bluuebunny/crossref_metadata_2025 huggingface dataset:
Case Series of Primary Breast Diffuse Large B-Cll Lymphoma in Ampang Hospital, Malaysia
Authors: Kuan Jew Win , Lau Ngee-Siang , Wong Lily L , Lau Lee-gong , Chang Kian Meng
Published: October 2019

Abstract 5113

INTRODUCTION

Breast is a rare extranodal site of non-Hodgkin's lymphoma. Traditionally, primary breast lymphoma (PBL) is defined as disease localized to one or both breasts with or without regional lymph nodes involvement (ipsilateral axillary and/or supraclavicular lymph nodes). Recently, there is a suggestion to define breast lymphoma by the number of involved extranodal disease. The commonest histology of PBL is diffuse large B-cell lymphoma (DLBCL). Treatment with CHOP-like regime (cyclophosphamide, doxorubicin, vincristine and prednisolone) in PBL-DLBCL remains unsatisfactory with 5-year progressive free survival rates of approximately 50–65%, which is generally worse than the localized nodal or other extranodal disease. The reason to the poorer outcome might be explained by the genotype. About 80% of the PBL-DLBCL is non-germinal center B-cell or activated B-cell (ABC) subtype, a poorer prognostic genotype compared to germinal center B-cell (GCB) subtype. The data of rituximab (R) overcoming genotypic effect of PBL-DLBCL is still inconclusive. However, the recent matched pair analysis showed similar 3-year overall survival rate to nodal DLBCL if using rituximab and CHOP. Immunoblast morphology appeared to be one of the prognostic factors besides the bulkiness of the disease and the high proliferation index (PI). We present our case series of DLBCL who presented with primary breast swelling from 2009 till June 2012.

METHOD

All the DLBCL cases diagnosed from 1st January 2009 till 30th June 2012 were filtered for breast involvement from database. Only cases presented with primary breast swelling and biopsy-proved DLBCL breast lesion were studied. Six cases were identified. All are female.

CONCLUSION

Looking at the six cases of primary breast DLBCL, the outcome is poor despite rituximab based chemotherapy. Better chemotherapy regime other than CHOP and agent like bortezomib and upfront autologous HSCT should be explored.

Disclosures:

No relevant conflicts of interest to declare.
Thrombin Generation Responses to Human Factor XIa in Plasma of Animal Species
Authors: Liang Yideng , Struble Evi B , Ma Li , Woodle Samuel A , Jha Naveen , Scott Dorothy , Lee Timothy K , Ovanesov Mikhail V
Published: October 2019

Abstract 5142

Thromboembolic events (TEE), including deep venous thrombosis and myocardial infarction, have been reported in patients receiving immunoglobulin intravenous (IVIG). Recent research showed that many TEE-induced IVIG samples were contaminated with human coagulation factor XIa (FXIa). At present, there is no systematic investigation of animal models that can be used to assess FXIa dependent thrombogenicity risk in animals and be predictive for human response. To assist in species selection for in vivo animal studies, we compared the magnitude and kinetic parameters of thrombin generation in commercially available human and animal plasma samples in response to human FXIa. A low sample volume, highly reproducible thrombin generation test was developed for this study. In our assay, thrombin generation in plasma (20 microliters) is initiated by physiological activator tissue factor in the presence of procoagulant phospholipids, sample (FXIa), buffer and calcium (combined volume of all additions to plasma is 20 microliters). The thrombin generation response to human FXIa was evident from increased thrombin peak height and/or shortened time to thrombin peak. The minimal effective FXIa concentration was species-dependent and ranged from below 0. 1 pM in monkey plasma to over 100 nM in BalbC mouse. The FXIa sensitivity of commercial plasma samples was aligned in the following order: Rhesus and Cynomolgus Monkeys > Human Donors > New Zealand White Rabbit and Sprague Dawley Rat > Hartley Guinea Pig and CD1 Mouse > Lewis Rat > Wistar and Fisher 344 Rats > C57BL6 and Blab C Mice. However, further experiments on freshly collected guinea pig plasma demonstrated that preanalytical variables, notably, blood collection and plasma freezing and thawing techniques, are critical to achieve optimal sensitivity of the thrombin generation test to FXIa. Therefore, variable quality of commercial plasma may have affected observed interspecies differences in thrombin generation. Collectively, these findings will help to clarify differences in minimal thrombogenic doses of FXIa observed in various animal models and levels of FXIa associated with thrombotic events in humans.

This research was supported by the FDA Office of Women's Health grant (2012).

Disclosures:

No relevant conflicts of interest to declare.
Cerebral Sinovenous Thrombosis Related to Iron Deficiency Anemia As a Result of Severe Menorrhagia: A Case Report
Authors: Corrales-Medina Fernando F. , Rodriguez Nidra , Egas-Bejar Daniela E , Valdivia-Ascuna Zoila , Grant Leon , Mancias Pedro
Published: October 2019

Abstract 5143

Cerebral sinovenous thrombosis (CSVT) is a rare condition with a wide spectrum of non-specific symptoms, which makes it occasionally difficult to diagnose. CSVT has been associated with various etiologies including dehydration, hypercoagulable states, neoplasmic invasion of a venous sinus, intracranial and systemic infections, use of oral contraceptives, puerperium and pregnancy. Although iron deficiency anemia (IDA) has been reported as a cause of CSVT in several pediatric cases, this association is extremely rare. To our knowledge, IDA-associated CSVT in teenagers has been rarely reported.

Here, we present the case of a 13-year old female with complete thrombosis of the vein of Galen, the straight sinus, and the left internal cerebral vein related to iron deficiency anemia as a result of severe menorrhagia. She initially presented with three-week history of headaches, nausea and vomiting. She was initially diagnosed and treated for bacterial sinusitis by her primary pediatrician, without symptom relief. She then suffered a syncopal episode, reason why she was evaluated at our institution. Initial laboratories revealed a slightly increased PT (15. 6 secs), hemoglobin of 5. 1 g/dL, MCV 63. 6 and D-dimer 1. 31. Non-contrast brain CT demonstrated no evidence of stroke, hemorrhage or mass. However, an abnormal hyperdensity in the straight sinus, inferior sagittal sinus, vein of Galen, and deep cerebral veins, were suggestive of venous thrombosis. Findings were then confirmed by brain MRI/MRV/MRA and CT angiogram. A bleeding work-up performed based on her history of heavy menses was normal. Her ferritin level was significantly decreased (6 ng/mL). Iron supplementation was initiated as well as progestin-only hormonal therapy for menstrual cycle regulation. The patient's clinical status improved back to baseline during her 8-day admission. She was discharged home on anticoagulation with scheduled follow up. Brain MRI/MRV six months later showed patency of the cerebral veins and dural venous sinuses.

IDA should be considered as an underlying cause of CSVT in pediatric patients, particularly in patients with other risk factors for thrombosis including dehydration, regardless of their age. Early recognition and management of CSVT is critical to minimize the possibility of permanent neurologic damage. Physicians should be aware of the non-specific signs and symptoms of CSVT and should have a high index of suspicion in patients with anemia.

Disclosures:

No relevant conflicts of interest to declare.
Prevalence of 25-Hydroxyvitamin D Deficiency in Korean Patients with Anemia
Authors: Yoo Eun-Hyung , Cho Hyun-Jung
Published: October 2019

Abstract 5164

Background:

Vitamin D deficiency is a very common health problem in Korea. Vitamin D has been suggested to have an important role on nonskeletal functions including cellular proliferation and differentiation, muscle function, immunity and erythropoiesis. Recent studies have been reported that vitamin D deficiency had associated with iron deficiency anemia, anemia of chronic kidney disease, and anemia of inflammation. In this study, we investigated the prevalence of vitamin D deficiency in Korean patients with anemia and also analyzed the association between vitamin D status and specific subtypes of anemia.

Methods:

A total of 147 anemic patients (median age 66 years, range 19∼91 years) and 300 nonanemic controls (median age 60 years, range 29∼87 years) were included. Anemia was defined according to World Health Organization (WHO) criteria. Serum 25- hydroxyvitamin D [25(OH)D] was measured using electrochemiluminescence immunoassay. The deficiency of 25(OH)D was defined as <20 ng/mL and severe deficiency was defined as <10 ng/mL. We compared serum 25(OH)D levels based on the presence and subtypes of anemia.

Results:

The prevalence of 25(OH)D deficiency was 74% (109/147) and 70% (210/300) in anemic (median Hb 9. 6 g/dL) and nonanemic group (median Hb 14. 3 g/dL), respectively. The prevalence of severe 25(OH)D deficiency was significantly higher in anemic group than in nonanemic group [44. 8% (66/147) vs 11. 7% (35/300), P<0. 0001] Odds ratio for severe 25(OH)D deficiency in anemic patients was 6. 17 (95% CI 3. 820–9. 965, P<0. 0001). The prevalence of 25(OH)D deficiency was not different between iron deficiency anemia (IDA) group and anemia of chronic disease (ACD) group. However, the serum 25(OH)D levels of ACD patients were lower than those of IDA patients in male (median 25(OH)D 14. 34 ng/mL vs 23. 04 ng/mL, P=0. 04).

Conclusion:

This study demonstrates that severe vitamin D deficiency is associated with anemia in Korea. Although vitamin D deficiency is also very common in nonanemic Korean population, anemia is related to much worse vitamin D deficient status. Multiple factors including poor nutritional status and potential roles of vitamin D on inflammation and erythropoiesis might be considered.

Disclosures:

No relevant conflicts of interest to declare.
Significance of Low Levels of Glucose-6-Phosphate Dehydrogenase Levels in Pregnancy
Authors: Chintapatla Rangaswamy , Melinscak Hrvoje , Varma Mala
Published: October 2019

Abstract 5165

Purpose of the study:

To characterize the significance of Glucose-6- Phosphate Dehydrogenase (G6PD) levels measured in pregnancy.

Case series and results:

We saw 3 patients referred to Hematology clinic over the past 1 year. The G6PD levels were measured in pregnancy by the obstetrician while working up the anemia (Table 1). They did not have any evidence of hemolysis during the time of workup and it is unclear as to why the G6PD levels were sent. The G6PD levels were found to be low and all 3 patients had iron deficiency anemia, that improved with iron supplementation. There was no evidence of pregnancy induced hypertension, urinary tract infections, or infections during pregnancy. All the pregnancies were uneventful resulting in healthy babies and the G6PD levels normalized after the delivery in all the patients. We did a literature search to see the significance of such an association and if there were any recommendations for further testing in these individuals.

Discussion:

G6PD deficiency is one of the most common inherited red cell disorders transmitted in an X-linked recessive fashion affecting approximately 400 million people worldwide. In the United States, black males are most commonly affected, with a prevalence of approximately 10 percent. Acute hemolysis is caused by infection, ingestion of fava beans, or exposure to an oxidative drug. G6PD catalyzes nicotinamide adenine dinucleotide phosphate (NADP) to its reduced form, NADPH, in the pentose phosphate pathway. NADPH protects cells from oxidative damage. G6pd deficiency predisposes cells to increased oxidative damage predisposing to hemolysis. G6PD deficiency in pregnancy may manifest as increased urinary tract infections, preeclamsia, neonatal jaundice, hydrops fetalis and still birth. Vergnes et al. [Lancet 1968] reported that low erythrocyte G6PD levels were found in 25% of women in early pregnancy and in up to 65% of women in late pregnancy. In their study G6PD levels normalized after delivery. The reason for this decrease is unclear. Pregnancy is shown to cause increased oxidative stress, alteration in neutrophil G6PD trafficking [ Kindzelskii et al. J Clin Invest 2002] though it is unclear if any of these mechanisms are implicated in the decreased levels of G6PD during pregnancy.

Conclusions:

There are no clear guidelines for the subsequent testing and management of low levels of G6PD diagnosed in the setting of pregnancy. Evidence based guidelines would be helpful towards identifying the subset of patients with true G6PD deficiency.

Disclosures:

No relevant conflicts of interest to declare.
Chronic Iron Deficiency Anemia and Cancer Risk
Authors: Liu Chia-Jen , Chen Kuo-Wei , Hu Yu-Wen , Hong Ying-Chung , Huang Yu-Chung , Chiou Tzeon-Jye , Tzeng Cheng-Hwai
Published: October 2019

Abstract 5172

Background

Iron deficiency anemia (IDA) is a common initial manifestation of gastrointestinal malignancies, but the relationship between chronic IDA and further cancer development is not established.

Aims

We would like to investigate the cancer risk of chronic IDA over a 10-year period in Taiwan.

Methods

We enrolled patients with iron deficiency anemia from a nationwide health registry in Taiwan during the period 2000–2009. All patients received at least 2 related examinations and were treated with iron supplement. Since IDA is an initial manifestation of many underlying malignancies, we excluded patients whose cancer developed within 1 year after IDA diagnosis. Standardized incidence ratios (SIRs) of cancers were calculated to compare the cancer incidence of chronic IDA patients to general population. The specific types of cancer were addressed.

Results

A total of 4, 373 patient with IDA were identified, and 132 (3. 02%) IDA patients developed cancer 1 year after enrollment (SIR 1. 52; 95% confidence interval [CI] 1. 27 – 1. 80, p < 0. 001). The risks of kidney (SIR 5. 47, 95% CI 2. 73 – 9. 78), bladder (SIR 3. 6, 95% CI 1. 64 – 6. 83), liver (SIR 2. 80, 95% CI 1. 82 – 4. 13), and colorectal malignancies (SIR 1. 93, 95% CI 1. 22 – 2. 90) elevated. The risk of hematological malignancies were not increased in current study (SIR 1. 13, 95% CI 0. 31 – 2. 89, p = 0. 758).

Conclusions

After exclusion of cancer development within the first year, IDA patients still had increased risk of cancer, especially kidney, bladder, liver, and colorectal malignancies.

Disclosures:

No relevant conflicts of interest to declare.
Effect of Vitamin D Supplements On Hemoglobin Concentration in Patients with Iron Deficiency Anemia and Concurrent Vitamin D Deficiency:A Randomized Single Blind Placebo Controlled Trial
Authors: Malhotra Pankaj , GS Basavaraj , Sachdeva Naresh , Ahluwalia Jasmina , Bhadada Sanjay
Published: October 2019

Abstract 5173

Background:

The effect of vitamin D replacement on hemoglobin concentration in subjects with concurrent iron deficiency anemia and vitamin D deficiency is not known.

Methods:

In a single blinded randomized placebo controlled study, thirty subjects with iron deficiency anemia (serum ferritin < 15 ug/dl) were randomized to intervention arm (vitamin D 6 lakh IU IM once) or placebo. In all subjects, iron deficiency was corrected with parental iron. All the causes other than iron deficiency were excluded with appropriate investigation. The primary end point was rise in hemoglobin concentration.

Results:

Baseline age, BMI, hemogram, levels of serum ferritin, 25(OH)D and PTH were similar in both the arms. Twelve weeks after vitamin D replacement, there was significant increase in 25 (OH) D levels and decrease in PTH levels in subjects with intervention arm compared to placebo arm (p<0. 01). The increment in serum ferritin in both the group was similar. There was no further increase in hemoglobin concentration in intervention arm compared to placebo arm.

Conclusion:

Vitamin D replacement in subjects with iron deficiency anemia after iron correction does not improve hemoglobin concentration further.

Disclosures:

No relevant conflicts of interest to declare.
Regulation of Mesenchymal Progenitor Cell-Induced Neo-Vascularization by Endothelial Progenitor Cell-Derived Exosomes
Authors: Lener Thomas , Peckl-Schmid Doris , Gimona Mario , Oeller Michaela , Rohde Eva
Published: October 2019

Abstract 5188

Background and rationale

Despite advances in the application of autologous mesenchymal stem/progenitor cells (MSPCs) and endothelial colony forming cells (ECFCs) for novel regenerative therapies, our understanding of the basic mechanisms involved in therapeutic neovascularization remains incomplete. Specifically, the individual contribution of the cell types to vessel formation and maintenance is poorly understood. Here we investigate if MSPC-derived exosomes (small vesicles that are secreted into the extracellular space and fuse with other cells to deliver mRNAs, regulatory miRNAs and proteins) can promote ECFC proliferation, migration and differentiation.

Results

We have generated exosome-enriched fractions from conditioned culture media of human umbilical cord or bone marrow-derived MSCPs propagated in human platelet lysate. These purified fractions were added to the culture media of ECFCs for 48 – 72 hours. Employing FACS analysis, microscopy, and the automated xCELLigence system (ROCHE) we uncovered that MSPC exosomes significantly alter the morphology and the proliferative index of ECFCs, as well as their specific marker profile. Moreover we identified that exosome enriched fractions are sufficient for modifying the expression pattern of the collagen sensor DDR1 at the mRNA level and also influence the levels of the DDR1 regulatory miRNA 199. Significantly, exosome-enriched fractions from ECFCs did not elicit any detectable alterations in ECFCs themselves, whereas we could see changes in morphology, migratory potential, proliferative activity and marker expression in MSPCs.

Conclusions

Taken together our data suggest that progenitor cell-derived exosomes contribute to the neo-vascularization process induced by pairwise injection of ECFCs and MSPCs, and that at least part of the role executed by MSPCs could be delivered in a cell-free manner through exosome-enriched preparations.

Disclosures:

No relevant conflicts of interest to declare.
Anti-Human VWF Monoclonal Antibody SZ-123 Prevents Arterial Thrombus Formation by Inhibiting VWF–collagen and VWF-Platelet Interactions in Rhesus Monkeys
Authors: Zhao Yiming , Ruan Changgeng
Published: October 2019

Abstract 5194

Objective:

To investigate the in vivo antithrombotic efficacy of an anti-VWF monoclonal antibody SZ-123, and its potential underlying mechanism.

Methods and Results:

Cyclic flow reductions (CFRs) were measured in the femoral artery of monkeys before and after intravenous administration of SZ-123. Ex vivo VWF binding to collagen, platelet aggregation, platelet count and template bleeding time were performed as measurements of antithrombotic activity. In addition, plasma VWF, SZ-123 levels, and VWF occupancy were measured by ELISA. Administration of 0. 1, 0. 3, and 0. 6 mg/kg SZ-123 resulted in 45. 3%, 78. 2%, and 100% reduction in CFRs, respectively. When 0. 3 and 0. 6 mg/kg SZ-123 were administrated, 100% of VWF was occupied by the antibody. Moreover, 100% ex vivo inhibition of VWF-collagen binding and 60–95% inhibition of platelet aggregation were observed from 15 min to 1h. None of the doses resulted in significant prolongation of bleeding time. In vitro experiment also revealed that SZ-123 not only blocks collagen-VWF A3 interaction but also inhibits indirectly VWF A1 binding to GPIba induced by ristocetin.

Conclusions:

SZ-123 prevents in vivo arterial thrombus formation under high shear conditions by inhibiting VWF A3–collagen and VWF A1-platelet interactions and does not prolong bleeding time.

Disclosures:

No relevant conflicts of interest to declare.
Gas6 and TAM Receptors in Mouse Platelets
Authors: Kucuk Bahadir , Bingol-Ozakpinar Ozlem , Demir Muzaffer , Uras Fikriye
Published: October 2019

Abstract 5199

Objective:

Growth Arrest Specific-6 (Gas6) is a new vitamin K-dependent protein and is a ligand for TAM receptors, three receptors (Tyro, Axl and Mer) belonging to the receptor tyrosine kinase subfamily. The number of publications concerning the physiological importance of the new vitamin-K dependent proteins, which are not members of the coagulation cascade, is growing. Gas6 has a structural homology to natural anticoagulant of protein S, but has no anticoagulant activity. Some research groups have reported contradictory results concerning the presence of Gas6 and its receptors in mouse platelets. The purpose of this study is to investigate the presence of Gas6 and its receptors in mouse platelets as mRNA and protein levels.

Material and Methods:

25 specific pathogen-free, 8–10 week old, 25–30 g in weight, race of Balb-C male/female mice were anesthetized under light ether anesthesia and blood samples were taken from their hearts. Total RNAs were isolated from platelets, then mRNAs encoding Gas6 and TAM receptors were detected by Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Protein concentrations of Gas6 and TAM receptors in platelets were measured by ELISA. We were unable to measure the Mer protein level because of the absence of any commercial ELISA kit for mouse Mer.

Results:

RT-PCR results indicated the presence of mRNAs encoding to Gas6 and Mer in mouse platelets. However, although RT-PCR reactions were performed at various temperatures and cycles, we could not detect the presence of mRNAs encoding Axl and Tyro. Receptor levels of Axl and Tyro were below the detection limits of the ELISA method. Plasma Gas6 concentration was found to be 26 ng/ml.

Conclusion:

We found the presence of mRNAs encoding Gas6 and only receptor Mer in mouse platelets but not Axl and Tyro. Protein Gas6, Axl and Tyro levels were below the detection limits of the ELISA methods. The presence of mRNA is not evidence of protein expression in platelets because they have no DNA. Further studies are required to clarify the presence of Gas6 and TAM receptors in platelets using Real Time PCR, and more sensitive immunological methods, and then, as studies of their effects on mechanisms. Original results from these further studies will provide reliable information as to whether Gas6 and its TAM receptors may be a pharmacological target in thrombosis, hemostasis, restenosis and atherosclerosis.

Disclosures:

No relevant conflicts of interest to declare.
The Leukemic Fusion Gene NUP98-HOXD13 Suppresses Non-Homologous End Joining Factors and Impairs DNA Double Strand Break Repair
Authors: Caudell David L. , Puthiyaveetil Abdul Gafoor A.
Published: October 2019

Abstract 521

Chromosomal translocations are hallmark features of hematologic malignancies that often require collaborating mutations for malignant transformation. These secondary mutations can occur spontaneously, or rather, be induced by the primary translocation. Mutations can occur as a result of DNA damage and misrepair with DNA double strand breaks being one of the most serious types of cell damage. Double strand breaks are classically repaired by the non-homologous end joining (NHEJ) mechanism and impaired NHEJ has been shown to promote mutagenesis. Transgenic mice expressing the myeloid leukemic fusion gene NUP98-HOXD13 (NHD13) develop Myelodysplastic syndrome and progress to acute leukemia after acquiring secondary mutations. Our studies have shown that B lymphocyte development and class switch recombination are impaired in stimulated B lymphocytes from NHD13 mice. Based on this, we used in vitro class switch recombination (CSR) to delineate the DNA break induction and repair mechanisms in NHD13 B lymphocytes. Naïve B lymphocytes were harvested from wild type (WT) and NHD13 spleens and cultured in the presence of E.coli Lipopolysaccharide (LPS) and IL-4 to induce CSR. The DNA break induction pattern was determined using phosphorylated H2AX labeling combined with confocal microscopy and flow cytometry. Our results showed that NHD13 B lymphocytes had a comparable break induction pattern, but significantly reduced DNA repair. Analysis of the cell cycle pattern of stimulated B cells at 24 hour intervals showed cell cycle arrest at the G2/M phase at 72 hours following stimulation—a hallmark feature of impaired DNA break repair. We analyzed the expression of classical NHEJ and alternative end joining factors including Ku70, Ku80, DNA Protein Kinase catalytic subunit (DNAPKcs), Xrcc4, DNA ligase 4, Ligase 1, and Ligase 3. Our results showed reduced expression of DNAPKcs, Ligase 4 and Xrcc4 in NHD13 B lymphocytes at 72 hours following stimulation, suggesting that cells failed to initiate NHEJ-mediated DNA repair. Our results suggest that a myeloid leukemic gene can impair the DNA repair mechanism and may indirectly promote mutations necessary for malignant transformation.

Disclosures:

No relevant conflicts of interest to declare.
Up-Regulation of Heme Oxygenase-1 Is Induced by the Leukemia Stroma-Cell Interaction and Rescues Acute Myeloid Leukaemia (AML) Cells From Cytarabin Induced Cell Death
Authors: Brendel Cornelia A. , Wang Xiaonan , Henkenius Katharina , Pali Mihaela , Haferlach Torsten , Stiewe Thorsten , Neubauer Andreas
Published: October 2019

Abstract 533

Although the majority of patients achieve a complete remission after induction chemotherapy with cytarabine and antracyclines relapse is still the major cause of death in acute myeloid leukemia (AML). In order to address the question of resistance mechanisms in residual leukemic cells we employed a murine leukemia model system. We transplanted Rag2−/−gc−/− mice with human KG1a leukemia cells and treated them with four doses of cytarabine at the onset of AML. Residual human leukemic cells were collected from murine marrow and spleen and RNA was further subjected to whole genome microarray analysis. Heme Oxygenase-1 (HO-1) was found to be highly up-regulated in the persistent cell fraction that had survived cytarabine chemotherapy in vivo. The clinical relevance of HO-1 up-regulation was confirmed with two AML data sets. In a small set of 31 unselected AML patients treated in one institution, overall survival (OS) was significantly worse in patients with high HO-1 expression as determined by qPCR analysis (log rank: p < 0.0001). A second patient cohort of 286 patients was therefore retrospectively analyzed for HO-1 expression based on whole genome expression profiling. Again, OS was inferior in patients expressing HO-1 at high levels (p<0.005).

HO-1 is a well-known stress-response protein also termed heat-shock protein 32 (hsp32) that catalyzes the enzymatic cleavage of heme into biliverdin, carbon monoxide and iron, but also decreases cellular levels of reactive oxygen species (ROS). Employing an in vitro co-culture system we show that mesenchymal stem cells (MSC) increase HO-1 expression in leukemic cells causing a decrease in ROS levels and protection from apoptosis particularly in the adherent fraction of leukemic cells. The cytoprotective effect induced by leukemia stromal cell interaction can be overcome by concomitant treatment of cytarabin together with zinc protophoryrin (ZnPP), a specific competitive inhibitor of HO-1. Taken together we demonstrate that expression of HO-1 may play a critical role for chemotherapy resistance in AML in particular with regard to the leukemia stromal cell interaction. ZnPP is a specific HO-1 inhibitor that has already proven anti-tumor activity in vivo and might therefore be a promising candidate for further targeted drug therapy strategies that intend to eradicate persistent leukemic cells.

Disclosures:

No relevant conflicts of interest to declare.
A Novel Rat Model of Hereditary Hemochromatosis Due to a Mutation in Transferrin Receptor 2
Authors: Bartnikas Thomas Benedict , Wildt Sheryl , Wineinger Amy , Schmitz-Abe Klaus , Markianos Kyriacos , Cooper Dale , Fleming Mark D.
Published: October 2019

Abstract 612

Sporadic iron overload has been reported previously in rats but the underlying cause has not been ascertained. In this study, phenotypic analysis of a subpopulation of Wistar rats designated Hsd:HHCL revealed a low incidence of histologically detected liver iron overload. One rat out of 132 screened animals exhibited liver iron accumulation in a predominantly periportal, hepatocellular distribution; this male rat expressed low RNA levels of the iron regulatory hormone hepcidin and low protein levels of transferrin receptor 2, a membrane protein essential for hepcidin expression in humans and mice and mutated in forms of hereditary hemochromatosis, a disease of excessive intestinal iron absorption and progressive tissue iron overload. Sequencing of the transferrin receptor 2 gene in the iron-overloaded rat revealed a novel Ala679Gly polymorphism affecting a highly conserved residue. Quantitative trait locus mapping revealed that a transferrin receptor 2 polymorphism correlated strongly with serum iron and transferrin saturations in male rats. Transfection of Tfr2 expression constructs into tissue culture cell lines revealed that the Gly679 Tfr2 variant is expressed at a lower level than the Ala679 variant. Selective breeding of rats carrying this polymorphism and characterization of iron metabolism in the resulting progeny indicated that homozygosity for the Ala679Gly allele leads to a hemochromatosis phenotype. The Hsd:HHCL rat is the first genetic rat model of hereditary hemochromatosis and may prove useful for understanding the molecular mechanisms underlying the regulation of iron metabolism and the pathogenesis of hereditary hemochromatosis.

Disclosures:

No relevant conflicts of interest to declare.
IRAK4 Kinase As A Novel Therapeutic Target in the ABC Subtype of Diffuse Large B Cell Lymphoma
Authors: Lim Kian-Huat , Romero Donna L. , Chaudhary Divya , Robinson Shaughnessy D. , Staudt Louis M.
Published: October 2019

Abstract 62

Activating mutations of MyD88, particularly L265P, occur in about 30% of activated B cell-like diffuse large B cell lymphomas (ABC DLBCLs), the more malignant molecular subtype of DLBCL that responds poorly to standard chemotherapy. We found that the oncogenic signal of mutant MyD88 is transduced predominantly through the Interleukin-1 Receptor-Associated Kinase (IRAK) cascade consisting of IRAK4 and IRAK1, resulting in constitutive activation of classical NF-κB signaling. Importantly, using knockdown-rescue experiments, we found that the kinase activity of IRAK4, but not IRAK1, is required for the oncogenic effect of mutant MyD88 in the survival of ABC DLBCL cell lines. As such, we have proposed that inhibitors of IRAK4 kinase activity could have a therapeutic impact in lymphomas with MyD88 mutations. We have identified two potent small molecule IRAK4 inhibitors, ND-2158 and ND-2110, that are highly selective across a panel of more than 300 kinases. When administered to rodents, both compounds lead to >90% suppression of LPS-induced TNF-α release in serum. Both inhibitors are universally toxic towards ABC DLBCL but not GCB DLBCL cell lines, consistent with the highly specific mechanism of action. The molecules demonstrate good pharmacologic drug-like properties and are expected to have a suitable safety profile for clinical evaluation. Mechanistic studies indicate that both inhibitors potently abrogate IRAK4-mediated phosphorylation of IRAK1 and NF-κB activity resulting from the MyD88 (L265P) mutation in ABC DLBCL. Additionally, these agents suppress secretion of the pro-inflammatory cytokines IL-6 and IL-10 by ABC DLBCL cells. A second, parallel survival pathway in ABC DLBCL is engaged by “chronic active” B cell receptor signaling, which can be blocked by inhibiting Bruton's tyrosine kinase (BTK) either genetically or pharmacologically. Notably, the IRAK4 inhibitors strongly synergized with BTK knockdown in killing multiple ABC DLBCL cell lines. Our results provide a solid rationale for the further development of IRAK4 inhibitors for the therapy of ABC DLBCL and suggest that simultaneous inhibition of B cell receptor signaling may provide superior clinical responses.

Disclosures:

Romero: NIMBUS Discovery: Employment. Chaudhary:NIMBUS Discovery: Employment. Robinson:NIMBUS Discovery: Consultancy, Employment.
High Dose Therapy Improves Survival in Systemic Light Chain Amyloidosis: 14 Year Follow up
Authors: Parmar Simrit , Howell Joshua , Wang Michael , Khan Mubeen A , Bashir Qaiser , Shah Jatin J. , Shah Nina , Popat Uday R. , Giralt Sergio A , Orlowski Robert Z. , Qazilbash Muzaffar
Published: October 2019

Abstract 750

Background:

Treatment remains a challenge for systemic light chain amyloidosis (AL). Autologous stem cell transplant (AutoSCT) has been associated with long term survival. However, a recent multicenter randomized study failed to show survival benefit for AutoSCT perhaps due to high non-relapse mortality (NRM). Here we present a comparison of AutoSCT to other conventional therapies in AL patients treated at our institution with a 14-year follow up.

Methods:

A total of 2018 cases were identified upon pathology review from 1998–2012. AL was confirmed in 264 patients; primary amyloidosis (PA) in 147 pts and multiple myeloma with amyloidosis (AM) in 110 patients; solitary amyloidoma in 7 patients. AutoSCT was performed in 126 patients (PA=79 and AM=47).

Results:

The day 100 NRM was 5% and 1-year NRM was 8%. With a follow up of 14 years in surviving patients, the 10-year overall survival (OS) of AL patients was significantly better in those undergoing AutoSCT (41% vs. 17%; p<0.0001; figure 1). Involvement of more than one organ (6-yr OS 36% vs. 55%; p=0.04) and cardiac involvement (2-yr OS of 57% vs. 78%; p=0.01) were associated with poor outcome. In the patients undergoing AutoSCT: PA vs. AM, Mayo staging, Boston University (BU) staging or bone marrow plasma cells >10 % at the time of autoSCT did not have an impact on OS. Cardiac biomarkers including NT-ProBNP and Troponin-I and T levels were available in a limited number of patients and were not analyzed for survival outcomes. In multivariate analysis, superior OS was associated with: age <60yrs (HR 2.1, p=0.022); and induction treatment before AutoSCT (HR 2.7, p=0.02). Involvement of kidney as the only end organ showed a trend toward improved survival (HR 1.6, p=0.06) (Table 1). Specifically for PA patients (n=79); treatment before autoSCT was associated with improved 3-yr OS: 85% vs. 66%; p=0.02.

Conclusions:

AL patients should be evaluated for AutoSCT and selected patients should undergo induction therapy to decrease amyloid burden prior to AutoSCT.

Disclosures:

No relevant conflicts of interest to declare.
CD4 Cells Engineered to Express an MHC Class I Restricted TCR Can Rescue CD8 Cells Tolerized to Tumour-Associated Antigens
Authors: Ghorashian Sara , Carpenter Ben , Holler Angelika , Nicholson Emma , Ahmadi Maryam , Zech Mathias , Bennett Clare , Xue Shao-An , Morris Emma , Stauss Hans , Chakraverty Ronjon
Published: October 2019

Abstract 952

Background:

The efficacy of T cell therapies for cancer may be limited when targeting tumour-associated antigens (TAA) which are also self-antigens. Ongoing exposure to TAA on normal cells may lead to tolerance via anergy or exhaustion of antigen-specific T cells.

Methods:

We have designed a model of tolerance to TAA in which T cell receptor (TCR)-transduced CD8 T cells recognise pMDM2, a TAA that is also a ubiquitous self-antigen. CD8+ T cells were transduced with pMDM2-specific TCR (MDM-CD8) and transferred to sub-lethally irradiated B6 mice that express pMDM2 in the context of MHC Class I (H2-Kb). MDM-CD8 cells are detectable 4 weeks after transfer but show defective in vivo killing of target cells pulsed with MDM2 peptide. We have used this model to determine the mechanism of tolerance and to evaluate whether tolerant CD8+ T cells can be rescued by CD4 help.

Results:

To determine whether tolerance of MDM-CD8 cells was dependent upon recognition of cognate antigen, we transferred MDM-CD8 cells into mice of a different MHC background (BALB/c) which lack H2-Kb required for presentation of the TCR-recognised MDM2 peptide. When BALB/c MDM-CD8 cells were transferred to BALBc hosts their functions were preserved and they retained efficient antigen-specific cytolysis.

To determine whether tolerance could be modified by provision of CD4+ T cell help, we co-transferred MDM-CD8 with transgenic OT-II CD4+ cells. OT-II cells were primed with dendritic cells (DCs) loaded with cognate pOVA323-339 or irrelevant peptide. When activated through their TCR, OT-II cells increased both the frequency of MDM2-specific CD8 cells and their cytotoxic functions, indicating that CD4 help can overcome CD8 tolerance to TAA.

Ineffective antigen presentation to CD4 cells and lack of known MHC class II-restricted TAA are major limitations to providing CD4 help in T cell therapy for cancer. We therefore tested whether transfer of the MHC Class I-restricted MDM2 TCR into CD4 cells could provide help upon transfer to antigen-expressing hosts. Co-transfer of MDM2-TCR-transduced CD4 cells with CD8 cells improved antigen-specific killing of target cells when compared to single transfer of either TCR-transduced CD8 or CD4 cells.

Conclusion:

CD4 cells rendered capable of responding to an MHC class I restricted TAA by TCR transfer can rescue tolerance developing in a CD8 population with the same specificity. This is potentially a novel way to circumvent defective immune responses arising in adoptively transferred effector cells due to prolonged exposure to cognate antigen on normal host cells.

Disclosures:

Stauss: Cell Medica: Scientific Advisor Other.
Pathway Mutations in the Splicing Machinery in Myeloid Neoplasms
Authors: Ogawa Seishi
Published: October 2019

Abstract SCI-14

During the past decade, significant progress has been made in our understanding of the molecular pathogenesis of myelodysplastic syndromes (MDS) and related myeloid neoplasms, in which one of the major findings was frequent mutations of genes in epigenetic regulation, such as DNA methylation (DNMT3A, TET2, and IDH1/2) and chromatin modifications (ASXL1, EZH2, EED, and SUZ12). They are also found in comparable or even higher fractions of other myeloid neoplasms, underscoring the common impact of deregulated epigenetic regulation on myeloid leukemogenesis. On the other hand, a new class of pathway mutations has been uncovered recently that commonly involve the RNA splicing machinery (1, 2, 3). Thus, at least eight different components of the machinery, invariably engaged in the 3' splice site recognition, have been reported to be mutated in as high as 45 percent to 85 percent of cases with different subtypes of MDS and related myeloid neoplasms mostly in a mutually exclusive manner. This indicates that the 3' splice site recognition is the functional target of these mutations. There exist discrete mutational hotspots in three out of four major targets, including SF3B1, SRSF2, U2AF35, and ZRSR2, and these mutant alleles can induce abnormal RNA splicing, indicating the gain-of-function nature of the mutations (2). Splicing factor mutations are largely specific to myelodysplasia phenotypes but relatively rare in acute myeloid leukemia and myeloproliferative neoplasms (2), suggesting their primary roles in the pathogenesis of myelodysplasia. The genotype-phenotype association is especially prominent in the case of SF3B1 mutations, which were found in 76 percent to 83 percent of cases of refractory anemia with ringed sideroblasts (RARS), refractory anemia with ringed sideroblasts associated with marked thrombocytosis (RARS-T), and refractory cytopenia with multilineage dysplasia and ringed sideroblasts (RCMD-RS) (1, 2). In this session, the updated findings on the spliceosome mutations found in myelodysplasia, including their clinical and functional aspects, will be discussed.

Disclosures:

No relevant conflicts of interest to declare.
Primitive and Definitive Erythropoiesis
Authors: Palis James
Published: October 2019

Abstract SCI-37

Studies in mammalian and nonmammalian vertebrate embryos indicate that erythropoiesis comes in two flavors: primitive and definitive. The primitive erythroid lineage in mammalian embryos is characterized by a transient wave of lineage-committed progenitors that emerge from the yolk sac and generate a wave of precursors that synchronously mature in the bloodstream. Primitive erythroid precursors dynamically regulate embryonic globin gene expression and ultimately enucleate to form erythrocytes. Primitive erythropoiesis is superseded by definitive erythroid cells that mature extravascularly in association with macrophage cells. Studies in the mouse embryo indicate that definitive erythropoiesis has two distinct developmental origins. The first is a transient wave of erythro-myeloid progenitors (EMP) that emerge from the yolk sac and seed the early fetal liver. The second is a long-term program of erythropoiesis derived from hematopoietic stem cells. Erythropoietin is the central regulator of definitive erythropoiesis, in part by regulating the survival of committed progenitors. In contrast, the role of erythropoietin in primitive erythropoiesis remains poorly understood. Recent studies indicate that erythropoietin does not regulate the primitive erythroid progenitor compartment, but rather plays a critical role in establishing an antiapoptotic state during the terminal maturation of primitive erythroblasts. EMP-derived proerythroblasts are capable of extensive self-renewal in vitro, while primitive erythroid progenitors are incapable of self-renewal under the same conditions. These studies, taken together, indicate that the primitive and definitive forms of erythropoiesis have fundamental differences in the regulation of red cell output. The overlapping emergence of primitive and definitive erythroid lineages in differentiating embryonic stem cells suggests that the transient yolk-sac-derived primitive and EMP-derived definitive erythroid programs are recapitulated in vitro. These studies offer the hope that human embryonic stem cells can serve as a source of functional definitive erythroid cells for transfusion therapy.

Disclosures:

No relevant conflicts of interest to declare.
Autoantibody Epitope Mapping By Hydrogen-Deuterium Exchange Mass Spectrometry at Nearly Single Amino Acid Residue Resolution Reveals Novel Exosites on ADAMTS13 Critical for Substrate Recognition and Mechanism of Autoimmune Thrombotic Thrombocytopenic Purpura
Authors: Casina Veronica C. , Hu Wenbing , Hanby Hayley A. , Pickens Brandy , Mayne Leland , Ostertag Eric , Kacir Stephen , Siegel Don L. , Englander S. Walter , Zheng X. Long
Published: October 2019

Acquired thrombotic thrombocytopenic purpura (TTP), a potentially fatal arterial thrombotic disorder, is primarily caused by autoantibodies that bind and inhibit plasma von Willebrand factor (VWF)-cleaving metalloprotease (ADAMTS13) activity. However, the mechanisms underlying autoantibody-mediated inhibition of ADAMTS13 activity and acquired TTP are not fully understood. By a hydrogen-deuterium exchange coupled with mass spectrometry (HX-MS) technique, we found that human monoclonal anti-ADAMTS13 antibodies, the single chain variable region fragments (scFvs)4-20, 4-16, and 3-1 that were, isolated by phage display from two patients with acquired TTP, predominantly bound to a discontinuous and conformational epitope in the spacer domain of ADAMTS13 with a subtle difference. The epitope for scFvs4-20 and 4-16 comprises five small flexible loops, including a previously described motif A (or exosite 3, R659-E664), motif B (exosite 4, L632-R639), and several other outlying residues (F592, Y658, and Y665), while scFv3-1 bound all other residues except for those in motif A. Site-directed mutagenesis and biochemical analysis demonstrated that both motifs A and B were found to be critical for recognition and proteolysis of VWF73 and multimeric VWF. Deletion of motif A or motif B in full-length ADAMTS13 abolished the binding of scFvs4-20 and 4-16 but not 3-1 (which did not bind motif A). Our findings demonstrate the powerful use of HX-MS for mapping antibody epitopes at nearly single amino acid resolution. This provides a new way to reveal mechanisms of autoantibody-mediated inhibition of plasma ADAMTS13 activity and acquired TTP.

Figure 1 Figure 1.

Disclosures

No relevant conflicts of interest to declare.
Von Willebrand Factor Is a Cofactor in Complement Regulation
Authors: Feng Shuju , Kroll Michael H. , Afshar-Kharghan Vahid
Published: October 2019

Complement, besides its involvement in eliminating microbes, participates in such diverse processes as synapse maturation, clearance of immune complexes, angiogenesis and tissue regeneration. Delicate balance between complement activation and regulation contributes to complement’s role in physiology. Any trigger that tips this balance can induce self-attack. Atypical hemolytic uremic syndrome (aHUS) is a systemic disease characterized by non-immune hemolytic anemia, thrombocytopenia, and renal impairment. In over 50% of cases, aHUS is known to be caused by uncontrolled activation of the complements.

Von Willebrand factor (VWF) is a large multimeric glycoprotein that plays an important role in stopping the escape of blood from vessels following vascular injury. VWF and VWF cleavage enzyme ADAMTS13 gene defects have been identified in patients with aHUS, raising the possibility that VWF could have contributed to complement regulation. To examine the role of VWF in complement activation, we investigated whether VWF functions as a cofactor for FI-mediated C3b cleavage through in vitro assay. We found that C3b binds to VWF. In the presence of plasma-purified VWF (pVWF), FI cleaves C3b to 68kD and 43kD degradation products (iC3b) (Figure (A)). VWF alone, or FI alone, did not have any effect on C3b cleavage. C3b, not C3 or iC3b, was the mainly substrate for FI/VWF proteolysis. Increasing VWF concentration or prolonging the incubation time with VWF enhanced FI-mediated C3b cleavage. To remove the possibility that another plasma protein co-purified with pVWF that affects our results, we used recombinant VWF dimers purified from human embryonic kidney (HEK) 293 cell expressing VWF-Dpro cDNA, and detected a similar cofactor activity (Figure (B)).To investigate whether the size of VWF multimers have any effect on C3b cleavage, we compared the cofactor activity of pVWF, recombinant VWF dimers, and ULVWF multimers. While pVWF and dimer VWF enhanced C3b cleavage by FI, ULVWF did not have any effect on C3b cleavage (Figure (B)). In plasma, complement proteins Factor B and Factor D coexist with the inhibitory protein FI. To investigate the effect of VWF on complement activity in the presence of both pro-activation and inhibitory complement proteins, we incubated C3, FB, FD, and FI with VWF. In the presence of FB and FD, C3 be activated and resulted in the generation of C3a and C3b. Addition of recombinant VWF put a brake on complement activation and shifted C3 toward the generation of iC3b (Figure (C)). We conclude that normal plasma VWF, function as a cofactor, prevents complement activation through steers the complement pathway toward the generation of inactive iC3b. ULVWF multimers, as are present in patients with thrombotic microangiopathy, lack an inhibitory effect on complement and permit complement activation.

Figure 1 Figure 1.

Disclosures

No relevant conflicts of interest to declare.