Protocol for quantification of circulating immature granulocytes in patients with a chronic myeloid malignancy using flow cytometry.

Immature granulocytes (iGRANs) circulating in the peripheral blood of patients with a chronic myeloid malignancy, such as chronic myelomonocytic leukemia, are immunosuppressive cells that promote disease progression. Here, we present a multiparameter flow cytometry-based protocol to quantify iGRANs among peripheral blood mononucleated cells (PBMCs). We describe steps for collecting and labeling PBMCs.

Monocytes generated by interleukin-6-treated human hematopoietic stem and progenitor cells secrete calprotectin that inhibits erythropoiesis.

Elevated circulating levels of calprotectin (CAL), the S100A8/A9 heterodimer, are biomarkers of severe systemic inflammation. Here, we investigate the effects of CAL on early human hematopoiesis. CAL demonstrates limited impact on gene expression in stem and progenitor cells, in contrast with interleukin-6 (IL6), which promotes the expression of the and genes in hematopoietic progenitors and the generation of monocytes that release CAL.

Targeting heterochromatin eliminates chronic myelomonocytic leukemia malignant stem cells through reactivation of retroelements and immune pathways.

Chronic myelomonocytic leukemia (CMML) is a severe myeloid malignancy affecting the elderly, for which therapeutic options are limited. DNA hypomethylating agents (HMAs) provide transient responses, failing to eradicate the malignant clone. Hematopoietic stem cell (HSC) aging involves heterochromatin reorganization, evidenced by alterations in histone marks H3K9me2 and H3K9me3.

CXCL8 secreted by immature granulocytes inhibits WT hematopoiesis in chronic myelomonocytic leukemia.

Chronic myelomonocytic leukemia (CMML) is a severe myeloid malignancy with limited therapeutic options. Single-cell analysis of clonal architecture demonstrates early clonal dominance with few residual WT hematopoietic stem cells. Circulating myeloid cells of the leukemic clone and the cytokines they produce generate a deleterious inflammatory climate.

Effective requesting method to detect fusion transcripts in chronic myelomonocytic leukemia RNA-seq.

RNA sequencing technology combining short read and long read analysis can be used to detect chimeric RNAs in malignant cells. Here, we propose an integrated approach that uses k-mers to analyze indexed datasets. This approach is used to identify chimeric RNA in chronic myelomonocytic leukemia (CMML) cells, a myeloid malignancy that associates features of myelodysplastic and myeloproliferative neoplasms.

Technical, gating and interpretation recommendations for the partitioning of circulating monocyte subsets assessed by flow cytometry.

The monocyte subset partitioning by flow cytometry, known as "monocyte assay," is now integrated into the new classifications as a supporting criterion for CMML diagnosis, if a relative accumulation of classical monocytes above 94% of total circulating monocytes is observed. Here we provide clinical flow cytometry laboratories with technical support adapted for the most commonly used cytometers. Step-by-step explanations of the gating strategy developed on whole peripheral blood are presented while underlining the most common difficulties.

CDKN1A is a target for phagocytosis-mediated cellular immunotherapy in acute leukemia.

Targeting the reprogramming and phagocytic capacities of tumor-associated macrophages (TAMs) has emerged as a therapeutic opportunity for cancer treatment. Here, we demonstrate that tumor cell phagocytosis drives the pro-inflammatory activation of TAMs and identify a key role for the cyclin-dependent kinase inhibitor CDKN1A (p21). Through the transcriptional repression of Signal-Regularity Protein α (SIRPα), p21 promotes leukemia cell phagocytosis and, subsequently, the pro-inflammatory reprogramming of phagocytic macrophages that extends to surrounding macrophages through Interferon γ.

Macrophage migration inhibitory factor is overproduced through EGR1 in TET2 resting monocytes.

Somatic mutation in TET2 gene is one of the most common clonal genetic events detected in age-related clonal hematopoiesis as well as in chronic myelomonocytic leukemia (CMML). In addition to being a pre-malignant state, TET2 mutated clones are associated with an increased risk of death from cardiovascular disease, which could involve cytokine/chemokine overproduction by monocytic cells. Here, we show in mice and in human cells that, in the absence of any inflammatory challenge, TET2 downregulation promotes the production of MIF (macrophage migration inhibitory factor), a pivotal mediator of atherosclerotic lesion formation.

Incorporating flow cytometry and next-generation sequencing in the diagnosis of CMML. Are we ready for prime?

In the last version of the WHO classification of myeloid malignancies, flow cytometry and molecular investigation are listed as potentially useful, yet non-essential diagnostic tools in hard-to-recognize chronic myelomonocytic leukemias (CMML). Flow recognition of CMML was initially based on an increase in the fraction of peripheral blood, CD14,CD16 classical monocytes ≥94% of total monocytes. An associated inflammatory disease can preclude the detection of classical monocyte fraction increase by inducing accumulation of CD14,CD16 intermediate monocytes.

A miR-150/TET3 pathway regulates the generation of mouse and human non-classical monocyte subset.

Non-classical monocyte subsets may derive from classical monocyte differentiation and the proportion of each subset is tightly controlled. Deregulation of this repartition is observed in diverse human diseases, including chronic myelomonocytic leukemia (CMML) in which non-classical monocyte numbers are significantly decreased relative to healthy controls. Here, we identify a down-regulation of hsa-miR-150 through methylation of a lineage-specific promoter in CMML monocytes.

Multicenter validation of the flow measurement of classical monocyte fraction for chronic myelomonocytic leukemia diagnosis.

Peripheral blood monocytes include three subsets defined by CD14 and CD16 surface markers. An increase in the CD14CD16 classical monocyte fraction ≥ 94% of the total monocytes was proposed to rapidly and efficiently distinguish chronic myelomonocytic leukemia from reactive monocytosis. The robustness of this assay required a multicenter validation.

MUB Binds to Lactoferrin and Stands as a Specific Neutrophil Marker.

Neutrophils represent the most abundant immune cells recruited to inflamed tissues. A lack of dedicated tools has hampered their detection and study. We show that a synthesized peptide, MUB, binds to lactoferrin, the most abundant protein stored in neutrophil-specific and tertiary granules.

Engraftment of chronic myelomonocytic leukemia cells in immunocompromised mice supports disease dependency on cytokines.

Chronic myelomonocytic leukemia (CMML) is a clonal hematopoietic stem cell disorder that typically associates with mutations in epigenetic, splicing, and signaling genes. Genetically modified mouse models only partially recapitulate the disease phenotype, whereas xenotransplantation of CMML cells in immunocompromised mice has been rarely successful so far. Here, CMML CD34 cells sorted from patient bone marrow (BM) or peripheral blood (PB) were injected intravenously into NSG (NOD/LtSz-scid IL2rγnull) mice and NSG mice engineered to express human granulo-monocyte colony-stimulating factor, stem cell factor, and interleukin-3 (NSGS mice).

High sensitivity of the Hematoflow™ solution for chronic myelomonocytic leukemia screening.

Background: Accumulation of classical monocytes CD14 CD16 (also called MO1) ≥ 94% can accurately distinguish chronic myelomonocytic leukemia (CMML) from reactive monocytosis. The HematoFlow™ solution, able to quantify CD16 negative monocytes, could be a useful tool to manage monocytosis which remains a common issue in routine laboratories.

Methods: Classical monocytes were quantified from 153 whole blood samples collected on EDTA using both flow cytometry methods, either MO1 percentage determination by the multiparameter assay previously published and regarded here as the reference method, or CD16 negative monocyte percentage determination by the means of HematoFlow™.

Eosinophil-rich tissue infiltrates in chronic myelomonocytic leukemia patients.

Chronic myelomonocytic leukemia (CMML) is a clonal hematopoietic disorder that associates dysplastic and proliferative features. Tissue inflammatory disorders occur in a fraction of CMML patients during the course of their disease. Here, we describe the occurrence of eosinophil-rich tissue inflammation, including eosinophilic pneumonia, chondritis, and cystitis, in CMML patients.

Mutation allele burden remains unchanged in chronic myelomonocytic leukaemia responding to hypomethylating agents.

The cytidine analogues azacytidine and 5-aza-2'-deoxycytidine (decitabine) are commonly used to treat myelodysplastic syndromes, with or without a myeloproliferative component. It remains unclear whether the response to these hypomethylating agents results from a cytotoxic or an epigenetic effect. In this study, we address this question in chronic myelomonocytic leukaemia.

The PRKAA1/AMPKα1 pathway triggers autophagy during CSF1-induced human monocyte differentiation and is a potential target in CMML.

Autophagy is induced during differentiation of human monocytes into macrophages that is mediated by CSF1/CSF-1/M-CSF (colony stimulating factor 1 [macrophage]). However, little is known about the molecular mechanisms that link CSF1 receptor engagement to the induction of autophagy. Here we show that the CAMKK2-PRKAA1-ULK1 pathway is required for CSF1-induced autophagy and human monocyte differentiation.

Characteristic repartition of monocyte subsets as a diagnostic signature of chronic myelomonocytic leukemia.

Chronic myelomonocytic leukemia (CMML) is a myelodysplastic syndrome/ myeloproliferative neoplasm whose diagnosis is currently based on the elevation of peripheral blood monocytes to >1 × 10(9)/L, measured for ≥3 months. Diagnosis can be ambiguous; for example, with prefibrotic myelofibrosis or reactive monocytosis. We set up a multiparameter flow cytometry assay to distinguish CD14(+)/CD16(-) classical from CD14(+)/CD16(+) intermediate and CD14(low)/CD16(+) nonclassical monocyte subsets in peripheral blood mononucleated cells and in total blood samples.