bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of the Binding Between the Polymeric Platelet Adhesive Proteins, Multimerin 1 and Von Willebrand Factor
Authors: Parker D'Andra , Tasneem Subia , Fuller Nola , Sadler J. Evan , de Groot Philip G , Hayward Catherine PM
Published: October 2019

Abstract 1144

Introduction:

Multimerin 1 (MMRN1) is a massive variably-sized homopolymeric protein that is stored in platelet and endothelial cell secretion granules, for release with vascular injury. Recently, MMRN1 was identified to support platelet adhesion in vitro and in vivo. At high shear, MMRN1 supports platelet adhesion by a von Willebrand factor (VWF)-dependent, but integrin-independent mechanism, involving platelet glycoprotein (GP) Ibα. Direct binding of MMRN1 to GP Ibα has not been demonstrated. These data led us to postulate that VWF binds MMRN1 at site(s) distinct from the GP Ibα binding site, and test the roles of VWF A domains in MMRN1 binding.

Methods:

Modified enzyme linked immunosorbent assays (ELISA) and surface plasmon resonance (SPR) were used to assess binding interactions between wildtype (WT) MMRN1 and WT or domain deleted VWF constructs, and VWF polypeptides. Protein constructs tested included: multimeric VWF deletion constructs ΔA1A2A3-VWF, ΔA1A3-VWF, and ΔA1-VWF, and monomeric VWF polypeptides A1A2A3, A1A2, A1 and A3. Bovine serum albumin (BSA) coated surfaces were used as the negative control.

Results:

Unlike WT-VWF, VWF lacking the A domains (ΔA1A2A3-VWF) or the combination of the A1 and A3 domains (ΔA1A3-VWF) did not detectably bind to MMRN1 (p < 0.001). VWF lacking the A1 domain (ΔA1-VWF) showed MMRN1 binding comparable to WT-VWF (p = 0.39), excluding the possibility that MMRN1 binding site is located in VWF A1 domain (the region that binds GP Ibα). VWF polypeptides A1A2A3, A1A2 and A3 bound to MMRN1 (p < 0.001), unlike the VWF polypeptide A1 (p = 0.137), although the A1A2 polypeptide showed reduced binding compared to A1A2A3 (p < 0.001). SPR analyses confirmed that MMRN1 binding was supported by VWF peptides containing the A3 and/or A2 domains.

Conclusions:

The regions of VWF that support MMRN1 includes the A3, and possibly A2 domains, which respectively contain binding sites for collagen and ADAMTS-13. Our data suggest that the mechanism by which GP Ibα and VWF support platelet adhesion to MMRN1 at high shear include: VWF binding to GP Ibα via the A1 domain, and to MMRN1 via the A3 and possibly A2 domains. These findings have implications for the molecular mechanisms that support platelet adhesion at sites of vessel injury.

Disclosures:

No relevant conflicts of interest to declare.
Sustained Responses to Thrombopoetin Analogs in HIV Patients with Refractory ITP
Authors: Ochoa Roberto , Akunyili Ikechukwu , Milone Mary Anne , Harrington Thomas J. , Byrnes John J.
Published: October 2019

Abstract 1165

Thrombocytopenia(TP) is common in patients with Human Immunodeficiency Virus (HIV) infection, reported in as many as 10–30% of patients and seen more often in IV drug users and advanced HIV infection even in post-HAART era. Both decreased production of platelets and increased destruction have been proposed as mechanisms of thrombocytopenia and at least 2 different clinical scenarios have been described: patients with very advanced AIDS, being thrombocytopenia another manifestation of bone marrow suppression; and patients without significant T-cell depletion in whom thrombocytopenia develops likely as an immune phenomenom, “ITP-like” HIV-related thrombocytopenia (HIV-TP).

In HIV-TP, common treatments for classical ITP have been reported including corticosteroids, danazol, splenectomy, Intravenous Gammaglobulin, interferon, Anti-D, dapsone and splenic radiation with varying degrees of success with the highest responses seen with the use of HAART.

Romiplostim (RMP) and Eltrombopag (ETP) are thrombopoeitin agonists (TA) approved in 2009 for refractory ITP and recently proposed to be used as first-line agents. No reports of the use of TA in patients with HIV-ITP are available to date.

5 patients with HIV-related ITP were treated with TP in our centers between 2009 to 2011. Mean age was 48 years (25–61), 4 males and 1 female. All of the patients had failed or relapsed after prednisone, 3 of the patients had also failed multiple treatments including splenectomy (1 patient), IVIG, Rho(D) immune globulin, Rituximab, danazole and vincristine. All but 1 of the patients were on HAART by the time of treatment and CD4 counts were <200 cells/μL in 2 patients (10–157 cells/ μL).

All 5 patients were treated initially with RMP at the starting dose of 1 mcg/Kg and titrated as per guidelines. One patient requested to be switched to ETP and developed an acute lower extremity DVT and symptomatic PE 1 week after starting the medication.

Responses to treatment are shown in graph 1. Mean baseline Platelet count was 27 × 103/μL (13–40 × 103/μL) and it increased to 66 × 103/μL (45–86 × 103/μL) within 1 week and 76 × 103/μL (45–107 × 103/μL) after 1 month of therapy, 2.8 times the baseline (2.1 – 3.4 × baseline value). 4 patients achieved a platelet count ≥50 × 103/μL within 1 month. 4 of the patients had durable responses for ≥ 12 months not requiring dose adjustments or modifications; the other patient had several compliance issues and missed several doses of RMP, his platelet count improved every time the medication was restarted.

We present the first report of the successful and sustained use of TA in patient with HIV- ITP.

Disclosures:

Byrnes: Amgen: Honoraria, Speakers Bureau.
Critical Role for Stat5 in the Initiation and Maintenance of Polycythemia Vera in a Jak2V617F Knock-In Mouse Model
Authors: Yan Dongqing , Mohi Golam
Published: October 2019

Abstract 121

Version:1.0 StartHTML:0000000207 EndHTML:0000006199 StartFragment:0000002599 EndFragment:0000006163 SourceURL:file://localhost/Users/mohim/Desktop/ASH%202011/Dongqing%20Yan%202011%20ASH%20Abstract.doc

The JAK2V617F mutation has been identified in most cases of Ph-negative myeloproliferative neoplasms (MPNs) including polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). Expression of JAK2V617F results in constitutive activation of multiple signaling molecules/pathways. However, the key signaling downstream of JAK2V617F required for transformation, induction and maintenance of MPNs remains elusive. Using a mouse genetic strategy, we found that Stat5 is absolutely required for the pathogenesis of PV induced by Jak2V617F. Whereas inducible expression of Jak2V617F in mice resulted in all the features of human PV, including increase in red blood cells, hemoglobin, hematocrit, white blood cells, platelets, and splenomegaly, deletion of Stat5 in the Jak2V617F knock-in mice normalized all the blood parameters and the spleen size. Histopathologic analyses revealed that Stat5 deficiency blocked the development of PV in mice expressing Jak2V617F. In addition, deletion of Stat5 completely abrogated erythropoietin (Epo)-independent erythroid colony formation evoked by Jak2V617F, a hallmark feature of PV. Flow cytometric analysis revealed that concomitant deletion of Stat5 reduced the Jak2V617F-induced expansion of LSK (lin−Sca-1+c-kit+) and MEP (megakaryocyte-erythroid progenitors) as well as CD71+Ter119+ and Gr-1+Mac-1+ populations to normal levels. Unlike Jak2V617F knock-in mice, which developed myelofibrosis at old age, Stat5-deficient Jak2V617F-expressing mice failed to develop myelofibrosis. Re-expression of Stat5 in Stat5-deficient Jak2V617F knock-in mice bone marrow by retroviral transduction completely rescued the defects in transformation of hematopoietic progenitors and the PV phenotype. Furthermore, deletion of Stat5 after establishment of PV disease in the transplanted animals expressing Jak2V617F by injection with polyinosine:polycytosine (pI:pC) normalized the blood parameters and inhibited the progression of the disease. Together, these results indicate a critical function for Stat5 in the induction and maintenance of PV. Biochemical analyses revealed that Stat5 deficiency significantly inhibited constitutive phosphorylation of p70S6 kinase and markedly reduced expression of Bcl-xL, Cyclin-D2 and Pim-1 mediated by Jak2V617F. These suggest that p70S6 kinase, Bcl-xL, Cyclin-D2 and Pim-1 are downstream targets of Jak2V617F-Stat5 signaling, and they may play a role in hematopoietic transformation induced by Jak2V617F. These findings provide strong support for the development of Stat5 inhibitors as targeted therapies for the treatment of PV and other JAK2V617F-positive MPNs.

Disclosures:

No relevant conflicts of interest to declare.
Effects of Strong P2Y12 Receptor Inhibition by Prasugrel on Platelet Inhibition During Endotoxemia: A Randomized Controlled Trial
Authors: Spiel Alexander O , Derhaschnig Ulla , Jilma-Stohlawetz Petra , Jilma Bernd
Published: October 2019

Abstract 1245

Background:

P2Y12 receptor antagonists have become a mainstay for the treatment of cardiovascular diseases. Yet, they have rarely been evaluated under pathophysiological conditions apart from arterial diseases.

Objectives:

We hypothesized interactions between prasugrel and enhanced von Willebrand Factor (VWF) release in a model of systemic inflammation, and compared the pharmacodynamic effects of prasugrel versus placebo on agonist-induced platelet aggregation and shear-induced platelet plug formation.

Subjects/Methods: Twenty healthy male volunteers were enrolled in a double-blind, placebo-controlled two-way cross-over trial. Each volunteer received either placebo or a 60 mg-loading dose of prasugrel two hours before endotoxin infusion. Platelet inhibition was measured with Multiple Electrode Aggregometry (MEA), the Platelet Function Analyzer-100 (PFA-100) and the Vasodilator Stimulated Phosphoprotein (VASP) phosphorylation assay, respectively. Results: Prasugrel reduced the platelet reactivity index in the VASP assay from 79% to 5–7%, and unequivocally prolonged the closure times of the Innovance cartridge to >300s, but also the CADP-CT to >300s in the majority of subjects. Prasugrel not only blunted platelet aggregation induced by ADP (−81%), but also other pathways including arachidonic acid (−60%), ristocetin (−75%; p<0.001 for all), and to a lesser degree collagen or thrombin receptor activating peptide (TRAP). Prasugrel decreased shear-induced platelet plug formation but VWF release during endotoxemia partly antagonized the inhibitory effect of prasugrel as measured with the PFA-100. Endotoxemia acutely decreased ristocetin and TRAP induced platelet aggregation, and enhanced ristocetin induced aggregation after 24h.

Conclusions:

These data for the first time demonstrate that strong in vivo blockade of P2Y12 by prasugrel inhibits a broad spectrum of platelet aggregation pathways. However, VWF release may reduce prasugrel's effects under high shear conditions.

Disclosures:

No relevant conflicts of interest to declare.
Carbon Monoxide Therapy Modulates Hematopoietic Stem Cell Development in Heme-Oxygenase-1 Knockout Mice
Authors: Beckman Joan Denise , Marker Paul H , Nguyen Julia , Belcher John D , Croatt Anthony J. , Nath Karl A. , Hebbel Robert P , Vercellotti Gregory M
Published: October 2019

Abstract 1318

Previous studies have demonstrated that heme-oxygenase-1 (HO-1), the rate limiting enzyme in the catabolism of heme, is a regulator of the balance between hematopoiesis and stem cell preservation under stress. HO-1−/− mice display oxidative stress, anemia, and leukocytosis with chronic inflammation. We hypothesize that therapy with inhaled carbon monoxide (CO), a by-product of HO-1 activity with antioxidant and anti-apoptotic properties; will reduce stress hematopoiesis in HO-1−/− mice, reducing inflammation and redistributing hematopoietic potential. In order to test this hypothesis we treated 25 week old HO-1−/− mice and HO-1+/+ mice (n=7/strain) with 250 ppm inhaled CO for 1 h/day, 3 days/week for eight weeks and compared them to an equal number of untreated HO-1−/− and HO-1+/+ mice. After 8-weeks of treatment the mice were sacrificed and flow cytometry was performed on bone marrow to assess hematopoietic potential. Cell cycle analysis of the bone marrow demonstrates that untreated HO-1−/− mice have a significantly decreased percent of cells in S-phase compared to untreated HO-1+/+ mice. Treatment with CO significantly (p<0.05) increases the percent of cells in S-phase in HO-1−/− mice but not HO-1+/+ mice. Reactive oxygen species (ROS) production in lineage−, c-kit+, Sca-1+ (KLS) cell population was assessed using 5-(and 6-)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate dye. HO-1−/− mice have a significantly (p<0.001) increased proportion of ROS positive KLS cells compared with HO-1+/+ mice. Analysis of long-term (LT), short-term, and multipotent (MPP) progenitor cell populations was conducted. Treatment with CO significantly (p<0.05) increases the percent of LT-HSC and MPP progenitor cells in HO-1−/− mice but not HO-1+/+ mice. Concordantly, the total white blood cell count of the CO-treated mice increased significantly. The differential of the mature blood cells demonstrates significant shift in cell maturation, with significant increase in red blood cells, platelets, and lymphocytes and a significant decrease in monocytes. Combined this data indicates that CO therapy is able to modify the hematopoietic potential of HO-1−/− mice leading to a change in mature cell populations. We propose a model in which CO-mediated signaling initiates a homeostatic conditioning program in stem cells to balance hematopoiesis and stem cell preservation, ultimately leading to a change in the inflammatory milieu of the mice.

Disclosures:

Belcher: Sangart, Inc: Research Funding. Vercellotti:Sangart, Inc: Research Funding.
Regulatory Networks Governed by MicroRNAs in T-ALL Oncogenesis and Normal T-Cell Development
Authors: Van der Meulen Joni , Mets Evelien , Mestdagh Pieter , Pipelers Peter , Taghon Tom , Trujillo David Camacho , Avran David , Van Peer Gert , Van Roy Nadine , Thas Olivier , Benoit Yves , Vandesompele Jo , Poppe Bruce , Van Vlierberghe Pieter , Rondou Pieter , Soulier Jean , Speleman Frank
Published: October 2019

Abstract 1366

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy of thymocytes affecting children, adolescents and adults. T-ALL is characterized by a differentiation arrest at specific stages of T-cell development, primarily originating from the ectopic expression of T-cell oncogenes. Based on the mutation status of these genes and mRNA profiles, at least 5 different molecular-cytogenetic subgroups have been delineated. In addition, genetic abnormalities affecting cell cycle, proliferation, survival and self-renewal are present in all T-ALL subgroups. In a recent study, we discovered five oncogenic microRNAs (miRNAs) co-regulating the expression of key tumor suppressor genes implicated in the pathogenesis of T-ALL (Mavrakis et al., Nature Genetics, 2011). In this study, we established the extended miRNAome (756 miRNAs) in a genetically well characterized T-ALL patient cohort (n=65), 20 T-ALL cell lines as well as 9 different subsets of sorted T-cell populations from 3 human donors in parallel to mRNA and gene copy number profiles thus allowing integrative data mining. Cross-comparison between the different datasets resulted in the identification of miRNAs with presumed function in normal T-cell development as well as novel candidate miRNAs in T-ALL pathogenesis. The candidates were validated in an independent patient series (n=50). To better understand our previously established miRNA-mRNA regulatory T-cell/T-ALL network, we performed miRNA-mRNA correlation analysis followed by gene set enrichment analysis in order to assign putative functions to the selected miRNAs (http://www.mirnabodymap.org/; Mestdagh et al., Nucleic Acids Research, in press). In addition, the regulatory network was consolidated and extended through identification of miRNAs targeting selected key T-ALL oncogenes and tumor suppressor genes using a robust high throughput 3'UTR screening assay. In conclusion, this study further details the regulatory networks controlling normal T-cell development and T-ALL oncogenesis.

Disclosures:

No relevant conflicts of interest to declare.
Significance of Quantification of the PML-RARa Transcript in Children with Acute Promyelocytic Leukemia: A Single Center Experience
Authors: Zhang Li , Cao Zeng , Zou Yao , Ruan Min , Li Qinghua , Wang Jianxiang , Zhu Xiaofan
Published: October 2019

Abstract 1474

Objective:

Minimal residual disease (MRD) monitoring based on the detection of PML/RARa transcripts employing PCR technology has clearly demonstrated its benefit in the diagnosis and follow-up of acute promyelocytic leukemia (APL) patients. So far, real-time quantitative PCR (RQ-PCR) has been investigated to provide prognostic indexes for APL management in many adults studies. However, there are still no data on the use of such assays for child APL therapy. The aim of this study was conducted to clarify the relationship between the level of MRD and outcome in children with newly diagnosed PML/RARa-positive APL and identify the subgroups at low-risk of relapse.

Methods:

Since January 2004, we have analyzed 40 child patients treated with all-trans-retinoic acid (ATRA)±arsenic trioxide (ATO) in induction, with a median follow-up of 47 months. They were monitored by RQ-PCR. Hematologic and molecular relapses were recorded. We then looked for associations between relapse risk and RQ-PCR results in children.

Results:

The pretreatment characteristics of the 40 patients are listed in Table 1. Thirty-nine patients (97.5%) entered complete remission (CR). The 5-year probabilities of disease-free survival (DFS) and overall survival (OS) were 73.1% and 91.4%, respectively. By employing the standardized real-time quantitative polymerase chain reaction (RQ-PCR) for minimal residual disease (MRD) monitoring, no significant difference were observed in the PML/RARa normalized copy number (NCN) between patients in continuous complete remission and those who relapsed (neither at diagnosis nor after induction). After induction therapy, eight out of 25 cases with positive RQ-PCR (more than 1 NCN) relapsed in contrast to none out of 13 patients with negative RQ-PCR(100% and 55.2% DFS at 5 years in the negative and positive RQ-PCR groups, respectively; P=0.018, Fig.1). Also of note, in the positive RQ-PCR group, the patients treated with ATRA+ATO in induction had a lower relapse rate when compared with those treated with ATRA alone (P=0.03).

Conclusions:

PML/RARa - based MRD monitoring by RQ-PCR might allow us to identify subgroups of patients at low risk of relapse after induction in childen. Patients with a low risk of relapse could be monitored less frequently. Combination of ATO and ATRA might decrease the relapse rate compared with ATRA alone in induction therapy for childhood APL.

Disclosures:

No relevant conflicts of interest to declare.
Early Assessment of Minimal Residual Disease (MRD) by Qualitative PCR for NPM-ALK Identifies Patients At Very High Risk of Relapse in Anaplastic Large Cell Lymphoma
Authors: Damm-Welk Christine , Mussolin Lara , Pillon Marta , Zimmermann Martin , Reiter Alfred , Woessmann Wilhelm , Rosolen Angelo
Published: October 2019

Abstract 1565

25–35% of children and adolescents with ALK-positive anaplastic large cell lymphoma (ALCL) suffer a relapse. Detection of minimal disseminated disease (MDD) by qualitative RT-PCR for NPM-ALK in bone marrow (BM) or blood (PB) confers a relapse risk of 50%. We set out to address the issue of whether detection of minimal residual disease (MRD) by qualitative RT-PCR for NPM-ALK in BM or PB early during treatment allows identification of patients at highest risk of relapse.

ALCL-patients were treated according to protocols NHL-BFM95, LNH97 or ALCL99, all BFM-type protocols consisting of comparable cytoreductive prephase and chemotherapy courses between 8/1998 and 12/2008. The qualitative RT-PCR for NPM-ALK with a sensitivity of 10−5 was applied as previously described. PB and/or BM of 184 patients with NPM-ALK-positive ALCL were screened for the presence of NPM-ALK transcripts (MDD) at diagnosis. BM or PB was requested from those patients with detectable MDD in BM or PB for the measurement of MRD before the second course of chemotherapy (course 2).

MDD was detected in BM or PB or both media in 103 of 184 patients with NPM-ALK positive ALCL. The cumulative incidence of relapse (CIR) of 81 MDD-negative patients was 16±5% compared to 51±5% of 103 patients who were MDD-positive in either BM or PB or both (p<.001). MRD before course 2 could be evaluated in either BM or PB of 52 MDD-positive patients. CIR of the 26 MRD-positive patients (81±8%) was significantly higher than CIR of the 26 MDD-positive, but MRD-negative patients (31±9%) (p<.001). CIR of MDD-positive patients without material for MRD available was 35±7%. 5-year survival of MDD-negative patients and MDD-positive but MRD-negative patients were similar (92±2% and 92±5%, respectively) whereas survival of MRD-positive patients was 65±9% (p<.001).

Our data suggest that early evaluation of MRD by a sensitive RT-PCR in NPM-ALK positive ALCL with detectable MDD at diagnosis identifies patients with a very high risk of relapse.

Disclosures:

No relevant conflicts of interest to declare.
Fludarabine and Mitoxantrone Followed by Yttrium-90 Ibritumumab Tiuxetan in Untreated Patients with Follicular Lymphoma. Long Term Efficacy and Toxicity Results of the FLUMIZ Trial.
Authors: Zinzani Pier Luigi , Derenzini Enrico , Pellegrini Cinzia , Rigacci Luigi , Fabbri Alberto , Quirini Federica , Stefoni Vittorio , Gandolfi Letizia , Argnani Lisa , Casadei Beatrice , Broccoli Alessandro , Pulsoni Alessandro , Gobbi Marco , Perrotti Alessio , Zaccaria Alfonso , Voso Maria Teresa , Cabras Maria Giuseppina , Renzo Amalia De , Baccarani Michele
Published: October 2019

Abstract 1604

We previously reported the results of a multicenter non-randomized phase II trial of fludarabine and mitoxantrone plus radioimmunotherapy (RIT) [FLUMIZ (Fludarabine, Mitoxantrone, Zevalin) trial], demonstrating that this combination was safe and very effective in untreated patients with follicular non-Hodgkin lymphoma. We are now providing long term efficacy and toxicity results of this combination strategy.

Sixty-one patients with stage III and IV untreated follicular lymphoma were enrolled between June 2004 and April 2006, at 13 Italian institutions. Briefly, treatment schedule was the following: oral fludarabine 40 mg/m2 on days 1–3, intravenous mitoxantrone 10 mg/m2 on day 1 every 28 days for six cycles, followed by one course of yttrium-90 (90Y)-labelled ibritumumab tiuxetan (Zevalin), which consisted in two weekly infusions of Rituximab 250 mg/m2 followed by a weight based dose of 90Y-ibritumumab tiuxetan. Primary endpoints at the time of the first analysis were complete response and hematological toxic effects, secondary endpoints were overall survival (OS) and progression free survival (PFS). Fifty-seven patients were treated with RIT after the completion of six courses of fludarabine and mitoxantrone (FN) regimen. Four patients were excluded because of disease progression (n=1) and bone marrow infiltration > 25% (n=3) at the end of the FN regimen. Median follow up at the time of the last analysis was 52 months (range 24–75). Five-year PFS was estimated to be 68%, 5-year OS was estimated to be 93.0%. Noteworthy, late hematological side effects such as myelodisplastic syndromes or acute myeloid leukemias have not been observed so far. All patients had a complete hematological recovery after the completion of the sequential treatment. 16 patients relapsed during the follow-up period and 4 patients died due to disease progression. 22 patients (38%) are in first complete remission after more than 4 years of follow-up. All relapsed patients underwent second line chemotherapy and high dose chemotherapy with stem cell rescue was performed in 4 patients.

These results confirm the long term efficacy and safety of 6 cycles of fludarabine and mitoxantrone followed by consolidation with 90Y-ibritumumab tiuxetan: the 5-year PFS and OS compare favourably with the results of chemoimmunotherapy alone in untreated follicular lymphoma, with no increased incidence of secondary hematologic malignancie

Disclosures:

No relevant conflicts of interest to declare.
Inflammatory and Thrombotic Mediators in Small Cell Lung Carcinoma: Potential Role in Thromboembolic Complications
Authors: Hoppensteadt Debra , Khan Hussein , Thethi Indermohan , Demir Muzaffer , Adiguzel Cafer , Rahman Saud , Fareed Jawed
Published: October 2019

Abstract 2294

Introduction:

Small Cell Lung Carcinoma (SCLC) patients exhibit a higher prevalence of thromboembolic complications. We hypothesized that in this malignancy, procoagulant and inflammatory mediators contribute to the pathogenesis of such complications and warfarin treatment may down regulate these levels. Methods: In a prospective, randomized, controlled study, patients with inoperable lung cancer (n=100) were randomized to receive chemotherapy and radiation with and without warfarin (INR 1.5 – 2.5). Blood samples were drawn prior to and after the 2nd treatment cycle with warfarin or control and retrospectively analyzed for microparticles and thrombin generation markers such as fibrinopeptide A (FPA), thrombin-antithrombin complex (TAT) and prothrombin fragment F1.2 (F1.2). In addition, biochip array for C-reactive protein (CRP), D Dimer, neuron specific enolase (NSE), neutrophil gelatinase associated lipocalin (NGAL), tumor necrosis factor receptor 1 (TNFR1), and thrombomodulin (TM) were measured. The results were compared with a normal population (N=50).Results: The results are shown in the following table.

The microparticles were markedly increased in the SCLC patients (3 fold increase) at baseline. Similarly, the thrombin generation markers showed variable increase (1.5–3.2 fold increase). In the biochip array analysis, variable increase was noted. CRP (2.4 fold), D DIMER (11.6 fold), NSE (1.8 fold), NGAL (1.7 fold), TNFR1 (2 fold) and TM (1.3 fold) were all increased as compared to normal controls. All of the markers exhibited a decrease after warfarin treatment with a most pronounced decrease in the D Dimer and TNFR1.

Conclusions:

These results validate the hypothesis that SCLC patients exhibit a hypercoagulable state that is associated with simultaneous upregulation of inflammatory mediators. Warfarin treatment results in a down regulation of these mediators. Our results provide a rationale for prophylactic anticoagulant therapy in this group of patients with conventional and newer oral anticoagulant drugs.

Disclosures:

No relevant conflicts of interest to declare.
A Novel Role of Histone Deacetylase 11 (HDAC11) in Regulation of Myeloid-Derived Suppressor Cell (MDSC) Expansion
Authors: Sahakian Eva , Powers John , Rock-Klotz Jennifer , Adriani Marsilio , Woan Karrune V. , Merino Oscar , Gill Ajaypal S. , Deng Susan , Cheng Fengdong , Villagra Alejandro , Luetteke Noreen , Seto Edward , Sotomayor Eduardo M. , Borrello Ivan M. , Pinilla-Ibarz Javier
Published: October 2019

Abstract 2439

HDAC11 is the newest member of the HDAC family. The physiological role of this HDAC was largely unknown until the discovery by our group that HDAC11 regulates IL-10 gene expression in myeloid cells in-vitro1. To better elucidate the role of HDAC11 in these cells, we have utilized an HDAC11 promoter-driven eGFP reporter transgenic mice (TgHDAC11-eGFP) which allow us to “visualize” dynamic changes in HDAC11 gene expression /transcriptional activity in immune cells in vivo.

Immature myeloid cells (IMCs) differentiate into dendritic cells, macrophages, and neutrophils and are also considered to be precursors of MDSCs in tumor-bearing hosts. Here, we show for the first time that HDAC11 plays an important role in this process. First, IMCs from the bone marrow and spleen of TgHDAC11-eGFP mice display high expression of eGFP indicative of HDAC11 transcriptional activation in these cells in the steady state. Subcutaneous injection of PANCO2 tumor cells into these mice resulted in expansion of MDSCs (identified by the expression of CD11b+/GR1+ [Ly6G and Ly6C] with variable expression of CD49d and CD115) in their lymphoid organs which was similar in magnitude to the expansion observed in tumor-bearing wild type (WT) mice. Of note, flow cytometric analysis revealed that expression of eGFP was significantly decreased in the myeloid compartment of tumor bearing TgHDAC11-eGFP mice, suggesting that the transition of IMC into MDSCs might require a decrease in HDAC11 expression. Reminiscent of our findings in the eGFP mice, studies in non-transgenic mice also demonstrated that tumor derived CD11b+ Ly6G+ MDSCs display less HDAC11 mRNA expression. Additional support for the regulatory role of HDAC11 in MDSC expansion/function has been recently provided by our studies in HDAC11KO mice, demonstrating the acquisition of a suppressive cell phenotype, by myeloid cells identical to MDSCs, in the steady state and in the absence of tumor challenge.

Taken together, HDAC11 might function as a negative regulator of MDSC expansion/function in vivo. A better understanding of this previously unknown role of HDAC11 in MDSC biology might lead to targeted epigenetic therapies to influence the suppressive abilities of these cells and augment the efficacy of immunotherapeutic approaches against hematologic malignancies.

1. Villagra A, et al. Nat Immunol. 2009 Jan;10(1):92-100

Disclosures:

No relevant conflicts of interest to declare.
Expression and Immunomodulatory Function of RANKL in Leukemia
Authors: Schmiedel Benjamin J , Baessler Tina , Azuma Miyuki , Kanz Lothar , Salih Helmut R.
Published: October 2019

Abstract 245

The TNF family member RANKL and its receptors RANK and osteoprotegerin (OPG) are key regulators of bone remodelling, but have also been shown to influence progression of malignancies like breast cancer (Tan et al., Nature 2011), myeloma (Sordillo et al., Cancer 2003) and CLL (Secchiero et al. J Cell Physiol. 2006). NK cells are cytotoxic lymphocytes that play an important role in tumor immune surveillance especially of hematopoietic malignancies. Their reactivity is influenced by a variety of activating and inhibitory molecules expressed by their target cells including several members of the TNF family. Recently, we reported that RANK, upon interaction with RANKL which can be expressed by malignant hematopoietic cells, mediates signals that impair NK reactivity (Schmiedel et al., Blood 2010 116,21:893–893). Here we extended these analyses and comprehensively studied the expression and immunomodulatory function of RANKL in leukemia. Analysis of primary leukemia cells revealed substantial RANKL surface expression in a high proportion of the investigated cases (AML, 47 of 65 (72%); ALL, 16 of 21 (76%); CML, 6 of 10 (60%); CLL, all 54 (100%)). Signaling via surface-expressed RANKL into the malignant cells mediated the release of cytokines like TNF, IL-6, IL-8 and IL-10 which have been shown to act as autocrine and paracrine growth and survival factors in leukemia. Moreover, the factors released upon RANKL signaling upregulated RANK expression on NK cells. In line, NK cells from leukemia patients (n=75) displayed significantly (p<0.001, Mann-Whitney U-test) higher RANK expression compared to healthy controls (n=30) confirming our notion that RANK-RANKL interaction may contribute to leukemia pathophysiology. We further found that RANK-RANKL interaction, beyond directly inhibiting NK cell function via RANK, may contribute to evasion of leukemia cells from NK immunosurveillance by creating an NK inhibitory cytokine milieu. This was revealed by impaired cytotoxicity and degranulation in response to leukemia targets following exposure of the NK cells to the factors released upon RANKL signaling by leukemia cells. Notably, the RANKL-mediated cytokine release of leukemia cells could be disrupted by the clinically approved RANKL antibody Denosumab/AMG162. Thus, RANKL signaling may trigger a “vicious cycle” comprising of release of immunosuppressive cytokines and also upregulation of RANK on NK cells. The latter both directly inhibits NK reactivity and may result in augmented RANKL signaling into leukemia cells. Our data suggest that therapeutic modulation of the RANK/RANKL system e.g. with Denosumab/AMG162, which is approved for treatment of osteolysis, may be a promising strategy to reinforce NK reactivity against hematopoietic malignancies.

Disclosures:

No relevant conflicts of interest to declare.
Result of FDG PET Imaging After Chemotherapy +/− Immunotherapy Is a Significant and Independent Prognostic Indicator of Outcome for Patients with Follicular Lymphoma: Survey From a Single Institution
Authors: Magagnoli Massimo , Mazza Rita , Gandolfi Sara , Rodari Marcello , Giordano Laura , Anastasia Antonella , Lopci Egesta , Chiti Arturo , Santoro Armando , Balzarotti Monica
Published: October 2019

Abstract 2674

Aim

FDG positron emission tomography (PET) is the mainstay of response evaluation in some lymphoma subgroups such as DLBCL or HL according to Cheson criteria 2007. Due to its indolent behaviour, PET restaging has been poorly explored in Follicular Lymphoma (FL)

Methods:

The analysis was retrospectively conducted in pts with FL who underwent whole-body 18F-FDG-PET as part of response evaluation at the end of first line or savage treatment program at our institution from August 2001 to June 2010.

Results:

Seventy five patients were identified. Main clinical characteristics: median age 58 (range 26– 78); male 37 pts; B symptoms 6 pts; bulky disease 5 pts; stage IV 42 pts; bone marrow involvement 32 pts. Fifty-five pts were evaluated after first line treatment program, while 20 pts after salvage therapy program. Main chemotherapy regimen: CHOP or CHOP-like 38, CVP 11, polichemotherapy containing high-dose cytosine-arabinoside 11, fludarabine containing regimens 4. Furthermore, 19 pts underwent autologous peripheral stem-cell transplantation after either first line or salvage therapy. Eleven pts also received consolidation radiotherapy. Forty-two pts received Rituximab during chemotherapy. At the end of treatment, 54 pts reached complete remission as confirmed by PET. With a median follow-up of 53 months, a significantly lower actuarial 4yr PFS was observed in post-treatment PET+ versus. PET- patients: 35.1% vs. 74.8% (log rank p<0.01). Among all other prognostic factors analyzed (B-symptoms, bulky disease, chemotherapy regimens, chemotherapy vs chemoimmunotherapy, first line therapy vs salvage chemotherapy), only stage IV showed a correlation with PFS.

In a multivariate Cox model including all patients, post-treatment PET+ (HR 2.20; C.I. 95%: 1.02–4.76 p<0.04) and Stage IV (HR 3.08; C.I. 95%: 1.23–7.73 p<0.01) were unfavorable predictors for PFS.

Conclusion:

This retrospective study demonstrates that post-treatment negative PET is a powerful predictor for PFS. Patients who are PET- can expect a prolonged PFS either in first line or successive lines of treatment. Future clinical trials are needed to evaluate a PET oriented approach focused on improving outcomes according to PET response.

Disclosures:

No relevant conflicts of interest to declare.
B Cell Lymphoma, Unclassifiable: Survival Advantage for Aggressive Therapy
Authors: Cobain Erin , Ahmadi Tahamtan , Hoffmann Marc , Loren Alison W. , Frey Noelle V. , Svoboda Jakub , Luger Selina , Stadtmauer Edward A. , Nasta Sunita D.
Published: October 2019

Abstract 2689

Background:

Burkitt lymphoma (BL) and B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma (BLL) are mature B-cell non-Hodgkin lymphomas with an aggressive clinical course. Whereas the advent of short, intensive, multi-agent chemoimmunotherapy regimens has significantly improved the outcomes for BL patients (pts), questions remain regarding the optimal therapy for BLL.

Patients and Methods: We analyzed 25 patients (pts), aged 20 to 73 (median: 51 years), treated at the University of Pennsylvania for either BL or BLL from 2005 to 2010. All pts had c- myc+ lymphoma, with a Ki-67>98% and a negative HIV test. In order to be classified as BLL, expression of bcl-6 was required. Bcl-2 expression was noticed in 7/14 (50%) of BLL cases. All patients had Stage III/IV disease. All but two pts had high-risk disease by IPI. All 10 pts with BL were treated with the R-HyperCVAD regimen. Of the 14 pts with BLL, 7 where treated with R-HyperCVAD and 7 with R-CHOP (+ intrathecal prophylaxis in five out of seven pts). No differences in IPI, PS or co-morbidities were noted between the three groups. Notably, patients treated with R-CHOP were slightly younger with median of 52 years vs. median age of 62 years for those with BLL, treated with R-HyperCVAD.

Results:

Overall response rate (ORR) to R-HyperCVAD in BL was 70%, with two treatment-related deaths. At median follow up of 24 months, progression-free survival (PFS) was 72% (95% CI: 36–90%). ORR in BLL treated with R-HyperCVAD was 86% with one treatment-related death vs.57% when treated with R-CHOP, with two treatment-related deaths. At median follow up of 9 months for both groups, PFS for BLL treated with R-HyperCVAD was 54% (95% CI: 14–83%), whereas only one pt treated with R-CHOP was free of disease after 9 months with a median PFS of 8 months and median overall survival (OS) of 9 months. Within our data set, only one of the six pts with primary refractory disease and one of four relapses responded to salvage therapy. None of the BL/BLL pts treated with R-HyperCVAD, who achieved a CR, relapsed. Four of the seven relapses after R-CHOP presented with CNS involvement.

Conclusion:

Our data suggest that R-HyperCVAD allows for long-term disease control in both BL and BLL, with a somewhat lower PFS in BLL. In contrast, R-CHOP appears to be inferior with no long-term disease control in BLL.

Disclosures:

No relevant conflicts of interest to declare.
Gemcitabine, Dexamethasone and Cisplatin (GDP regimen) As First Salvage Treatment of Patients with Refractory or Relapsed Diffuse large B Cell Non Hodgkin Lymphoma (DLBCL)
Authors: Hamid Thoraya Mohamed Abdel , Ramadan Mona fawzy , Bahnassy Abeer , Taleb Fouad Abu- , Saber Magdy
Published: October 2019

Abstract 2696

Background:

Many chemotherapy regimens have been used for patients with refractory or relapsed DLBCL. No regimen has demonstrated superiority to another in this setting. Specific markers could predict the response to certain agents. Aim: to evaluate the response of GDP regimen in relapsed and refractory DLBCL patients and to assess ribonucleotide reductase subunit M1 (RRM1) as a possible predictor marker to Gemcitabine response. Patients and method: Patients with Relapsed or refractory DLBCL after one previous anthracycline-containing chemotherapy regimen were treated with the GDP regimen. RRM1 was assessed by immunohistochemistry in 55 cases and its expression was correlated to treatment outcome. Patients who could not proceed to stem cell transplantation (SCT) were followed for chemotherapy response and their results are presented. Results: The study included 70 patients with a median age of 40 years (range 18–73). At start of GDP, 19 patients (27.1%) were refractory and 51 (72.9%) were relapsed. After 4 cycles of treatment, 42 patients achieved CR, with a CR rate of 60% (95% CI: 53–68%). The median DFS was 6 months (95% CI: 5 to7 months), this DFS didn't include patients who underwent auto SCT. After a median follow-up of 20 months (range 6 to 30 months), the median OS of the patients who achieved CR was not reached, while those who didn't achieve CR had a median OS of 27.4 months (p= 0.01). Correlation between response and the pretreatment prognostic factors including IPI score or any of its elements, previous line of chemotherapy, time to relapse and status at time of salvage were studied with only significant difference in response to GDP between patients with refractory and those with relapsed disease (CR= 21.1% versus 60% respectively, p < 0.001). There was significant correlation between RRMI study results and response to GDP, 30/31 cases with low RRM1 expression achieved CR (96.8%), while only 7/24 cases with high expression achieved CR (29.2%), (p=0.001). No significant relation could be found between RRM1 expression and DFS. Conclusion: GDP regimen is active for patients with refractory or relapsed DLBCL however, the duration of response is short and high-dose therapy with SCT support is the reference postremission treatment. RRM1expression can predict response to Gemcitabine-based chemotherapy.

Disclosures:

No relevant conflicts of interest to declare.
Lenalidomide, Bendamustine, and Rituximab As First-Line Therapy for Patients > 65 Years with Mantle Cell Lymphoma: Results From the Phase I Portion of the Nordic Lymphoma Group MCL4 (LENA-BERIT) Trial
Authors: Jerkeman Mats , Kolstad Arne , Laurell Anna , Räty Riikka , Grønbæk Kirsten , Pedersen Lone Bredo , Geisler Christian H.
Published: October 2019

Abstract 2700

Background:

Mantle cell lymphoma is a disease of the elderly, with a median age of 70 years. Younger patients may be treated with potentially curative treatment including high dose chemotherapy. For elderly patients, however, no standard therapy has been defined. In a randomized comparison between R-CHOP and R-bendamustine (R-B) by the German StiL Group, R-B was associated with less toxicity and improved outcomes, making R-B a preferable first-line treatment option. Lenalidomide (LEN) is another active agent in MCL, with a response rate of 53% as a single agent in relapsed/refractory MCL. In the current trial, we investigate if the addition of lenalidomide to R-B may enhance efficacy, with manageable toxicity, for the older population of MCL patients.

Methods:

In phase I, the MTD of LEN was to be determined, starting with 5 mg/day increasing up to 25 mg/day in a sequential dose escalation using a 3+3 design. LEN, bendamustine and rituximab are given in 6 cycles/28 days. LEN D1-21, B 90 mg/m2 D1-2 and R 375 mg/m2 D1. The maintenance phase consists of LEN 25 mg/day, D1-21, for 7 cycles. Eligibility criteria are age > 65 years or ≤ 65 years, unable to tolerate high dose chemotherapy, with stage II-IV untreated mantle cell lymphoma.

Results:

The trial was commenced in October 2009.The phase I portion initially recruited 12 pts according to the original protocol design in 3 cohorts with LEN dose from 5–15 mg d 1–21. Median age was 72.5 years, range 66–85. MIPI high risk n=8, intermediate risk n=4. Response after 6 cycles: CR/CRu n=9/10, PR n=1/10 (ORR 100%). Molecular remission in BM: 5/9 pts. Toxicity was more profound than expected, mostly during cycle 1 (SAE n=9, AE Grade III/IV n=14). Notable was a high incidence of cutaneous and allergic AE. No patients could receive more than 10 cycles, median 6.5. A dose limiting toxicity (DLT) was noted at the dose of 15 mg. This led to a modification of the phase I protocol: Cohort A: No LEN in cycle 1, cycles 2–6: 10 mg LEN days 1–14. During maintenance, cycles 7–8: LEN 10 mg days 1–21, cycles 9–13: LEN 15 mg days 1–21. Cohort B: Same as Cohort A, but reducing B to 70 mg/m2 in cycles 2–6. Cohort C: as Cohort B, but reducing LEN to 5 mg days 1–14 cycles 2–6. In Cohort A, 2 of 6 patients experienced a DLT. Evaluation of Cohort B (6 pts) is ongoing.

Conclusions:

The addition of LEN to the R-B regimen leads to increased toxicity in elderly patients with MCL. Early data indicate a high response rate.

Disclosures:

No relevant conflicts of interest to declare.
The Pro-Metastasis Tyrosine Phosphatase, PRL-3 (PTP4A3), Is a Novel Target of BCR-ABL Signaling Involved in Human Chronic Myeloid Leukemia
Authors: Zhou Jianbiao , Cheong Lip-Lee , Mahara Sylvia , Liu Shaw-Cheng , Chong Phyllis SY , Bi Chonglei , Koh Tze-Loong , Ong Kelly OK , Zeng Qi , Chng Wee-Joo
Published: October 2019

Abstract 2741

Chronic myeloid leukemia (CML) is the best and most successful disease model for tyrosine kinase inhibitor (TKI). The mechanism of BCR-ABL leading transformation and signaling transduction networks have been intensively characterized over decades. However, resistance to TKIs remains a challenge in management of patients with CML. A better understanding BCR-ABL signaling network will lead to a better therapy.

Here we report the discovery of a novel downstream target of BCR-ABL signalling, PRL-3 (PTP4A3), an oncogenic tyrosine phosphatase. Analysis of CML cancer cell lines and CML patient samples reveals the upregulation of PRL-3. A search of Gene Expression Atlas (http://www.ebi.ac.uk/gxa/gene/ENSG00000184489) identified the expression level of PRL-3 was highest in CML among 950 human cancer cell lines crossing 32 different types of cancers (Dataset code: E-MTAB-37), suggesting a potential role of PRL-3 in CML pathogenesis. Inhibition of BCR-ABL signalling either by Imatinib or by RNAi silencing BRC-ABL in CML cell line K562, KCL-22 and primary patient samples reduces PRL-3, in parallel with suppression of signal transducer and activator of transcription (STAT) pathway activities and increased cleavage of PARP, a hallmark of apoptosis. In contrast, the amount of PRL-3 protein remains constant or even increased in response to Imatinib treatment in drug resistant cells expressing BaF3-P210 T315I. Finally, analysis with specific shRNA demonstrated K562-shPRL-3 (shP) cells proliferated as much as 2-time lesser than K562-shControl (shC) at day 8 (p < 0.001). Colony-forming efficiency is an indicator of self-renewal capacity of leukemic cell. K562-shP cells also showed significantly impaired colony generating capacity by 3-fold compared to K562-shC (p < 0.001). These results indicate a critical role for PRL-3 in CML cell expansion and self-renewal.

In summary, the present study demonstrates that PRL-3 is remarkably upregulated in human CML cell lines, BCR-ABL transformed cell lines and primary CML patient samples. Our results highlight that PRL-3 is a novel downstream target of BCR-ABL pathway, which is crucial for BRC-ABL-mediated cell survival and self-renewal. These data support a functionally important role of PRL-3 in CML biology downstream of BCR-ABL and maybe a viable therapeutic target in BCR-ABL positive cells even in those with Imatinib resistant mutations.

Disclosures:

Off Label Use: Imatinib will be used as a tool to dissect BCR-ABL signaling.