bluuebunny/crossref_metadata_2025 huggingface dataset:
Therapy for Chronic ITP - the Patients' Perspective: US and Germany.
Authors: Matzdorff Axel C. , Arnold Gabriele , Hummler Simone S. , Grotzinger Kelly M. , Horblyuk Ruslan V.
Published: October 2019

Abstract 4451

Background

Chronic ITP is a rare disorder. Steroids are the usual 1st line therapy. Despite published guidelines there is considerable variation in 2nd and 3rd line therapies. Objective of this study was to compare therapies and patient-perceived treatment effects between the US and a European country (Germany).

Methods

Members of a US and a German patient support group >18 y with self-reported history of chronic ITP (>6 mo) were surveyed. A questionnaire was developed from literature review with clinician and patient input, and administered on-line. Demographics, patient histories, ITP therapy-related effects and side effects were recorded.

Results

712 subjects participated in both surveys (US 589, GER 123). Most were female (US 78%, GER 63%), and diagnosed with ITP for ≥5 years (US 59%, GER 70%). Many subjects had a minimum platelet count under 50.000/μl during the last year (US 68%, GER 66%), dry (US 79%, GER 72%) and wet purpura (US 46%, GER 64%). Corticosteroids were the most frequently reported therapy (USA 92%, GER 86%) followed by IVIg (USA 55%, GER 55%), splenectomy (USA 39%, GER 28%), rituximab (USA 36%, GER 22%, note: GER public health insurance does not reimburse rituximab). Patients experienced steroid treatment as particularly bothersome (both US and GER 90% report weight gain, moon face). For rituximab US patients reported most commonly fatigue, tiredness, weakness (47%), GER patients infusion reactions (55%). For splenectomy US patients reported most commonly scarring (67%), GER patients antibiotic use (38%). 56% of splenectomized GER pts. would not recommend this procedure to other patients.

Conclusions

Responding patients in the US and GER survey are both heavily pre-treated and report comparable side effects of those treatments. 1st line (steroid) and emergency treatment (IVIg) does not differ while 2nd/3rd line therapies vary. US patients are more likely to receive invasive (splenectomy) or even “off-label” therapies (rituximab) to have a chance of cure, presumably to reduce the financial burden of their disease. GER public health insurance covers all costs (except rituximab) and this might explain why GER patients are more willing to delay or even forego splenectomy. This implies that GER patients might be more inclined to try thrombopoietin receptor agonists before splenectomy. Future studies need to consider the effect of different health care systems on treatment decisions.

Disclosures:

Matzdorff: GlaxoSmithKline: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; AMGEN: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Baxter: Consultancy, Honoraria. Off Label Use: Rituximab for chronic ITP. Hummler:GlaxoSmithKline: Employment. Grotzinger:GlaxoSmithKline: Employment. Horblyuk:GlaxoSmithKline: Employment.
Alemtuzumab and Rituximab in the treatment of refractory autoimmune Cytopenias.
Authors: Gomez-Almaguer David , Solano-Genesta Manuel , Cantu-Rodriguez Olga , Gutierrez-Aguilar Cesar Homero , Jaime-Perez Jose Carlos , Tarin-Arzaga Luz Carmen , Gomez-Guijosa Miguel Angel
Published: October 2019

Abstract 4458

Introduction

The combination of rituximab and alemtuzumab have been used to treat CLL. This combination is feasible without excessive toxicity. Both antibodies have been used as monotherapy for the treatment of autoimmune diseases, including autoimmune hemolytic anemia (AHA) and Immune thrombocytopenic purpura (ITP).

Objective

Evaluate the efficacy and safety of the combination of low dose alemtuzumab and rituximab in patients with AHA and ITP.

Material and methods

patients diagnosed with AHA (clinical and biochemical data of hemolysis) or ITP (less than < 50 K/μl) who failed or relapsed after first line therapy (steroids) were included. Basal lymphocyte count and immunoglobulin levels were assessed. Patients received alemtuzumab 10 mg SC on days 1,2 and 3 followed by rituximab 100 mg IV on days 4,11,18 and 25. Complete response (CR) was defined as normal hemoglobin in the absence of hemolysis and platelet count >150K/μl; and partial response (PR): increase of 2 g/dl of hemoglobin and platelets 50-149K/μl The effect of the antibodies combination was evaluated by lymphocyte B, T and NK count 8 and 24 weeks after their administration. Prophylactic bactrim, acyclovir and itraconazole were given for the first 2 months.

Results

We included 13 patients, and eleven were evaluable, 5 patients in the AHA group and 6 in the ITP group, demographic data is shown on table 1. For AHA the 5 patients achieved CR with a median follow up of 37 weeks; only 1 patient relapsed after the 49th week of follow up. For the ITP group 2 patients achieved CR, 2 patients PR and 2 were classified as non responders. Alemtuzumab and rituximab combination quickly depleted B lymphocites falling to zero or near zero at the 8th week determination, with a sustained decrease at 24th week, in which levels did not return to pre-treatment levels.

Conclusions

These initial results indicate that low dose alemtuzumab and rituximab combination is safe and demonstrate promising activity for AHA and ITP refractory patients including durable complete responses and as a steroid spring therapy.

Disclosures:

Off Label Use: alemtuzumab for refractory autoimmune cytopenias rituximab for refractory autoimmune cytopenias.
Racial Disparities in Pediatric Immune Thrombocytopenic Purpura.
Authors: Perkins Tiffany , Jayabose Somasundaram , Ozkaynak Mehmet , Tugal Oya , Sandoval Claudio
Published: October 2019

Abstract 4464

Background

Immune thrombocytopenic purpura (ITP) occurs in children with an estimated incidence of 5/100,000; however, it is not known whether racial disparities in the incidence of pediatric ITP exist. We hypothesize that because petechiae may go unrecognized in black children the observed incidence of ITP in this race may be less. The purpose of this study was to analyze an established pediatric ITP population at New York Medical College in order to assess whether such disparities exist amongst races and to determine the presenting features of ITP in black children.

Methods

Retrospective chart review of 630 pediatric ITP patients diagnosed and treated at New York Medical College from 1985 to 2009 was performed to determine the incidence in various races and to study the presenting features in black children. The patients' race was determined by review of medical records, physician recollection and when necessary by contacting patients by phone. Observed rates of ITP in various races were compared to expected rates given the incidence of ITP and the area's demographic construct. Census of children younger than 19 years residing in the Hudson Valley (Westchester, Putnam, Dutchess, and Rockland counties and Danbury, Connecticut) was obtained from the United States Census Bureau. We used a χ2 “goodness-of-fit” test to determine whether the sample distribution differed from the population distribution.

Results

Racial demographics were available for 530 of 630 (84%) ITP patients. The estimated racial demographics of children younger than 19 years residing in the Hudson Valley are as follows: total population, 468,550; 75% Caucasian; 10% Hispanic 10% black; and 5% Asian. Of the 530 identified, 406 (76.6%) were found to be Caucasian, 55 (10%) Hispanic, 36 (7.5%) Black, and 33 (6.2%) Asian. If ITP is detected equally amongst races, one would expect to find 397.5 White patients (observed expected difference [O – E] 8.5 with a residual of 0.426), 53 Hispanic (O - E 2 with a residual of 0.275), 53 black patients (O – E -17 with a residual of -2.335), and 26 Asian (O – E 6.5 with a residual of 1.263). The distribution of our sample over race groups compared with the distribution of the population over race groups had a p-value of 0.063 with the largest contribution due to under-representation of black children in our sample. Of the 36 black children with ITP 18 (50%) were found to present with bruising/petechiae, 4 (11%) presented with a combination of wet purpura and bruising/petechiae, 3 (8%) presented with wet purpura alone, and 11 (31%) were asymptomatic.

Conclusion

The incidence of ITP in black children may be less than in Caucasian, Hispanic, and Asian children.

Disclosures:

No relevant conflicts of interest to declare.
Heparin Induced Thrombocytopenia; A Diagnostic Dilemma.
Authors: Reagan John L. , Dalia Samir , Butera James N.
Published: October 2019

Abstract 4468

Introduction

The diagnosis of HIT can be challenging. Short of the serotonin release assay, which can be difficult to get in a timely manner, the 4T method developed by Lo et al., in conjunction with the HIT elisa assay (EIA), is the best established criteria for the rapid diagnosis of Heparin Induced Thrombocytopenia (HIT). We sought to determine how often the 4Ts were being applied at a large academic institution. In addition, we sought to understand the outcome of patients started on direct thrombin inhibitors (DTIs) who did not meet the 4T/EIA criteria for HIT.

Methods

A retrospective case series identified patients placed on DTI for HIT treatment from 10/2006 to 5/2008. The 4T pretest probability was then applied retrospectively. HIT EIA Ab testing, optical densities, duration of DTI treatment, and bleeding incidence were recorded. Liberal and modified conservative criteria from Lo et al.'s study were used to define patients with HIT. Overdiagnosis of HIT was then recorded based on the above criteria.

Results

76 patients were included for analysis. The average age was 61.4 years. Based on the 4T pretest probability, 30 patients were classified as low risk, 37 intermediate risk, and 9 high risk for HIT. The average HIT OD for low, intermediate, and high pretest probability was 0.49, 1.17, and 2.05.

76% and 36% of patients treated with DTIs for HIT met the liberal and modified conservative HIT definitions, respectively. 18 patients (24%) did not meet liberal HIT criteria but remained on DTI treatment for 1 to 60 days. Cost of DTI overuse in this subset was $84,960. Cost of DTI use in patients who did not meet modified conservative criteria was $462,246. All 16 patients who developed bleeding complications fulfilled a liberal diagnosis of HIT while only 6 of these patients fulfilled the modified conservative definition of HIT.

Conclusions

1) The 4T method for diagnosing HIT is not being applied to a large percentage of patients in a tertiary academic center. 2) There is an overdiagnosis of HIT regardless of definition criteria with 24% of patients treated for HIT not meeting the liberal definition while 64% of patients did not meet the modified conservative definition. This overdiagnosis has both a significant monetary and morbidity cost.

Disclosures:

No relevant conflicts of interest to declare.
The Significance of Anti-Platelet Antibodies Associated with Antibiotic Use, A Descriptive Analysis.
Authors: Shah Ruta M , Szczepiorkowski Zbigniew M , Leach, MS, MT Miriam K , Zuckerman Richard A
Published: October 2019

Abstract 4470

Introduction

Antibiotics (Abx) have been implicated in immune thrombocytopenia via drug dependent platelet antibodies (DDPA). Our institution has had a DDPA assay available since 1994 which is used to guide clinical decisions. We performed a retrospective review to determine the significance of DDPA.

Methods

We reviewed the medical records of patients (pts) who tested positive for abx DDPA between 1994 and 2006 and performed a descriptive analysis. Detection of DDPAs was performed using a previously described, modified solid phase red cell adherence assay that detects hapten or immune complex reactions.

Results

A total of 71 pts were included in this analysis. Multiple classes of abx were tested. Platelet nadir was <50 in 70%, between 50 and 100 in 26% and over 100 in 4% of pts. Pts had between 1 and 4 abx tested: 37% had 1, 37% had 2, 15% had 3, and 11% had 4 tested; 65% of pts had one abx positive and 35% had >1 abx positive for DDPA. Of those with >1 abx tested (n=45), 14 (31%) had all tested abx positive. 53% of abx testing took place on or after the abx stop date and 32% of abx tested were administered for <=3 days. Only 29 of 38 pts receiving heparin were tested for heparin-associated antibodies, and 14 had positive results. 49 pts had other non-abx drugs tested, 19 with positive DDPA. Thus, 35% had alternative non-abx testing positive for DDPA. 31% of pts had bleeding complications and 35% of pts died during hospitalization. Excluding pts who died and those with non-abx positive DDPA left 25 pts, median platelet counts were: abx start=142; nadir=22, abx discontinuation=46, hospital discharge=192.

Conclusions

Antibiotic DDPA testing is usually performed in ill pts with multiple medical complications and comorbidities. Abx were stopped for concern of DDPA in a number of pts for whom typical immune thrombocytopenia was not present or alternative explanations could be found. Though some pts exhibited improvement in platelet count after abx were stopped, a clear understanding of which pts may benefit from DDPA testing could not be determined based on the retrospective nature of this study and the complexity of pts histories. Further research is necessary to clarify the clinical applicability of DPPA testing.

Disclosures:

Szczepiorkowski: Cersus Corporation: Research Funding; CaridianBCT: Research Funding; BASF: Research Funding; Terumo Corporation: Research Funding; Fenwel, Inc - Scientific Advisory Board: Research Funding.
Bone Marrow Transplant Induces Pulmonary Vascular Remodeling in Mice.
Authors: Aliotta Jason M , Del Tatto Michael , Dooner Mark , Pereira Mandy , Quesenberry Peter J.
Published: October 2019

Abstract 4480

Objective

Pulmonary complications are common in bone marrow transplant (BMT) recipients and often of an infectious etiology or attributed to intensive conditioning regimens. Cases of pulmonary arterial hypertension have been described in the setting of autologous BMT but are believed to be the result of acquired infections or previously-administered chemotherapy. Whether transplanted marrow cells themselves are toxic to the recipient's lungs is unknown.

Methods

To address this, non-irradiated female BALB/C mice were transplanted with 5-6×107 whole bone marrow (WBM) cells from male BALB/C mice daily for four days on week zero, then again on week eight (4.5×108 cells total/mouse), or an equal volume of diluent (control). On week 24, transplanted mice received 1000 cGy of chest-only radiation or no radiation. On week 32, histochemical and immunohistochemical analyses were performed on recipient's lungs.

Results

Bone marrow chimerism was not significantly different in the irradiated and non-irradiated cohorts at the time of sacrifice (average 45.11+6.25% Y chromosome+, all mice). Recipient lungs contained few non-hematopoietic donor marrow-derived cells. These cells were exclusively epithelial (Y+/cytokeratin+), primarily type II pneumocytes (Y+/prosurfactant C+), while no endothelial (Y+/von Willebrand+) or vascular smooth muscle (Y+/alpha-actin+) cells were identified. Irradiated and non-irradiated cohorts had similar quantities of these cells (0.80+0.22% vs. 0.51+0.08% Y+/cytokeratin+; 0.37+0.08% vs. 0.32+0.12% Y+/prosurfactant C+, p>0.2). Pulmonary vessel wall thickness-to-blood vessel diameter ratios (PVWT/D) were significantly increased in the non-irradiated cohort compared with controls and these ratios were further increased in the irradiated cohort (141+5.75%, 161+5.34% of control, p<0.05 comparing all groups).

Conclusions

These data indicated that marrow cell infusion alone results in pulmonary vascular remodeling and this effect is augmented by radiation injury. These changes are independent of transplanted marrow cell conversion to pulmonary vascular endothelial, smooth muscle cells. These studies suggest that transplanted cells may be lung toxic entities in the setting clinical BMT.

Disclosures:

No relevant conflicts of interest to declare.
Gene Therapy of Chronic Myelogenous Leukemia Using Pseudotyped Recombinant Adeno-Associated Viral Vectors.
Authors: Sellner Leopold , Veldwijk Marlon R , Kleinschmidt Jürgen A. , Laufs Stephanie , Topaly Julian , Zeller W. Jens , Wenz Frederik , Fruehauf Stefan
Published: October 2019

Abstract 4506

Introduction

Gene transfer into malignant leukemia cells can be of relevance to overcome conventional therapy-resistance. Either suicide or immune stimulating gene therapy vectors may be a tool for second line treatment of imatinib-resistant chronic myelogenous leukemia (CML). Unfortunately, for gene transfer into CML cells, most current vector systems either lack sufficient transduction efficiency or an acceptable safety profile. Conventional adeno-associated virus (AAV) based vectors have an advantageous safety profile, yet lack the required efficiency.

Methods

Pseudotyped recombinant adeno-associated viruses of the serotypes 2/1 to 2/6 (rAAV2/1 to rAAV2/6) were screened on a panel of human CML cell lines and primary CML cells to determine their gene transfer efficacy. Additionally, double-stranded self complementary rAAV (scAAV) were used to determine possible second strand synthesis limitations.

Results

On CML cell lines, generally rAAV2/2 and rAAV2/6 were most efficient (Fig. 1). For both, an interesting difference in transduction efficiency between the imatinib-resistant LAMA84-R and imatinib-sensitive LAMA84-S cells were observed. On primary human CML cells, rAAV2/6 proved to be significantly more efficient than the other tested vectors (4.6% ± 5.3% GFP-positive cells, p=0.011). Additionally, the transduction efficiency could be significantly increased by using scAAV vectors (scAAV2/6: 43.1% ± 25.9% GFP-positive cells, p<0.001 vs rAAV2/6; Fig. 2). Our data suggest that both the conversion of the single stranded AAV genome to double stranded DNA, as well as cell binding/entry are rate limiting steps in efficient transduction of primary human CML cells with AAV vectors. Furthermore, donor-dependent differences in gene transfer efficiency were observed. Of note, data from experiments on CML cell lines seems to provide limited information about the transduction efficiency of rAAV vectors on primary CML cells.

Conclusions

Overall, pseudotyped rAAV and scAAV vectors offer efficient clinically relevant gene transfer into human CML cells with a potential for future clinical application.

Disclosures:

No relevant conflicts of interest to declare.
Influence of Magnetic Fe3O4 Nanoparticle On the Lymphocyte and Macrophage Function of Mice.
Authors: Chen Bao-An , Jin Nan , Wang Jun , Ding Jia-Hua , Gao Chong , Cheng Jian , Chen Ning-Na , Zhao Gang , Wang Xue-Mei , Pei Xiao-Ping
Published: October 2019

Abstract 4519

Objective

In this paper we investigate effects of magnetic nanoparticle of Fe3O4 (MNP-Fe3O4) on the murine immune system especially in lymphocyte and macrophage function.

Methods

ICR mice were assigned randomly into four groups which were treated with normal saline, the dose of 300mg/kg, 600mg/kg and 1200mg/kg MNP-Fe3O4. The mice were killed when they were exposed by intragastric administration after 2 weeks. The ratios of spleen to body weight, lymphocyte transformation rate in spleen suspension, phagocytic index of macrophage in belly cavity were detected.

Results

The ratios of spleen to body weight in all experimental groups were not significantly difference in comparison to the control group(P>0. 05). The lymphocyte transformation rate in spleen suspension in experimental groups were all higher than that of the control group, especially in the media dose group(600mg/kg)(P < 0. 05). And the results of phagocytic index of macrophage in belly cavity indicated that though all experimental groups showed that they enhanced the phagocytic function of macrophage more than the control group, media dose group(600mg/kg) could increase the phagocytic index more than the other two experimental group(P < 0. 05).

Conclusion

It is concluded that suitable dose of MNP-Fe3O4 supplement enhance cellular immune function and phagocytic funtion of macrophage of mice.

Disclosures:

No relevant conflicts of interest to declare.
Erythropoietin Stimulating Agents (ESAs): Reassessment of Use in Cancer and Chronic Kidney Disease (CKD) Settings in the United States, Canada, and Europe.
Authors: Bennett Charles L , Samaras Athena T , McKoy June M , Edwards Beatrice J , Zheng Mi , Fisher Matthew J , Boyle Simone N , Trifilio Steven M , Boen Monica , Deleon Dianne , West Dennis P
Published: October 2019

Abstract 4538

Introduction

International reassessments of erythropoiesis stimulating agents (ESAs) for anemia have recently occurred. While ESAs prevent blood transfusions and, improve select quality of life domains among chronic kidney disease (CKD) patients, 8 trials individually identified increased mortality and/or tumor progression among cancer patients treated with ESAs. For CKD, one study of non-dialysis CKD patients targeted to higher versus lower hemoglobin (Hb) levels identified 34% increased relative risk (17.5% vs. 13.5%) for death, acute myocardial infarction, congestive heart failure hospitalization, or stroke with a higher Hb target.

Methods

Guidelines, notifications from regulatory agencies and manufacturers, reimbursement policies, and utilization for ESAs in the cancer and CKD settings within the U.S., Europe, and Canada were reviewed.

Results: Cancer Setting

In 2008, the Food and Drug Administration (FDA) restricted ESAs from cancer patients seeking cure. Reimbursement is limited to Hb levels < 10 g/dL. In the U.S., ESA usage increased 340% between 2001 and 2006, and decreased 60% since 2007. The European Medicines Agency (EMEA) reports that ESA benefits do not outweigh risks. In Europe, ESA reimbursement is included in global provider payments. Between 2001 and 2006, ESA use increased 51%; since 2006, use has decreased 10%. Canadian manufacturers recommend ESA usage based on patient preference. In Canada, reimbursement for myelosuppressive chemotherapy with ESAs is restricted to Hb levels of 10-11 g/dL in Alberta and 11-12.5 g/dL in British Columbia. Usage increased 20% between 2004 and 2007.

CKD Setting

The FDA recommends Hb levels < 12 g/dL and Medicare reimbursement is restricted to Hb levels ≥ 13 g/dL. Between 2001 and 2006, usage increased 480% (non-dialysis) and 40% (dialysis) in the U.S. Since 2007, ESA use has decreased 30% (non-dialysis) and 17% (dialysis). The EMEA recommends target Hb levels of 10-12 g/dL. In Europe, ESA usage has increased 10% since 2001. The 2009 Canadian label recommends Hb levels < 12 g/dL. Within Canada, ESA usage increased 30% since 2004.

Conclusions

While reassessments of ESA safety have occurred internationally, safety concerns in the U.S. have resulted in marked decrements in ESA use among cancer and CKD patients, whereas in Europe and Canada ESA use has increased over time.

Disclosures:

No relevant conflicts of interest to declare.
From Bench to Bedside: Hematopoietic Growth Factors (HGFs)- a Cause of Serious and Unanticipated Class Toxicities.
Authors: Bennett Charles L. , Polish Ari , Samaras Athena T , Zheng Mi , West Dennis P , Trifilio Steven M , Edwards Beatrice J , Boyle Simone N , Fisher Matthew J , McKoy June M
Published: October 2019

Abstract 4555

Introduction

HGFs have vastly improved the management of anemia, neutropenia, and thrombocytopenia, but are associated with unexpected toxicities.

Methods

Toxicities associated with granulocyte, erythroid, and thrombopoietic growth factors were reviewed. Data sources included systematic reviews, clinical trials, guidelines, and materials disseminated by the Food and Drug Administration (FDA), the European Medicines Agency (EMEA), and Canada Health and their advisory committees; product labels and safety notifications disseminated by manufacturers; and utilization data. Data abstracted were thromboembolic complications, immune-mediated cytopenias, bone marrow toxicities, systematic and synergistic toxicities, and regulatory responses.

Results

Granulopoiesis agents

Pulmonary infiltrates were associated with administration of HGFs with bleomycin or chest radiation. Increased myelodysplasia (MDS) or acute myeloid leukemia (AML) risks among chemotherapy (chemo)-treated breast cancer patients were reported.

ESAs

Increased mortality and tumor progression were seen when cancer patients received ESAs versus placebo; increased mortality and cardiovascular events were reported when chronic kidney disease (CKD) patients received ESAs targeted to complete anemia correction. Between 1998 and 2003, 191 CKD patients developed neutralizing antibodies and pure red cell aplasia (PRCA) following recombinant erythropoietin administration.

Thrombopoietins (TPO)

TPO receptor agonist administration is associated with bone marrow reticulin and collagen deposition. 3% of healthy volunteers who received repeated rHuMGDF developed neutralizing autoantibodies and severe thrombocytopenia.

Conclusions

While ESAs, granulopoietic factors, and thrombopoietins have important clinical benefits, they also retain the potential to exhibit serious and unanticipated toxicities.

Disclosures:

No relevant conflicts of interest to declare.
AML FAB Subtypes Shows Different Proportion of Leukemia Initiating Cell Which Has Lower Single Cell Dividing Property.
Authors: Kim Hye Ran , Shin Myung-Geun , Kim Yester , Kim Hyeoung Joon , Cho Duck , Kee Seung-Jung , Kim Soo-Hyun , Shin Jong-Hee , Suh Soon-Pal , Ryang Dong-Wook
Published: October 2019

Abstract 4568

Background

Acute myeloid leukemias (AML) are maintained by a subpopulation of leukemia initiating cell that can regenerate themselves as well as give rise to more differentiated and less proliferative cells that constitute the bulk of the disease. Recent studies discovered both the nature of leukemia stem cells and their origin. Immunophenotype of AML stem cell (ASC) is known as CD34+CD38– leukemic cells. Yet, despite their critical importance, much remains to be learned about the frequency, proportion and dividing activity of ASC in a single cell levels. To address these issues, the current study investigated the frequency, proportion according to AML FAB subtypes and single cell plating efficiency of ASC from bone marrow (BM) at the initial diagnosis.

Materials and methods

The proportion and frequency of ASC were examined on BM samples of twenty AML patients using single cell sorter (BD FACS Aria cell sorter, BD Biosciences, USA). Growth and proliferation capacities of single normal hematopoietic stem cells and LSC were determined using plating efficiency (number of the wells in which more than two cells grew / total number of cells in 96-well plate culture x 100). Individual single cells were cultured in 96-well plates with each well containing 100 uL of serum media with each 100 ng/mL of stem cell factor, Flt-3, thrombopoietin and G-CSF for five days.

Results

The frequency and proportion of ASC varied according to FAB subtypes. The mean proportion of ASC from AML M2, M4 and M3 showed 25.1±22.5% (mean±SD), 15.1±16.6% and 6.5±3.3%, respectively. The mean proportion of remaining other subtypes was 13.6±16.2%. CD34 percentage in BM at the initial diagnosis closely correlated with the proportion of ASC. ASC proportion was statistically significant different between patient's group with more than 20% of CD34 and those with less than 20% (P=0.003). Single cell plating efficiency of ASC was markedly reduced compared with non-ASC (CD34+CD38+ leukemic cell population) (7.2 ± 4.7% in ASC vs. 12.6 ± 7.6% in non-ASC).

Conclusion

ASC proportion and frequency in BM samples were very diverse according to FAB subtypes. This study directly proved lower dividing activity of single ASC, and also implicated strategies for single ASC cloning to develop new therapeutics targeting ASC.

Disclosures:

No relevant conflicts of interest to declare.
Mesenchymal Stem Cell Grafting to Treat Cutaneous Radiation Syndrome: Developement of a New Minipig Model.
Authors: Agay Diane , Forcheron Fabien , Scherthan Harry , Grenier Nancy , Meineke Viktor , Herodin Francis , Drouet Michel
Published: October 2019

Abstract 4574

Cutaneous radiation syndrome (CRS) is the delayed consequence of localized skin exposure to high doses of ionizing radiations. Recent grafting of 3 burned patients has suggested the benefit of local injection of bone marrow multipotent mesenchymal stem cells to favour wound healing. However, further studies are required to standardize recommendations. The aim of this study was to set up a pig model of severe CRS in order to clarify some of these points. Göttingen minipigs were locally irradiated (20 cm x 3 cm area at the back) using a 60Co gamma source. This model corresponds to a 50-60 Gy entrance and a 15-25 Gy exit dose delivery. Skin damage was associated to deeper lesions especially muscular. Experimental groups were as followed: ungrafted 50 Gy (n=2) and 60 Gy (n=1); grafted 50 Gy (minipig GM5) and 60 Gy (minipig GM4). Humeral bone marrow collected before irradiation was put into culture in minimal essential medium with 10% Fetal Calf Serum and Fibroblast Growth Factor (2ng/ml) at 37° in air with 5% CO2. Pig GM5 was intradermally injected at days 48, 69 and 96 for a total amount of 128.5 ×106 MSC, pig GM4 at days 48 and 69 for a total amount of 99 ×106 MSC (range 2 to 3×106 MSC per point). All animals exhibited an early erythema on day 1 and a hair loss marked within 28 days. Minipig GM4, grafted after the onset of macroscopic damage (day 40), exhibited a similar evolution to controls. In minipig GM5, local inflammation and macroscopic damages were delayed in entrance area when compared with ungrafted 50 Gy animals (thermal camera signal at day 96 versus day 53; no macroscopic damage in entrance area until day 222 euthanasia versus day 85).

Immunolabellings showed severe late skin damages in all animals (reduced keratinisation, deep ingrowth of epidermis, small cavities and apoptotic cells). Finally this study establishes a new minipig model close to human for the improvement of cell therapy strategies to treat high local irradiations. Work is going on to document the homing of injected cells (eGFP labelled MSC) and the molecular response of grafted tissues in order to optimize graft efficacy.

Disclosures:

No relevant conflicts of interest to declare.
Human Mesenchymal Stem Cells Elicit Complement Activation in Human Blood.
Authors: Le Blanc Katarina , Moll Guido , Rasmusson Ida , Ekdahl Kristina Nilsson , Elgue Graciela , Sundberg Berit , Nilsson Bo
Published: October 2019

Abstract 4580

Infusion of third-party mesenchymal stem cells (MSCs) appears to be a promising therapy for steroid-refractory acute graft-versus-host disease (GvHD). Little is known about how MSCs interact with the innate immune system after clinical infusion. In this study, we show that exposure of MSCs to ABO-compatible human blood activates the complement system, which triggers complement-mediated effector cell functions, and correlates with the immunosuppressive properties of MSCs. We found deposition of the complement component 3 (C3) derived opsonins iC3b and C3dg on MSCs, and fluid-phase generation of the chemotactic anaphylatoxins C3a and C5a. These events triggered complement receptor 3 (CD11b/CD18)-mediated effector cell activation; but could be prevented by culturing MSCs in human ABserum or by blocking complement function. Our study demonstrates the important role of the complement system as a possible mediator of immune modulation in clinical applications using MSCs, and implies that complement activation may substantially affect the treatment efficiency.

Disclosures:

No relevant conflicts of interest to declare.
The Critical Function of c-Myb and CD9 On Megakaryopoiesis.
Authors: Mukai Harumi Yamamoto , Kouno Tomoko , Motohashi Hozumi , Kozuma Yukinori , Kojima Hiroshi , Ninomiya Haruhiko , Yamamoto Masayuki
Published: October 2019

Abstract 4587

The nuclear proto-oncogene c-myb plays crucial roles in the growth, survival and differentiation of hematopoietic cells. We previously reported that the c-Myb knock down (KD) mice exhibited anemia and thrombocytosis. To better understand the transcriptional regulatory program that accompanies the decrease of c-Myb, we performed a transcriptome analysis of megakaryocyte-erythrocyte lineage-restricted progenitors (MEPs). We found that CD9 expression was dramatically increased in the MEPs from c-Myb KD mice. CD9 belongs to a tetraspanin family and regulates cell motility and adhesion. Several reports described that CD9 is involved in platelet function by forming a complex with integrin family members including platelet fibrinogen receptor integrin aIIb-b III. To elucidate the functional contribution of CD9 to the thrombogenesis within the regulatory hierarchy mediated by c-Myb, we first examine the relation between c-Myb and CD9. We found the recruitment of c-Myb to the promoter region of CD9 by chromatin immunoprecipitation assay. A reporter assay showed that c-Myb represses CD9 promoter activity. These results indicate that CD9 is directly repressed by c-Myb. Since an agonistic antibody against CD9 stimulated megakaryocytic colony formation, we investigated the role of CD9 during megakaryopoiesis in vivo by using CD9-null mice. Numbers of megakaryocytes and platelets, CFU-Meg, and ploidy were not different between wild-type and CD9-null mice. However, proplatelet formation (PPF) was significantly impaired in CD9-null megakaryocytes, and the size of proplatelets was smaller than those generated by wild-type megakaryocytes. Consistent with this result, the recovery phase of platelet counts were delayed in the CD9-null mice after the bone marrow suppression with 5-fluorouracil, indicating the impaired platelet production. In CD9-null mice, the proliferation of megakaryocytes was promoted and circulating megakaryocytes in the peripheral blood were increased, which might compensate the PPF impairment of CD9-null megakaryocytes. Our study suggests that c-Myb suppresses CD9 expression under the steady state, while, in the stress megakaryopoiesis, CD9 is derepressed and acts to enhance platelet production. Elucidation of c-Myb-CD9 regulatory function seems to be important to understand the stress megakaryopoiesis.

Disclosures:

Ninomiya: Alexion: Research Funding.
Filamin A Controls Embryonic Stem Cell Differentiation.
Authors: Kanaji Taisuke , Okamura Takashi , Newman Peter J.
Published: October 2019

Abstract 4599

Filamin A is a major non-muscle actin binding protein that plays an important role in cross-linking cortical actin filaments into three-dimensional networks. In addition to its role as a cytoskeletal scaffolding molecule, Filamin A is also known to bind more than 30 other proteins, regulating their subcellular location and coordinating their ability to signal. To analyze the role of filamin A in mouse embryonic stem (ES) cell maturation, we generated filamin ALow ES cells by introducing a micro-RNA that specifically downregulates filamin A expression under the control of a cytomegalovirus promoter. Filamin ALow ES cells exhibited a more rounded morphology than did their wild-type filamin ANormal counterparts, and expressed increased levels of the ES cell transcription factor Nanog. In contrast, non-transfected cells in the same culture dish retained normal expression of filamin A, expressed low levels of Nanog, and exhibited a more elongated and spread phenotype characteristic of differentiating cells. Further evidence for a role for filamin A in ES cell differentiation was provided by the observation that withdrawing leukemia inhibitory factor (LIF) to induce ES cell differentiation was accompanied by increased expression of filamin A, a concomitant loss of Nanog expression, and acquisition of a differentiated morphology. Filamin ALow ES cells were able to retain their undifferentiated phenotype, as evaluated by alkaline phosphatase (Alp) activity, in the presence of a 10-fold lower concentration of LIF than was permissive for filamin ANormal ES cells, or following exposure to the differentiating agent, bone morphogenic protein 4 (BMP4). LIF-induced phosphorylation of ERK was decreased in filamin ALow relative to filamin ANormal ES cells, as was BMP-induced phosphorylation of Smad1/5 - two signaling pathways that initiate ES cell differentiation. Finally, embryoid bodies comprised of filamin ALow ES cells were unable to differentiate into CD41+ hematopoietic progenitor cells. Taken together, these data demonstrate that filamin A plays a previously unrecognized, but critical, scaffolding function that support both the LIF - ERK and BMP4 - Smad1/5 signaling pathways leading to ES and hematopoietic cell differentiation. Manipulation of filamin levels might be useful in the future to modulate the differentiation requirements for a variety of clinically-and therapeutically-useful stem cells.

Disclosures:

Newman: Novo Nordisk: Consultancy; New York Blood Center: Membership on an entity's Board of Directors or advisory committees.
Hedgehog Signaling and Hematopoiesis Regulation: Preliminary Study in Myelosuppressed Adult Non Human Primate model.
Authors: Drouet Michel , Garrigou Philippe , Grenier Nancy , Delaunay Christophe , Mayol Jean-François , Herodin Francis
Published: October 2019

Abstract 4603

The precise role of hedgehog signaling (Shh) in adult hematopoiesis is still debated. One one hand its implication in differentiation and proliferation of hematopoietic stem cells has been reported by different teams. Thus Bhatia et al showed using an immunocompromized mouse model that Shh allows in vitro amplification of CD34+/38- pool. Shh may be especially invoved in megakaryocytopoiesis. In addition transgenic mice (ptc1/-) exhibiting an increased Shh activity has been shown to recover more rapidly than controls following myelosuppression and prolonged Shh stimulation appeared to deplete the stem cell pool due to cell cycle acceleration. On the other hand, recent studies also suggest that hedgehog signaling could be dispensable for adult hematopoieisis (Hoffmann-Zhang, Blood 2008 abstr 1391. The goal of this study was to set up an adult non human primate model to clarify some of these points. We chose a gene therapy strategy based on short term expression of Shh in bone marrow of highly irradiated monkeys (efficient stimulation and reduction of side effects). We first demonstrated the feasibility of transducing multipotent fibroblastic stem cells as vector cells using a piRES-2-Shh-eGFP plasmid and Nucleofector technology (Amaxa). As bone marrow nonhuman primate mesenchymal stem cells are known to be frequently contaminated by retroviruses, we used adipocyte derived stem cells as vector cells. In this model, Shh protein was detected during 5 days in the transduced cells and was secreted during 3 days in accordance with our short term secretion model. Then Shh-ASC were injected to rhesus monkeys the day after a global and frontal irradiation at the dose of 8Gy 60Co gamma. At this dose, monkeys exhibited a severe and prolonged neutropenia and thrombocytopenia allowing us to evaluate the therapeutic benefit of the grafts. In a first grafted monkey (9kg), 2×106 Shh-ASC (about 43% GFP positive cells) were injected in the femur. Blood cell counts did not significantly differ from irradiated control animals (n=4). Further experiments will contribute to determine whether Hedgehog signaling is involved in adult primate recovery following myelosuppression.

Disclosures:

No relevant conflicts of interest to declare.
Activity of CNTO 530 in a Murine Model of Sickle Cell Anemia.
Authors: Makropoulos Dorie , Achuthanandam Ram , Rafferty Patricia , Chroscinski Denise , Brosnan Kerry , Ogunkua Lawrence , Miller Andrew , Avery Justin , Walker Mindi , Bugelski Peter
Published: October 2019

Abstract 4606

CNTO 530 is an erythropoietin receptor agonist MIMETIBODYTM construct. CNTO 530 has been shown to be active in a number of rodent models of acquired anemia (e.g. renal insufficiency and chemotherapy induced anemia) and in a model of beta-thalassemia. Here we report on the activity of CNTO 530 in murine globin KO-human HbS transgenic (sickle cell) mice. Berkeley mice, as originally described by Pászty et al (Pászty et al. Science 1997), are knocked out for murine alpha and beta globin and are transgenic for human alpha, beta (sickle) and gamma globin genes. Thus, these mice express exclusively human hemoglobin A (HbAsickle) (or sickle hemoglobin, HbS) and can also express human fetal hemoglobin (HbF). To test the effectiveness of CNTO 530 in this model, mice received a single subcutaneous (s.c.) dose of CNTO 530 (0.3 mg/kg), epoetin-alpha (0.1 mg/kg) or darbepoetin-alpha (0.03 mg/kg) (as comparators) at 10,000 U/kg, a dose previously shown to increase hemoglobin in normal mice. Hematology, histology of the spleen, F-cells and expression of HbF were then evaluated over time. We show that these mice express a severe compensated hypochromic microcytic anemia and display the sickle cell phenotype. In addition, splenomegaly and extramedullary erythropoiesis were observed. Ten days after dosing, CNTO 530 but not epoetin-alpha or darbepoetin-alpha caused an increase in reticulocytes, red blood cells (RBC), F-cells, total hemoglobin (Hgb) and HbF. In conclusion, these results demonstrate that a single dose of CNTO 530 can increase expression of HbF in sickle cell mice and suggest that human treatment with CNTO 530 may have beneficial effects in sickle cell anemia patients.

Disclosures:

Makropoulos: Centocor, R&D, Inc: Employment, Equity Ownership. Achuthanandam:Centocor, R&D, Inc: Employment, Equity Ownership. Rafferty:Centocor, R&D, Inc: Employment, Equity Ownership. Chroscinski:ACE Animals, Inc: CRO. Brosnan:Centocor, R&D, Inc: Employment, Equity Ownership. Ogunkua:Chroscinski: Employment, Equity Ownership. Miller:Centocor, R&D, Inc: Employment, Equity Ownership. Avery:Centocor, R&D, Inc: Employment, Equity Ownership. Walker:Centocor, R&D, Inc: Employment, Equity Ownership. Bugelski:Centocor, R&D, Inc: Employment, Equity Ownership.
Asthma IN SICKLE CELL DISEASE as A RISK FACTOR for Acute Chest Syndrome IN Pediatric PATIENTS.
Authors: Borns Andrea P , Marquez Kristy , Amendah Djesika , Whiteman Alan , Jordan Lanetta B , Grant Althea
Published: October 2019

Abstract 4610

OBJECTIVES

Both asthma and sickle cell disease are major Public Health concerns. Previous studies have demonstrated that asthma among children with sickle cell disease (SCD) may increase the risk of developing acute chest syndrome (ACS), which can be life threatening. These respiratory complications may increase emergency department (ED) utilization, increase health care costs, and reduce life span among persons with SCD. The purpose of this study is to determine whether children with SCD and asthma have significantly more ED visits and if they are at higher risk of developing ACS compared to children with SCD who do not have asthma.

METHODS

We used MarketScan® Multi-State Medicaid Databases from Thompson Reuters (Ann Arbor, Michigan) for the years 2001 to 2005. These are proprietary datasets covering eight unidentified states. We used International Classification of Disease, 9th Division, Clinical Modifications (ICD-9-CM) codes to identify SCD, asthma and ACS.

RESULTS

In 2005, there were 2428 children with SCD continuously enrolled in Medicaid. Among those, 369 (15.2%) patients were identified as having asthma. The mean number of emergency department visits was significantly higher among children with both SCD and asthma compared to children with SCD without asthma (p<0.05). In addition, children with SCD and asthma were more likely to have at least one episode of ACS than children with SCD without asthma (28.2% vs. 7.8%, respectively).

CONCLUSIONS

Among children with SCD who are enrolled in Medicaid, asthma is a common comorbidity, which is associated with high incidence of ACS. Therefore asthma should be aggressively managed among SCD patients. Proper management of asthma may result not only in reduced pulmonary complications, but also reduced costs related to emergency department utilization and progression of lung disease into adulthood.

Disclosures:

No relevant conflicts of interest to declare.
Adding Hydroxyurea to Chronic Transfusion Therapy for Stroke in Sickle Cell Disease.
Authors: Claster Susan , Quirolo Keith C.
Published: October 2019

Abstract 4620

Introduction

Transfusions have clearly been shown to prevent stroke in patients with Sickle Cell Disease(SCD). The usual goal of transfusion therapy is to keep the percent sickle Hgb (%S) less than 30. %S greater than 30 has been associated with stroke reoccurance. Inability to transfuse adequately may result from poor vascular access or high hemolytic rate. Hydroxyurea, a drug which requires active erythropoeisis, is usually not given to patients who are chronically transfused. However, the presence of an elevated reticulocyte count in a transfused patient indicates ongoing sickle hematopoiesis. We added Hydroxyurea therapy to two patients who were unable to achieve %S levels less than 30 and who had elevated reticulocyte counts to assess whether this drug could improve their hematological parameters.

Results

We treated two post stroke patients, one receiving pheresis therapy, and the other on straight transfusion therapy who were unable to achieve 30% Hgb S despite regular transfusions. Patient 1, a 33 y/o female with Hgb SS had been on transfusions since childhood for multiple CVA's and resulting moya-moya. Her last stroke had occurred while she was being chronically transfused and had a % S of 40. Due to venous access issues the patient was receiving straight transfusion only and was unable to adequately suppress her marrow. In 8/07 her %S was greater than 40 and her reticulocyte count was 11.3 %. Hydroxyurea was started at 1500 mg daily(22 mg/kg). Over the next 24 months her Bilirubin dropped from 8.7mg/dl to 1.9 mg/dl, her reticulocyte count dropped to 4%(figure below) and her MCV increased to 104.6 from 90. Her Hgb F rose to 30%.Her %S has not changed. She has remained clinically stable with no new neurological findings. The second patient, a 17 y/o male had been on chronic transfusions since childhood for a stroke which resulted in a dense left hemiparesis. He has been on a pheresis program for 13 years. Despite this his % S remained greater than 40% and had been as high as 60% prepheresis. Hydroxyurea was added in 5/08 at a dose of 25mg/kg. Within 6 months his pretransfusion %S had dropped to 22-27%. No increase in fetal Hgb was detected, possibly due to the exchange transfusion process. His bilirubin, which had been running between 12-16 mg/dl dropped to 7.6-8.9 mg/dl and his reticulocyte count went from an average of 19.6% to 11.2% in the same time frame. He has also remained stable.

Discussion

These two patients demonstrate that the addition of Hydroxyurea to patients on chronic transfusion can be useful in decreasing hemolytic rate and improving the efficacy of transfusions by decreasing sickle erythropoiesis and increasing fetal hemoglobin synthesis. Further studies of the combination of these two modalities may be warranted in those patients who cannot achieve adequate suppression of sickle erythropoeisis.

Disclosures:

No relevant conflicts of interest to declare.