bluuebunny/crossref_metadata_2025 huggingface dataset:
Deletion of the 17p Chromosomal Region Is Associated with a Very Poor Outcome in Multiple Myeloma Independently of the Type of Treatment.
Authors: Avet-Loiseau Hervé , Leleu Xavier , Roussel Murielle , Mathiot Claire , Caillot Denis , Hulin Cyrille , Marit Gerald , Facon Thierry , Attal Michel , Harousseau Jean-Luc , Minvielle Stéphane , Campion Loic , Moreau Philippe
Published: October 2019

Abstract 1817

Poster Board I-843

Deletion of the short arm of chromosome 17 [del(17p)] is known to confer a poor prognosis in multiple myeloma (MM). However, no large study has been specifically dedicated to this purpose, especially in the novel drug era. We analyzed a series of 1324 patients with MM at diagnosis, treated within or according to IFM trials, and analyzed for del(17p). Most of the patients were under 65 years of age (1122 of the 1324 patients), and were treates either with a VAD-based induction or a Velcade®/Dexamethasone-based induction, followed by one or two courses of high-dose melphalan. Del(17p) was correlated to the major other prognostic parameters, both for event free survival and overall survival. Del(13) was observed in 71% of the patients with del(17p). A significant association was observed with anemia < 10g/dl (p=.05), with thrombocytopenia < 130 G/l (p<.0001), with hypercalcemia (p=.001), and b2-microglobulin>3.5 mg/l (p=.006). No specific association was observed with t(4;14) (17% of the del(17p)-positive patients displayed t(4;14)). Del(17p) was observed in 10% of the patients. Del(17p) was associated with a very poor outcome, both in young and elderly patients. Actually, the prognostic value was observed only in patients displaying del(17p) in at least 70% of their plasma cells. The median EFS and OS were 18 and 28 months respectively, versus 30 and 69 months for patients lacking the del(17p). A particularly poor outcome was observed in patients presenting both del17p and t(4;14), with a median EFS of 4.5 months and a median OS of 12 months. Of particular importance, none of the treatment modality (high-dose melphalan, thalidomide, bortezomib) overcame the poor prognosis associated with del(17p), although patients lacking del(17p) displayed a better outcome when treated with Vel/Dex or MP-Thalidomide. In conclusion, del(17p) is associated with an especially poor outcome, independently of the type of treatment, including novel drugs.

Disclosures

No relevant conflicts of interest to declare.
Mtor and PI3K Inhibitors Block Myeloma Cell Growth in a Synergistic Manner.
Authors: Guenther Andreas , Burger Renate , Klapper Wolfram , Tiemann Markus , Bakker Frank , Brocke-Heidrich Katja , Horn Friedemann , Gramatzki Martin
Published: October 2019

Abstract 1843

Poster Board I-869

Introduction: The mammalian target of rapamycin (mToR) plays a crucial role in cell growth due to its role as nutrient dependent regulator of important cytokine signaling pathways. In multiple myeloma, mToR is involved in the phosphoinositide-3-kinase (PI3K)-AKT pathway which can be activated by the loss of the tumor suppressor phosphatase and tensin homolog (PTEN) or by stimulation with growth and survival factors such as interleukin-6 (IL-6) and insulin-like growth factor-1 (IGF-1). Inhibitors of the mToR pathway (sirolimus/rapamycin, everolimus and temsirolimus) are approved for immunosuppression and/or cancer treatment. However, the clinical activity of mToR inhibitors may be limited by the fact that, after inhibition of the rapamycin-sensitive mToR-Raptor complex, AKT is activated by the rapamycin-insensitive mToR-Rictor complex. In this regard, the inhibitory effect of mToR inhibitors was evaluated in combination with PI3K inhibitors in vitro and in vivo. Results: Rapamycin and everolimus induced a dose-dependent growth inhibition in six human malignant plasma cell lines. Growth inhibition was mediated by G1 cell cycle arrest and in a subset of cell lines by induction of apoptosis. Overexpression of Bcl-XL or Mcl-1 proteins did not prevent from apoptosis induction by mToR inhibitors, nor did sensitivity to rapamycin-induced apoptosis correlate with the p53 mutation status. In the INA-6 SCID mouse xenograft model, treatment with rapamycin resulted in a significant survival benefit compared to untreated mice. Six out of 14 rapamycin treated mice did not develop plasmacytomas during the observation period of 149 days. Remarkably, short term treatment of plasmacytoma bearing mice led to a significant shrinkage of the plasma cell tumor. Explanted tissue showed apoptotic plasma cells, a finding confirmed by immunohistological staining using an antibody specific for the human cleaved form of poly (ADP-ribose) polymerase (PARP). The combination of rapamycin and the PI3K inhibitor Ly294002 led to an increase of growth inhibition in all tested plasma cell lines. The additional growth inhibition by Ly294002 appeared to be due to AKT activation upon mToR inhibition by the rapamycin-insensitive Rictor complex, indicated by increased AKT phosphorylation at Ser473 as determined by Western blot analysis. Conclusion: Clinical trials currently evaluate mToR inhibitors for their potential to expand treatment options for myeloma patients. The data presented here suggest that a combination of mToR inhibitors with PI3K inhibitors may lead to additive therapeutic chances.

Disclosures:

Guenther: Novartis: Consultancy, Research Funding. Gramatzki:Novartis: Consultancy, Research Funding, Speakers Bureau.
Low Dose Subcutaneous Alemtuzumab and Prophylactic Donor Lymphocyte Infusion Results in Favorable Outcomes for High Risk Recipients of Unrelated Donor Allogeneic Transplants.
Authors: Solomon Scott R. , Bashey Asad , Morris Lawrence E. , Manion Karen L , Holland H. Kent
Published: October 2019

Abstract 2272

Poster Board II-249

Non-relapse mortality (NRM) remains a major obstacle to success of unrelated donor (UD) allogeneic transplantation, mostly due to the increased morbidity from graft-versus-host disease (GVHD). Unfortunately, efforts to reduce GVHD mortality with ex vivo or in vivo T cell depletion techniques have often been associated with increased risk of relapse and infectious complications. We hypothesized that pre-transplant low-dose Alemtuzumab would reduce early NRM following UD transplantation, coupled with the early use of donor lymphocyte infusion (DLI) to correct incomplete donor chimerism (DC) to improve disease control. We targeted a high risk patient population (age>40 years or prior transplant) to test this hypothesis. Twenty-seven patients (median age 56 years) received PBSC (n=25) or BM (n=2) allografts from matched (n=25) or one-antigen mismatched (n=2) unrelated donors for high-risk hematologic malignancies (CIBMTR disease risk high [n=14], intermediate [n=6], low [n=7]). Conditioning included Fludarabine and Busulfan (myeloid) or Cyclophosphamide ± Rituximab (lymphoid). Subcutaneous Alemtuzumab was administered at a dose of 43mg over 3 days, day -11, -10, and -9. Twenty-six patients (96%) achieved sustained engraftment. With a median follow-up of 1.6 years, the estimated 2-year overall survival is 73.5%. NRM at 100 days and 2 years is 0% and 4.2%, respectively. Twenty-two patients received early prophylactic DLI for incomplete DC, while 7 received ≥2 DLI. Risk of Grade II-IV and III-IV acute GVHD is estimated to be 48% and 11%, respectively, with chronic GVHD occurring in 78%. Relapse/progression was documented in 41.9% of patients with current disease-free survival (DFS) estimated to be 65.2%. Compared to contemporaneously treated UD transplant recipients not receiving pre-transplant Alemtuzumab, study patients demonstrated delayed achievement of complete DC (median 148 vs. 61 days, p=0.002), delayed acute GVHD onset (median 113 vs. 30 days, p=001), and increased requirement for DLI (81% vs. 10%, p<0.001). Overall rates of acute and chronic GVHD were similar. Despite being a decade older in age (median 56 vs. 44 years), study patients had comparable outcomes with a non-significant trend to lower 2-year NRM (4.2 vs. 21.5%) and higher current DFS (65.2 vs. 46.8%) (see figure). Low dose subcutaneous Alemtuzumab with prophylactic DLI results in favorable transplant outcomes and modest rates of NRM in an older patient population with high-risk hematologic malignancies receiving UD transplantation.

Disclosures:

Off Label Use: Alemtuzumab is being used off-label as part of the preparative regimen for unrelated donor allogeneic transplantation..
Visualizing the Molecular and Cellular Basis of Heparin Induced Thrombocytopenia.
Authors: Rauova Lubica , Hirsch Jessica , Greene Teshell , Nagaswami Chandrasekaran , Cines Douglas B. , Weisel John W. , Poncz Mortimer
Published: October 2019

Abstract 228

Heparin-induced thrombocytopenia (HIT) is an iatrogenic complication of heparin therapy caused by antibodies that recognize the platelet chemokine, Platelet Factor 4 (PF4), complexed to heparin or to cellular glycosaminoglycans (GAG). The origin of the sentinel feature of HIT - high propensity for thrombosis in the setting of mild thrombocytopenia - remains undefined. We have shown previously that monocytes are particularly susceptible to activation by HIT antibody because unlike platelets, whose surface glycosaminoglycans (GAGs) consist almost entirely of chondroitin sulfates (CS), monocytes express heparan and dermatan sulfates that bind to PF4 with greater affinity. Therefore, antigenic complexes form at lower concentrations of PF4 on monocytes than on platelets and are more resistant to removal by exogenous heparin. We now provide 2 lines of visual evidence in support of this model of the prothrombotic state in HIT: 1) We demonstrate by scanning electron microscopy that spheroidal particles indicative of PF4/GAG complexes are present on the surface of human monocytes upon addition of PF4. 2) Thrombus formation has been visualized in a previously described murine HIT model involving the infusion of the HIT-like monoclonal antibody KKO into mice double-transgenic for human PF4 (hPF4+) and FcγRIIA receptor (FcγRIIA+) on their platelets. After KKO infusion, hPF4+/FcγRIIA+ mice developed vigorous and concurrent accumulation of HIT antigenic complexes and fibrin at sites of laser-induced endothelial injury, not present in similarly treated single transgenic mice. Accumulation of PF4 and fibrin at sites of injury was not increased by a polyclonal anti-hPF4 antibody that does not induce thrombocytopenia in hPF4+/FcγRIIA+ mice. Monocytes and/or monocyte microparticles accumulated at sites of laser-induced injury in KKO-infused hPF4+/FcγRIIA+ mice, but not in KKO-infused single transgenic mice or hPF4+/FcγRIIA+ mice infused with an isotype control, beginning 2.5 min after formation of HIT antigenic complexes and fibrin deposition, accumulating preferentially at the upstream end of developing thrombi. These results provide new insights into the potential role of monocytes in the prothrombotic sequelae of HIT. Antigenic complexes form on monocytes exposed to PF4 and accumulate in HIT antibody-induced thrombi. Monocytes appear to reinforce thrombus formation initiated by injured endothelial cells and activated platelets.

Disclosures:

Poncz: Diagnostica Stago: Patents & Royalties.
Non T-Depleted Bone Marrow Transplantation From Haploidentical Related Donor in Hematological Malignancies.
Authors: Santarone Stella , De Angelis Gottardo , Di Bartolomeo Erminia , Bavaro Pasqua , Montanari Mauro , Leoni Pietro , Cerretti Raffaella , Di Bartolomeo Paolo , Arcese William
Published: October 2019

Abstract 2291

Poster Board II-268

Haploidentical bone marrow transplantation (BMT) is an alternative treatment to patients with high-risk hematologic malignancy lacking a HLA-matched donor and those urgently need transplantation. We used a haploidentical-BMT protocol without ex vivo T cell depleted based on the knowledge that marrow grafts have 10 times fewer lymphocytes compared to peripheral blood stem cell grafts and granulocyte colony-stimulating factor (G-CSF) donor priming reduce the incidence of acute GvHD.

Materials and Methods:

40 patients (median age of 32, 12-63) with advanced disease or leukemia with poor prognostic features underwent unmanipulated haplo-BMT: 22 with AML, 9 with ALL, 3 with CML, 3 with Hodgkin lymphoma and 3 with plasmacell leukemia. Status at disease: 22 early (first or second comple! te remission), 18 advanced (progressive or refractory disease). All pairs of donors and recipients were identical for one HLA haplotype and incompatible at 2 or 3 loci.The myeloablative conditioning regimens used were different; antithymocyte globuline, cyclosporine, metotrexate, mycophenolate mofetil and basiliximab were used for GvHD prophylaxis. Donors were primed with filgrastim at 4 micrograms/Kg/d for 7 consecutive days. Bone marrow cells were harvest on the 8 day and were infused unmanipulated.

Results:

the median dose of total nucleated, CD34+ and CD3+cells was 7×10e8/Kg (1.01-28.7), 2.3×10e6/Kg (1.17-6.0) and 23.3×10e6/Kg (9.7-66.6) respectively. 1 patients had a primary graft failure and 5 patients died early prior to engraftment. In the remaining 34 patients, engraftment was seen with median time to granulocyte and platelet recovery of 22 and 27 days respectively; acute GvHD was grade 0 in 17 patients (50%), grade I in 9 (26%), grade II in 7 (20%) and grade IV in 1 (3%). In 29 evaluable patients, chronic GvHD was limited in 3 (10%) and extensive in 1 (3%). Transplant-related mortality at 6 months for early and advantage stage was 22% and 35% respectively. After a median follow up of 18 (3-42) months, 8 patients relapsed; 11 patients (50%) in the early stage and 4 (22%) in advanced phase are now living in haematological remission. The 1-year Kaplan-Meyer probability of disease-free survival is 45% for all patients.

Conclusion:

the high engraftment rate, low incidence of grade II-IV acute GvHD and an acceptable TRM suggest that G-CSF-primed marrow grafting along with sequential immunosuppression could provide an excellent alternative for patients who lack matched donors.

Disclosures:

No relevant conflicts of interest to declare.
A Randomized Phase II Trial of High-Dose Melphalan, Ascorbic Acid and Arsenic Trioxide with or without Bortezomib in Multiple Myeloma.
Authors: Sharma Manish , Thall Peter , Wang Xuemei , Hosing Chitra , Mendoza Floralyn L , Han Eric , Wang Michael , Weber Donna , Shah Jatin , Alousi Amin , Anderlini Paolo , Kebriaei Partow , Khouri Issa F , Popat Uday , de Lima Marcos , Champlin Richard E , Giralt Sergio , Qazilbash Muzaffar
Published: October 2019

Abstract 2309

Poster Board II-286

Background

There is a role for novel preparative regimens in multiple myeloma to improve the outcome after high-dose chemotherapy and autologous hematopoietic stem cell transplantation (auto HCT). Bortezomib is an active agent in newly diagnosed or relapsed multiple myeloma, and has synergistic activity with melphalan. We conducted a randomized phase II trial to determine the safety and efficacy of adding bortezomib to a preparative regimen of arsenic trioxide (ATO), ascorbic acid (AA) and melphalan.

Methods

auto HCT, with preparative regimen melphalan 100 mg/m2 IV on days -4 and -3, AA 1000 mg/day IV on days -9 to -3 and ATO 0.25 mg/kg IV on days -9 to -3. Patients were randomized to 3 arms; no bortezomib (arm 1), bortezomib 1 mg/m2 on days -9, -6 and -3(arm 2), and bortezomib 1.5 mg/m2 on days -9, -6 and -3 (arm 3). Primary endpoints were complete response (CR), NCI grade 4 toxicity, and overall survival (OS).

Results

In arms 1, 2 and 3, median intervals between diagnosis and auto HCT were 12.2, 9.6 and 8.8 months; median follow up in all surviving patients was 20 months (range 10 to 31). CR+near CR rates in arms 1, 2 and 3 were 35%, 30% and 25%. Grade 3–4 toxicity was seen in 6 patients in arm 1 (mucositis 3, dyspnea 1, acute renal failure 1, pleural effusion 1), 6 patients in arm 2 (mucositis 3, diarrhea 1, pneumonia 1 and hydronephrosis 1) and 6 patients in arm 3 (pulmonary edema 2, mucositis 1, intestinal obstruction 1, low back pain 1, elevated transaminases 1). The most common adverse events were nausea, diarrhea and pedal edema. Grade 1–2 weight gain due to fluid retention was seen in 84%, 70% and 95% of patients in arms 1, 2 and 3. Median time to neutrophil engraftment (ANC >500/dl) was 10 days in each arm. Median OS has not been reached in any of the 3 arms. Median progression-free survival (PFS) times were18.6, 13.2 and 17.5 months. OS was significantly shorter in patients with relapsed disease (0.00001) and cytogenetic abnormalities at auto HCT (0.0002).

Conclusions

Adding bortezomib to a preparative regimen of ATO, AA and high dose melphalan is safe and well tolerated in patients with multiple myeloma. There was no significant impact of adding bortezomib at either dose on the CR rate, grade 3-4 toxicity or OS.

Disclosures:

Shah: Celgene, Amgen, Novartis, Elan: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Qazilbash:Cephalon: Speakers Bureau.
Status of Minimal Residual Disease Determines Outcome of Autologous HSCT in Adults with High-Risk Acute Lymphoblastic Leukemia.
Authors: Giebel Sebastian , Stella-Holowiecka Beata , Krawczyk-Kulis Malgorzata , Goekbuget Nicola , Hoelzer Dieter , Doubek Michael , Mayer Jiri , Piatkowska-Jakubas Beata , Skotnicki Aleksander , Dombret Herve , Ribera Josep-Maria , Piccaluga Pier Paolo , Czerw Tomasz , Sadus-Wojciechowska Maria , Kyrcz-Krzemien Slawomira , Holowiecki Jerzy
Published: October 2019

Abstract 2321

Poster Board II-298

The role of autologous hematopoietic stem cell transplantation (autoHSCT) in the treatment of adult acute lymphoblastic leukemia (ALL) is a subject of controversies as several prospective studies failed to prove its advantage over maintenance chemotherapy. Those studies, however, did not take into account the status of minimal residual disease (MRD), which is now recognized a potent predictor for relapse among patients treated with conventional-dose chemotherapy. The goal of this analysis was to determine the impact of MRD on outcome of autoHSCT.

Data on 123 autoHSCT recipients collected from 6 study groups cooperating in the European Leukemia Net were analyzed. Median age of 77 B-lineage and 46 T-lineage high-risk ALL patients was 31 (16-59) years. Ph+ ALL was recognized in 20 cases. All patients were in first complete remission (CR) lasting 6 (1.5-22) months. Peripheral blood was used as a source of stem cells in 67 patients whereas bone marrow, in 56 cases. Conditioning was based on chemotherapy alone (n=76) or total body irradiation (n=47). MRD was evaluated in bone marrow with the use of either multiparametric flow cytometry (n=79) or molecular techniques (n=44). MRD level of 0.1% bone marrow cells was used as a cut-off point for the purpose of this study. At the time of autoHSCT MRD was &0.1% in 93 patients and ≧0.1% in 30 cases.

With the median follow up of 5 years, the probability of leukemia-free survival (LFS) at 5 years for the whole group equaled 48% (+/-5). Three patients died of transplantation-related complications. The LFS rate was significantly higher for patients with the MRD level at transplantation &0.1% compared to those with MRD ≧0.1% (57% vs. 19%, p=0.0002). The difference was particularly pronounced for peripheral blood HSCT (66% vs. 20%, p=0.0006) and for T-lineage ALL (62% vs. 8%, p=0.001). In a multivariate analysis adjusted for other potential prognostic factors (age, CR duration, Ph+ ALL, immunophenotype, source of stem cells, type of conditioning), the MRD status &0.1% remained the only independent factor associated with increased LFS (HR=2.5, p=0.0009).

CONCLUSIONS: MRD status is the most important predictor for LFS after autoHSCT in adults with ALL. More than half of patients with high risk disease and low MRD level at the time of transplantation may be cured. This observation may contribute to re-evaluation of the role of autoHSCT in the therapy of adult ALL.

Disclosures:

No relevant conflicts of interest to declare.
Rituximab Maintenance Following Chemoimmunotherapy Improves Outcome in B-Cell Chronic Lymphocytic Leukemia.
Authors: Del Poeta Giovanni , Del Principe Maria Ilaria , Ragusa Dario , Maurillo Luca , Buccisano Francesco , Ottaviani Licia , Giannì Laura , Luciano Fabrizio , Bulian Pietro , Rossi Francesca Maria , Venditti Adriano , de Fabritiis Paolo , Gattei Valter , Amadori Sergio
Published: October 2019

Abstract 2364

Poster Board II-341

The treatment goal of B-cell chronic lymphocytic leukemia (B-CLL) has already shifted from symptom palliation to the attainment of maximal disease control combining purine analogs with monoclonal antibodies. This immunochemotherapeutic approach resulted both in more complete responses (CR) and longer response duration, often remaining only a minimal residual disease (MRD) detectable by flow cytometry. We treated in first line 120 B-CLL symptomatic patients (pts), median age 62 years, with six monthly courses of intravenous or oral fludarabine at conventional doses and then, after a median time of 31 days, with four weekly doses (375 mg/sqm) of rituximab (rtx). Fourteen pts had a low Rai stage, 103 an intermediate stage and 3 a high stage. We defined as high risk pts having at least two of these markers: unmutated IgVH, CD38>30%, ZAP-70>20%, intermediate unfavorable cytogenetics (trisomy 12 or del11q or del17p). Based on NCI criteria, 92/120 (77%) pts achieved a CR, 24/120 (20%) a partial remission (PR) and 4/120 (3%) no response or progression. Ten pts underwent grade 3 (WHO) infective lung toxicity, 1 patient acute fatal B hepatitis and 2 pts progressed towards Richter's syndrome. Hematologic toxicity included mainly neutropenia (grade 3 and/or 4 in 56 pts) and thrombocytopenia (grade 3 and/or 4 in 8 pts). Fifty-four pts either in CR with B-CLL bone marrow cells >1% (MRD+, n=16 pts) or in CR MRD negative, but with B-CLL peripheral cells going up >1000/microl within 1 year after induction (n=22 pts) or in PR (n=16 pts), underwent consolidation and maintenance therapy with four monthly cycles of rtx at 375 mg/sqm followed by twelve monthly low doses of rtx (150 mg/sqm). The median follow-up duration was 50 months. All treated pts experienced a long progression-free survival from the end of induction treatment (40% at 9 years). On the other hand, global overall survival (OS) was 54% at 10 years. Nevertheless, CLL pts undergoing consolidation and maintenance therapy (n=54) showed a longer response duration vs MRD+ not consolidated pts (n=16; 75% vs 9% at 4 years; P<0.00001, Figure). Noteworthy, persistently MRD negative (>1 year) pts (n=43) showed a very long response duration (79% at 6 years, Figure). Moreover, OS was shorter in MRD+ not consolidated pts (0% vs 79% at 15 years; P=0.0007). Noteworthy, within the high risk subset (n=48), consolidated pts (n=17) showed a longer response duration (56% vs 0% at 2.5 years, P=0.003) vs MRD+ not consolidated pts (n=11). Therefore, rituximab consolidation and maintenance immunotherapy improve response duration in B-CLL, thus potentially increasing OS, also within the high risk subset.

Disclosures:

No relevant conflicts of interest to declare.
Dramatic Reduction of Chronic Lymphocytic Leukemia (CLL) Cells Following Adoptive Transfer of Cord Blood (CB) Natural Killer (NK) Cells Using CB-Engrafted NOD-SCID IL2Rγnull (NSG) Mice as a Model.
Authors: Xing Dongxia , Ramsay Alan G. , Decker William , Li Sufang , Robinson Simon , Yang Hong , Shah Nina , Hosing Chitra , Bollard Catherine M. , Gribben John G. , Shpall Elizabeth J.
Published: October 2019

Abstract 2370

Poster Board II-347

Adoptive transfer of NK cells in human B-CLL has been limited by the lack of suitable animal models to test the clinical efficacy of this immune therapy strategy. Primary patient B-CLL cells are difficult to engraft in NOD-SCID mice as these mice lack an immune microenvironment that provides essential accessory cells for tumor development. In the current study, we utilize a novel 2-step engraftment protocol using NOD-SCID γnull mice (NSG). Firstly, human cord blood (CB) derived CD34+ stem cells were engrafted to generate humanized chimeric mice capable of supporting B-CLL cells. By week 12, the human engraftment level reached 30% to 60% as detected in peripheral blood. Secondly, these mice were infused with human primary B-CLL cells labeled with CFSE. Our results show that following tail vein injection of 5E7 primary patient CLL cells, chimeric mice exhibited proliferation of CFSE+CD5+ B cells in the spleen and bone marrow, with disease development resembling human CLL. This allowed us to investigate the efficacy of ex vivo IL-2 expanded CB NK cell therapy using this novel mouse model system. As early as 24 hours post-infusion of IL-2 expanded human CD56+CD3- CB NK cells (10E7 per mouse) there was a detectable reduction of CD5+ leukemia cells in the peripheral circulation of CLL-engrafted mice by flow cytometry analysis. Moreover, by day 7 there was a dramatic 99.5 % reduction of CD5+ B-CLL cells in the blood, bone marrow, and spleen of experimental animals compared to the non-treated control group (P<0.001). Of note, expanded CB-NK cells exhibited high anti-leukemic specificity as healthy B cells from the original CB transplant were spared post-infusion. Overall, our studies suggest that the chimeric NSG mouse has utility as an in vivo model for testing immunotherapeutic strategies in aggressive B-CLL. Here, our results highlight the strong anti-leukemic response of infused ex vivo expanded CB-NK cells.

Disclosures:

No relevant conflicts of interest to declare.
Antitumor Activity of Small-Molecule SYK Inhibitor R788 and Fludarabinin Mono- and Combined Therapy in a Human B-CLL Xenograft Model.
Authors: Schüler Julia , Buchner Maike , Fiebig Heinz-Herbert , Veelken Hendrik , Zirlik Katja
Published: October 2019

Abstract 2378

Poster Board II-355

B cell chronic lymphocytic leukemia (B-CLL), one of the most common leukemia in adults, is characterized by the accumulation of mature B cells expressing CD19 and CD5. Improved understanding of CLL has lead to new prognostic tools and therapeutic options. The protein kinase Syk as a key mediator of proximal B-cell receptor (BCR) signalling is constitutively phosphorylated in CLL B cells. For these novel therapeutics, functional in vivo models are highly valuable. We have established a cell line-based, disseminated B-CLL model in NOD/SCID-IL2-receptor-gamma-chain-/- (NSG) mice. In the current study, SYK inhibitor R788 (60mg/kg/day (d); applied daily) was evaluated in mono- and in combined therapy with Fludarabin (8mg/kg/day; applied 5 consecutive days in two cycles) as a well established compound in B-CLL treatment regimens in comparison with a control group. Equal parts of MEC1 cells were injected intravenously and into the peritoneal cavity of NSG mice and respective therapies were started 7 days after implantation. Tumor growth was monitored with flow-cytometry (FACS), daily monitoring of B-CLL symptoms and fluorescence-based in vivo imaging (FI). Tumor inhibition was calculated as the proportional reduction of mean B-CLL cell infiltration at the respective compartment of the test- compared to the control-group (in %). MEC1 cells engrafted predominantly in bone marrow (BM; take rate = 100%), but were as well detectable in spleen (33%) and peripheral blood (PB; 8%). At the respective doses and schedules the examined compounds were well tolerated in tumor-bearing mice. No acute toxicity could be observed and maximal body weight loss was below 15% in mono- and combined therapy. Tumor development was clearly reduced by R788 (optimal T/C of 48% on day 18), albeit to a lesser extend then standard anticancer agent Fludarabin (optimal T/C of 19% on day 18). Combined therapy induced no synergistic effects showing an optimal T/C value of 42% on day 19. Thus, MEC1 engraftment into NSG mice is a valuable in vivo model for B-CLL which exhibits high reproducibility and take-rates in relevant compartments closely mimicking the clinical situation. Collection of whole-body FI data proved to be a time- and animal-saving analysis that allows to closely monitor B-CLL growth. Further investigations will optimize the very promising antitumor activity of R788 and evaluate the potentially synergistic effect of R788 with additional well-established B-CLL therapeutics.

Disclosures:

No relevant conflicts of interest to declare.
Oversulfated Chondroitin Sulfate Does Not Cause Augmentation in HIT Antibody Mediated Heparin-Induced Platelet Aggregation (HIPA).
Authors: Iqbal Omer , Plamoottil George , Sadeghi Nasir , Hoppensteadt Debra , Fareed Jawed
Published: October 2019

Abstract 2417

Poster Board II-394

Heparin is one of the widely used anticoagulants for the prophylaxis and treatment of thrombotic disorders. Heparin, a linear polysaccharide belonging to a family of glycosaminoglycans (GAGS) consists of repeating disaccharide units of 1-4 linked iduronic acid and glucosamine residues. Heparin is a mixture of low, medium and high molecular fractions and is heterogeneous not only in terms of molecular weight but also sites and degree of sulfation. Oversulfated chondroitin sulfate, one of the important toxic contaminants in certain batches of heparins manufactured during 2007 to 2008 resulted in deaths of some patients following intravenous administration that led to the recall of these heparins by the FDA. An increased incidence of heparin-induced thrombocytopenia (HIT) and thrombosis syndrome (HITTS) was reported as a result of contaminated heparins. It was hypothesized that OSCS interacts with heparins and other LMWHs increasing their HIT/HITTS potential. In order to validate this hypothesis blood was drawn from healthy volunteers (n=5) and mixed with 3.2% sodium citrate solution (9 parts of whole blood to 1 part of citrate). The citrated blood samples were centrifuged at 800 rpm to obtain platelet rich plasma (PRP) and further centrifuged at 3000 rpm to obtain platelet poor plasma (PPP). Apheresis fluid was collected from confirmed cases of HIT and pooled. The platelet aggregometers (Biodata) were blanked with individual PPP. The PRP (300ml) was mixed with HIT positive pooled apheresis fluid (200ml) and platelet aggregation was recorded using Gentium Heparin, Enoxaparin, and Bemiparin at a final concentration of 10mg/ml, either alone or in mixtures of 10%, 20% and 30% OSCS. The Heparin-induced platelet aggregations (HIPA) were run for a period of 30 minutes and the tracings obtained. While OSCS alone at a final concentration of 10 mg/ml caused an average of 29.5% platelet aggregation, it did not cause an augmentation of platelet aggregation in 10%, 20% and 30% mixtures with Heparin, Enoxaparin, Bemiparin and AVE 5026 (see table). This data suggests that drugs like heparin, and low molecular weight heparins such as Enoxaparin, and Bemiparin did not augment HIT/HITTS response when combined in various proportions with OSCS. Based on HIPA the reported increase in HIT response may therefore be due to in vivo interactions of the OSCS with platelets and endothelial cells. Corroborative evidence from clinical studies are warranted to validate these preliminary results.

Disclosures:

No relevant conflicts of interest to declare.
Improved Engraftment without Graft-Versus-Host Disease After MHC-Mismatched Cord Blood Transplantation with Photochemically Treated Donor Lymphocytes.
Authors: Kanathezhath Bindu , Mizokami Myra , Neumayr Lynne , Guo Hua , Walters Mark C. , Kuypers Frans A.
Published: October 2019

Abstract 2425

Poster Board II-402

Introduction:

Unrelated cord blood transplantation (CBT) is associated with a risk of graft rejection due in part to a limiting cellular content of the CB unit. Increasing the cellular content of the CB unit mitigates the graft rejection risk, but methods to use adjuvant immuno-modulatory cell co-infusions have also been tested with some success. We have investigated the co-infusion of photochemically (psoralen S59) treated mature donor T lymphocytes in a major histocompatibility complex (MHC) [H2-haplotype] mismatched murine transplant model as a new method to facilitate engraftment of donor CB cells.

Methods:

We analyzed the rates of donor hematopoietic cell engraftment, graft versus host disease (GVHD), and long-term survival in H2 haplotype disparate (C57BL/6®AKR) mice after CBT. Three different experimental groups were transplanted after sublethal radiation. Group 1 received allogeneic full term newborn peripheral blood alone, group 2 was transplanted with the same donor cells and unmanipulated donor T cells, and group 3 was transplanted with the similar donor cells and psoralen (S-59) treated donor T cells.

Results:

We observed a low rate of donor engraftment after transplantation with cord blood alone (Group 1). There was better engraftment but a high rate of fatal GVHD after transplantation with cord blood and unmodified donor T-cells (Group 2). The best results were observed after transplantation with 3 × 106 nucleated cord blood cells and 9 ×106 S-59 treated T cells (Group 3b). The engraftment rate was 75% compared to 12.5% after transplantation with 6 × 106 CB cells alone (p=0.04). The long-term survival in group 3 was 100% and the rate and severity of GVHD were minimal. Engraftment observed after CBT with unmodified donor T-cells (group 2) was accompanied by severe GVHD and poor survival. Donor myeloid, B cells and T cells were documented in the spleen and bone marrow of Group 3 mice by 30 days after CBT, although full hematological recovery was delayed until 50-60 days after CBT.

Conclusions:

These results show improved cord blood engraftment kinetics across a disparate H2 haplotype by adding psoralen-treated donor T lymphocytes. Co-transplantation of psoralen treated lymphocytes with CB cells facilitated durable engraftment of donor MHC high/c-kit+ cells in the marrow and splenic compartments with complete but delayed hematopoietic recovery. The low GVHD risk and excellent long-term survival observed in this murine model suggests the potential for clinical application.

Disclosures:

No relevant conflicts of interest to declare.
Safety of Excluding Suspected Deep Vein Thrombosis with a Single Whole-Leg Compression Ultrasound: Systematic Review and Meta-Analysis.
Authors: Johnson Stacy A , Stevens Scott M , Woller Scott C , Lake Erica , Donadini Marco , Douketis James D
Published: October 2019

Abstract 243

Background:

In patients with suspected lower extremity deep vein thrombosis (DVT), compression ultrasonography (CUS) is typically used as the initial test to confirm or exclude DVT. Patients with a negative CUS and either a moderate-to-high probability and/or a positive D-dimer usually require repeat CUS testing after 5–7 days to exclude proximal propagation of distal DVT, reducing diagnostic efficiency. Whole-leg compression ultrasound may safely exclude proximal and distal DVT in a single evaluation.

Purpose:

To assess the safety of withholding anticoagulation in patients presenting with suspected lower extremity DVT following a single negative whole-leg CUS.

Methods:

MEDLINE, EMBASE, CINAHL, LILACS, Cochrane, Health Technology Assessments databases were searched from January 1970 to April 2009 without language restrictions. This search was supplemented by reviewing Google, Google Scholar, clinicaltrials.gov, meeting abstracts, conference proceedings, reference lists, and by contacting content experts.

Study Selection and Data Extraction:

Randomized controlled trials and prospective cohort studies of patients with suspected DVT with a negative whole-leg CUS, not treated with anticoagulation, and followed at least 90 days for occurrence of venous thromboembolism (VTE). Studies required objective confirmation of VTE events during follow-up. Two authors independently reviewed articles and extracted data.

Results:

Six studies were included totaling 4,229 patients with negative whole-leg CUS exams and not receiving anticoagulation. VTE or suspected VTE-related death occurred within the follow-up period in 24 (0.6%) patients. Of these 24 events, 9 (37.5%) were distal DVT, 7 (29.2%) were proximal DVT, 6 (25.0%) were non-fatal pulmonary embolism, and 2 (8.3%) were deaths, possibly related to VTE. Combined VTE event rate at 3 months was 0.46% (95% CI 0.22, 0.70).

Limitations:

Pretest probability assessment was not available for all analyzed patients.

Conclusions:

Withholding anticoagulation in patients with suspected DVT based on a single whole-leg CUS is associated with a low risk of VTE (0.46%) during 3 months of follow-up. This strategy represents a safe and efficient alternative to serial CUS testing in patients with suspected DVT.

Disclosures:

No relevant conflicts of interest to declare.
Prevalence of Venous Thromboembolism Across Ethnic Groups in the United States.
Authors: Deitelzweig Steven B , Lin Jay , Johnson Barbara H , Schulman Kathy L.
Published: October 2019

Abstract 2492

Poster Board II-469

Objective:

Understanding the overall prevalence of VTE is paramount in estimating the burden of illness associated with this disease. This study aims to assess the number of VTE cases in the United States across ethnic groups based on recent data.

Methods:

Data from the Marketscan® Medicaid database from Thomson Reuters (Jan 2002–Dec 2005) were extracted for patients aged ≥18 years. Patients were evaluated for VTE in each year, defined by the presence of a VTE diagnosis on an inpatient claim or on ≥1 outpatient claim with evidence of anticoagulant administration. Age-, sex- and race-specific VTE prevalence rates were derived by dividing the number of VTE cases identified by the number of individuals in the underlying MarketScan populations during that time. These rates were then multiplied by 100,000 to obtain a VTE prevalence rate per 100,000.

Results:

In 2002, there were 276 individuals with evidence of VTE per 100,000 plan enrollees. In 2005, that number grew to 358, a 30% increase. African American males had the highest overall observed prevalence rate of 584 per 100,000 enrollees in 2002, growing to 785 in 2005 (Table). Caucasian males had the next highest observed prevalence at 457 per 100,000 enrollees in 2002, growing to 643 per 100,000 in 2005. Females generally had lower observed prevalence of VTE than males, although Hispanic females had similar prevalence's to Hispanic males (94 vs 93 in 2002, 149 vs 154 in 2005). Following multivariate adjustment for comorbidities and conditions known to be strong risk factors associated with developing VTE, African Americans were at significantly increased risk for VTE than Caucasians (Odds Ratio 1.04, 95% CI 1.00–1.07, p<0.05).

Conclusions:

VTE prevalence increased during the study period for the overall US Medicaid population. African Americans had the highest rate of VTE, followed by Caucasians and Hispanics. There is a need for improved VTE awareness and prevention across all ethnic groups.

Disclosures:

Deitelzweig: sanofi-aventis: Honoraria, Research Funding, Speakers Bureau, The authors received editorial/writing support in the preparation of this abstract funded by sanofi-aventis U.S., Inc.; Bristol-Myers Squibb: Honoraria, Research Funding, Speakers Bureau; Scios: Honoraria, Research Funding, Speakers Bureau; Pfizer: Speakers Bureau. Lin:sanofi-aventis: Employment. Johnson:sanofi-aventis: Research Funding. Schulman:sanofi-aventis: Research Funding.
Osteoclasts From Human Embryonic Stem Cell-Derived Hematopoietic Progenitors Support Development of An in Vitro Niche Microenvironment.
Authors: Islam Mohammad Saiful , Allred Jeremy R , Hill Katherine L , Hexum Melinda K , Kaufman Dan S.
Published: October 2019

Abstract 2517

Poster Board II-494

Osteoclasts are bone resorbing cells located in the bone marrow that play a key role in hematopoiesis and formation of the hematopoietic niche. Previous studies demonstrate osteoclasts arise from the monocyte-macrophage lineage of cells within the bone marrow microenvironment. Osteoclast progenitor cells typically express the monocyte marker CD14, though relatively little is known about the developmental history of these osteoclastogeneic cells in humans. Osteoclast progenitors have been previously isolated from peripheral blood and bone marrow. Previous studies by our group and others demonstrate human embryonic stem cells (hESCs) can differentiate into CD34+CD45+ cells that can be supported to differentiate into many types of hematopoietic cells, though development of osteoclasts have not been previously demonstrated. To better evaluate osteoclastogenesis, hESCs were allowed to differentiate on M210 stromal cells for 19–21 days then sorted for CD34+CD45+ cells. These cells were placed into a secondary co-culture with M210 stromal cells using osteoclast differentiation factors macrophage colony-stimulating factor (hM-CSF) and receptor activator of nuclear factor–kB ligand (hRANKL) for 14–21 days. During this time, mononuclear hematopoietic cells expanded on top of the stromal cells, followed by appearance of multinucleated cells that became adherent and embedded within the stromal cell layer. These adherent multi-nucleated cells were TRAP-positive, consistent with osteoclasts. Q-RT-PCR analysis demonstrated expression of osteoclast genes NFATC1, TRAP, CATHEPSIN K AND MMP-9. Culturing these putative osteoclasts on dentin discs demonstrated dentin resorption and pit formation, consistent with osteoclasts cultured from peripheral blood. No pit formation observed when the same experiments were done without hRANKL and hMCSF. Most remarkable was the extensive proliferation of CD45+CD33+ myeloid cells around the periphery of the adherent, multinucleated osteoclasts in culture, resembling a multi-cellular hematopoietic niche environment. Further studies are underway to better evaluate the interaction of these osteoclast cells with other hESC-derived hematopoietic cells. Together, these studies demonstrate development of functional osteoclasts directly from hESC-derived CD34+CD45+ hematopoietic progenitor population. We also show development of a novel in vitro HSC niche microenvironment utilizing hESC-derived osteoclasts. This system has the potential to now incorporate other hESC-derived cell populations as a model to better define cellular and extracellular mechanisms that mediate human hematopoietic development.

Disclosures:

No relevant conflicts of interest to declare.
Roles of Histone Acetyltransferase MORF and MOZ in Hematopoiesis.
Authors: Katsumoto Takuo , Kitabayashi Issay
Published: October 2019

Abstract 2525

Poster Board II-502

MOZ (MOnocytic leukemia Zinc finger protein) and MORF (MOz Related Factor), Myst-type histone acetyltransferases, are involved in chromosome translocations associated with FAB-M4/5 subtypes of acute myeloid leukemia. We have reported that MOZ is essential for hematopoietic cell development and self-renewal of hematopoietic stem cells. To explore the possibility MORF also plays important roles in hematopoiesis, we generated Morf-deficient mice with homologous recombination methods. Morf−/− mice were smaller than their wildtype littermates and died within 4 weeks after birth on C57BL/6 background. In MORF−/− fetal liver, Flt3-negative KSL (c-Kit+ Sca-1+ Lineage-) cells containing hematopoietic stem cells were decreased. When fetal liver cells were transplanted into irradiated recipient mice, MORF−/− cells less efficiently reconstituted hematopoiesis than wild-type cells. Additionally, bone marrow cells reconstituted with MORF−/− cells rarely contributed to hematopoiesis in secondary transplants. To reveal relationship between MORF and MOZ in hematopoiesis, we generated double heterozygous (Moz+/− Morf+/−) mouse. Double heterozygous mice were smaller than wild-type littermates and died at least 4 weeks after birth. Numbers of KSL cells, especially Flt3- KSL cells and common myeloid progenitors were decreased in the double heterozygous embryos. The double heterozygous fetal liver cells also displayed less activity to reconstitute hematopoiesis than MOZ+/− or MORF+/− cells. Since MORF−/− mice and MOZ/MORF double heterozygous mice were alive at adult on a mixed C57BL/6/DBA2 genetic background, we investigated adult hematopoiesis in these mice. MORF−/− or MOZ/MORF double heterozygous mice were smaller than their wild-type littermates and had small numbers of thymocytes and splenocytes. However, there were no significant differences in number of bone marrow cells and hematopoietic lineage population in MORF−/− or MOZ/MORF double heterozygous mice. These results suggest that MORF as well as MOZ plays important roles in self-renewal of hematopoietic stem cells.

Disclosures:

No relevant conflicts of interest to declare.
FGFR1 Is Required for and Elucidates Multiple Mechanisms of HSC Mobilization.
Authors: Ross Jason T.
Published: October 2019

Abstract 2532

Poster Board II-509

Though fibroblast growth factor receptor 1 (FGFR1) is known to be expressed in hematopoietic stem cells (HSCs), and FGF ligands can support their in vitro culture, the function of FGFR1 in HSCs in vivo has not been elucidated. We examined mouse models in which Fgfr1 is conditionally deleted in adult hematopoietic tissues. While they exhibit normal hematopoiesis, FGFR1-null HSCs fail to mobilize to peripheral tissues in response to 5-fluorouracil (5FU) and AMD3100; however, they mobilize at levels similar to control animals when treated with granulocyte-colony stimulating factor (G-CSF). Further investigation revealed that FGFR1-null HSCs cannot migrate in response to the CXCR4 ligand SDF-1 in vitro, and after 5FU treatment they display diminished expression of CXCR4 in bone marrow at time points prior to mobilization. Additionally, HSCs lacking FGFR1 cannot be expanded in culture, indicating they bear proliferation defects. Together, these data support a novel role for FGFR1 in mobilization of HSCs and indicate that activation of FGF signaling may distinguish stress-responsive HSC mobilization from more physiologic mechanisms triggered by cytokines such as G-CSF.

Disclosures:

No relevant conflicts of interest to declare.