Immunophenotyping myelodysplastic neoplasms: the role of flow cytometry in the molecular classification era

The unique heterogenous landscape of myelodysplastic syndromes/neoplasms (MDS) has resulted in continuous redefinition of disease sub-entities, in view of the novel translational research data that have clarified several areas of the pathogenesis and the progression of the disease. The new international classifications (WHO 2022, ICC 2022) have incorporated genomic data defining phenotypical alterations, that guide clinical management of specific patient subgroups. On the other hand, for over a decade, multiparameter flow cytometry (MFC) has proven its value as a complementary diagnostic tool for these diseases and although it has never been established as a mandatory test for the baseline evaluation of MDS patients in international guidelines, it is almost universally adopted in everyday clinical practice for the assessment of suspected cytopenias through simplified scoring systems or elaborate analytical strategies for the detection of immunophenotypical dysplastic features in every hematopoietic cell lineage in the bone marrow (BM). In this review, we explore the clinically meaningful interplay of MFC data and genetic profiles of MDS patients, to reveal the currently existing and the potential future role of each methodology for routine clinical practice, and the benefit of the patients. We reviewed the existing knowledge and recent advances in the field and discuss how an integrated approach could lead to patient re-stratification and guide personalized management

Fetal hemoglobin induction in azacytidine responders enlightens methylation patterns related to blast clearance in higher-risk MDS and CMML

Background: As new treatment options for patients with higher-risk myelodysplastic syndromes are emerging, identification of prognostic markers for hypomethylating agent (HMA) treatment and understanding mechanisms of their delayed and short-term responses are essential. Early fetal hemoglobin (HbF) induction has been suggested as a prognostic indicator for decitabine-treated patients. Although epigenetic mechanisms are assumed, responding patients’ epigenomes have not been thoroughly examined. We aimed to clarify HbF kinetics and prognostic value for azacytidine treated patients, as well as the epigenetic landscape that might influence HbF re-expression and its clinical relevance. Results: Serial HbF measurements by high-performance liquid chromatography (n = 20) showed induction of HbF only among responders (p = 0.030). Moreover, HbF increase immediately after the first azacytidine cycle demonstrated prognostic value for progression-free survival (PFS) (p = 0.032, HR = 0.19, CI 0.24–1.63). Changes in methylation patterns were revealed with methylated DNA genome-wide sequencing analysis (n = 7) for FOG-1, RCOR-1, ZBTB7A and genes of the NuRD-complex components. Targeted pyrosequencing methodology (n = 28) revealed a strong inverse correlation between the degree of γ-globin gene (HBG2) promoter methylation and baseline HbF levels (p = 0.003, rs = − 0.663). A potential epigenetic mechanism of HbF re-expression in azacytidine responders was enlightened by targeted methylation analysis, through hypomethylation of site -53 of HBG2 promoter (p = 0.039, rs = − 0.504), which corresponds to MBD2-NuRD binding site, and to hypermethylation of the CpG326 island of ZBTB7A (p = 0.05, rs = 0.482), a known HbF repressor. These changes were associated to blast cell clearance (pHBG2 = 0.011, rs = 0.480/pZBTB7A = 0.026, rs = 0.427) and showed prognostic value for PFS (pZBTB7A = 0.037, HR = 1.14, CI 0.34–3.8). Conclusions: Early HbF induction is featured as an accessible prognostic indicator for HMA treatment and the proposed potential epigenetic mechanism of HbF re-expression in azacytidine responders includes hypomethylation of the γ-globin gene promoter region and hypermethylation of the CpG326 island of ZBTB7A. The association of these methylation patterns with blast clearance and their prognostic value for PFS paves the way to discuss in-depth azacytidine epigenetic mechanism of action. Graphical abstract: (Figure presented.)</p

An integrated diagnostic approach of neutropenia and pancytopenia

Background: Myelodysplastic Syndromes (MDS) are a group of clonal hematopoietic stem cell disorders with significant heterogeneity in their clinical presentation and the prognosis of the patients. Several attempts have been made to incorporate flow cytometry (FC) findings into the diagnostic and/or prognostic criteria of dysplasia, but bone marrow (BM) aspirate morphology evaluation remains the gold-standard for diagnosis. The purpose of this study was to provide a diagnostic tool for MDS that relies on BM immunophenotyping and objectifies the interpretation of FC analysis and to validate its capacity to discriminate MDS from other causes of cytopenias. Methods: To that purpose, a mathematical formula was developed which incorporates granulocytic maturation markers and the percentage of selected myeloid populations and translates them into a single parameter that quantifies the maturation and differentiation defects of BM granulocytes, named Dysmyelopoiesis Index (DMI). Bone marrow samples from 84 MDS patients and 47 non-MDS cytopenic patients were analyzed with FC and DMI was calculated for every patient. Results: DMI detected clonal dysplasia with 84.5% sensitivity and 93.6% specificity, identified as MDS 77.2% of low grade patients and revealed multilineage dysplasia for a number of RA and RARS cases. It discriminated prognostic sub-groups of MDS patients (P< .005) and negatively correlated with IPSS (r= - .472, P= .000), WPSS (r= - .481, P= .000) and IPSS-R (r= -.395, P= .000). Conclusions: DMI represents an accurate quantification of dysmyelopoiesis and an effective stand-alone diagnostic test for MDS, facilitating FC analysis and daily clinical practice.Τα Μυελοδυσπλαστικά Σύνδρομα (ΜΔΣ) αποτελούν ένα σύνολο κλωνικών νοσημάτων του αρχέγονου αιμοποιητικού κυττάρου με σημαντική ετερογένεια ως προς την κλινική τους εμφάνιση και την πρόγνωση των ασθενών. Έχουν γίνει πολλές απόπειρες να ενταχθούν τα ευρήματα της κυτταρομετρίας ροής (ΚΡ) στα διαγνωστικά και/ή προγνωστικά κριτήρια της δυσπλασίας, αλλά η μορφολογική εκτίμηση του επιχρίσματος του μυελού των οστών (ΜτΟ) παραμένει η μέθοδος εκλογής για την διάγνωση. Ο σκοπός αυτής της μελέτης ήταν να παρέχει ένα εργαλείο για την διαγνωστική προσπέλαση των κυτταροπενικών ασθενών, το οποίο θα βασίζεται στην ανοσοφαινοτύπηση του ΜτΟ και θα αντικειμενικοποιεί την ερμηνεία των αποτελεσμάτων της ανάλυσης της ΚΡ, και να αξιολογήσει την ικανότητά του να διακρίνει τα ΜΔΣ από άλλες αιτίες κυτταροπενιών του περιφερικού αίματος. Σε αυτή την κατεύθυνση, αναπτύχθηκε ένα μαθηματικό μοντέλο, το οποίο ενσωματώνει την ποσοτικοποιημένη έκφραση δεικτών ωρίμανσης των κοκκιοκυττάρων με τα ποσοστά επιλεγμένων υποπληθυσμών μυελικής διαφοροποίησης και αποδίδει μία παράμετρο η οποία ποσοτικοποιεί τον βαθμό δυσμυελοποίησης των κοκκιοκυττάρων του ΜτΟ και η οποία ονομάζεται δείκτης δυσμυελοποίησης (Dysmyelopoiesis Index - DMI). Δείγματα ΜτΟ 84 ασθενών με ΜΔΣ και 47 κυτταροπενικών ασθενών-μαρτύρων συλλέχθηκαν, παρασκευάστηκαν και αναλύθηκαν με ΚΡ και ο DMI υπολογίστηκε για κάθε ασθενή. Ο DMI ανέδειξε τις περιπτώσεις ΜΔΣ με 84.5% ευαισθησία και 93.6% ειδικότητα, ταυτοποίησε το 77.2% των ΜΔΣ χαμηλού βαθμού και ανέδειξε πολυγραμμική δυσπλασία σε ασθενείς με διαγνώσεις RA και RARS. Διέκρινε προγνωστικές υπο-ομάδες των ασθενών με ΜΔΣ (P< .005) και είχε στατιστικά σημαντική αρνητική συσχέτιση με τα προγνωστικά σκορ των ασθενών IPSS (r= - .472, P= .000), WPSS (r= - .481, P= .000) και IPSS-R (r= -.395, P= .000). Ο DMI αντιπροσωπεύει μία ακριβή ποσοτικοποίηση του βαθμού δυσμυελοποίησης και μία αποτελεσματική διαγνωστική δοκιμασία για τα ΜΔΣ, απλοποιώντας την ανάλυση της ΚΡ και την καθημερινή κλινική πρακτική

Characterization Of T Follicular Helper Cells In Patients With Low Risk Myelodysplastic Syndromes

A subpopulation of CD4+helper T cells, T-follicular helper cells (TFH), are characterized by the surface expression of CXCR5, ICOS, and PD1, the transcription factor Bcl-6, and produce mainly IL-21, but also IL-17, IL-4, and IFN-γ. They represent the major population that helps B cells to turn into plasma cells and they are implicated in the pathogenesis of different autoimmune diseases. Peripheral CXCR5+CD4+ T helper cells (p-TFH) are the circulating component of TFH. p-TFH cells have been extensively studied in the context of inflammation and autoimmunity. Patients with systemic lupus erythematosus and rheumatoid arthritis have increased p-TFH. Immune dysregulation characterizes low risk myelodysplastic syndromes (MDS). The expression of p-TFH in patients in low risk MDS has not been previously evaluated. To examine the expression of p-TFH in this disease we isolated peripheral blood mononuclear cells (PBMCs) from patients with low risk MDS (n=20, Refractory Anemia,RA; and Refractory Cytopenias with Multilineage Dysplasia, RCMD) and ten healthy, age- matched controls. Written informed consent was obtained from all study subjects. PBMCs were left untreated or activated with PMA and Ionomycin, in the presence of Brefeldin A, for 5 hours. Subsequently, cells were stained with the surface markers CXCR5, CD4, CD45RO, ICOS and intracellular IL-21, and analyzed by flow cytometry. Patients with MDS show decreased CD4+ICOS+ cells compared to healthy controls (5,34±1,44% vs 8,69±4,08% respectively, p=0.028). The median percentages of CXCR5+CD4+ cells (estimated on CD4+ cells) before stimulation were lower in MDS patients although not statistically significant different from the control subjects. The expansion of the CXCR5+CD4+ population after stimulation was higher in MDS patients compared to healthy individuals (ratio of stimulated to unstimulated CXCR5+CD4+ cells of MDS patients: 1.44±0.47 vs control: 1.23± 0.32, p=0.027). MDS patients at diagnosis showed increased median levels of IL-21 producing CXCR5+CD4+ cells compared to patients previously treated with erythropoietin (EPO), (21,34±10,14% vs 17,55±12,91, respectively). Additionally, patients with RA showed increased CXCR5+CD4+IL-21+ cells compared to the RCMD patients (22,39±11,76% vs 17,84±10,70%, respectively). Collectively, the percentage of IL-21 producing CXCR5+CD4+ cells did not differ significantly between the MDS and control groups (20,57± 10,12% vs 19,86± 11,03%, respectively). The presence of marrow fibrosis did not affect the differences observed and described above. The p-TFH cell subset was identified in every case as a memory component of CD4+ T helper cells, as previously described. Although the number of patients analyzed is limited, our results suggest that low risk MDS patients show a trend of lower CXCR5+CD4+ cells compared to age–matched control subjects. EPO treatment eliminates IL-21 production from CXCR5+CD4+ cells compared to treatment-naïve patients. Further analysis of a larger pool of subjects along with the examination of the specific transcription factor Bcl-6 will reveal the role of p-TFH cells in low risk MDS. Disclosures: No relevant conflicts of interest to declare. </jats:sec

Increased T Follicular Helper Cells in Patients with Aplastic Anemia

Abstract Introduction:T-follicular helper cells represent a sub-population of CD4+helper T cells (TFH), and they are characterized by the surface expression of CXCR5, ICOS, and PD1, the transcription factor Bcl-6, and produce mainly IL-21, but also IL-17, IL-4, and IFN-γ. They represent the major population that helps B cells to turn into plasma cells and they are implicated in the pathogenesis of different autoimmune diseases. Peripheral CXCR5+CD4+ T helper cells (p-TFH) are the circulating component of TFH. p-TFH cells have been extensively studied in the context of inflammation and autoimmunity. Patients with systemic lupus erythematosus and rheumatoid arthritis have increased p-TFH. T follicular regulatory T cells (Tfr) express CXCR5, ICOS and PD1, CD25 but also FOXP3, and they can inhibit B cell responses. Immune dysregulation characterizes patients with aplastic anemia. We and others have previously shown that aplastic anemia patients have decreased levels of regulatory T cells (Tregs) at presentation and increase if they respond to treatment. Additionally, these patients display increased Th17 cells at presentation. Specific aim: In this study we wanted to examine the expression of p-TFH and Tfr cells in patients with aplastic anemia. We isolated peripheral blood mononuclear cells (PBMCs) from patients with aplastic anemia (n=8) and ten healthy, age- matched controls. Written informed consent was obtained from all study subjects. PBMCs were left untreated or activated with PMA and Ionomycin, in the presence of Brefeldin A, for 5 hours. Subsequently, cells were stained with the surface markers CXCR5, CD4, CD57, CD25, CD127, CD278, CD279, IFN-g,IL-21, and IL-17, and subsequently analyzed by flow cytometry. Results: Patients with aplastic anemia showed increased expression of CD4+CXCR5+ cells (Tfh) compared to healthy controls (3,03±0,55% vs 1,3±0,52% respectively, p=0.08). Additionally, these patients also showed decreased expression of Treg cells compared to healthy controls (0,74±0,27% vs 2,25±0,70% respectively, p=0.03). When analyzed for Tfr cells, patients with aplastic anemia showed minimal levels of these cells compared to helathy controls (0,05±0,02% vs 1,14±0,16% respectively, p&lt;0.001) (Fig. 1). Conclusion: Although the number of patients analyzed is limited, our preliminary results suggest that aplastic anemia patients show increased CXCR5+CD4+ cells compared to age-matched control subjects, and decreased Tfr cells. Further analysis of a larger pool of subjects is underway along with the examination of the specific transcription factor Bcl-6, to reveal the role of p-TFH and Tfr cells in the immune pathogenesis of aplastic anemia. Figure Figure. Disclosures No relevant conflicts of interest to declare. </jats:sec

Prognostic Value of PSMB5 and Correlations with LC3II and Reactive Oxygen Species Levels in the Bone Marrow Mononuclear Cells of Bortezomib-Resistant Multiple Myeloma Patients

Proteasome inhibitors (PIs) constitute the most common type of induction treatment for multiple myeloma. Interactions between the proteasome, autophagy, and reactive oxygen species (ROS) have been shown in the past, thus emphasizing the need for a better understanding of the underlying pathophysiology. For this study, bone marrow mononuclear cells from 110 myeloma patients were collected at different disease stages. PSMB5 and LC3I/II protein levels were determined using Western blot, proteasome proteolytic activity (PPA) with spectrofluorometry, and ROS with flow cytometry. PSMB5 accumulation was found to diminish after PI treatment (p-value = 0.014), and the same pattern was observed in PPA (p-value &lt; 0.001). Conversely, LC3II protein levels were elevated at both remission and relapse compared to baseline levels (p-value = 0.041). Patients with a baseline PSMB5 accumulation lower than 1.06 units had longer disease-free survival compared to those with values above 1.06 units (12.0 &plusmn; 6.7 vs. 36 &plusmn; 12.1 months; p-value &lt; 0.001). Median ROS levels in plasma cells were significantly higher at relapse compared to both baseline and remission levels (p-value &lt; 0.001), implying poor prognosis. Overall, post-treatment PSMB5 reduction could indicate a shift from proteasomal to autophagic degradation as a main proteostatic mechanism, thus explaining resistance. The elevated oxidative stress in PI-treated patients could possibly serve as an additional compensatory mechanism

Platelet-Induced T Cell Activation in Patients with Myocardial Infarction; The Role of miR155

Abstract Specific aim: We have previously shown that platelets from patients with myocardial infarction (with ST elevation-STEMI) can activate T cells in vitro. In this study we wanted to expand our initial observation and explore the mechanism of this T cell activation. Methods: After written informed consent was obtained, peripheral blood mononuclear cells (PBMCs) were isolated from heparinized venous blood obtained from 30 patients with STEMI (28 men and 2 women) at the time of hospital admission at diagnosis, before receiving any treatment, as well as 5 days and 30 days later. We also analyzed 15 healthy subjects (12 men and 3 women), and 7 patients with unstable angina who served as the disease control group. PBMCs were analyzed by flow cytometry with the following markers and their isotypic controls: CD4, CD25, CD127, FOXP3, and CD69. We also isolated platelet rich plasma or plasma alone from the patients and the healthy subjects, and used in mixed cultures with PBMCs. miR 155 levels were examined with real-time PCR. Results: Initially, we incubated T cells with platelets from patients with STEMI and examined T cell activation by CD69 expression. These T cells showed increased expression of CD69 compared to T cells treated with platelets from healthy subjects (p&lt;0.05). No T cell activation was observed following incubation with plasma alone from patients or healthy controls. Next, the percentages of CD4+CD25+hi CD127low FOXP3+ (regulatory T cells, Tregs) were examined in all study subjects. Patients at presentation showed comparable Treg levels with the controls (healthy subjects and disease control group). Five days later, patients with STEMI displayed increased levels of Tregs compared with the controls (p&lt;0.05) (Fig.2). To explore the mechanism of Treg up-regulation the miR155 levels through real time PCR were evaluated. Patients with STEMI showed comparable levels of miR155 at presentation, but five days later showed decreased miR155 levels compared to controls (p&lt;0.001) (Fig. 1); we observed an inverse correlation between miR155 levels and Treg numbers in patients with STEMI. At re-evaluation, one month later, patients with STEMI displayed Treg numbers comparable to the initial presentation levels Conclusion: Herein, we show that platelets from patients with STEMI can activate T cells in vitro. This platelet activation in patients with STEMI, results in an increase in Tregs through down-regulation of miR155, that return to normal levels a month later. This Treg increase reflects possibly an effort to suppress immune system activation secondary to platelet activation, shortly after the infarct. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare. </jats:sec

Platelets from Patients with Myocardial Infarction Can Activate T Cells in Vitro

Abstract Specific aim: Our aim was to examine whether T cells can be activated by the circulating activated platelets from patients with myocardial infarction (with ST elevation-STEMI) Methods: After written informed consent was obtained, peripheral blood mononuclear cells (PBMCs) were isolated from heparinized venous blood obtained from 20 patients with STEMI (18 men and 2 women) at the time of hospital admission at diagnosis, before receiving any treatment, as well as 5 days and 30 days later. We also analyzed 10 healthy subjects (8 men and 2 women), and 5 patients with unstable angina who served as the disease control group. PBMCs were analyzed by flow cytometry with the following markers and their isotypic controls: CD4, CD25, CD69, and FOXP3. We also isolated platelet rich plasma or plasma alone from the patients and the healthy subjects, and used in mixed cultures with PBMCs. Results: We first examined T cell activation by measuring CD69 expression on CD4 T cells following incubation with platelets obtained from patients with STEMI. T cells treated with platelets from patients with STEMI showed increased expression of CD69 (as an activation marker) compared with T cells treated with platelets from healthy subjects (p&lt;0.05, Figure 1). There was no T cell activation following incubation with plasma alone from patients or healthy controls. We then examined the percentages of CD4+CD25+hi (regulatory T cells). There was no statistical difference in Tregs between patients at presentation and controls (healthy subjects and disease control group). Five days later, patients with STEMI displayed increased levels of Tregs compared with the 2 control groups; one month later, Treg numbers returned to the initial presentation levels (p&lt;0.05, Figure 2) Conclusion: To our knowledge we describe for the first time that platelets from patients with STEMI can activate T cells in vitro. In patients with STEMI, an increase in Tregs possibly in an effort to suppress immune system activation secondary to platelet activation, appears shortly after the infarct and normalizes a month later. Figure 1. T cell activation after treatment with platelets from patients with STEMI compared with the platelets from healthy controls or plasma alone Figure 1. T cell activation after treatment with platelets from patients with STEMI compared with the platelets from healthy controls or plasma alone Figure 2. Increased T regs in patients with STEMI , 5 days after admission (STEMI EXIT) ( UA;unstable angina, STEMI 1mfup; STEMI after one month) Figure 2. Increased T regs in patients with STEMI , 5 days after admission (STEMI EXIT) ( UA;unstable angina, STEMI 1mfup; STEMI after one month) Disclosures No relevant conflicts of interest to declare. </jats:sec