bluuebunny/crossref_metadata_2025_split huggingface dataset:
A New Procoagulant Mechanism Mediated by Fucoidans
Authors: Dockal Michael , Panholzer Erwin , Hartmann Rudolf , Ehrlich Hartmut J. , Scheiflinger Friedrich 
Published: October 2019

Abstract
Abstract 4420
Fucoidans are heterogeneous, polyanionic molecules with procoagulant activities in a wide concentration range. They have been described as non-anticoagulant sulfated polysaccharides (NASP) and shown to improve clotting in FVIII- and FIX-deficient plasma. In vitro characterization has suggested that fucoidans exert their procoagulant activity by inhibiting tissue factor pathway inhibitor (Liu et al. Thromb Haemost 2006; 95:68) and by accelerating thrombin-dependent FVa formation (Mutch et al. J Thromb Haemost 2007; 5 Suppl2). In our study we describe a new, previously unrecognized mechanism by which fucoidans act as procoagulant agents. The procoagulant activity of several fucoidans was characterized by calibrated automated thrombography in tissue factor (TF)-dependent experiments and by using coagulation factor-deficient plasmas. Spiking experiments with purified coagulation factors or inhibitory antibodies verified the mechanism.
Stimulation of thrombin generation (TG) by fucoidans requires anionic lipid surfaces like synthetic phospholipid vesicles which contain phosphatidylserine and is TF-dependent (0-20pM). However, stimulatory activity was most pronounced in the absence of TF. Control experiments with corn trypsin inhibitor or FXII-deficient plasma excluded any involvement of the contact system. Plasmas from patients with congenital coagulation factor deficiencies were screened for TG to identify the target coagulation factor by which fucoidans exert their procoagulant activities. In the absence of TF, plasmas deficient in coagulation factors from the common pathway do not support fucoidan-mediated thrombin generation, whereas FVII-deficient plasma does. FXI was identified as the most upstream factor of the intrinsic pathway which is required for fucoidan-stimulated thrombin generation, suggesting it to be the target for the procoagulant activities of fucoidan. Spiking 30nM FXI to FXI-deficient plasma restored fucoidan-mediated TG and addition of polyclonal FXI inhibitory antibodies to normal plasma abrogated TG. Fucoidan-dependent TG did not improve when FXIa (60pM) was added to FXI-deficient plasma, suggesting activation of FXI by fucoidan. The relevance of this mechanism in hemophilia A plasma was studied by addition of low levels of FVIII (0.2-10%) resulting in a FVIII concentration-dependent increase in fucoidan-mediated TG. These results highlight the requirement of a functional intrinsic pathway for this new mechanism of fucoidans. Our findings present FXI activation at low TF concentrations as a possible mechanism for fucoidan.

Disclosures:
Dockal: Baxter Innovations GmbH: Employment. Panholzer:Baxter Innovations GmbH: Employment. Hartmann:Baxter Innovations GmbH: Employment. Ehrlich:Baxter Innovations GmbH: Employment. Scheiflinger:Baxter Innovations GmbH: Employment.
Preliminary Study of the Autoantigens on the Membrane of Erythropoietic Cells of the Patients with BMMNC- Coomb's Test(+) Hemocytopenia
Authors: Fu Rong , Liu Hui , Shao Zonghong , Wang Jun , Li Lijuan 
Published: October 2019

Abstract
Abstract 4435

Objective
To observe the relationship between EPO receptor(EPOR) and autoantibodies-IgG/IgM (auto-Ab) on the membrane of erythropoietic cells of the patients with bone marrow mononuclear cell Coomb's (BMMNC-Coomb's) test(+) immuno-related pancytopenia(IRP), and then explore the probable autoantigens of auto-Ab in IRP.

Methods
46 newly diagnosed IRP patients (15 with auto-Ab on erythropoietic cells and 31 without auto-Ab on erythropoietic cells) and 18 healthy donors as controls were enrolled in this study. EPOR expression on their nuclear erythrocytes were tested with flow cytometry to observe the relationship between EPOR and auto-Ab; After sorting erythropoietic cells in bone marrow, EPOR mRNA and protein Stat5,P-Stat5 were investigated by RT-PCR and Western blot to observe the production of EPOR and EPO/EPOR signal transduction; Finally, EPOR expression on the membrane were tested again after stripping auto-Ab with glycine buffer.

Results
(1) EPOR of auto-Ab(+) arm(1.59±0.87)% was significantly lower than that of auto-Ab (-) arm(4.58±4.09)%(P<0.01), and the latter was significantly higher than that of normal controls (2.27±1.76)%(P<0.05); EPOR of IRP patients was negatively correlated with their auto-Ab (r=-0.543,P=0.000) and its regression equation was Y(EPOR)=0.040-0.335X(auto-Ab);(2)EPOR mRNA of auto-Ab(+) arm(0.685±0.136)was significantly higher than that of auto-Ab (-) arm(0.554±0.116)(P<0.01)and normal controls (0.580±0.119)(P<0.05);(3)Protein Stat5 of auto-Ab(+) arm(1.45±0.94) was significantly higher than that of normal controls (0.54±0.36)(P<0.05); While P-Stat5 of auto-Ab(+) arm(0.42±0.18) was significantly lower than that of normal controls (0.85±0.38)(P<0.05); (4) EPOR expression became higher while auto-Ab became lower after stripping with glycine buffer.

Conclusion
The auto-Ab of some IRP patients might block or competitively inhibit the EPOR on the membrane of erythropoietic cells. EPOR was one of autoantigens in IRP.

Disclosures:
No relevant conflicts of interest to declare.
Characterization of CDR3 Structure of Vβ21 T Cell Clones In Patients with P210BCR-ABL Positive CML and B-ALL
Authors: Zha Xianfeng , Chen Shaohua , Yang Lijian , Li Bo , Chen Yu , Yan Xiaojuan , Li Yangqiu 
Published: October 2019

Abstract
Abstract 4455
The clonally expanded T cells identified in most cancer patients which were considered to respond to tumour association antigen (TAA) have definitely specific anti-tumor cytotoxicity. P210BCR-ABL protein regards as the hallmark of CML and known to be causative of the disease, and also expresses in partial acute lymphoblastic leukaemia (ALL) can induce to yield tumor-specific cytotoxic T lymphocytes (CTL) which can kill bar-abl+ tumor cells in many trials. In our previous study, we found the oligoclonal expansion of TCR Vβ21 subfamily in the peripheral blood (PB) of 8 cases with P210BCR-ABL positive CML and 3 cases with P210BCR-ABL positive B-ALL patients and 1 case with P210BCR-ABL negative B-ALL patients using the genescan technology. To further elucidate the oligoclonal TCR Vβ21 features, we carried out sequencing analysis of CDR3 region of each oligoclonal TCR Vβ21 and determined the primary structure of the CDR3 region. The 12 TCR Vβ21 clones used different Jβ segments, Jβ2.3 segment was used in 4 clones, both Jβ2.1 and Jβ1.1 segments were used in 2 clones respectively, Jβ1.2, Jβ1.3, Jβ2.2 and Jβ2.7 segments were used in 1 clone. Only a conserved amino acid motif (SLxxV) was found within the CDR3 region from 3 patients with CML. Then we constructed the three-dimensional structures of the CDR3 sequences from all 12 clones by homology modeling methods and through pairwise comparing with the three-dimensional structures of all CDR3 sequences, the results showed that the conformation of the CDR3 region either containing the conserved amino acid motif or using the same Jβ segment displayed low similarity. Interesting, the conformation among some clones whose CDR3 region contain different amino acid motif and did not use the same Jβ segment were high resemble. In conclusions, our findings described that the high frequency of TCR Vβ21 subfamily expansion emerged in p210BCR-ABL positive CML and B-ALL patients, and according to perplexing features of CDR3 region, it could not easy to confirmed that the oligoclonal expanded TCR Vβ21 subfamily must recognize to p210BCR-ABL protein, however, primary structure characterization of CDR3 region, in combination with spatial structure features, ultimately might help to identify the tumor-associated antigen immune attack on tumor cell in p210BCR-ABL positive CML or B-ALL patients.

Disclosures:
Li: The study was supported by grants from National “863” project (No. 2006AA02Z114) and Key project of Natural Science Foundation of Guangdong province, China (No. 05103293, 9251063201000001).: Research Funding.
ABL Mutations In Early Chronic Phase Chronic Myeloid Leukemia (CP-CML) Are Associated with a Greater Likelihood of Progression and Shorter Survival
Authors: Carella A. M. , Garuti A. , Cirmena G. , Catania G. , Pica G. , Beltrami G. , Patrone F. 
Published: October 2019

Abstract
Abstract 4458
Acquired resistance to Imatinib in advanced phase of CML has been associated with mutations in the kinase domain (KD) of BCR-ABL. We have recently reviewed the status of the mutations in 52 patients with early CP-CML on the samples collected at diagnosis. Mutations were identified by direct sequency (DS) with BidDye Terminator V1.1. cycle sequencing kit and analyzed with a 3130 AB capillary electrophoresis system. Twenty-eight patients had low risk, 10 intermediate risk and 14 high risk, according to Sokal/Euro. Ten out of 14 high Sokal risk patients showed the following mutations: Y253C, S265R, E255K, F359Y, N374S, E255V, E255V, E255V, R332L, E334G. Three of these patients progressed during Imatinib and second-line TKIs and died of blastic phase CML at 23, 33 and 69 months. Curiously, S265R and N374S mutations disappeared during Imatinib treatment but were substituted during follow-up by other two mutations: E255L and H396R. The patient carrying E255L mutation died in blastic phase at 33 months and the one with mutation H396R was well controlled by Nilotinib and he is now alive in CMR 26 months after. Only one out of the 10 intermediate Sokal risk carried KD mutations at diagnosis (D363G). This patient is alive in MMR at 26 months after diagnosis under Imatinib. None of the 28 low Sokal risk patients carried KD mutations at diagnosis and no patients developed cytogenetic evolution while on treatment. In conclusion, the fact that KD mutations were more present in patients with high Sokal risk supports the hypothesis that the probability developing a mutation is related to the basic biology of the disease rather than being merely a random event.

Disclosures:
No relevant conflicts of interest to declare.
High Levels of Nm23-H2 Protein Are a Specific and Invariable Feature of CML-CP Cells Due to Post Transcriptional Control Via a Bcr-Abl Dependent Pathway
Authors: Tschiedel Sabine , Cross Michael , Müller Enrica , Jilo Annette , Wang Song-Yau , ucinic Vladan , Lange Thoralf , Al-Ali Haifa Kathrin , Niederwieser Dietger W 
Published: October 2019

Abstract
Abstract 4461

Introduction:
We have previously identified Nm23-H2 as a tumor antigen in a case of chronic myeloid leukemia. In the following project we investigated the molecular background for the increased expression of Nm23-H2 in CML cells from a large number of patients.

Material and Methods:
Clinical samples from 30 patients with CML in chronic phase at diagnosis [Bcr-Abl/Abl transcript levels from 69.5 to 93% IS (international scale)], in addition to patients with molecular response to imatinib [n=10; median 2 (range 0.1–11) % IS Bcr-Abl/Abl transcript level] and to imatinib resistant patients [n=10; median 66 (range 37–100) % IS Bcr-Abl/Abl transcript level] were investigated. Subpopulations were isolated from the blood (CD3+, CD14+) or bone marrow (CD34+) by FACS sorting. AML samples were obtained from 12 patients with de novo AML and 3 patients with secondary AML at diagnosis. Ba/F3 cells expressing either wild-type Bcr-Abl (Bcr-Ablwt Ba/F3), the T315I mutant of Bcr-Abl (Bcr-AblT315I Ba/F3) or the E255K mutant of Bcr-Abl (Bcr-AblE255K Ba/F3) were used in the cell line model.
Messenger RNA levels of Nm23-H2 were measured using the QuantiTect Primer Assay (Qiagen). Results were normalized to mRNA for the ribosomal protein RPLP0. Western blotting for Nm23-H2 was performed by standard techniques using chemiluminescence and immunocytochemistry was performed using Nm23-H2 and ß-Actin antibodies. Mean fluorescence intensity was determined using the LSM Image Browser software.

Results:
Nm23-H2 protein is over-expressed in the cytoplasm of mononuclear cells from 30/30 CML patients at diagnosis and 10/10 patients with resistance to imatinib. In contrast, patients responding to Imatinib had markedly reduced levels of Nm23-H2 protein. Over-expression is limited to Bcr-Abl+ progenitor and myeloid populations and is absent from (Bcr-Abl-) T lymphoid cells, normal donors and 14/15 acute myeloid leukemia samples. The increase in Nm23-H2 protein is due to post-transcriptional regulation, with no detectable increase in mRNA levels. The increase in Nm23-H2 can be reversed by imatinib or nilotinib treatment of Ba/F3 cells expressing wild-type Bcr-Abl, but not of cells expressing the respective resistant Bcr-Abl mutants.

Conclusions:
The post-transcriptional up-regulation of Nm23-H2 is therefore a common and specific property of CML closely associated with Bcr-Abl activity, making Nm23-H2 a potential target for the development of novel immunotherapeutic or pharmaceutical therapies for CML.

Disclosures:
Al-Ali: Novartis: Consultancy, Honoraria. Niederwieser:Bristol-Myers Squibb: Speakers Bureau; Novartis: Speakers Bureau.
Treatment with TKIs Overcomes Imatinib Resistance through the PLCgamma-1 Signaling Pathway In Imatinib Resistant Human CML Cell Lines
Authors: Romano Alessandra , Vigneri Paolo , Romano Chiara , Vetro Calogero , Stella Stefania , Massimino Michele , Stagno Fabio , Raimondo Francesco Di , Liotta Lance , Espina Virginia 
Published: October 2019

Abstract
Abstract 4468
Suppression of BCR-ABL1 catalytic activity by the tyrosine kinase inhibitor (TKI) Imatinib Mesylate (IM) has dramatically improved the natural history of Chronic Myeloid Leukemia (CML) and - to date - represents the first and most successful example of effective anti-cancer targeted therapy {Druker, 2009}. Despite the success, patients can become resistant. Since IM-resistance in CML patients occurs despite adequate suppression of BCR-ABL activity, it is likely due to the activation of other pathways, and for this reason we need to discover novel Bcr-Abl independent pathways than can become the targets of resistant cells.
Therefore we used immortalized CML human cell lines K562 and LAM84, both sensitive and resistant to Imatinib, to study the signaling in sensitive/resistant cell lines following treatment with 4 different compounds. Reverse-Phase protein microarrays were used to quantitatively map 35 cell signaling pathway endpoints, including survival, proliferation, drug resistance, apoptosis, and autophagy. Cells were incubated with IM 1uM, Dasatinib 1uM, Nilotinib 1uM or LY-29400210 uM and after 2 or 12 hours were placed in a preservative that suppresses fluctuations in kinase pathway proteins (Espina, 2008).
5/35 protein endpoints were linked together and suppressed by Dasatinib, even in the resistant cell line: PLC-y-1-Tyr783, and its upstream (ShCTyr317 SrcTyr416) and downstream targets (mTORSer2448, STAT5Tyr694, ERKThr202/Tyr204) without interfering with AKT activation status on Ser473 compared to Imatinib (p=0.0031 for K562, p= 0.042 for LAM84), Nilotinib (p=0.0034 for K562, p= 0.043 for LAM84), LY-294002 (p=0.0009 for K562, p= 0.015 for LAM84).
In Imatinib-sensitive cell line LAM84 there were no differences between Dasatinib and Imatinib in the modulation of the pathway, compared to IM-sensitive K562 cell line (p=0.005), thus confirming the different profile among these two CML models.
Dasatinib showed a greater suppression of the PLC-y-1 pathway compared to LY-294002 in both resistant cell lines (K562, p=0.0009 and LAM84, p=0.015).
These data confirm and extend the conclusions of Markova et al. (Oncogene 2010) showing PLC-y-1 as a mechanism of death in sensitive cells. Our data showed the mechanism by which second generation TKIs (dasatinib) can overcome Imatinib resistance by suppressing the PLC-y-1 pathway.

Disclosures:
No relevant conflicts of interest to declare.
Validation of a New Flow Cytometry-Based Method to Discriminate Malignant From Normal Stem Cells In CML
Authors: del Castillo Santiago , García Regina , Hernandez Agustin M , Entrena Laura , Llano Ma Paz Queipo De , Campos Arturo , Rosell Ana , Cerda Maria , Ramirez Gemma 
Published: October 2019

Abstract
Abstract 4474

Introdution:
Previously, leukemic stem cells in Chronic Myelogenous Leukemia could only be identified indirectly by using culture techniques. Jeroen J.W et al have developed a new flow cytometric approach that allows a direct discrimination of CML stem cells from their normal counterparts within single patient samples. CD34+CD38- leukemic stem cells could be discriminated from normal stem cells by high CD90 expression, which is a specific feature of CML stem cells, while CD90 expression is low on their normal counterparts.
Material: A total of 8 patients and 3 health controls were included in this study.

Result:
Health controls did not present CD90 molecular expression in stem cells. 100% of the patients presented different levels of CD90 in CD34+/CD38- cells. Our patients seemed to show an asociation between CD34+/CD38- stem cells which express CD90+ and bcr-abl levels. We have evolutive studies of 4 patients who are under treatment with imatinib; in those patients we apreciate decrease in the level of CD90 related to good response.

Conclusion:
Our results confirm Jeroen's data; we think that this new technique will expand our possibilities to identify CML stem cell specific targets and may improve efficacy assessment of CML treatment as well.

Disclosures:
García: Celgene: Research Funding.
Phase II Study of Dasatinib In Patients with Relapsed Chronic Lymphocytic Leukemia.
Authors: Al-Ameri Ali M , Badoux Xavier , Ferrajoli Alessandra , Wierda William G. , Fayad Luis , Estrov Zeev , Bickel Susan , Keating Michael J , O'Brien Susan 
Published: October 2019

Abstract
Abstract 4488
Src-family kinases (SFK) are described to be over expressed in CLL resulting in increased BCR signaling. Aberrant activation of the SFK, Lyn, leads to defective apoptosis of CLL cells in-vitro. C-Abl kinase is also overexpressed in CLL cells compared to normal B-lymphocytes. Since dasatinib is a dual Src and c-abl kinase inhibitor with in-vitro pro-apoptotic properties in CLL cells, we investigated the activity of dasatinib in patients (pts) with CLL. We designed a phase II study of dasatinib in pts with relapsed CLL/SLL and T-PLL. Pts were eligible if they were previously treated and had indication for therapy according to NCI-working group criteria. All pts had adequate performance status, renal and liver function prior to therapy. Treatment consisted of dasatinib 50mg given orally twice daily. In case of suboptimal response the dose of dasatinib could be increased up to a maximum dose of 70mg twice daily. Dose reductions to 20mg twice daily were permitted for toxicity. Pts were assessed for treatment response according to 1996 NCI-WG criteria. Seventeen pts have been enrolled in this study. The median age was 67 years (42-83 years), 9 (52%) had Rai stage III-IV, median beta-2 microglobulin levels was 5.9 (3.0 – 11.8) mg/L. The median number of prior treatments was 4 (1 – 8). An objective response (PR) was observed in 1 patient, 13 pts had no objective response and 3 pts were not evaluable for response due to early discontinuation of therapy (0-3 days). Fourteen pts remained on therapy for a median of 2 (0-19) months with 4 pts discontinuing due to disease progression and 9 pts discontinuing due to adverse events and lack of response. Hematological toxicities consisted of grade 3–4 neutropenia in 76% of the pts, grade 3–4 thrombocytopenia in 44% of pts and grade 1–2 anemia in 80%. Non-hematological toxicity consisted of grade 3–4 fatigue in 1 patient and grade 3 pleural effusions in another patient. Grade 1 and 2, toxicities included flushing 38%, headache 38%, fatigue 46% anorexia and nausea 46%, and diarrhea 23%. Several pts showed evidence of biological activity. Treatment with dasatinib lacks efficacy in pts with heavily pretreated CLL. Responses occurred in only 6% of pts and dasatinib administration was associated with a high incidence of neutropenia.

Disclosures:
O'Brien: Bristol-Myers Squibb: Research Funding.
Dasatinib for Chronic Phase Chronic Myeloid Leukemia After Failure of Imatinib In Korean Patients.
Authors: Lee Ha Yeon , Chang Myung-Hee , Jung Chul Won , Kim Dong Hwan (Dennis) , Kim Won Seog , Kim Seok Jin , Kim Kihyun , Jang Jun Ho , Yoon Sung-Soo , Lee Kyoo-Hyung , Kim Hyeoung Joon , Chung Joo-Seop , Sohn Sang Kyun 
Published: October 2019

Abstract
Abstract 4494

Background:
Although imatinib could induce efficacious and stable responses in patients with chronic myeloid leukemia (CML), resistance is increasingly problematic. Dasatinib, a BCR-ABL inhibitor with 325-fold greater potency in vitro than imatinib, induced MCyR 59%, CCyR 48%, PFS 90% and OS 96% for patients who can not tolerate or are resistant to imatinib in START-C.

Method:
We assessed retrospectively the efficacy and safety of dasatinib in patients with chronic phase (CP)-CML, resistant or intolerant to imatinib who received dasatinib 70 mg to 140 mg per day. Dose was adjusted according to toxicity.

Result:
Between 21 June 2005 and 31 March 2010, medical records of 47 patients from 6 centers in Korea were reviewed: 22 with imatinib-resistant and 1 with imatinib-intolerant CP-CML. 8 Imatinib–resistant ABL kinase domain mutations were found including E255K, T315I, F317L. Median duration of dasatinib therapy was 20.6 months. CHR, MCyR, CCyR and MMR was attained in 91%, 79%, 56% and 44% of patients, respectively. 18 month MMR rate was 71%, 67% in imatinib-intolerant and 53% in imatinib-resistant group. There was no difference in PFS according to the Sokal score, to the best response of imatinib to the type of mutation. 3-year PFS was 71% and OS was 79% with a median follow up of 20.6 months. There was no disease progression or death in imatinib intolerant group. Grade 3/4 anemia, neutropenia and thrombocytopenia were reported in 36%, 49% and 45% of patients, respectively. Non-hematologic toxicity (grade 3/4) consisted of infection(15%), dyspnea(4%), pleural effusion(6%), abdominal pain(2%) and skin rash(2%).

Conclusion:
Dasatinib showed promising efficacy and tolerability in imatinib-resistant or -intolerant CP-CML in Korean patients.

Disclosures:
No relevant conflicts of interest to declare.