Recently, we described B-cell precursor acute lymphoblastic leukemia (BCP-ALL) subtype with early switch to the monocytic lineage and loss of the B-cell immunophenotype, including CD19 expression. Thus far, the genetic background has remained unknown. Among 726 children consecutively diagnosed with BCP-ALL, 8% patients experienced switch detectable by flow cytometry (FC). Using exome and RNA sequencing, switch was found to positively correlate with three different genetic subtypes: PAX5-P80R mutation (5 cases with switch out of 5), rearranged DUX4 (DUX4r; 30 cases of 41) and rearranged ZNF384 (ZNF384r; 4 cases of 10). Expression profiles or phenotypic patterns correlated with genotypes, but within each genotype they could not identify cases who subsequently switched. If switching was not taken into account, the B-cell-oriented FC assessment underestimated the minimal residual disease level. For patients with PAX5-P80R, a discordance between FC-determined and PCR-determined MRD was found on day 15, resulting from a rapid loss of the B-cell phenotype. Discordance on day 33 was observed in all the DUX4r, PAX5-P80R and ZNF384r subtypes. Importantly, despite the substantial phenotypic changes, possibly even challenging the appropriateness of BCP-ALL therapy, the monocytic switch was not associated with a higher incidence of relapse and poorer prognosis in patients undergoing standard ALL treatment.

Cerebrospinal fluid (CSF) flow cytometry has a crucial role in the diagnosis of leptomeningeal disease in onco-hematology. This report describes the flow cytometry characterization of 138 CSF samples from patients affected by non-Hodgkin lymphoma, negative for disease infiltration. The aim was to focus on the CSF non-neoplastic population, to compare the cellular composition of the CSF with paired peripheral blood samples and to document the feasibility of flow cytometry in hypocellular samples. Despite the extremely low cell count (1 cell/µl, range 1.0-35) the study was successfully conducted in 95% of the samples. T lymphocytes were the most abundant subset in CSF (77%; range 20-100%) with a predominance of CD4-positive over CD8-positive T cells (CD4/CD8 ratio = 2) together with a minority of monocytes (15%; range 0-70%). No B cells were identified in 90% of samples. Of relevance, a normal, non-clonal B-cell population was documented in 5/7 (71%) patients with primary central nervous system lymphoma at diagnosis (p<0.0001), suggesting a possible involvement of blood-brain barrier cell permeability in the pathogenesis of cerebral B-cell lymphomas. The highly significant differences between CSF and paired peripheral blood lymphoid phenotype (p<0.0001) confirms the existence of an active mechanism of lymphoid migration through the meninges.
Copyright © 2021 Cordone, Masi, Giannarelli, Pasquale, Conti, Telera, Pace, Papa, Marino, de Fabritiis and Mengarelli.

Exosomes are small (30- to 100-nm) vesicles secreted by all cell types in culture and found in most body fluids. A mean of 1 mL of blood serum, derived from healthy donors, contains approximately 10(12) exosomes. Depending on the disease, the number of exosomes can fluctuate. Concentration of exosomes in the bloodstream and all other body fluids is extremely high. Several B-cell surface antigens (CD19, CD20, CD22, CD23, CD24, CD37, CD40, and HLA-DR) and the common leukocyte antigen CD45 are interesting in terms of immunotherapy of hematologic malignant neoplasms. The established standard for exosome isolation is ultracentrifugation. However, this method cannot discriminate between exosome subpopulations and other nanovesicles. The main purpose of this study was to characterize CD81(+) and CD63(+) subpopulations of exosomes in terms of these surface markers after release from various types of B-cell lymphoma cell lines using an easy and reliable method of immunomagnetic separation.
Western blotting, flow cytometry, and electron microscopy were used to compare the total preenriched extracellular vesicle (EV) pool to each fraction of vesicles after specific isolation, using magnetic beads conjugated with antibodies raised against the exosome markers CD63 and CD81.
Magnetic bead-based isolation is a convenient method to study and compare subpopulations of exosomes released from B-cell lymphoma cells. The data indicated that the specifically isolated vesicles differed from the total preenriched EV pool. CD19, CD20, CD24, CD37, and HLA-DR, but not CD22, CD23, CD40, and CD45, are expressed on exosomes from B-cell lymphoma cell lines with large heterogeneity among the different B-cell lymphoma cell lines. Interestingly, these B-cell lymphoma-derived EVs are able to rescue lymphoma cells from rituximab-induced complement-dependent cytotoxicity.
Distribution of exosomes that contain CD19, CD20, CD24, CD37, and HLA-DR may intercept immunotherapy directed against these antigens, which is important to be aware of for optimal treatment. The use of an immunomagnetic separation platform enables easy isolation and characterization of exosome subpopulations for further studies of the exosome biology to understand the potential for therapeutic and diagnostic use.
Copyright © 2014 The Authors. Published by EM Inc USA.. All rights reserved.

Most consensus leukemia & lymphoma antibody panels consist of lists of markers based on expert opinions, but they have not been validated. Here we present the validated EuroFlow 8-color antibody panels for immunophenotyping of hematological malignancies. The single-tube screening panels and multi-tube classification panels fit into the EuroFlow diagnostic algorithm with entries defined by clinical and laboratory parameters. The panels were constructed in 2-7 sequential design-evaluation-redesign rounds, using novel Infinicyt software tools for multivariate data analysis. Two groups of markers are combined in each 8-color tube: (i) backbone markers to identify distinct cell populations in a sample, and (ii) markers for characterization of specific cell populations. In multi-tube panels, the backbone markers were optimally placed at the same fluorochrome position in every tube, to provide identical multidimensional localization of the target cell population(s). The characterization markers were positioned according to the diagnostic utility of the combined markers. Each proposed antibody combination was tested against reference databases of normal and malignant cells from healthy subjects and WHO-based disease entities, respectively. The EuroFlow studies resulted in validated and flexible 8-color antibody panels for multidimensional identification and characterization of normal and aberrant cells, optimally suited for immunophenotypic screening and classification of hematological malignancies.

Acute promyelocytic leukemia (APL) results from a blockade of granulocyte differentiation at the promyelocytic stage. All-trans retinoic acid (ATRA) induces clinical remission in APL patients by enhancing the rapid differentiation of APL cells and the clearance of PML-RARα, APL's hallmark oncoprotein. In the present study, we demonstrated that both autophagy and Beclin 1, an autophagic protein, are upregulated during the course of ATRA-induced neutrophil/granulocyte differentiation of an APL-derived cell line named NB4 cells. This induction of autophagy is associated with downregulation of Bcl-2 and inhibition of mTOR activity. Small interfering RNA-mediated knockdown of BECN1 expression enhances apoptosis triggered by ATRA in NB4 cells but does not affect the differentiation process. These results provide evidence that the upregulation of Beclin 1 by ATRA constitutes an anti-apoptotic signal for maintaining the viability of mature APL cells, but has no crucial effect on the granulocytic differentiation. This finding may help to elucidate the mechanisms involved in ATRA resistance of APL patients, and in the ATRA syndrome caused by an accumulation of mature APL cells.