bluuebunny/crossref_metadata_2025 huggingface dataset:
Life History of Normal Human Lymphocytes Revealed By Somatic Mutations
Authors: Machado Heather E , Øbro Nina Friesgaard , Mitchell Emily , Davies Megan , Green Anthony R , Saeb-Parsy Kourosh , Hodson Daniel James , Kent David , Campbell Peter J
Published: November 2019

Introduction: Mature blood cells harbor a mixture of mutations inherited from ancestral hematopoietic stem cells (HSCs) and mutations accumulated after maturation. The landscape of these somatic mutations in normal blood is poorly mapped, with questions as simple as "how many mutations does a memory T cell accumulate throughout life?" remaining unanswered. This gap in our knowledge is particularly relevant for hematopoietic malignancy- while we know that lymphomas derive from lymphocytes of particular stages of differentiation, we do not know if the patterns we see are reflected in their normal counterparts.

Results: In order to characterize normal somatic mutation in lymphocytes, we performed single-cell expansion and whole genome sequencing of over 600 T and B lymphocytes and 200 HSC and progenitor cells across 5 individuals (ages 0-85). All lymphocyte subsets show increased mutation burden with respect to HSCs across all classes of variants (Figure 1). Some of this increase can explained by lymphocyte-specific mutational processes, such as the activity of RAG, accounting for at least 20% of observed structural variants. We also find a striking variation in mutation burden within and between lymphocyte subsets. Microenvironment specific mutational processes dominate the observed differences. Examples of this include germinal center ("non-canonical AID") mutations in memory B cells and UV-like mutations in memory T cells (putatively skin resident cells). Naive B and T cells show a lack of variation in discrete mutational patterns relative to their memory counterparts, and have mutational profiles and mutation burdens more similar to that of HSCs. We also observe differences in the mutational patterns between B and T cells that are indicative of the increased divergence of T lymphocytes from the HSC pool. In general, the mutation burden we observe in normal lymphocytes approach those seen in lymphoma.

Conclusions: Our work highlights the substantial genetic diversity in normal lymphocytes, with some cells accumulating thousands of mutations on top of those inherited from the HSC compartment. These mutations can be used to describe the life history of each individual lymphocyte including their exposure to specific microenvironments. Our findings shed light on the biology of these cells and will help differentiate between normal and disease processes.

Figure 1

Disclosures

No relevant conflicts of interest to declare.
iPLA2β Contributes to Activation of Human and Mouse Platelets Via ITAM Receptors Fcγ RIIA and GPVI
Authors: Abraham Shaji , Patel Pravin , Naik Ulhas P. , McKenzie Steven Edward
Published: November 2019

Platelet activation by ITAM receptors contributes to hemostasis, thrombosis, vascular integrity and host defense. In the course of our studies of FcγRIIA-mediated platelet activation, we became interested in those mechanisms that require neither full Syk activation nor changes in intracellular calcium. Calcium-independent phospholipase family member iPLA2β has been observed to modulate phospholipid remodeling and second messenger generation in human platelets (Beckett, Thromb Res 2007; Duvernay, Biochem 2015), while iPLA2γ has been studied in knockout mouse platelets (Yoda, JTH 2014) with modest effects noted on thrombosis and hemostasis. These enzymes do not require increased cytoplasmic calcium for their activity in cleaving the acyl group from the sn2 position of phospholipids to yield a free fatty acid and a lysophospholipid. However, the precise role of iPLA2β in human and mouse platelet activation has not been elucidated. Neither has the contribution of iPLA2β in the response to FcγRIIA-mediated activation been reported.

We identified the presence of iPLA2β protein in western blots of human and FcγRIIA transgenic mouse platelets. Of interest, multiple isoforms arising from proteolytic cleavage were detected. We treated washed human and FcγRIIA transgenic mouse platelets with agonists to FcγRIIA (IV.3 + GAM) and to GPVI (collagen or collagen-related peptide) in the absence and presence of pharmacologic inhibitors of iPLA2β. At a range of agonist doses up to 3X threshold, we observed significant inhibition of aggregation, dense granule secretion and alpha granule secretion (p<0.05 vs. vehicle only, n = 3 to 4 each). Inhibition occurred with either S-BEL (bromo-enolactone) or with FKGK18 (a fluoroketone), two chemically distinct iPLA2β inhibitor molecules with different modes of action. The IC50 for S-BEL was found to be 1.02 uM for human FcγRIIA, 2.04 uM for human GPVI, and 2.76 uM for transgenic mouse FcγRIIA activated platelets. FKGK18 was less potent, with IC50s at 7.88 uM for human FcγRIIA. In contrast, iPLA2γ inhibitor R-BEL was able to inhibit FcγRIIA -mediated activation, but at an IC50 of 2.62 uM. Notably, iPLA2β inhibition could eliminate ATP secretion from dense granules downstream of FcγRIIA and GPVI activation. When we added ADP to FcγRIIA stimulation in the presence of inhibitory doses of S-BEL, we overcame the inhibition.

We have identified for the first time that iPLA2β contributes to aggregation and secretion of both human and FcγRIIA transgenic mouse platelets. The platelets were slightly more sensitive to FcγRIIA than to GPVI inhibition. In other activatable secretory cells, iPLA2β plays both a homeostatic and signaling role. The mechanisms of iPLA2β action in platelets merit further study. Studies are in progress with genetic knockdown and knockout of the enzyme, to complement the findings with inhibitors.

Disclosures

No relevant conflicts of interest to declare.
Clinical Evidence Against the Continuum of Low-Primed Uncommitted Hematopoietic and Progenitor Cells (CLOUD-HSPC) Concept for Hematopoiesis
Authors: Mohanlal Ramon , Blayney Douglas W. , Huang Lan
Published: November 2019

Introduction: The classical model of blood stem cell differentiation is branch-like linear HSPC progression from progenitor multi/oligo potent stem cells to differentiated mature cell types. An alternative model, referred to as CLOUD-HSPC, proposes bone marrow (BM) HSPCs continuous and direct differentiation into unipotent cells of myeloid lineage (neutrophils (N), basophils (B), eosinophils (E), monocytes (M), mast cells, megakaryocytes (platelets) and erythrocytes without linear stem cell commitment (Velten Nat Cell Biol 2017). Pegfilgrastim (Peg), and the non-colony stimulating agent Plinabulin (Plin) prevent chemotherapy (chemo) induced neutropenia (CIN) in cancer patients (pts), and both exert BM effects leading to CD34+ cell mobilization (Blayney ASH 2017, 2018). In mice, Plin enhances LSK (Lin-Sca+cKit3+) cell differentiation in BM (Ghosh, AACR 2018). Here we clinically validated the CLOUD-HSPC concept by analyzing peripheral blood mononuclear cell (PBMC) counts derived from two bifurcating progenitor cells 1. Granulocyte-Monocyte Progenitor (GMP) producing N,M,E and B and 2. Common Lymphoid Progenitor (CLP) producing lymphocytes (L), after exposure to Peg or Plin in a CIN setting. With linear stem cell commitment, increase in the differentiated mature cell counts would positively inter-correlate, and with CLOUD-HSPC, they would not.

Methods: In the Phase 3 portion of study BPI-2358-105 (NCT03102606), pts with NSCLC, BC or HRPC received pre-medication with dexamethasone (Dex) on day (D) -1,0, and 1 and docetaxel (Doc) on day (D) 1. Pts were randomized 1:1 to either Plin 40 mg (n=52), given 30 min after Doc infusion D1, or Peg 6 mg (n=53), D2. Central laboratory (Covance Laboratory) PBMC counts were obtained at screening, D 1,2, 6,7,8,9,10, 15 in Cycle 1, and correlated with each other at a pre-planned interim analysis. D1 and D2 data points were omitted due to confounding effects of Dex and demargination. Data of Plin and Peg were combined, since both drugs exert BM effects leading to increased N cell counts and showed similar correlation trends.

Results: Maximum increases in % from screening value on D5-D15 of each N,M,E,B and L counts correlated with each other. Pearson correlation coefficients (r) after linear regression and corresponding p-value are summarized below.

Conclusions: There is a linear and positive inter-correlation between differentiated mature cells derived from the GMP and CLP lineage, proving strong clinical evidence against CLOUD-HSPC, but in favor of the classical linear commitment model.

Table

Disclosures

Mohanlal: BeyondSpring Pharmaceuticals: Employment. Blayney:BeyondSpring Pharmaceuticals: Research Funding. Huang:BeyondSpring Pharmaceuticals: Employment.
Fast Onset of Action of Subcutaneously Administered Marzeptacog Alfa (Activated) Supports on-Demand Treatment in Hemophilia a Mice
Authors: Del Greco Frank , Neuman Linda , Levy Howard , Reckless Jill , Knudsen Tom , Blouse Grant E.
Published: November 2019

Background: Patients with Hemophilia A or B with and without inhibitors lack treatment options that are fast and easy to use for treating acute bleeding. Currently, their only options are intravenous administration of replacement or bypass therapies. Marzeptacog alfa (activated) (MarzAA) is a novel increased potency recombinant FVIIa in clinical development. We hypothesized that, in addition to its clinically demonstrated efficacy in SC prophylaxis, MarzAA could potentially be used SC on-demand to control acute hemorrhage after bleeding has started.

Objective: To demonstrate the procoagulant effect of dosing MarzAA subcutaneously at timepoints both before and after injury using a tail clip bleeding model in hemophilia A mice.

Methods: Each mouse was initially weighed and briefly anesthetized with isoflurane for collection of 5 µL blood to assess baseline hemoglobin levels. Later, anaesthetized (100 mg/kg ketamine + 10 mg/kg xylazine) F8-/- (Haem A, B6;129S4-F8tm1Kaz/J) mice were submitted to a tail clip injury model completely transecting the tail at a diameter of 1.25 mm from the tip using a sharp razor blade. Following injury, blood loss was monitored with the tail submerged in warm saline for 20 minutes and quantified by hemoglobin content. Mice were dosed with MarzAA SC (1.5 mg/kg) or vehicle at 15, 30, 60, or 90 minutes before or one minute after injury (1.5 and 4.5 mg/kg). An ED50 for MarzAA was determined using various doses ranging from 0.15 mg/kg to 3.0 mg/kg. Statistical significance was set at α = 0.05.

Results: SC administration of MarzAA resulted in a dose dependent reduction in bleeding when administered to hemophilia A mice 15 minutes before injury. At the highest dose of MarzAA, full efficacy was achieved as bleeding in the treated mice was comparable to the blood loss observed in normal hemostatically competent mice. The ED50 for MarzAA was calculated to be 387 µg/kg after SC administration in this model. More importantly, when dosed as a rescue therapy one minute after injury, SC MarzAA significantly reduced bleeding to 350±46 µL from 635±50 µL (vehicle), p = 0.02.

Conclusion: MarzAA demonstrated efficacy when administered SC both before and after injury. Taken together these data suggest that SC MarzAA could potentially be used on-demand to treat patients with acute bleeding. These results provide a basis for further clinical investigation of the acute treatment of a bleed with SC MarzAA in hemophilia patients.

Disclosures

Del Greco: Catalyst Biosciences: Consultancy, Equity Ownership. Neuman:Catalyst Biosciences: Employment, Equity Ownership. Levy:Catalyst Biosciences: Employment, Equity Ownership. Reckless:RxCelerate: Employment. Knudsen:Catalyst Biosciences: Employment, Equity Ownership. Blouse:Catalyst Biosciences: Employment, Equity Ownership.
Quality of Life in Patients with Multiple Sclerosis after Autologous Hematopoietic Stem Cell Transplantation
Authors: Olivares-Gazca Juan Carlos , Murrieta-Álvarez Iván , Olivares-Gazca Jesús Mauricio , García-Navarrete Yarely Itzayana , Cantero-Fortiz Yahveth , León-Peña Andrés A. , Ruiz-Delgado Guillermo J. , Ruiz-Arguelles Guillermo J.
Published: November 2019

Introduction

Multiple sclerosis (MS) is an inflammatory, demyelinating and neurodegenerative disease of the central nervous system (CNS) that causes a whole spectrum of neurological disorders associated with a profound decrease in the quality of life of affected patients. Currently, autologous hematopoietic cell transplantation (ASCT) is a validated therapeutic approach and has been shown to be superior to new immunomodulatory agents. However, the impact of these therapies on the quality of life of patients with MS is unknown.

Objective

Identify the impact on the quality of life in patients with multiple sclerosis after ASCT at our center.

Methods

A quasi-experimental, longitudinal, prospective and single-center study was conducted in which the quality of life was determined in patients with MS before and after ASCT. Patients who could not answer the questionnaire themselves were excluded and incomplete questionnaires were eliminated. The quality of life was determined by applying the MS-QoL 29 instrument which is validated instrument for this pathology (Cronbach 0.88-0.90 and Pearson with high correlation with MS-QoL56). The variables related to the physical and mental components of the instrument as well as demographic characteristics were studied. The statistical analysis of the data included measures of central tendency as well as inferential for the comparison of means and proportions (NC 95%, p <0.05).

Results

We included 52 patients prospectively from October 2018 to June 2019, 71% of the patients were women and the remaining 29% men. The median of age of the subset is 50 years (Interval 27-65). Of the selected patients, 45% has PPMS, 39% has SPMS and 16% has PPMS. Twenty six patients were followed at 3 months and seventeen were followed 6 months after ASCT. The statistical differences between the quality of life in the patients prior to the ASCT and the follow-up at 3 and 6 months in both the physical and mental components was analyzed. In the physical component the differences at 3 months (A) were significant (p = 0.019, 95% NC) as well as the differences at 6 months (b) after ASCT (p = 0.0024, 95% NC). In the mental component the differences were significant at 3 months (C) (p = 0.0012, NC 95%) as well as the differences at 6 months (D) after ASCT (p = 0.0007, NC 95%).

Conclusions

The study suggests that ASCT is a feasible and safe therapeutic alternative to improve the quality of life in patients with multiple sclerosis.

Figure

Disclosures

No relevant conflicts of interest to declare.
An Institutional Survey of Warfarin Reversal in the Setting of Life-Threatening Bleeds
Authors: Garrahy Ian , Pawar Tushar , Donato Anthony , Stevenson Amber , Forman Daniel
Published: November 2019

Introduction:

The bleeding risk of warfarin is well known. Therapeutic options for warfarin reversal in life-threatening bleeds include fresh frozen plasma (FFP), recombinant factor VIIa (rFVIIa), and prothrombin complex concentrate (PCC). Despite the theoretical advantage and clinical evidence supporting PCC, it is not widely used in the US.

Methods:

An online anonymous questionnaire was sent to all providers in the Tower Health System asking them about their practice, specialty, degree, years in practice, and basic questions regarding their comfort and frequency of prescribing anticoagulants. The questionnaire also asked the providers how they would manage ten various clinical scenarios related to anticoagulation management. One question specifically addressed the management of warfarin reversal in an 85-year-old presenting with hemorrhagic shock. Analysis of variance was used to compare the scoring means between groups while linear regression and Pearson's correlation coefficient measured the relationship between years of practice and test scores.

Results:

Out of 404 responders, 232 (57.4%) selected an incorrect answer and 212 (52.5%) incorrectly selected fresh frozen plasma as the answer to the question (see uploaded image) that addressed warfarin reversal in the setting of major bleeding. Those providers who answered this question correctly and those who answered it incorrectly had mean scores of 68.26% and 52.16% respectively on the overall survey (p value 0.000).

Conclusion:

As compared to FFP, the use of PCC for warfarin reversal is associated with a significant reduction in all-cause mortality. This project demonstrates a professional practice gap and serves to highlight an area in medicine where many providers are not practicing in accordance with evidence-based practice. Furthermore, this particular question from the survey discovered that providers who performed better on the survey were more likely to answer questions regarding anticoagulation reversal correctly.

Figure

Disclosures

No relevant conflicts of interest to declare.
Molecular Profiling of Aggressive Non-Hodgkin Lymphoma - Results from a Phase 1/2 Preben Study
Authors: Leivonen Suvi-Katri , Jørgensen Judit , Larsen Thomas Stauffer , Pasanen Annika , Karjalainen-Lindsberg Marja-Liisa , Toldbod Helle M , d'Amore Francesco , Leppa Sirpa
Published: November 2019

Background: Aggressive non-Hodgkin lymphoma (NHL) relapsing after standard first line chemotherapy represents an unmet clinical need. Currently, a phase 1/2 study with the combination of pixantrone, etoposide, bendamustine, and in CD20 positive tumors, rituximab, in patients with relapsed aggressive NHL of B- or T- cell phenotype (the PREBEN study) is ongoing. Here our aim was to molecularly characterize samples from the PREBEN trial and find clinical correlates for predicting treatment response.

Methods: The profiling cohort consisted of 21 patients with pre-treatment RNA samples and clinical data. Nanostring PanCancer Pathways and PanCancer Immune profiling panels (altogether 1348 genes) were utilized for the gene expression analyses. The findings from gene expression analyses were correlated with clinical parameters.

Results: Fourteen patients had diffuse large B-cell lymphoma (DLBCL), whereas seven had peripheral T-cell lymphoma (PTCL). In general, the expression of DNA replication genes distinguished DLBCL from PTCL. Additionally, gene expression analyses identified genes having differential expression based on the response to the treatment. Supervised hierarchical clustering of the ten most differentially expressed genes could separate the responding (n=4) and non-responding (n=10) DLBCL patients into two distinct subgroups (Fig. 1A). Similarly, the responding (n=3) and non-responding (n=4) PTCL patients could be separated into distinct subgroups by supervised clustering with the ten most differentially expressed genes (Fig. 1B).

Conclusion: Molecular profiles of aggressive NHL are heterogeneous and may be utilized for predicting the treatment response. More detailed molecular analyses are currently ongoing.

Disclosures

Jørgensen: Gilead: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees. d'Amore:Servier: Research Funding. Leppa:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen-Cilag: Research Funding; Bayer: Research Funding; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Honoraria, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding.

OffLabel Disclosure:

combination of bendamustine and pixantrone for relapsed NHL
GRK6 Regulates the Hemostatic Response to Injury through Its Rate-Limiting Effects on GPCR Signaling in Platelets
Authors: Chen Xi , Gupta Shuchi , Cooper Matthew , Dehelian Daniel , Zhao Xuefei , Brass Lawrence F. , Naik Ulhas P. , Naik Meghna Ulhas , Benovic Jeffrey , Ma Peisong
Published: November 2019

Inappropriate platelet activation remains a major cause of cardiovascular and cerebrovascular diseases. Most agonists activate platelets through G protein-coupled receptors (GPCRs). However, questions remain about mechanisms that provide negative feedback towards activated GPCRs to limit platelet activation and thrombus formation.

Here we provide the first evidence that GPCR kinase 6 (GRK6) serves this role in platelets, using GRK6-/- mice generated by CRISPR-Cas9 genome editing to examine the consequences of GRK6 knockout on GPCR-dependent signaling. Hemostatic thrombi formed in GRK6-/- mice are larger than in WT controls during the early stages of thrombus formation, with a rapid increase of platelet accumulation at site of injury. Platelet activation in the absence of GRK6 is enhanced, but in an agonist-selective manner. Responses to PAR4 agonist peptide or ADP stimulation in GRK6-/- platelets are increased compared to WT control littermates, while the response to TxA2 is normal. Underlying these changes in GRK6-/- platelets is an increase in Ca2+ mobilization, Akt activation, and granule secretion. Furthermore, deletion of GRK6 in human MEG-01 cells causes an increase in Ca2+ response and PAR1 surface expression in response to thrombin. Finally, we show that in human platelets, platelet activation in response to thrombin causes an increase in binding of GRK6 to PAR1, as well as an increase of the phosphorylation of PAR1. Deletion of GRK6 in MEG-01 cells causes a decrease in PAR1 phosphorylation.

Collectively, these observations, for the first time, show that 1) GRK6 regulates the hemostatic response to injury by thrombin and ADP, 2) it mediates platelet activation by reducing PAR1/4- and P2Y12-dependent signaling, and 3) GRK6 limits the rate of platelet activation during early stage of thrombus growth and helps prevent inappropriate platelet activation.

Disclosures

No relevant conflicts of interest to declare.
Dual Anticoagulant Facilitied Peripheral Blood Stem Cell Harvest from Childhood Donors with Low Weight and Low MCV
Authors: Yue Li , Bu Chaoke , Ma Zhirui , Li Chunfu , Wang Wenjun , Zhou Jun
Published: November 2019

Background:Low weight children can sometimes be selected as donor in HSCT. Donor with thalassemia minor has low MCV, which affects peripheral blood stem cell (PBSC) harvest. It is worthwhile to explore a harvest program for donors with low body weight and low MCV.

Objective:The aim of this study is to find out an appropriate program of PBSC collection in children with low body weight and low MCV.

Methods:10 children, with median age of 2 (1-6) years, average weight of 13.4 (9.5- 20) kg and average MVC value of 63fl (54-78)) were hospitalized in our center from October 2018 to July 2019 for stem cell mobilization. Donor weight were less than recipients in 7 cases. 5 donors have 5 kg less than recipient, 3 have 10 kg less and 1 has 18 kg less, respectively. They received G-CSF of 5 mg/kg/d twice a day for 5 days before harvest. 500 ml ACD-A solution and 3000 IU heparin were mixed and given by the ratio of 1 ml mixed anticoagulant versus 24 ml whole blood. Blood-flow was 25-40 ml/min.

Results:More 20/ul CD34+ cells were harvested in 9 cases (90%), and 19.2/ul in 1 case (10%). Median nucleated cells were13.2(6.7-22.4)x108/kg in donor weightand 9.8 (8.3-13.9) x108/kg in recipient weight. The median CD34+ cells were 5 (1.9-10.2) x106/kg in donor weight and 3.4 (1.4-9.7) x106/kg in recipient weight. The coagulation function was normal in reexamination. PBSC were harvested only once in all donors. Average circulating volume was 2.48 (1.14-5.79) total body volume, average harvesting time was 2.75 (2-6) hours, and average PBSC volume was 89 (63-160) ml. Donor peripheral blood calcium and potassium ion concentration showed no obvious abnormality.

Conclusion:Combination of citrate and heparin can be well used to collect PBSC in donors with low body weight and low MCV. All children were well tolerance to the program, which was safe and effective for large volume collection of PBSC.

Disclosures

No relevant conflicts of interest to declare.
Outcome Among Adolescents and Young Adults with Acute Myeloid Leukemia at Pediatric Versus Adult Centers: A Population-Based Study Using the IMPACT Cohort
Authors: Gupta Sumit , Baxter Nancy , Pole Jason , Lau Cindy , Sutradhar Rinku , Nagamuthu Chenthila , Nathan Paul
Published: November 2019

Background: Survival outcomes among adolescents and young adults (AYA) with acute myeloid leukemia (AML) remain poor. In AYA with acute lymphoblastic leukemia, outcomes differ between patients treated in pediatric vs. adult centers. This has not been well evaluated in AML. We therefore compared outcomes between AYA with AML treated at pediatric vs. adult centers using a population-based clinical database. In addition, we determined other predictors of outcome within this population.

Methods: The IMPACT Cohort comprises all Ontario, Canada AYA aged 15-21 years diagnosed with one of six common cancers (including AML) between 1992-2012. Detailed demographic, disease, treatment, and outcome data were collected through chart abstraction and validated by content experts. Locus of cancer care (LOC - pediatric vs. adult center) was determined based on where the majority of therapy was delivered in the first three months after diagnosis. Linkage to population-based health administrative data identified additional cancer events (second cancers, relapse, death). Event-free (EFS) and overall survival (OS) were determined using Kaplan-Meier methods. The impact of LOC on EFS and OS was determined using multivariable Cox proportional hazard models, adjusting for demographic, disease, and treatment variables. Events included disease progression, relapse, death, and second malignancies.

Results: Among 140 AYA with AML, 89 (63.6%) received therapy at an adult center. AYA treated in pediatric centers were younger than those treated at adult centers (median 16 years vs. 19 years; p<0.001) and were more likely to live in higher-income neighborhoods [37/51 (72.5%) vs. 47/89 (52.8%); p=0.02]. Disease markers such as presenting white blood cell count and AML subtype did not differ by LOC. The 5-year EFS and OS for the whole cohort were 35.0%±4.0% and 53.6%±4.2%. Neither EFS nor OS differed by LOC (Table 1). In multivariable analyses adjusting for disease characteristics, LOC was not predictive of either EFS [adult vs. pediatric center hazard ratio (HR) 1.3, 95thconfidence interval (CI) 0.8-2.2, p=0.27] or OS (HR 1.0, CI 0.6-1.6, p=0.97). AYA with AML living in rural areas however experienced significantly inferior outcomes as compared to their urban counterparts (EFS: HR 2.5, CI 1.3-4.7, p=0.005; OS: HR 2.0, CI 1.1-3.8, p=0.04).

Conclusions: In this population-based cohort, outcomes did not differ between AYA with AML treated at pediatric vs. adult centers, unlike what has been previously shown in AYA with acute lymphoblastic leukemia. However, rural AYA experienced substantially inferior outcomes than urban AYA, suggesting that even within a universal single payer system of healthcare, socioeconomic disparities persist in this population.

Disclosures

No relevant conflicts of interest to declare.
Effect of Extended Platelet Storage on Platelet Metabolism and Clot Retraction
Authors: Nair Prajeeda M , Meledeo Michael Adam , Wu Xiaowu , Peltier Grantham , Liu Bin , Reddoch-Cardenas Kristin M , Bynum James , Cap Andrew P.
Published: November 2019

Background: Platelet (PLT) hemostatic responses are heavily dependent on PLT bioenergetics which drives the generation of PLT contractile forces and contribute to mechanical hemostasis. Although effective clot retraction (CR) requires PLT adhesion, aggregation and formation of strong clots, CR remains an understudied PLT function. In this work, we hypothesize that well-conserved PLT bioenergetics during storage is crucial for highly energy-dependent PLT functions including CR. To further investigate this correlation, we characterized the effect of extended PLT storage for up to 15 days on PLT bioenergetics and CR at room temperature (22°C, RT) and refrigeration (4°C, 4C).

Methods: Apheresis PLT concentrates in plasma were collected from 5 healthy donors and stored for 21 days at RT with agitation, or stored stationary in the cold (4C). Fresh PLTs were treated with metabolic inhibitors for 1 hour at 37°C, recalcified with 20mM calcium chloride, and incubated at 37°C to initiate CR. The clot weights were measured post retraction, and the clots were prepared for immunohistochemistry to assess clot structure. Similarly, extent of retraction and mitochondrial respiration levels were estimated in PLTs stored for 5, 10 and 15 days. Additionally, Platelet additive solution (PAS) stored PLTs were tested as a viable alternative to plasma storage to improve metabolic exhaustion during storage.

Results: 4C-stored PLTs in plasma retracted similar to fresh PLTs for up to 15 days. In comparison, RT-stored PLTs in plasma did not retract after 10 days, whereas PLTs stored in PAS at RT or 4C seemed to retain their contractile function for up to 15 days (Fig 1A). 4C-stored and fresh PLTs yielded heavier clots with a highly organized structure compared to the disorganized structure of clots from RT-stored PLTs. Mitochondrial respiration was significantly reduced over 10 days in RT- plasma stored PLTs, which was mitigated by PAS storage. Similarly, 4C storage in PAS mitigated the reduction in respiration seen in 15 days 4C- stored PLTs in plasma (Fig 1B).

Conclusion: Retraction properties of 4C-stored PLTs were similar to those of fresh PLTs, together with superior clot structure and function compared to RT- stored PLTs. This suggests that 4C storage could be a suitable alternative for extended storage. In RT-stored PLTs, reduction in mitochondrial respiration did not show any correlation with the reduced ability of PLTs to retract, suggesting that preserved glycolysis in PAS- stored RT-stored PLTs enabled CR. Thus, storing PLTs in PAS at RT or 4C could potentially mitigate mitochondrial dysfunction during extended storage.

Disclosures

No relevant conflicts of interest to declare.
Molecular Background of Double Protein Expressor B-Cell Lymphomas
Authors: Meriranta Leo , Alkodsi Amjad , Pasanen Annika , Haukka Jari , Karjalainen-Lindsberg Marja-Liisa , Leppa Sirpa
Published: November 2019

Introduction: Concomitant deregulation of MYC and BCL2, whether at the genomic or protein level, comprises clinically a significant biological high-risk feature in aggressive B-cell lymphomas. Yet, the molecular background of these lymphomas is not fully understood. We sought to comprehensively characterize BCL2 and MYC aberrations and to identify clinically feasible determinants of the recent genomic subtypes and clinical outcome in diffuse large B-cell lymphoma (DLBCL).

Patients and Methods: We analyzed BCL2, BCL6 and MYC rearrangements and corresponding protein expression in the context of somatic exome-wide driver gene alterations, copy number annotations, transcriptome and clinical data in 181 patients with primary DLBCL.

Results: We found that germinal center B-cell (GCB) and activated B-cell (ABC)-like DLBCLs utilized different mechanisms to deregulate MYC and BCL2 expression. While the structural variations of BCL2 were subtype-specific and specifically increased antiapoptotic BCL2 expression, MYC deregulation was associated with gene expression signatures including biosynthetic pathways and telomerase activity and depletion of JAK-STAT, cytokine and immune cell signatures. Genomic dissection of MYC translocations and protein overexpression revealed associations with other lymphoma drivers, of which we highlight a novel interplay between MYC deregulation and TP53 alterations.

Double protein expression (DPE) arose from different molecular backgrounds in a subtype-dependent manner. In GCB-DLBCL, concurrent alterations of MYC and BCL2 loci gave rise to the majority of DPE DLBCLs. Importantly, we recognized more GCB lymphomas with double hit -like clinical behavior when BCL2 copy number alterations were considered. In the non-GCB DLBCLs in turn, concurrent alterations were rare and clinical behavior of DPE was dependent on the molecular context. Interestingly, BN2/C1 -like non-GCB lymphomas with BCL6 translocations had excellent outcomes regardless of the DPE status. Overall, DPE DLBCL defined a prognostic entity, which was independent of International Prognostic Index (IPI) and cell-of-origin, and together with TP53 alterations had synergistic dismal impact on survival.

Conclusion: Our findings elucidate the pathogenesis and modern biological determinants of high-risk DLBCL and reveal subtype-specific and clinically feasible improvements to risk-stratification and predictors of outcome.

Disclosures

Leppa: Celgene: Consultancy; Takeda: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Roche: Consultancy, Honoraria, Research Funding; Bayer: Research Funding.
Infection Rates in Autologous Hsct with Prbc Transfusion
Authors: Woodard Christopher Ryan , Locantore-Ford Patricia
Published: November 2019

Introduction: Infection remains a dominant source of morbidity/mortality for patients undergoing myeloablative chemotherapy and autologous stem cell transplant (ASCT). We sought to determine the relative risk of infection with the use of packed red blood cell (pRBC) transfusion to treat chemotherapy associated anemia.

Methods: Patients who underwent ASCT at Pennsylvania Hospital between 1995 and 2019 were identified. Data on transfusion status and infection were obtained from patient discharge and transplant summary forms including: Participation in bloodless medicine program, number of units of pRBCs transfused, and documented post-transplant infection. Standard 2 variable relative risk analysis and odds ratio were calculated to assess association between transfusion use and post-transplant infection rate.

Results: 306 patients were identified to have undergone ASCT with 176 (57.7%) participating in the bloodless medicine program, 46 (15.1%) having no need of pRBC, and 83 (27.1%) receiving at least 1 unit of pRBC. Median number of pRBC in transfused patients was 2 units. Rate of infection among those who did not receive pRBC was 33.3% while it was significantly higher in those who did at 49.4%. Patients who received pRBC were at higher risk of developing infection (Relative risk (RR) 1.48, odds ratio (OR) 1.95, 95% confidence interval (CI) 1.17-3.26, p=0.01) than those who did not. Further analysis showed that higher quantity of pRBC units further increased infection risk in patients who got 4 or more units of pRBC (RR=1.97, OR=4.14, CI=1.814-9.450, P=.0007).

Conclusion: Infection after ASCT was more common with the use of pRBC transfusion to treat anemia while number of pRBC units was positively correlated with rates of infection. This area needs further study to better plot timelines of events as well multiple regression analysis to control for confounding variables such as relative levels of neutropenia before onset of infection.

Disclosures

No relevant conflicts of interest to declare.
Trends and Outcomes of Venous Thromboembolism in Hospitalized Patients with Pancreatic Cancer: Results from National Inpatient Sample Database 1998-2016
Authors: Alhasson Hussam , Cai Peng , Gong Zimu , Saad Anas , Al-Husseini Muneer , Yang Pei , Alhasson Bassam , Tirumanisetty Pratyusha , Syed Tausif , Zhao Yu
Published: November 2019

Introduction:

Pancreatic cancer (PC) has a known association with venous thromboembolism (VTE), with incidence of approximately 17%. There is limited published data about trends and outcomes of PC patients with VTE. The purpose of this study was to describe the prevalence and mortality trends in PC with VTE and analyze VTE impact on hospitalized PC patients from 1998 to 2016.

Methods:

We analyzed data from the National Inpatient Sample (NIS) database of the Agency of Healthcare Research and Quality (AHRQ). Adults≥18 years with PC as well as presence of VTE were identified by using ICD-9 or ICD-10 codes. Cost of hospitalization was adjusted for inflation in reference to 2016. Comorbidities were classified using the Elixhauser comorbidity index. Demographic characteristics, trends and in-hospital outcomes between PC with and without VTE were compared. Multiple logistic regression was used to obtain risk-adjusted odds ratio (OR) to compare inpatient mortality, length of stay (LOS), total charges, and disability at discharge between PC patients with and without VTE. The regression model was adjusted for age, sex, primary expected payer, teaching status of the hospital, hospital location, and presence of comorbid conditions.

Results:

96,777 (6.5%) of a total of 1,488,543 hospitalized PC patients had an accompanying diagnosis of VTE. Mean age of the study population was 67 years. African Americans, younger age, and metastatic disease are associated with higher VTE prevalence rate. After adjusting for potential confounders, compared with those without VTE, PC patients with VTE had significantly higher inpatient mortality (12.6% vs 9.7%; OR, 1.41 [confidence interval (CI), 1.34-1.49]; P<0.001), longer LOS (8.04 vs 7.98 days; OR, 1.27 [CI, 1.23-1.32]; P<0.001), higher average cost of hospitalization (US $71,332 vs US $57,117; OR, 1.4 [CI, 1.34-1.46]; P<0.001), and greater likelihood of moderate to severe disability (defined as any beyond routine home discharge; ranging from short-term stay to skilled nursing facility to death upon discharge) (62.2% vs 50.6%, OR, 1.71 [CI, 1.65-1.78]; P<0.001). Although the annual prevalence of VTE among PC increased from 2.1% to 8.8%, in-hospital mortality declined from 23.3% in 1998 to 12.9% in 2016 (P<0.001).

Conclusion:

In the NIS cohort of hospitalized patients with PC and VTE from 1998-2016, annual prevalence increased while mortality overall decreased. When compared to patients without VTE, PC patients with VTE had higher inpatient mortality, longer length of stay, higher hospital cost and higher degree of disability upon discharge. Consideration for anticoagulation and interventions to limit VTE in PC patients may improve in-hospital outcomes.

Figure

Disclosures

No relevant conflicts of interest to declare.
The pan-PIM inhibitor INCB053914 displays potent synergy in combination with ruxolitinib in models of MPN
Authors: Mazzacurati Lucia , Collins Robert J. , Pandey Garima , Lambert-Showers Que T. , Amin Narmin E. , Zhang Ling , Stubbs Matthew C. , Epling-Burnette Pearlie K. , Koblish Holly K. , Reuther Gary W.
Published: November 2019

Key Points

INCB053914 and ruxolitinib synergize to induce apoptosis of JAK2V617F-driven cells and to inhibit neoplastic growth of primary MPN cells. INCB053914 antagonizes ruxolitinib persistence in an in vivo MPN model.
Larotrectinib in TRK fusion–positive pediatric B-cell acute lymphoblastic leukemia
Authors: Schewe Denis M. , Lenk Lennart , Vogiatzi Fotini , Winterberg Dorothee , Rademacher Annika V. , Buchmann Swantje , Henry Dahlia , Bergmann Anke K. , Cario Gunnar , Cox Michael C.
Published: November 2019

Key Points

Larotrectinib can be effective in patients with ETV6-NTRK3–positive B-cell lymphoblastic leukemia, inducing prolonged molecular remission. Single-agent tyrosine kinase inhibitor treatment could be a valuable treatment option in subgroups of kinase fusion–positive ALL patients.
Association of radiation risk in the second and third generations with polymorphisms in the genes CYP1A1, CYP2E1, GSTP1 and changes in the thyroid
Authors: Massabayeva Meruyert , Chaizhunusova Nailya , Aukenov Nurlan , Bulegenov Tolkyn , Apsalikov Bakytbek , Shapihanova Aigerim , Zhunussov Yersin
Published: November 2019

Background

To study the association of radiation risk in the 2nd –3rd generations with polymorphisms in the genes CYP1A1, CYP2E1, GSTP1 and changes in the thyroid.

Methods

5 polymorphic gene variants (rs1048943, rs4646421, rs2070676, rs3813867, rs1695) were studied in 399 people living in the East Kazakhstan region in this research. 248 people of the 2nd - 3rd generation lived in the territory with radiation exposure in Abai, Borodulikha areas, and 151 people the comparison group lived in Kurchum district without radiation exposure comparable in sex and age with control group.

Results

The results show that there is a significant association of rs1048943 in exposed and unexposed groups (p < 0.003), and the absence of association of rs4646421, rs2070676, rs3813867, rs1695 in the studied groups. The mean value of thyroxine in carriers of the AG + GG genotype of rs4646421 is significantly lower than in AA genotype carriers (p = 0.04); no significant changes were found in genotypes’ distribution with thyroid-stimulating hormone and anti-thyroid peroxidase indicators. Significant changes were in levels of anti-thyroid peroxidase between exposed and unexposed groups (p = 0.007). The thyroxine - thyroid-stimulating hormone levels were not significantly different in exposed and unexposed groups (p > 0.3).

Conclusions

This study demonstrated the association of rs1048943 polymorphism with living in the radiation zone in the 2nd and 3rd generations for the first time. Thyroxine levels decrease was identified in the 2nd and 3rd generation residents of the exposed area, as well as a significant increase of anti-thyroid peroxidase occurs in individuals of the 2nd and 3rd generation living in areas with radiation exposure.
Association of diet and headache
Authors:   On behalf of the School of Advanced Studies of the European Headache Federation (EHF-SAS), Razeghi Jahromi Soodeh , Ghorbani Zeinab , Martelletti Paolo , Lampl Christian , Togha Mansoureh
Published: November 2019

The global prevalence of migraine as a primary headache has been estimated as 14.4% in both sexes. Migraine headache has been ranked as the highest contributor to disability in under 50 years old population in the world. Extensive research has been conducted in order to clarify the pathological mechanisms of migraine. Although uncertainties remains, it has been indicated that vascular dysfunction, cortical spreading depression (CSD), activation of the trigeminovascular pathway, pro-inflammatory and oxidative state may play a putative role in migraine pain generation. Knowledge about pathophysiological mechanisms of migraine should be integrated into a multimodal treatment approach to increase quality of life in patients. With respect to this, within the integrative health studies growing interest pertains to dietary interventions. Although the number of studies concerning effects of diet on headache/migraine is not yet very large, the current article will review the available evidence in this area. All publications on headache/migraine and dietary interventions up to May 2019 were included in the present review through a PubMed/MEDLINE and ScienceDirect database search. According to the current findings, Ketogenic diet and modified Atkins diet are thought to play a role in neuroprotection, improving mitochondrial function and energy metabolism, compensating serotoninergic dysfunction, decreasing calcitonin gene-related peptide (CGRP) level and suppressing neuro-inflammation. It can also be speculated that prescription of low glycemic diet may be promising in headache/migraine control through attenuating the inflammatory state. Moreover, obesity and headaches including migraine could be attributed to each other through mechanisms like inflammation, and irregular hypothalamic function. Thereby, applying dietary strategies for weight loss may also ameliorate headache/migraine. Another important dietary intervention that might be effective in headache/migraine improvement is related to balance between the intake of essential fatty acids, omega-6 and omega-3 which also affect inflammatory responses, platelet function and regulation of vascular tone. Regarding elimination diets, it appears that targeted these diets in migraine patients with food sensitivities could be effective in headache/migraine prevention. Taken together, dietary approaches that could be considered as effective strategies in headache/migraine prophylaxis include weight loss diets in obese headache patients, ketogenic and low-calorie diets, reducing omega-6 and increasing omega-3 fatty acid intakes.
Single-molecule real-time transcript sequencing identified flowering regulatory genes in Crocus sativus
Authors: Qian Xiaodong , Sun Youping , Zhou Guifen , Yuan Yumei , Li Jing , Huang Huilian , Xu Limin , Li Liqin
Published: November 2019

Background

Saffron crocus (Crocus sativus) is a valuable spice with medicinal uses in gynaecopathia and nervous system diseases. Identify flowering regulatory genes plays a vital role in increasing flower numbers, thereby resulting in high saffron yield.

Results

Two full length transcriptome gene sets of flowering and non-flowering saffron crocus were established separately using the single-molecule real-time (SMRT) sequencing method. A total of sixteen SMRT cells generated 22.85 GB data and 75,351 full-length saffron crocus unigenes on the PacBio RS II panel and further obtained 79,028 SSRs, 72,603 lncRNAs and 25,400 alternative splicing (AS) events. Using an Illumina RNA-seq platform, an additional fifteen corms with different flower numbers were sequenced. Many differential expression unigenes (DEGs) were screened separately between flowering and matched non-flowering top buds with cold treatment (1677), flowering top buds of 20 g corms and non-flowering top buds of 6 g corms (1086), and flowering and matched non-flowering lateral buds (267). A total of 62 putative flower-related genes that played important roles in vernalization (VRNs), gibberellins (G3OX, G2OX), photoperiod (PHYB, TEM1, PIF4), autonomous (FCA) and age (SPLs) pathways were identified and a schematic representation of the flowering gene regulatory network in saffron crocus was reported for the first time. After validation by real-time qPCR in 30 samples, two novel genes, PB.20221.2 (p = 0.004, r = 0.52) and PB.38952.1 (p = 0.023, r = 0.41), showed significantly higher expression levels in flowering plants. Tissue distribution showed specifically high expression in flower organs and time course expression analysis suggested that the transcripts increasingly accumulated during the flower development period.

Conclusions

Full-length transcriptomes of flowering and non-flowering saffron crocus were obtained using a combined NGS short-read and SMRT long-read sequencing approach. This report is the first to describe the flowering gene regulatory network of saffron crocus and establishes a reference full-length transcriptome for future studies on saffron crocus and other Iridaceae plants.