Accelerated DNA replication fork speed due to loss of R-loops in myelodysplastic syndromes with SF3B1 mutation.

Myelodysplastic syndromes (MDS) with mutated SF3B1 gene present features including a favourable outcome distinct from MDS with mutations in other splicing factor genes SRSF2 or U2AF1. Molecular bases of these divergences are poorly understood. Here we find that SF3B1-mutated MDS show reduced R-loop formation predominating in gene bodies associated with intron retention reduction, not found in U2AF1- or SRSF2-mutated MDS. Compared to erythroblasts from SRSF2- or U2AF1-mutated patients, SF3B1-mutated erythroblasts exhibit augmented DNA synthesis, accelerated replication forks, and single-stranded DNA exposure upon differentiation. Importantly, histone deacetylase inhibition using vorinostat restores R-loop formation, slows down DNA replication forks and improves SF3B1-mutated erythroblast differentiation. In conclusion, loss of R-loops with associated DNA replication stress represents a hallmark of SF3B1-mutated MDS ineffective erythropoiesis, which could be used as a therapeutic target.

Loss of hematopoietic progenitors heterogeneity is an adverse prognostic factor in lower-risk myelodysplastic neoplasms.

Myelodysplastic neoplasms (MDS) are characterized by clonal evolution starting from the compartment of hematopoietic stem and progenitors cells (HSPCs), leading in some cases to leukemic transformation. We hypothesized that deciphering the diversity of the HSPCs compartment may allow for the early detection of an emergent sub-clone that drives disease progression. Deep analysis of HSPCs repartition by multiparametric flow cytometry revealed a strong disorder of the hematopoietic branching system in most patients at diagnosis with different phenotypic signatures closely related to specific MDS features. In two independent cohorts of 131 and 584 MDS, the HSPCs heterogeneity quantified through entropy calculation was decreased in 47% and 46% of cases, reflecting a more advanced state of the disease with deeper cytopenias, higher IPSS-R risk and accumulation of somatic mutations. We demonstrated that patients with lower-risk MDS and low CD34 + CD38+HSPCs entropy had an adverse outcome and that this parameter is as an independent predictive biomarker for progression free survival, leukemia free survival and overall survival. Analysis of HSPCs repartition at diagnosis represents therefore a very powerful tool to identify lower-risk MDS patients with a worse outcome and valuable for clinical decision-making, which could be fully integrated in the MDS diagnostic workflow.

White blood cells scattergram as a valuable tool for COVID-19 screening: A multicentric study.

INTRODUCTION: New tools have been developed to distinguish the COVID-19 diagnosis from other viral infections presenting similar symptomatology and mitigate the lack of sensitivity of molecular testing. We previously identified a specific "sandglass" aspect on the white blood cells (WBC) scattergram of COVID-19 patients, as a highly reliable COVID-19 screening test (sensitivity: 85.9%, specificity: 83.5% and positive predictive value: 94.3%). We then decided to validate our previous data in a multicentric study. METHODS: This retrospective study involved 817 patients with flu-like illness, among 20 centers, using the same CBC instrument (XN analyzer, SYSMEX, Japan). After training, one specialist per center independently evaluated, under the same conditions, the presence of the "sandglass" aspect of the WDF scattergram, likely representing plasmacytoid lymphocytes. RESULTS: Overall, this approach showed sensitivity: 59.0%, specificity: 72.9% and positive predictive value: 77.7%. Sensitivity improved with subgroup analysis, including in patients with lymphopenia (65.2%), patients presenting symptoms for more than 5 days (72.3%) and in patients with ARDS (70.1%). COVID-19 patients with larger plasmacytoid lymphocyte cluster (>15 cells) more often have severe outcomes (70% vs. 15% in the control group). CONCLUSION: Our findings confirm that the WBC scattergram analysis could be added to a diagnostic algorithm for screening and quickly categorizing symptomatic patients as either COVID-19 probable or improbable, especially during COVID-19 resurgence and overlapping with future influenza epidemics. The observed large size of the plasmacytoid lymphocytes cluster appears to be a hallmark of COVID-19 patients and was indicative of a severe outcome. Furthers studies are ongoing to evaluate the value of the new hematological parameters in combination with WDF analysis.

Along with PaO2/FiO2 ratio and lymphopenia, low HLA-DR monocytes are the only additional parameter that independently predicts the clinical course of undifferentiated SARS-CoV-2 patients in Emergency Departments.

Since 1/3 of patients deteriorate after their admission to the emergency department (ED), assessing the prognosis of COVID-19 patients is of great importance. But to date, only lymphopenia and PaO2/FiO2 (P/F) ratio have been reported as partly predictive of COVID-19 further deterioration and their association has not been evaluated. We asked whether other key biomarkers of SARS-CoV2 immunologic defects - increase in circulating immature granulocytes (IGs), loss of monocyte HLA-DR (mHLA-DR) expression and monocyte differentiation blockade - could also predict further COVID-19 deterioration. A series of 284 consecutive COVID-19 patients with, as sole inclusion criterion of being an adult, were prospectively enrolled at ED admission (D0) of two different hospitals: one for the exploratory (180 patients) and one for the confirmatory cohort (104 patients). Deterioration was assessed over the next seven days. Neither increased IG levels nor monocyte differentiation blockade predicted patient worsening. Among more than 30 clinical, biological and radiological parameters, the value of decreased PaO2/FiO2 (P/F) ratio and lymphopenia for prediction of further COVID-19 deterioration was strongly confirmed and the loss of mHLA-DR was the only additional independent marker. Combined together in a simple OxyLymphoMono score, the three variables perfectly predicted patients who did not worsen and correctly predicted worsening in 59% of cases.By highlighting lymphocyte and monocyte defects as preceding COVID-19 deterioration, these results point on early immunosuppression in COVID-19 deterioration. Combining P/F ratio, lymphopenia and loss of mHLA-DR together in a simple and robust score could offer a pragmatic method for COVID-19 patient stratification.

Neutralizing Antibody Levels as a Correlate of Protection Against SARS-CoV-2 Infection: A Modeling Analysis.

Although anti-severe acute respiratory syndrome-coronavirus 2 antibody kinetics have been described in large populations of vaccinated individuals, we still poorly understand how they evolve during a natural infection and how this impacts viral clearance. For that purpose, we analyzed the kinetics of both viral load and neutralizing antibody levels in a prospective cohort of individuals during acute infection with alpha variant. Using a mathematical model, we show that the progressive increase in neutralizing antibodies leads to a shortening of the half-life of both infected cells and infectious viral particles. We estimated that the neutralizing activity reached 90% of its maximal level within 11 days after symptom onset and could reduce the half-life of both infected cells and circulating virus by a 6-fold factor, thus playing a key role to achieve rapid viral clearance. Using this model, we conducted a simulation study to predict in a more general context the protection conferred by pre-existing neutralization titers, due to either vaccination or prior infection. We predicted that a neutralizing activity, as measured by 50% effective dose > 103 , could reduce by 46% the risk of having viral load detectable by standard polymerase chain reaction assays and by 98% the risk of having viral load above the threshold of infectiousness. Our model shows that neutralizing activity could be used to define correlates of protection against infection and transmission.

Enhanced fetal hematopoiesis in response to symptomatic SARS-CoV-2 infection during pregnancy.

BACKGROUND: Pregnant women and their fetuses are particularly susceptible to respiratory pathogens. How they respond to SARS-CoV-2 infection is still under investigation. METHODS: We studied the transcriptome and phenotype of umbilical cord blood cells in pregnant women infected or not with SARS-CoV-2. RESULTS: Here we show that symptomatic maternal COVID-19 is associated with a transcriptional erythroid cell signature as compared with asymptomatic and uninfected mothers. We observe an expansion of fetal hematopoietic multipotent progenitors skewed towards erythroid differentiation that display increased clonogenicity. There was no difference in inflammatory cytokines levels in the cord blood upon maternal SARS-CoV-2 infection. Interestingly, we show an activation of hypoxia pathway in cord blood cells from symptomatic COVID-19 mothers, suggesting that maternal hypoxia may be triggering this fetal stress hematopoiesis. CONCLUSIONS: Overall, these results show a fetal hematopoietic response to symptomatic COVID-19 in pregnant mothers in the absence of vertically transmitted SARS-CoV-2 infection which is likely to be a mechanism of fetal adaptation to the maternal infection and reduced oxygen supply.

During pregnancy, women are more prone to respiratory infectious diseases. It is not known if COVID-19 infection has an adverse effect on the growing fetus. Here, we aimed to identify any potential effects of COVID-19 infection on the fetus by taking measurements from the umbilical cord blood cells. In mothers who displayed symptomatic COVID-19 infection, we observed an increased production of hematopoietic progenitor cells, especially the ones that are responsible for producing red blood cells. We think this might be a coping mechanism for the fetus, as the mother's body deals with the infection. Therefore, our work shows that growing fetuses do respond to maternal COVID-19 symptoms, even when they are protected in the womb from the infection and may never get infected by the mother.

[Pathophysiology of myelodysplastic syndromes]. / Physiopathologie des syndromes myélodysplasiques.

During aging, the onset of mutations at low frequency in hematopoietic cells or clonal hematopoiesis of indeterminate significance favors the evolution towards hemopathies such as myelodysplastic syndromes or acute leukemias, but also cardiovascular diseases and other pathologies. Acute or chronic inflammation related to age influences the clonal evolution and the immune response. Conversely, mutated hematopoietic cells create an inflammatory bone marrow environment facilitating their expansion. Various pathophysiological mechanisms depending on the type of mutation produce the diversity of phenotypes. Identifying factors affecting clonal selection is mandatory to improve patient care.

Artificial intelligence to empower diagnosis of myelodysplastic syndromes by multiparametric flow cytometry.

The diagnosis of myelodysplastic syndromes (MDS) might be challenging and relies on the convergence of cytological, cytogenetic, and molecular factors. Multiparametric flow cytometry (MFC) helps diagnose MDS, especially when other features do not contribute to the decision-making process, but its usefulness remains underestimated, mostly due to a lack of standardization of cytometers. We present here an innovative model integrating artificial intelligence (AI) with MFC to improve the diagnosis and the classification of MDS. We develop a machine learning model through an elasticnet algorithm directed on a cohort of 191 patients, only based on flow cytometry parameters selected by the Boruta algorithm, to build a simple but reliable prediction score with five parameters. Our AI-assisted MDS prediction score greatly improves the sensitivity of the Ogata score while keeping an excellent specificity validated on an external cohort of 89 patients with an Area Under the Curve of 0.935. This model allows the diagnosis of both high- and low-risk MDS with 91.8% sensitivity and 92.5% specificity. Interestingly, it highlights a progressive evolution of the score from clonal hematopoiesis of indeterminate potential (CHIP) to highrisk MDS, suggesting a linear evolution between these different stages. By significantly decreasing the overall misclassification of 52% for patients with MDS and of 31.3% for those without MDS (P=0.02), our AI-assisted prediction score outperforms the Ogata score and positions itself as a reliable tool to help diagnose MDS.

Multiparameter flow cytometry in the evaluation of myelodysplasia: Analytical issues: Recommendations from the European LeukemiaNet/International Myelodysplastic Syndrome Flow Cytometry Working Group.

Multiparameter flow cytometry (MFC) is one of the essential ancillary methods in bone marrow (BM) investigation of patients with cytopenia and suspected myelodysplastic syndrome (MDS). MFC can also be applied in the follow-up of MDS patients undergoing treatment. This document summarizes recommendations from the International/European Leukemia Net Working Group for Flow Cytometry in Myelodysplastic Syndromes (ELN iMDS Flow) on the analytical issues in MFC for the diagnostic work-up of MDS. Recommendations for the analysis of several BM cell subsets such as myeloid precursors, maturing granulocytic and monocytic components and erythropoiesis are given. A core set of 17 markers identified as independently related to a cytomorphologic diagnosis of myelodysplasia is suggested as mandatory for MFC evaluation of BM in a patient with cytopenia. A myeloid precursor cell (CD34+ CD19- ) count >3% should be considered immunophenotypically indicative of myelodysplasia. However, MFC results should always be evaluated as part of an integrated hematopathology work-up. Looking forward, several machine-learning-based analytical tools of interest should be applied in parallel to conventional analytical methods to investigate their usefulness in integrated diagnostics, risk stratification, and potentially even in the evaluation of response to therapy, based on MFC data. In addition, compiling large uniform datasets is desirable, as most of the machine-learning-based methods tend to perform better with larger numbers of investigated samples, especially in such a heterogeneous disease as MDS.

Flow cytometric analysis of myelodysplasia: Pre-analytical and technical issues-Recommendations from the European LeukemiaNet.

BACKGROUND: Flow cytometry (FCM) aids the diagnosis and prognostic stratification of patients with suspected or confirmed myelodysplastic syndrome (MDS). Over the past few years, significant progress has been made in the FCM field concerning technical issues (including software and hardware) and pre-analytical procedures. METHODS: Recommendations are made based on the data and expert discussions generated from 13 yearly meetings of the European LeukemiaNet international MDS Flow working group. RESULTS: We report here on the experiences and recommendations concerning (1) the optimal methods of sample processing and handling, (2) antibody panels and fluorochromes, and (3) current hardware technologies. CONCLUSIONS: These recommendations will support and facilitate the appropriate application of FCM assays in the diagnostic workup of MDS patients. Further standardization and harmonization will be required to integrate FCM in MDS diagnostic evaluations in daily practice.

Clinical application of flow cytometry in patients with unexplained cytopenia and suspected myelodysplastic syndrome: A report of the European LeukemiaNet International MDS-Flow Cytometry Working Group.

This article discusses the rationale for inclusion of flow cytometry (FCM) in the diagnostic investigation and evaluation of cytopenias of uncertain origin and suspected myelodysplastic syndromes (MDS) by the European LeukemiaNet international MDS Flow Working Group (ELN iMDS Flow WG). The WHO 2016 classification recognizes that FCM contributes to the diagnosis of MDS and may be useful for prognostication, prediction, and evaluation of response to therapy and follow-up of MDS patients.

Plasma Endocan as a Biomarker of Thrombotic Events in COVID-19 Patients.

(1) Background: Endocan is a marker of endothelial dysfunction that may be associated with thrombotic events. The aim of the study was to investigate the performance of endocan as a marker of thrombotic events in COVID-19 patients. (2) Methods: We measured endocan in plasma from 79 documented COVID-19 patients classified according to disease severity (from mild to critical). Thrombotic events were recorded. (3) Results: Endocan concentrations at admission were significantly increased according to COVID-19 severity. Levels of endocan were significantly increased in patients experiencing thrombotic events in comparison with those without (16.2 (5.5−26.7) vs. 1.81 (0.71−10.5) ng/mL, p < 0.001). However, endocan concentrations were not different between pulmonary embolism and other thrombotic events. The Receiver Operating Characteristic (ROC) analysis for the identification of thrombotic events showed an area under the ROC curve (AUC) of 0.776 with an optimal threshold at 2.83 ng/mL (93.8% sensitivity and 54.7% specificity). When combining an endocan measurement with D-dimers, the AUC increased to 0.853. When considering both biomarkers, the Kaplan−Meier survival curves showed that the combination of endocan and D-dimers better discriminated patients with thrombotic events than those without. The combination of D-dimers and endocan was independently associated with thrombotic events. (4) Conclusions: Endocan might be a useful and informative biomarker to better identify thrombotic events in COVID-19 patients.

Diagnosis of Myelodysplastic Syndromes: From Immunological Observations to Clinical Applications.

Myelodysplastic syndromes (MDS) constitute a very heterogeneous group of diseases with a high prevalence in elderly patients and a propensity for progression to acute myeloid leukemia. The complexity of these hematopoietic malignancies is revealed by the multiple recurrent somatic mutations involved in MDS pathogenesis and the paradoxical common phenotype observed in these patients characterized by ineffective hematopoiesis and cytopenia. In the context of population aging, the incidence of MDS will strongly increase in the future. Thus, precise diagnosis and evaluation of the progression risk of these diseases are imperative to adapt the treatment. Dysregulations of both innate and adaptive immune systems are frequently detected in MDS patients, and their critical role in MDS pathogenesis is now commonly accepted. However, different immune dysregulations and/or dysfunctions can be dynamically observed during the course of the disease. Monitoring the immune system therefore represents a new attractive tool for a more precise characterization of MDS at diagnosis and for identifying patients who may benefit from immunotherapy. We review here the current knowledge of the critical role of immune dysfunctions in both MDS and MDS precursor conditions and discuss the opportunities offered by the detection of these dysregulations for patient stratification.

Dynamics of circulating calprotectin accurately predict the outcome of moderate COVID-19 patients.

BACKGROUND: Severe COVID-19 is associated with a high circulating level of calprotectin, the S100A8/S100A9 alarmin heterodimer. Baseline calprotectin amount measured in peripheral blood at diagnosis correlates with disease severity. The optimal use of this biomarker along COVID-19 course remains to be delineated. METHODS: We focused on patients with a WHO-defined moderate COVID-19 requiring hospitalization in a medical ward. We collected plasma and serum from three independent cohorts (N = 626 patients) and measured calprotectin amount at admission. We performed longitudinal measures of calprotectin in 457 of these patients (1461 samples) and used a joint latent class mixture model in which classes were defined by age, body mass index and comorbidities to identify calprotectin trajectories predicting the risk of transfer into an intensive care unit or death. FINDINGS: After adjustment for age, sex, body mass index and comorbidities, the predictive value of baseline calprotectin in patients with moderate COVID19 could be refined by serial monitoring of the biomarker. We discriminated three calprotectin trajectories associated with low, moderate, and high risk of poor outcome, and we designed an algorithm available as online software (https://calpla.gustaveroussy.fr:8443/) to monitor the probability of a poor outcome in individual patients with moderate COVID-19. INTERPRETATION: These results emphasize the clinical interest of serial monitoring of calprotectin amount in the peripheral blood to anticipate the risk of poor outcomes in patients with moderate COVID-19 hospitalized in a standard care unit. FUNDING: The study received support (research grants) from ThermoFisher immunodiagnostics (France) and Gustave Roussy Foundation.

RAS activation induces synthetic lethality of MEK inhibition with mitochondrial oxidative metabolism in acute myeloid leukemia.

Despite recent advances in acute myeloid leukemia (AML) molecular characterization and targeted therapies, a majority of AML cases still lack therapeutically actionable targets. In 127 AML cases with unmet therapeutic needs, as defined by the exclusion of ELN favorable cases and of FLT3-ITD mutations, we identified 51 (40%) cases with alterations in RAS pathway genes (RAS+, mostly NF1, NRAS, KRAS, and PTPN11 genes). In 79 homogeneously treated AML patients from this cohort, RAS+ status were associated with higher white blood cell count, higher LDH, and reduced survival. In AML models of oncogenic addiction to RAS-MEK signaling, the MEK inhibitor trametinib demonstrated antileukemic activity in vitro and in vivo. However, the efficacy of trametinib was heterogeneous in ex vivo cultures of primary RAS+ AML patient specimens. From repurposing drug screens in RAS-activated AML cells, we identified pyrvinium pamoate, an anti-helminthic agent efficiently inhibiting the growth of RAS+ primary AML cells ex vivo, preferentially in trametinib-resistant PTPN11- or KRAS-mutated samples. Metabolic and genetic complementarity between trametinib and pyrvinium pamoate translated into anti-AML synergy in vitro. Moreover, this combination inhibited the propagation of RA+ AML cells in vivo in mice, indicating a potential for future clinical development of this strategy in AML.