Platelet mass cytometry reveals dysregulation of prothrombotic pathways in essential thrombocythemia.

Thromboembolic events are common in patients with essential thrombocythemia (ET). However, the pathophysiological mechanisms underlying the increased thrombotic risk remain to be determined. Here, we perform the first phenotypical characterization of platelet expression using single-cell mass cytometry in six ET patients and six age- and sex-matched healthy individuals. A large panel of 18 transmembrane regulators of platelet function and activation were analyzed, at baseline and after ex-vivo stimulation with thrombin receptor-activating peptide (TRAP). We detected a significant overexpression of the activation marker CD62P (p-Selectin) (p = .049) and the collagen receptor GPVI (p = .044) in non-stimulated ET platelets. In contrast, ET platelets had a lower expression of the integrin subunits of the fibrinogen receptor GPIIb/IIIa CD41 (p = .036) and CD61 (p = .044) and of the von Willebrand factor receptor CD42b (p = .044). Using the FlowSOM algorithm, we identified 2 subclusters of ET platelets with a prothrombotic expression profile, one of them (cluster 3) significantly overrepresented in ET (22.13% of the total platelets in ET, 2.94% in controls, p = .035). Platelet counts were significantly increased in ET compared to controls (p = .0123). In ET, MPV inversely correlated with platelet count (r=-0.96). These data highlight the prothrombotic phenotype of ET and postulate GPVI as a potential target to prevent thrombosis in these patients.

Essential thrombocythemia (ET) is a rare disease characterized by an increased number of platelets in the blood. As a complication, many of these patients develop a blood clot, which can be life-threatening. So far, the reason behind the higher risk of blood clots is unclear. In this study, we analyzed platelet surface markers that play a critical role in platelet function and platelet activation using a modern technology called mass cytometry. For this purpose, blood samples from 6 patients with ET and 6 healthy control individuals were analyzed. We found significant differences between ET platelets and healthy platelets. ET platelets had higher expression levels of p-Selectin (CD62P), a key marker of platelet activation, and of the collagen receptor GPVI, which is important for clot formation. These results may be driven by a specific platelet subcluster overrepresented in ET. Other surface markers, such as the fibrinogen receptor GPIIb/IIIa CD41, CD61, and the von Willebrand factor receptor CD42b, were lower expressed in ET platelets. When ET platelets were treated with the clotting factor thrombin (thrombin receptor-activating peptide, TRAP), we found a differential response in platelet activation compared to healthy platelets. In conclusion, our results show an increased activation and clotting potential of ET platelets. The platelet surface protein GPVI may be a potential drug target to prevent abnormal blood clotting in ET patients.

Comparison of two autologous hematopoietic stem cell mobilization strategies in patients with multiple myeloma: CE plus G-CSF versus G-CSF only: A single-center retrospective analysis.

BACKGROUND: Despite recent advances in the treatment of multiple myeloma, high-dose chemotherapy followed by autologous hematopoietic stem cell transplantation (ASCT) remains an essential therapeutic keystone. As for the stem cell mobilization procedure, different regimens have been established, usually consisting of a cycle of chemotherapy followed by application of granulocyte-colony stimulating factor (G-CSF), although febrile neutropenia is a common complication. Following national guidelines, our institution decided to primarily use G-CSF only mobilization during the COVID-19 pandemic to minimize the patients' risk of infection and to reduce the burden on the health system. STUDY DESIGN AND METHODS: In this retrospective single-center analysis, the efficacy and safety of G-CSF only mobilization was evaluated and compared to a historic control cohort undergoing chemotherapy-based mobilization by cyclophosphamide and etoposide (CE) plus G-CSF. RESULTS: Although G-CSF only was associated with a higher need for plerixafor administration (p < .0001) and a higher number of apheresis sessions per patient (p = .0002), we were able to collect the target dose of hematopoietic stem cells in the majority of our patients. CE mobilization achieved higher hematopoietic stem cell yields (p = .0015) and shorter apheresis sessions (p < .0001) yet was accompanied by an increased risk of febrile neutropenia (p < .0001). There was no difference in engraftment after ASCT. DISCUSSION: G-CSF only mobilization is a useful option in selected patients with comorbidities and an increased risk of serious infections, especially in the wintertime or in future pandemics.

Clonal hematopoiesis as a pitfall in germline variant interpretation in the context of Mendelian disorders.

Clonal hematopoiesis because of somatic mutations in hematopoietic stem/progenitor cells is an age-related phenomenon and commonly observed when sequencing blood DNA in elderly individuals. Several genes that are implicated in clonal hematopoiesis are also associated with Mendelian disorders when mutated in the germline, potentially leading to variant misinterpretation. We performed a literature search to identify genes associated with age-related clonal hematopoiesis followed by an OMIM query to identify the subset of genes in which germline variants are associated with Mendelian disorders. We retrospectively screened for diagnostic cases in which the presence of age-related clonal hematopoiesis confounded exome sequencing data interpretation. We found 58 genes in which somatic mutations are implicated in clonal hematopoiesis, while germline variants in the same genes are associated with Mendelian (mostly neurodevelopmental) disorders. Using five selected cases of individuals with suspected monogenic disorders, we illustrate how clonal hematopoiesis in either variant databases or exome sequencing datasets poses a pitfall, potentially leading to variant misclassification and erroneous conclusions regarding gene-disease associations.

Comprehensive characterization of central BCL-2 family members in aberrant eosinophils and their impact on therapeutic strategies.

PURPOSE: Hypereosinophilia represents a heterogenous group of severe medical conditions characterized by elevated numbers of eosinophil granulocytes in peripheral blood, bone marrow or tissue. Treatment options for hypereosinophilia remain limited despite recent approaches including IL-5-targeted monoclonal antibodies and tyrosine kinase inhibitors. METHODS: To understand aberrant survival patterns and options for pharmacologic intervention, we characterized BCL-2-regulated apoptosis signaling by testing for BCL-2 family expression levels as well as pharmacologic inhibition using primary patient samples from diverse subtypes of hypereosinophilia (hypereosinophilic syndrome n = 18, chronic eosinophilic leukemia not otherwise specified n = 9, lymphocyte-variant hypereosinophilia n = 2, myeloproliferative neoplasm with eosinophilia n = 2, eosinophilic granulomatosis with polyangiitis n = 11, reactive eosinophilia n = 3). RESULTS: Contrary to published literature, we found no difference in the levels of the lncRNA Morrbid and its target BIM. Yet, we identified a near complete loss of expression of pro-apoptotic PUMA as well as a reduction in anti-apoptotic BCL-2. Accordingly, BCL-2 inhibition using venetoclax failed to achieve cell death induction in eosinophil granulocytes and bone marrow mononuclear cells from patients with hypereosinophilia. In contrast, MCL1 inhibition using S63845 specifically decreased the viability of bone marrow progenitor cells in patients with hypereosinophilia. In patients diagnosed with Chronic Eosinophilic Leukemia (CEL-NOS) or Myeloid and Lymphatic Neoplasia with hypereosinophilia (MLN-Eo) repression of survival was specifically powerful. CONCLUSION: Our study shows that MCL1 inhibition might be a promising therapeutic option for hypereosinophilia patients specifically for CEL-NOS and MLN-Eo.

In vivo inducible reverse genetics in patients' tumors to identify individual therapeutic targets.

High-throughput sequencing describes multiple alterations in individual tumors, but their functional relevance is often unclear. Clinic-close, individualized molecular model systems are required for functional validation and to identify therapeutic targets of high significance for each patient. Here, we establish a Cre-ERT2-loxP (causes recombination, estrogen receptor mutant T2, locus of X-over P1) based inducible RNAi- (ribonucleic acid interference) mediated gene silencing system in patient-derived xenograft (PDX) models of acute leukemias in vivo. Mimicking anti-cancer therapy in patients, gene inhibition is initiated in mice harboring orthotopic tumors. In fluorochrome guided, competitive in vivo trials, silencing of the apoptosis regulator MCL1 (myeloid cell leukemia sequence 1) correlates to pharmacological MCL1 inhibition in patients´ tumors, demonstrating the ability of the method to detect therapeutic vulnerabilities. The technique identifies a major tumor-maintaining potency of the MLL-AF4 (mixed lineage leukemia, ALL1-fused gene from chromosome 4) fusion, restricted to samples carrying the translocation. DUX4 (double homeobox 4) plays an essential role in patients' leukemias carrying the recently described DUX4-IGH (immunoglobulin heavy chain) translocation, while the downstream mediator DDIT4L (DNA-damage-inducible transcript 4 like) is identified as therapeutic vulnerability. By individualizing functional genomics in established tumors in vivo, our technique decisively complements the value chain of precision oncology. Being broadly applicable to tumors of all kinds, it will considerably reinforce personalizing anti-cancer treatment in the future.

Cyclophosphamide plus etoposide is a safe and effective mobilization regimen in patients with multiple myeloma.

High-dose chemotherapy followed by autologous stem cell transplantation is a major component in the treatment of patients with multiple myeloma. As a prerequisite, the successful collection of a sufficient number of viable peripheral blood hematopoietic CD34+ cells is critical. A common standard protocol for mobilization is currently not defined and critically discussed especially in German-speaking Europe. In times of the Covid-19 pandemic, safe and effective strategies have to be chosen to minimize hospitalization times and severe courses. In this single-center retrospective analysis, safety and efficacy of cyclophosphamide plus etoposide (CE) and growth-factor support (n = 33) was compared to cyclophosphamide mono treatment and growth-factor support (n = 49) in 82 patients with multiple myeloma at first diagnosis. CE was superior to cyclophosphamide mono with a significantly higher number of collected CD34+ cells (15.46 × 106 CD34+ cells/kg vs. 9.92 × 106 CD34+ cells/kg), significantly faster engraftment of granulocytes after stem cell transplantation (day 10.5 vs. day 11.6), shorter duration of the inpatient stay (17.47 days vs. 19.16 days) and significantly less transfusions (8.82 % vs. 30.61 % patients receiving transfusions). The safety profile was comparable in both groups and in line with published data. We conclude that CE is a safe and highly effective mobilization protocol in patients with multiple myeloma at first diagnosis and appears to be superior to the commonly used cyclophosphamide mono regimen.

MLKL promotes cellular differentiation in myeloid leukemia by facilitating the release of G-CSF.

The blockade of cellular differentiation represents a hallmark of acute myeloid leukemia (AML), which is largely attributed to the dysfunction of lineage-specific transcription factors controlling cellular differentiation. However, alternative mechanisms of cellular differentiation programs in AML remain largely unexplored. Here we report that mixed lineage kinase domain-like protein (MLKL) contributes to the cellular differentiation of transformed hematopoietic progenitor cells in AML. Using gene-targeted mice, we show that MLKL facilitates the release of granulocyte colony-stimulating factor (G-CSF) by controlling membrane permeabilization in leukemic cells. Mlkl-/- hematopoietic stem and progenitor cells released reduced amounts of G-CSF while retaining their capacity for CSF3 (G-CSF) mRNA expression, G-CSF protein translation, and G-CSF receptor signaling. MLKL associates with early endosomes and controls G-CSF release from intracellular storage by plasma membrane pore formation, whereas cell death remained unaffected by loss of MLKL. Of note, MLKL expression was significantly reduced in AML patients, specifically in those with a poor-risk AML subtype. Our data provide evidence that MLKL controls myeloid differentiation in AML by controlling the release of G-CSF from leukemic progenitor cells.

Open-flow respirometry under field conditions: How does the airflow through the nest influence our results?

Open-flow respirometry is a common method to measure oxygen-uptake as a proxy of energy expenditure of organisms in real-time. Although most often used in the laboratory it has seen increasing application under field conditions. Air is drawn or pushed through a metabolic chamber or the nest with the animal, and the O2 depletion and/or CO2 accumulation in the air is analysed to calculate metabolic rate and energy expenditure. Under field conditions, animals are often measured within the microclimate of their nest and in contrast to laboratory work, the temperature of the air entering the nest cannot be controlled. Thus, the aim of our study was to determine the explanatory power of respirometry in a set-up mimicking field conditions. We measured O2 consumption of 14 laboratory mice (Mus musculus) using three different flow rates [50 L*h-1 (834 mL*min-1), 60 L*h-1 (1000 mL*min-1) and 70 L*h-1 (1167 mL*min-1)] and two different temperatures of the inflowing air; either the same as the temperature inside the metabolic chamber (no temperature differential; 20 °C), or cooler (temperature differential of 10 °C). Our results show that the energy expenditure of the mice did not change significantly in relation to a cooler airflow, nor was it affected by different flow rates, despite a slight, but significant decrease of about 1.5 °C in chamber temperature with the cooler airflow. Our study emphasises the validity of the results obtained by open-flow respirometry when investigating energy budgets and physiological responses of animals to ambient conditions. Nevertheless, subtle changes in chamber temperature in response to changes in the temperature and flow rate of the air pulled or pushed through the system were detectable. Thus, constant airflow during open-flow respirometry and consequent changes in nest/chamber temperature should be measured.

Inhibition of PLK1 by capped-dose volasertib exerts substantial efficacy in MDS and sAML while sparing healthy haematopoiesis.

INTRODUCTION: Targeting the cell cycle machinery represents a rational therapeutic approach in myelodysplastic syndromes (MDS) and secondary acute myeloid leukemia (sAML). Despite substantial response rates, clinical use of the PLK inhibitor volasertib has been hampered by elevated side effects such as neutropenia and infections. OBJECTIVES: The primary objective was to analyse whether a reduced dose of volasertib was able to limit toxic effects on the healthy haematopoiesis while retaining its therapeutic effect. METHODS: Bone marrow mononuclear cells (BMMNCs) of patients with MDS/sAML (n = 73) and healthy controls (n = 28) were treated with volasertib (1 µM to 1 nM) or vehicle control. Short-term viability analysis was performed by flow cytometry after 72 hours. For long-term viability analysis, colony-forming capacity was assessed after 14 days. Protein expression of RIPK3 and MCL-1 was quantified via flow cytometry. RESULTS: Reduced dose levels of volasertib retained high cell death-inducing efficacy in primary human stem and progenitor cells of MDS/sAML patients without affecting healthy haematopoiesis in vitro. Interestingly, volasertib reduced colony-forming capacity and cell survival independent of clinical stage or mutational status. CONCLUSIONS: Volasertib offers a promising therapeutic approach in patients with adverse prognostic profile. RIPK3 and MCL-1 might be potential biomarkers for sensitivity to volasertib treatment.

Venetoclax with azacitidine targets refractory MDS but spares healthy hematopoiesis at tailored dose.

Patients with Myelodysplastic Syndromes (MDS) and secondary Acute Myeloid Leukemia (sAML) have a very poor prognosis after failure of hypomethylating agents (HMA). Stem cell transplantation is the only effective salvage therapy, for which only a limited number of patients are eligible due to age and comorbidity. Combination therapy of venetoclax and azacitidine (5-AZA) seems to be a promising approach in myeloid malignancies, but data from patients with HMA failure are lacking. Furthermore, a considerable concern of combination regimens in elderly AML and MDS patients is the toxicity on the remaining healthy hematopoiesis. Here, we report in vitro data showing the impact of venetoclax and 5-AZA, alone or in combination, in a larger cohort of MDS/sAML patients (n = 21), even after HMA failure (n = 13). We especially focused on the effects on healthy hematopoiesis and the impact on colony forming capacity as a parameter for long-term effects. To the best of our knowledge, we show for the first time that venetoclax in combination with capped dose of 5-AZA targets cell malignancies, while sparing healthy hematopoiesis.