bluuebunny/crossref_metadata_2025_split huggingface dataset:
Lung-Resident Megakaryocytes Display Distinct Transcriptional and Functional Properties
Authors: Yeung Anthony K , Villacorta-Martin Carlos , Hon Stephanie , Dowrey Todd W , Murphy George J 
Published: November 2019

Megakaryocytes (MKs) are responsible for producing anucleate platelets which are indispensable due to their roles in hemostasis, growth and development. Recent studies have also demonstrated the influence of platelets in regeneration and immune protection, thus highlighting the functionality of platelets beyond their classically defined roles. Standard models of thrombopoiesis emphasize that MK development occurs initially in the fetal liver during development, followed by long-term residency in sites of adult hematopoiesis such as the bone marrow sinusoids. Outside of the hematopoietic system, MKs have been shown to reside in the interstitium of the lung, with emerging evidence pointing to the lung microvasculature as a specialized environment that may support a significant bulk of platelet production. However, little is known about how MKs traffic to the lung and whether they are influenced by the lung microenvironment to become functionally distinct from their hematopoietic counterparts. To characterize the transcriptional signature of lung-resident MKs, we performed single cell RNA sequencing (scRNA-Seq) on primary MKs derived from syngeneic mouse lung and hematopoietic tissues. Samples were processed to obtain single cell suspensions, followed by flow cytometry to enrich for CD41 positive cells and prepared for scRNA-Seq using the 10x genomics platform. scRNA-Seq analysis revealed a developmental trajectory starting from megakaryocyte erythroid progenitors (MEPs) and ending in two distinct clusters of mature MKs which were made up of mostly lung derived MKs (Fig. 1). One of these mature clusters was made up of 91% lung MKs and possessed a unique gene enrichment profile, including marked upregulation of platelet factor 4 (PF4) and enrichment for cellular processes related to ATP production and translation. To further profile lung-resident MKs, we performed flow cytometric assessment of ploidy and maturation markers. In comparison to bone marrow derived MKs, lung derived MKs displayed a distinct ploidy profile with a significantly increased number of cells expressing CD42, a marker of MK maturation. Collectively, these findings suggest that the subset of MKs found within the lung may be in a more mature and metabolically active state to potentially facilitate increased platelet output. These studies may shed new light on the process of MK maturation, including what triggers MK endoreplication, and may advance methodologies for in-vitro platelet production. Based on the symbiotic relationship between the lung and the hematopoietic system, our ongoing studies are focused upon the interplay between the lung and resident MKs in the developmental and functional patterning of both tissue types.

Disclosures
No relevant conflicts of interest to declare.
Clinical Evidence of Granulocyte-Monocyte Progenitor (GMP) Stem Cell Involvement in Plinabulin's Mechanism of Action (MoA) for the Prevention of Docetaxel (Doc) Chemotherapy (Chemo)-Induced Neutropenia (CIN)
Authors: Blayney Douglas W. , Huang Lan , Mohanlal Ramon 
Published: November 2019

Introduction:
Plinabulin (Plin) is being developed as a novel non-Granulocyte colony stimulating factor (G-CSF) for CIN prevention. Previously we reported that Plin was equally effective as Pegfilgrastim for the prevention of grade 4 CIN in a Phase (Ph) 2 trial in NSCLC patients (Pts) receiving 75 mg/m2 Doc (Blayney, ASH 2017, 2018). Plin is single dose per cycle, on the same day of (and 30 min after) Chemo, does not cause bone pain and has anticancer activity. Plin is separately developed as an anti-cancer agent in a global Ph3 trial in NSCLC pts (NCT02504489). Preclinical studies showed that Plin reversed Chemo-induced inhibition of progenitor LSK differentiation in bone marrow (Ghosh, AACR 2018). We have shown that peripheral blood CD34+ hematopoietic stem cell counts increase in chemo plus plin treated patients (Blayney ASH, 2018). Since normal differentiation of GMPs leads to increases in peripheral blood myelo-monocytic (M) and granulocytic cells [neutrophils(N), eosinophils (E) and basophils (B)], we hypothesize that if GMPs are involved in Plin's CIN MoA, increases in N count would positively correlate with increases in M, E and B counts.
Method: The Ph 3 portion of study BPI-2358-105 (NCT03102606) evaluated Plin (40mg) head-to-head with Pegfilgrastim (6mg) for the prevention of Doc-induced CIN in patients (pts) with NSCLC, BC or HRPC. Blood N,M,B and E counts were taken at screening, pre-and post-dose during Cycle 1 and analyzed by a central laboratory (Covance). Pts received dexamethasone (Dex) pre-medication on day (D) 0,1,2, and on D1 Docetaxel (Doc) 75 mg/m2 followed by Plin (40 mg), 30 min after completion of Doc. Here we only include data from a pre-planned interim analysis, and from the Plin arm only (n=52), and we calculated the maximum increase in N,M,E and B on day (D) 5,6,7,8,9,10 15 as % of screening counts, since N,M,E and B counts on D1-D5 are confounded by demargination and Dex effects on these counts.
Results: Pearson correlation coefficients (r) with associated p-values were calculated for maximum increases in N and compared with those for M,E and B, and summarized below.
Conclusions: Correlation of M, E, and B with neutrophil count increase, further confirms our proposed MoA for Plin prevention of CIN: Plin protects the neutrophil precursor from chemotherapy induced damage in bone marrow. We will further explore this MoA in other CIN studies.
Table

Disclosures
Blayney: BeyondSpring Pharmaceuticals: Research Funding. Huang:BeyondSpring Pharmaceuticals: Employment. Mohanlal:BeyondSpring Pharmaceuticals: Employment.
Determination of Appropriate Deferral Time for Repeat Apharesis Platelets Concentrate Donation in Abuja, Nigeria
Authors: Ukoma Chi-Kadibia Theophilus , Ogbe Oche P. , Wakama Tamunomieibi T. , Njoku Ogbonnaya S. 
Published: November 2019

Background: Plateletpheresis is the process by which a therapeutic adult dose of platelet concentrate is produced from a single donor using automated cell separator equipment. Recently, the demand for Apheresis Platelets concentrate transfusions has been increasing in our hospitals. This has been attributed to increased cases of malignancy associated bone marrow suppression and various other causes of thrombocytopaenia and thrombopathies. The Increasing use of myeloablative chemotherapy in treatment of haematological and other malignancies also results in transient severe fall in platelet count. The demand generally outstrips the supply necessitating the need to search for additional donor sources to increase the supply of platelets for transfusion. Is it therefore possible to reduce the time interval between plateletpheresis while maintaining donor safety?
Aims and Objectives: The aim of the study was to determine the platelets recovery time after platelets concentrate donation with a view to determining the appropriate deferral time for repeat plateletpheresis
Methods: The study was conducted at the National Hospital Abuja. Fifty seven healthy plateletpheresis donors were recruited over a period of seven months and their haematological parameters were analyzed on days 0, 2 and 7 post donation. We excluded donors whose pre-donation platelet count were less than 200 x 109/L
Results and Analysis: Analysis was done in only forty eight (48) donors as nine (9) participants withdrew before the end of the study. The results show There was a significant decrease in the platelets counts (p <0.001), with subsequent good platelets recovery post donation (Pre: 259.83±44.61 x 109/L; Post - Day 0: 205.29±36.77 x 109/L, day 2: 216.88±32.71 x 109/L and day 7: 240.3±41.83 x 109/L). The apheresis donors recovered 64.29% of the lost platelets by day 2 of donation and subsequently attain 92.49% of pre-donation count within 7 days of donation. There was a statistically significant transient increase in the immediate post donation haemoglobin concentration (14.72±1.32)g/dL but this gradually reduced on day 2 (13.75±1.04)g/dL and 13.83±1.04g/dL on day 7 showing no significant different from the pre-donation Hb of 13.96±1.02g/dL
Conclusion: This study has shown statistically significant platelets recovery 7 days after plateletpheresis making a 7 day deferral for repeat plateletpheresis feasible in our environment. The study also shows feasibility of a 2 days deferral as recommended by AABB. Strict monitoring and pre-donation evaluation of donors should be done to prevent any adverse events such as anaemia and thrombocytopenia.

Disclosures
No relevant conflicts of interest to declare.
Study of Diagnosis, Classification and Prognostic Factors Determination of Acute Leukemia By Flow Cytometry Immunophenotyping
Authors: Silva Alessandra Suelen Jardim , Freire Juliana Mendonça , Júnior Lenilton Silva DA Silva , Oliveira Gustavo Henrique de Medeiros , Silva Antonia Eduarda Martins Oliveira Elói , Lima João Pedro Andrade , Soares Victor lima , Freitas Rodrigo Villar , Bahia Frank , Oliveira Taissa Moura , Silva Dany GKC , Sales Valéria SF , Araujo Rafael Santiago , Paiva Hugo Diogenes De Oliveira , Paiva Aldair Sousa , Cavalcanti Geraldo Barroso 
Published: November 2019

Introduction: Acute leukemias (AL) are clonal diseases classified into two large groups: acute lymphoid leukemia (ALL), more common in children, and acute myeloid leukemia (AML), more rare in childhood, besides the rare phenotype leukemias such as acute biphenotypic leukemia (ABL) and undifferentiated acute leukemia (UAL). Although the cytomorphology still be relevant in theses leukemias diagnoses, the immunophenotyping by flow cytometry (FC) have become essential in the diagnosis, classification and follow-up of these neoplasms, standing out as a modern and practical methodology, presenting characteristically as a method of multiparametric and quantitative analysis of the leukemic cells. Objective: The objective of this study was realize a retrospective study of immunophenotyping in 371 patients with AL. Methodology: Immunophenotyping was performed biological samples by FC after labeling with a panel of monoclonal antibodies specific for AL directed against lymphoid antigens (B, T and NK cells), myeloid, and markers related to other cell immaturity. At the same time, information was obtained regarding patients such as age, sex, clinical data related to the disease, and previous hematological analysis. Results: From 371 cases, 127 were ALL (71 B lineage and 55 T=ALL), 239 AML, 04 ABL and 2 UAL. In the ALL, it was observed a higher frequency in children, contrasting with the cases of AML, ABL and UAL more prevalent in adults. In ALL, clinical signs and laboratorial data related to disease were more present in the ALL-T and mature-B corroborating with information in the literature. In AML, ABL and UAL the most clinical parameters observed were splenomegaly, hepatomegaly and bleeding. The classification FAB subtypes of AML more predominant were M1, M2 and M4 and lower incidence of the M7 subtype. Conclusion: These data demonstrate the importance of FC technology in the diagnosis, classification and establishment of prognostic factors of these neoplasms.

Disclosures
No relevant conflicts of interest to declare.
Risk Factors and Trends for Incidence of Acute Coronary Syndrome after Hematopoietic Stem Cell Transplant - a 15-Year Experience with National Inpatient Sample
Authors: Sidhu Jasdeep Singh , Virk Jeevanjot Kaur , Handa Shivani , Mandal Amrendra , Rajeeve Sridevi 
Published: November 2019

Background:Significant improvement has been noted in the outcome of patients with advanced hematologic malignancies with the advances in hematopoietic cell transplantation (HCT) techniques. However, it has been observed that patients receiving HCT have increased risk of cardiovascular disease (CVD) over time with increased risk of cardiovascular mortality.
Materials and Methods:This was a retrospective observational analysis. We queried the National Inpatient Sample database from 1998 to 2012 for patients aged ≥18 years who had received HCTin the past and were admitted for non-ST-elevation acute coronary syndrome (NSTE-ACS) or ST-elevation myocardial infarction (STEMI). We performed univariate logistic regression followed by multivariate logistic regression analysis to study various demographic factors and comorbiditiesand temporal trends of ACS in these patients.
Results:A total of 150,072 patients with prior history of HCT were identified, out of which 952 hospitalizations were for ACS.47.16% of these patients underwent Percutaneous CoronaryIntervention.Mean age for ACS patients was 56.98 years and 71.75% patients were male. The demographic factors found to significantly affect the incidence of ACS were increasing Age (OR 1.02, p=0.01) and Insurance(Medicare as reference)[Medicaid(OR 0.3, p=0.04), private(OR 1.66, p=0.01). Charlson Comorbidity Index (CCI) had significant correlation with incidence of ACS (CCI=1 as reference) [CCI=2(OR 0.12, p=0.00), CCI³3(OR 0.60, p=0.01)]. The Medical comorbidities found to significantly affect the outcome were Congestive Heart Failure (OR 1.53, p=0.04), COPD(OR 0.54, p=0.02), smoking(OR 2.96, p=0.00), underlying CAD (OR 39.65, p=0.00) and Pulmonary Hypertension (OR 4.01, p=0.00). A trend analysis for Incidence of ACS in patients with History of HCT showed overall decline in ACS incidence which was found to be statistically significant. (Trend p-Value 0.003).
Conclusion:Our study identifiedvarious factors affecting incidence of ACS in HCT patients. We also discoveredan overall downward trend in incidence of ACSin HCT patients.Further studies need to be conducted to confirm these findings.

Disclosures
Rajeeve: ASH-HONORS Grant: Research Funding.
Residual TP53 Mutation Contributes to Chemoresistance through Clonal Expansion in Acute Myeloid Leukemia
Authors: Yan Bowen , Chen Qinwei , Xu Jianfeng , Li Wei , Xu Bing , Huang Suming , Qiu Yi 
Published: November 2019

TP53 mutation (TP53mut) associates with poor survival in AML patients. However, the risk of residual TP53mut has not fully characterized. We found there are around 3% of patients have detectable residual (less than 25%) TP53mut reads from TCGA AML patient exon sequencing data under current sequencing depth. Importantly, these patients also showed worse outcome as compared to the TP53 wild type patients. We therefore studied the effect of residual p53 mutation using two AML cell lines, OCI-AML2 and MV4-11. Both cell lines have been characterized as p53 wild type cells. However, a distinct p53 single hotspot mutation transcripts were detected in low frequency with RNA-seq analysis in both cells. TP53mut cells isolated from the parental cells show higher drug tolerance and have higher population of stem cell maker positive cells, a characteristic of chemoresistant cells. When mixed with p53 wild type cells, the TP53mut cells also show survival advantage under chemo-drug pressure in vitro and vivo (Fig. A), suggesting its advantage to become chemoresistant cells. Interestingly, the chemo-drug resistant cell lines derived from both lines have homogeneous TP53mut at same spot as parental residual mutation cells. Further, we found two p53 target genes p21 and GADD45A contribute to cell survival and chemoresistance. Decrease of these genes leads to cell less sensitive upon chemo-drug treatment. We previously showed that histone deacetylase inhibitor Romidepsin can resensitize chemoresistant cells. Here we further show that Romidepsin can reactivate p53 targeted genes which are failed to be activated by mutant p53 (Fig. B), including p21 and GADD45A, through increasing gene promotor histone acetylation. Moreover, it can preferentially target p53 mutant cell subpopulation (Fig. C). Therefore, either single treatment or combination with chemotherapy drug, Romidepsin can potentially cure or prevent residual p53 mutation caused chemoresistance and relapse in patient. Taking together, our study raise importance on previous under looked residual p53 mutation in AML patient and shed lights on therapeutic strategies for treatments on chemoresistance.
Figure

Disclosures
No relevant conflicts of interest to declare.
BRAFV600E-Positive Precursors As Molecular Markers of Bone Marrow Involvement in Pediatric Langerhans Cell Histiocytosis
Authors: Kudo Ko , Kanezaki Rika , Kobayashi Akie , Sato Tomohiko , Kamio Takuya , Sasaki Shinya , Masaru Imamura , Imai Chihaya , Irie Masahiro , Sasahara Yoji , Ando Kumiko , Kakuda Harumi , Doi Takehiko , Kawaguchi Hiroshi , Kudo Kazuko , Kanegane Hirokazu , Terui Kiminori , Toki Tsutomu , Ito Etsuro 
Published: November 2019

Introduction: The BRAF mutation V600E, the most common somatic mutation in Langerhans cell histiocytosis (LCH), has been reported in approximately 50% of LCH patients and is associated with certain high-risk clinical features. Precursors harboring this mutation can differentiate into Langerhans cells resulting in infiltrates in multiple organs under inflammatory conditions. However, BRAF status in the bone marrow of pediatric LCH patients is unclear. The present study examined somatic mutations in paired tumor and bone marrow samples, using a highly sensitive assay involving next-generation targeted sequencing and droplet digital polymerase chain reaction (PCR) for pediatric LCH patients.
Methods: Between 1996 and 2019, in total of 17 Japanese pediatric patients with LCH were enrolled. The male/female ratio was 7/11. Ages of onset of LCH were median 13 months (range 5-193 months). At diagnosis of LCH, 2 patients were positive for risk organ involvement, 15 were negative. We retrospectively performed mutational analyses of 17 LCH cases using formalin-fixed paraffin-embedded LCH tumor specimens to provide templates for PCR-based targeted amplicon sequencing with customized primers to detect mutations in exons 12 and 15 in BRAF, and exons 2 and 3 in MAP2K1. Thereafter, we identified somatic mutations in the 17 paired bone marrow samples via droplet digital allele-specific PCR, targeting BRAF V600E and BRAF exon 12 in-frame deletion 496-500 (Ex12 in-del).
Results: We detected BRAF V600E in 11 of 17 tumor samples (65%) and the BRAF Ex 12 in-del in 3 of 17 tumors (18%). We identified BRAF V600E in bone marrow samples in 10 of the 11 cases (90%) with the mutation in the tumor at low variant allele frequency (median 0.25%, range 0.14-7.0%). BRAF Ex 12 in-del was not detected in the bone marrow. Cases with detectable bone marrow involvement included eight patients with multi-system disease affecting multiple organs, one patient with multi-focal bone disease, and one patient with single-system disease. Clinical phenotypes including relapse did not correlate with BRAF V600E upon detection in the bone marrow.
Conclusion: We established the sensitive assay based on PCR-based targeted NGS for detecting somatic mutations in LCH even accessible for formalin-fixed, paraffin-embedded clinical specimens. Bone marrow involvement is frequently detectable at the molecular level in pediatric LCH with the BRAF V600E mutation. A prospective study is warranted to evaluate the clinical impact of mutational burden in bone marrow.

Disclosures
Kudo: Unum Therapeutics: Patents & Royalties. Imai:Juno Therapeutics: Patents & Royalties.
HOXA11 Regulates Chemoresistance By Modulating p53 Gene Expression in Acute Myeloid Leukemia
Authors: Guo Xutao , Yan Bowen , Qiu Yi 
Published: November 2019

Acute myeloid leukemia (AML) exhibits large intrinsic variation in drug responsiveness due to its inherent heterogeneity. Therefore, it is important to understand the resistant mechanism in order to improve the treatment. In our previously study, the OCI-AML2-resistant cell lines were established to resist cytarabine (Ara-C) in the concentration of 50 µM (OCI-AML2 R50). The RNA-seq results showed that many genes changed in the resistant cells compared to wild type OCI-AML2 cells. One of the most remarkably decreased gene in resistant cells was HOXA11 (Homeobox A11). It is the part of the A cluster on chromosome 7 and encodes a DNA-binding transcription factor which regulates gene expression, morphogenesis, and differentiation. In this study, we have evaluated the importance of HOXA11 in AML chemoresistance. We found that knockdown of HOXA11 repressed the WT OCI-AML2 cell proliferation and increased the population of cells expressing CD123 and CD47 LSC (Leukemia stem cell) markers and enhanced the resistance to Ara-C in vitro, while overexpression of HOXA11 showed the reverse effect. These results support the idea that HOXA11 promotes drug sensitivity and apoptosis in AML. However, the result also showed that overexpression of HOXA11 repressed the OCI-AML2 R50 cell proliferation and enhanced the resistance. Therefore, HOXA11 plays opposite role in sensitive cells and resistant cells. We further investigated the mechanism for these effects. We found that knockdown of HOXA11 decreased the p53 gene expression and overexpression of HOXA11 increased the expression of p53 in OCI-AML2 and R50 cells. Further, in OCI-AML2 R50 cells p53 has a hotspot mutation in DNA binding site and studies have shown that p53 mutation enhance cancer cell survival and chemoresistance. Therefore, our study shows dual roles for HOXA11 in cell survival. In p53 wild type parental AML2 cells, HOXA11 induces wild type p53 expression to enhance drug sensitivity while in resistant cell, HOXA11 promotes mutant p53 expression and enhances the resistance of chemotherapy.

Disclosures
No relevant conflicts of interest to declare.