Subclones with variants of uncertain clinical significance might contribute to ineffective hemopoiesis and leukemia predisposition.

BACKGROUND: Splicing modifications, genomic instability, and hypomethylation are central mechanisms promoting myelodysplasia and acute myeloid leukemia (AML). In this real-life retrospective study, to elucidate pathophysiology of clonal hemopoiesis in hematological malignancies, we investigated clinical significance of mutations in leukemia-related genes of known pathogenetic significance and of variants of uncertain clinical significance (VUS) in a cohort of patients with MDS and AML. METHODS: A total of 59 consecutive subjects diagnosed with MDS, 48 with AML, and 17 with clonal cytopenia with unknown significance were screened for somatic mutations in AML-related genes by next-generation sequencing. RESULTS: We showed that TET2, SETBP1, ASXL1, EZH2, RUNX1, SRSF2, DNMT3A, and IDH1/2 were commonly mutated. MDS patients also showed a high genetic complexity, especially for SETBP1. Moreover, the presence of SETBP1 wild-type or two or more simultaneous VUS variants identified a subgroup of AML and MDS patients with better outcome, while the presence of single SETBP1 VUS variant was related to a worse prognosis, regardless TET2 mutational status. CONCLUSIONS: In conclusions, we linked both pathogenic and VUS variants in AML-related genes to clonal hematopoiesis; therefore, we proposed to consider those variants as prognostic markers in leukemia and myelodysplasia. However, further studies in larger prospective cohorts are required to validate our results.

Implementation of International Prognostic Index with flow cytometry immunophenotyping for better risk stratification of chronic lymphocytic leukemia.

BACKGROUND: Current chronic lymphocytic leukemia (CLL) International Prognostic Index (IPI) stratifies patients based on clinical, molecular, and biochemical features; however, B-cell markers also influence CLL outcomes. Here, prognostic roles of CD11c, CD38, and CD49d were first evaluated, and then an immunophenotypic score was combined with CLL-IPI for risk stratification of CLL patients. METHODS: A total of 171 CLL subjects were included, and surface marker expression was assessed by flow cytometry. Levels ≥30% were chosen as cut-off of positivity to a marker; then values of 1 (for CD11c and CD38) or 3 (for CD49d) were assigned and scores determined for each patient's clone immunophenotype. RESULTS: CD49d positivity was significantly associated with simultaneous expression of CD11c and/or CD38, unmutated IGHV status, and higher ß2-microglobulin levels compared to those with CD49d negativity. Moreover, CD49d+ patients experienced a shorter progression-free survival and time to treatment. When the immunophenotypic score was combined with CLL-IPI, patients with high-risk immunophenotype had a significantly lower time-to-treatment regardless CLL-IPI. CONCLUSIONS: Our results suggested clinical utility of an integrated prognostic score for better risk stratification of CLL patients. These results require further validation in prospective larger studies.

Biomimetic proteolipid vesicles for reverting GPI deficiency in paroxysmal nocturnal hemoglobinuria.

Nano-vesicular carriers are promising tissue-specific drug delivery platforms. Here, biomimetic proteolipid vesicles (BPLVs) were used for delivery of glycosylphosphatidylinositol (GPI)-anchored proteins to GPI deficient paroxysmal nocturnal hemoglobinuria (PNH) cells. BPLVs were assembled as single unilamellar monodispersed (polydispersity index, 0.1) negatively charged (ζ-potential, -28.6 ± 5.6 mV) system using microfluidic technique equipped with Y-shaped chip. GPI-anchored and not-GPI proteins on BPLV surface were detected by flow cytometry. Peripheral blood mononuclear cells (PBMCs) from healthy and PNH subjects were treated with BPLVs (final concentration, 0.5 mg/mL), and cells displayed an excellent protein uptake, documented by flow cytometry immunophenotyping and confocal microscopy. BPLV-treated cells stressed with complement components showed an increased resistance to complement-mediated lysis, both healthy and PNH PBMCs. In conclusion, BPLVs could be effective nanocarriers for protein transfer to targeted cells to revert protein deficiency, like in PNH disease. However, further in vivo studies are required to validate our preclinical in vitro results.

Hodgkin Lymphoma and Hairy Cell Leukemia Arising from Chronic Lymphocytic Leukemia: Case Reports and Literature Review.

Richter's syndrome represents the progression of chronic lymphocytic leukemia (CLL) to more aggressive diseases, most frequently diffuse large B-cell lymphoma, while Hodgkin's lymphoma (HL) and hairy cell leukemia (HCL) are rarely described. The first case involved a 67-year-old man with a diagnosis of a high-risk stage-II CLL treated with rituximab and ibrutinib, developed a HL nodular sclerosis variant after three months of therapy for CLL. After achieving a complete remission for HL and ibrutinib cessation because of drug-related cardiotoxicity, the patient relapsed after five months off-therapy and died due to disease progression after two cycles of brentuximab-vedotin. The second case involved an 83-year-old female with a diagnosis of stage-IV CLL treated with rituximab plus bendamustine who developed a HCL eight years later. Pentostatin was unsuccessfully employed as upfront HCL therapy, and the patient was then switched to rituximab while in remission for CLL. In conclusion, Richter's transformation risk rate might be higher in patients treated with novel targeted therapies, and multiparametric flow cytometry and lymph node biopsy at relapse could help in early identifying small clones. The treatment of predominant neoplasia is mandatory, and disease-specific drugs are administered; however, clinical efficacy might be lower in these patients.

Inter-intra instrument comparison and standardization of a 10-color immunophenotyping for B and T cell non-Hodgkin lymphoma diagnosis and monitoring.

Harmonization of flow cytometry protocols from instrument settings to antibody panel and reagents is highly encouraged for inter-laboratory data comparison in both research and clinical settings, especially for minimal residual disease monitoring evaluation in hematological diseases across centers. Here, we described inter-intra instrument comparison of two standardized 10-color staining dried tubes for B- and T-cell lymphoproliferative disorder diagnosis and monitoring on two different flow cytometers, a Beckman Coulter NaviosEx and a Beckman Coulter DxFlex. A total of 47 consecutive patients were enrolled, and 39 of them were evaluable for further studies. We show highly comparable results between the two cytometers for cell frequency and fluorescence intensity signals for both standardized 10-color staining dried tubes. For this latter, fluorescence of each antibody and subject was normalized on the mean value obtained from the entire study cohort thus reducing the effects of biological variability and allowing comparison between instruments with different detector sensitivity. In summary, dried tubes were confirmed as an optimal standardized diagnostic tool, especially when associated with EuroFlow standardized procedures by minimizing technical and biological variability. However, data analysis is still operator-dependent, and more efforts are needed to develop automated or semi-automated software for flow cytometry data analysis for diagnostic purposes.

c-Kit M541L variant is related to ineffective hemopoiesis predisposing to clonal evolution in 3D in vitro biomimetic co-culture model of bone marrow niche.

Hematopoietic stem cell (HSC) maintenance in vitro is challenging because stem cell survival relies on cell-to-cell contacts and paracrine signals from bone marrow (BM) microenvironment. Indeed, HSCs easily differentiate in conventional culture systems, and in vitro study of stem cell biology, leukemogenesis, and evolutionary trajectories is limited. 3D-culture systems can mimic tissue architecture and microenvironment thus preserving HSC phenotype. In this study, we developed a calcium alginate hydrogel-based 3D co-culture system of BM mononuclear cells (BMMCs) and BM-derived mesenchymal stem cells (BM-MSCs) to study hemopoiesis in health and disease, such as biological roles of c-Kit M541L somatic mutation of unknown significance. BMMCs and peripheral blood stem cells were obtained from an acute myeloid leukemia patient who experienced graft failure and his haploidentical donor, and from a healthy donor. Cells embedded in alginate scaffolds were cultured for up to 21 days, and flow cytometry immunophenotyping was performed at baseline and every seven days. Our results showed suitability of our 3D culture system in preserving HSC vitality and phenotype throughout the culture period, and also in maintaining composition and vitality of total BMMCs. Moreover, 3D in vitro culture results suggested that M541L c-Kit somatic mutation could be a loss-of-function alteration by reducing HSC maintenance ability thus quickly promoting differentiation, as documented by in vivo graft failure and in vitro absence of long-term culture stability. In conclusions, our 3D BM-like biomimetic culture system allowed long-term stemness maintenance, making it a valid and effective tool for in vitro study of physiological and pathological hemopoiesis.

Clinical efficacy of azacytidine and venetoclax and prognostic impact of Tim-3 and galectin-9 in acute myeloid leukemia and high-risk myelodysplastic syndromes: A single-center real-life experience.

Treatment of acute myeloid leukemia (AML) and high-risk myelodysplastic syndromes (MDS) is difficult in older patients with comorbidities and high-risk disease factors. Venetoclax, the first-in-class Bcl-2 inhibitor, has proven efficacy and safety in combination with azacytidine for treatment of high-risk myeloid diseases. In this single-center real-life retrospective study, a total of 27 consecutive patients treated with azacytidine plus venetoclax were included, and clinical outcomes, hematological improvements, and biomarkers of responsiveness to therapy were compared to those observed in an historical cohort of 95 consecutive patients treated with azacytidine as single agent. Azacytidine plus venetoclax was effective and safe in older and frail AML and high-risk MDS patients, with median overall survival of 22.3 months, higher than that reported in phase III trial (14.7 months), and higher than that of historical cohort (5.94 months). Progression-free survival was higher in patients treated with the drug combination compared to those treated with azacytidine as single agent (p = 0.0065). Clinical benefits might increase when azacytidine and venetoclax are administered as upfront therapy (p = 0.0500). We showed that Tim-3 expression could be a promising therapeutic target in refractory/relapsed patients, and galectin-9 a biomarker of responsiveness to therapy. Moreover, patients treated with azacytidine and venetoclax displayed a higher overall survival regardless the presence of negative prognostic markers at diagnosis (e.g., increased WT1 copies and/or normalized blast count). These encouraging results in a real-world setting supported efficacy and safety of azacytidine plus venetoclax as upfront therapy in AML and high-risk MDS, with clinical outcomes comparable to those of clinical trials when an appropriate venetoclax management with bone marrow assessment at every first, second, fourth, and eighth cycle, and dose adjustments for toxicities are performed.

WT1 Expression Levels Combined with Flow Cytometry Blast Counts for Risk Stratification of Acute Myeloid Leukemia and Myelodysplastic Syndromes.

Wilm's tumor 1 (WT1), a zinc-finger transcription factor and an epigenetic modifier, is frequently overexpressed in several hematologic disorders and solid tumors, and it has been proposed as diagnostic and prognostic marker of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). However, the exact role of WT1 in leukemogenesis and disease progression remains unclear. In this real-world evidence retrospective study, we investigated prognostic role of WT1-mRNA expression levels in AML and MDS patients and correlations with complete blood counts, flow cytometry counts, and molecular features. A total of 71 patients (AML, n = 46; and MDS, n = 25) were included in this study, and WT1 levels were assessed at diagnosis, during treatment and follow-up. We showed that WT1 expression levels were inversely correlated with normal hemopoiesis in both AML and MDS, and positively associated with blast counts. Flow cytometry was more sensitive and specific in distinguishing normal myeloid cells from neoplastic counterpart even just using linear parameters and CD45 expression. Moreover, we showed that a simple integrated approach combining blast counts by flow cytometry, FLT3 mutational status, and WT1 expression levels might be a useful tool for a better prognostic definition in both AML and MDS patients.