ALBERT

Description: Background: Intensive care unit (ICU) patients undergo frequent blood sampling for laboratory testing resulting in blood loss that contributes to anemia and red blood cell (RBC) transfusion. Only 10% of the blood collected is used for testing suggesting that lower sample volumes may reduce the incidence/severity of anemia and use of RBC transfusion without compromising care. Small-volume (soft-draw) Vacutainer® tubes for blood collection (2-3 mL) have the same cost, physical dimensions, and blood draw technique as standard-volume tubes (4-10 mL) and are compatible with laboratory analyzers. We previously conducted a mixed-methods pilot study (n=369) in which small-volume tubes were implemented in the ICU resulting in a 45% reduction in blood loss with acceptability to end-users and no increased frequency of inadequate samples. We are currently conducting the STRATUS trial, a multi-center stepped wedge cluster randomized trial to determine whether a policy of using small-volume blood collection tubes reduces RBC transfusion compared to standard-volume tubes in adult ICU patients. Using this design an intervention can be incorporated into everyday practice at a site of care (such as an ICU) where individual randomization is not feasible. It is ideal for studying the effectiveness and implementation of interventions that have a high likelihood of benefit and low risk of harm. Study Design and Methods: STRATUS is a pragmatic stepped wedge cluster randomized trial being conducted at 25 ICUs (clusters) in Canada over 18 months. At 6-week intervals (steps), 2 ICUs are randomly selected to switch from standard-volume tubes (control) to small-volume tubes (intervention) for blood sampling. All patients admitted to the ICU during the study period are enrolled with waived consent and undergo blood sampling using the allocated tubes. To be eligible for STRATUS, ICUs met the following criteria: (i) adult care only (≥18 years), (ii) unit size ≥14 beds, (iii) capacity for mechanical ventilation; (iv) use of standard-volume Vacutainer® blood collection tubes at baseline; and (v) availability of electronic administrative and laboratory information. The primary outcome is the number of RBC units transfused per patient during their ICU stay in patients admitted to ICU for ≥48 hours. Secondary outcomes include (i) number of RBC units transfused per patient for the entire cohort, (ii) percentage change in estimated blood loss; (iii) change in hemoglobin concentration from ICU admission to discharge adjusted for RBC transfusion; (iv) number of samples with inadequate volume for testing; (v) ICU and hospital mortality; and (vi) ICU and hospital length of stay. The study is approved by local and provincial research ethics boards. Data are collected from electronic administrative and health record sources. Assuming a power of 90% with a type I error of 5% (2-sided) and intra-cluster correlation coefficient (ICC) of 0.01, 25 sites with 729 patients per site (total 18,240 ICU patients) are required to detect a minimum difference of 5 units per 100 patients (or 2.5% reduction). As the interventions are applied in hospital, we expect minimal loss to follow-up. From trial initiation January 2, 2019 to August 1, 2019, 6 sites have been switched to small-volume blood collection tubes on schedule. Up to May 2, 2019 enrolment was 4,186 patients. Significance: STRATUS will determine whether the use of small-volume blood collection tubes reduces RBC transfusion in ICU patients using an innovative cluster trial design that incorporates the intervention into routine ICU practice. With electronic administrative and health record data collection and a clinically relevant primary outcome, this pragmatic trial has the potential to change blood collection practices widely. Disclosures Siegal: Portola: Honoraria; BMS-Pfizer: Honoraria; Novartis: Honoraria; Bayer: Honoraria; Leo Pharma: Honoraria; Aspen Pharma: Honoraria. Arnold:Novartis: Honoraria, Research Funding; Rigel: Consultancy, Research Funding; Bristol-Myers Squibb: Research Funding; Principia: Consultancy. Connolly:Bayer Healthcare: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Portola Pharmaceuticals: Consultancy, Research Funding; Daiichi Sankyo: Consultancy, Research Funding. Crowther:Octapharma: Membership on an entity's Board of Directors or advisory committees; Shionogi: Membership on an entity's Board of Directors or advisory committees; Alexion: Speakers Bureau; Asahi Kasei: Membership on an entity's Board of Directors or advisory committees; CSL Behring: Other: preparing educational material and/or providing educational presentations; Pfizer: Other: preparing educational material and/or providing educational presentations; Servier Canada: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; BMS Canada: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Other: Data and Safety Monitoring Board, Research Funding, Speakers Bureau; Diagnostica Stago: Other: preparing educational material and/or providing educational presentations, Research Funding; Alnylam: Equity Ownership.

Description: A large unmet medical need exists for safer antithrombotic drugs because all currently approved anticoagulant agents interfere with hemostasis, leading to an increased risk of bleeding. Genetic and pharmacologic evidence in humans and animals suggests that reducing factor XI (FXI) levels has the potential to effectively prevent and treat thrombosis with a minimal risk of bleeding. We generated a fully human antibody (MAA868) that binds the catalytic domain of both FXI (zymogen) and activated FXI. Our structural studies show that MAA868 traps FXI and activated FXI in an inactive, zymogen-like conformation, explaining its equally high binding affinity for both forms of the enzyme. This binding mode allows the enzyme to be neutralized before entering the coagulation process, revealing a particularly attractive anticoagulant profile of the antibody. MAA868 exhibited favorable anticoagulant activity in mice with a dose-dependent protection from carotid occlusion in a ferric chloride–induced thrombosis model. MAA868 also caused robust and sustained anticoagulant activity in cynomolgus monkeys as assessed by activated partial thromboplastin time without any evidence of bleeding. Based on these preclinical findings, we conducted a first-in-human study in healthy subjects and showed that single subcutaneous doses of MAA868 were safe and well tolerated. MAA868 resulted in dose- and time-dependent robust and sustained prolongation of activated partial thromboplastin time and FXI suppression for up to 4 weeks or longer, supporting further clinical investigation as a potential once-monthly subcutaneous anticoagulant therapy.

Description: Introduction: Achievement of MRD negativity in MM is associated with prolonged progression-free survival (PFS) and is being investigated as a potential surrogate for established clinical endpoints, such as PFS and overall survival (OS). Here, we evaluated the predictive utility of MRD in patients (pts) with MM for PFS and OS using a systematic literature review (SLR) and meta-analysis, and investigated how hazard ratios (HR) for PFS and OS, stratified by MRD status, changed for various pt subgroups. Methods: A SLR was conducted to identify all studies in MM reporting survival outcomes by MRD status (through 8 June 2019). In these studies, MRD was assessed by various assays (multiparametric flow cytometry [MFC], next generation sequencing [NGS], and polymerase chain reaction [PCR]), sensitivity thresholds (10-4, 10-5, and 10-6), and disease settings (relapsed/refractory MM [RRMM], transplant-eligible [TE] and transplant-ineligible [TIE] newly diagnosed MM [NDMM]). Studies with allogeneic transplant, where MRD was measured in peripheral blood or using PET-CT, or from which PFS and OS data could not be extracted were excluded from the analysis. To obtain a pooled effect estimate of MRD status on PFS and OS HRs, a meta-analysis was performed. Subgroup analyses were performed to adjust for variables expected to impact the association of MRD and PFS/OS outcomes. Variables were selected based on available qualitative evidence from studies. Statistical analyses were performed using the 'metafor' R package for meta-analyses. Results: 143 publications met the inclusion criteria; 86 publications were included in the meta-analysis based on data availability (65 PFS and 28 OS HRs). Outcomes for PFS (N = 8590) and OS (N = 3392) were significantly improved for MRD-negative pts: PFS HR 0.35 (95% confidence interval [CI], 0.31-0.39) and OS HR 0.48 (95% CI, 0.41-0.55; P

Description: Pulmonary arterial hypertension (PAH) is a progressive disease characterized by lung endothelial dysfunction and vascular remodeling. Recently, bone marrow progenitor cells have been localized to PAH lungs, raising the question of their role in disease progression. Independently, serotonin (5-HT) and its receptors have been identified as contributors to the PAH pathogenesis. We hypothesized that 1 of these receptors, 5-HT2B, is involved in bone marrow stem cell mobilization that participates in the development of PAH and pulmonary vascular remodeling. A first study revealed expression of 5-HT2B receptors by circulating c-kit+ precursor cells, whereas mice lacking 5-HT2B receptors showed alterations in platelets and monocyte-macrophage numbers, and in myeloid lineages of bone marrow. Strikingly, mice with restricted expression of 5-HT2B receptors in bone marrow cells developed hypoxia or monocrotaline-induced increase in pulmonary pressure and vascular remodeling, whereas restricted elimination of 5-HT2B receptors on bone marrow cells confers a complete resistance. Moreover, ex vivo culture of human CD34+ or mice c-kit+ progenitor cells in the presence of a 5-HT2B receptor antagonist resulted in altered myeloid differentiation potential. Thus, we demonstrate that activation of 5-HT2B receptors on bone marrow lineage progenitors is critical for the development of PAH.

Description: Angiogenic sprouting requires that cell-cell contacts be maintained during migration of endothelial cells. Angiopoietin-1 (Ang-1) and vascular endothelial growth factor act oppositely on endothelial cell junctions. We found that Ang-1 promotes collective and directional migration and, in contrast to VEGF, induces the formation of a complex formed of atypical protein kinase C (PKC)-ζ and β-catenin at cell-cell junctions and at the leading edge of migrating endothelial cells. This complex brings Par3, Par6, and adherens junction proteins at the front of migrating cells to locally activate Rac1 in response to Ang-1. The colocalization of PKCζ and β-catenin at leading edge along with PKCζ-dependent stabilization of cell-cell contacts promotes directed and collective endothelial cell migration. Consistent with these results, down-regulation of PKCζ in endothelial cells alters Ang-1–induced sprouting in vitro and knockdown in developing zebrafish results in intersegmental vessel defects caused by a perturbed directionality of tip cells and by loss of cell contacts between tip and stalk cells. These results reveal that PKCζ and β-catenin function in a complex at adherens junctions and at the leading edge of migrating endothelial cells to modulate collective and directional migration during angiogenesis.

Description: Background : Polycythemia vera (PV) is a myeloproliferative neoplasm driven by JAK/STAT activation and is associated with erythrocytosis and a broad symptom burden that may negatively impact patient quality of life (QoL). Hydroxyurea (HU) is often used as first-line therapy for high-risk patients but may not effectively control or reduce symptom burden. RESPONSE is a phase III trial comparing ruxolitinib (RUX) with best available therapy (BAT) in patients with PV who were intolerant of or resistant to HU according to modified European LeukemiaNet (ELN) criteria. The primary study endpoint (a composite of hematocrit control and ≥35% spleen volume reduction at Week 32) was achieved by 21% of patients in the RUX arm vs 1% in the BAT arm (P

Description: Introduction: Published trial data shows that ruxolitinib improves both splenomegaly-related and nonsplenomegaly-related constitutional symptoms in patients with intermediate-2 and high risk myelofibrosis (MF). However, few trial-based assessments of ruxolitinib in lower-risk MF patients have been conducted and no studies to date have made such assessments in a real-world population. In this study, we assessed changes in spleen size and constitutional symptoms during ruxolitinib treatment for lower-risk MF patients in real-world clinical settings. Methods: This was a retrospective, observational review of anonymized medical record data collected in January 2014 by 49 hematologists and oncologists in the United States. Patient inclusion criteria were: (1) diagnosed with lower-risk MF (International Prognostic Scoring System score of 0 or 1); (2) first treated with ruxolitinib ≥3 months before the medical record abstraction date; (3) ≥18 years of age at ruxolitinib initiation; (4) complete medical history from MF diagnosis until the medical record abstraction date; and (5) never enrolled in an MF-related interventional trial. Minimum quotas of n=50 and n=25 were set for intermediate-1 and low-risk patients, respectively, with a predetermined maximum of 110 patients in the combined total. Spleen size and constitutional symptoms were retrospectively observed at MF diagnosis, at ruxolitinib initiation, and at best response while on ruxolitinib. Spleen size was captured via predefined categories of no splenomegaly (spleen not palpable), very mild or mild splenomegaly (20 cm palpated). Symptoms of interest included those captured in the MPN Symptom Assessment Form (MPN-SAF), which were categorized as mild, moderate, or severe based on medical notes recorded at each time point. For this abstract, we present findings on the 7 most commonly observed MPN-SAF symptoms in our sample. This study was exploratory and used only descriptive analyses. Results: A total of 108 patients were included in the study (25 low-risk and 83 intermediate-1 patients). Ruxolitinib start dates spanned January 2012 – November 2013. All low-risk patients were ≤65 years of age, and nearly 80% of intermediate-1 patients were ≤65 years of age. The majority of patients in both risk groups (60% and 69%, respectively) were male. A higher proportion of intermediate-1 patients were positive for JAK2 V617F mutation (72%) than low-risk patients (56%). Most patients (92% of low-risk, 77% of intermediate-1) were still receiving ruxolitinib treatment at the medical record abstraction date. Median observed ruxolitinib exposure time was approximately 8 months in both risk groups. In low-risk patients, the combined proportion of patients with moderate or severe splenomegaly (≥10 cm palpated spleen) decreased from 64% at MF diagnosis to 16% at best response during ruxolitinib treatment (Figure 1a). Similar findings were observed for intermediate-1 patients: the proportion with moderate or severe splenomegaly decreased from 53% at MF diagnosis to 10% at best response (Figure 1b). General fatigue was the most commonly observed constitutional symptom in both low-risk and intermediate-1 patients (Figures 2a and 2b). Shifts in symptom severity from more severe to less severe were observed in both low-risk and intermediate-1 patients. Among low-risk patients with fatigue, the proportion with moderate or severe fatigue decreased from 90% at MF diagnosis to 37% at best ruxolitinib response; in intermediate-1 patients, the decrease was from 76% at MF diagnosis to 42% at best response. For most other symptoms, similar improvements in severity distribution were observed. Conclusions: Both low-risk and intermediate-1 MF patients experienced a substantial decrease in spleen size from MF diagnosis through ruxolitinib treatment in real-world clinical settings. Furthermore, for most symptoms examined, there was a distinct improvement in the distribution of symptom severity at the time of best response during ruxolitinib treatment. These findings suggest that lower-risk MF patients may benefit clinically from ruxolitinib treatment. Further studies are needed to assess adverse effects and evaluate the benefit-risk tradeoff of ruxolitinib. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures Davis: Novartis Pharmaceuticals Corporation: Research Funding. Kaye:Novartis Pharmaceuticals Corporation: Research Funding. Cote:Novartis: Employment. Off Label Use: The study discusses use of ruxolitinib in patients with lower-risk (IPSS 0 or 1) myelofibrosis (MF) in real-world practice; currently ruxolitinib is only indicated in higher-risk MF (IPSS 〉1).. Gao:Novartis: Employment. Ronco:Novartis: Employment. Seifeldin:Novartis: Employment. Mendelson:Novartis: Employment.

Description: Background Adherence to iron chelation therapy (ICT) is critical in patients with transfusion dependent anemias (TDA) (e.g., sickle cell disease [SCD], thalassemia), myelodysplastic syndromes (MDS), aplastic anemia, and myelofibrosis (MF). One of the biggest challenges is poor adherence to therapy, which in turn could cause tissue damage in vital organs such as the heart, liver, and endocrine organs, leading to end-organ dysfunction and eventually organ failure. Some causes of poor adherence to ICT therapy in the literature include inconvenience and palatability issues. The literature also suggests that satisfaction with medication impacts adherence to medication regimens, and that satisfaction is an umbrella term for specific components such as convenience, and efficacy. There is limited information on the factors that influence adherence with ICT, however, from the patient and caregiver perspective. Aim The objective of this study was to explore the factors that influence adherence with ICT from the perspective of patients and caregivers of patients through qualitative research. Methods Semi-structured interviews including an open-ended component were conducted to elicit subjects’ (patients with TDA, MDS, aplastic anemia, or MF or caregivers thereof) experiences with ICT. Institutional Review Board approval was granted and subjects were screened by clinicians to meet pre-defined inclusion and exclusion criteria. Audio recordings of the interviews were transcribed and a code book that contained definitions of all codes was developed through a consensus process. Transcripts were coded and data were analyzed using Atlas ti, a qualitative data analysis software package. Several concepts related to adherence and satisfaction were identified. Results Four interviewers conducted 11 patient and 10 caregiver interviews in 6 US cities. The children of the caregivers were not interviewed as they were below the age of nine. Eight of the eleven patients were male and three were female, aged 14 to 81 years (mean=43). Nine of the ten caregivers were female, aged 35 to 65 years (mean=48). Six of the ten children of caregivers were female and four were male, with a mean age of 8.9 years (range 1.56-16.81 years). Conditions across pediatric and adult patients included: MDS (n=3), SCD (n=13), thalassemia (n=3), MF (n=1), and aplastic anemia (n=1). Reported reasons for adherence included perceived effect on health and longevity, support from clinicians and caregivers, and an established routine for taking their treatment. Reported reasons for non-adherence included palatability related issues of taste and aftertaste, texture of the ICT formulation, solubility, side effects such as GI symptoms, and mealtime restrictions. Caregivers reported better adherence if their children had a better understanding of treatment benefit, which usually “gets better as the child gets older”; physician involvement in explaining the importance of ICT; and patients witnessing the decrease in their iron levels. Caregivers indicated that if they were satisfied with the efficacy of the ICT, their children were more likely to adhere to their medications. Coping mechanisms included modifying how the medicine was taken to change or mask the taste, aftertaste, or texture and “dose splitting” during the day. Conclusions Factors associated with adherence and non-adherence to ICT and satisfaction with current therapies identified by patients and caregivers of patients provide information and insight for clinicians to provide optimal treatment with ICT and gain better adherence. These factors can be used to develop measures of adherence, palatability, GI symptoms, and satisfaction with ICT and design interventions to improve adherence to ICT. Disclosures Bal: Novartis Pharmaceutical Corporation: Employment, Equity Ownership. Cote:Novartis Pharmaceutical Corporation: Employment, Equity Ownership. Lasch:Pharmerit International: Employment, Research Funding. Huang:Novartis Pharmaceutical Corporation: Employment, Equity Ownership.

Description: Background. Hematopoiesis is a highly regulated system where multiple, yet undiscovered, factors orchestrate the self-renewal of bone marrow stem cells and their differentiation into blood cells. Following acute stresses like infections, inflammation, chemotherapy or radiation, the hematopoietic system quickly adapts by a process termed "emergency" or "stress" hematopoiesis. For instance, switches from steady state to emergency granulopoiesis or emergency erythropoiesis have been described in response to infection or bleeding. We recently identified, both in human and murine erythroid progenitors, a functional cell-autonomous serotonergic network with pro-survival and proliferative functions. Furthermore, pharmacologic restoration of serotonin levels using selective serotonin reuptake inhibitors (SSRIs) such as fluoxetine, a common antidepressant, was demonstrated to rescue the anemic phenotype in mice models. Here, we hypothesized that serotonin also has a role on other hematopoietic lineages, and that the serotonergic system could be a valuable therapeutic target in radiation or chemotherapy-induced cytopenia. Moreover, as our previous work suggested a cross-talk between serotonin and erythropoietin, we postulate that serotonin could act in cooperation with known hematopoietic growth factors. Material and method. For mice models, we submitted C57BL/6 wild-type 8-10 weeks old mice to sub-lethal irradiation and monitored hematopoietic recovery through complete blood counts. We compared mice treated with fluoxetine administrated orally (~ 20 mg/kg/day in drinking water) to a control group, with or without hematopoietic growth factors. For the retrospective human cohort, we used a computerized database to identify patients who underwent autologous hematopoietic stem cell transplantation (ASCT) in the adult hematology department of Necker hospital between 2008 and 2018. We compared 22 patients treated with an SSRI to 66 controls, matched according to number of injected CD34+ cells/kg, age, sex, conditioning chemotherapy, pathology, depth of response before transplantation, previous lines of chemotherapy, and year of transplantation. The study was conducted according to the Declaration of Helsinki. Results. First, we confirmed that, following sub-lethal irradiation, pharmacologic restoration of 5-HT levels in mice using fluoxetine improved normalization of the erythroid lineage (at day 17 post-irradiation, mean hemoglobin was 8.1 versus 2.8 g/dL in the fluoxetine versus control group (p=0.0002)). Second, the data revealed that the use of SSRI lead to a more rapid restoration of the leukocytes and platelets levels (at day 17 post-irradiation, mean leukocyte level was 1740 versus 314/mm3 (p

Description: Acute myeloid leukemia (AML) is a heterogeneous myeloid malignancy characterized by mutational and clonal heterogeneity. Mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) are common, occurring in approximately 15-20% of patients, and actionable, with recently approved inhibitors for both mutations. These inhibitors lead to leukemia cell differentiation in vitro, in vivo and in patients. Healthy myeloid differentiation is governed by precise regulation of intracellular signaling, but this regulation is disrupted in AML. Given that signal transducer and activator of transcription (STAT) proteins are involved in both leukemogenesis and myeloid differentiation, we sought to determine the role of phosphorylated STATs and other signaling proteins in mIDH AML and inhibitor-induced differentiation of mIDH2 AML. To simultaneously dissect single cell signaling, differentiation and epigenetic changes, we employed fluorescence flow cytometry and mass cytometry to study an in vitro model of mIDH2R140Q leukemia, consisting of a parental TF-1 leukemia cell line and a CRISPR/Cas9 gene-edited mIDH2R140Q-mutated cell line. In order to generate provoked signaling profiles, we stimulated both the wild-type IDH2 and mutant IDH2 cells with nine cytokines. Additionally, we treated both cell lines and primary mIDH2 AML samples ex vivo with enasidenib and measured changes in high-dimensional single cell phenotype and phospho-protein expression via mass cytometry. mIDH2 leukemia cells displayed elevated basal pSTAT1, pSTAT3 and pNFkB-s529 expression, and concomitant low basal IkBa levels when compared to parental cells. Further, mIDH2 cells had increased PMA induced pS6 and IL1β induced pNFkB-s529 than the parental cell line, while wtIDH2 cells had higher levels of cytokine-induced pSTAT1, pSTAT3 and pSTAT5. After prolonged enasidenib treatment (28 days), model mIDH2 cells expressed lower basal pSTAT1, pSTAT3 and pNFkB-s529 than vehicle-treated mIDH2 cells. Further, when stimulated with GM-CSF, enasidenib-treated mIDH2 leukemia cells showed increased response at pSTAT3 and pSTAT5 as compared to vehicle-treated leukemia cells. We dissected enasidenib-induced differentiation using 18 cell surface markers and visualized results using t-distributed stochastic neighbor embedding (tSNE). Presence of mIDH2 leads to baseline expression differences including higher CD90 and CD71. Following treatment with enasidenib, mIDH2 leukemia cells had increased CD45 and CD11b expression as compared to vehicle-treated controls. Additionally, prolonged treatment with enasidenib increased proliferation as shown by increased Ki67 and decreased histone hypermethylation at suppressive histone marks, H3K27 and H3K9, while 7 days of enasidenib did not result in changes to these marks. We further explored the impact of mIDH2 in primary leukemia samples. First, we developed an ex vivo stromal co-culture system that allowed for treatment and expansion of four primary mIDH2 AML samples for 16 days. While the basal levels of signaling markers were sample dependent, consistently across all samples enasidenib-treated primary AML was more sensitive to IL-6 and GM-CSF-induced pSTAT1, pSTAT3 and pSTAT5 signaling. The enasidenib-treated AML also showed increased expression of mature myeloid markers, including CD33 and CD11c. Here we have shown the presence of mIDH2 mutations leads to decreased STAT signaling in response to cytokine stimulation as compared to wtIDH2 AML cells. We have also demonstrated increased response of pNFkB-s529 and pS6 to cytokine stimulation in mIDH2 AML cells as compared to wtIDH2 AML cells. Moreover, in both mIDH2 AML cell line and primary samples, IDH2 inhibitor-induced differentiation restored sensitivity to cytokine responses and reduced histone hypermethylation. Future work exploring these aberrant signaling events may reveal precise connections between mutated IDH, associated epigenetic changes, and intracellular signaling, potentially uncovering synergistic therapeutic strategies for mIDH AML. Disclosures Ferrell: Incyte: Research Funding.