Cytokine-like protein 1-induced survival of monocytes suggests a combined strategy targeting MCL1 and MAPK in CMML.

Mouse models of chronic myeloid malignancies suggest that targeting mature cells of the malignant clone disrupts feedback loops that promote disease expansion. Here, we show that in chronic myelomonocytic leukemia (CMML), monocytes that accumulate in the peripheral blood show a decreased propensity to die by apoptosis. BH3 profiling demonstrates their addiction to myeloid cell leukemia-1 (MCL1), which can be targeted with the small molecule inhibitor S63845. RNA sequencing and DNA methylation pattern analysis both point to the implication of the mitogen-activated protein kinase (MAPK) pathway in the resistance of CMML monocytes to death and reveal an autocrine pathway in which the secreted cytokine-like protein 1 (CYTL1) promotes extracellular signal-regulated kinase (ERK) activation through C-C chemokine receptor type 2 (CCR2). Combined MAPK and MCL1 inhibition restores apoptosis of monocytes from patients with CMML and reduces the expansion of patient-derived xenografts in mice. These results show that the combined inhibition of MCL1 and MAPK is a promising approach to slow down CMML progression by inducing leukemic monocyte apoptosis.

Plasmacytoid dendritic cells proliferation associated with acute myeloid leukemia: phenotype profile and mutation landscape

Neoplasms involving plasmacytoid Dendritic Cells (pDCs) include Blastic pDC Neoplasms (BPDCN) and other pDC proliferations, where pDCs are associated with myeloid malignancies: most frequently Chronic MyeloMonocytic Leukemia (CMML) but also Acute Myeloid Leukemia (AML), hereafter named pDC-AML. We aimed to determine the reactive or neoplastic origin of pDCs in pDC-AML, and their link with the CD34+ blasts, monocytes or conventional DCs (cDCs) associated in the same sample, by phenotypic and molecular analyses (targeted NGS, 70 genes). We compared 15 pDC-AML at diagnosis with 21 BPDCN and 11 normal pDCs from healthy donors. CD45low CD34+ blasts were found in all cases (10-80% of medullar cells), associated with pDCs (4-36%), monocytes in 14 cases (1-10%) and cDCs (2 cases, 4.8-19%). pDCs in pDC-AML harbor a clearly different phenotype from BPDCN: CD4+ CD56- in 100% of cases, most frequently CD303+, CD304+ and CD34+; lower expression of cTCL1 and CD123 with isolated lymphoid markers (CD22/CD7/CD5) in some cases, suggesting a pre-pDC stage. In all cases, pDCs, monocytes and cDC are neoplastic since they harbor the same mutations as CD34+ blasts. RUNX1 is the most commonly mutated gene: detected in all AML with minimal differentiation (M0-AML) but not in the other cases. Despite low number of cases, the systematic association between M0-AML, RUNX1 mutations and an excess of pDC is puzzling. Further evaluation in a larger cohort is required to confirm RUNX1 mutations in pDC-AML with minimal differentiation and to investigate whether it represents a proliferation of blasts with macrophage and DC progenitor potential.

Dyserythropoiesis evaluated by the RED score and hepcidin:ferritin ratio predicts response to erythropoietin in lower-risk myelodysplastic syndromes.

Erythropoiesis-stimulating agents are generally the first line of treatment of anemia in patients with lower-risk myelodysplastic syndrome. We prospectively investigated the predictive value of somatic mutations, and biomarkers of ineffective erythropoiesis including the flow cytometry RED score, serum growth-differentiation factor-15, and hepcidin levels. Inclusion criteria were no prior treatment with erythropoiesis-stimulating agents, low- or intermediate-1-risk myelodysplastic syndrome according to the International Prognostic Scoring System, and a hemoglobin level <10 g/dL. Patients could be red blood cell transfusion-dependent or not and were given epoetin zeta 40 000 IU/week. Serum erythropoietin level, iron parameters, hepcidin, flow cytometry Ogata and RED scores, and growth-differentiation factor-15 levels were determined at baseline, and molecular analysis by next-generation sequencing was also conducted. Erythroid response (defined according to the International Working Group 2006 criteria) was assessed at week 12. Seventy patients, with a median age of 78 years, were included in the study. There were 22 patients with refractory cytopenia with multilineage dysplasia, 19 with refractory cytopenia with unilineage dysplasia, 14 with refractory anemia with ring sideroblasts, four with refractory anemia with excess blasts-1, six with chronic myelomonocytic leukemia, two with del5q-and three with unclassifiable myelodysplastic syndrome. According to the revised International Prognostic Scoring System, 13 had very low risk, 47 had low risk, nine intermediate risk and one had high-risk disease. Twenty patients were transfusion dependent. Forty-eight percent had an erythroid response and the median duration of the response was 26 months. At baseline, non-responders had significantly higher RED scores and lower hepcidin:ferritin ratios. In multivariate analysis, only a RED score >4 (P=0.05) and a hepcidin:ferritin ratio <9 (P=0.02) were statistically significantly associated with worse erythroid response. The median response duration was shorter in patients with growth-differentiation factor-15 >2000 pg/mL and a hepcidin:ferritin ratio <9 (P=0.0008 and P=0.01, respectively). In multivariate analysis, both variables were associated with shorter response duration. Erythroid response to epoetin zeta was similar to that obtained with other erythropoiesis-stimulating agents and was correlated with higher baseline hepcidin:ferritin ratio and lower RED score. ClinicalTrials.gov registration: NCT 03598582.

Iron deficiency, an unusual cause of thrombocytopenia: results from a multicenter retrospective case-controlled study.

Iron deficiency anemia (IDA) is often associated with mild to moderate thrombocytosis, and iron deficiency-associated thrombocytopenia (IDAT) is much more uncommon and often misdiagnosed as immune thrombocytopenia (ITP). To better describe the features of IDAT, we conducted a retrospective multicenter case-control study. We identified 10 patients (9 women) with a definite diagnosis IDAT, with a median age of 43.5 [range, 16-72] years and a median platelet count of 30.5 × 109/L [range, 21-80], and 7 patients with a possible diagnosis of IDAT. Bleeding manifestations were absent in all patients but one. All the patients recovered (platelet count ≥ 150 × 109/L) upon iron therapy ± red blood cell transfusion after a median time of 6 [4-39] days. When compared with 30 randomly newly diagnosed ITP patients matched on age, the baseline platelet count was significantly lower in ITP (median = 7 × 109/L [4-59], p < 0.001) whereas MPV was higher (10.5 fL [9,4-13,8] vs 8.2 fL, for IDAT p < 0.001). The median platelet count on day 7 was 337 × 109/L [113-1000] for IDAT cases vs 72 × 109/L [13-212] for ITP controls (p < 0.001). IDAT is potentially an under-recognized cause of thrombocytopenia that may be easily managed with iron therapy.

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. 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.

Six Months of Hydroxyurea Reduces Albuminuria in Patients with Sickle Cell Disease.

The earliest symptom of glomerular injury in patients with sickle cell disease (SCD) is microalbuminuria. The effect of hydroxyurea (HU) on urine albumin-to-creatinine ratio (ACR) is unclear and should be determined, because increasing numbers of patients with SCD take this drug to improve red blood cell function. In this cohort study of 58 SS-homozygous adults with SCD who initiated HU therapy, we evaluated ACR changes and relationships of these changes with demographic, clinical, and biologic parameters at HU initiation (baseline) and 6 months later (follow-up). Between baseline and follow-up, ACR declined significantly for the entire population (3.0-1.7 mg/mmol; P<0.01), but this was primarily driven by the ACR reduction in the microalbuminuria subgroup (8.1-2.3 mg/mmol; P=0.03; n=23). According to bivariate analyses on 39 patients who did not receive a blood transfusion during the study period, the baseline to follow-up ACR decline was strongly associated with decreases in levels of hemolysis markers, percentage of dense red blood cells, and systolic BP. Bivariate analysis also revealed a close association between the ACR decrease and high baseline levels of hemolysis markers and percentage of dense red blood cells. These results show that urine ACR decreased significantly after 6 months of HU and confirm a close relationship between ACR and hemolysis evolution in patients with SCD.

Haematological determinants of cardiac involvement in adults with sickle cell disease.

AIMS: Cardiac involvement is common in sickle cell disease (SCD). Studies are needed to establish haematological determinants of this involvement and prognostic markers. The aim of the study was to identify haematological factors associated with cardiac involvement in SCD and their impact on prognosis. METHODS AND RESULTS: This longitudinal observational study was performed on 1780 SCD patients with SS or S-ß(0)-thalassemia referred to our centre. Six hundred fifty-six met our inclusion criteria (availability of a blood-workup and echocardiogram obtained <1 year apart, no heart valve surgery and no current pregnancy). Median age was 31 (interquartile range, 25-40) years, and median haemoglobin (Hb) was 87 (80-95)g/L. Left ventricular (LV) dilation, left atrial dilation, cardiac index (CI) >4 L/min/m(2), LV ejection fraction <55%, and tricuspid regurgitant velocity (TRV) ≥2.5 m/s were found in 35, 78, 23, 8.5, and 17% of patients, respectively. Compared with other patients, those in the fourth quartiles (Q4) of LV end-diastolic dimension index (LVEDDind) and left atrial dimension index (LADind) and those with high CI had significantly lower Hb, % foetal Hb (HbF), and red blood cell (RBC) counts; and significantly higher lactate dehydrogenase, bilirubin, and %dense RBCs. Independent haematologic determinants of Q4 LVEDDind and LADind were low RBC count and %HbF; high %dense RBCs were associated with LADind. Low %HbF and RBC count were associated with high CI. High %dense RBCs or no α-thalassemia gene deletion was associated with greater severity of anaemia and cardiac dilation and with higher CI. During the median follow-up of 48 (32-59) months, 50 (7.6%) patients died. Tricuspid regurgitant velocity ≥ 2.5 m/s was a predictor of mortality. The risk of death increased four-fold when left ventricular ejection fraction <55% was present also (P = 0.0001). CONCLUSION: Cardiac dilation and CI elevation in patients with SCD are associated with haematologic variables reflecting haemolysis, RBC rigidity, and blood viscosity. Tricuspid regurgitant velocity ≥ 2.5 and LV dysfunction (even mild) predict mortality.

Morbidity and mortality of sickle cell disease patients starting intermittent haemodialysis: a comparative cohort study with non- Sickle dialysis patients.

We performed a retrospective study to assess the changes in clinical, biological and heart echocardiographic parameters in 32 sickle cell disease (SCD) patients beginning haemodialysis. Acute SCD-related complications were similar at 6 months before and 6 months after the initiation of haemodialysis. Median haemoglobin level did not change significantly, but the need for blood transfusions increased (P < 0·001). The 5-year incidence of death was higher in SCD patients (P < 0·0001). The 5-year likelihood of receiving a renal graft was lower in SCD patients (P = 0·022). Our findings suggest that SCD patients have poorer survival and a lower likelihood of receiving a renal graft.

Spectrum of adult Parvovirus B19 infection according to the underlying predisposing condition and proposals for clinical practice.

The virological diagnosis of Parvovirus B19 (PvB19) infection is currently based on sero-diagnosis, molecular methods or both, yet without clear recommendations. We retrospectively identified patients with polymerase chain reaction-positive PvB19 and/or positive serological assay between 2007 and 2013. Eighty-two adults with at least one diagnostic criterion of recent PvB19 infection (IgM antibodies, viral DNA in blood and/or in marrow) were included and classified into three homogeneous groups: 30 patients had no underlying predisposing condition, 25 a hereditary haemolytic anaemia, 27 an underlying immunodeficiency. The classical PvB19-related manifestations were less frequent in immunocompromised than in immunocompetent patients (arthromyalgia: 5 vs. 14; erythema: 4 vs. 17, respectively). Only 41·4% of patients with no underlying disease were anaemic. Bicytopenia and pancytopenia were observed mainly in immunocompromised patients. Classical pure red cell aplasia was observed in only 9 of the 27 marrow smears performed. Specific IgM were found in 93% of immunocompetent patients, whereas only 58% had detectable viral DNA in blood. IgM and DNA were present alone or together in all patients with hereditary haemolytic anaemia. In immunocompromised patients, the diagnosis was confirmed by marrow analysis in 91% of cases. We make some proposals based on this large series of PvB19-infected patients.

Multicentric study underlining the interest of adding CD5, CD7 and CD56 expression assessment to the flow cytometric Ogata score in myelodysplastic syndromes and myelodysplastic/myeloproliferative neoplasms.

Although numerous recent publications have demonstrated interest in multiparameter flow cytometry in the investigation of myelodysplastic disorders, it is perceived by many laboratory hematologists as difficult and expensive, requiring a high level of expertise. We report a multicentric open real-life study aimed at evaluating the added value of the technically simple flow cytometry score described by the Ogata group for the diagnosis of myelodysplastic syndromes. A total of 652 patients were recruited prospectively in four different centers: 346 myelodysplastic syndromes, 53 myelodysplastic/myeloproliferative neoplasms, and 253 controls. The Ogata score was assessed using CD45 and CD34 staining, with the addition of CD10 and CD19. Moreover, labeling of CD5, CD7 and CD56 for the evaluation of myeloid progenitors and monocytes was tested on a subset of 294 patients. On the whole series, the specificity of Ogata score reached 89%. Respective sensitivities were 54% for low-risk myelodysplastic syndromes, 68% and 84% for type 1 and type 2 refractory anemia with excess of blasts, and 72% for myelodysplastic/myeloproliferative neoplasms. CD5 expression was poorly informative. When adding CD56 or CD7 labeling to the Ogata score, sensitivity rose to 66% for low-risk myelodysplastic syndromes, to 89% for myelodysplastic/myeloproliferative neoplasms and to 97% for refractory anemia with excess of blasts. This large multicenter study confirms the feasibility of Ogata scoring in routine flow cytometry diagnosis but highlights its poor sensitivity in low-risk myelodysplastic syndromes. The addition of CD7 and CD56 in flow cytometry panels improves the sensitivity but more sophisticated panels would be more informative.

Chemokine (C-X-C motif) receptor 4 blockade by AMD3100 inhibits experimental abdominal aortic aneurysm expansion through anti-inflammatory effects.

OBJECTIVE: Inflammation plays a critical role in the development of abdominal aortic aneurysms (AAAs). Because stromal cell-derived factor 1 (SDF-1) is known for its ability to attract inflammatory cells, we investigated whether SDF-1/chemokine (C-X-C motif) receptor 4 (CXCR4) axis is expressed in aneurysmal aortic wall and plays a role in AAA physiopathology and asked whether its blockade modulates AAA formation and expansion. APPROACH AND RESULTS: Quantitative real-time polymerase chain reaction analysis showed that SDF-1α and CXCR4 mRNA levels are increased in both human and CaCl2-induced mouse AAA wall and are positively correlated to the aortic diameter in mice. ELISA quantification and immunostaining demonstrated that, in mice, aortic SDF-1α is rapidly induced during AAA formation, first by apoptotic vascular smooth muscle cells in the injured media and then by adventitial macrophages once AAA is fully established. Using green fluorescent protein-positive (GFP(+/-)) bone marrow transplantation experiments, we demonstrated that aortic SDF-1 overexpression is implicated in the recruitment of bone marrow-derived macrophages within the AAA wall. Furthermore, in mice, blockade of CXCR4 by AMD3100 decreases the infiltration of adventitial macrophages, inhibits AAA formation, and prevents aortic wall destruction. AMD3100 reduces the mRNA levels of MMP-12 and MMP-14 as well as that of inflammatory effectors MCP-1, MIP-1ß, MIP-2α, RANTES, IL-1ß, IL-6, TNF-α, and E-selectin. Finally, AMD3100 stabilizes the diameter of formed, expanding AAAs in 2 experimental models. CONCLUSIONS: SDF-1/CXCR4 axis is upregulated in human and mouse AAAs. Blockade of CXCR4 with AMD3100 suppresses AAA formation and progression in two rodent models. Blockade of SDF-1/CXCR4 axis may represent a new strategy to limit progression of small human AAAs.