bluuebunny/crossref_metadata_2025_split huggingface dataset:
Dysfunctional CLEC-2 and GPVI-Mediated Signaling in Syk Y342F Knock-in Mouse Platelets
Authors: Kostyak John C , Mauri Benjamin R , Dangelmaier Carol A , Patel Akruti , Wright Monica , Reddy Haritha G , Eble Johannes A , Kunapuli Satya P. 
Published: October 2019

Abstract
Platelet activation is essential for hemostasis. Central to platelet activation are the signals transmitted through surface receptors like GPVI, the protease activated receptors (PARs), and C-type lectin-like receptor 2 (CLEC-2). GPVI is an ITAM-bearing receptor, while Clec-2 is a HemITAM-bearing receptor. Both ITAM and hemITAM-bearing receptors signal through spleen tyrosine kinase (Syk). Syk activity is dependent on phosphorylation of several residues including Y342, Y346, and Y519/520 (Y348, Y352, and Y525/526 in human). However, the importance of each residue on either phosphorylation of other residues or activation of Syk is unknown. Therefore, we produced a Syk Y342F knock-in mouse line via CRISPR/Cas9 to investigate the importance of Y342 on Syk-mediated signaling using a platelet system. Syk Y342F mice are viable, have no gross abnormalities, and their blood cell counts are normal. We were unable to detect any Syk Y342 phosphorylation following stimulation of Y342F platelets with the GPVI agonist collagen-related peptide (CRP) (Figure 1). Further, Platelet reactivity is reduced in response to the GPVI agonists CRP (Figure 2) and collagen, as well as the CLEC-2 agonist rhodocytin. When using CLEC-2 antibody as an agonist no response is observed in Syk Y342F mouse platelets even though WT littermate control mouse platelets responded well (Figure 3). Signaling initiated by either GPVI or CLEC-2 is also inhibited, as Syk Y519/520 phosphorylation is impaired. Furthermore, PLCg2 and SLP-76 phosphorylation are reduced in Syk Y342F platelets compared to platelets from littermate control mice following stimulation of either GPVI or CLEC-2. Although reactivity of Y342F platelets is clearly reduced compared to WT platelets, there was no difference in occlusion time following ferric chloride injury of the carotid artery. Similarly, there was no difference in mortality rate following pulmonary thromboembolism using Syk Y342F mice compared to WT littermate control mice. Nevertheless, these data demonstrate that phosphorylation of Y342 on Syk following stimulation of either ITAM or hemITAM-bearing receptors is important for Syk activation.

Disclosures
No relevant conflicts of interest to declare.
Impact of T-Cell Dose on Gvhd Risk after Allogeneic HLA-Matched PBSC Transplantation
Authors: Saad Ayman , Wang Tao , Hemmer Michael T. , Spellman Stephen R. , Arora Mukta , Lamb Lawrence S. , Hashmi Shahrukh K. 
Published: October 2019

Abstract
Background: Conflicting data exists on whether T-cell dose of allogeneic stem cell product influences transplant outcome.
Methods: Using CIBMTR database, we identified 2,736 adult patients who underwent first allogeneic HCT for AML/ALL/MDS (between 2008-2014) with PBSC from an HLA-identical sibling donor (MSD) or 8/8-matched unrelated donor (MUD). This cohort excluded ex-vivo and in-vivo T-cell depletion transplants. Correlative analysis was done between CD3 cell dose and risk of GVHD, relapse, NRM, DFS and OS.
Results: T-cell dose cutoff values were identified (using maximum likelihood estimation method) to identify differential risk of aGVHD (grade II-IV) in both MSD and MUD groups. A CD3 cell dose cutoff of 14 x107 cells/kg identified MSD/low CD3 (n=223) and MSD/high CD3 (n=1214) and a dose of 15 x107 cells/kg identified MUD/low CD3 (n=197) and MUD/high CD3 (n=1102). CD3 and CD34 cell doses did not correlate. Median CD3 cell dose were 11 and 29 in the MSD/Low and MSD/High groups, respectively, and 10 and 28 in the MUD/Low and MUD/High groups, respectively. In MSD/high CD3, cumulative incidence (CI) of D100-aGVHD (GII-IV) was higher (33% vs 25%) (P value =0.009) without influence on engraftment, severe aGVHD (GIII-IV), cGVHD, NRM, relapse, DFS, or OS. In MUD/high CD3, CI of D100-aGVHD (GII-IV) was higher (50% vs 40%) (p value=0.009) and so was the CI of 6-month-cGVHD (31% vs23%) (p value = 0.02) with no influence on engraftment, severe aGVHD, 2-year-GVHD, NRM, relapse, DFS, or OS. On multivariate analysis, both MSD (table 1) and MUD (table 2) groups failed to show a correlation between CD3 cell dose and aGVHD (GII-IV) (p value =0.1 and 0.07), cGVHD (p value=0.8 and 0.3) for both groups respectviely. Sub-analysis of CD4, CD8 and CD4/CD8 ratio failed to identify cutoff values predictive of transplant outcome. Using log-rank test, the sample size was, however, suboptimal to identify difference (with 80% power at 0.05% significant level) at these cutoff cell dose.
Conclusion: In this relatively small registry study, the CD3 cell dose in the PBSCT product did not influence the risk of acute or chronic GVHD or other transplant outcome when using matched sibling or 8/8 unrelated donors.

Disclosures
Saad: Actinium: Consultancy. Lamb:Incysus Therapeutics Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding.
A Prospective Phase II Study of Thalidomide, Cyclophosphamide and Prednisone in Newly Diagnosed Idiopathic Multicentric Castleman's Disease
Authors: Zhang Lu , Zhao Ai-lin , Feng Jun , Cao Xinxin , Zhang Yan , Zhu Tienan , Duan Minghui , Zhou Daobin , Li Jian 
Published: October 2019

Abstract
【Introduction】Idiopathic Multicentric Castleman's disease (iMCD) is a rare lymphoproliferative disorder without 'standard' treatment. In current study, we aimed to investigate the efficacy and safety of TCP regimen (thalidomide, cyclophosphamide and prednisone) in newly diagnosed iMCD patients.
【Methods】From Jun, 2015 to Jun, 2018, thirty-six newly diagnosed iMCD patients were enrolled. TCP regimen (thalidomide 100mg qn for 1 year and maintained with 100mg qn for the second year; cyclophosphamide 300mg/m2 weekly on d1, 8, 15, 22 out of a 28-day cycle; prednisone 1mg/kg twice a week on d1-2, d8-9, d15-16, d22-23 out of a 28-day cycle) was administrated until death or 'treatment failure'. Data from twenty-five patients whose were followed for at least 24 weeks were extracted and analyzed.
【Results】Among these 25 patients, the median age was 40 years (range 20-63 years), with a male : female ratio of 2.1:1. Two patients fulfilled the diagnostic criteria of TAFRO syndrome. After TCP treatment, 48.0% (n=12) patients achieved 'durable tumor and symptomatic response' for at least 24 weeks; 40.0% (n=10) patients were evaluated as 'treatment failure'; 12.0% (n=3) patients were considered as stable disease. The median symptom score was reduced from 14 (range 6-41) to 3 (range 0-20); the median Interleukin-6 level decreased from 21.3 pg/ml (range 7.4-865) to 9.15pg/ml (range 2.0-36.2). As for safety issues, one patient died from pulmonary infection and one patient suffered from Grade 3 rash. No other patients suffered from Grade 3 or above adverse events. With a median follow-up of 14 months (range 6-36), 8.0% (n=2) patients died: one patient died from progression of disease (severe anemia) and the other died from pulmonary infection after TCP administration. Median overall survival (OS) and progression-free survival (PFS) were not reached, with the estimated 3-year PFS and OS of 60.0% and 88.0%, respectively (Figure 1).
【Conclusions】TCP regimen, an inexpensive oral therapy, is an effective and safe first-line treatment option for newly diagnosed iMCD patients.
Figure 1. Figure 1.

Disclosures
No relevant conflicts of interest to declare.
Increased Longevity of Continuous Bone Marrow Cultures and Radioresistance of Bone Marrow Stromal Cells from SOD193A ALS (Amyotrophic Lateral Sclerosis) Mice
Authors: Henderson Andrew , Fisher Renee , Epperly Michael , Shields Donna , Rigatti Lora , Donnelly Christopher , Watkins Simon , Greenberger Joel S. 
Published: October 2019

Abstract
Introduction: The SOD1G93A mouse model of ALS, demonstrates hind limb paralysis beginning at 90 - 100 days of age with stage 4 paralysis at 125 days of age and progressive neuromuscular loss.
Materials & Methods: To determine whether deficiency of functional SOD1 influenced parameters of hematopoiesis, long-term bone marrow cultures were established from ALS and control mice. Bone marrow stromal cell lines derived from LTBMCs were tested for clonogenic radiation survival. We tested the effect of bone marrow transplant after total body irradiation on delay of paralysis.
Results: SOD1G93A marrow cultures demonstrated significant increase in production of hematopoietic progenitor cells (p < 0.0001) and overall longevity of production of hematopoietic cells (p = 0.0354), and bone marrow stromal cell lines were significantly radioresistant (D0 = 1.33 ± 0.09, and ñ = 8.57 ± 1.8) compared to control C57BL/6J mice (D0 = 1.59 ± 0.11, p = 0.117; and ñ = 3.4 ± 0.4, p= 0.0466).
Total body irradiation and bone marrow transplantation with GFP+ donor marrow demonstrated a significant increase in paralysis free interval from 129.2 ± 3.0 to 240.7 ± 21.1 days (p = 0.0010), normalization of blood/brain barrier permeability, and increase in M2 marrow origin microglial cells in proximity to degenerating anterior horn cell/motor neurons. Isolated brain and spinal cord irradiation did not prolong the paralysis free interval (129.0 ± 2.7 days, p = 0.7748).
Conclusions: The results showing increased longevity of hematopoiesis in LTBMCs of marrow from mice displaying an absence of SOD1 and the radioresistance of derived bone marrow stromal cell lines represent two unexpected pleiotrophic effects of the SOD1 G93A genotype. Further studies will be required to determine how marrow transplant after TBI prolonged the paralysis free interval in these ALS mice.

Disclosures
No relevant conflicts of interest to declare.
Acquired Secondary Erythrocytosis in Long-Term Survivors of Allogeneic Hematopoietic Stem Cell Transplantation
Authors: Kibirova Albina , Najeeb Sidra , Craig Michael 
Published: October 2019

Abstract
Background: Erythrocytosis is a condition of increased red blood cell mass, as reflected by laboratory findings of elevated hemoglobin (HGB) and hematocrit (HCT). Erythrocytosis is classified into primary (polycythemia) and secondary, depending on its etiology. Secondary polycythemia most commonly develops in the setting of chronic hypoxemia which triggers increased production of erythropoietin by the kidneys. We have observed several patients in our hematology clinic who were long-term survivors after allogeneic hematopoietic stem cell transplant (allo-HSCT), and subsequently developederythrocytosisduring their follow up without obvious secondary causes. On literature review we found two single-institutional studies with estimated incidence of this unexpected complication reported as 0.45 and 1.1% respectively. Majority of described patients in the literature were young males with aplastic anemia (AA).
Methods: To establish the incidence of post-transplant erythrocytosis in long ter m survivors of allo-HSCT, we conducted a retrospective single site chart review study using the WVU database . All patients above age of 18 at the time of transplantation regardless the initial diagnosis that lead to transplantation and type of allo-HSCT were included. Patients with disease relapse and follow up less than 12 months were excluded. We also excluded patients with underlying conditions that are commonly associated with secondary erythrocytosis. Following definition of polycythemia or erythrocytosis was used: a) HCT >48% in women or >49% in men and/or HGB >16.0 g/dL in women or >16.5 g/dL in men (as extrapolated from WVO 2016 PV criteria); b) HGB/HCT elevation consistent on at least two measurements 3 months apart.
Results: We identified 133 eligible patients for final analysis. Among them, 12 (9.0% ) developed erythrocytosis in a median time from initial allo-HSCT 44.5 months (11.8-60.1). Peripheral blood Jak2V617F mutation was tested in 8/12 patients - negative in all. Serum erythropoietin (EPO) level was tested for 9/12 patients and found to be not suppressed in all cases to the level of HGB/HCT, median 11.1 mU/ml (4.9-18.6). All patients were non-smokers and 11/12 patients had different degree of GVHD requiring immunosuppressive therapy. The highest levels of HGB/HCT were observed in two patients with aplastic anemia and one of them required periodic therapeutic phlebotomies. In rest of the patients erythrocytosis was relatively mild but persistent. Details on our findings are presented in a table below.
Conclusions: Acquired secondary erythrocytosis is not uncommon in long-term survivors of allo-HSCT, estimated at 9.0% in our patient cohort. The exact mechanisms of erythrocytosis in this setting are unknown but probably related to dysregulated EPO production and response.

Disclosures
Craig: Celgene: Research Funding; Novartis: Research Funding; Actinium Pharmaceuticals: Research Funding.
Longitudinal Flow Cytometry Identified "Minimal Residual Disease" (MRD) Evolution Patterns for Predicting the Prognosis of Patients with Transplant-Eligible Multiple Myeloma
Authors: Gu Jingli , Liu Junru , Chen Meilan , Huang Beihui , Li Juan 
Published: October 2019

Abstract
Background: Many questions about "minimal residual disease" (MRD) still need to be answered for multiple myeloma (MM). Methods: Flow MRD were monitored in 104 consecutive patients with MM after induction and at the 3rd, 6th, 9th, 12th, 18th, and 24th months post-transplant. Results: Four MRD evolution patterns were revealed: pattern 1 patients had persistent MRD-negative after post-induction with no progression; pattern 2 patients had MRD-positive status post-induction but became MRD negative within 24 months post-transplant; pattern 3 patients had MRD-negative post-induction but became MRD-positive within 24 months post-transplant; and pattern 4 patients had persistent MRD-positive after post-induction. Patients with MRD evolution pattern 1 had a better time to progression (TTP) than did the patients with the other evolution patterns (not reached vs. not reached, vs. 15.4 ± 2.4 months, vs. 16.9 ± 3.0 months; log-rank test, p=0.003, 0.000, 0.000). Patients with MRD pattern 1 had a significantly longer OS than did patients with pattern 3 (not reached vs. 35.2 ± 18.6 months; log-rank test, p=0.000) and pattern 4 (not reached vs. 23.8 ± 15.0 months, log-rank test, p=0.000), but had a similar OS as did pattern 2 patients (not reached vs. not reached; log-rank test, p=0.229). For progressing patients with MRD evolution pattern 2 or 3, the median interval of a sustained MRD-negative status was only 17 months and the median time from MRD reappearance to disease progression was only 4.6 months. Conclusions: (1) A more complete MRD evolution pattern was developed to predict the outcomes for patients with MM. (2) The optimal time of MRD assessment should include post-induction, 3rd month and 24th month post-transplant. (3) Regular MRD assessments will help detect relapse early. (4) A sustained negative MRD status should last for at least 24 months.

Disclosures
No relevant conflicts of interest to declare.
Mutational Phasing: Clinical Relevance in Tyrosine Kinase Domain Mutations Using Next Generation Sequencing in Chronic Myeloid Leukemia
Authors: Bhatwadekar Seema S. , Shah Parth 
Published: October 2019

Abstract
Background: Tyrosine kinase mutation analysis in BCR/ABL1 gene is important for management of patients with chronic myeloid leukemia. Sanger Sequencing has been the mainstay for testing with Next Generation Sequencing (NGS) now becoming the primary technology. In this study we show a comparison between NGS versus Sanger Seqencing based ABL kinase domain mutation analysis with a likely trend of clinical relevance based on a compound versus polyclonal state of mutational distribution which may also need to be considered for patient management and therapy.
Methodology:
A total of 213 Imatinib-resistant patients with CML for BCR-ABL1 mutation analysis were processed on both technologies.Initial blood counts were assessed and RNA was extractedfollowed by cDNA conversion. NGS libraries were prepared with 400bp multiplexed amplicons to allow optimal phasing.
Results:
179 samples were negative by both technologies. A total of only 20 samples were positive and concordant by both technologies(58.2%). Mutations in 14 other samples however were only detected in NGS(41.17%). In these 14 samples (41.17%), NGS was able to detect 23 mutations with mutation frequencies of 3-28%, which were missed by Sanger.
Conclusions:
Moreover 11/34 patients had 2 or >2 mutations. An inhouse script delineated mutations as compound or polyclonal from NGS data. 2/11 cases demonstrated compound mutations (Mutations in the same clone) while 7/11 cases were polyclonal per NGS. Sanger sequencing cannot differentiate between polyclonal and compound mutations. 2/11 cases appeared to have polyclonal and compound mutations. 4/11 patients presented in a blast crisis or accelerated phase CML. Interestingly, most of these patients hadat leasttwo mutations and were polyclonal(3/4).
Significantly previously archived samples patients with polyclonal mutations showed polyclonality at extremely low frequency percentages in initial samples.
None of the single mutation patients had presented in a blast crisis or an accelerated phase.

Disclosures
No relevant conflicts of interest to declare.
Oncogenic KRASG12D in the Hematopoietic System Causes NLRP3 Inflammasome Activation Leading to Myeloproliferative Syndrome
Authors: Hamarsheh Shaima'A , Erlacher Miriam , Osswald Lena , Gorka Oliver , Duyster Justus , Groß Olaf , Lengerke Claudia , Börries Melanie , Brummer Tilman , Zeiser Robert 
Published: October 2019

Abstract
Oncogenic Ras mutations occur frequently in myelodysplastic (MDS) and myeloproliferative syndromes (MPN). It was unclear if besides the direct transforming effect via constant RAS/MEK/ERK signaling, an inflammation related effect of KRAS contributes to the development of the disease.
To address this question we performed a microarray based analysis of bone marrow derived from Rosa26CreERT2; LSL-KrasG12D mice after induction of KrasG12D with tamoxifen. We found that the NLRP3 inflammasome and related genes were upregulated. In agreement with increased NLRP3 sensitivity, KrasG12D bone marrow derived dendritic cells (BMDCs) showed increased inflammasome activation upon stimulation with LPS and ATP as quantified by intracellular levels of IL-1β and caspase-1 p20 subunit.
To validate the functional role of the NLRP3 inflammasome for the MDS phenotype in vivo we generated Rosa26CreERT2; LSL-KrasG12D; NLRP3-/- mice. We transferred BM (CD45.2) from WT mice, Rosa26CreERT2; LSL-KrasG12D or Rosa26CreERT2; LSL-KrasG12D; NLRP3-/- mice into C57BL/6 WT recipients (CD45.1) after myeloablative conditioning and induced KrasG12D by tamoxifen treatment.
Rosa26CreERT2; LSL-KrasG12D BM recipient mice developed MPN stigmata including anemia, leukopenia and thrombocytopenia. The mice also presented weight loss, splenomegaly, and an increase in myeloid cells in peripheral blood, spleen and BM, as well as increased CD11b+ckit+ immature myeloid progenitors. In contrast, Rosa26CreERT2; LSL-KrasG12D; NLRP3-/- recipient mice developed no disease phenotype.
BMDCs isolated from Rosa26CreERT2; LSL-KrasG12D recipient mice exhibited strong NLRP3 inflammasome activation and high IL-1β levels which was not found in BMDCs of Rosa26CreERT2; LSL-KrasG12D; NLRP3-/- mice.
CD11b+ cells of a Juvenile myelomonocytic leukemia (JMML) patient with an activating KRAS mutation showed increased inflammasome activation as compared to a non-mutant patient, which is in agreement with our observations in mouse models.
Our findings support the concept that oncogenic KrasG12D does not only act via its oncogenic driver function, but also enhances activation of the NLRP3/IL-1β axis. This could lead to novel therapeutic approaches combining inhibition of oncogenic signaling and immune modulation via IL-1β blockade.

Disclosures
No relevant conflicts of interest to declare.
Temporal Trends in Early Mortality in Acute Promyelocytic Leukemia in the United States
Authors: Xavier Ana C. , Kutny Matthew A. , Jamy Omer , Costa Luciano J 
Published: October 2019

Abstract

Background: Survival of patients with acute promyelocytic leukemia (APL) has dramatically improved with the use of all-trans retinoic acid (ATRA) and arsenic trioxide (ATO). Despite this, due to the complexity of initial management and the high risk of fatal thrombotic and hemorrhagic complications at presentation, early mortality (EM) remains the major contributor for treatment failure. It is less known whether advances in treatment, improvements in supportive measures, urgent access to specialized care and broad availability of ATRA and ATO have reduced EM in the last two decades.
Methods: We used data from the National Cancer Institute's Surveillance Epidemiology and End Results program (SEER-13) to determine the rates of EM (death within the first 30 days from diagnosis) and overall survival (OS) in patients with APL. Inclusion criteria was the diagnosis of APL as first malignant neoplasm among patients of all ages between 1992 and 2015. Follow up was updated to the end of 2015. Cases were grouped and analyzed according to age, children, adolescent and young adults (< 40 years) and older adults (≥ 40 years), race/ethnicity, gender, county-level income and educational achievement, and residency in rural or urban county. Trends in EM and OS were analyzed across consecutive 4-year eras.
Results: A total of 2,224 APL cases (895 <40 and 1,329 ≥ 40 years) were included in the analysis with median follow up of 41 months (range 0-287 months). Median age of patients was 45, and 1,090 (49%) were male. Most patients were White (1,228; 55.2%), 199 (9%) Black, 785 (35.3%) other ethnicity, and 12 (0.5%) unknown. Three-year OS for APL patients diagnosed in 1992-1995 was 49.2% ± 3.5% vs. 76.4 ± 2.1% for patients diagnosed in 2012-2015 (p < 0.001), Figure 1. Early mortality improved for patients < 40 years (27.4% in 1992-1995 vs. 5.4% in 2012-2015, p < 0.001), but not at the same extent for patients ≥ 40 years (35.2% in 1992-1995 vs. 22.2% in 2012-2015, p = 0.02), Figure 2. Improvements in EM are displayed in Table 1. Importantly, improvements in EM were not seen among patients of residents of rural counties.
Conclusion: These findings confirm consistent improvements in EM and OS for APL patients in the US and point to the challenge of further extending these improvements in EM rates to older patients and for those living in rural areas.

Disclosures
Costa: Celgene: Honoraria, Research Funding; Sanofi: Honoraria; Karyopharm: Research Funding; Janssen: Research Funding; BMS: Research Funding; Abbvie: Research Funding; Amgen: Honoraria, Research Funding.