Chronic myelomonocytic leukemia (CMML) is a myelodysplastic syndrome/ myeloproliferative neoplasm whose diagnosis is currently based on the elevation of peripheral blood monocytes to >1 × 109/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 CD14low/CD16+ nonclassical monocyte subsets in peripheral blood mononucleated cells and in total blood samples. Compared with healthy donors and patients with reactive monocytosis or another hematologic malignancy, CMML patients demonstrate a characteristic increase in the fraction of CD14+/CD16− cells (cutoff value, 94.0%). The associated specificity and sensitivity values were 95.1% and 90.6% in the learning cohort (175 samples) and 94.1% and 91.9% in the validation cohort (307 samples), respectively. The accumulation of classical monocytes, which demonstrate a distinct gene expression pattern, is independent of the mutational background. Importantly, this increase disappears in patients who respond to hypomethylating agents. We conclude that an increase in the fraction of classical monocytes to >94.0% of total monocytes is a highly sensitive and specific diagnostic marker that rapidly and accurately distinguishes CMML from confounding diagnoses.
•An increase in the classical monocyte subset to >94% of total monocytes discriminates CMML from other monocytoses with high specificity.•This characteristic increase in classical monocytes disappears in CMML patients who respond to hypomethylating agents.
Despite their location at the cell surface, several receptor tyrosine kinases (RTK) are also found in the nucleus, as either intracellular domains or full length proteins. However, their potential ...nuclear functions remain poorly understood. Here we find that a fraction of full length Colony Stimulating Factor-1 Receptor (CSF-1R), an RTK involved in monocyte/macrophage generation, migrates to the nucleus upon CSF-1 stimulation in human primary monocytes. Chromatin-immunoprecipitation identifies the preferential recruitment of CSF-1R to intergenic regions, where it co-localizes with H3K4me1 and interacts with the transcription factor EGR1. When monocytes are differentiated into macrophages with CSF-1, CSF-1R is redirected to transcription starting sites, colocalizes with H3K4me3, and interacts with ELK and YY1 transcription factors. CSF-1R expression and chromatin recruitment is modulated by small molecule CSF-1R inhibitors and altered in monocytes from chronic myelomonocytic leukemia patients. Unraveling this dynamic non-canonical CSF-1R function suggests new avenues to explore the poorly understood functions of this receptor and its ligands.
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. Mir150 knock-out mice demonstrate a cell-autonomous defect in non-classical monocytes. Our pulldown experiments point to Ten-Eleven-Translocation-3 (TET3) mRNA as a hsa-miR-150 target in classical human monocytes. We show that Tet3 knockout mice generate an increased number of non-classical monocytes. Our results identify the miR-150/TET3 axis as being involved in the generation of non-classical monocytes.
Introduction: Hemolytic anemia combines 3 components to various extents: extravascular hemolysis, intravascular hemolysis and dyserythropoiesis. Global hemolysis in excess to defense lines induces ...oxidative and inflammatory syndromes and vascular damages in various organs. Therefore, accurate hemolysis biomarkers are required for a better evaluation of hemolytic disorders, such as sickle cell disease (SCD) or thalassemia syndromes and to evaluate the efficacy of various therapies. Accordingly, we have developed a new spectrophotometric method to measure and calculate several hemolysis biomarkers in plasma or serum including Hemoglobin in various forms (HbO2, HbCO, MetHb), Heme or Hemin bound to albumin or to hemopexin, total bilirubin and total hemopexin.
Patients and Method : Blood samples were collected at steady-state for 77 SCD adults (mean age 39.8 ± 10.2 yrs, M/F ratio 0.64) and 23 beta thalassemia patients (mean age 42.7 ± 16 yrs, M/F ratio 0.91); SCD patients (SS or Sb0-Thalassemia) were either treated with Hydroxycarbamide (HU: 27) or not (NT: 50). For comparison, plasma samples from healthy volunteers (HV) were also analyzed. Continuous variables were expressed as means ± SD or medians interquartile range, depending on their normal or asymmetric distributions. Categorical variables were expressed as numbers (%). Univariate analyses were done using Student's t-test or Mann-Whitney non-parametric test, depending on the distribution. Correlation were analyzed using a spearman test.
The dosage methodology is based on the light absorption spectrophotometry of plasma samples using an appropriate mathematical conversion of the signal, reference spectra of the different species and some chemical modifications of the iron redox and ligation states.
Results: The levels of plasma Hb were statistically higher in homozygous SCD patients compared to beta-thalassemia patients (p=0.001) and healthy volunteers, with median of 6.3 3.4-11, 2.6 1-5.4 and 1.7 0.5-3 µM respectively (Table 1). Interestingly levels of plasma heme were higher in beta thalassemia patient compared to SCD patients (p=0.0001) and HV, with a mean of 1.05 0.05-3, 10.5 3.5-24 and ≤ 0.2 µM level of detection respectively (Table 1). A significant negative correlation was found between heme and hemopexin levels in both diseases (p<0.0001; r=0.85). Among 77 SS or Sb0-Thalassemia patients, 29 were treated by HU without any difference for plasma Hb, plasma Heme and Hemopexin values compared to non-treated patients.
Discussion and Conclusions: plasma Hb is a more accurate dosage for intra vascular hemolysis than other biomarkers which are not specific of intra vascular hemolysis and could be biased by other pathological conditions: LDH or ASAT can be increased in hepatic or muscular cytolysis, bilirubin is dependent on the heme oxygenase and glycuronyl transferase activities, and reticulocytes are dependent on erythropoiesis. Our results showed that the intra-vascular hemolysis is more pronounced in SCD compared to beta-thalassemia based on the plasma Hb levels. The elevated plasma heme concentration in beta thalassemia is a new finding that should be investigated in more details. It could reflect the ineffective erythropoiesis or heme export from erythroblasts or macrophages involving hemopexin scavenging.
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Bencheikh:Hemanext: Research Funding. Bartolucci:HEMANEXT: Membership on an entity's Board of Directors or advisory committees; AddMedica: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.
Polymerization of the sickle hemoglobin (HbS) is a key determinant of sickle cell disease (SCD), an inherited blood disorder. Fetal hemoglobin (HbF) is a major modulator of the disease severity by ...both decreasing HbS intracellular concentration and inhibiting its polymerization. However, heterocellular distribution of HbF is common in SCD. For HbS polymerization inhibition, the hypothesis of an “HbF per red blood cell (HbF/RBC) threshold” requires accurate measurement of HbF in individual RBC. To date, HbF detection methods are limited to a qualitative measurement of RBC populations containing HbF ‐ the F cells, which are variable. We developed an accurate method for HbF quantification in individual RBC. A linear association between mean HbF content and mean RBC fluorescence by flow cytometry, using an anti‐Human‐HbF antibody, was obtained from non‐SCD subjects presenting homogeneous HbF distribution. This correlation was then used to measure HbF/RBC. Hydroxyurea (HU) improves SCD clinical manifestations, mainly through its ability to induce HbF synthesis. The HbF distribution was analyzed in 14 SCD patients before and during HU treatment. A significant decrease in RBC population containing less than 2 pg of HbF/RBC was observed. Therefore, we tested associations for %RBC above different HbF/RBC thresholds and showed a decrease in the pathognomonic vaso‐occlusive crisis incidence from the threshold of 4 pg. This quantity was also correlated with the level of sickle RBC after in vitro deoxygenation. This new method allows the comparison of HbF/RBC distributions and could be a useful tool to characterize baseline patients HbF distribution and therapeutic response to HbF inducers.
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