bluuebunny/crossref_metadata_2025 huggingface dataset:
Baseline Characteristics of Patients with Sickle Cell Disease in An Ongoing 5-Year, Prospective, Noninterventional Registry Trial
Authors: Heeney Matthew M. , Mueller Brigitta U. , Baltz Brad , Paley Carole , Esposito Jason , Sharma Surabhi , Vichinsky Elliott
Published: October 2019

Abstract 1060

Introduction:

The baseline characteristics and therapeutic management of sickle cell disease (SCD) can be quite variable depending on disease severity. This report describes baseline assessments and therapies of 420 patients in an ongoing registry designed to study current treatment patterns, natural history, and outcomes in patients with SCD.

Methods:

This registry trial involving 57 US hematology centers had a sequential recruitment target of 600 patients with SCD subdivided into 3 age groups (2-<12 yr, 12-<18 yr, ≥18 yr). Patients '2 years of age with HbSS, HbS/β-thalassemia, or HbSC were eligible. Follow-up visits were every 6 months for a maximum of 5 years. Assessments included demographics, SCD history, Karnofsky Performance Status, medical history, types of crises, frequency of crises and hospitalization, treatment history, transfusion practices and use of iron chelation. Differences between pediatric (<18 yrs) and adult (≥18 yrs) patients were examined. (ClinicalTrials.gov identifier, NCT01220115).

Results:

Enrollment: 420 patients (254 <18 yrs, 166 ≥18 yrs) completed the initial baseline visit. Baseline characteristics and medical history are shown in the Table and Figure.

Conclusions:

These data indicate differences in baseline characteristics and treatment patterns between adult and pediatric patients with SCD in this registry. Karnofsky performance status was worse, and serum ferritin levels were higher in adult versus pediatric patients. The percentage of patients having regular transfusions was higher in pediatric patients, while adult patients had higher rates of intermittent transfusions. As expected, the lifetime chelation exposure was more frequent in adult patients. Hospitalizations and use of hydroxyurea were also greater in pediatric patients. In terms of medical history related to SCDs, pediatric patients had higher frequencies of asthma, abnormal TCDs, dactylitis and splenic sequestration, while adults had more frequent evidence of avascular necrosis, gall bladder disease, leg ulcers and pulmonary hypertension. Pain was the most common crisis type in both cohorts, although this accounted for a greater relative proportion of crises in the adult cohort. The relative proportion of ACS events was lower in the adult versus pediatric patients; all other crisis types occurred in similar relative proportions in both cohorts. This ongoing registry will provide information about disease patterns, current treatment practices, and outcomes, thus contributing to a better understanding of the appropriate therapeutic management of patients with SCD.

Disclosures:

Heeney: Novartis: Consultancy, Research Funding. Baltz:Novartis: Research Funding. Paley:Novartis: Employment. Esposito:Novartis: Employment. Sharma:Novartis: Employment. Vichinsky:Novartis, Apotex, Ferrokin: Research Funding.
Cancer Patients Requiring Interruption of Long-Term Anticoagulant Therapy: The Use of Fixed Sub-Therapeutic Doses of Low-Molecular Weight Heparin
Authors: Saccullo Giorgia , Malato Alessandra , Coco Lucio Lo , Barbello Ilenia , Caracciolo Clementina , Raso Simona , Santoro Marco , Di Bartolo Orazio Graziano , Piazza Ambra Di , Abbadessa Vincenzo , Siragusa Sergio
Published: October 2019

Abstract 1244

Introduction.

We tested the efficacy and safety of fixed doses of Low-Molecular Weight Heparin (LMWH) in cancer patients requiring interruption of Vitamin-k Antagonist (VKA) because of invasive procedures (defined as major and non major surgery) or chemotherapy inducing platelets depletion. Methodology. Cancer patients were defined to be at high (atrial fibrillation [AF] with previous stroke, prosthetic mitralic valves and venous thromboembolism [VTE] lasting < 3months) or low risk of thrombosis (AF without previous stroke, VTE lasted > 3 months, and prosthetic aortic valves). They discontinued VKA 5 + 1days before surgery or chemotherapy; in those at low-risk for thrombosis, LMWH was given at a prophylactic dosage of 3.800 U.I. (nadroparin) or 4.000 U.I. (enoxaparin) anti-FXa once daily the night before the procedure or the beginning of chemotherapy. In patients at high-risk for thrombosis, LMWH was started early after VKA cessation (when an INR < 1.5) and given at fixed sub-therapeutic doses (3.800 or 4.000 UI anti-FXa twice daily) until procedure or beginning of chemotherapy. In patients undergoing procedures, LMWH was reinitiated 12 hours after procedure while VKA the day after; in patients receiving chemotherapy, LMWH was reinitiated 12 h after a stable platelet count > 30.000 mmc3 and VKA 12 h after a stable platelet count > 50.000 mmc3. Heparin was continued until a therapeutic INR value was reached. The primary efficacy endpoints were the incidence of thromboembolism and major bleeding from VKA suspension to 30 + 2 days post-procedure or next chemotherapy. Results. A total of 156 patients (68, 53.9% at low-risk and 58, 46.1% at high-risk for thrombosis) were enrolled (Table 1); 44 (28.2%) underwent major surgery, 53 (33.9%) non-major surgery and 29 (18.5%) chemotherapy. Overall, thromboembolic events occurred in 5 patients (3.2%, 95% confidence intervals 0.5–5.9), 4 belonging to high-risk and 1 to low-risk group. Overall, major bleeding occurred in 5 patients (3.2%, 95 CI 0.5–5.9), all belonging to high-risk group (3 during major surgery and 2 during chemotherapy). The mean time of anticoagulation with LMWH was 7.5 days in patients underwent procedures and 13.2 days in those who received chemotherapy. Conclusion. LMWH given at fixed sub-therapeutic doses appears to be a feasible and relatively safe approach for bridging therapy in chronic anticoagulated cancer patients requiring VKA interruption.

Disclosures:

No relevant conflicts of interest to declare.
Hemophagocytic Lymphohistiocytosis in Adults: Updates in Diagnosis and Treatment
Authors: Kleynberg Roman Leonid , Schiller Gary J.
Published: October 2019

Abstract 5037

Hemophagocytic lymphohistiocytosis (HLH) is rare potentially life-threatening disorder that must be recognized and treated early to improve clinical response rates and outcomes. We describe a 36 year-old male with a longstanding history of ulcerative colitis who presented with a one-week history of lymphadenopathy, splenomegaly, fevers, and chills. Workup at an outside institution had been intensive - he had a long complicated hospital course that included hypoxemic respiratory failure requiring intubation and mechanical ventilation, acute kidney injury, hyperbilirubinemia, pancytopenia, coagulopathy, hypofibrinogenemia, CMV viremia, and Clostridium difficile colitis for which he was treated. At our facility, the patient presented with a white blood count of 0.19 109/L (N: 5%, L: 95%, M: 0%, E: 0%, B: 0%), hemoglobin 9.0 mg/L, platelets 54 x109/L, ferritin 538 ng/dL, and triglycerides of 280 mg/dL. A bone marrow biopsy revealed CMV positivity by immunohistochemistry and marked hemophagocytic histiocytosis. The patient was treated with the HLH-2004 chemo-immunotherapy protocol with dexamethasone, etoposide (VP-16), and cyclosporine A. He responded well and is now eight months status-post initial treatment.

HLH in adults is a disorder that little is known about. Acquired forms of HLH can occur in otherwise healthy adults, whereas in children, HLH often presents in its inherited (familial) form. In adults it is often associated with infections (i.e. Epstein-Barr virus, herpes simplex, cytomegalovirus), rheumatologic diseases (i.e. rheumatoid arthritis, systemic lupus erythematosus, adult onset systemic Still's disease), malignancy (i.e. natural killer cell leukemia, peripheral T-cell lymphoma, EBV in T-cell lymphoma, B-cell lymphoma, and a variety of other lymphomas). HLH in adults is characterized by cytokine dysfunction and results in the systemic accumulation of activated T-lymphocytes and activated histiocytes (macrophages). Natural killer (NK)-cell and cytotoxic T cell activity in HLH patients is commonly depressed. The standard chemo-immunotherapy protocol as recommended by the Histiocyte Society (HLH-2004) includes therapy with dexamethasone, etoposide (VP-16), cyclosporine A upfront and, in selected patients, cortiocosteriods and intrathecal methotrexate. Thus, early recognition and treatment of this disorder is essential to decrease associated morbidity/mortality. Hemophagocytic lymphohistiocytosis in adults is a diagnosis that must be in the differential whenever a patient presents with fever, cytopenia, hepatosplenomegaly, hypertriglyceridemia and/or hypofibrinogenemia.

Disclosures:

Schiller: BMS: Research Funding; Celgene: Research Funding; Ambit: Research Funding; Novartis: Research Funding; Sunesis: Research Funding.
Platelet Activation in Cardiovascular Disease
Authors: Corinaldesi Giorgio
Published: October 2019

Abstract 5242

Platelets play a pivotal role in the pathogenesis of coronary artery disease (CAD). Among a number of platelet integrin receptors ITGA2B and ITGB3 appear to be fundamental mediators of platelet aggregation as they interact with fibrinogen, VWF, fibronectin, vitronectin, and thrombospondin; for these major properties these two receptors are risk-determining factors for increased platelet thrombogenicity, enhancement of leukocytes trafficking and activation by CD11a/CD18 (LFA-1) and CD11b/CD18 (MAC-1), thus leading to CAD.

Receptor clustering promote cell adhesion by increasing the number of ligand receptor bonds GPIb-IX-VWF, fibrinogen /alfaIIb-beta 3 which promote platelet aggregation; this is showed by various parameters including increased platelet-monocyte aggregates, P-selectin (CD62P), CD42b, CD63, and EMMPRIN (CD147). The link between platelet activation, leukocyte recruitment, endothelial dysfunction, progression of atherosclerosis plaque and CAD has been demonstrated through the involvement of inflammatory cytokines that play a central role in the development of atherosclerotic plaques with MCP-1, CCL-2, Rantes, IL-8, and CD40L.

In this study, we investigated a total of 38 subjects (mean age of 62 +/− 6 years) with confirmed CAD (unstable angina: 18 patients; ST elevation myocardial infarction: 20 patients) on coronary angiography and 32 controls. They were examined for CD62P, CD63, CD42b, and CD147 expression, troponin level, creatine-kinase-MB level (indicator of myocardial injury), C-reactive protein, and IL-6 (being both indicators of systemic inflammation).

Patients with CAD had significantly higher markers of platelet activation (p<0.001) which were constituted by the expression of GP receptors (CD62P/PSGL-1, CD162, CD61/63, CD147). This activity closely correlates with a significant increase of acute phase reactant in cases than in controls (p<0.0001), and mostly, it confirmed that these associations may be largely responsible of cardiovascular events.

Disclosures:

No relevant conflicts of interest to declare.
Podia in Multiple Myeloma (MM) Cells Promote Adhesion with Bone Marrow (BM) Fibroblastic Stromal Cells
Authors: Ramasamy Karthik , Anwar Amna , Kazmi Majid A. , Schey Steve , Calle Yolanda
Published: October 2019

Abstract 626

The physical contact between MM cells and the BM microenvironment can lead to cell adhesion mediated drug resistance. Although some components of adhesive structures in MM cells have been identified very little is known about their exact organisation and dynamics. We have observed formation of proteoglycan CD138 and F-actin containing membrane extensions in MM cells in in vitro cultures of BM aspirates as well as in sections of BM trephines of MM patients. MM membrane extensions elongated on the surface of BM stromal cells or interconnected MM cells forming seemingly cellular networks. Morphologically, MM membrane extensions appear similar to other structures in haematopoietic cells such as nanotubes formed by T-cells involved in cell communication and podia in CD34+ cells and leukemia cell lines whose function remains unknown. Dexamethasone (a drug commonly used clinically against MM) increased the percentage of MM cells displaying membrane extensions and the length of these structures. This correlated with enhanced expression of surface CXCR4, increased adhesion of MM cells on fibroblastic stromal cells and protection of MM cells against Dexamethasone-mediated apoptosis. Treatment with Bortezomib at clinically achievable doses decreased CXCR4 levels in MM cells and did not induce podia formation/extension and correlated with inhibition of MM cell adhesion and induction of MM cell apoptosis in a dose dependent manner. Blocking CXCR4 signalling with Plerixafor (AMD3100) inhibited the rate of formation and length of MM membrane extensions and correlated with sensitisation of MM cells to Dexamethasone. Similar inhibitory results were obtained using Dexamethasone in combination with the c-Abl/Src kinase inhibitor Dasatinib. Neither Dasatinib nor Plerixafor at doses achievable in patients directly induced apoptosis in MM or BM stromal cells. We conclude that membrane extensions/podia formed by MM cells are involved in the interaction with BM stromal cells, require CXCR4 and Src and/or c-Abl activity and could be involved in resistance to treatment with Dexamethasone.

Disclosures:

No relevant conflicts of interest to declare.
Local Treg Immunomodulation Cures Metastatic Lymphoma Including CNS Sites
Authors: Marabelle Aurelien , Kohrt Holbrook E , Brody Joshua , Torchia James Anthony , Zhou Gang , Luong Richard , Rajapaksa Ranjani , Tse Victor , Levy Ronald
Published: October 2019

Abstract LBA-2

Background:

CD4+CD25+FOXP3+ regulatory T-cells (Tregs) protect cancers from the immune system. They express important functional molecules such as CTLA-4 and Ox40 on their surface. Anti-CTLA4 monoclonal antibody (mAb) is the first drug to improve survival of refractory metastatic melanoma. Its recent approval by the FDA/EMEA inaugurates a paradigm shift in cancer therapy where the immune system is targeted rather than the tumor itself.

Scientific question:

Does local immunomodulation of tumor specific Tregs trigger a systemic anti-tumor immune response and cure disseminated lymphoma?

Results:

The Tregs that infiltrate the tumor site preferentially express CTLA4 and OX40 compared to their counterparts in the blood and other lymphoid organs, both in mice and in humans. Upon injection of low doses of anti-CTLA4 and anti-OX40 together with CpG, a TLR-9 agonist, directly into the tumor, tumor-specific Tregs are depleted from the tumor-infiltrate. This immunomodulatory combination therapy triggers an anti-tumor immune response able to cure mice with established disseminated disease. The triple combination is uniquely required as neither CpG alone nor mAbs without CpG are effective. The local Treg immunomodulation with anti-CTLA4+anti-OX40+CpG in a single sub-cutaneous tumor eradicates disease in mice with established CNS lymphoma, even with leptomeningeal and spinal cord metastases, whereas chemotherapies or mAb therapy directed against the tumor cells have little effect in the CNS site. Moreover, these cured mice have a long term intra-cranial anti-tumor immunity since they are protected against late intra-cranial re-challenge.

Significance:

Immunomodulatory antibodies are currently under clinical development for cancer treatment, and their major side effect is the triggering of auto-immune diseases. We show here that injecting very little doses of these antibodies in combination with CpG at one tumor site is sufficient to trigger a systemic anti-tumor response able to eradicate distant sites, including in the CNS which is usually considered a sanctuary site for conventional systemic therapies.

Impact:

We recently have published positive results of intra-tumoral CpG in patients with follicular Lymphoma (Brody,Levy, et al. JCO, 2010). Anti-CTLA4 has just been FDA approved in patients with metastatic melanoma, and anti-Ox40 antibodies are currently being tested in phase I/II clinical trials. Therefore, the combination described here can be tested in patients with injectable sites of lymphoma, even if they have CNS disease.

Disclosures:

No relevant conflicts of interest to declare.
Distress Screening Scores of Malignant and Benign Hematology Patients: Results of a Pilot Project.
Authors: McLellan Linda , Pohlman Brad , Rybicki Lisa , Foster Larry , Tench Shawnda , Cooper Michele , McKenzie Millie , Kilbane Megan , Wright Cheryl , Dimitrov Julie , Bauer Julianne , Dabney Jane , Savage Barbara , Poole Christa , Barrat-Gordon Rene' , Fireman Karen , Bolwell Brian J.
Published: October 2019

Abstract 3173

The Cleveland Clinic Taussig Cancer Institute completed a pilot project on distress screening as part of the plan to address the American College of Surgeons Commission on Cancer requirement (Standard 3.2: Psychosocial Distress Screening) to screen malignant and benign hematology patients for distress and psychosocial health needs. Over a two week period every hematology patient seeing a physician or mid-level provider completed the distress screening instrument Emotions Thermometer (ET), a five dimensional tool that includes four predictor domains: distress, anxiety, depression, anger and one outcome domain of need for help. Each domain is rated on a 0 to 10 Likert type scale, in a thermometer format. With respect to validity and reliability, the sensitivity and specificity of the ET tool has been found comparable to known validity and reliability of other accepted measures of distress, depression, and anxiety (Mitchell, et al, 2009). Per National Comprehensive Cancer Network (NCCN) guidelines, a score of 4 or higher on distress screening warrants a referral to a psychosocial professional. Distress scores 4 and over were compared among the diagnoses using the Chi-Square test. When the overall P-value was significant (P <0.05), at least two groups differed, and pairwise comparisons were conducted to determine which diagnostic groups differed.

Of the five thermometer domains, patients scored highest on anxiety. There were no differences among diagnoses on anger (P=0.51), but there were differences in the other four domains (P<0.001 distress, depression, and need for help; P=0.026 anxiety). For all four, pairwise comparisons indicated that multiple myeloma patients had higher (worse) scores on anxiety, distress, depression, and need for help than all other diagnoses; there were no differences among benign hematology, leukemia/MDS/CML, or lymphoma/CLL.

While this pilot project does not answer why multiple myeloma patients report levels of psychosocial distress significantly higher than other hematology patients, increased attention needs to be given to address these patients' experience of anxiety, depression, and distress. This project provides valuable information about the levels of distress experienced among all hematology patients and is useful for determining staffing levels of psychosocial professionals needed to address distress.

Percentage of Emotion Thermometer Scores Over 4 Diagnosis Distress Anxiety Depression Anger Help Benign Hem 18% 27% 18% 12% 7% Leuk/MDS/CML 19% 28% 17% 9% 14% Lymph/CLL 18% 31% 16% 14% 13% Myeloma 38% 44% 35% 15% 27% P-value <0.001 0.026 <0.001 0.51 <0.001

Disclosures:

No relevant conflicts of interest to declare.
Partial Reversal of Dabigatran Effect by a Prothrombin Complex Concentrate in a Model of Thrombin Generation
Authors: Hoffman Maureane R. , Volovyk Zoya , Monroe Dougald M.
Published: October 2019

Abstract 3420

Dabigatran etexilate (Pradaxa) is a prodrug that is metabolised to a small molecule direct thrombin inhibitor. It has some more desirable charactersitics than warfarin for long-term anticoagulation of patients with atrial fibrillation, particularly the fact that it does not routinely need laboratory monitoring. However, it has the drawback that it is not readily reversable. While the package insert suggests administration of fresh frozen plasma or recombinant FVIIa as “antidotes”, there is no good theoretical or experimental basis to support this recommendation. Several sutdies have suggested that prothrombin complex concentrates (PCCs) can partially reverse the effects of dabigatran on laboratory assays. However, there is still much controversy over the potential of PPCs to reverse dabigatran anti-coagulation. The goal of our studies was to assess the effects of a 4-component PCC (Beriplex, CSL-Behring) on parameters of thrombin generation in PRP and a cell-based model. This allow us to use varying levels of dabigatran and varying levels of PCC, a cell-associated source of tissue factor (TF), fresh platelets, and either plasma or plasma levels of coagulation factors. Thrombin generation was assessed continuously using a fluorogenic thrombin substrate in a fluorescent plate reader. Dabigatran (Selleck Chemicals) was dissolved in DMSO and added to the test plasma at concentrations from 189–944 ng/mL. Beriplex was added to test plasma at concentrations from 0.25 FIX units/mL to 2.0 FIX units/mL. As others have reported for conventional calibrated automated thrombogram (CAT) assays, we found that dabigatran prolonged the lag before onset of thrombin generation. It also progressively depressed the rate and peak level of thrombin production. We found that the addition of Beriplex increased the rate and peak level of thrombin generation in a dose-dependent manner, but had little effect on the lag. This effect was seen at all levels of dabigatran tested, with a level of 1 U/mL restoring the rate and peak levels of thrombin generation, as well as the area-under-the-curve, to normal levels. The addition of FIX alone at 1 U/mL had no significant effect on thrombin generation in the presence of dabigatran. The effects of Beriplex were similar, though not identical, to the pattern seen when prothrombin alone was added to this model. Thus, we hypothesize that Beriplex has its dominant effect on reversal of dabigatran effects by a prothrombin-mediated mechanism. This increases the burst of platelet surface thrombin generation. However, this unactivated PCC does not reverse the inhibitory effect of dabigatran on the amplification phase of hemostasis as reflected in the lag time before the onset of thrombin generation. Thus, Beriplex corrects some, but not all, parameters all of thrombin generation affected by dabigatran in this model.

Disclosures:

Hoffman: CSL Behring: Research Funding.
Regulatory Mutations in GATA2 Associated with Aplastic Anemia
Authors: Townsley Danielle M. , Hsu Amy , Dumitriu Bogdan , Holland Steven M. , Young Neal S.
Published: October 2019

Abstract 3488

Germline heterozygous mutations in GATA2 have been reported to cause familial myelodysplasia–acute myeloid leukemia (MDS/AML), monocytopenia and mycobacterial infection (monoMAC syndrome), dendritic cell, myeloid and NK cell lymphopenia (DCML), and Emberger syndrome (lymphedema and MDS). GATA2 is a zinc finger transcription factor that plays a crucial role in regulating growth of hematopoietic progenitors. In some pedigrees, patients or family members have manifestations of bone marrow failure. We hypothesized that patients with aplastic anemia (AA) may harbor mutations in GATA2. The coding regions and regulatory regions of GATA2 were sequenced in 99 patients with confirmed AA. Sequences from 100 normal individuals as well as published human genomes from unaffected individuals (dbSNP build 135 and 1000 Genomes Project) were used as controls. Genetic variants were confirmed in hematopoietic and somatic tissues.

We identified 4 heterozygous mutations in regulatory regions of GATA2 in 5 patients. In two patients, a mutation at nucleotide 59T>G in exon 1 of isoform 2 was identified; both had severe AA in early adulthood refractory to immunosuppressive therapy. We noted this 59T>G mutation in two unrelated individuals with severe disseminated mycobacterial disease. We identified a mutation at nucleotide 20G>A in exon 2 of isoform 1, in a 3 year-old male with hepatitis-associated severe AA whose disease was refractory to multiple rounds of immunosuppressive therapy. Another mutation was present in 38G>A in exon 2 of isoform 1 in a 32 year old male with moderate AA and paroxysmal nocturnal hemoglobinuria (PNH). We also identified the exon 2 38G>A mutation in a patient with disseminated mycobacterial disease where reduced transcription of the mutant 38G>A allele was noted on RT-PCR. Finally, an intron 5, c.512+573 G>A variant was identified in an 18 year old male with severe AA who progressed after immunosuppressive therapy to MDS/AML. This variant, which causes a disruption of the FLI1 binding site, has also been found to be pathogenic in monoMAC syndrome.

In summary, a subset of patients with AA were found to have mutations in GATA2 suggesting a role for the gene in the pathogenesis of bone marrow failure. It also may identify patients at higher risk of infectious complications, those who may have less advantageous responses to immune suppression, and command earlier bone marrow transplantation.

Disclosures:

No relevant conflicts of interest to declare.
APS From the Root of Angelica Sinesis Promotes Megakaryocytopoiesis and Thrombopoiesis Through PI3k/Akt Pathway
Authors: Yang Mo , Meng Fanyi , Ye Jie Yu , Xu Yue , Xiao Bin , Chong Beng , Liu Chang
Published: October 2019

Abstract 4648

We have demonstrated the thrombopoietic effects of Chinese Medicine Dang Gui Bu Xue Tong (DBT), a formula composed of Radix Angelicae Sinensis and Radix Astragali in a thrombocypenic mouse model (Yang M et al, J Ethnopharm 2009). In this study, we further test the effect of polysaccharide (APS) from the root of Radix Angelicae Sinensis (APS) in in-vivo thrombopoiesis and in in-vitro megakaryocytopoiesis. A myelosuppression and thrombocytopenia mouse model was treated with APS (10 mg/kg/day), thrombopoietin (TPO) and saline as control. Peripheral blood cells from APS, TPO and vehicle-treated samples were then counted in different time-points. Using the colony-forming unit (CFU) assays, we determined the effects of APS on the hematopoietic stem/progenitor cells and megakaryocytic lineages. Analyses of Annexin V, Caspase-3, and Mitochondrial Membrane Potential were also conducted in megakaryocytic cell line M-07e. Lastly, the effects of APS on cells treated with Ly294002, a Phosphatidylinositol 3-Kinse inhibitor and the effect of APS on the phosphorylation of AKT were further studied in megakaryocytic cells. Our results showed that APS significantly enhanced the platelets and other blood cells recovery and CFU formation in this model. Morphological examination of bone marrows showed that APS treatment significantly increased the recovery of the megakaryocytic series and other hematopoietic progenitor cells. We observed the antiapoptotic effects of APS on M-07e cells. Addition of Ly294002 alone increases the percentage of cells undergoing apoptosis. In contrast, additional of APS to Ly294002-treated cells reversed the percentage of cells undergoing apoptosis. Furthermore, addition of APS significantly increased the phosphorylation of AKT. Our studies demonstrate that APS promotes platelet recovery in the mouse model and has anti-apoptosis effect in megakaryocytic cells. This effect is likely to be mediated by the PI3K/AKT pathway.

Disclosures:

No relevant conflicts of interest to declare.
Effects of Small-Molecule Inhibitors of TNFR2 Signaling Pathways On TNF-Mediated Expansion of CD4+FoxP3+ Regulatory T Cells
Authors: Wu Xueqiang , Chen Yan , Han Chunsheng , Gong Yiwen , Bu Dingfang , Zhang Yin , Xia Junyan , Liu Qian , Chen Xue , Hu Yang , Zhu Ping
Published: October 2019

Abstract 4722

TNF is a pleiotropic cytokine with biphasic proinflammatory and immunosuppressive effects. Previous work clearly demonstrated that TNF has the capacity to preferentially activate and promote the proliferative expansion of CD4+FoxP3+ regulatory T cells (Tregs), which represent a basis for the paradoxical anti-inflammatory action of TNF. Our studies also indicate that TNFR2, one of the TNF receptor which is preferentially expressed by Tregs, mediates the Treg-activating effect of TNF. However, the molecular mechanism and signaling pathways mediated Treg-activating effect of TNF remain to be understood.

In this study, we first further verified that TNFR2 transduces the activating signal of TNF on Tregs, based on the evidence that 1) human TNF, known to only bind to mouse TNFR1, did not activate mouse Tregs; 2) a blocking anti mouse TNFR2 Ab, but not TNFR1 Ab, dose-dependently inhibited TNF-mediated proliferation of mouse Tregs at concentrations of 2.5–500 ng/ml. We next examined the signaling pathways of TNFR2 leading to the proliferation of Tregs, by using specific small-molecule inhibitors. It is well established that the activation of IKK-NFkB is a major consequence of activation of TNFR2. However, small molecule inhibitors of NFkB signaling pathway, namely BAY11–7082 and Sulfasalazine, did not block TNF-mediated proliferation of Tregs. In contrast, small molecule inhibitors of MAPK signaling pathway, SB203580 (P38 MAPK inhibitor), SP600125 (JNK inhibitor) and PD98059 (Erk1/2 inhibitor), potently suppressed TNF-induced replication of Tregs in a dose-dependent manner.

Our results indicate that TNF selectively stimulates the expansion of FoxP3+ Tregs through TNFR2. Activation of MAPK (ERK1/2,P38 and JNK), rather than NFkB, is responsible for this activity of TNF.

Disclosures:

No relevant conflicts of interest to declare.
Preclinical Data In Cynomolgus (cyn) Monkeys Of ASTX727, a Novel Oral Hypomethylating Agent (HMA) Composed Of Low-Dose Oral Decitabine Combined With a Novel Cytidine Deaminase Inhibitor (CDAi) E7727
Authors: Oganesian Aram , Redkar Sanjeev , Taverna Pietro , Joshi-Hangal Rajashree , Azab Mohammad
Published: October 2019

Background

Hypomethylating agents (HMAs) such as decitabine and azacitidine have been successfully used in the clinic to treat hematological malignancies by parenteral administration (IV or SC). Due to low oral bioavailability of these agents, relatively high doses are required to achieve therapeutic systemic exposures by the oral route. Higher oral doses can be associated with significant GI toxicities due to intestinal enterocytes being exposed to high concentrations of these agents in the lumen. One of the main factors contributing to low oral bioavailability of HMAs is the high first-pass effect due to cytidine deaminase in GI and liver.

Methods

In this study, E7727, a novel CDA inhibitor (CDAi) was combined with decitabine (3 mg/kg) for oral administration in cynomolgus (cyn) monkeys at E7727 doses of 0.1-10 mg/kg.

Results

Systemic AUC exposures for decitabine increased from 21.7 ng*hr/mL (without E7727) to as high as 1,494 ng*hr/mL at 10mg/kg E7727. At a dose combination of 1 mg/kg CDAi and 3 mg/kg decitabine (corresponding to a human equivalent dose of 36 mg/m2), AUC exposures were 301±94 ng*hr/mL, which are slightly higher than the published clinical therapeutic range after decitabine IV infusion of 20 mg/m2 (115-220 ng*hr/mL), suggesting that a combination approach for oral CDAi+decitabine is feasible to achieve required clinical exposures at a range of relatively low-dose(s). In addition, the concentration-time profile of decitabine when combined with CDAi resembled that achieved with IV decitabine over 1 hour infusion. The effect of drug-drug interaction between CDAi and decitabine was modeled based on a relationship between CDAi Ki (∼ 0.14 µM based on IC50) and Cmaxlevels and appeared to predict the observed fold-increases in decitabine AUC with high concordance. In addition, low dose oral decitabine+CDAi achieved decitabine exposures that produce potent hypomethylation as observed by LINE-1 assay.

Conclusion

Pronounced increase in oral decitabine exposures was achieved when combined with the novel CDA inhibitor E7727. Cyn monkeys appear to be a relevant model for human PK based on similar background of circulating serum cytidine levels reflective of similar CDA status between humans and cyn monkeys. These data support the initiation of clinical First in Human (FIH) study of ASTX727, a novel oral HMA combining oral decitabine with the CDAi E7727.

Disclosures:

Oganesian: Astex Pharmaceuticals: Employment. Redkar:Astex Pharmaceuticals: Employment. Taverna:Astex Pharmaceuticals Inc.: Employment. Joshi-Hangal:Astex Pharmaceuticals, Inc.: Employment. Azab:Astex Pharmaceuticals Inc.: Employment.
LY2928057, An Antibody Targeting Ferroportin, Is a Potent Inhibitor Of Hepcidin Activity and Increases Iron Mobilization In Normal Cynomolgus Monkeys
Authors: Witcher Derrick R , Leung Donmienne , Hill Karen A , De Rosa David C , Xu Jianghuai , Manetta Joseph , Wroblewski Victor J , Benschop Robert J
Published: October 2019

Hepcidin, a 25-amino acid peptide hormone which is primarily synthesized and secreted from the liver, is a key regulator of iron homeostasis. It regulates dietary iron absorption, plasma iron concentrations, and tissue iron distribution through interactions with ferroportin, the only known mammalian cellular iron exporter. Hepcidin induces the internalization and subsequent degradation of ferroportin. The reduction in iron release caused by the loss of ferroportin, combined with the continuing demand for iron by erythropoietic precursors, results in a decrease in circulating iron levels. Dysregulation of the hepcidin-ferroportin axis contributes to the pathogenesis of different anemias. Decreased synthesis of hepcidin may cause systemic iron overload in iron-loading anemias such as beta-thalassemia; whereas overproduction of hepcidin may contribute to the development of anemia in inflammatory disorders, malignancies, and chronic kidney disease. LY2928057 is a novel humanized IgG4 monoclonal antibody that binds to human ferroportin with a high affinity, blocks the binding of human hepcidin to ferroportin, and is a potent inhibitor of hepcidin activity in a recombinant ferroportin expressing HEK 293 cell-based assay. In addition, this antibody was able to significantly inhibit hepcidin-induced increase in ferritin levels using Caco-2 cells, a human enterocyte cell line that naturally expresses ferroportin. LY2928057 does not block the efflux of iron from ferroportin, nor does this antibody cause the internalization of this transporter in vitro. Administration of LY2928057 results in a dose dependent increase in serum iron and hepcidin in normal cynomolgus monkeys. LY2928057 may provide therapeutic benefit for patients with hepcidin-related anemia by stabilizing ferroportin located on the cell surface, thus restoring iron export and erythropoiesis. LY2928057 is currently in clinical evaluation.

Disclosures:

Witcher: Eli Lilly and Company: Employment, Equity Ownership. Leung:Eli Lilly and Company: Employment, Equity Ownership. Hill:Eli Lilly and Company: Employment, Equity Ownership. De Rosa:Eli Lilly and Company: Employment, Equity Ownership. Xu:Eli Lilly and Company: Employment, Equity Ownership. Manetta:Eli Lilly and Company: Employment, Equity Ownership. Wroblewski:Eli Lilly and Company: Employment, Equity Ownership. Benschop:Eli Lilly and Company: Employment, Equity Ownership.
Establishment Of a Reference Range For The Pbac-Score
Authors: Halimeh Susan , Rott Hannelore , Kappert Guenther , Siebert Manuela
Published: October 2019

Introduction

Heavy menstrual bleeding (HMB) is defined as bleeding that lasts for more than seven days or as the loss of more than 80mL of blood per mentrual cycle (1)The menstrual blood loss can be quantified by the use of a pictorial bleeding assessment chart (PBAC).

The PBAC-Score was initially validated by Higham et al. (2) With the PBAC-Score the women can capture the number of pads or tampons and also state the intensity through the assessment of the drenching.

The aim of this study was to establish a reference range for the PBAC-Score.

Samples and Methods

We analysed samples of 310 women with menorrhagia and 108 controls by conducting the following tests: Blood count, VWF:RCo, VWF:Ag, VWF:CB, VWF:multimers, Fibrinogen (Clauss), activities of FII, FV, FVII, FVIII (clotting and chromogenic), FIX, FX, FXI, FXII, FXIII. In all women the menstrual blood loss was quantified usind the PBAC-Score and the results were compared.

Results

In 202 of 310 women (65.1%) a bleeding disorder could be detected. In those with a bleeding disorder, the distribution was as followed: 64% of these women had a von Willebrand disease, 7.2% FVII-deficiency, 7.7% FXIII-deficiency and the remaining 21.1% other mild factor dificiencies. The mean PBAC-Score in women with menorrhagia was 262 (range 31 – 4212) in our control group the mean PBAC-Score was 60 (range 8 – 97).

Discussion/Conclusion

Attemps to measure the quantity of menstrual blood loss can be useful in clinical practice.

In our opinion the best cut of for the PBAC-Score is 100 with a sensitifity of 90% and a specifity of 100%.

We found a high correlation of a PBAC >100 and an inherited bleeding disorder, especially von Willebrand diesease. In 65.1% of our patients an abnormal coagulation was found. In 4 (3.7%) women of the control group slightly abnormal von Willebrand parameters could been dectected.

We validated the reference range for the PBAC-Score as 0 - 100.

Disclosures:

Halimeh: Octapharma AG: Investigator Other, Research Funding.
Factor XIII Deficiency As Underlying Cause Of Unexplained Bleeding
Authors: Mercado Marta Ruiz , Cozzarelli Silvia Verdesoto , Calderón-Cabrera Cristina , Vázquez Ramiro Núñez , Bárcenas Reyes Jiménez , Garrido Rosario Pérez , Pérez-Simón Jose Antonio , Rodríguez Martorell Francisco Javier
Published: October 2019

Introduction

Factor XIII deficiency (FXIII) is an uncommon coagulation disorder. Congenital FXIII deficiency, generally due to mutations in F13A1 gene, presents with early life- threating hemorrhages in homozygotes. Acquired deficiency, a more rare state, has been associated with certain drugs and inhibitors against FXIII.

Materials and methods

We retrospectively analyzed 47 patients (male:female 1:1.3). Study criteria were unexplained hemorrhage, mainly after surgery, or spontaneous intracranial bleeding at perinatal period and no relevant findings in conventional haemostasia assays, from 01/01/2010 to 15/07/2013. FXIII measure was performed by a functional method.

Results

20 out of 47 patients (42.5%) other abnormalities that might contribute to bleeding were detected: 13 had primary haemostasis disorders and 7 had low levels of a coagulation factor different from FXIII. In 10 patients, FXIII deficiency was observed: 4 congenital and 6 acquired. In all acquired deficiencies, the presence of an inhibitor was discarded. Patients with congenital FXIII deficiency, had mucocutaneous bleeding in 75% of cases and hemorrhage after surgery in 50%. However, there was no intracranial hemorrhage. In acquired deficiency (median FXIII 44.5 U/dl at first diagnostic work up), mucocutaneous bleeding appeared in 40% patients and postoperative and intracranial hemorrhage in 100%. In 8/ 10 patients FXIII concentrate was administered, achieving bleeding control in 7. In 3 cases of congenital deficiency, prophylactic substitutive therapy was started enabling a cessation of new bleeding episodes, except for a posttraumatic muscle hematoma; median FXIII levels reached 6.8 U/ dL. 2 patients with acquired deficiency died of non-hemorrhagic complications, 3 patients developed spontaneous remission of the deficiency in a median time of 2 months and 1 is still receiving substitutive therapy.

Conclusions

In congenital deficiency, maintenance of FXIII through levels in the range of 5-10% is enough to avoid bleeding manifestations. The acquired deficiencies have at least the same frequency as congenital, develop hemorrhage episodes at higher levels of factor and respond to therapy in a thrifty way. For those reasons, tests for FXIII are essential for diagnosis in high index clinical suspicious cases, such as unexplained bleeding.

Disclosures:

No relevant conflicts of interest to declare.
Molecular Analysis Of The Largest Group Of Patients With Factor XIII Deficiency In Southeast Of Iran
Authors: Naderi Majid , Dorgalaleh Akbar , Alizadeh Shaban , Tabibian Shadi , Bamedi Taregh , Karimi Mehran
Published: October 2019

Background

Factor XIII deficiency is an extremely rare hemorrhagic disorder with estimated incidence of 1/3000000 million. Sistan and Baluchistan, southeast of Iran has the highest prevalence of disease worldwide. Thus the aim of this study was to assess molecular characteristics of this large group of patients with factor XIII deficiency.

Methods

This descriptive study was conducted on one hundred and nineteen patients from same number of unrelated families. Genotype analysis for factor XIII subunit A Val34Leu, Tyr204Phe, and Pro564Leu and Trp187Arg was performed for all patients.

Results

Molecular analysis of this large group of patients with severe factor XIII deficiency revealed that all studied patients were homozygous for TGG CGG mutation at codon 187, in exon 4 of FXIII-A1 gene. Val34Leu, Tyr204Phe, and Pro564Leu FXIII-A subunit gene polymorphisms were not observed in any of patients.

In the study population, umbilical cord bleeding, deep soft tissue and hematoma were the most common clinical manifestations and was observed in 82.5%, 53% and 31% respectively.

Conclusion

Trp187Arg is the most common mutation of FXIII-A subunit in southeast of Iran and probably due to the large number of patients in this area (352 patients), is the most common mutation of factor XIII worldwide.

Disclosures:

No relevant conflicts of interest to declare.
Engagement of interleukin-7 receptor stimulates tyrosine phosphorylation, phosphoinositide turnover, and clonal proliferation of human T-lineage acute lymphoblastic leukemia cells
Authors: Dibirdik I , Langlie MC , Ledbetter JA , Tuel-Ahlgren L , Obuz V , Waddick KG , Gajl-Peczalska K , Schieven GL , Uckun FM
Published: October 2019

The purposes of this study were to examine the biologic effects of the engagement of the interleukin-7 receptor (IL-7R) with recombinant human interleukin-7 (rhIL-7) in immunophenotypically distinct T-lineage acute lymphoblastic leukemia (ALL) blasts and to elucidate the biochemical nature of the IL-7R-linked transmembrane signal in rhIL-7-responsive T- lineage ALL blast populations. In the absence of costimulants, rhIL-7 stimulated the in vitro proliferation and colony formation of freshly isolated leukemic blasts from six to eight T-lineage ALL patients with a mean plating efficiency of 196 +/- 53 (background subtracted) colonies/10(5) blasts plated. Stimulation of T-lineage ALL blasts with rhIL-7 resulted in markedly enhanced tyrosine phosphorylation of six distinct phosphoproteins with molecular weights of 57, 72, 98, 123, 150, and 190 Kd, and induced a rapid increase in the production of inositol-1,4,5-trisphosphate (Ins-1,4,5-P3), which was inhibitable by the tyrosine-specific protein kinase inhibitor genistein, but not by the serine/threonine-specific protein kinase C inhibitor H7. Similarly, rhIL-7 stimulated Ins-1,4,5-P3 production in CEM-1.3 T-lineage ALL cells and this stimulation was inhibitable by the tyrosine-specific protein kinase inhibitors genistein and herbimycin A, but not by H-7. Thus, the transmembrane signal triggered by engagement of the IL-7R is intimately linked to a functional tyrosine-specific protein kinase pathway and stimulates the phosphoinositide (PI) turnover and proliferation of T-lineage ALL blasts. The presented data confirm and extend previous studies on the expression of functional IL-7R on T- lineage ALL blasts and support the hypothesis that IL-7 may play an important regulatory role in the biology of T-lineage ALL.
Autoantibody activity of immunoglobulins isolated from B-cell follicular lymphomas
Authors: Dighiero G , Hart S , Lim A , Borche L , Levy R , Miller RA
Published: October 2019

Previous work with monoclonal Igs (MIgs) has demonstrated that a high proportion of paraproteins bind to self-antigens such as the Fc fragment of IgG, Ii blood group antigens, cytoskeleton proteins, DNA, and myelin-associated glycoprotein (MAG). Recent work in CLL indicates that CD5+ B lymphocytes are frequently committed to production of autoantibodies. We have examined the antibody specificity of MIgs derived from the tumor cells of 31 different patients with CD5- B-cell lymphomas. Our results indicate that the tumor cells from 8 of these 31 patients (25.8%) express Igs with autoantibody activity. In two cases antibody activity was multispecific. In four cases, antibody activity was exclusively directed against the Fc fragment of IgG, whereas the two other cases bound to both Fc fragment of IgG and nuclear antigens. Most non-Hodgkin's lymphomas (NHL) are derived from CD5- B cells. These results indicate that like CLL, NHL also express Igs that frequently have autoantibody activity.
Selective expression of CD45 isoforms defines CALLA+ monoclonal B- lineage cells in peripheral blood from myeloma patients as late stage B cells
Authors: Jensen GS , Mant MJ , Belch AJ , Berenson JR , Ruether BA , Pilarski LM
Published: October 2019

The peripheral blood lymphocytes from 42 patients with multiple myeloma (MM) and 13 patients with monoclonal gammopathy of undetermined significance (MGUS) were studied by three-color immunofluorescence (IF) using antibodies directed to a broad range of B-cell markers (CD19, CD20, CD21, CD24), CALLA (CD10), PCA-1 (a plasma cell marker), and to the high and low molecular weight isoforms of the leukocyte common antigen, CD45RA (p205/220) and CD45RO (p 180). CD45RA is expressed on pre-B and B cells, and a transition from CD45RA to CD45RO defines differentiation towards plasma cells. Peripheral blood mononuclear cells (PBMC) from patients with myeloma included a large subset of B- lineage cells (mean of 39% to 45%) that were CALLA+ and PCA-1+ in all patients studied, including newly diagnosed patients and patients undergoing chemotherapy. Southern blot analysis indicated the presence of monoclonal Ig rearrangements in PBMC and a substantial reduction in the germ-line bands consistent with the presence of a large monoclonal B-cell subset. Avoidance of purification methods involving depletion of adherent cells was essential for detection of the abnormal B cells. Phenotypically, this abnormal B-cell population corresponded to late B or early pre-plasma cells (20% to 80% of PBMC), as defined by the concomitant expression of low densities of CD19 and CD20, moderate densities of CALLA and PCA-1, and strong expression of CD45RO on all B cells, with weakly coexpressed CD45RA on a small proportion. Heterogeneity in the expression of CD45RA and CD45RO within the abnormal B-cell population from any given patient suggested multiple differentiation stages. Abnormal B cells similar to those in MM were also detected in MGUS, although as a lower proportion of PBMC (26%). Abnormal B cells from patients with MGUS expressed predominantly the CD45RO isoform, but had a lower proportion of CALLA+ and PCA-1+ cells than were found on B cells from MM. This work indicates that the large subset of circulating monoclonal B lymphocytes from myeloma patients are at a late stage in B-cell differentiation, continuously progressing towards the plasma cell stage.
Recombinant human MCP-1/JE induces chemotaxis, calcium flux, and the respiratory burst in human monocytes
Authors: Rollins BJ , Walz A , Baggiolini M
Published: October 2019

The JE gene was first described as a platelet-derived growth factor (PDGF)-inducible gene in mouse 3T3 cells. The human homologue of JE encodes a protein whose predicted amino acid sequence is identical to that of the monocyte chemoattractant MCP-1 (also called MCAF and SMC- CF), which belongs to a recently identified family of small secretory proteins with cytokine properties. We purified recombinant human MCP- 1/JE (hMCP-1/JE) produced in COS cells and demonstrated that it is chemotactic for human monocytes with a specific activity similar to natural MCP-1. In addition, pure recombinant hMCP-1/JE stimulates monocytes, inducing an increase in cytosolic free calcium and the respiratory burst, but is completely inactive on human neutrophils. These results help to define functionally a well-known growth factor- inducible gene and a member of a new family of cytokines.