bluuebunny/crossref_metadata_2025_split huggingface dataset:
Regulation of Von Willebrand Factor A1 Domain by Mechanical Force and Neighboring Domains
Authors: Thomas Wendy E , Penkala Rebecca A , Hillenmeyer Elaine , Whitfield Matthew , Tu An-yue , Munday Adam D , Lenting Peter J , Kulman John , López José A. 
Published: October 2019

Abstract
Abstract 2117
Regulation of the bond between platelet glycoprotein (GP) Ibα of the GPIb-IX-V complex, and the von Willebrand Factor (VWF) A1 domain is critical to the balance between hemostasis and thrombosis, particularly in high shear conditions. The GPIbα-A1 interaction is known to be activated by shear stress and inhibited by neighboring domains in VWF, but the role of neighboring domains in the shear-dependence remained unknown. Here it is shown that platelet aggregation required shear stress in the presence of VWF proteins that contain the neighboring D′D3 domain (Plus D′D3 or plasma VWF) but that platelets aggregate spontaneously with a protein that lacks this region (Delta D′D3). Moreover, platelets and microspheres coated with the N-terminal 300 amino acids of GPIbα (GC300) bind to immobilized VWF in a shear-enhanced manner for Plus D′D3 but not for Delta D′D3. In single-molecule force spectroscopy experiments, the D′D3 domain decreased the number of GPIbα-A1 bonds that formed, but did not alter bond rupture force, consistent with the hypothesis that D′D3 shields the A1 domain. By expressing recombinant VWF fragments that contain the A1 domain and various lengths of the N-terminal region, we determined that most of the inhibition by the D′D3 domain was conferred by 23 amino acids in the linker between the A1 domain and the D′D3 domain. By anchoring the fragments to the surface in an oriented manner, we demonstrated that binding was much stronger when force was applied between GPIbα and the A1 C-terminus, than when force was applied between GPIbα and the A1 N-terminus, similar to what has been observed for integrins. Based on these results, we propose the following model for regulation of VWF by mechanical force. When multimeric VWF is stretched in flow, the D′D3 domains are pulled away from the A1 domains, exposing the latter to bind platelets. When force is applied between GPIbα and the C-terminus of A1, it induces an activating conformational change that could be analogous to that seen in integrins.

Disclosures:
No relevant conflicts of interest to declare.
Dynamics of Minimal Residual Leukemia After Combinations of the HyperCVAD Regimen with Imatinib or Dasatinib In Patients with Philadelphia-Chromosome Positive Acute Lymphoblastic Leukemia
Authors: Ravandi Farhad , Thomas Deborah A , O'Brien Susan , Faderl Stefan , Jorgensen Jeffrey , Luthra Rajyalakshmi , Jones Dan , Garris Rebecca , Cortes Jorge E. , Kantarjian Hagop 
Published: October 2019

Abstract
Abstract 2127

Background:
The addition of tyrosine kinase inhibitors imatinib and dasatinib to combination chemotherapy regimens has improved the outcome of patients (pts) with Philadelphia-chromosme-positive (Ph+) acute lymphoblastic leukemia (ALL). We examined whether the dynamics of minimal residual disease (MRD) measured by multiparameter flow cytometry (MFC) or reverse transcription quantitative polymerase chain reaction (RQ-PCR) was different among pts treated with the combination of hyperCVAD with imatinib or dasatinib.

Materials and Methods:
From April 2001 to September 2006, 54 pts with newly diagnosed Ph+ ALL were treated with the combination of hyperCVAD and imatinib; from October 2006 to July 2009, 42 pts were treated with the hyperCVAD and dasatinib regimen. The median ages for the two groups were 50 and 51 years [ranges, (27 - 84) and (21 - 78)]. Fifty one (94%) and 40 (95%) achieved complete remission (CR) on the two regimen and were followed by serial bone marrow assessments for MRD. MFC was performed using 4 or 6 color combinations of antibodies to lymphoblast and myeloid antigens (e.g., CD10, CD13, CD15, CD19, CD20, CD22, CD25, CD33, CD34, CD38, CD58, CD66c, and CD81), with a sensitivity of 0.01%. RQ-PCR for BCR-ABL was performed using TaqMan primer/probes for the e1a2, e13a2 (b2a2), and e14a2 (b3a2) BCR-ABL transcripts in a single tube with normalization to total ABL transcripts.

Results:
The dynamics of the MRD by RQ-PCR and MFC over the initial 18 month period after achieving CR are shown in the attached figures 1 and 2, respectively. The decline in the median value of BCR-ABL/ABL was more rapid with dasatinib, with more patients reaching the level of <0.01 at 3 and 6 months (p <0.05 and p=0.05, respectively). Similarly, a higher proportion of patients receiving dasatinib were negative by MFC at 3 and 6 months after therapy; 82% and 92% were negative at 3 months, and 94% and 100% negative at 6 months for imatinib and dasatinib, respectively.

Conclusions:
Dasatinib containing regimen produces a deeper and more rapid decline in the BCR-ABL and MRD by MFC in the first 6 months of treatment. Whether this would translate to an improved long-term disease-free and overall survival remains to be investigated.

Disclosures:
Ravandi: Bristol Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Speakers Bureau. Off Label Use: frontline use of dasatinib in acute lymphoblastic leukemia. Thomas: Novartis: Honoraria; Bristol-Meyer-Squibb: Honoraria; Pfizer:; Amgen: Research Funding. Cortes: Bristol Myers Squibb: Research Funding; Novartis: Research Funding. Kantarjian: Novartis Pharmaceuticals: Research Funding; Bristol Myers Squibb: Research Funding.
The Prophylactic Use of G-CSF During Induction and Consolidation of Adults with Acute Lymphoblastic Leukemia Is Associated with Reduced Risk of Relapse and Improved Leukemia-Free Survival. A Joint Analysis of Five Randomized Trials on Behalf of the European Working Group for Adult ALL (EWALL)
Authors: Giebel Sebastian , Thomas Xavier , Hallbook Helene , Geissler Klaus , Boiron Jean-Michel , Huguet Françoise , Smedmyr Bengt , Koller Elisabeth , Hellmann Andrzej , Holowiecki Jerzy 
Published: October 2019

Abstract
Abstract 2130
The use of granulocyte colony stimulating factor (G-CSF), either filgrastim or lenograstim, along with induction and consolidation of adults with acute lymphoblastic leukemia (ALL) was a subject of several trials documenting significant shortening of neutropenia, reduced rate of infections and better adherence to chemotherapy protocol. The above studies, however, were not powered to detect differences with regard to long-term outcome.
We performed a joint analysis of follow-up data from five multicenter prospective, randomized trials performed in France, Poland, Sweden, Austria and Australia. Among 347 adults with ALL (median age 33 years), 185 were assigned to receive prophylactically G-CSF while 162 patients were treated without G-CSF support. G-CSF was administered either in parallel to chemotherapy or in a sequential mode.
With the median follow-up of 3.2 years, the remission duration was significantly longer for patients receiving G-CSF compared to controls (33 vs. 17 months, p=0.007). The difference was particularly pronounced for T-ALL (median not reached vs. 13 m., p=0.01) and a trend was observed for B-ALL (22 vs. 17 m., p=0.11). The 5-years probability of leukemia-free survival (LFS) was 38% for patients assigned to the G-CSF arm and 23% for the remaining subjects (p=0.01). There was also a tendency to increased overall survival (OS) rate in favor of patients receiving G-CSF (45% vs. 39%, p=0.17), which reached statistical significance in a subgroup of T-ALL (54% vs. 33%, p=0.03). In a multivariate analysis adjusted to age, initial WBC and the presence of Ph chromosome the prophylactic use of G-CSF was associated with reduced risk of relapse (HR=0.65, p=0.01) and treatment failure (HR=0.7, p=0.02). The differences remained significant when the observations were censored at the time of alloHSCT.
We conclude that the prophylactic use of G-CSF during induction and consolidation of adults with ALL is associated with reduced risk of relapse and improved LFS. The possible explanation could be better adherence to chemotherapy schedules, as demonstrated in previous studies. Patients with T-ALL appear to particularly benefit from the use of G-CSF.

Disclosures:
No relevant conflicts of interest to declare.
VWF Binding to Types I, III or VI Collagen In the ZPMCB-VWD with Identification of Subjects with Selective Reduced Plasma VWF:CB
Authors: Flood Veronica H , Haberichter Sandra L , Friedman Kenneth D , Lentz Steven R , Gill Joan Cox , Montgomery Robert R 
Published: October 2019

Abstract 235
Von Willebrand Disease (VWD) is a common bleeding disorder caused by quantitative (types 1 and 3) and qualitative (type 2) abnormalities in von Willebrand factor (VWF). Defective VWF binding to collagen (VWF:CB) has been identified in VWD patients, but the type(s) and amount of collagen vary between recommended assays. We measured VWF:CB using separate assays for types I, III, and VI collagen in plasmas from 233 healthy controls and 315 VWD index cases (261 type 1, 37 type 2 and 17 type 3) recruited into the Zimmerman Program for the Molecular and Clinical Biology of VWD (ZPMCB-VWD). Additional studies included VWF antigen (Ag), VWF ristocetin-cofactor (RCo), VWF multimer, and VWF gene sequence analysis. VWF:CB was tested using ELISA assays with type I human placental collagen (5 ug/ml), type III human placental collagen (1 ug/ml), and by a commercial human placental type VI VWF:CB assay (Technoclone, Austria). Normal ranges were established for VWF:CB and VWF:CB/Ag ratios with the ZPMCB-VWD healthy control population. Three subjects (2 healthy controls and 1 type 1 VWD subject) were identified with reduced VWF:CB and reduced VWF:CB/Ag ratios to type VI collagen, despite normal results with type I and type III collagen. The two control subjects had VWF:CB/Ag ratios of 0.43 and 0.51 for type VI collagen. The patient with type 1 VWD had absent type VI VWF:CB and a VWF:Ag of 54 IU/dL with a history of clinical epistaxis and an EU Bleeding Score of 8 (normal less than or equal to 3). VWF sequencing demonstrated that these three individuals had an A1 loop polymorphism, R1399H, which has been previously reported as a VWF polymorphism. No other individuals in our study had this sequence change. Of particular note was that the VWF:CB/Ag ratios for types I and III collagen were each normal – suggesting a selective abnormality. As anticipated, collagen binding with all collagens was undetectable in the type 3 VWD subjects. Type 2A and 2B VWD subjects demonstrated reduced VWF:CB and VWF:CB/Ag ratios to all three types of collagen. Type 2M and 2N VWD subjects exhibited normal VWF:CB/Ag ratios to all three types of collagen, but one type 2M patient with an I1425F A1 loop mutation had a reduced VWF:CB/Ag ratio of 0.58. In the VWF A1 domain crystal structure, amino acid 1425 is in close proximity to amino acid 1399, suggesting the conformation of that region may be critical to type VI collagen binding to VWF. Since the frequency of the R1399H polymorphism is estimated to be 2% of the population (Sadler and Ginsburg, 1993), defective binding to type VI collagen may be an important contributor to the variability in the bleeding phenotype of VWD.

Disclosures:
Lentz: Novo Nordisk: Consultancy. Montgomery:GTI Diagnostics, Inc: Consultancy.
Autologous Stem Cell Transplantion for Adolescents and Young Adults with Relapsed and Refractory Hodgkin Lymphoma
Authors: Ali Muhammad , Schechter Tal , Panzarella Tony , Villa Diego , Kuruvilla John , Kukreti Vishal , Tsang Richard , Doyle John J , Crump Michael 
Published: October 2019

Abstract
Abstract 2402
Adolescents and young adults with Hodgkin lymphoma (HL) represent a unique patient population, facing cancer and the toxicities of therapy during an important, transformative period of their lives. The long term outcomes of AYA undergoing intensive chemotherapy and autologous stem cell transplantation (ASCT), both medical and psychosocial, have not been well studied. We assessed the outcome of AYAs undergoing ASCT to define the results of treatment, and assemble a cohort in which we can evaluate the effect of treatment for relapsed disease on personal developmental and psychological outcomes.
Between 1988 and 2009, 181 AYAs (median age 24, range 15–30y) with relapsed or refractory HL received salvage therapy followed by ASCT at Princess Margaret Hospital (PMH) and the Hospital For Sick Children (HSC). Patient characteristics: M:F 94:87; advanced stage (IIB-IV) at diagnosis 84%; first progression or relapse: 90%. All patients had complete or partial response to 2–3 cycles salvage chemotherapy (platinum-base, mini-BEAM or ifosfamide-vinorelbine), and underwent bone marrow harvest (n=83) or stem cell mobilization with chemotherapy and filgrastim (n=98). Intensive therapy: etoposide + melphalan (PMH) or cyclophosphamide, carmustine + etoposide (HSC). Patients with disease bulk >5cm at relapse received post-ASCT involved field radiation if tissue tolerance allowed.

Results:
After a median follow-up of surviving patients of 4 years (maximum 16 years), of 181 pts, there have been 105 events for progression-free survival (PFS) and 81 deaths. PFS at 5 years is 41% +/&minus;4% (+/&minus; standard error) and at 10 years is 36% +/&minus;4% (median 1.6 years). Overall survival is 52% +/&minus;5% at 5 years and 39% +/&minus; 5% at 10 years(median 5.6 years). There have been no HL relapses beyond 39 months post-ASCT. Twenty patients (11%) died from treatment related-toxicity (10), second malignancies (5 AML, 2 NHL, 1 lung cancer) or unknown causes (2). Nine patients received an allogeneic stem cell transplant for relapse; 8 have died from progressive HL or treatment-related toxicity and one is alive in relapse.

Conclusions:
While highlighting the clear need for improved strategies for disease control for AYAs undergoing ASCT, this analysis has provided a cohort of patients in which to evaluate the long-term impact of intensive therapy on personal and social adjustment, and to identify opportunities for psychosocial intervention and health promotion.

Disclosures:
Kuruvilla: Hoffman LaRoche: Honoraria, Research Funding; Celgene: Research Funding; Amgen: Honoraria; Otsuka: Honoraria; Genzyme: Honoraria. Kukreti: Celgene: Honoraria.
MLL/AF4 Positive Acute Lymphoblastic Leukemia Has Resistance to Tumor Necrosis Factor-Alpha Caused by up-Regration of S100A6
Authors: Tamai Hayato , Miyake Koichi , Yamaguchi Hiroki , Okabe Masahiro , Dan Kazuo , Inokuchi Koiti , Shimada Takashi 
Published: October 2019

Abstract
Abstract 2477
Mixed-lineage leukemia (MLL)/AF4 positive acute lymphoblastic leukemia (ALL), which is common in infant leukemia, is associated with a poor prognosis even after allogeneic hematopoietic stem cell transplantation (allo-HSCT). The resistance to graft versus leukemia (GVL) effect may be the possible reason of the poor effect of allo-HSCT on MLL/AF4 positive ALL. Cytotoxic effector mechanisms, which are mediated by tumor necrosis factor-alpha (TNF-alpha), Fas ligand (FasL), or perforin were reported to contribute to GVL effect. We analyzed the sensitivity to TNF-alpha of MLL/AF4 positive ALL cell lines (SEM and RS4;11) together with MLL/AF4 negative leukemia cell lines (MOLT4, Raji and K562) and found that the growth inhibition by TNF-alpha of MLL/AF4 positive ALL cell lines group was significantly lower than those of another MLL/AF4 negative leukemia cell lines group (P<0.01). To examine the possible mechanism of resistance to TNF-alpha of MLL/AF4 positive leukemia, we focused on S100A6 as a possible factor, since it was reported that expression of S100A6, which is a calcium-binding protein implicated in many cellular processes and often up-regulated in cancers limits apoptosis induced by TNF-alpha through p53 inactivation in cardiac myocytes. Western Blot analysis showed that significant up-regulation of S100A6 expression and inhibition of acetyl p53/p53 expression were observed only in MLL/AF4 positive ALL cell lines in the presence of TNF-alpha (5ng/ml)(P<0.01 for S100A6 and P<0.01 for acetyl-P53/p53). To confirm the effect of S100A6 on the resistance to TNF-alpha of MLL/AF4 positive ALL cell lines, we examined the apoptosis rate of MLL/AF4 positive ALL cell lines treated with small interfering RNA (siRNA) against S100A6. TUNEL assay showed that significantly increased rate of apoptosis under s100A6 siRNA in the presence of TNF-alpha (control siRNA VS s100A6 siRNA; 42.5%&mnplus;4.5% VS 90.0%&mnplus;5.0%, P<0.01 for SEM, 32.5%&mnplus;3.5% VS 87.5%&mnplus;5.0%, P<0.01 for RS4;11). Western Blotting also showed that these apoptosis were mediated by p53-Caspase8 pathway. Up-reguration of S100A6 and inhibition of p53-Caspase8 pathway were also confirmed in previously established MLL/AF4 transgenic mice. These results suggest that MLL/AF4 positive ALL escapes from TNF-alpha mediated apoptosis by up-regulation of S100A6 expression followed by interfering with p53 acetylation. Our present results suggest that S100A6 may be a promising therapeutic target for MLL/AF4 positive ALL by combinating with allo-HSCT.

Disclosures:
No relevant conflicts of interest to declare.
Impact of Protein Phosphatase PP2A on ATRA-Induced Activation of FoxO3a In Acute Promyelocytic Leukemia Cells
Authors: Sakoe Yasuhiko , Sakoe Kumi , Shimazaki Haruo , Kirito Keita , Komatsu Norio 
Published: October 2019

Abstract
Abstract 2492
Acute promyelocytic leukemia (APL) is a subtype of acute myeloid leukemia caused by reciprocal translocations of the long arms of chromosomes 15 and 17, which prevent cellular differentiation into mature neutrophils. The translocation of the promyelocytic leukemia (PML) gene on chromosome 15 and a retinoic acid receptor α (RARα) gene on chromosome 17 generates a PML-RARα fusion protein that inhibits PML-dependent apoptotic pathways in a dominant negative fashion. This fusion protein also blocks granulocytic differentiation by direct transcriptional inhibition of retinoic acid target genes. All-trans retinoic acid (ATRA) arrests cell growth, granulocytic differentiation, and apoptosis in APL cells via proteasome-dependent degradation of PML-RARα fusion protein and subsequent PML-nuclear body (NB) formation. Although PML is the essential component of PML-NBs and functions as a tumor suppressor, disruption of PML-NBs by the PML-RARα fusion protein inhibits endogenous PML tumor-suppressive functions in APL cells. Therefore, degradation of PML-RARα fusion protein and reorganization of PML-NBs during ATRA treatment are regarded as critical cellular responses, similar to the cell growth arrest and apoptosis of leukemia cells. Recently we demonstrated that FoxO3a (also named FKHRL1), a member of the Forkhead family of transcription factors, is a key molecule for the ATRA-induced cellular responses in APL cells (Blood 2010; 115: 3787–3795). In this study, we investigated the mechanism by which FoxO3a is activated by ATRA treatment in a human promyelocytic leukemia cell line NB4. Okadaic acid, a potent PP2A inhibitor, cancelled ATRA-induced dephosphorylation of AKT and its downstream molecule FoxO3a in NB4 cells. Knockdown of endogenous PP2A by siRNA significantly enhanced phosphorylation of both AKT and FoxO3a. These results suggested that PP2A is involved in ATRA-induced dephosphorylation of AKT and FoxO3a. Concomitantly, PP2AC, a catalytic subunit of PP2A, was dephoshorylated at tyrosine 307, and phosphatase activity of PP2A increased after ATRA treatment. Co-immunoprecipitation assay revealed that PP2A constitutively and directly binds to FoxO3a. Using artificial oligopeptides, we demonstrated that enhanced PP2A activity by ATRA directly dephosphorylates phosphothreonine 32 on FoxO3a. In addition, we found that 14-3-3 epsilon binded to phosphorylated FoxO3a in the cytoplasm in the absence of ATRA. After ATRA treatment, however, dephosphorylated FoxO3a dissociated from 14-3-3 epsilon and moved into the nucleus. Confocal microscopic analysis revealed that PP2A-FoxO3a complex partially co-localized with PML-NBs in the nucleus after ATRA treatment. Together, PML orchestrates nuclear networking with PP2A and FoxO3a for ATRA-induced granulocytic differentiation and apoptosis of APL cells.

Disclosures:
No relevant conflicts of interest to declare.
A Novel PHD2 Mutation Associated with Tibetan Genetic Adaptation to High Altitude Hypoxia.
Authors: Lorenzo V Felipe R , Simonson Tatum S , Yang Yingzhong , Ge Rili , Prchal Josef T. 
Published: October 2019

Abstract
Abstract 2602
The ability of the body to maintain oxygen hemostasis is essential for its survival. The body is required to adapt to the physiologic constraints imposed by a hypoxic environment in order to ensure optimal physiological functions. The study of indigenous high-altitude residents provides an opportunity to identify genes that may have play a role in hypoxia adaptation, and recent studies indicate that Tibetans have genetically adapted to the extremely hypoxic environment at high altitude. Interestingly, Tibetan adaption includes protection from polycythemia. We recently reported genome-wide scans of selection (Affymetrix Human SNP chips v6.0) for Tibetans (Simonson, Science July, 2010). At least 10 chromosomal regions were identified as likely targets for high-altitude adaptation, and two of these regions, EGLN1 (PHD2) and PPARA, were associated with protection against high-altitude polycythemia which is unique to this indigenous highland population. PHD2 is a negative regulator of the alpha subunits of HIF-1 and HIF-2 transcription factors regulating many essential functions including erythropoiesis, iron metabolism, development, in addition to other functions. A gain of function mutation of the alpha subunit of HIF-2 is associated with congenital polycythemia. We sequenced the exons of PPARA and PHD2 by direct sequencing and found two missense mutations in exon 1 of the PHD2 gene: the first is located within codon 4, and changes Aspartate to Glutamate (Asp4Glu) and the second is an unvalidated SNP (rs12097901) at codon 127 that changes Cysteine to Serine (Cy127Ser). The samples with high Hb show a heterozygous change, while the subject with normal Hb exhibited a homozygous change in both codons of the PHD2 gene.
The association of these two missense PHD2 mutations to hemoglobin level is now being evaluated by studies of a larger population of Tibetan natives and the functional effects on levels and activities of HIF-1 and HIF-2 genes evaluated by transfection of these mutated PHD2 cDNA constructs in reporter cells.
Table 1. List of samples and controls screened for mutation in PHD2. Sample Race Sex Hb PHD2 Mutation Asp4Glu Cys127Ser T03 Tibetan F 194 Hetero Hetero T43 Tibetan M 194 Hetero Hetero T48 Tibetan M 101 Hetero Homo T53 Tibetan M 159 Homo Homo Control Caucasian M ? WT WT Control Caucasian F ? WT WT

Disclosure:
No relevant conflicts of interest to declare.