bluuebunny/crossref_metadata_2025 huggingface dataset:
In Vivo Monitoring of Venous Thrombosis in Mice.
Authors: Aghourian Meghedi , Blostein Mark
Published: October 2019

Abstract 5061

Venous thromboembolism afflicts 117 people per 100,000 each year and is an important cause of morbidity and mortality. There has been extensive research dedicated to the clinical aspect of venous thrombosis, especially with regards to its diagnosis and treatment. However, animal models studying this phenomenon are scarce and, in most cases, very crude. Developing a murine model of venous thrombosis using techniques similar to the ones used to detect thrombosis in humans can be a constructive step in studying this phenomenon in more detail. The model developed in our lab uses ultrasound imaging to visualize venous clots in the Inferior Vena Cava (IVC) of mice, allowing for precise measurements of the formed clot. Ligation of the IVC is one of the well established models for studying thrombosis in mice. We ligated the IVC of wild type C57B6 mice, and allowed them to recover. We then followed clot formation at several time points after the operation using micro-ultrasonography, the Vevo 770®, a novel imaging ultrasound technology designed to monitor murine vasculature. To assess the precision of the clot measurements, we then sacrificed the mice, and dissected out the thrombi in order to precisely measure and weigh them. A thrombosis develops only after 5 hours of ligation post surgery when a clot is visualized in the IVC. The clot increases slightly over the next 24 hours. The measurements of the clot after dissection correlates favourably with the measurements done by ultrasonagraphy using the Vevo770®. These data suggest that the Vevo770® can be used as a reliable technique for non-invasive assessment of venous thromboembolism in mice. Developing a murine model for thrombosis using more accurate, and clinically more relevant techniques such as ultrasonography, is a step towards better understanding and treatment of venous thromboembolism.

Disclosures

No relevant conflicts of interest to declare.
Elevated Levels of Cytokines and Chemokines in Patients with Antiphospholipid (aPL) Antibodies.
Authors: Pierangeli Silvia S , Martinez-Martinez Laura Aline , Aguilar-Valenzuela Renan , Gwin Gerald Mc , Doan Ellis , Papalardo Elizabeth , Vega Jo Ann , Gonzalez Emilio B. , Murthy Vijaya , SEif Alan , Erkan Doruk
Published: October 2019

Abstract 5062

Purpose

Systemic Lupus Erythematosus (SLE) and Antiphospholipid Syndrome (APS) are two closely related diseases. APL antibodies are present in 30-40% of SLE patients. Studies have indicated that certain cytokines, chemokines, tissue factor (TF), vascular endothelial growth factor (VEGF), soluble (s) E-selectin (sE-sel), tumor necrosis (TNF)-a, may be associated with APS and/or SLE. However, whether those biomarkers are elevated in plasma of APS patients with or without SLE is uncertain. Here we examined whether the levels of cytokines/chemokines in aPL antibody-positive patients positive are different from controls.

Methods

Twenty-two sera/plasma of patients with significantly and persistently elevated levels aPL antibodies (IgG and/or IgM aCL, and/or IgG and/or IgM anti-β2glycoprotein I (β2GPI) and/or positive lupus anticoagulant (LAC), demographic and clinical data were obtained from an on-going pilot clinical study (clinical trials.gov#NCT00674297). Patients that were on more than 10 mg prednisone/day, or on other immunosuppressive therapy, or on hydroxychloroquine or on statins were excluded. Thirty-two healthy donors with no evidence of autoimmune, infectious or inflammatory diseases were used as controls. .Levels of IL1b, IL6, IL8, TNF-a, VEGF, IP-10, sCD40L were measured in serum using a Millipore Millliplex” Multiplex Assay; titers of sE-sel, and sTF were detected by ELISA. The Kruskal-Wallis test was used to compare levels of biomarkers in aPL positive subjects vs. controls and Spearman tests to correlate levels of the biomarkers in the different subgroups of patients.

Results

Significant number of aPL-positive samples had elevated levels of IL1b, IL6, TNF-a, VEGF, IP-10, sCD40L, sTF, sCD40L and sE-sel and their titers were significantly different when compared to controls. APS /SLE patients (n=8) showed significantly higher levels of TNF-a compared to APS without SLE (n=6), to “asymptomatic” aPL positive (n=4), to “asymptomatic” aPL positive with SLE (n=2) or with “catastrophic” APS (n=2) patients (p=0.00630).

Conclusions

This study underscores the importance of identifying biomarkers of disease that may help to better understand pathogenic mechanisms, predict disease activity and possibly to better address treatment of patients with aPL antibodies.

Disclosures

No relevant conflicts of interest to declare.
Characteristics and Outcomes of Pulmonary Embolism IN CANCER PATIENTS and IN PATIENTS without CANCER.
Authors: Karippot Asha , Maroules Michael , DeBari Vincent A
Published: October 2019

Abstract 5065

Background

Venous thromboembolism (VTE), a frequent complication in cancer and, occasionally, a harbinger of occult cancer, is one of the leading causes of death in cancer patients. VTE recurs with a three-fold greater frequency in cancer patients than in patients who do not have cancer, and requires long-term anticoagulation. There is a two-fold greater risk of bleeding complications in these patients compared to patients who do not have cancer. Because pulmonary embolism (PE) is a life-threatening sequel of VTE, we sought to evaluate the hypothesis that cancer patients suffer from extensive pulmonary embolism (involving central pulmonary artery or bilateral pulmonary arteries) and to determine if we could detect differences in two clinical outcomes, length of stay (LOS) and mortality.

Patients and Methods

This retrospective cohort of subjects with pulmonary embolism (PE) was developed using data beginning in January 2003 to December 2007 at a 680 bed, urban teaching hospital. The cohort of 118 patients with PE diagnosed with CT angiogram yielded 41 patients in cancer group (20 males and 21 females) and 47 (23 males and 24 females) patients in non cancer group. Other baseline characteristics gathered for the two groups included age, gender, race, co-morbidities, ambulatory status and therapeutic interventions. Criteria for being assigned to the “central PE” were a filling defect in the pulmonary arteries or the main stem. Patients who showed filling defects in both central and peripheral vessels were also assigned to the “central PE”. All other patients with filling defects in one or more peripheral vessels were assigned to the “peripheral PE” group.

Results

The baseline characteristics did not show significant differences between the groups. Our data demonstrated that central PE was strongly associated with the cancer group (26/41; 63.4%) compared with 15 of 47 (31.9%) in the non-cancer group (OR = 3.70; 95% CI: 1.53 to 8.95; p = 0.0051). The common cancers associated with VTE were genito–urinary (13) followed by gastro-intestinal (10), then lung and breast (6 in each group). LOS was significantly greater in the cancer group (median: 9d; IQR: 7-19d) compared to non-cancer group (median: 7d; IQR: 5-11d; p = 0.032. We detected no significant difference in overall mortality between the two groups (p = 0.11). Kaplan-Meier analysis of survival, similarly, demonstrated no significant difference (95% CI of HR: 0.62 to 3.18; p = 0.42)

Conclusion

Cancer patients appear to be at a higher risk for central PE and have longer stay in the hospital after diagnosis than patients without cancer. However, in this study we did not detect increased mortality over a twelve-month observation period.

Disclosures

No relevant conflicts of interest to declare.
Statins Reduce Disease Activity (SLAM-R) and Organ Damage Accrual (SLICC Damage Index, SDI) Scores in SLE Patients From A Multi-Center, Multi-Ethnic, US Cohort.
Authors: Pierangeli Silvia S , SEif Alan , Aguilar-Valenzuela Renan , Martinez-Martinez Laura Aline , Doan Ellis , Dang Neha , Papalardo Elizabeth , Reveille John D. , Alarcon Graciela S.
Published: October 2019

Abstract 5066

Purpose

Statins, in addition to lower cholesterol levels, have anti-thrombotic, anti-inflammatory and cardiovascular protective effects. Cardiovascular morbidity and accelerated atherosclerosis are important SLE features, but It is unclear whether statins have beneficial effects in these patients.

Objectives

To assess longitudinally the impact of statins on levels of biomarkers (cytokines/chemokines), SLAM-R and SDI.

Methods

Sera from 21 SLE patients (ACR criteria) from a multi-ethnic, multi-center cohort were assessed at baseline and at a subsequent visit after statin treatment started. Patients on >10 mg prednisone/day, or on other immunosuppressant were excluded (age range: 16-67; mean age: 44.6 yrs); 38% African American, 9% Caucasians and 4% Puerto Rican Hispanics; 86% female. Serum levels of IL1b, IL6, IL8, IFN- α,, IP-10, MCP-1. VEGF, sCD40L and TNF- α, were measured using a Millipore Millliplex™ Multiplex Assay and titers of soluble (s) E-selectin (E-sel), intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), were detected by ELISA. High sensitive C reactive protein (hsCRP) was measured by nephelometry. Changes in the levels of biomarkers were tested using a signed rank test. Changes in levels of biomarkers and those in SLAM-R or SDI scores were examined with Spearman correlations.

Results

SLAM-R and SDI scores were significantly decreased by statins (p=0.0199 and 0.039, respectively). IL1b, IL6, IL8, TNF-α, VEGF, IP-10, sCD40L and sVCAM-1 levels were reduced after treatment with statins in 28%, 28%, 52%. 43%, 57%, 42%, 52% and 62% of the subjects that had detectable levels of the biomarkers at baseline, respectively, but the overall reduction was not statistically significant (see Table). hsCRP was increased in 50% of the subjects at baseline (over the cut-off point) and reduced by 37% after treatment with statins. Importantly, the changes in IL-6 after treatment with statins correlated with changes in SLICC scores (p=0.036

Conclusions

Significant number of SLE patients' samples showed a decrease in levels of some cytokines and chemokines and of hsCRP after statin treatment. Importantly, SLAM-R and SDI scores were significantly reduced by statins and the changes in IL-6 correlated with a significant decrease in organ damage accrual (SLICC).

Disclosures

No relevant conflicts of interest to declare.
The Diagnosis of the Antiphospholipid Syndrome in Our Medical University Center for the Year 2007.
Authors: Boisjoly Josie-Anne , Lepine-Martin M. D , St-Onge Carole
Published: October 2019

Abstract 5068

Introduction

The antiphospholipid syndrome (APS) is associated with arterial and venous thrombosis and with obstetrical complications. According to the recommendations published in 2006, the diagnosis of SAP requires at least one clinical manifestation and, at least, one abnormal laboratory test: lupus anticoagulant (LA), anti-cardiolipin antibodies (ACL) or anti-B2-Glycoprotein antibodies (anti-B2GP1). More, the abnormal test has to be repeated and still abnormal 3 months later. In this study, we evaluated the process leading to a diagnosis of APS in our hospital during a whole year period. The clinicians had previously received a copy of published recommendations.

Method

We registered the identification number of all patients tested for 1 or more test (LA, ACL, anti-B2-GP1) in our laboratory during the year 2007. We then, retrospectively, analyzed the medical files of the patients who had their medical evaluation in our Center. We retained the following data: sex, age, symptoms at the presentation, final diagnosis, results of the specific tests for APS with the interval between the positive tests and the control tests.

Results

During 2007, 331 patients were tested for APS in our laboratory. We analyzed the medical files of 259 patients, 184 women and 75 men, with a mean age of 43,9 years. The indications for testing were: venous thrombosis (36, 7%), arterial thrombosis (14,3%), obstetrical complications (15,7%), collagenosis (17%), prolonged TTPa (3,1%), others (13,9%). In the laboratory, we found 91 positive tests in 70 patients. For 5 patients, all the three tests were positive. Only 48 positive tests were controlled after a period of 6 to 12 weeks. In total, 24 patients had at least one positive and controlled test; from them, 10 had all the criteria for the APS and 13 had abnormal tests without the clinical criteria for the APS.

Conclusion

In this study, 3, 9% of tested patients had the diagnostic criteria for the APS. The indications for ordering the tests seemed adequate in 86% of the cases. Only 52.7% of the positive tests were controlled. We have modified our interpretation of the positive tests, suggesting a control after 3 months.

Disclosures

No relevant conflicts of interest to declare.
The Association of 14-3-3ζ and Filamin A with the Central Cytoplasmic Domain of GPIbα Involves in Regulation of the Von Willebrand Factor Binding and Signaling Functions of Platelet Glycoprotein Ib-IX.
Authors: Zhang Weilin , Yuan Yanhong , Zhao Lili , Liao Yi , Ruan Changgeng , Dai Kesheng
Published: October 2019

Abstract 5073

Introduction

Conformational changes in von Willebrand factor (VWF) exposed at the injured vessel wall initiate platelet tethering and simultaneously trigger signaling cascades leading to platelet adhesion, spreading, and thrombus formation. Although the intracellular signaling protein 14-3-3ζ and the membrane skeleton protein filamin A have been confirmed to interact with the cytoplasmic domain of GPIbα and involve in the regulation of VWF binding function, whereas, the mechanisms still remain unclear. We have recently identified a novel 14-3-3ζ binding site with partial phosphorylation of Ser559 in resting platelets located in the cytoplasmic filamin A binding domain of GPIbα. Disruption of the central cytoplasmic 14-3-3ζ binding site of GPIbα did not abolish the interaction between 14-3-3ζ and GPIb-IX, whereas obviously diminished VWF binding function. Here we show that disruption of 14-3-3ζ binding to GPIbα by GPIbα mutants or cell permeable peptides enhances the association of filamin A with GPIb-IX and the expression of GPIb-IX in the plasma membrane. In contrast, disruption of 14-3-3ζ binding to GPIbβ, which would enhance the opportunity for 14-3-3ζ monomer to bind to GPIbα, reduces filamin A-GPIb-IX interaction and GPIb-IX membrane expression. Compared with a GPIb-IX mutant truncating the whole cytoplasmic domain of GPIbα (Δ551), GPIb-IX mutant retaining only the central 14-3-3ζ binding site of the cytoplasmic domain of GPIbα (Δ565) showed an enhanced cell adhesion to VWF under flow and ristocetin-induced VWF binding. Furthermore, the two cell permeable peptides, MP-P and MPαC, which had been confirmed to block the central 14-3-3ζ binding site of GPIbα (MP-P) or diminished the association of 14-3-3ζ with GPIb-IX complex (MPαC), obviously inhibited ristocetin induced wild-type GPIb-IX cell aggregation. In addition, MP-P and MPαC inhibited botrocetin-induced platelet spreading and lamellipodia formation on VWF matrix, and GPIb-IX-VWF interaction induced co-immunoprecipitation of Src with GPIb-IX. Taken together, these data indicate that the binding of 14-3-3ζ and filamin A to the central cytoplasmic domain of GPIbα involves in regulation of surface expression, VWF binding and signaling functions of glycoprotein Ib-IX.

Disclosures

No relevant conflicts of interest to declare.
Remission of Paroxysmal Atrial Fibrillation with Iron Reduction in Hemophilia A.
Authors: Zacharski Leo R. , McKernan Laurel J , Metzger Michael E , Malone Maura G , Samnotra Vivek , Bhargava Archana , Steiner Paul R , Rauwerdink Cocav , Ornstein Deborah L , Cornell Cornelius J.
Published: October 2019

Abstract 5100

Two male first cousins with mild hemophilia A had symptomatic electrocardiographically documented paroxysmal atrial fibrillation for several years that had become resistant to pharmacologic suppression. Both had baseline factor VIII levels of 12 to 15% and experienced bleeding requiring coagulation factor infusion therapy in response to trauma and surgical procedures. Radiofrequency ablation of ectopic electrical foci was considered in both cases but deferred because anticoagulation required to prevent thromboembolic complications of radiofrequency ablation carried an unacceptably high risk of bleeding. Iron accumulation with aging has been implicated in the pathogenesis of cardiovascular disease in general and arrhythmias in particular. Uncompensated iron-catalyzed oxygen free radical formation accounts for such toxicity. Remission of arrhythmias has been reported with iron reduction therapy in patients with iron overload disorders. Effects of iron reduction on arrhythmias in settings other than iron overload syndromes are unknown. Body iron stores as assessed by serum ferritin levels were elevated in both men (389 and 305 ng/ml respectively) but neither had the C282Y or H63D genes for hemochromatosis. Calibrated reduction of iron stores by serial phlebotomy, avoiding iron deficiency, was performed and was followed by sustained remission of PAF in both cases. These findings suggest that iron reduction with maintenance of ferritin levels below about 100 ng/ml may be effective treatment for arrhythmias apart from the classic iron overload syndromes. Effects of primary prevention of iron accumulation over time and reduction of existing elevated iron stores deserve further study in patients with bleeding disorders in whom standard treatment of diseases of aging may be costly and carry high risk. The efficacy, safety, simplicity and cost effectiveness of iron reduction therapy commends application of this investigational approach to disease prevention and treatment in the general population.

Disclosures

No relevant conflicts of interest to declare.
Serum Pro-Hepcidin Level in Helicobacter Pylori-Infected Iron Deficiency Anemia Patients.
Authors: Lee Mark , Song Eun Young , Yun Yeo Min , Yoon So Young , Cho Yo Han , Lee Sun-Young , Kim Sung-Yong
Published: October 2019

Abstract 5101

Background

Helicobacter pylori infection seems to subvert the human iron regulatory mechanism, and thus up-regulate hepcidin that results in unexplained iron deficiency anemia (IDA). We evaluated serum pro-hepcidin levels before and after H. pylori eradication in IDA patients to assess whether it plays a role in H. pylori-related IDA.

Materials and Methods

Subjects diagnosed as unexplained IDA underwent upper gastrointestinal endoscopy and colonoscopy to diagnose H. pylori infection and to exclude gastrointestinal bleeding. Blood sampling were done before H. pylori eradication and after a month. Serum pro-hepcidin level was measured by a commercialized enzyme-linked Immunosorbent assay kit.

Results

Initial serum pro-hepcidin levels were not different between 23 H. pylori-infected subjects (212.9 ± 88.2 ng/ml) and nine non-infected subjects (217.8 ± 56.2 ng/ml) (p=0.879). Serum pro-hepcidin level decreased after either dual oral iron replacement with H. pylori eradication (p=0.011) or H. pylori eradication without iron replacement (p=0.075). It also decreased after iron replacement in non-infected subjects (p=0.086). The reduction ratio of serum pro-hepcidin level after the treatment was not different between three groups (p=0.972).

Conclusions

Serum pro-hepcidin level decreases after either H. pylori eradication or oral iron administration with IDA improvement. Serum pro-hepcidin is related to the status of anemia rather than the presence of H. pylori itself.

Disclosures

No relevant conflicts of interest to declare.
A NOVEL Mutation of the Erythroid-Specific Aminolevulinate Synthase 2 Gene IN A Patient with Pyridoxine Responsive Sideroblastic Anemia and Deferasirox Responsive Hemochromatosis.
Authors: Caliskan Umran , Tokgoz Huseyin , Yuksekkaya Hasan
Published: October 2019

Abstract 5105

A-14 years-old man, admitted to our clinic with weakness and paleness since one month. He has hepatosplenomegaly. Blood tests and peripheral blood smear showed anemia that severe hypochromic, microcytic anemia. There is ringed sideroblasts without dysplastic hematopoiesis in bone marrow cytology. Liver tests were normal. A liver biopsy showed heavy parenchymal iron deposition and grade-III fibrosis. Screening for HFE gene mutations was negative. MR imaging demonstrated that severe iron accumulation in liver and heart. ALAS2 gene screening showed that novel mutation in exon 7 (Gly390Gly, c.1170, C□T). Eventually, was diagnosed as sideroblastic anemia and hemochromatosis. He was treated successfully with pyridoxine and chelating agent (deferasirox, IGL-670). The findings suggest that the Gly390Gly in ALAS2 mutation causes sideroblastic anemia and hemochromatosis, without hereditary hemochromatosis gene mutations. This mutation cause sideroblastic anemia is clinically pyridoxine-responsive. Deferasirox is effective agent for reduce hepatic iron loading in this condition.

Disclosures

Off Label Use: deferasiroks was used for hemochromatosis secondary to congenital syderoblastic anemia.
Cancer Treatment with Gemcitabine Does Not Increase Fetal Hemoglobin Levels.
Authors: Saylor Philip J , Bornhauser Chris , Read William
Published: October 2019

Abstract 5112

Background

Pharmacological induction of fetal hemoglobin (Hb F) can benefit patients with beta-hemoglobinopathies. Hydroxyurea (HU) is the drug most commonly used to induce Hb F. HU is converted to a free radical scavenger in vivo and inhibits the R2 subunit of ribonucleotide reductase. When used in the treatment of myeloproliferative syndromes, HU has been described to increase Hb F in people without hemoglobinopathies (Alter et al, Blood 66(2) 373-5). Gemcitabine is a nucleoside analog chemotherapeutic that inhibits the large R1 subunit of ribonucleotide reductase. We hypothesized that like HU, gemcitabine treatment would cause Hb F percentage to rise. Though oncology patients are a distinct population, gemcitabine induction of Hb F during the treatment of cancer would suggest a potential role for gemcitabine in the management of beta-hemoglobinopathies such as sickle cell disease and beta-thalassemia.

Methods

Participants for this prospective observational cohort study were recruited from the Moores UCSD Cancer Center upon documentation of a cancer diagnosis and oncologist-initiated plans for treatment with single agent gemcitabine. Enrolled subjects were gemcitabine naïve and had not received any chemotherapy for the preceding 4 weeks. Serum Hb F was quantified by electrophoresis prior to each of the first 4 gemcitabine treatments. All other laboratory evaluation was standard-of-care. The primary objective was to assess changes in Hb F among cancer patients in response to their initial 3 treatments with gemcitabine. Enrollment was halted early given the absence of a substantial treatment effect among the first 6 subjects.

Results

Six subjects were eligible. Three were men. Five were Caucasian and one was an Asian/Pacific Islander. All subjects received gemcitabine monotherapy for the treatment of adenocarcinoma of the pancreas or breast at a weekly dose of 1000 mg/m2. Baseline Hb F range was 0 – 1.0%. Four of the six subjects (three women, one man; all Caucasian) experienced no change in Hb F from baseline to the final measurement. Two subjects experienced a 0.4% increase in Hb F from baseline to the final measurement 4 weeks later. Final Hb F range was 0 – 1.4%.

Conclusions

We found that gemcitabine monotherapy for the treatment of cancer did not induce Hb F in this small study cohort. Gemcitabine may lack this ability. Alternative explanations for the absence of clinically meaningful Hb F induction include the small population studied, differences between oncology patients and the general population, short duration of treatment, and intermittent dosing of gemcitabine.

Disclosures

No relevant conflicts of interest to declare.
Immunisation against Human c-MYC Elicits a T-Cell Response and Influences Growth of High Grade B-Cell Lymphoma in Mice.
Authors: Helm Florian , Wilke Andrea , Kammertoens Thomas , Friese Christian , Mautner Josef , Loddenkemper Christoph , Bornkamm Georg , Gerbitz Armin
Published: October 2019

Abstract 5120

Overexpression of the proto-oncogene c-myc due to chromosomal translocation is the hallmark of Burkitt-lymphoma. The evolving high grade lymphoma is dependent on the overexpression of c-myc, which provides the necessary signal to drive uncontrolled proliferation. Therefore loss of function or recognition of c-myc overexpressing cells by c-MYC specific T-cells should result in killing of the target and a halt to lymphoma progression. C-myc is also expressed in a variety of other human malignancies. Peptide prediction reveals several potential foreign epitopes in the context of murine H2b due to 87% homology between human and mouse c-MYC. In this study we explored whether the human c-myc gene product can be a target for T-cell therapy. Wildtype C57BL/6 mice were immunized with recombinant human c-MYC protein in combination with incomplete Freund′s adjuvans and CpG, and were boosted at various time points thereafter using either c-MYC protein or 40mer peptides encompassing the non homologous regions. Control animals were vaccinated with recombinant GFP or OVA protein. C-MYC vaccinated animals displayed a higher IFNg release upon re-stimulation with c-MYC pulsed dendritic cells compared to control vaccinated animals. In ELISPOT assays we observed a higher number of IFNg positive cells (299±17 vs. 122±8.5 (GFP vaccinated) vs. 66±8.5 (OVA vaccinated)). Vaccination using single peptides revealed that peptides spanning the region from amino acid 87-123, 216-255 and 334-376 produced similar results. In addition, using a human c-MYC specific ELISA we were able to detect c-MYC specific antibodies in serum from immunized mice in a concentration up to 40mg/l. Using established cell lines from l-hu-c-myc transgenic mice, where the human c-myc gene is overexpressed due to the juxtaposition of elements of the immunoglobuline lambda locus as found in t(8;22) of Burkitt's lymphoma, we investigated whether vaccination with human c-MYC protein would influence lymphoma growth in a lymphoma transfer model. Animals were s.c. challenged with 0.1 Mio 291cells overexpressing human c-MYC and were monitored for lymphoma growth. C-MYC vaccinated animals (n=15) displayed a delay in tumor onset and a significantly better disease free survival (28 vs. 22 days, p=0.012) compared to control (OVA) vaccinated animals (n=10). This delayed growth was associated with an increased number of infiltrating CD3+/Perforin+ cells. However, all mice eventually succumbed to lymphoma growth, indicating that the T-cell response was not sufficient to control lymphoma growth in the long term. From these data we conclude that the human c-MYC is a possible target antigen for T-cells, but responses are weak and presumably low in frequency.

Disclosures

No relevant conflicts of interest to declare.
Low Absolute CD4 Central Memory Count On Day 30 Is Associated with An Increased Incidence of Chronic Graft-Versus-Host Disease.
Authors: McIver Zachariah A. , Grim Andrew , Naguib Nicolas , Barrett A. John
Published: October 2019

Abstract 516

Early lymphocyte recovery is an important determinant of hematopoietic stem cell transplant (SCT) outcome. We found that robust day 30 lymphocyte counts are associated with a decreased incidence of relapse, decreased rates of both acute and chronic graft-versus-host disease (a-GvHD, c-GvHD), and increased survival. Natural killer (NK) cells contribute to lymphocyte recovery and may be associated with a strong graft-versus-leukemia and anti-GvHD effect. However, the contribution of the recovery of specific T cell subsets to transplant outcome has not been fully explored. We studied 43 patients undergoing a myeloablative matched-sibling T-cell depleted or selectively depleted SCT for a variety of hematological malignancies including AML, ALL, CML, and MDS. Median age was 43 years (range, 13–68), and median CD34 cell dose was 6.1×106/kg (range, 3.1-10.1). Thirty-one patients developed a-GvHD (12 grade I, 18 grade II, 1 grade II). Twelve patients had c-GvHD (7 limited, 5 extensive). On day 30 after SCT a peripheral blood sample was collected on all patients and cryopreserved. In preparation for analysis, mononuclear cells from these samples were thawed and rested overnight. Flow cytometry was then performed on a BD FACS CantoII flow cytometer with multicolor fluorochrome antibodies to CD3, CD4, CD8, CD27, and CD45RO. Subsets were defined as follows: Naive (N) CD27+CD45RO-, Central Memory (CM) CD27+CD45RO+, Effector Memory (EM) CD27-CD45RO+, and Effectors (E) CD27-CD45RO-. Data was analyzed by BD FACSDiva software, and Kaplan-Meier survival statistical analysis was performed on the readouts. Median subset frequencies were CD3+: 254 /μL (range, 75-2085), CD4+ : 132 /μL (range, 3-501), CD8+ :101 /μL (range, 9-1361), CD4+ EM 49/μL (range, 2-252), CD4+CM 60/μL (range, 1-253), CD8+ EM: 50 cells/μL (range 2-977), CD8+ CM 28 /μL (range, 2-401), Naïve and Effector cells were minimally or non-detected in both CD4+ and CD8+ compartments. When T cell subset recovery was correlated with transplant outcomes (a-GVHD, c-GVHD, relapse and survival) one significant association was identified: low CD4+ CM counts correlated with a higher incidence of c-GvHD. Patients that had less than the median value of 60 CD4+ CM cells/μL had a significantly higher likelihood of developing c-GvHD (HR 4.28, p=0.039, see figure). Additionally, when considering degree of disease, low CD4+ CM counts were associated with the severest manifestations of c-GvHD (p=0.021). As a result, we conclude that low absolute concentrations of CD4+ CM cells on day 30 after SCT reflects a deficiency in regulatory mechanisms important in the control of alloreactivity, and may be used as a surrogate marker for individuals at risk of developing c-GvHD.

Disclosures:

No relevant conflicts of interest to declare.
Arsenic Binding to RBCC Domain Modulates Oncogenic PML-RARa and Wild-Type PML as a Key Molecular Mechanism of Arsenic Trioxide Treatment for APL.
Authors: Zhang Xiaowei , Yan Xiaojing , Yang Feifei , Zhou Ziren , Wu Ziyu , Sun Hongbin , Liang Wenxue , Song Aixin , Huang Qiuhua , Zhou Guangbiao , Tong Jianhua , Zhang Yan , Wu Jihui , Hu Hongyu , Chen Saijuan , Chen Zhu
Published: October 2019

Abstract 592

Arsenicals represent one group of the oldest drugs used in both traditional Chinese medicine (TCM) and Western medicine since 2,000 years ago to treat a variety of ailments from periodic fever to cancer. Recently, this ancient remedy has been revived due to its remarkable therapeutic efficacy for acute promyelocytic leukemia (APL) through selective degradation of the leukemogenic PML-RARaƒn as well as the wild-type PML protein. However, the precise molecular mechanism leading to arsenic-initiated modulationƒn of the target proteins remained unclear. Here we show that arsenic directly binds to PML and PML-RARaƒn through their RBCC (RING-B box-coiled coil) domain which contains conserved cysteine/histidine residues with metal-binding ability. Among RBCC domain, the RING and B2 motif are responsible for arsenic binding in cells, with recombinant RING motif showing the highest affinity to arsenic binding in vitro. We also observed that arsenic tends to coordinate with three sulfur atoms from the three conserved cysteines in the RING zinc finger. Arsenic binding alters the native structure of RING coordinated with zinc and induces its oligomerization through arsenic-mediated conformational change by intramolecular coordination as well as cross-linking between two RING motifs. Following conformational change and oligomerization of PML RBCC with arsenic binding, PML and PML-RARa undergoes SUMOylation through enhanced interaction with Ubc9, the E2 ligase for SUMOylation. Our findings provide the evidence that the PML RBCC domain is the direct target of arsenic and that the structural change of PML RBCC induced by arsenic binding facilitates the enhanced interaction with the cellular enzymatic machinery for protein SUMOylation/ubiquitination, which ultimately leads to the degradation of PML-RARa and cell differentiation and/or apoptosis. This mechanism sheds new insights into the mechanism of action of As2O3 for APL treatment, a model of targeted cancer therapy.

Disclosures:

No relevant conflicts of interest to declare.
Platelet ADP Receptor P2Y12 Stabilizes Atherosclerosis Plaques by Regulating Alpha Granule Release and Inflammatory Cell Recruitment
Authors: Li Ding , Wang Yanhua , Wang Kemin , Zhang Lin , Chai Weiran , Sun Yueping , Ruan Changgeng , Chen Guo-Qiang , Gartner T. Kent , Liu Junling
Published: October 2019

Abstract 652

Atherosclerosis, a major cause of cardiovascular disease, is a complicated chronic inflammatory process. A variety of data demonstrate that platelets are crucial players in this pathological process. Platelet activation results in a-granule secretion and the secreted materials include adhesive proteins, chemokines and growth factors, which attract and mediate continuous inflammatory cell attachment to, and infiltration of dysfunctional endothelium and atherosclerotic plaques. Here, we show that the ablation of P2Y12, an ADP receptor highly expressed in platelets and smooth muscle cells, can significantly decrease monocyte infiltration of the aortic root, and thereby negatively affect enlargement of the atherosclerotic lesion in ApoE−/− mice fed on a high fat diet for 5 months. Ablation of P2Y12 in ApoE−/− mice also stabilizes the atherosclerotic plaques that do form in the double knockout mice. Plaque stability was demonstrated by Immunohistochemistry studies that revealed greater fibrous and diminished macrophage content in the atherosclerotic plaques. Bone marrow transplantation experiments confirmed that platelet P2Y12, but not smooth muscle P2Y12, is the key factor responsible for atherosclerosis formation. Further work demonstrated that P2Y12, through inhibition of the cAMP/PKA signaling pathway, critically regulates platelet expression of p-selectin and the release of PF4. This demonstration is crucial because PF4 is a chemokine documented to be important for atherosclerosis development. The results of in vitro assays demonstrated that under standard conditions, P2Y12+/+ platelets, but not P2Y12−/− platelets, released their a-granule contents during platelet activation and thereby enhanced monocyte migration. Compared to results obtained using P2Y12+/+ApoE−/− mice under the same conditions, the basal levels of PF4 and SDF-1a in platelet-poor plasma were significantly lower in P2Y12−/−ApoE−/− double knockout mice fed a high-fat diet, thereby providing in vivo support for our thesis that P2Y12 affects atherogenesis by regulating a-granule secretion. Our results point out that platelet P2Y12 modulates atherogenesis primarily through regulation of a-granule release, and secondarily by the resulting inflammatory cell recruitment, and therefore serves as a potential drug target for atherosclerosis prevention, in addition to being the target for prevention of thrombus formation in response to plaque rupture.

Disclosures:

No relevant conflicts of interest to declare.
Cytogenetics Abnormalities Predict the Outcome of Allogeneic Transplantation In AML: A CIBMTR Study
Authors: Armand Philippe , Perez Waleska , Zhang Mei-Jie , Kim Haesook , Klumpp Thomas R. , Cin Paola Dal , Waller Edmund K , Litzow Mark R. , Liesveld Jane L. , Lazarus Hillard M. , Artz Andrew , Gupta Vikas , Savani Bipin N. , McCarthy Philip L. , Cahn Jean-Yves , Schouten Harry , Finke Jurgen , Ball Edward D. , Aljurf Mahmoud , Cutler Corey , Rowe Jacob M. , Antin Joseph H. , Isola Luis M , Bartolomeo Paolo Di , Camitta Bruce , Miller Alan M , Cairo Mitchell S. , Stockerl-Goldstein Keith E. , Sierra Jorge , Savoie Mary Lynn , Halter Joerg , Stiff Patrick J , Nabhan Chadi , Jakubowski Ann A. , Bunjes Donald , Petersdorf Effie W. , Devine Steven M , Maziarz Richard , Bornhauser Martin , Lewis Victor A , Marks David I. , Bredeson Christopher N , Soiffer Robert , Weisdorf Daniel J.
Published: October 2019

Abstract 680

Cytogenetics play an essential role in determining the prognosis of patients with AML. However, there is still no validated cytogenetics grouping scheme that specifically applies to patients undergoing allogeneic stem cell transplantation, which hampers accurate prognostication and risk stratification. We studied 821 adult patients (median age 41, range 18–74) who underwent SCT between 1999 and 2004 for AML (excluding APL) in CR1 or CR2 and who were reported to the CIBMTR from centers with >20 patients meeting study criteria. 75% of patients received a myeloablative conditioning. 496 patients had a normal karyotype. The primary cytogenetics reports were manually reviewed for 92% of the patients with an abnormal karyotype. We compared the ability of the 6 existing grouping schemes (MRC, CALGB, EORTC/GIMEMA, SWOG/ECOG, DFCI, and Monosomal Karyotype (MK) classifications) to stratify patients, using both the Akaike Information Criterion in multivariable models and the C-statistic. Among all existing schemes, the DFCI system provided a marginally superior stratification for overall and leukemia-free survival. We also built a new classification using individual cytogenetic abnormalities in a Cox model that included other significant covariates (performance status, therapy-related disease, conditioning intensity, graft source, donor match, duration of CR1, and gender match). This CIBMTR scheme (see Table), which is similar to the DFCI scheme except for the inclusion of patients with t(8;21) in the intermediate group, could stratify patients into 3 groups with similar treatment-related mortality but significantly different overall survival, leukemia-free survival, and incidence of relapse. This scheme appeared to apply to both patients in CR1 and in CR2 (see FiguresF2). This transplant-specific scheme could be adopted for prognostication purposes and to stratify patients with karyotypic abnormalities entering transplantation clinical trials. Future studies may clarify the relative outcome of patients with t(8;21) and refine this scheme with the inclusion of molecular abnormalities.

Disclosures:

No relevant conflicts of interest to declare.
A Minor Genetic Variation In the Granzyme B Gene Predicts Relapse After HLA-Fully-Matched Unrelated Bone Marrow Transplantation for Hematologic Malignancies
Authors: Espinoza Luis J. , Takami Akiyoshi , Yamada Kayoko , Yamada Mayu , Motohashi Yuki , Nakata Katsuya , Morishita Eriko , Ohtake Shigeki , Nakao Shinji
Published: October 2019

Abstract 681

Granzyme B (GZMB) is a serine protease with important roles mediating target-cell apoptosis induced by natural killer cells and cytotoxic CD8+ T cells. The polymorphism rs8192917 (A55G) in the GZMB gene has been reportedly associated with the ability of effector cells to secrete GZMB. In this study we analyzed the impact of GZMB polymorphism on transplant outcomes in patients undergoing unrelated HLA-fully-matched myeloablative bone marrow transplantation (BMT) through the Japan Marrow Donation Program. The GZMB genotypes were retrospectively analyzed in a cohort of 360 pairs of patients with hematologic malignancies and their unrelated donors. The presence of the G/G genotype in the donor side was associated with a significantly higher incidence of relapse (62% vs. 32%, P=0.04; Fig 1). Multivariate analysis revealed that the donor G/G genotype, which is expected to produce lower levels of GZMB after stimulation, (Girnita et al, Transplantation 2009) was an independent risk factor for relapse (hazard ratio, 4.17; 95% confidence interval, 1.63 to 10.70; P=0.003). The host GZMB genotypes did not significantly influence the transplant outcomes. These results suggest an association of low GZMB secretors with disease relapse. These could therefore be useful in selecting the donor and creating therapeutic strategies for improving the final outcome of allogeneic BMT.

Disclosures:

No relevant conflicts of interest to declare.
Platelet Factor 4 Levels Inversely Correlate with Platelet Transfusion Needs In Pediatric Patients Treated for Standard Risk Acute Lymphoblastic Leukemia
Authors: Lambert Michele , Reznikov Alisa , Nguyen Yvonne , Rauova Lubica , Poncz Mortimer
Published: October 2019

Abstract 725

Platelet factor 4 (PF4) is a chemokine found almost exclusively in megakaryocytes and platelets. PF4 has been previously shown to be a negative paracrine inhibiting megakaryopoiesis in vitro in humans and mice. We confirmed this finding and also found an inverse correlation between PF4 and steady-state platelet counts in mice. In both chemotherapy- and radiation-induced thrombocytopenia, platelet PF4 levels inversely correlated with platelet count recovery after bone marrow injury, and blocking this effect ameliorated the thrombocytopenia. We now asked whether platelet PF4 levels are of clinical relevance on human platelet biology in patients undergoing chemotherapy. We selected pediatric patients who had completed treatment for standard risk acute lymphoblastic leukemia at the Children's Hospital of Philadelphia, as this was a fairly large population that have reached remission after relatively uniform therapy. Enrolled patients had completed therapy since January 1999. Blood samples were obtained and medical records were retrospectively reviewed for platelet counts, platelet transfusion requirements and duration of therapy during delayed intensification (DI). DI was chosen for investigation as a preliminary study showed that 35% of our patients require platelet transfusion during DI and need for transfusion at that point in therapy is unlikely to be related to primary underlying bone marrow disease. To date, 68 subjects have been enrolled. Sixty-two subjects had evaluable PF4 levels. PF4 levels were independent of age and sex. Leukemia survivors did not have significantly different PF4 levels when compared to a pediatric control population. There was a direct relationship between measured total PF4 level and platelet count (Pearson r 0.36, p<0.006) although contrary to animal studies, there was no correlation between PF4 per platelet and platelet count. Transfusion data from the first 22 patients have been evaluated. Patients who did not require platelet transfusion during DI had markedly lower PF4 per platelet (6.35 ± 1.85 SE) when compared to patients who required transfusion (13.26 ± 1.89 SE, p<0.02). In addition, duration of therapy (for girls) was inversely correlated with PF4 per platelet (r 0.689, p=0.04), consistent with animal data in which platelet count recovery was inversely correlated with PF4 per platelet. These data suggest that PF4 may be an important in vivo regulator of human platelet counts in the setting of bone marrow injury. Further clinical studies will confirm these findings and begin to explore potential interventions to allow for intensified chemotherapy regimens in subjects at risk for more severe chemotherapy-induced thrombocytopenia based on their level of this negative paracrine of megakaryopoiesis.

Disclosures:

No relevant conflicts of interest to declare.
Next-Generation Sequencing Analysis of 23 Therapy-Related Acute Myeloid Leukemia Transcriptomes
Authors: McNerney Megan E. , Brown Christopher D. , White Kevin P.
Published: October 2019

Abstract 850

Therapy-related acute myeloid leukemia/myelodysplastic syndrome (t-AML) is a fatal complication that occurs in up to 5% of patients treated with chemotherapy or radiation. As more cancer patients achieve longer remissions, t-AML has increased in incidence, yet remains refractory to current therapy, with a median survival of 8–10 months. T-AML is thought to be a direct product of genetic mutations induced by alkylating agents, topoisomerase-II inhibitors, and radiation. Two main subtypes of t-AML have been described that differ in their morphology, cytogenetic karyotype, and prognosis: alkylator-agent-induced and topoisomerase-II inhibitor-induced. To identify expressed genetic variants that distinguish the two subtypes of t-AML, we have sequenced the leukemic mRNA of 23 t-AML samples using Illumina paired-end technology, yielding over 1 billion base pairs of aligned sequence per sample. The alkylator agent-related group is comprised of 13 samples with deletions of part or all of chromosomes 5 and/or 7, or with complex karyotypes. 10 samples with other karyotypes, including normal karyotype and balanced translocations, constitute the topoisomerase-II-related group. In addition, 13 de novo AML patient samples with a spectrum of cytogenetic abnormalities similar to the t-AML samples have been similarly sequenced. We have found over 13,000 single nucleotide variants (SNVs) per sample. SNP array analysis on 2 samples demonstrates 92% sensitivity and 98% specificity for SNP identification. Phenotypically relevant SNVs are prioritized by population-level allele frequency estimates, evolutionary constraint estimates, and the predicted coding region change. This analysis has yielded approximately 200 rare, predicted deleterious SNVs per sample. Some of these variants include those previously reported to be aberrant in AML (for example, FLT3, P53, RUNX1, PTPN11, CBL, and TET2), validating our analysis pipeline. Putative disease-relevant SNVs are being compared between the two main subtypes of t-AML, as well as between therapy-related and de novo AML by analyzing the frequency of the aberrant gene or biological pathway within the groups. We have found that SNVs frequently occur in the same gene or pathway in multiple samples. These findings are being validated within tumor and paired normal DNA, as well as in a larger cohort of 100 t-AML leukemia samples and 50 de novo AML leukemia samples using single-base primer extension assays. Furthermore, comparisons of transcript expression level and isoform usage are being made between groups and cytogenetic types. This information will allow the identification of pathways altered in t-AML, correlation of mutations with clinical outcome, identification of potential biomarkers, and generation of candidate targets for leukemia therapeutics.

Disclosures:

No relevant conflicts of interest to declare.
The COPII Pathway and Hematologic Disease
Authors: Ginsburg David
Published: October 2019

Abstract SCI-18

Combined deficiency of factor V and factor VIII is a rare autosomal recessive bleeding disorder associated with coordinately decreased levels of two key blood coagulation factors. Genetic studies of affected families identified a novel pathway for the selective transport of a subset of secreted proteins, including factors V and VIII, from the ER to the Golgi. Patients with this disorder carry mutations in one of two components of this pathway, LMAN1 or MCFD2, which together form a receptor for the recruitment of specific cargo to the COPII vesicle for export from the ER. Mutations in other COPII components have recently been identified in other human genetic disorders, including deficiency of SEC23B in patients with congenital dyserythropoietic anemia type II. The molecular pathogenesis of these disorders will be discussed, along with the insights these findings have provided into the function of the mammalian COPII pathway.

Disclosures:

No relevant conflicts of interest to declare.