bluuebunny/crossref_metadata_2025 huggingface dataset:
ITK-SYK Induces T-Cell Lymphoma with Accompanying Inflammatory Disease in Mice.
Authors: Dierks Christine , Ma Hong , Veelken Hendrik , Warmuth Markus , Adrian Francisco
Published: October 2019

The ITK-SYK fusion protein was previously described to be present in 17% of unspecified peripheral T-cell lymphomas. Here we demonstrate that expression of ITK-SYK in the bone marrow of Balb\c mice causes T-cell lymphomas in mice with a latency of only 3–4 weeks. The disease is characterized by infiltration of the spleen, lymph nodes, bone marrow and the skin with malignant T-cells and progredient destruction of these organs. The mice die about 2 months after the transplantation due to dramatic weight loss caused by infiltration of T-cells into the colon and because of progredient anemia end neutropenia due to progredient infiltration of the bone marrow. The malignant T-cells were characterized as a mixed population of CD3+, CD4+, CD8- T cells and CD3+, CD4-, CD8- T-cells. The malignant disease was accompanied by a generalized inflammatory reaction including upregulation of the inflammatory cytokines IL-5 and INF-γ. Modulation of the membrane binding of ITK-SYK or its binding to Cbl by point mutations in the pleckstrin homology domain of ITK could alter the transforming activity of ITK-SYK. The intact kinase domain was essential for the transformation process and the disease could be reversed by treatment of diseased mice with the Syk-inhibitor Curcumine. Our results demonstrate that the fusion protein ITK-SYK causes T-cell lymphomas in mice and mimics the human disease. Therefore pharmacological inhibition of Syk in patients with U-PTCL carrying the ITK-SYK fusion protein might be a new and effective treatment strategy.
ABT-737 Is a Useful Component of Combinatory Chemotherapies for Chronic Myelogenous Leukemias with Diverse Drug Resistance Mechanisms.
Authors: Kuroda Junya , Kimura Shinya , Andreeff Michael , Ashihara Eishi , Kamitsuji Yuri , Strasser Andreas , Taniwaki Masafumi , Maekawa Taira
Published: October 2019

Chronic myelogenous leukemia (CML) is characterized by its refractoriness to various apoptotic insults by Bcr-Abl tyrosine kinase (TK)-mediated signalling. Although imatinib mesylate (IM), a Bcr-Abl TK inhibitor, has markedly improved the therapeutic outcomes of CML, additional or alternative molecular targeting strategies are still needed. Since the interplay of anti-apoptotic Bcl-2 proteins and BH3-only proteins, such as Bim and Bad, is crucial for regulating the cellular fate of Bcr-Abl+ leukemic cells (Kuroda J et al, PNAS , 2006; Cell Death Differ, 2007), the direct targeting of anti-apoptotic Bcl-2 proteins by the use of a BH3-only protein mimetic is an attractive approach for treating CML. We here investigated the activity of ABT-737, a mimic of BH3-only proteins that inhibit anti-apoptotic Bcl-2, Bcl-XL and Bcl-w, but not Mcl-1 or A1, against CML. The Annexin-V-staining study, the assay for mitochondrial outer membrane potential and the internucleosomal fragmentation assay revealed that ABT-737 potently induced apoptosis in CML cell lines (BV173, K562, KCL22, KT-1, MEG-01 and MYL) with the IC50 for induction of cell death by 48 h of treatment ranging from 0.04 to 4.06 μM. ABT-737 was also effective in killing primary CML samples in vitro. ABT-737 prolonged the survival of mice xenografted with a CML cell line, BV173, demonstrating its in vivo bioactivity. Higher expression of Bcl-2, Bcl-XL, or Mcl-1 reduced cell killing by ABT-737 in each cell line, but we found no correlation between the sensitivities to ABT-737 and the specific expression patterns of Bcl-2 family proteins among different cell lines. The levels of Bcr-Abl and Lyn, a member of Src kinase family associated with apoptosis resistance in CML, also varied among the cell lines, and we found no consistent relationship between the sensitivity to ABT-737 and the expression levels of these proteins Thus, the cell killing effect of ABT-737 in CML must be determined in part by other drug resistance mechanisms, such as high expression of Bcr-Abl, overexpression of P-glycoprotein (P-gp), a drug-efflux pump, and/or their combination. Importantly, ABT-737 augmented the cell killing effect of IM in CML cell lines with high levels of anti-apoptotic Bcl-2 family proteins (Bcl-2, Bcl-XL, or even Mcl-1), Bcr-Abl, P-gp, or Lyn, unless leukemic cells harboured IM-insensitive Abl kinase domain (KD) mutations. Moreover, the combination of ABT-737 with homoharringtonine, an herbal-derived anti-CML therapeutic that potently reduces Mcl-1 within 6 hours in vitro, dramatically enhanced the killing by ABT-737 in CML cells with diverse drug resistance mechanisms, including IM-insensitive Abl KD mutations, such as T315I mutation. These results suggest that ABT-737 could be a useful component of combinatory chemotherapies for CML.
10 Year Musculoskeletal Outcomes with Tailored Primary Prophylaxis: The Canadian Hemophilia Prophylaxis Study.
Authors: Blanchette Victor S. , Rivard Georges E. , Pai Mohan K. , Israels Sara J. , McLimont Marjorie , Feldman Brian M.
Published: October 2019

High-dose prophylaxis for severe hemophilia A [25–40 factor (F) VIII Units (u) kg−1 on alternate days] reduces arthropathy, but does so at great cost. (NEJM2007; 357: 535–544). Less intensive prophylaxis regimens might be cost-effective while reducing the need for indwelling venous access devices and their attendant complications. Tailored prophylaxis starts with FVIII, 50 u kg−1 weekly (Step1). If bleeding frequency is unacceptable (4 joint/soft tissue bleeds or 5 bleeds into a joint on a step of protocol), or if a target joint develops (≥3 bleeds into a single index joint-ankle, elbow or knee) over a consecutive 3-month period the dose of FVIII is escalated to 30 u kg−1 twice weekly (Step 2); a third escalation to 25 u kg−1 on alternate days if bleeding frequency remains unacceptable (Step3).

Objectives: To assess the degree of joint damage in the 6 index joints for subjects receiving tailored prophylaxis.

Methods: We studied an inception cohort of boys ages 1–2.5 years with severe hemophilia A (<2% FVIII) and no inhibitors treated with tailored prophylaxis. Patients were followed at 6 monthly intervals and parents have kept detailed diaries. Joint damage was measured using a validated physiotherapy joint score and function was assessed using the Child Health Assessment Questionnaire (CHAQ)

Results: 54 boys were enrolled from 11 Canadian hemophilia treatment centers. The total follow-up is 3423 patient months (mean 62.2 months, median 54 months and range 5–117 months). 16 (29.6%) boys have been escalated to Step 2 and 14 (25.9%) have escalated to Step 3. 11 of the escalations were due to target joint bleeding. Subjects on study had a mean of 1.4 joint bleeds/patient-year. Physiotherapy Scores Maximum Possible Score Median Observed Range Ankles 56 2 0–20 Elbows 48 0 0–13 Knees 56 0 0–15 All Joints Swelling 18 0 0–4 Muscle Atrophy 18 0 0–3 CHAQ 3 0 0–2 89% of participants were compliant with the primary prophylaxis protocol. Non compliant subjects had the poorest outcomes. No treatment related adverse events have occurred.

Conclusion: Tailored primary prophylaxis should be considered as a valid option for long term prophylaxis in young boys with severe hemophilia A.

Maximum Possible Score Median Observed Range Ankles 56 2 0–20 Elbows 48 0 0–13 Knees 56 0 0–15 All Joints Swelling 18 0 0–4 Muscle Atrophy 18 0 0–3 CHAQ 3 0 0–2
The mTOR Inhibitor RAD001 (Everolimus) Improves Survival in Preclinical Models of Primary Human ALL.
Authors: Crazzolara Roman , Cisterne Adam , Thien Marilyn , Hewson John , Bradstock Kenneth Francis , Bendall Linda J.
Published: October 2019

The Akt/mammalian target of rapamycin (mTOR) signaling pathway is a potential target in acute lymphoblastic leukemia (ALL) due to its constitutive expression and critical role in regulating leukemic cell growth and survival. In this study, we investigated whether RAD001 (Everolimus, Novartis), is effective as a therapeutic agent in a murine model of human ALL. Leukemic cells from 5 separate patient samples were injected into sub-lethally irradiated NOD/SCID mice and treatment was started after leukemia was clearly detectable in the blood (>5% peripheral blasts) and continued for 4 weeks. In 2 of 3 cases examined, animals treated with RAD001 as a single agent (5mg/kg, 3 times weekly) showed 2 to12-fold reduction in the number of circulating blasts and 2.5 to 30-fold decrease in splenomegaly after only 7 days of treatment (p<0.01). In 1 case, blasts completely disappeared by 14 days of treatment accompanied by recovery of normal hematopoiesis. In contrast vehicle treated mice showed progressive leukemia. In all 5 cases examined, decreased leukemic burden was associated with increased survival, with a median survival of 52 days in the RAD001 treated mice compared to 24 days in the control group (p<0.02 for all samples). Combination treatment with RAD001 and Vincristine (0.15–0.25 mg/kg, weekly) was more effective than either agent alone in reducing leukemic cell count and prolonging survival (p<0.02 compared to Vincristine alone). In one case, all mice treated with either RAD001 alone or RAD001 plus Vincristine survived for greater than 10 weeks following the completion of treatment and only one animal (RAD001 treated) showed signs of disease on elective sacrifice. Cell cycle analysis of cells recovered from the spleens of RAD001 treated animals revealed an arrest in the G0/G1 phase. Furthermore, electron microscopy demonstrated the predominant appearance of autophagy as well as limited apoptosis in cells within the sternums of treated animals. In conclusion, we have demonstrated the efficacy of RAD001 alone or with a subtherapeutic dose of Vincristine in an in vivo leukemia model. These preclinical results support further clinical development of mTOR inhibitors for the treatment of ALL patients. RAD001 may provide significant therapeutic benefit when used alone, or in combination with standard chemotherapy agents such as Vincristine.
Wolinella Asparaginase as an Immunologically Distinct, Efficacious, and Potentially Less Toxic Asparaginase for Use in Lymphoid Malignancies.
Authors: Watt Tanya C. , Keller Frank G. , Durden Donald L.
Published: October 2019

L-asparaginase targets the protein translational mechanism and is an essential component in the treatment of acute lymphoblastic leukemia (ALL) and lymphoid malignancies. The current available preparations, E. coli and Erwinia have significant toxicities, often limiting their clinical use. In order to provide patients with a novel form of asparaginase, a recombinant form of Wolinella (rWS) asparaginase was developed. C3H mice were transplanted with 2 × 106 6C3HED lymphoma cells. Mice treated with native or rWS asparaginase demonstrated a significant decrease in tumor volume, and they remained tumor free for greater than 30 days after treatment. Human ALL cell lines were tested for sensitivity to rWS asparaginase and the molecular basis for rWS asparaginase sensitivity was examined in these cells. We hypothesized that rWS asparaginase would be immunologically distinct to that of E. coli. The sera of 6 patients previously identified to have high titer inhibitors to E. coli asparaginase were tested for antibodies to rWS asparaginase. The antibodies to E. coli did not bind to the rWS asparaginase, therefore demonstrating that there is no cross-reactivity to rWS asparaginase. Thus, antibodies or hypersensitivity reactions to previous forms of asparaginase will not prohibit therapy with rWS asparaginase. Therefore rWS asparaginase represents the first recombinant form of asparaginase and shows promise as a novel form of asparaginase which is immunologically distinct from E. coli and Erwinia asparaginase. In addition, it appears to be equally efficacious, and in previously published data, may have less toxicity. Asparaginase is an important component of treatment regimens for lymphoid malignancies, and therefore future studies will include a Phase I/II trial utilizing rWS asparaginase in relapsed lymphoid malignancies.
Non-Compliance Is the Predominant Cause of Aspirin Resistance.
Authors: Schwartz Kenneth A. , Schwartz Dianne E. , Barber Kimberly , Reeves Matt , DeFranco Anthony
Published: October 2019

Classifying patients as resistant to aspirin is an arbitrary designation with up to 60% of patients reported to be aspirin resistant. Our previous publication showed that 16 or 9% of 192 patients with a history of M.I. could be labeled as aspirin resistant and that all of these patients were aspirin resistant because of non-compliance. This report compares the relative frequency of aspirin resistance between known compliant and non-compliance subjects to demonstrate that non-compliance is the predominant cause of aspirin resistance. Platelet prostaglandin agonist (PPA) stimulated light aggregometry was used to measure amount of aspirin induced platelet inhibition and arachidonic acid (AA) stimulated platelet aggregation to classify platelets as either aspirin inhibited or not. After not taking aspirin for 7 days blood for aggregation studies was drawn, off aspirin time point, which was followed by observed ingestion of 365mg of aspirin. Two hours after observed aspirin ingestion aggregation studies were repeated, on aspirin time point. The difference in the slopes of the PPA light aggregation curves on and off aspirin was defined as the net aspirin response. After supposedly refraining from aspirin for 7 days, 45 subjects were judged non-compliant with the protocol and removed from the study. Of the remaining 185 subjects 39 were normals and 146 had a past history of myocardial infarction (MI). There was no difference in net aspirin response between normal and MI subjects. The net aspirin response in known compliant patients was statistically normally distributed. Only 4% of complaint subjects (2 normals and 5 MI) had a net aspirin response of less than one standard deviation which would qualify as a conservative designation of aspirin resistance. Of the 230 enrolled subjects 16 were noncompliant with their daily prescribed aspirin, 45 were non-compliant with the protocol not to take aspirin for 7 days and one patient had NANSAID interference with aspirin’s effect. In total a maximum of 30% of the 230 patients could be classified as aspirin resistant and or non-compliant with 9% aspirin resistant because of non-compliance 18% non-compliant with the protocol and only 3% because of a decreased net aspirin response in known compliant patients. We conclude that:

the predominant cause of aspirin resistance is noncompliance; net aspirin response in compliant patients is normally distributed; in compliant patients no difference in net aspirin response is observed between normals and MI patients; and PPA stimulated light aggregometry measured off aspirin and 2 hours after observed aspirin ingestion is a useful technique for measuring net aspirin response.
Rearrangement and Deletion of the PAX5 Gene in Pediatric Acute B-Cell Lineage Lymphoblastic Leukemia.
Authors: Kawamata Norihiko , Ogawa Seishi , Zimmermann Martin , Sanada Masashi , Hemminki Kari , Yamatomo Go , Nannya Yasuhito , Koehler Rolf , Flohr Thomas , Miller Carl W. , Harbott Jochen , Ludwig Wolf-Dieter , Stanulla Martin , Schrappe Martin , Bartram Claus R. , Koeffler Phillip H.
Published: October 2019

Pediatric acute lymphoblastic leukemia (ALL) is the most common malignant disease in children. The disease results from accumulation of mutations of tumor suppressor genes and oncogenes. Recently, higher resolution SNP-chips (50,000–500,000 probes) have been developed allowing us to identify genes involved at the start sites of deletions/duplications. This permitted us both to identify unbalanced translocations involving t(1;19)(q23;p13) (TCF3/PBX1) and t(12;21)(p13;q11) (ETV6/RUNX1), as well as, to find novel fusion genes involving PAX5 in B-cell lineage ALL. PAX5 gene was rearranged to a variety of partner genes including ETV6, FOXP1, AUTS2 and C20orf112. In each case, tthe C-terminal end of the PAX5 gene was replaced by the partner gene. The PAX5 fusion gene products suppressed transcriptional activity of PAX5 in a dominant negative fashion. We also found a point mutation of PAX5 at codon 26 (Val 26 Gly); and this mutated PAX5 had attenuated transcriptional activity. Expression of PAX5/C20orf112 fusion gene in a B-cell line suppressed endgenous expression of PAX5 target genes including BLK1 and CD19. Furthermore, deletion of PAX5 was common in B-cell lineage ALL (34/339 cases). PAX5 gene is localized on chromosome 9p and concurrent deletion of PAX5 and INK4A genes were frequently detected in B-cell linage ALL. PAX5 gene may behave as a tumor suppressor gene during early development of B-cells and its alteration by either fusion to another gene, point mutation, or deletion may be associated with leukemogenesis of B-cell lineage ALL.
Placental Growth Factor Is Elevated in Patients with Sickle Cell Disease and Associated with Pulmonary Hypertension, Hemolysis, Inflammation, Iron Overload, and Hepatic Dysfunction.
Authors: Mendelsohn Laurel , Tailor Anitaben , Kato Gregory J
Published: October 2019

Abstract 1531

Poster Board I-554

Placental Growth Factor (PlGF) is a functional cytokine in the vascular endothelial growth factor (VEGF) family that generally promotes angiogenesis, depending on the specific context, and can also promote atherogenesis. Produced in erythroid cells, its level in patients with sickle cell disease (SCD) has been previously related to the rate of erythropoiesis. We evaluated PlGF plasma levels in SCD patients by ELISA, and related it to biomarkers of pulmonary hypertension (PH), an emerging and serious complication of SCD linked to early mortality. We find that PlGF levels are significantly higher in SCD (n=95) than healthy African American control subjects (n=19) (median 16.6 vs. 2.1 pg/mL, p<0.001). PlGF levels were higher in SCD patients with elevated pulmonary pressure (normal pulmonary pressure vs. mildly elevated vs. highly elevated: medians 13.7 vs. 16.7 vs. 19.8 pg/mL, p<0.0001). Supporting a linkage to rate of hemolysis, PlGF correlated with LDH (p=0.001) and inversely with hemoglobin level (p<0.0001). Suggesting a link to inflammation, PlGF correlated significantly with C-reactive protein (p=0.001) and erythrocyte sedimentation rate (p<0.001). PlGF correlated with markers of iron overload, including ferritin, transferrin saturation and inversely with transferrin (all p<0.001). Finally, PlGF correlated with markers of hepatic dysfunction, including low albumin and high direct bilirubin (p<0.001).

We found significantly higher PlGF levels in SCD patients taking hydroxyurea compared to those not taking it (median 17.4 vs. 14.0 pg/ml, p<0.01). Confirming that hydroxyurea increases PlGF levels, in a separate cohort of seven patients, PlGF levels rose significantly from their baseline values after initiating hydroxyurea (median approx 22 vs. 27, p<0.05). Our data suggest that elevated PlGF level is associated with PH in patients with SCD, and PlGF is correlated with severity of hemolysis, inflammation, iron overload and hepatic dysfunction. Considering the variable evidence in the literature for either stimulating or inhibiting angiogenesis, it is not clear whether pathologic elevation of PlGF may be mediating pulmonary hypertension, or perhaps conversely providing an adaptive response to vascular damage. It has been suggested by Perelman et al. that PlGF may mediate leukocyte activation that might promote disease severity in SCD. However, hydroxyurea, which tends to ameliorate SCD complications, stimulates PlGF level in an unexpected manner, possibly related to the ability of hydroxyurea to stimulate erythropoietin production, which might in turn induce PlGF. Further research is needed to reconcile the role of PlGF in PH in SCD.

Disclosures

Tailor: Mesoscale: Employment.
Peroxisome Proliferator-Activated Receptor (PPAR)-Gamma Agonists Inhibit Dectin-1 mediated Activation of Human Dendritic Cells (DC).
Authors: Kock Grethe , Bringmann Anita , Held Stefanie , von Schwarzenberg Karin , Brossart Peter
Published: October 2019

Abstract 1645

Poster Board I-671

Human Dectin-1 (hDectin-1) is a member of the C-type lectin-like receptor family that was shown to be the major receptor for fungal beta-glucans and to play an important role in the cellular responses mediated by these pathogens. Activation of Dectin-1 via extracts such as zymosan or more specifically with curdlan can lead to DC activation characterized by upregulation of surface molecules or secretion of cytokines. Futhermore, Dectin-1 is important for recruitment of leukocytes and production of inflammatory mediators at the site of infection.. Peroxisome proliferator-activated receptor (PPAR) and its ligands, cyclopentenone prostaglandins or thiazolidinediones, have been shown to have profound modulatory effects on B and T lymphocytes as well as DCs. Cyclopentenone prostaglandins are produced during the late phase of inflammation due to up-regulation of cyclooxygenase 2 (COX2), a key enzyme for the synthesis of cyclopentenone prostaglandins, that mediate their effects by activation of PPAR-gamma dependent and independent pathways. In our study we analyzed the effects of troglitazone (TGZ), a high affinity synthetic ligand of PPAR-gamma, on the Dectin-1 mediated activation of antigen presenting cells (APC). TGZ was added to the cell culture medium during the differention of monocyte derived DC. On day 5-6 of culture the cells were additionally treated with curdlan or zymosan and phenotypical and functional analyses were performed. Activation of PPAR-gamma resulted in a reduced stimulation of APC via Dectin-1 characterized by down-regulation of CD1a, CD83 and costimulatory molecules on the cell surface and reduced secretion of cytokines and chemokines involved in T-lymphocyte activation and recruitment including IL-1beta, TNF-a, IL-6, IL-12, MIP-1a and Rantes. Western blot analyses revealed that these inhibitory effects were mediated by the inhibition of the mitogen-activated protein (MAP) kinase pathways characterized by a reduced phosphorylation of ERK1/2 and p38. Furthermore, we found that Dectn-1 induced c-Jun phosphorylation was reduced by pretreatment with TGZ. In addition, Dectin-1 induced nuclear translocation of NF-kappaB family members RelB and cRel was dramatically reduced in APC incubated with TGZ. The observed inhibition of signaling pathways was not due to a reduced expression of Dectin-1, phosphorylation of Syk or increased secretion of IL-10 and TGF-beta. TGZ had no effect on the Dectin-1 induced phosphorylation and nuclear expression of NFAT. Our data demonstrate that PPAR-gamma ligands inhibit Dectin-1 mediated activation by interfering with the MAP kinase, c-Jun and NF-kappaB signaling pathways, thus confirming their important role as a negative feedback mechanisms in the regulation of potentially harmful inflammation signaling pathways.

Disclosures

No relevant conflicts of interest to declare.
A Reduced Rate of Leukaemia Relapse After HLA-Identical Sibling Stem Cell Transplantation for Acute Myeloid Leukaemia but Not Other Haematological Malignancies Is Associated with Donor KIR Genes 2DL5A, 2DS1 and 3DS1.
Authors: Stringaris Kate , Adams Sharon , Uribe Marcela , Eniafe Rhoda , Wu Colin , Savani Bipin N. , Barrett A. John
Published: October 2019

Abstract 2282

Poster Board II-259

Stem cell transplantation (SCT) from a healthy donor can be curative for patients with haematological malignancies resistant to other treatments. Elimination of malignant cells through a graft-versus-leukaemia (GVL) effect involves donor T- and NK-cells but their relative contribution to this process is poorly defined. NK-cell alloreactivity and GVL effects are controlled by the nature of the interaction of NK activation receptors and killer-immunoglobulin-like-receptors (KIR) with major-histocompatibility locus class I antigens on thetarget cell. We performed KIR-genotyping of HLA-matched donors in two hundred and forty eight T-celldepleted SCTs to identify genetic factors affectingtransplant outcome (transplant-related mortality (TRM), leukaemic relapse and survival). Transplant-related factors and donor KIRgenotyping significant in univariate analysis were entered into a multivariateanalysis to identify independent variables predictive of outcome for differentforms of leukaemia.

Patients studied were transplanted at a single center between 1993-2008.They included 70 AML, 99 CML, 40 ALL, 39 MDS patients and 13 patients with CLL, NHL or Myeloma. Median age was 36 years (range 9-66). All received aT cell depleted SCT from an HLA identical sibling. Patients were conditioned with totalbody irradiation 12-13.5Gy (4Gy for patients over 55y) and cyclophosphamide120mg/kg +/- fludarabine 125mg/m2. Patients received 0.2-5 × 10 5 CD3 cells /kg and amedian CD34 cell dose of 3 × 10 6/kg. Cyclosporine was used as the sole GVHDprophylaxis.

Multivariate analysis confirmed known predictive factors; for survival: highCD34 cell dose(RR 0.51, p <0.001) and standard rather than high risk disease (RR 3.1, p <0.001), for TRM: poor lymphocyte recovery at day 30 (RR 0.4, p < 0.02), for relapse: high risk disease (RR 4.65, p<0.002). In addition, the presence of donor B haplotype KIR genes: 2DL5A, 2DS1 and 3DS1was associated with significantly less relapse in patients with AML (13% vs 57% ) but not inpatients with other myeloid or lymphoid malignancies. AMLpatients receiving SCT from donors with these KIR genesspan style=“font-size: 10pt;”>relapsed five times less frequently than patientstransplanted from donors with other KIR genotypes.No other factors relating to NK alloreactivity including (missing donor KIR ligand, presence of donor haplotype B, total number of inhibitory KIR and total number of activatory KIR) was foundto be predictive for transplant outcomes. Thesefindings suggest specific, genetically determined,interactions between NK-cells and AML cells whichfacilitate the graft-versus-leukaemia effect and haveimplications for donor selection for AML patients.

Disclosures:

No relevant conflicts of interest to declare.
Second Autologous Peripheral Blood Stem Cell Transplantation with High Dose Melphalan (HDM/SCT) in Patients Relapsing After An Initial Course of HDM/SCT for the Treatment of AL Amyloidosis.
Authors: Quillen Karen , Seldin David C. , Finn Kathleen T. , Sanchorawala Vaishali
Published: October 2019

Abstract 2318

Poster Board II-295

High-dose melphalan and autologous stem cell transplant (HDM/SCT) can induce complete hematologic responses (CR), defined as disappearance of the underlying monoclonal gammopathy from serum and urine by immunofixation electrophoresis, and of the clonal plasma cell dyscrasia by bone marrow immunohistochemistry, and extend survival in patients with AL amyloidosis. HDM/SCT results in a CR in 40% of patients, and leads to clinical improvements in organ function in >70% of those who achieve a CR. However, hematologic and clinical relapses occur in ∼8% of patients who initially achieve a CR. Tandem cycles of HDM/SCT, which are typically performed within 12 months of each other, have been shown to achieve a higher ultimate CR rate of >60%. Among patients who do not achieve a CR following a single cycle of HDM/SCT, 30% nonetheless experience improvement in organ function. However, in this latter group, clinical improvement is not durable. We designed a study to explore the feasibility, and efficacy, of a second cycle of HDM/SCT in patients who relapse after initially responding to a first cycle of HDM/SCT.

Results:

Eleven patients, median age 55 (range 39-62), M:F 7:4, who had achieved hematologic and clinical responses after an initial cycle of HDM/SCT, were treated with a second cycle of HDM/SCT when a hematologic and/or clinical relapse occurred after a median time interval of 34 months (range 12-63). Five patients underwent a second course of G-CSF mobilization and a mean of 5.1 million (range 3.4-7.6 million) CD34 cells/kg was collected in a median of 2 days; the other patients had cells saved from the first mobilization. Six patients received 200 mg/m2 HDM; 5 patients received modified high-dose HDM at 140 mg/m2. Engraftment occurred at a median of 10 days for neutrophils, and 12 days for platelets (two days without platelet transfusion support); this engraftment timing is similar to that following the initial transplants (10 days for neutrophils, 13 days for platelets). There was no treatment-related mortality, but toxicity was moderate; almost all patients (except one) experienced grade III/IV non-hematologic toxicities. Of the 11 patients, 3 achieved hematologic CR at one year; these patients are alive and in continuous remission at 2-6 yr after the second transplant, including one patient who received a subsequent renal transplant. Three patients died of progressive disease at 1-2 years after the second transplant. Five patients are alive at 1-3 years post second transplant, in partial remission.

Conclusion:

27% (3/11) of patients with AL amyloidosis who experience a hematologic or clinical relapse after responding to initial HDM/SCT can achieve a hematologic CR with a second course of HDM/SCT.

Disclosures:

No relevant conflicts of interest to declare.
Increased Levels of Histone In Human Plasma In Septic Patients with DIC.
Authors: Kawasugi Kazuo , Yamamoto Tadashi , Shirasaki Ryosuke , Tashiro Haruko , Shirafuji Naoki
Published: October 2019

Abstract 1422

Sepsis is a life-threatening condition that is characterized by a whole-body inflammatory state (called a systemic inflammatory response syndrome, SIRS). Histones are essential for packing DNA into the cell nucleus and also play role in regulating gene expression but the new study (Jun Xu et al, Nature Medicine 15. 1318, 2009) have shown histones released into blood stream at the onset of sepsis can have devastating effects (endothelial damage and organ failure etc). However, about extracellular histones in the plasma from human, they have shown only one patient's data. Therefore, we measured extracellular hisotones in the plasma from septic patients with DIC (n=20). Also, we investigated extracellular hisotones in the plasma from septic patients without DIC (n=5). Extracellular histones in the plasma from human were examined by western blot analysis for histone H3 (H3).

The thrombin antithrombin complexes (TAT) levels were higher in septic patients with DIC as reported by others. We detected high levels of extracellular hisotones (H3) in the plasma of all septic patients with DIC by western blotting. However, we did not find any extracellular histones (H3) in the plasma of fiver septic patients without DIC by western blotting. Moreover, high-mobility group box 1 (HMGB1) concentrations in the human plasma were determined using ELISA kit (Shino-Test, Japan). Plasma levels of HMBG1 were negative for four cases in 20 septic patients with DIC. Also, Plasma levels of HMBG1 were negative in septic patients without DIC.

These results suggest that assess of extracellular hisotones in the plasma may be helpful in the making the diagnosis in septic patients with DIC. It appears that extracellular hisotones in the plasma may contribute to develop DIC in patients with sepsis.

Disclosures:

No relevant conflicts of interest to declare.
Daily Pain Is Unrelated to Admission Rate in Adult Patients with Sickle Cell Disease
Authors: van Tuijn Charlotte F.J. , Biemond Bart J.
Published: October 2019

Abstract 1016

Background and aims

Sickle cell disease is characterised by frequent episodes of vaso-occlusive pain resulting in frequent health care utilisation and cumulating organ damage. Disease severity is often quantified by the number of hospital admissions. However, sickle cell patients frequently experience episodes of pain without health care utilisation. In the present study, we analysed whether daily self-reported sickle cell pain in adult patients is related to disease severity quantified by frequency of hospital admission or disease severity defined by genotype.

Methods

We conducted a prospective cohort study in order to analyse daily pain in adult sickle cell patients in a tertiary teaching hospital in the Netherlands. Patients were divided in having severe (HbSS/HbS-β0) or milder (HbSC/HbS-β+) disease according to genotype. Daily pain was assessed for a period of 6 months by the use of pain diaries. Pain intensity was measured with different validated pain assessment instruments (Visual Analog Scale (VAS), McGill Pain Questionnaire and Brief Pain Inventory). Daily pain scores were correlated with genotype and frequency of hospital admission.

Results

110 consecutive outpatients with sickle cell disease were approached. 16 patients were excluded for different reasons (pregnancy, unable to fill in a diary or declined participation), 19 patients missed their appointments in the outpatient clinic and 26 patients completed less than a month of their diaries. 49 patients (mean age 33 years (SD 12.6); 67% female) completed at least two month of the diaries (response rate: 65%). No differences in disease severity according to genotype or age were observed between responders/non-responders.

Pain was reported in 18% of the total 5016 patient-days. Overall median pain intensity was 3.6 (VAS). 41% of the patients reported pain in 5% or fewer diary days and 5% of all patients reported pain in more than 95% of the days. The frequency of hospital admission was unrelated to self-reported pain and no difference in daily pain was observed between patients with severe or milder disease according to genotype. Furthermore, lab results (hemoglobin and fetal hemoglobin levels, leukocyte counts, lactate dehydrogenase (LDH) and the presence of alfa-thalassemia) were not associated with daily pain.

Conclusions

Using daily pain diaries, adult sickle cell patients report pain in 18% of their total diary days. Interestingly, our patient population reported less daily pain in comparison to daily pain in adults reported previously (over 50%). Daily pain appeared to be unrelated to disease severity according to genotype or defined by frequency of hospital admission. Our results further stress that daily pain is an important problem in sickle cell patients and appears to be unrelated to the traditional definitions of disease severity.

Disclosures:

No relevant conflicts of interest to declare.
Critical Role for Histone Deacetylase 6 (HDAC6) in the Regulation of IL-6, and the JAK/STA3 Signaling Cascade in Macrophages
Authors: Lienlaf Maritza , Perez-Villarroel Patricio , Cheng Fengdong , Wang Hongwei , Marante Danay , Yoder Sean , V. Woan Karrune , Knox Tessa , Lagor Stephen , Pinilla-Ibarz Javier , Seto Edward , Sotomayor Eduardo M. , Villagra Alejandro
Published: October 2019

Abstract 1039

Background

Histone Deacetylases (HDACs) have divergent effects over the production of anti- and pro-inflammatory cytokines in Antigen Presenting Cells (APCs). We have previously shown that modulation of specific HDACs can alter the immunogenicity of APCs, either to an activating or tolerogenic phenotype. We recently identified HDAC6 to positively regulate IL-10 production. However, the participation of this HDAC in other immune related cellular processes remains unknown. In this work we are presenting evidence of the important role of HDAC6 in the regulation of IL-6 via activation of the JAK/STAT3 pathway.

Methods

Stable knockdown clones of HDAC6 (KDHDAC6) and Non-target (NT) cells were generated in RAW264.7 murine macrophages using lentiviral shRNA for HDAC6 or non-target (NT) respectively. Two KDHADC6 and two NT clones were treated with LPS or untreated and then analyzed by microarray using the Affymetrix GeneChip Mouse 430 2.0. Significantly down- or up-regulated genes were analyzed by their ontology distribution and selected genes were validated by quantitative real-time RT-PCR, ELISA, or immunoblots. Additionally, changes in the expression of these selected genes were tested in cells treated with selective HDAC6 inhibitors.

Results

1542 genes were down-regulated and 775 up-regulated in KDHDAC6 cells. Their ontology distribution revealed significant changes in immune-related (632) and apoptosis/cell cycle control (47) genes. Importantly, IL-6 was one of the most highly down-regulated genes in KDHDAC6 cells. Therefore, we next analyzed the relevance of these findings by studying the tolerogenic JAK/STAT3 signaling pathway which is known to be activated by IL-6 and critical in the final outcome of APCs in response to stimuli. We observed a complete abrogation in the phosphorylation of JAK2 and STAT3 proteins in KDHDAC6 cells in response to LPS, which was reverted when these cells were treated with exogenous IL-6.

Conclusions

These results demonstrate the requirement of HDAC6 in the production of IL-6 in response to LPS, and therefore positions this deacetylase as an important regulator of the JAK/STAT3 pathway. These findings provide insight into the molecular mechanisms controlling the immunogenicity of APCs, supporting the use of HDAC6 inhibitors to enhance immune activation.

Disclosures:

No relevant conflicts of interest to declare.
A GM-CSF and IL-4 Fusion Cytokine Triggers Conversion of B-Cells to Tumoricidal Effectors
Authors: Deng Jiusheng , Li Pingxin , Pennati Andrea , Yuan Shala , Hsieh Hsiang-Chuan (Jeremy) , Wu Jian Hui , Galipeau Jacques
Published: October 2019

Abstract 1048

We have previously demonstrated that coupling of GMCSF at the N-terminus of common γ-chain interleukins IL2, IL15 and IL21 leads to meaningful gain-of function activity in interleukin-responsive lymphomyeloid cells. Considering the physiological and immunological importance of IL4 that is a member of γ-chain interleukins, we tested the bioactivity of a novel fusion cytokine consisting of a fusion between GM-CSF and IL-4 (GIFT4). We observed that GIFT4 leads to a pan-STAT hyper-phosphorylation response in resting splenic B-cells distinct from IL4 only and that treated B-cells up-regulated expression of MHCI/II, CD80 and CD86, secreted IL-12, IL-1a, IL-6, and substantial amounts of CCL3 and GM-CSF, akin to recently described innate response activator (IRA) B-cells (Science 335, 597, 2012). In vivo delivery of recombinant GIFT4 protein to normal mice leads to homeostatic expansion of splenic B cells and plasma cells as well as humoral hyper-responsiveness to antigenic challenge. We further showed that B16F0 melanoma cells engineered to secrete GIFT4 are immune-rejected in a B-cell dependent manner. The clinical effect was abolished when B16F0-GIFT4 cells were implanted in B-cell deficient μMT, CD4−/−, CD8−/− or FcγR−/− mice consistent with a pivotal for B cells, their T-cell helper function and antibody-dependent cell-mediated cytotoxicity for the observed melanoma-specific therapeutic effect. Thus, GIFT4 defines a novel engineered cytokine that mediates endogenous expansion of B-cells with potent immune helper and antigen-specific effector function. We propose that GIFT4 protein could serve as a novel immunotherapeutic agent and defines a previously unrecognized potential of B-cells as tumoricidal effectors.

Disclosures:

No relevant conflicts of interest to declare.
Is a Pbac-Score a Good Tool to Quantify Menorrhagia in Women with Von Willebrand Disease and Rare Bleeding Disorders?
Authors: Halimeh Susan , Rott Hannelore , Siebert Manuela , Kappert Guenther
Published: October 2019

Abstract 1133

Introduction:

Von Willebrand disease (VWD) is the most common inherited bleeding disorder. VWD and other autosomal inherited bleeding disorders equally affect women and men.

Menorrhagia or severe menstrual bleeding (HMB) is the most common symptom of women with bleeding disorders. HMB is defined as bleeding that lasts for more than seven days or as the loss of more than 80 mL of blood per menstrual cycle.

The menstrual blood loss can be quantified by the use of a pictorial bleeding assessement chart (PBAC).

Samples and methods:

In 195 women with menorrhagia and in 45 controls menstrual blood loss was quantified using pictorial blood assesment charts (PBAC) and results were compared.

Results:

In 169 of 195 women (86%) a bleeding disorder could be detected. In those with a bleeding disorder, the distribution was as followed: 62% had a von Willebrand disease, 14,4% had a factor-VII-deficiency (F7D), 5% had a factor-XIII-deficiency (F13D) and the remaining 18,6% had other beedling disorders (e. g. hypofibrinogenaemia and other mild factor deficiencies). The median PBAC-Score of all patients was 268 (range: 10–4212). In our controi group of 45 women the median PBAC-Score was 46,5 (3- 137).ROC-Analysation shows that the PBAC (AUC=0.977) is much more useful than the number of bleeding days (AUC=0.855) in order to distingish controls from patients suffering from menorrhagia due to a coagulation disorder. We found that the best cutoff for the PBAC is 100 with an sensitifity of 88% and a specifity of 97%.

Discussion:

Attempts to measure the quantity of menstrual blood loss can be useful in clinic practice. One study found that variables predicting a blood loss higher than 80ml per menses were clots greater than one inch, low ferritin levels, or changing a pad or tampon more than hourly (flooding). A prospective method of quantifying menstrual blood loss includes the use of a pictorial bleeding assessement calendar (PBAC). We are of the opinion, that we would have detected more bleeding disorder also in the patients, where we did not find any diagnosis until now, if we would have controlled them more than one time during the cycle period.

Conclusions:

Women with hyermenorrhagia frequently suffer from a bleeding disorder, in 86% of our patients an abnormal coagulation was found.

The PBAC-Score is an easy tool to quantify menstrual blood loss in women.

In our study a PBAC-Score above 100 was suspicious of having a bleeding disorder.

Disclosures:

No relevant conflicts of interest to declare.
Risk of Venous Thrombosis Associated with White Cell Count On Peripheral Blood and Its Interrelationship with Other Environmental Risk Factors
Authors: Rezende Suely M , Lijfering Willem M. , Rosendaal Frits R. , Cannegieter Suzanne C.
Published: October 2019

Abstract 1149

Background:

Antithrombotic prophylaxis needs to be balanced against bleeding risk. Therefore, risk stratification is useful to identify patients who would benefit most. In this study, we analyzed how VT risk relates to different leukocyte count. We also analyzed the effect of high leukocyte counts in groups with high risk of VT.

Methods:

In the MEGA case-control study, blood leukocyte count and information on environmental risk factors were collected from 2443 patients with VT and 1459 partner controls. Logistic regression analyses were adjusted for age and sex.

Results:

Population characteristics are detailed in Table 1. Risk of VT increased for measurements above the 97.5th percentile for total white cells, granulocytes, lymphocytes and monocytes. Adjusted odds ratio, [OR] for 99th percentile was 1.37 (95% confidence interval [CI], 0.73–2.56), 1.48 (95% CI, 0.79–2.77), 1.20 (95% CI, 0.63–2.31) and 1.88 (95% CI, 1.02–3.46), for total white cells, granulocytes lymphocytes and monocytes, respectively, compared to the reference percentile (5th-95th) (Table 2). We also analysed the effect of high leukocyte counts on VT in high risk groups, including surgical patients, hospitalized patients, and patients with cancer. Adjusted OR for high leukocyte counts (white cell, lymphocyte, monocyte or granulocyte counts > 97.5th percentile) was 6.3 (95% CI, 0.84–47.1) for groups who had surgery or were hospitalized and 2.2 (95% CI, 0.6–8.2) for patients with cancer, respectively, compared with cell counts within the percentile between 5th and 97.5th.

Conclusions:

High blood leukocyte count is associated with increased risk of VT. The risk is further increased in high risk groups. These results may assist in individually tailored thromboprophylaxis in high risk groups.

Disclosures:

No relevant conflicts of interest to declare.
Hematopoietic Stem Cells Are Dependent On Homologous Recombination Only During Active Cell Cycle in Nuclease-Mutant Exonuclease1mut mice
Authors: Desai Amar , Gerson Stanton L. , Qing Yulan
Published: October 2019

Abstract 1225

Hematopoietic stem cell (HSC) maintenance and self-renewal is crucial for long term tissue repopulation and immune function. HSC populations require functional DNA repair pathways in order to maintain their reconstitution capabilities but the pathways involved and the mechanisms of regulation are still being elucidated. It has been proposed that quiescent HSCs rely on the error prone non homologous end joining pathway for DNA double strand break (DSB) repair while HSCs in cycle use both NHEJ and the high fidelity homologous recombination (HR), but functional in vivo studies have not yet been completed. Exonuclease 1 participates in homologous recombination. We used Exo1mut fibroblasts to demonstrate that loss of Exo1 function results in a defective HR response, increased sensitivity to DSB inducing agents, and aberrant DNA damage signaling. However, Exo1mut mice did not appear to require HR to maintain quiescent HSCs at steady state or to respond to DNA damage. Exo1mutmice were able to sustain long term serial repopulation, displayed no defect in competitive repopulation or quiescence maintenance, and did not display increased sensitivity to whole body ionizing radiation (IR). In contrast, when Exo1mut HSCs were pushed into cell cycle with 5-Fluorouracil, the hematopoietic population and HSCs became hypersensitive to IR stress relative to WT B6 mice, as shown by decreased bone marrow cellularity, colony forming unit defects, loss of the HSC population, and finally animal death. Thus, loss of Exo1, and in turn fully functional HR, in quiescent HSC is not critical to stem cell function, survival, or recovery after DNA damage, whereas HR mediated repair of DNA damage is essential for HSC maintenance after cell cycle entry. In HSCs, DNA damage repair response, and sensitivity is dependent on cell cycle.

Disclosures:

No relevant conflicts of interest to declare.
Elevation of Mitochondrial DNA Copy Number in Bone Marrow Cells Associated with Increased Risk of Childhood Acute Myeloid Leukemia
Authors: Kim Yu-Na , Kim Hye-Ran , Kim Hwan-Young , Dai Trang Nguyen Thi , Han Dong-Kyun , Baek Hee-Jo , Kook Hoon , Shin Myung-Geun
Published: October 2019

Abstract 1423

Background:

Alteration of mitochondrial DNA (mtDNA) is now implicated in tumorigenesis as well as poor clinical outcomes. mtDNA copy number is altered in patients with various hematological malignancies such as lymphocytic leukemia, lymphoma and other disorders. Moreover, recent studies has reported that mtDNA copy number is associated with increased risk of non-hodgkin lymphoma (Blood 2008;112:4247–9). Therefore, the present study was fist to validate the hypothesis that alteration of mtDNA copy number in bone marrow cell would be associated with risk of childhood acute myeloid leukemia (AML).

Patients and Methods:

Direct sequencing of mtDNA control region, analysis of mtDNA minisatellites and mtDNA copy number were performed in 48 patients with initially diagnosed childhood AML cases and 48 matched case controls from Environmental Health Center for Childhood Leukemia and Cancer cohort. Molecular analysis of mtDNA including quantitation of mtDNA copy number was determined using our published protocol (Int J Cancer 2012;131:1332–41).

Results:

Numerous sequence alterations of mtDNA were only found in patient's bone marrow cells. Instability of mtDNA minisatellites was mainly occurred in the 303 polyC and 16184 polyC minisatellite. mtDNA copy number was significantly elevated in patients with childhood AML compared with those of control group (p=0.000075, Fig. 1). There was a dose-response relationship between tertiles of mtDNA copy number and risk of childhood AML (odds ratio [OR], 95% confidence interval [CI]: 1.0; 1.145 [0.412–3.183]; and 6.818 [2.244–20.712], respectively; p(trend) =.000). The effect was most pronounced for childhood AML harboring normal karyotype (OR: 1.0; 1.75[0.369–8.302]; 9.00 [1.888–42.904]; p(trend) =.008) (Table 1).

Conclusion:

mtDNA minisatellite inability was frequently observed in bone marrow cells of childhood AML. Elevation of mtDNA copy number could be associated with increased risk of childhood AML, particularly normal karyotype childhood AML.

Disclosures:

No relevant conflicts of interest to declare.