bluuebunny/crossref_metadata_2025 huggingface dataset:
Thrombosis and Inflammation Are Coupled by a Common Thioredoxin Reductase Pathway Downstream of the P2×7 Receptor.
Authors: Rothmeier Andrea S , Furlan-Freguia Christian , Petrich Brian G. , Marchese Patrizia , Ruggeri Zaverio M. , Ginsberg Mark H. , Ruf Wolfram
Published: October 2019

Abstract 2218

The purinergic P2X7 receptor contributes to thrombosis by promoting tissue factor (TF) activation and the release of prothrombotic microparticles (MP). On primed macrophages, P2X7 stimulation induces the procoagulant activity of cell surface TF and the release of MP that carry TF, integrin β1 and protein disulfide isomerase (PDI). The generation of TF+ MP is dependent on extracellular thiol-disulfide exchange, but intermediates leading to MP generation downstream of P2X7 signaling are incompletely defined. Tracking of cell surface TF by confocal microscopy shows that constitutive internalization of TF is prevented by P2X7 activation. In non-stimulated cells, inhibition of dynamin-dependent endocytosis retains TF on the cell surface, rapidly upregulates TF activity, and releases procoagulant MP carrying TF and integrin β1. Integrin recycling is dependent on the small GTPase ARF6 that is found incorporated into MP released when internalization is blocked. In contrast, activation of P2X7 releases ARF6 into the MP-depleted supernatant rather than associated with MP. Decreased ARF6 GTP loading in P2X7-stimulated cells and additional data in integrin α4 S988A mutant mice with reduced ARF6 activity support the conclusion that P2X7 interrupts constitutive ARF6 recycling to increase cell surface availability of TF-integrin complexes for incorporation into MP. Confocal imaging shows that P2X7 stimulation promotes filopodia formation and the peripheral transport of TF to the tips of filipodia. Proteomics identifies γ-actin as a thiol-regulated MP protein that is released by P2X7 signaling. We show that specific inhibition of thioredoxin reductase attenuates P2X7-induced actin remodeling and filopodia formation, the generation of highly procoagulant MP carrying γ-actin and PDI, and the increased availability of extracellular thioredoxin and free thiols. Importantly, inhibition of thioredoxin reductase also prevented the processing and release of the pro-inflammatory cytokine IL1β. Thus, the thioredoxin reductase system is a crucial intermediate downstream of P2X7 activation that may couple coagulation and inflammation in cardiovascular diseases.

Disclosures:

No relevant conflicts of interest to declare.
Efficiency of RBC Antibody Detection Methods: Comparison of Manual Indirect Antiglobulin Test (IAT) to a Method Using Very Low Ionic Strength Conditions (vLIS/IAT).
Authors: Szymanski Irma O.
Published: October 2019

Abstract 2276

Is has long been known that RBC antibody detection is enhanced following a decrease in the ionic concentration. In the past we added into our routine pre-transfusion antibody screen a new test, which employs ionic strength conditions between 15 to 17% of the ionic strength of 0.9% NaCl (vLIS/IAT): 2 drops of patient's plasma and one drop of 3% RBC are incubated for 15 minutes in 0.5 mL of 5% mannitol, pH 6.0 with phosphate buffer, containing 8mM EDTA. The RBCs are washed with normal saline and tested for agglutination by an anti-IgG reagent. Other published low ionic strength methods decrease the ionic concentration less, up to about 50%.

In the process of pre-transfusion testing we found 711 patients with one, two or more blood group antibodies. The following table shows the specificities and quantities of clinically significant antibodies detected by these two methods. The following antibody specificities were not included: those that were present only in small quantities, directly agglutinating IgM antibodies and anti-Lea and Lebantibodies which are usually considered to be clinically insignificant.

Antibody specificity Total Number detected % Detected by IAT % Detected by vLIS/IAT C 71 71.8% 93.0% c 36 80.5% 92.0% D 147 68.7% 97.3% E 220 40.3% 95.5% Fya 44 70.5% 95.5% Jka 60 60.0% 96.7% K 154 89.0% 76.0% M 23 34.8% 87.0% S 18 50.0% 100% Total 773 Avg. 62.8% Avg. 92.6%

Overall, vLIS/IAT detected an increase in clinically relevant antibodies compared to IAT. This is clearly seen in the case of anti-E, anti-D, anti-Jka, anti-M and anti-S antibodies. Anti-K antibodies, however, were detected preferably by IAT. It should be noted that more anti-Le antibodies, although not shown here, were detected either by IAT or by direct agglutination (IgM antibodies).

In summary, these data show that vLIS/IAT improved antibody detection when incorporated into routine pre-transfusion tests. They also indicate that one method does not detect all clinically significant antibodies; therefore it is beneficial to use two methods which in combination cover the varying spectra of RBC antibodies.

Disclosures:

No relevant conflicts of interest to declare.
Validation of a Non-Invasive Hemoglobin Estimation in Whole Blood Donors.
Authors: Al-Khabori Murtadha K. , Farsi Khalil Al , Al-Huneini Mohammed , Daar Shahina , Al-Hashim Abdulhakeem , Al-Kemyani Nasser , Al-Riyami Arwa Z.
Published: October 2019

Abstract 2280

Introduction:

Pre-donation screening of blood donors are currently based on venous or capillary blood sampling. Adoption of a non-invasive hemoglobin estimation may increase blood donor recruitment. Masimo Pronto-7 Pulse CO-oximetry device is a spectrophotometry based device used to estimate the hemoglobin (Hb) level non-invasively, waiving the need of blood sampling. It has not been validated in normal blood donors. The primary objective of our study was to validate the pulse CO-oximetry based hemoglobin estimation in normal blood donors.

Methods:

We conducted a prospective observational study on 106 whole blood donors attending the blood bank of a tertiary care center over 4 weeks. We estimated a Spot Hemoglobin (Sp Hb) concentration using Masimo Pronto-7 Pulse CO-oximetry device (two measurements per donor) and compared it to a venous sample Hb (Reference Hemoglobin; Ref Hb) measured using Abbott CELL-DYN Sapphire hematology analyzer. We calculated Pearson correlation coefficient and coefficient of determination (R2). The multivariable linear regression model of predicting the estimation differences included age, gender, weight, height, blood pressure and reference hemoglobin.

Results:

We enrolled 106 donors (98 males, 8 females) with a mean age of 27 years (SD 6.2; 18–49) and a mean Ref Hb of 14.2 g/dL (SD 1.2; 11.5–17). The mean Sp Hb was 14.4 g/dL (SD 1.2; 11.3–16.7). The correlation coefficient between the Sp Hb and Ref Hb was 0.46 (R2 = 21%) with a mean difference of 0.2 g/dL (SD 1.2; −4.5 to 3). In the multivariable model, height (p =0.015) and Ref Hb level (p <0.001) were statistically significant predictors of the difference in measurement. There was a strong correlation between the two CO-oximetry Hb measurements (correlation coefficient 0.78, R2 = 60%).

Conclusions:

Our study demonstrated the validity of the CO-oximetry Hb measurement in normal blood donors and with good reproducibility. Larger prospective studies are needed to confirm our findings.

Disclosures:

No relevant conflicts of interest to declare.
A Phase II Trial of Azacitidine (NSC-102816) and Gemtuzumab Ozogamicin (NSC-720568) As Induction and Post-Remission Therapy in Patients of Age 60 and Older with Previously Untreated Non-M3 Acute Myeloid Leukemia (SWOG S0703): Report On the Poor Risk Patients
Authors: Nand Sucha , Othus Megan , Godwin John E. , Willman Cheryl L , Norwood Thomas , Erba Harry Paul , Howard Dianna , Coutre Steven E. , Appelbaum Frederick R.
Published: October 2019

Abstract 3584

Background:

Increasing age and worsening performance status (PS) are associated with low complete remission (CR) rates and high early death rates in patients (pts) with acute myeloid leukemia (AML). Data from 4 SWOG trials show that in patients ≥70 and PS of ≥ 2, the CR rate with standard chemotherapy is 29% and 30-day death rate 48%. Preliminary data suggest that a regimen combining azacitidine (AZA) and gemtuzumab ozogamicin (GO) has significant activity and low toxicity in this group of patients. The current trial was designed to test this regimen in a larger group of patients in a cooperative group setting.

Methods:

Newly diagnosed pts, ≥60 years of age, with de novo or secondary non-M3 AML were treated as follows: Induction: Hydroxyurea 1500 mg twice daily till WBC <10,000/mcL, followed by azacitidine 75 mg/m2/day s/cu or iv days 1–7, gemtuzumab ozogamicin 3mg/m2 D8. If D14 marrow showed residual disease, induction treatment was repeated. Those achieving CR received one consolidation treatment which was identical to the induction treatment. This was followed by 4 cycles of azacitidine 75/m2/day, D1–7, given every 4 weeks. Subsequent management was left to the treating physicians. Patients were prospectively entered into good risk (age 60–69 or PS 0–1) and poor risk (age ≥70 and PS 2 or 3) cohorts. Based on our previous experience, we concluded that the regimen would be worth further study if CR+CRi was ≥ 30% and a 30 day survival was ≥ 70%. Promoter and global methylation studies were performed at defined time points.

Results:

Data on 83 good risk pts were presented at ASCO 2012. The results presented here are from the poor risk cohort. A total of 54 poor risk pts were treated. Median age was 76 (70.3–87) and 33 were males. Five pts had pre-existing MDS. Of the 54 evaluable pts, 19 (35%) achieved a CR or CRi. One additional pt achieved a CR with continued AZA therapy after being removed from the study for persistent disease on D28. Median progression free survival is 7 mo and median overall survival 6 months. There were 31 grade 3 or 4 toxicities. Seven (14 %) pts died early, with a 30 day survival of 86%. An estimated 30% of the pts (in good risk and poor risk groups) were able to receive their induction therapy in the outpatient setting.

Conclusions:

The combination of hydroxyurea, azacitidine and GO is associated with lower induction mortality, can be given in the outpatient setting and results in a CR rate better than that seen in poor risk pts with AML treated with standard chemotherapy. These results are sufficiently encouraging to warrant further studies with this approach.

Clinical Trials.govIdentifier: NCT00658814.

Disclosures:

Nand: Celgene: Research Funding.
Immune Complexes Enhance Uptake of Blood Coagulation Factor VIII by Antigen Presenting Cells
Authors: Wroblewska Aleksandra , Herczenik Eszter , ten Brinke Anja , Voorberg Jan
Published: October 2019

Abstract 37

The X-chromosome-linked bleeding disorder hemophilia A is caused by the absence or dysfunction of blood coagulation factor VIII (FVIII) which can be corrected by regular intravenous administration of FVIII. Development of antibodies directed against FVIII (referred to as FVIII inhibitors) is common complication in hemophilia care. Immune tolerance induction (ITI) comprising frequent administration of high doses of FVIII is the treatment of choice for hemophilia A patients with inhibitors. Thus far, the molecular mechanisms contributing to tolerance induction have not been defined. Here we sought to study the role of immune complex (IC) formation in endocytosis and functional presentation of FVIII by antigen presenting cells. Bone marrow-derived dendritic cells (DC) were used as model antigen-presenting cells, as DCs are known for their ability to take up and process immune complexes via FcγR. Purified recombinant FVIII was labeled using the Microscale Alexa Fluor 488 protein-labeling kit. For T-cell proliferation assays, FVIII−/− mice were injected intravenously 5× weekly with 1 μg of recombinant FVIII. Subsequently, spleens collected after weekly injections were processed into single-cell suspensions and used as a source of FVIII-specific T cells. Bone marrow-derived murine DCs were able to efficiently take up FVIII pre-complexed with anti-FVIII antibodies (FVIII-IC) in a dose-dependent manner. Endocytosis of FVIII-IC was 3–6 fold more efficient when compared to equimolar concentrations of soluble FVIII. Uptake of FVIII-IC, but not FVIII alone, could be inhibited with 2.4G2 antibody indicating functional involvement of FcγRII/III in this process. These results were confirmed using murine DCs isolated from FcγR-deficient mice. Moreover, antibodies used for IC formation were analysed based on isotype – IgG2a antibodies were most efficient in mediating IC endocytosis. Experiments performed with FVIII-specific T cells showed that in the low concentration range (<1 nM), FVIII-IC induced stronger T cell proliferation when compared to soluble FVIII. Collectively, these data provide further insight and understanding of modulation of anti-FVIII immune response in hemophilia A patients with pre-existing FVIII inhibitors.

Disclosures:

No relevant conflicts of interest to declare.
Inhibition of Histone Deacetylase 6 (HDAC6) Disrupts the Tolerogenic STAT3 Signaling Pathway and Augments Antitumor Immune Responses in Mantle Cell Lymphoma (MCL)
Authors: Wang Hongwei , Cheng Fengdong , Wang Zi , Lienlaf Maritza , Perez-Villarroel Patricio , Knox Tessa , Sahakian Eva , Pinilla-Ibarz Javier , Horna Pedro , Chen-Kiang Selina , Martin Peter , Bergman Joel , Kozikowski Alan , Villagra Alejandro , Tao Jianguo , Shah Bijal D. , Sotomayor Eduardo M.
Published: October 2019

Abstract 3724

MCL is an aggressive and incurable subtype of B-cell Non-Hodgkin's lymphomas. Although patients often respond to first-line chemotherapy plus monoclonal antibodies, relapse and decrease response to further lines of treatment eventually occurs. Manipulation of the immune system to unleash its specificity and long-lasting protective effect might provide a unique opportunity to induce more durable responses or prevent relapse in MCL.

Previous studies by our group have shown that HDAC6 is required for production of the immunosuppressive cytokine, IL-10 in antigen-presenting cells (APCs) and that genetic or pharmacologic disruption of HDAC6 in these cells triggers potent antigen-specific T-cell responses. Given the role of IL-10 in suppressing the immunogenicity of B-cells, we asked therefore whether inhibition of HDAC6 with the isotype-selective inhibitor, Tubastatin-A (Tub-A) could reverse the tolerogenic properties of malignant B-cells in a murine model of MCL1. First, in vitro treatment of FC-muMCL1 cells with Tub-A resulted in increased acetylation of a-tubulin, a known HDAC6 target. Treated B-cells also displayed an enhanced expression of MHC class II and the co-stimulatory molecules B7.1, B7.2 and CD40 relative to untreated cells. Such changes resulted in more immunogenic MCL cells able to effectively activate naïve antigen-specific CD4+ T-cells and more importantly, capable of restoring the responsiveness of anergic T-cells from lymphoma-bearing mice. Second, in vivo treatment of FC-muMCL1-bearing C57BL/6 mice with Tub-A resulted in lymphoma rejection. This antitumor effect was not observed in immunodeficient (SCID) C57BL/6 mice treated with Tub-A, pointing to the immunological effects triggered by HDAC6 inhibition in B-cells as playing a dominant role in Tub-A induced anti-MCL activity. Third, mechanistically we have found that HDAC6 physically interacts with STAT3 and it is required for STAT3 phosphorylation and recruitment to the IL-10 gene promoter. Furthermore, Tub-A treated MCL cells displayed a diminished STAT3 phosphorylation and abrogation of IL-10 gene transcriptional activity.

Taken together, a concerted regulatory mechanism involving HDAC6 and STAT3 influence the immunogenicity of MCL cells. Targeting HDAC6 with specific inhibitors to disrupt the tolerogenic STAT3/IL-10 axis represents a novel strategy to trigger effective anti-MCL immunity.

Disclosures:

Martin: Millennium Pharmaceuticals, Inc.: Speakers Bureau.
Human ROR1 Activates AKT and Accelerates Leukemia Cell Proliferation
Authors: Zhang Suping , Chen Liguang , Zhang Ling , Yu Jian , Rassenti Laura Z. , Kipps Thomas J.
Published: October 2019

Abstract 3872

ROR1 is an orphan-receptor-tyrosine-kinase-like developmental surface-antigen that is expressed by embryonic cells, chronic lymphocytic leukemia (CLL) cells, and neoplastic cells of many other types of cancer, but not by virtually all normal adult tissues. ROR1 can serve as a receptor for Wnt5a and potentially complex with other surface receptors for growth/survival factors elaborated by the tumor microenvironment or expressed by the tumor itself. In prior studies, we and others found that silencing ROR1 could impair tumor-cell growth and/or survival in vitro and in vivo. Although the CLL cells of ≥96% of all examined patients (N= 800) expressed ROR1, we found that a B-cell line derived from CLL cells, namely MEC1 (Leuk Res 23:127, 1999), lacked expression of this protein. This provided us with an opportunity to assess how introduction of ROR1 could affect the biology of this CLL-cell line, which has relatively slow doubling-time of ≈40 hours. We transfected MEC1 cells with either an expression vector encoding human ROR1 or a control vector, and then selected for stable transfectants in selection medium. MEC1 cells transfected with the ROR1 vector expressed high-levels of ROR1, but not MEC1 cells transfected with the control vector. MEC1 cells made to express ROR1 had higher levels of phosphorylated and activated-AKT and activated cAMP response-element-binding (CREB) protein than non-transfected MEC1 cells or cells transfected with the control vector. Furthermore, MEC1 made to express ROR1 had significantly greater proportions of cells in S/G2/M phase than did control-transfected cells 16 hours after transfer from serum-free medium to complete growth medium, indicating that MEC1 cells made to express ROR1 had increased relative proportions of cells undergoing cell division. Consistent with this, we found that ROR1+ MEC1 cells had significantly shorter doubling times in culture than did comparably cultured parental MEC1 cells or MEC1 cells transfected with the control vector. This study indicates that expression of ROR1 in the MEC1 CLL cell line can activate AKT/CREB and accelerate leukemia cell growth, providing us with a model system with which to interrogate the function of ROR1 in vitro.

Disclosures:

No relevant conflicts of interest to declare.
Targeting Mcl-1 for Multiple Myeloma (MM) Therapy: Drug-Induced Generation of Mcl-1 Fragment Mcl-1128–350 Triggers MM Cell Death Via c- Jun Upregulation
Authors: Fan Fengjuan , Tonon Giovanni , Sattler Martin , Schimming Alexander , Goldschmidt Hartmut , Opferman Joseph , Anderson Kenneth C. , Jaeger Dirk , Podar Klaus
Published: October 2019

Abstract 3959

Myeloid cell leukemia-1 (Mcl-1) is an anti-apoptotic protein of the Bcl-2 family located on chromosome 1q21. Gains of 1q21 are unfavorable genetic prognostic factors for Multiple Myeloma (MM) patients. Our present data confirm significant overexpression of Mcl-1 gene in tumor versus plasma cells derived from healthy donors. Moreover, a significant link between Mcl-1 overexpression and poor prognosis was found in a data set of 590 MM patients, supporting a key role for Mcl-1 in MM survival and drug resistance. Mcl-1 is therefore a potential therapeutic target in MM. Strategies to inhibit Mcl-1 activity in MM and other malignancies may not only include induction of proteasome-dependent Mcl-1 degradation and modulation of Mcl-1 interaction with BH3-only proteins, but also induction of proteasome- independent Mcl-1 degradation. Indeed, our own and other previous data demonstrate that in contrast to full length Mcl-1 a caspase- induced 28kD Mcl-1 fragment, Mcl-1D128–350, inhibits MM cell proliferation and triggers apoptosis. We therefore performed a large-scale drug screen for agents which induce generation of Mcl-1D128–350 in MM cells. Our results show that bortezomib and staurosporine (STS) but not melphalan, doxorubicin or thalidomide induce generation of Mcl-1D128–350. We next sought to identify molecular sequelae downstream of Mcl-1D128–350 which mediate its anti-proliferative and pro-apoptotic effect in MM cells. Surprisingly, we observed nuclear accumulation of drug- induced Mcl-1D128–350, enhanced AP-1 reporter activity as well as elevated protein levels of c-Jun. Conversely, drug- induced c-Jun upregulation was blocked after introduction of point mutations (A127D, A157D) into the two highly conserved Mcl-1 caspase-cleavage sites Asp127 and Asp157. Gene profiles on MM cells transfected with either Mcl-1wt or Mcl-1D128–350 identified a group of differentially expressed genes associated with MM cell proliferation, survival and drug resistance. Consistent with these data, treatment with bortezomib or STS triggered c-Jun upregulation followed by apoptosis in Mcl-1wt/wt but not Mcl-1d/null murine embryonic fibroblasts (MEFs). Importantly, STS-induced c-Jun upregulation and cell death was restored in Mcl-1d/null MEFs transfected with a plasmid carrying Mcl-1wt. In ongoing studies we are analyzing genome sequencing data in MM cells for the existence of point mutations in the Mcl-1 caspase cleavage sites. The presence of such mutations could represent a potential new mechanism of drug resistance. Taken together, our data add another facet to the complex cellular functions of Mcl-1 in MM and derive a promising therapeutic and potential prognostic role for Mcl-1D128–350 in MM.

Disclosures:

No relevant conflicts of interest to declare.
Down-Regulation of MMSET by RNA Interference Affects Cell Viability and Tumorigenicity in t(4;14) Myeloma Plasma Cells
Authors: Qian Shenxian , Gao Da-quan , Xie Yaping , Xu Yin , Huang Xilian
Published: October 2019

Abstract 3998

The recurrent immunoglobulin translocation, t(4;14)(p16;q32) occurs in 15% of multiple myeloma patients and is the worst prognostic subgroup in MM, although the basis for this poor prognosis is unknown. The t(4;14) is unusual in that it involves 2 potential target genes: fibroblast growth factor receptor 3 (FGFR3) and multiple myeloma SET domain (MMSET). MMSET is universally overexpressed in t(4;14) MM, whereas FGFR3 expression is lost in one-third of cases. Nonetheless, The involvement of MMSET in the pathogenesis of t(4;14) multiple myeloma and the mechanism or genes deregulated by MMSET upregulation are still unclear. Here we demonstrate a role for MMSET in t(4;14) MM cells. We found that Down-regulation of MMSET expression in KMS11 cell by RNA interference and by selective disruption of the translocated MMSET allele using gene targeting dramatically reduced DNA synthesis in KMS11 decreased to 20% of that of the control, whereas the viability of cells decreased to 2% (p_ 0.01) of that of the control and increase in apoptosis was twice that of the control. These findings were associated with reduced expression of genes (e.g. CCND2, CCNG1, MAF and VEGF). These results provide direct evidence that MMSET plays a significant cell survival and antiapoptotic role in t(4;14) MM and suggest that therapies targeting this gene could impact this particular subset of poor-prognosis patients.

Disclosures:

No relevant conflicts of interest to declare.
The Activity of MOR202, a Fully Human Anti-CD38 Antibody, Is Complemented by ADCP and Is Synergistically Enhanced by Lenalidomide in Vitro and in Vivo
Authors: Endell Jan , Boxhammer Rainer , Wurzenberger Cornelia , Ness Daniel , Steidl Stefan
Published: October 2019

Abstract 4018

Background:

MOR202, a HuCAL-derived fully human anti-CD38 antibody has proven to be highly effective in preclinical models of multiple myeloma (MM) and is currently evaluated in a phase I/II clinical trial. Having previously identified ADCC as potent effector mechanism of MOR202, the capability to induce killing of MM patient cells via antibody dependent cellular phagocytosis (ADCP) was assessed. Furthermore, the immunomodulatory agent lenalidomide (LEN), a commonly used agent in MM therapy was investigated for its potential to enhance the cytotoxicity of MOR202 in vitro and in vivo.

Methods:

Drug effects in presence or absence of LEN were characterized in vitro in a FACS-based phagocytosis assay using macrophages derived from M-CSF stimulated monocytes and a panel of CD38+ MM target cells. The therapeutic potential of MOR202 in vivo as a single agent and in combination with LEN was studied in a disseminated survival model of iv injected Ramos lymphoma cells. Synergy was determined using the fractional product concept.

Results:

MOR202 induced ADCP with high potency showing EC50 values between 40 and 130pM. Maximal killing was donor and cell line dependent ranging from 11 to 72%. The combination with LEN induced additive to synergistic enhancement of MOR202 activity which was mediated by several mechanisms identified to be direct cytotoxicity and increased CD38 expression levels on MM cells. In vivo, MOR202 at 1mg/kg increased median survival (MS) by 142% compared to vehicle control (MS day 48.5 vs. day 20), while LEN had no distinct effect at any of the tested doses between 25 and 100mg/kg (MS day 21–22). Intriguingly, despite the apparent lack of efficacy by LEN alone co-administration of 1mg/kg MOR202 and 100mg/kg LEN resulted in a superior increase of MS by 225% (MS day 65), thus demonstrating strong synergism. Of note, while no mouse survived beyond day 65 in any other group, 3/8 animals in the combo group were completely free of tumor until study termination on day 98.

Conclusions:

We here demonstrate ADCP to be an additional potent effector mechanism of MOR202. The combination of MOR202 and LEN enhanced ADCP-mediated killing in vitro and increased median survival in vivo in a synergistic manner. These results suggest a mechanistic rationale for MOR202 combinations with LEN including in particular LEN non-responders or relapse/refractory patients, thus warranting further evaluation in future clinical trials.

Disclosures:

Endell: MorphoSys AG: Employment. Boxhammer:MorphoSys AG: Employment. Wurzenberger:MorphoSys AG: Employment. Ness:MorphoSys AG: Employment. Steidl:MorphoSys AG: Employment.
STAT5A Regulates DNMT3 and Inactivates PTEN Tumor Suppressor Gene in CD34+/CD38− AML Cells
Authors: Takeuchi Asako , Nishioka Chie , Ikezoe Takayuki , Yang Jing , Yokoyama Akihito
Published: October 2019

Abstract 4087

Acute myelogenous leukemia (AML) is organized as a cellular hierarchy, initiated and maintained by a subset of self renewing leukemia stem cells (LSCs). We have recently reported that signal transducer and activator of transcription 5 (STAT5) is activated in CD34+/CD38− AML cells which contain abundant leukemia stem cells (LSCs) and inhibition of STAT5 induced apoptosis and sensitized these cells to the growth inhibition mediated by conventional chemotherapeutic agents (Int J Cancer. 2011;128:2317-25). This study attempted to identify molecules which are regulated by STAT5 and support survival of CD34+/CD38− AML cells. We lentivirally transduced a short hairpin (sh) RNA targeting STAT5 in CD34+/CD38− AML cells and compared their gene expression profiles with those of control shRNA transduced CD34+/CD38− AML cells by microarray analysis. Interestingly, DNA methyltransferase (DNMT) 3A, which catalyzes the transfer of a methyl group to DNA was differentially expressed between control and STAT5A down-regulated AML cells. In contrast, lentiviral transduction of STAT5A in CD34+/CD38− AML cells increased levels of DNMT3A. Reporter gene assays found that exposure of leukemia cells to IL-10 increased levels of the phosphorylated forms of STAT5, leading to transcriptional activation of DNMT3A. On the other hand, dephosphorylation of STAT5A by AZ960, a selective JAK2 inhibitor decreased this transcriptional activity in leukemia cells. Of note, forced expression of STAT5A in leukemia cells caused hypermethylation on the promoter region of tumor suppressor gene PTEN and down-regulated its mRNA levels as measured by methylation-specific PCR and real-time RT-PCR, respectively. Taken together, STAT5A may positively regulate levels of DNMT3A, resulting in inactivation of tumor suppressor gene by epigenetic mechanisms in LSCs.

Disclosures:

No relevant conflicts of interest to declare.
Constitutive Expression of CD40L by CAR-Modified Tumor Targeted T Cells Enhances Anti-Tumor Efficacy Both in Vitro and in Vivo
Authors: Curran Kevin J. , Seinstra Beatrijs , Nikhamin Yan , Yeh Raymond , Usachenko Yelena , Brentjens Renier J
Published: October 2019

Abstract 4120

T cells can be genetically modified to target tumor antigens through the expression of a chimeric antigen receptor (CAR). Recent reports have demonstrated the effectiveness of CAR modified T cells in patients with relapsed or refractory malignancies. However, CAR modified T cells have yet to demonstrate the ability to recruit an endogenous anti-tumor response which would greatly enhance their therapeutic benefit. To overcome these limitations we have developed a bi-cistronic gamma-retroviral vector allowing for constitutive co-expression of a CD19-specific CAR (19–28z) and human CD40 ligand (CD40L; CD154). The CD40 ligand/CD40 system has been demonstrated to activate dendritic cells (DCs) and alter the phenotype of B cells (upregulation of co-stimulatory and adhesion molecules and secretion of pro-inflammatory cytokines) with subsequent stimulation of CD8+ T cell activation and proliferation. We now demonstrate T cells genetically modified to constitutively express CD40L undergo enhanced proliferation and up-regulated secretion of pro-inflammatory cytokines including GM-CSF and INF-g. Furthermore, T cells modified to constitutively express CD40L, upon co-culture, will alter the phenotype of CD40+ B cell tumor cell lines by enhancing the expression co-stimulatory molecules (CD80/CD86), adhesion molecules (CD54/CD58/CD70) and death receptors (CD95; Fas). These findings were similarly evident in primary patient tumor samples (e.g. CLL cells) when co-cultured with autologous T cells modified to constitutively express CD40L. We further demonstrate maturation of monocyte derived DCs with subsequent secretion of IL-12 following co-culture with autologous T cells modified to constitutively express CD40L. T cells transduced with the bi-cistronic 19–28z/CD40L vector showed enhanced in vitro cytotoxicity against a panel of CD19+ tumor cell lines. Furthermore, infusion of 19–28z/CD40L modified T cells enhances the survival of CD19+ tumor bearing immunodeficient mice (SCID/Beige) when compared to mice treated with T cells modified to express the anti-CD19 19–28z CAR alone. We conclude that further genetic modification of CAR targeted T cells to constitutively express the co-stimulatory CD40L may enhance the anti-tumor efficacy of this adoptive T cell therapy. Our data suggests this enhanced T cell efficacy may be due to both autocrine and paracrine mediated mechanisms.

Disclosures:

No relevant conflicts of interest to declare.
Multicenter, Prospective Study of Tacrolimus Plus Methotrexate As Graft-Versus-Host Disease Prophylaxis in HLA-Matched Sibling Peripheral Blood Stem Cell Transplantation: Comparison with Cyclosporine Group As Control
Authors: Shin SeungHwan , Yoon JaeHo , Yahng SeungAh , Lee SungEun , Cho ByungSik , Eom KiSung , Kim YooJin , Lee Seok , Min ChangKi , Kim HeeJe , Cho SeokGoo , Kim DongWook , Min WooSung , Park ChongWon , Lee JongWook
Published: October 2019

Abstract 4177

Background

Graft-versus-host disease (GVHD) is an immunologic complication after allogeneic hematopoietic stem cell transplantation (HSCT) with significant mortality and morbidities. Allogeneic peripheral blood stem cell transplantation (PBSCT) has high risk of GVHD, especially chronic GVHD compared to bone marrow transplantation. Only limited data are available comparing the efficacy of FK506 with that of cyclosporine (CsA) in human leukocyte antigen (HLA)-matched sibling PBSCT.

Methods

Thirty-nine patients with various hematologic disease received PBSCT with FK506+methotrexate (MTX) as GVHD prophylaxis were compared to ninety-four historical control with CsA+MTX GVHD prophylaxis.

Results

The 1-year cumulative incidence of grade II-IV acute GVHD was significantly lower in patients who received FK506 than those in the CsA group (10.3% vs 28.2%, p=0.036). The female donor-male recipient pair (hazard ratio; 4.828, 95% CI; 17.84-13.06, p=0.002) and CsA prophylaxis (hazard ratio; 3.279, 95% CI; 1.14–9.43, p=0.027) were significant risk factor of acute GVHD in multivariate analysis. But, there was no difference in the 3-year cumulative incidence of chronic GVHD between the FK506 and the CsA group (77.6% vs 69.6%, p=0.793). The 3-year cumulative incidence of relapse (33.9% vs 23.1%, p=0.505) and 3-year treatment-related mortality (18.9% vs 28.4%, p=0.187) of the two groups were similar. The patients in the FK506 arm had a similar event-free survival (EFS) and overall survival (OS) with patients in the CsA arm (3-year EFS; 53.2% vs 55.1%, p=0.706, 3-year OS; 60.7 vs 61.5%, p=0.610). The age over 35 years (hazard ratio; 4.12, 95% CI; 1.95–8.70, p=0.001), female donor-male recipient pair (hazard ratio; 2.66, 95% CI; 1.52–4.65, p=0.001) and advanced pre-HSCT disease status (hazard ratio; 3.13, 95% CI; 1.72–5.71, p=0.001) were significant prognostic factors associated with OS in multivariate analysis.

Conclusion

These results demonstrated that the FK506+MTX can significantly reduce grade II-IV acute GVHD compared to CsA+MTX in sibling PBSCT with similar incidence of chronic GVHD, and comparable outcome in EFS, OS, relapse rate and TRM rate between two groups.

Disclosures:

No relevant conflicts of interest to declare.
Evaluating Prolonged Gvhd Based On Monocyte and T-Cell Activation, Leads to Early Detection of Immunological Changes Before Gvhd Exacerbations
Authors: Kuroda Rie , Fujiki Toshihiro , Mase Shintaro , Araki Raita , Maeba Hideaki , Koizumi Shoichi , Yachie Akihiro , Nishimura Ryosei
Published: October 2019

Abstract 4178

We have reported that evaluating the activation markers and homing molecules not only on T-cells but also on monocytes, combined with cytokine profiling, provides useful information for management of patients with prolonged GVHD (the 53th ASH). In the cases both T-cells and monocytes were highly activated as shown in Fig.1A, we have shown that steroid response was quite effective. When only T-cells were activated, administration of calcineurin inhibitors (CI) alone was sufficient to control the disease. However steroid refractory or steroid dependent patients are presented at a uniform rate. We have employed low dose MTX for these patients, which is widely used in autoimmune diseases because of its anti-inflammatory activity. In this report, we showed the transitional change of both T-cell and monocyte activation status in patients with prolonged GVHD after initiating immunosuppressive agents, especially in some steroid dependent cases given weekly low dose MTX.

Methods

We evaluated activation markers and homing molecules not only on T-cells but also on monocytes in peripheral blood obtained from 32 childhood patients (more than 700 samples at various time points) receiving hematopoietic stem cell transplantation (HSCT) by multicolor flowcytometry. The following markers were used: CD69, CD25, and HLA-DR for T cell activation, CCR4, CCR5, CXCR3, CCR9, and CLA for homing markers. Inflammatory monocytes were defined as CD14dimCD16+ cells or CD14+CD163+ cells. In addition we combined the data of cytokine profiles secreted mainly by T cells such as soluble interleukin 2 receptor (sIL-2R), or monocytes such as neopterin, or both such as tumor necrosis factor-ƒ¿ (TNF-ƒ¿), soluble TNF-ƒ¿RI, and soluble TNF-ƒ¿RII.

Results

Representative pattern of combined T-cells and monocytes activation was shown in Fig.1A. After initiating sufficient dose of steroid, both T-cells and monocytes were fully suppressed as shown in Fig.1B. When steroid was tapered just below the maintenance dose, both CD4+CCR5+ CXCR3+ and CD8+CCR5+ inflammatory T-cells were gradually increased without hypercytokinemia, and also increased frequency of CD4+CD45RO+ T-cell subpopulation was observed without any sign of GVHD exacerbations as shown in Fig.1C. In these condition, additional steroid or extra immunosuppressive drug such as weekly low dose MTX could gradually recover to fully suppressed state as shown in Fig.1D.

Discussion and conclusions

Evaluating prolonged GVHD by both T-cells and monocytes, combined with cytokine profiling, could lead to early detection of immunological changes before GVHD symptom develops. We can choose appropriate drug use such as weekly low dose MTX for steroid-sparing.

Disclosures:

No relevant conflicts of interest to declare.
Post-Allogeneic Monitoring with Molecular Markers Detected by Pre-Transplant Next Generation Sequencing (NGS) Predicts Clinical Relapse in Patients with Chronic Myelomonocytic Leukemia (CMML)
Authors: Kröger Nicolaus , Schroeder Thomas , Zabelina Tatjana , Badbaran Anita , Bacher Ulrike , Kobbe Guido , Schnittger Susanne , Kohlmann Alexander , Haferlach Torsten
Published: October 2019

Abstract 4212

Allogeneic stem cell transplantation remains the only curative treatment approach for chronic myelomonocytic leukemia (CMML), but relapse is the major cause of treatment failure. Here, we evaluated the impact of molecular mutations on outcome and the value of molecular monitoring post transplantation in patients with CMML. We screened 45 patients with a median age of 57 years (range: 24–73 years) who underwent allogeneic SCT pre transplant for molecular markers including a sensitive next-generation sequencing (NGS) technique. Of those, 7 were transformed to secondary acute myeloid leukemia, 5 were classified as MDS/MPN overlap and one patient as atypical CML. In 36 patients sufficient DNA was available for molecular analyses for ASXL1, CBL, NRAS and TET2, mutations. In particular, TET2 and CBL mutations were screened applying amplicon deep-sequencing (454 Life Sciences, Branford, CT).

In 32 patients (89%) at least one mutation could be detected: ASXL1: n=18: 50%; CBL: n=7: 19%; TET2: n=15:42%; NRAS: n= 11: 32%. Allogeneic SCT was performed after reduced intensity (66%) or standard myeloablative (33%) conditioning from related (22%) or unrelated donors (78%). The overall survival at 5 years was 46% (95% CI 28–64%) and the cumulative incidence of relapse at 2 year was 35%. Neither TET2, ASXL1, CBL nor NRAS mutations influenced overall survival significantly.

In 21 surviving patients with at least one positive marker pre transplantation, the corresponding marker was subsequently tested in peripheral blood after a median of 6 months (range 3–9 months) after transplantation.

In 6 (33%) of the patients one of the molecular markers was still detectable: ASXL1: n=3; CBL: n= 3; TET2: n=2, NRAS: n=3) and in 66% the markers were undetectable. In patients who became negative for any molecular marker the cumulative incidence of relapse at 1 year was significantly lower than in the positive tested patients. (15% versus 50%, p = 0.04).

In conclusion, this study shows that molecular monitoring post transplant with molecular markers detected also by NGS pre-transplant can help to predict the risk of relapse and may be used to guide post transplant immune-modulating therapies to prevent clinical relapse. In larger studies sequential monitoring of molecular markers after stem cell transplantation are mandatory to determine optimal timing for predicting relapse and for therapeutic intervention.

Disclosures:

Bacher: MLL Munich Leukemia Laboratory: Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Kohlmann:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.
Revisiting the Role of the Stat Laboratory: Is It Safe to Perform Activated Clotting Time Determinations (ACTs) Out of the Operating Room
Authors: Plietz Michael , Leifer Aaron , Padilla Vilma , Pineda Carole , Naraine Khrishan , Greenberg Mark , Fox Amy S
Published: October 2019

Abstract 4256

Background:

Activated clotting time (ACT) is determined during cardiac procedures in order to assess the level of anti-coagulation. There are three main anticoagulants used during cardiac procedures:heparin, angiomax and Plavix. Heparin is often used in the cardiac OR for major procedures and is neutralized, over time, by platelet factor 4(PF4). Angiomax, bivalirudin, has a quick onset of action, but a much shorter half-life when compared to Heparin. Plavix, clopidogrel, irreversibly inhibits platelets from clotting and is used on patients in need of anticoagulation for an extended period of time. The purpose of this study was to evaluate the stability of ACT results over time utilizing different coagulation regimens. If the ACT value remains stable over time, this would enable us to remove the device from the OR and place it in a stat laboratory.

Methods:

ACTs were determined by the ISTAT (Abbott), a hand held point of care device, using kaolin activated cartridges. A single blood sample was obtained and was evaluated at intervals of 0, 5, 10, 15, and 20 minutes. In addition, temperature, age, body surface area and platelet count of patient were recorded. The time 0 specimen was used to determine the patients ACT and interval testing was performed on the remnant.

Results:

36 samples representing 22 patients had ACT tests performed using the ISTAT. There was no significant change from 0 to 20 minutes over the entire dada set. However, when divided into different anti-clotting agents there were significant changes. Patients who had only taken Angiomax had a significant difference within the first 5 minutes (p value=.0094). Patients taking Angiomax and Plavix together had no change in ACT values at 20 minutes. Patients on Heparin alone demonstrated a loss of ACT stability at 10 minutes with values both increasing and decreasing.

Conclusions:

Patients on Angiomax alone demonstrated a significant difference in ACT value within the first 5 minutes and all intervals thereafter. Based on Angiomax short half-life the ACT should be performed as soon as possible. The ACT values in patients taking both Angiomax and Plavix achieved steady state throughout the 20 minutes and had little if any decrease in value. The results for the Heparin group were unreliable at 10 minutes. As a result of these findings, performing an ACT outside the OR is feasible assuming the test can be performed by 10 minutes.

Disclosures:

No relevant conflicts of interest to declare.
Reporting Bleeding Outcomes in Adult and Pediatric Studies of Primary Immune Thrombocytopenia: A Systematic Review
Authors: Noroozi Nastaran , Neunert Cindy , Goy Jennifer K , Buchanan George R. , Blanchette Victor S. , Arnold Donald M.
Published: October 2019

Abstract 4281

Background:

Bleeding is arguably the most clinically meaningful outcome in ITP clinical studies since it impacts morbidity, mortality and quality of life. Yet, the measurement of bleeding is not standardized, and its prevalence as a study outcome has not been well characterized.

Methods:

We performed a systematic review of all English language, prospective clinical studies enrolling 20 or more adults or children with primary ITP to determine if and how bleeding data were reported. In duplicate and independently, we searched MEDLINE, EMBASE, Cochrane and Cinahl databases until January 2012. Titles were screened for relevance and abstracts and full text articles were reviewed in duplicate for eligibility. Study selection and data abstraction were performed in duplicate. Patient demographics, use of a bleeding measurement tool and frequency of bleeding were collected from all studies.

Results:

We identified 167 prospective studies, including 29 (33%) randomized trials enrolling 13,976 patients with primary ITP (including 3,723 adults; 9,730 children; and 523 combined adults and children). Of 167 studies, 87 (52.1%) reported bleeding either as an efficacy endpoint (n=77), a safety endpoint (n=5) or both (n=5). Of the 87 studies that reported bleeding, 17 (19.5%) used an ITP-specific bleeding measurement tool. Such a tool was used in 4 of the 29 randomized trials. Less common were the Common Terminology Criteria for Adverse Events score (n=4) and the World Health Organization score (n=4); but the majority of studies did not use a predefined tool to measure bleeding. Bleeding was reported as present or absent (n=8 studies, 9.1%); by anatomical site only (n=39, 44%); by severity only (n=1, 1.1%); or by severity and anatomical site (n=29, 33%). Ten (29.8%) studies did not specify how bleeding was measured. Forty-three studies reported some association between platelet count and bleeding; however, none provided a statistical association with severity of thrombocytopenia.

This study represents a summative description of the English-language world literature on bleeding outcomes in ITP studies. Of all studies, just over half (52.1%) reported bleeding outcomes. Only 20% of studies used a bleeding measurement tool that was specific for ITP patients. A standardized approach to bleeding and other patient-centered outcomes will help direct future research in ITP.

Funding support from the McMaster Division of Hematology & Thromboembolism.

Disclosures:

Arnold: Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding; GlaxoSmithKline: Membership on an entity's Board of Directors or advisory committees, Research Funding; Hoffman-LaRoche: Research Funding.
Comparison of Complications Related to Peripherally Inserted Central Catheters, Infusion Ports, and Hickman Catheters in Acute Myeloid Leukemia Patients
Authors: Khalil Mohammad O. , Pierson Namali , Maymani Hossein , Holter Jennifer L. , Cherry Mohamad Ali
Published: October 2019

Abstract 4288

Background:

Intravenous catheters are widely used in acute myeloid leukemia (AML) patients. Complications associated with these catheters are frequently encountered and contribute to morbidity, mortality, and increased cost of treatment. Studies exploring and comparing complications in the different types of catheters in this unique patient population are lacking. We retrospectively studied infectious and thrombotic catheter-related complications in AML patients treated at the largest tertiary referral center for AML in Oklahoma.

Methods:

AML patients above the age of 18 who were referred to The University of Oklahoma Health Sciences Center from January, 2000 to June, 2012 were identified and medical records were reviewed. Patients were stratified according to type of first catheter inserted (peripherally inserted central catheter (PICC), infusion port (IP), or Hickman). First catheter-related blood stream infection and deep venous thrombosis (DVT) events were reported (subsequent catheter-related complications were not included). Statistical analysis was performed using SAS 9.2 software (SAS Institute Inc). Fisher exact test was used to compare patients with different types of catheters.

Results:

195 patients with AML were identified; of which 125 were included in the analysis (Patients referred for stem cell transplant (SCT) were excluded if not treated with prior chemotherapy at our institution). Median age at diagnosis was 51 years. 87 (70%) were males and 38 (30%) were females. 97 (78%) were White, 11 (9%) Native Americans, and 10 (8%) African Americans. 73 (58%) had PICC, 34 (27%) had Hickman, and 17 (14%) had IP. Blood stream infection occurred in 34% of all patients. Blood stream infection rates in each group were 32% in patients with PICC, 47% with IP, and 31% with Hickman (p=0.47). When divided by age group, infections occurred in 41% of patients 55 years of age or older and in 29% of those <55 years (p=0.19). Gram-positive cocci accounted for 57% and Gram-negative rods for 30% of all infections. DVT occurred in 14% of all cases. DVT rates in each group were 22% in patients with PICC, 6% with IP, and none with Hickman (p=0.002). When divided by age group, thrombosis rates were 9% in patients 55 years of age or older and 17% in those <55 years (p=0.29). 39% of patients with platelet count >20,000/microL at the time of event had DVT compared to only 8% of those with platelet count <20,000/microL (p=0.0005).

Conclusion:

Among AML patients referred to our institution, PICCs were associated with significantly higher rate of DVT compared to IPs and Hickmans. Platelet count >20,000/microL was also associated with significantly higher rate of thrombosis. There was no difference in blood stream infections across the different catheter types.

Disclosures:

No relevant conflicts of interest to declare.