-binding protein 2 (GATA2) is a transcription factor responsible for the regulation of blood cell proliferation, differentiation, and maintenance in hematopoietic stem cells. Here, we describe successful bone marrow transplantation in a carrier of a novel GATA2 pathogenic variant who was diagnosed with immunodeficiency a few years after completion of B-cell precursor acute lymphoblastic leukemia (BCP-ALL) treatment. At the age of 4 years, the patient was diagnosed with and treated for BCP-ALL. Antileukemic therapy was complicated by pulmonary cryptococcosis. Two years after completion of the maintenance therapy, the child was consulted by an immunologist because of recurrent respiratory tract infections and an episode of sepsis. Flow cytometry revealed deep monocytopenia, lymphopenia, absence of B lymphocytes, considerably reduced NK cells, poor thymic T lymphocyte production, minor defects in T cell maturation, and absence of TCRγδ+ T cells. The presence of the likely pathogenic, heterozygous missense variant within exon 5 of GATA2 (NM_032638.5: c.1047T>G, Cys349Trp) was identified in the proband and confirmed in the father of the patient, who underwent allogeneic hematopoietic stem cell transplantation (HSCT) from a matched unrelated donor due to myelodysplastic syndrome with excess blasts at the age of 22 years. An allogeneic hematopoietic stem cell transplantation with a reduced toxicity conditioning protocol was performed using a matched sibling donor. Pre-transplant conditioning included fludarabine (5 × 30 mg/m2), treosulfan (3 × 14 g/m2), and thiotepa (10 mg/kg). Complete donor chimerism was achieved on post-transplant day 17. During the 12 months of the posttransplant observation period, she remained free from symptoms of acute or chronic graft-versus-host disease, and immunosuppressive treatment was therefore stopped. This is the second reported case of BCP-ALL in a patient with GATA2 deficiency, and the first successfully treated with a reduced-toxicity conditioning HSCT protocol. The co-occurrence of lymphoid malignancies and primary immunodeficiencies points to the role of genetic counseling and family screening for possible cancer predisposition syndromes prior to the selection of related HSCT donors.

Multiomics to investigate the mechanisms contributing to repression of PTPRC and SOCS2 in pediatric T-ALL: Focus on miR-363-3p and promoter methylation.

T-cell acute lymphoblastic leukemia (T-ALL) is a heterogeneous and aggressive malignancy arising from T-cell precursors. MiRNAs are implicated in negative regulation of gene expression and when aberrantly expressed contribute to various cancer types, including T-ALL. Previously we demonstrated the oncogenic potential of miR-363-3p overexpression in a subgroup of T-ALL patients. Here, using combined proteomic and transcriptomic approaches, we show that miR-363-3p enhances cell growth of T-ALL in vitro via inhibition of PTPRC and SOCS2, which are implicated in repression of the JAK-STAT pathway. We propose that overexpression of miR-363-3p is a novel mechanism potentially contributing to overactivation of JAK-STAT pathway. Additionally, by combining the transcriptomic and methylation data of T-ALL patients, we show that promoter methylation may also contribute to downregulation of SOCS2 expression and thus potentially to JAK-STAT activation. In conclusion, we highlight aberrant miRNA expression and aberrant promoter methylation as mechanisms, alternative to mutations of JAK-STAT-related genes, which might lead to the upregulation of JAK-dependent signaling in T-ALL.

Bone Marrow Stromal Cell Regeneration Profile in Treated B-Cell Precursor Acute Lymphoblastic Leukemia Patients: Association with MRD Status and Patient Outcome.

For the last two decades, measurable residual disease (MRD) has become one of the most powerful independent prognostic factors in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). However, the effect of therapy on the bone marrow (BM) microenvironment and its potential relationship with the MRD status and disease free survival (DFS) still remain to be investigated. Here we analyzed the distribution of mesenchymal stem cells (MSC) and endothelial cells (EC) in the BM of treated BCP-ALL patients, and its relationship with the BM MRD status and patient outcome. For this purpose, the BM MRD status and EC/MSC regeneration profile were analyzed by multiparameter flow cytometry (MFC) in 16 control BM (10 children; 6 adults) and 1204 BM samples from 347 children and 100 adult BCP-ALL patients studied at diagnosis (129 children; 100 adults) and follow-up (824 childhood samples; 151 adult samples). Patients were grouped into a discovery cohort (116 pediatric BCP-ALL patients; 338 samples) and two validation cohorts (74 pediatric BCP-ALL, 211 samples; and 74 adult BCP-ALL patients; 134 samples). Stromal cells (i.e., EC and MSC) were detected at relatively low frequencies in all control BM (16/16; 100%) and in most BCP-ALL follow-up samples (874/975; 90%), while they were undetected in BCP-ALL BM at diagnosis. In control BM samples, the overall percentage of EC plus MSC was higher in children than adults (p = 0.011), but with a similar EC/MSC ratio in both groups. According to the MRD status similar frequencies of both types of BM stromal cells were detected in BCP-ALL BM studied at different time points during the follow-up. Univariate analysis (including all relevant prognostic factors together with the percentage of stromal cells) performed in the discovery cohort was used to select covariates for a multivariate Cox regression model for predicting patient DFS. Of note, an increased percentage of EC (>32%) within the BCP-ALL BM stromal cell compartment at day +78 of therapy emerged as an independent unfavorable prognostic factor for DFS in childhood BCP-ALL in the discovery cohort­hazard ratio (95% confidence interval) of 2.50 (1−9.66); p = 0.05­together with the BM MRD status (p = 0.031). Further investigation of the predictive value of the combination of these two variables (%EC within stromal cells and MRD status at day +78) allowed classification of BCP-ALL into three risk groups with median DFS of: 3.9, 3.1 and 1.1 years, respectively (p = 0.001). These results were confirmed in two validation cohorts of childhood BCP-ALL (n = 74) (p = 0.001) and adult BCP-ALL (n = 40) (p = 0.004) treated at different centers. In summary, our findings suggest that an imbalanced EC/MSC ratio in BM at day +78 of therapy is associated with a shorter DFS of BCP-ALL patients, independently of their MRD status. Further prospective studies are needed to better understand the pathogenic mechanisms involved.

Machine Learning Based Analysis of Relations between Antigen Expression and Genetic Aberrations in Childhood B-Cell Precursor Acute Lymphoblastic Leukaemia.

Flow cytometry technique (FC) is a standard diagnostic tool for diagnostics of B-cell precursor acute lymphoblastic leukemia (BCP-ALL) assessing the immunophenotype of blast cells. BCP-ALL is often associated with underlying genetic aberrations, that have evidenced prognostic significance and can impact the disease outcome. Since the determination of patient prognosis is already important at the initial phase of BCP-ALL diagnostics, we aimed to reveal specific genetic aberrations by finding specific multiple antigen expression patterns with FC immunophenotyping. The FC immunophenotype data were analysed using machine learning methods (gradient boosting, decision trees, classification rules). The obtained results were verified with the use of repeated cross-validation. The t(12;21)/ETV6-RUNX1 aberration occurs more often when blasts present high expression of CD10, CD38, low CD34, CD45 and specific low expression of CD81. The t(v;11q23)/KMT2A is associated with positive NG2 expression and low CD10, CD34, TdT and CD24. Hyperdiploidy is associated with CD123, CD66c and CD34 expression on blast cells. In turn, high expression of CD81, low expression of CD45, CD22 and lack of CD123 and NG2 indicates that none of the studied aberrations is present. Detecting aberrations in pediatric BCP-ALL, based on the expression of multiple markers, can be done with decent efficiency.

Expression of CD73 on leukemic blasts increases during follow-up - a promising candidate marker for minimal residual disease detection in pediatric B-cell precursor acute lymphoblastic leukemia.

Flow cytometry (FCM) is a precise and well-established tool to assess the minimal residual disease (MRD) level in childhood acute lymphoblastic leukemia (ALL). It is crucial to distinguish leukemic cells from their normal counterparts; thus new markers should be evaluated, to increase the accuracy of the analysis. The expression of CD73 on blast cells was measured and compared at the day of diagnosis and at days 15 and 33 of treatment. To determine antigen expression levels, a normalized scale based on median fluorescence intensity (nMFI) was used. The study group consisted of 188 patients from the Polish Pediatric Leukemia and Lymphoma Study Group. From 177 patients with positive MRD at day 15 of treatment, in 147 (83.1%) cases an increase of CD73 expression was observed (mean increase of +17 nMFI units). In addition, an increase of CD73 expression was noted in 26 of 31 (83.9%) patients at day 33 of treatment. In turn, a decrease of CD73 expression was observed only in 13/177 (7.3%) and 1/31 (3.2%) cases at days 15 and 33 of treatment, respectively. In 17 (9.6%) patients no change in expression of CD73 between diagnosis and day 15 of treatment was observed. In the great majority of cases the expression of CD73 is not only stable but increases during the early stages of treatment, which makes it a very useful marker to be used for MRD monitoring in childhood B-cell precursor (BCP)-ALL patients.

Impact of Pre-Analytical and Analytical Variables Associated with Sample Preparation on Flow Cytometric Stainings Obtained with EuroFlow Panels.

Objective interpretation of FC results may still be hampered by limited technical standardization. The EuroFlow consortium conducted a series of experiments to determine the impact of different variables on the relative distribution and the median fluorescence intensity (MFI) of markers stained on different cell populations, from both healthy donors and patients' samples with distinct hematological malignancies. The use of different anticoagulants; the time interval between sample collection, preparation, and acquisition; pH of washing buffers; and the use of cell surface membrane-only (SM) vs. cell surface plus intracytoplasmic (SM+CY) staining protocols, were evaluated. Our results showed that only monocytes were represented at higher percentages in EDTA- vs. heparin-anticoagulated samples. Application of SM or SM+CY protocols resulted in slight differences in the percentage of neutrophils and debris determined only with particular antibody combinations. In turn, storage of samples for 24 h at RT was associated with greater percentage of debris and cell doublets when the plasma cell disorder panel was used. Furthermore, 24 h storage of stained cells at RT was selectively detrimental for MFI levels of CD19 and CD45 on mature B- and T-cells (but not on leukemic blasts, clonal B- and plasma cells, neutrophils, and NK cells). The obtained results showed that the variables evaluated might need to be tailored for sample and cell type(s) as well as to the specific markers compared; however, defining of well-balanced boundaries for storage time, staining-to-acquisition delay, and pH of washing buffer would be a valid recommendation for most applications and circumstances described herein.

Prognostic significance of IKZF1 deletions and IKZF1plus profile in children with B-cell precursor acute lymphoblastic leukemia treated according to the ALL-IC BFM 2009 protocol.

The strongest predictors of outcome in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL) are minimal residual disease (MRD) and specific molecular abnormalities. One unfavorable prognostic factor is the presence of IKZF1 gene aberrations, particularly when co-occurring with high MRD level at the end of induction treatment. The present study determines the predictive value of a recently-defined IKZF1-plus (IKZF1plus ) microdeletion profile in 373 children with BCP-ALL treated according to the ALL-intercontinental Berlin-Frankfurt-Munster protocol 2009 protocol. IKZF1-wild type (IKZF1wt ) patients demonstrated lower leukemic burden parameters than those carrying IKZF1 deletion (IKZF1del [n = 26, 7.0%]) or IKZF1plus pattern (n = 34, 9.1%): (i) median blast percentage at diagnosis (78.0% vs. 86.9% vs. 86.0%; p = 0.021); (ii) median MRD level at day 15 of induction protocol (0.3% vs. 2.1% vs. 0.8%; p = 0.011); (iii) poor steroid response (7.6% vs. 26.5% vs. 12.5%; p = 0.010). Minimal residual disease level at day 33 (MRD33) exceeding 10-4 was more frequently observed in both the IKZF1del and IKZF1plus subgroups than in IKZF1wt patients (n = 9 [36.0%] vs. n = 13 [41.9%] vs. n = 70 [24.0%], p = 0.051). IKZF1plus individuals showed a tendency for a lower MRD reduction between day 15 and 33 compared to IKZF1del patients (p = 0.124). IKZF1del and IKZF1plus patients showed decreased relapse-free survival (HR [95%CI] for IKZF1wt as reference = 2.72 [1.21-6.11] and 2.00 [0.87-4.49], respectively, p = 0.023). Both genetic markers including IKZF1del and IKZF1plus microdeletion profile provide additional predictive value of treatment outcome in childhood BCP-ALL and may contribute to more efficient patient stratification; the same is true in MRD guided protocols, which are based on flow cytometric measurements on day 15 of induction protocol.

Flow cytometric minimal residual disease assessment in B-cell precursor acute lymphoblastic leukaemia patients treated with CD19-targeted therapies - a EuroFlow study.

The standardized EuroFlow protocol, including CD19 as primary B-cell marker, enables highly sensitive and reliable minimal residual disease (MRD) assessment in B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) patients treated with chemotherapy. We developed and validated an alternative gating strategy allowing reliable MRD analysis in BCP-ALL patients treated with CD19-targeting therapies. Concordant data were obtained in 92% of targeted therapy patients who remained CD19-positive, whereas this was 81% in patients that became (partially) CD19-negative. Nevertheless, in both groups median MRD values showed excellent correlation with the original MRD data, indicating that, despite higher interlaboratory variation, the overall MRD analysis was correct.

Multicolor flow cytometry immunophenotyping and characterization of aneuploidy in pediatric B-cell precursor acute lymphoblastic leukemia.

The aim of this study was to assess the incidence of DNA aneuploidy in Polish children with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) and the relationship between aneuploidy and immunological phenotype, age, leukocyte count, S-phase fraction (SPF) and early response to induction chemotherapy assessed by the percentage of residual blast cells in bone marrow aspirates. The study group consisted of 267 patients. DNA content and immunophenotype were assessed in the bone marrow before treatment using multicolor flow cytometry (FC). DNA aneuploidy was detected in 50/267 (19%) patients. High hyperdiploidy was found to be associated with lower leukocyte count (p = 0.006) and common ALL immunophenotype. Flow cytometry analysis revealed that high hyperdiploid BCP-ALL patients showed significantly higher expression of CD9, CD20, CD22, CD58, CD66c, CD86 and CD123 antigens as compared to other groups of ploidy. In contrast, CD45 showed decreased expression. The percentage of leukemic blasts at diagnosis was lower in high hyperdiploid BCP-ALL cases than in diploid (79% vs. 85.7%, p = 0.001). The difference in minimal residual disease (MRD) levels on day 15 and 33 of induction therapy between analyzed groups was not significant. This study showed that high hyperdiploidy is associated with lower WBC count and specific immunological phenotype. Flow cytometric evaluation of expression of selected antigens can be used for fast identification of markers of aneuploidy in pediatric BCP-ALL, before genetic tests results are available. Understanding the biological significance of aneuploidy in leukemia can potentially be exploited therapeutically using targeted therapies against specific blast cell subclones.

The effect of ethanolic extract of Brazilian green propolis and artepillin C on aFGF-1, Eselectin, and CD40L secreted by human gingival fibroblasts.

INTRODUCTION: Periodontal diseases are among the most common diseases of the oral cavity in the worldwide population. The prevention of gingivitis and periodontitis is based on the removal of bacterial plaque from the teeth with use of toothpaste containing active substances. Noteworthy is the ethanolic extract of Brazilian green propolis (EEP-B), which, due to the high content of artepillin C, has anti-inflammatory, antibacterial, or immunostimulatory effects. Little is known about interactions between EEP-B and gingival fibroblasts within the oral cavity. The purpose of the article is to determine the role of acidic fibroblast growth factor (aFGF-1), E-selectin, and ligand of CD40 (CD40L) secreted by human gingival fibroblasts (HGF-1) in the gingiva. MATERIAL AND METHODS: We performed our experiments on gingival fibroblasts (HGF-1), which are an ideal in vitro model for studying the processes taking place within the gingiva. We incubated cells with EEP-B or artepillin C at the concentrations of 1 µg/ml and 10 µg/ml. The aFGF-1, E-selectin, and CD40L were detected using the Bio-Plex Magnetic Luminex Assay and the Bio-Plex 200 System. RESULTS: Ethanolic extract of Brazilian green propolis and artepillin C increased the levels of aFGF-1 secreted by HGF-1. Moreover, EEP-B decreased the levels of E-selectin in both tested concentrations, which was not proved for artepillin C. No changes in the concentration of CD40L released by HGF-1 were observed. CONCLUSIONS: The obtained results may suggest that EEP-B, due to the mixture of various compounds including flavonoids, accelerates the wound healing effects and has anti-inflammatory activity.

Chemokine receptors on peripheral blood T lymphocytes in children on peritoneal dialysis.

BACKGROUND: Immune cell dysfunction is listed among complications resulting from chronic kidney disease (CKD). It could be associated with T-cells, which play a role in the lymphocytic migration and infiltration. However, the data on chemokine receptors expression on T-cells in patients with CKD particularly treated with peritoneal dialysis (PD) are still limited. METHODS: The study aimed at multiparameter flow-cytometric analysis of the absolute numbers and percentage of T-cell subsets with surface chemokine receptors (CCR4, CCR5, CCR7, CXCR3, and CXCR4) or receptors' combinations in 47 children treated with PD. RESULTS: We found lower absolute numbers of total T lymphocytes, lymphocytes with surface CCR5, CXCR4+CCR5, CXCR3+CCR5 antigens and T-cells with CCR4, CCR4+CD4, CXCR3, CXCR3+CD4, and CD8 receptors. Lymphocytes T with CD4, CCR7, CD28+CCR7, CXCR3+CD8 antigens showed higher percentage in children on PD as compared to healthy children and opposite percentage values of CCR4+, CCR4+CD4+, CXCR3+ T lymphocytes were diminished. Mean fluorescent intensity for CCR7+, CCR7+CD45RO+, CCR7+CD28+, CXCR4+CD4+, CCR5+CD4+, CCR4+, CCR4+CD4+ T-cells was lower in the PD group than in healthy children. The analysis of correlation between T lymphocyte subpopulations with chemokine receptors and other parameters revealed positive correlation of CCR7+ and CCR7+CD28+ T-cells and weekly creatinine clearance, negative correlation between the percentage of CD45RO+CCR7 antigen positive T-cells and KT/Vurea. SUMMARY: In conclusion, we could not confirm the phenomenon of earlier senescence of T-cells in children with CKD on PD treatment. This still requires further investigation. The higher percentage of T-cells with CCR7 surface receptor could be responsible for the increase of proliferation activity in this group of children.

Automated identification of leukocyte subsets improves standardization of database-guided expert-supervised diagnostic orientation in acute leukemia: a EuroFlow study.

Precise classification of acute leukemia (AL) is crucial for adequate treatment. EuroFlow has previously designed an AL orientation tube (ALOT) to guide toward the relevant classification panel and final diagnosis. In this study, we designed and validated an algorithm for automated (database-supported) gating and identification (AGI tool) of cell subsets within samples stained with ALOT. A reference database of normal peripheral blood (PB, n = 41) and bone marrow (BM; n = 45) samples analyzed with the ALOT was constructed, and served as a reference for the AGI tool to automatically identify normal cells. Populations not unequivocally identified as normal cells were labeled as checks and were classified by an expert. Additional normal BM (n = 25) and PB (n = 43) and leukemic samples (n = 109), analyzed in parallel by experts and the AGI tool, were used to evaluate the AGI tool. Analysis of normal PB and BM samples showed low percentages of checks (<3% in PB, <10% in BM), with variations between different laboratories. Manual analysis and AGI analysis of normal and leukemic samples showed high levels of correlation between cell numbers (r2 > 0.95 for all cell types in PB and r2 > 0.75 in BM) and resulted in highly concordant classification of leukemic cells by our previously published automated database-guided expert-supervised orientation tool for immunophenotypic diagnosis and classification of acute leukemia (Compass tool). Similar data were obtained using alternative, commercially available tubes, confirming the robustness of the developed tools. The AGI tool represents an innovative step in minimizing human intervention and requirements in expertise, toward a "sample-in and result-out" approach which may result in more objective and reproducible data analysis and diagnostics. The AGI tool may improve quality of immunophenotyping in individual laboratories, since high percentages of checks in normal samples are an alert on the quality of the internal procedures.