bluuebunny/crossref_metadata_2025 huggingface dataset:
Stem-Like Tumor Cells Confer Drug Resistant Properties to Mantle Cell Lymphoma
Authors: Jung Hyun Joo , McCarty Nami
Published: October 2019

Abstract 1840

Background:

We have recently identified clonogenic malignant stem cell populations in human mantle cell lymphoma (MCL), is a particularly deadly subtype of Non-Hodgkin's Lymphoma (NHL). We have discovered that CD45+CD19-MCL cells, which we termed MCL-initiating cells (MCL-ICs), are highly tumorigenic and display self-renewal capacities in vivo; in contrast, CD45+CD19+ MCL cells, which constitute the vast majority of cells within the tumors, show no self-renewal capacities and display greatly reduced tumorigenicity. Given the newly appreciated roles of cancer stem cells in the drug resistance of some cancers, it is critical to investigate whether MCL-ICs play roles in drug resistance of human MCL.

Methods:

CD45+CD19- and CD45+CD19+ cells isolated from MCL patients was tested for functional roles of MCL-ICs in drug-resistance of MCL using clinically-relevant chemotherapeutic regimens: R-CHOP, R-hyperCVAD, R-DHAP, R-HAD+B, and fludarabine-based regimen. We compared the cytotoxic effects and IC50 values of above regimens between two cell groups. To understand molecular mechanisms of drug resistance of MCL-ICs, we examined the cell cycle status of MCL-ICs using Rhodamine 123.

Results:

Cytotoxicity assay showed that MCL-ICs are more resistant to clinically relevant combined chemotherapeutic regimens in vitro as compared to bulk MCL tumor cells. Annexin V apoptosis assay supported this finding. IC50 values of chemotherapeutic agents that can induce the effective cytotoxicity of CD45+CD19-MCL-ICs were significantly higher than those of CD45+CD19+ MCL cells in most regimens. In contrast with CD45+CD19+ MCL cells, no significant growth inhibition was noted in CD45+CD19-MCL-ICs with combined drug treatments as well as a single agent. Rhodamine 123 efflux activity tests showed that MCL-ICs are maintained in a quiescent status.

Conclusions:

We have discovered MCL-ICs were more resistant to various clinically relevant chemotherapeutic agents in combination or in a single regimen compared to CD45+CD19+ MCL cells. The resistance of MCL-ICs to drugs was largely due to quiescent properties with enriched ABC drug transporters. In conclusion, designing novel therapies that can kill MCL-ICs may prevent relapse and increase patient survival.

Disclosures:

No relevant conflicts of interest to declare.
Clinical Experience with Azacitidine In Secondary Myelodysplastic Syndrome In Spain
Authors: García Regina , Sanz Guillermo F , Llorente Dunia de Miguel , Nomdedeu Benet , Romero M Mar , Bernal Teresa , Besalduch Juan , Amutio Elena , Brunet Salut , de Smd Grupo Andaluz
Published: October 2019

Abstract 1852

Introduction:

Secondary myelodysplastic syndromes (MDS) and acute leukemias are some of the most serious adverse side effects in the treatment with chemotherapy or radiotherapy, as those therapies affect the DNA methylation profile of different tissues. New data suggest that abnormalities in the DNA methylation profile would contribute to secondary leukemogenesis. In general, secondary MDS has a worse prognosis than de novo MDS. Azacitidine (AZA) is an hypomethylating agent approved in Europe for the treatment of MDS, based on the AZA-00 clinical trial. No secondary MDS was included in this trial. Until its approval in May 2009, AZA was used in Spain under compasionate use in clinical trials.

Materials and Methods:

Retrospective, longitudinal, multicenter Spanish study of data, promoted by the Hematology and Hematotherapy Andalusian Asociation and the Andalusian Group of MDS, on the course and management of patients treated with AZA under compasionate use. We present the analysis of clinical data of the 30 patients with Secondary MDS included in the registry.

Results:

Median age at diagnosed was 76 years. Male/female ratio: 13/17. ECOG performance status score 0–1 was 60%. The most frequently used initial dose of AZA was 75 mg/m2 in cycles of 28 days, administered subcutaneously (80%). The mean received cycles was 6. Overall response to treatment was 46.6% (International Working Group 2006 criteria): 16% complete response, 3.3% complete medullary response, 6.6% partial response and 20% hematological response. In addition, 23.3% had stable disease. AZA was generally tolerated well. The 3–4 grade adverse events documented in these patients were: Neutropenia (26%), thrombocytopenia (33%), anemia (20%), febrile neutropenia (3%), rash (6%), lumbar pain like colic type (3%) and subcutaneous reaction at the injection area (6%). Similar response ranges were found in patients with primary and secondary MDS, both within the registry as well as in the published literature.

Conclusion:

Our results show that patients with secondary MDS respond to AZA in the same manner as the primary patients do, although their prognosis is traditionally worse. These favorable results in secondary MDS suggest that these patients respond to the hypomethylating treatment, probably by the DNA methylation alterations involved in the leukemogenesis. More data from this registry and from different clinical trials are needed to confirm these data, and complete answers with survival analyses.

Disclosures:

García: Celgene : Research Funding.
Risk of Second Primary Malignancies in Patients with AL Amyloidosis Treated with Lenalidomide
Authors: Sanchorawala Vaishali , Shelton Anthony C , Doros Gheorghe , Seldin David C
Published: October 2019

Abstract 1873

An increase in second primary malignancies (SPM) has been reported in patients with myeloma treated with lenalidomide, particularly in association with melphalan. To determine the rate of second primary malignancies in AL amyloidosis patients treated with lenalidomide, we reviewed data on 82 patients treated on clinical trials at Boston Medical Center from 2004–2011. Data from lenalidomide and dexamethasone treatment in AL amyloidosis (ClinicalTrials.gov: NCT00091260) were analyzed in a post-hoc fashion for the incidence rate for SPM. The median age of these 82 patients was 62 years (range, 40–82); and 52 (63%) were male. There were 62 patients (76%) with lambda clonal plasma cell dyscrasia, and 43 (52%) had cardiac involvement by the consensus criteria from the Xth International Amyloidosis Symposium. Median duration for lenalidomide treatment was 9 cycles (range, 1–69); and 16 (20%) received lenalidomide for > 24 months. Of the 82 patients, 78 (95%) patients had prior therapies and 77 (94%) had prior alkylating agents based treatment. The median follow-up of the 82 patients was 28 months (range, 3–92). One patient, a smoker, treated with lenalidomide for 24 months, developed metastatic lung cancer. Additionally, there were 5 non-melanoma skin cancers. There was no case of hematologic malignancy or B-cell lymphoproliferative disorder in this group. This corresponds to an incidence rate of 0.44 per 100 patient-years for invasive cancer. The annual incidence of invasive cancers is 2.1 per 100 patient-years for the general population in the US. In conclusion, lenalidomide based treatment in AL amyloidosis did not appear to increase the incidence rate of SPM compared to incidence rate for the invasive cancers in general population. Longer prospective follow-up may be needed in a larger patient population to better define the risk.

Disclosures:

Sanchorawala: Celgene Corporation: Research Funding. Off Label Use: Use of lenalidomide in the treatment of AL amyloidosis. Seldin:Celgene Corporation: Research Funding.
DOCK2 Is Critical for FC to Induce Regulatory T Cells
Authors: Wen Yujie , Elliott Mary J , Huang Yiming , Corbin Deborah R , Miller Thomas O , Ildstad Suzanne T
Published: October 2019

Abstract 1885

CD8+/TCR− graft facilitating cells (FC) significantly enhance engraftment of hematopoietic stem cells (HSC) in both allogeneic and syngeneic recipients. We have shown that DOCK2−/− FC are compromised in cell migration in vitro and facilitating HSC engraftment in vivo. DOCK2 activates the small GTPase Rac and is indispensable for migration of neutrophils, lymphocytes and plasmacytoid dendritic cells (DC). A recent study demonstrated that DOCK2−/− DC were defective in antigen uptake and presentation. FC are a heterogeneous cell population, with a predominant subpopulation resembling plasmacytoid precursor DC. We previously found that FC induced antigen-specific regulatory T cells (Treg) in vitro and in vivo. In the present study, we evaluated whether DOCK2−/− FC could induce Treg. 5×105 wild-type (WT) sorted CD4+CD25− splenocyte T cells were incubated with 1×105 WT FC or DOCK2−/− FC at a 5:1 ratio in U bottom 96-well plates with long-term culture medium (IMEM with 20% horse serum, 10−6M hydrocortisone and 50μM β-mercaptoethanol) for up to 6 days. The co-cultures were harvested and CD4+CD25+ T cells were analyzed for FoxP3 by flow cytometry intracellular staining. WT CD4+CD25− splenocytes were cultured as negative control. Phagocytosis of APC-labeled polystyrene beads by WT and DOCK2−/− FC was assessed after incubating FC with the beads for 3 hours. We found that FC induced the generation of CD4+CD25+FoxP3+ Tregin vitro as expected, while DOCK2 deficiency completely abrogated the ability of FC to induce Treg generation. Moreover, the uptake of beads by DOCK2−/− FC was significantly impaired compared to their uptake by WT FC. In summary, DOCK2−/− FC were unable to induce the generation of CD4+CD25+FoxP3+ Tregin vitro and were compromised in phagocytosis.

Disclosures:

Ildstad: Regenerex, a biotech start-up company: Equity Ownership.
Abstract Title Submitted by Hollie Pegram to the 2012 ASH Annual Meeting: Expansion and Modification of Umbilical Cord Blood T Cells with a Chimeric Antigen Receptor and IL-12
Authors: Pegram Hollie J. , Barker Juliet , Giralt Sergio , Brentjens Renier J
Published: October 2019

Abstract 1907

Transplantation of unrelated umbilical cord blood (CB) derived stem cells is often used to treat adult patients with B-cell acute lymphoblastic leukemia (B-ALL). However, patients can relapse post transplant and prognosis of those with advanced or refractory disease is poor. We hypothesize that additional therapy involving adoptive transfer of CB derived T cells modified to express a CD19-specific chimeric antigen receptor (CAR) could improve patient outcome following CB transplantation in this population. We have previously demonstrated that human T cells which express the anti-CD19 1928z CAR, containing the signaling domains of the co-stimulatory CD28 receptor and CD3z chain, effectively eradicate CD19+ tumors in SCID-Beige mice, and have shown promise in early clinical trials.

In this study, we demonstrate the ability to effectively isolate and expand T cells from CB, necessary for application of adoptive CB T cell therapy. We compared T cell expansion in in vitro cultures with the addition of exogenous stimulatory cytokines, including IL-2, IL-7, IL-12, IL-15 or combinations thereof. We demonstrate that in vitro culture in the context of exogenous IL-12 combined with IL-15 resulted in optimal expansion of CB T cells (over 150-fold). In addition, expansion of T cells in the context of exogenous IL-12 and IL-15 resulted in a favorable phenotype, with maximal expression of memory T cell markers, CD62L and CCR7. Furthermore, these cells were shown to produce high levels of IFNγ and express high levels of CD107a following stimulation with PHA. CB T cells expanded in IL-12 and IL-15 were therefore shown to have a unique memory cell phenotype combined with effector T cell function.

Previous studies in our laboratory have demonstrated that expression of IL-12 in tumor targeted T cells resulted in increased anti-tumor function. Given these studies and the favorable effect of IL-12 on CB T cell expansion we used retroviral modification to express both 1928z CAR and IL-12 in CB T cells. CB T cells modified with both IL-12 and 1928z were shown to have increased cytokine secretion when cultured with CD19+ tumor cells, compared to UCB T cells modified with a CAR alone. Additionally, these cells were demonstrated to have increased lytic function, resulting in increased specific lysis of CD19+ tumor cells in a 51Cr release assay.

Our data suggest that CB T cells modified to express both CAR and IL-12 will have improved anti-tumor function, and could therefore decrease relapse and improve overall survival following CB transplant for the treatment of B-ALL.

Disclosures:

Giralt: Millenium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Onyx: Membership on an entity's Board of Directors or advisory committees, Research Funding.
Efficacy and Safety of Micafungin for the Prophylaxis of Invasive Fungal Disease During Neutropenia in Children and Adolescents Undergoing Allogeneic Hematopoietic Stem Cell Transplantation
Authors: Park Hyeon Jin , Park Meerim , Han Mira , Nam Byung Ho , Koh Kyung Nam , Im Ho Joon , Lee Jae Wook , Chung Nak Gyun , Cho Bin , Kim Hack Ki , Yoo Keon Hee , Koo Hong Hoe , Kang Hyoung Jin , Shin Hee Young , Ahn Hyo Seop , Lim Young Tak , Kook Hoon , Lyu Chuhl Joo , Hah Jeong Ok , Park Jun Eun , Seo Jong Jin
Published: October 2019

Abstract 1932

Introduction:

The use of micafungin, a member of the novel class of antifungal agents, the echinocandins, in adults has proven to be effective and safe for antifungal prophylaxis in allogeneic hematopoietic stem cell transplantation (HSCT) recipients. However, there are few reports describing its prophylactic use in pediatric patients. The objectives of this study were to evaluate the efficacy and safety of micafungin for the prevention of invasive fungal disease (IFD) in patients undergoing allogeneic HSCT exclusively focusing on children and adolescents.

Patients and Methods:

This was prospective, multi-center, open-label, single arm study. The study drug, micafungin, was administered intravenously at a dose of 1 mg/kg/day for patients < 50 kg and 50 mg/day for patients ≥50 kg from the beginning of conditioning until neutrophil engraftment. Treatment success was defined as the absence of suspected, probable, or proven IFD through the end of 4 weeks after therapy. Clinical and laboratory toxicities were graded according to the Common Terminology Criteria for Adverse Events version 4.0.

Results:

From April 2010 to December 2011, 152 patients were enrolled from 10 institutions in Korea, and a total of 144 patients were analyzed. The median age of the patients was 9.5 years (range, 0.4 – 19.8 years). Approximately 94% of patients received myeloablative conditioning regimen. Graft source included bone marrow (11.9%), peripheral blood (82.5%) and cord blood (5.6%). Most commonly, patients received HSCT for treatment of acute myeloid leukemia (27.8%), acute lymphocytic leukemia (27.1%), or severe aplastic anemia (19.4%). The median duration of prophylactic micafungin treatment was 23 days (range, 4 –169 days). Of the 144 patients, 117 patients completed micafungin prophylactic administration until neutrophil engraftment. Eleven patients developed suspected (n=5), possible (n=3), or probable (n=3) IFD. No patients died of IFD at any time during the study. Thirty-eight patients experienced adverse events (AEs) possibly related to micafungin. Only two patients discontinued micafungin prophylaxis due to AEs. Common AEs included hepatotoxicity, gastrointestinal discomfort, electrolyte imbalance and myelosuppression.

Conclusions:

This study shows the efficacy and safety of micafungin for the prevention of IFD after allogeneic HSCT in children and adolescents.

Disclosures:

No relevant conflicts of interest to declare.
Gemtuzumab Ozogamicin After Allogeneic Stem Cell Transplantation for Acute Leukemia
Authors: Kobayashi Takeshi , Shingai Naoki , Ikegawa Shuntaro , Takahashi Yukie , Aoki Jun , Yoshioka Kosuke , Mori Jinichi , Yasunaga Megumi , Umezawa Yoshio , Oshikawa Gaku , Doki Noriko , Kakihana Kazuhiko , Ohashi Kazuteru , Sakamaki Hisashi
Published: October 2019

Abstract 1970

Controversy abounds that anti-CD33 immunoconjugate, genutuzumab ozogamicin (GO) is really effective or not as a treatment for relapsed acute myeloid leukemia (AML). Consequently, GO is now commercially available in Japan, but not in USA or Europe. In this study, we have retrospectively analyzed the clinical impact of GO therapy as salvage or maintenance setting after allogeneic hematopoietic stem cell transplantation (allo-HSCT).

During last 5 years, GO was given to 19 patients with AML as salvage therapy for disease recurrence (n = 15 patients) or maintenance therapy (n = 4 patients) after allo-HSCT in our institution. Clinical characteristics of these 19 patients are summarized (see Table): Median age was 44 years (range, 21–70 years). GO was basically administered at a dose of 3 mg/m2. The median cycle of GO therapy was 3 cycles (range, 1–12 cycles) and 3 cycles (range, 1–4 cycles) as salvage and maintenance therapy, respectively. GO was administered at a median of 205 days after allo-HSCT (range, 38–3,111 days) in the setting of salvage therapy, while as maintenance therapy, patients with high risk AML received GO as early as 29 days after allo-HSCT (range, 24–71 days).

Two of 15 patients with recurrent disease achieved complete remission and 4 patients showed partial response (>50% decrease of bone marrow blast percentage). Thus, a total of 6 patients (40%) exhibited initial response to GO. However, 5 patients of them developed irreversible hepatic veno-occulusive disease (VOD) and eventually died at median of 146 days after GO therapy (range, 9–206 days). In view of 4 patients with maintenance therapy, 1 patient have faced to the subsequent disease relapse but no patients developed severe adverse effects including hepatic VOD and all patients are currently alive, albeit short follow-up. This small study demonstrates that GO offers an alternative tool for rescuing relapsed AML after allo-HSCT, but increases the risk of developing life-threatening hepatic VOD. Thus, further clarification is needed regarding which patients to treat with GO and at what dose of GO.

Disclosures:

No relevant conflicts of interest to declare.
Haploidentical Allogeneic Hematopoietic Stem Cell Transplantation Using the Donors with the HLA-B*5801-TNFα -308A Haplotype Produced Higher Frequency of Untoward Effects in Acute Lymphocytic Leukemia Patients
Authors: Yang Liyun , Chen Yan , Zhu Sainan , Bu Dingfang , Wang Lixiang , Jiao Xiaojie , Lu Xuechun , Liu Hongxing , Zhu Ping
Published: October 2019

Abstract 2002

In this study, we investigated the clinical characteristics of acute lymphoblastic leukemia (ALL) patients using sibling donors with HLA-B*5801-TNFα-308A haplotype (B5801-TNF2) for haploidentical or identical hematopoietic stem cell transplantation (HSCT). A total of 136 B-ALL cases and 29 T/NK-ALL cases were recruited. DNA samples from the patients and their siblings were assayed for genotyping of HLA and TNFα at -308 (rs1800629). B5801-TNF2 haplotype in donors was determined by analyzing TNFα -308 and HLA-B genotypes in patients and their family members. Outcomes within 2 years including overall mortality and non-relapse mortality, disease course and complications within 100 days were compared among patients using related donors with HLA-B*5801-TNFα-308A haplotype for haploidentical HSCT (B5801-TNF2+HID group), those using related donor without HLA-B*5801-TNFα-308A haplotype for haploidentical HSCT (B5801-TNF2-HID group), and those using related sibling either B5801-TNF2+ or B5801-TNF2- haplotype for identical HSCT (SID group). In the 165 sibling donors after HSCT, 35 were found to carry HLA-B*5801-TNFα-308A haplotype. There were 21 cases in B5801-TNF2+HID group, 100 cases in B5801-TNF2-HID group, and 44 cases in SID group. Compared patients in B5801-TNF2-HID and SID groups, patients in B5801-TNF2+HID group had higher overall mortality (adjusted P=0.039) and non-relapse mortality within 2 years (adjusted P=0.001), delayed platelet engraftment (adjusted P<0.0001), higher incidences of severe acute graft-versus-host disease (P=0.007), severe late onset hemorrhagic cystitis (P=0.002), blood stream infection (P=0.017), and invasive fungal disease (P=0.004) within 100 days. Therefore, sibling donors carrying HLA-B*5801-TNFα-308A haplotype for haploidentical HSCT caused higher mortality rate and higher frequency of severe complications in ALL recipients.

Keywords:

HLA-B*5801-TNFα-308A haplotype; acute lymphoblastic leukemia; allogeneic haploidentical hematopoietic stem cell transplantation; allogeneic identical hematopoietic stem cell transplantation

Disclosures:

No relevant conflicts of interest to declare.
Outcome of Stem Cell Transplantation in Adults with Acute Myelogenous Leukemia and Normal Cytogenetics (NC-AML) According to Presence of FLT-3 Internal Tandem Duplication (FLT-3ITD) At the Time of Diagnosis
Authors: Berman Ellin , Jakubowski Ann A , Maloy Molly , Jurcic Joseph G. , Jhanwar Suresh C. , Hedvat Cyrus V. , Giralt Sergio , Papadopoulos Esperanza B.
Published: October 2019

Abstract 2038

Background:

Cytogenetic abnormalities in leukemic blasts at time of diagnosis are the most important prognostic factors in acute myelogenous leukemia (AML). However, approximately 50% of patients (pts) with AML have normal cytogenetics (NC-AML). Within this population, the presence of somatic mutations further refines the prognosis. Mutations such as the internal tandem duplication of FLT-3 (FLT-3 ITD), and mutations in NPM1 and IDH1 have been all been shown to have significant prognostic value in determining response to chemotherapy and overall survival (OS). FLT-3 ITD is present in approximately 30% of pts with NC-AML and is considered the most significant predictor of poor prognosis following chemotherapy. However, it is not clear whether the presence of FLT-3 ITD influences the outcome of patients who undergo hematopoietic stem cell transplant (HSCT). We therefore performed a retrospective analysis of all adult patients with NC-AML and FLT-3 ITD present at time of diagnosis who underwent HSCT at MSKCC between 2007 and 2012.

Methods:

A total of 196 pts with AML underwent HSCT during this period. For this analysis, we excluded 112 pts with abnormal cytogenetics, 5 pts who had no cytogenetic testing performed, and 27 pts with normal cytogenetics who did not have molecular analysis performed.

Results:

A total of 52 pts with normal cytogenetics had molecular testing done; of these, 35 were FLT-3 negative (67%) and 17 were FLT-3 positive (33%). Characteristics of the 2 groups are shown below:

Conclusion:

Both groups had similar clinical and transplant characteristics, relapse rates, and OS (p=NS in all categories). We conclude that in this relatively small group of pts (1) HSCT appears to overcome the negative prognostic effect of FLT-3 ITD and (2) use of a T cell depleted graft does not influence transplant outcomes. A larger cooperative group analysis will be needed to confirm these findings.

Disclosures:

No relevant conflicts of interest to declare.
The Erythropoiesis Stimulating Agent Omontys/Peginesatide up-Modulates Erythroid Progenitor Formation, and Cell Surface Erythropoietin Receptor Levels.
Authors: Verma Rakesh , Green Jennifer M , Leu Karen , Mortensen Richard B , Young Peter R. , Schatz Peter J , Wojchowski Don M.
Published: October 2019

Abstract 2089

Peginesatide is a peptide-based erythropoietin receptor (EPOR) agonist with recent FDA approval for treating the anemia of chronic kidney disease among adult dialysis patients. Although peginesatide exhibits a 47.9h half-life when given IV in dialysis patients, it is administered once-monthly. Taken together, this predicts that additional functional properties contribute to peginesatide's durable erythropoiesis stimulating activity. Here we report on three such properties. During ex vivo murine bone marrow erythroid development, compared directly to rHuEPO, peginesatide first enhanced KitposCD71pos progenitor cell expansion (including KitposCD71lowCD36posCD13pos erythromyelo- progenitors). Second, peginesatide exhibited a 1300 minute EPOR residence time vs. 77 minutes for rHuEPO. Third, the culture of EPO-dependent human UT7epo cells in peginesatide led to substantial EPOR up-modulation, as well as an apparent lessening of the processing of mature EPOR's. Furthermore, in studies that compared peginesatide vs. rHuEPO effects on the erythroid development of human bone marrow-derived CD34pos progenitors, the following differences were observed. At days 6–10 of culture, peginesatide gave rise to increased frequencies (up to 200% as compared to rHuEPO) of KitposCD71pos co-positive erythroid progenitors. At early stages (d2-d4), cell-surface EPOR levels also were elevated among progenitors expanded in peginesatide. When levels of CD13posCD36pos co-positive cells were analyzed, frequencies of these erythromyelo-progenitors also were heightened up to three-fold. Analyses of cultures at later time-points (eg, d10 of culture) indicated that peginesatide and rHuEPO supported the formation of GPAhigh erythroblasts at similarly high frequencies. In addition, cytospin analyses revealed morphological distinctions for multicellular proerythroblast complexes formed in peginesatide. Peginesatide's persistent erythropoietic activity allowing for once-monthly dosing therefore is likely to involve novel effects on erythromyelo-progenitor recruitment, and increased EPOR cell surface expression including apparent increases in relative levels of full-length EPOR forms.

Disclosures:

Green: Affymax, Inc.: Employment. Leu:Affymax, Inc.: Employment. Mortensen:Affymax, Inc.: Employment. Young:Affymax, Inc.: Prior employment Other. Schatz:Affymax, Inc.: Employment. Wojchowski:Affymax, Inc.: Membership on an entity's Board of Directors or advisory committees.
Hydroxyurea up-Regulates Voltage-Dependent Anion Channels in Human Sickle Cell Erythrocytes.
Authors: Park Nick , Diaz Daniel , Nakagawa Peggy , Puthenveetil Geetha , Gershon Paul , Nugent Diane J
Published: October 2019

Abstract 2119

Background:

Hydroxyurea (HU) is clinically effective in reducing the frequency of pain crises in adults and children with sickle cell disease (SCD). In addition to increased fetal hemoglobin production, HU is known to induce macrocytosis in sickle cell erythrocytes and prolong red cell survival. However, the precise mechanism by which HU produces its varied effects is unknown. Isoelectric focusing has been applied to characterize changes in the red cell membrane following exposure to HU, but this technique has limitations in its ability to identify differences in specific membrane proteins. Mass spectrometry (MS) provides another proteomic approach in analyzing red cell proteins in SCD.

Methods:

Red blood cells were obtained from patients with SCD on HU therapy. After multiple wash and lysis steps, ghost membranes were obtained following ultracentrifugation. The membrane samples were then analyzed via tandem mass spectrometry and western immunoblot assay.

Results:

In comparison to a normal control, patients with SCD on HU therapy were found to have significantly elevated levels of voltage-dependent anion channels (VDAC) by MS, using a false discovery rate of less than five percent. The application of a stringent threshold initially identified 346 protein candidates. Out of these 346 proteins, 125 contained multiple peptides which all passed quantitation data filters according to quality parameters. Within the set of 125 proteins, 4 proteins were significantly up-regulated (up to 50-fold) in patients with SCD on HU therapy. VDAC2 and VDAC3 are members of the eukaryotic mitochondrial porin family which form channels through the mitochondrial outer membrane allowing diffusion of small hydrophilic molecules. VDAC1 can be found in both the mitochondrial outer membrane and the plasma membrane, where it is involved in cell volume regulation and apoptosis.

Conclusions:

Up-regulation of VDAC proteins may play an important role in altering the intracellular osmotic composition of the sickle cell erythrocyte resulting in decreased sickling and improved red cell survival in patients with SCD on HU therapy.

Disclosures:

No relevant conflicts of interest to declare.
Correlations Between Pancreatic Iron Burden and Heart Iron Overload and Function by MRI in a Large Cohort of Thalassemia Major Patients.
Authors: Meloni Antonella , Restaino Gennaro , Missere Massimiliano , Positano Vincenzo , Romano Nicola , Serra Giuseppe , Biguzzi Rino , De Franceschi Lucia , Favilli Brunella , Sallustio Giuseppina , Lombardi Massimo , Pepe Alessia
Published: October 2019

Abstract 2123

Background.

The leading cause of death in thalassemia major patients remains the heart failure, but impairment of the endocrine and exocrine function of the pancreas is a common complication. Multiecho T2* Magnetic Resonance Imaging (MRI) is a well established technique for heart and liver iron overload assessment. Little is known about the relationship between myocardial iron burden and cardiac function with pancreatic siderosis.

Aim of our study was to investigate using quantitative MRI the correlation between heart iron overload and function with pancreatic siderosis in thalassemia major patients.

Methods.

We studied 147 thalassemia major patients (233 males, mean age 31±9 years) consecutively enrolled in the Myocardial Iron Overload in Thalassemia network where MRI (1.5 T) was performed using standardized and validated procedures. Myocardial iron overload was measured by T2* multislice multiecho technique. Biventricular function parameters were quantitatively evaluated by cine images. Pancreatic iron burden was measured using a T2* gradient-echo multiecho sequence. The images were analyzed using a previously validated, custom-written software (HIPPO-MIOT IFC-CNR®).

Results.

Significant positive correlations of the global pancreas T2* values were demonstrated for global heart T2* values (r=0.355, P<0.0001; Figure 1), number of segments with normal T2* (r=0.389, P<0.0001) and T2* values in the mid-ventricular septum (r=0.323, P<0.0001). Of the 136 patients with pancreatic iron overload, 45 (33.1%) had a pathological global heart T2* value. No patients without pancreatic iron overload had cardiac iron overload. A normal global pancreas T2* value showed a negative predictive value of 100% for cardiac iron.

Pancreatic T2* values were positively related with left ventricular (r=0.21, P=0.028; Figure 2A) and right ventricular (r=0.23, P=0.015; Figure 2B) ejection fractions. No significant correlation was found between pancreatic T2* values and biventricular volumes.

Conclusions.

Pancreatic iron overload is positively correlated to myocardial iron overload and negatively correlated to bi-ventricular systolic function.

Disclosures:

No relevant conflicts of interest to declare.
Induced Pluripotent Stem Cells From a Patient with Reticular Dysgenesis Recapitulate Defective Myelopoiesis in-Vitro: A Disease Model to Enhance Our Understanding of a Rare Disease.
Authors: Weinacht Katja G. , Felgentreff Kerstin , Devine Alex , Schambach Axel , Al-herz Waleed , Notarangelo Luigi Daniele
Published: October 2019

Abstract 2142

Reticular dysgenesis (RD) is one of the most severe forms of combined immunodeficiency characterized by severe congenital neutropenia (SCN) and impaired lymphocyte development. Origin of the neutropenia is thought to be an early differentiation arrest at the promyelocte stage. Recently the disease has been mapped to mutations in the gene encoding for Adenylate Kinase 2 (AK2). Adenylate kinase 2 is a phosphokinase implicated in mitochondrial energy homeostasis. The exact mechanism, however, how a defect in this ubiquitously expressed enzyme selectively manifests in defective granulopoiesis and lymphopoiesis remains poorly understood. A significant obstacle to the study of rare diseases like Reticular Dysgenesis has been the inadequacy of animal models and limited availability of patient specimens.

We have successfully generated induced pluripotent stem cells (iPSCs) from a patient with Reticular Dysgenesis and proven genetic defect in Adenylate Kinase 2 (AK2). Myeloid precursors derived from the AK2-deficient iPSCs morphologically mimic the myeloid maturation arrest seen in the bone marrow of patients with this condition (Figure 1). We are currently using this in-vitro model as a platform to study the mechanisms underlying the maturation arrest on a cellular and molecular level.

Summary: Here we report a pluripotent stem cell (iPSC)-based disease model for Reticular Dysgenesis. This model does not only accurately recapitulate the myeloid disease phenotype observed in vivo but enables us to study previously unknown disease pathology. This is an example how iPSC technology enhances our understanding of a rare disease that has thus far been limited by the availability of study material.

Disclosures:

No relevant conflicts of interest to declare.
An ω-6 Fatty Acid, Dgla, Prevents Platelet Activation and Thrombosis in Vivo.
Authors: Yeung Jennifer , Ikei Kenneth , Vesci Joanne , Holman Theodore R , Holinstat Michael
Published: October 2019

Abstract 2169

Platelets play a pivotal role in thrombotic events leading to clot formation and clot stability in vivo. Uncontrolled signaling events in the platelet can result in unwanted thrombosis, which may eventually lead to the development of myocardial infarction or stroke. Previously, we have shown that ex vivo treatment of human platelets with the ω-6 fatty acid dihomo-γ-linolenic acid (DGLA) or its eicosanoid derived from 12-LOX oxidation, 12-hydroxyeicosatrienoic acid (12(S)-HETrE), inhibits PAR and collagen-induced platelet aggregation, clot retraction, and GPIIbIIIa activation. Since early studies have shown dietary supplementation of fatty acids increase fatty acid incorporation into the platelet lipid membrane (Barre, DE Lipids 1992; 27(5): 315–320; Marry, MJ Prostaglandins Leukot Essent Fatty Acids 1997; 56(3):223–223), we postulated that altering fatty acid composition in the platelet through dietary supplementation in vivo may be a viable approach to inhibiting platelet function. Therefore, a longitudinal study of wild-type mice on normal chow compared to mice supplemented with high (.5 g/kg) or low (.13 g/kg) DGLA diets was conducted. Each set of mice (7–8 mice) was given the designated diet for a period of 1, 2, or 3 months. At each time point, tail bleeding times and ex vivo platelet function in PRP were performed. Tail bleeding times from mice on the high DGLA diet were significantly prolonged by more than 15 minutes. Further, a smaller but statistically significant delay in clotting time was observed in mice on the low DGLA diet compared with control mice. Additionally, ex vivo aggregation response to collagen (1 μg/mL to 20 μg/mL) and PAR4-AP (50 μM to 500 μM) in platelets from mice on the high DGLA diet showed significant shifts to the right in their ability to induce platelet aggregation compared with control mice suggesting these mice were protected against thrombosis. JON/A and P-selectin binding to the PRP of high and low DGLA were also significantly attenuated in response to PAR4-AP. This study, which evaluated the in vivo and ex vivo effects of DGLA on regulation of platelet reactivity, supports DGLA as a potent, endogenous anti-thrombotic agent. Understanding the mechanistic details by which DGLA protects against thrombosis and maintains hemostasis through its COX-1 and 12-LOX-dependent bioactive metabolites will help to identify the potential viability of this target for anti-platelet intervention.

Disclosures:

No relevant conflicts of interest to declare.
Protective Role of ADAMTS13 for Myocardium in Mouse Model of Experimental Myocardial Infarction.
Authors: Doi Masaaki , Matsui Hideto , Takeda Yukiji , Saito Yoshihiko , Takeda Maiko , Matsunari Yasunori , Nishio Kenji , Shima Midori , Banno Fumiaki , Akiyama Masashi , Kokame Koichi , Miyata Toshiyuki , Sugimoto Mitsuhiko
Published: October 2019

Abstract 2175

The metalloprotease ADAMTS13 regulates the size of von Willebrand factor (VWF) multimers, controlling excessive VWF functions and preventing thrombotic occlusion of microvasculature. We previously reported that ADAMTS13 deficiency aggravated the extent of brain ischemic stroke in a mouse model of middle cerebral arterial occlusion, suggesting the relevant role of ADAMTS13 in the pathophysiology of brain stroke (Fujioka, et al. Blood, 2010; 115: 1650). These results raised the possibility that the functional regulation of VWF by ADAMTS13 could also play a role in coronary ischemic events such as myocardial infarction. To address this issue, we have used a mouse model of experimental myocardial infarction. The left anterior descending coronary artery in mice was ligated at 2 mm downstream from the origin under thoracotomy with ventilator-assisted respiration, and the cardiac function was evaluated with M-mode echocardiography after 7 days of operation. We compared 20 wild-type (WT) mice and 20 Adamts13 −/− (KO) mice, all of which were 12–14 weeks of age, healthy and fertile. Significantly (p < 0.01) decreased ejection fraction (EF; 44.0±6.7%) and increased left ventricular end-diastolic diameter (LVDd; 4.68±0.69 mm) of KO mice, as compared to WT (EF; 62.7±13.0% and LVDd; 3.77±0.56 mm, respectively), revealed that cardiac functions were apparently more impaired in KO mice. In addition, these reduced cardiac functions observed in KO mice were improved to an extent comparable to those of WT mice by the bolus injection of recombinant human ADAMTS13 (rhADAM; 3 μg/mouse, n=20) just after the operation (KO mice + rhADAM, EF; 58.2.±9.9% and LVDd; 3.16±0.52 mm). Consistent with echocardiography data, histological studies demonstrated the significantly (p < 0.01) higher infarct ratio in myocardium of KO mice (WT; 37.3±18.4%, KO; 59.1±16.3%, KO + rhADAM; 33.7±24.4%). Our results indicate that ADAMTS13, as seen in the case of brain ischemic stroke, plays a protective role for myocardium in coronary artery ischemia, improving myocardial functions and the severity of heart failure. Proper functional regulation of VWF-dependent thrombotic or inflammatory responses by ADAMTS13 could reduce thrombotic occlusion of microvasculature including leukocyte plugging, contributing to better local microcirculation which is crucial for tissue or organ functions.

Disclosures:

No relevant conflicts of interest to declare.
ADAMTS13 Reduces Vascular Inflammation and Early Development of Atherosclerosis Via VWF-Dependent Mechanism.
Authors: Gandhi Chintan , Lentz Steven R. , Chauhan Anil K
Published: October 2019

Abstract 2178

Background and objective:

The metalloprotease ADAMTS13 prevents spontaneous thrombosis in the microvasculature by cleaving hyperactive ultra large von Willebrand factor (ULVWF) multimers into smaller and less active forms. Recently, we and others have demonstrated that ADAMTS13-deficiency aggravates vascular inflammation and early atherosclerosis in apolipoprotein E-deficient (ApoE−/−) mice fed a high-fat Western diet. Although VWF is the only known substrate for ADAMTS13, it is not known if the effects of ADAMTS13 on vascular inflammation and atherosclerosis are mediated through its proteolytic effects on VWF or possibly another ADAMTS13 substrate. In this study, we determined whether the exacerbated atherosclerosis observed in the Adamts13−/−/ApoE−/− mice is dependent or independent of VWF.

Model and methods:

ApoE−/−, Adamts13−/−/ApoE−/−, Adamts13−/−/Vwf−/−/ApoE−/− and Vwf−/−/ApoE−/− male mice were fed a high-fat Western diet (20% fat, 0.2% cholesterol) beginning at 6 weeks of age until they were sacrificed at 4 months. We compared the extent of atherosclerosis in the cross section area of the aortic sinus using the VerHoeffs/Van Gieson stain. Inflammatory cells (neutrophils and macrophages) in the aortic lesions were quantitated by immunohistochemistry.

Results:

Similar to previous published reports by us and others, we found that the mean lesion area in the aortic sinus of the Adamts13−/−/ApoE−/− mice were significantly larger (mean ± SEM: 26.6 % ± 1.9 %, P<0.01) compared with ApoE−/− mice (mean ± SEM: 20.4 % ± 1.5 %). Next, we quantitated macrophage and neutrophil infiltration into lesions in the aortic sinus by immunohistochemistry. We observed significantly increased macrophages (mac-3 positive) and neutrophils (Ly6 B.2 positive) recruitment in the aortic sinus of Adamts13−/−/ApoE−/− mice compared with ApoE−/− mice. The mean lesion area in the aortic sinus of Adamts13−/−/Vwf−/−/ApoE−/− mice (mean ± SEM: 15.2 % ± 1.5 %) were similar to Vwf−/−/ApoE−/− mice (mean ± SEM: 15.6 % ± 0.9 %), suggesting that the accelerated atherosclerosis observed in ADAMTS13-deficient mice is VWF-dependent. Finally, macrophages and neutrophils recruitment in the aortic sinus of Adamts13−/−/Vwf−/−/ApoE−/− mice were similar to Vwf−/−/ApoE−/− mice, suggesting that increased vascular inflammation observed in ADAMTS13-deficient mice is also VWF-dependent. Total cholesterol and triglyceride levels were similar among groups fed a high-fat Western diet.

Conclusion:

These findings reveal that VWF-deficiency abrogates accelerated early atherosclerosis in ADAMTS13-deficient mice, suggesting that VWF the only relevant substrate for ADAMTS13 in murine atherosclerosis.

Disclosures:

Lentz: Novo Nordisk A/S: Consultancy, Investigator Other.
Vascular Disrupting Combretastatins Impair Bone Marrow Endothelial Cells by Depolymerizing the Microtubule Cytoskeleton.
Authors: Bosse Raphael C , Rohatgi Sunil , Meacham Amy , Wise Elizabeth , Scott Edward W , Cogle Christopher R
Published: October 2019

Abstract 2183

Background:

The vascular disrupting combretastatins CA1P (OXi4503) and CA4P (Zybrestat) are known to target solid tumor neovasculature by altering endothelial cell morphology and occluding cancer neovessels. In prior work, we and others have demonstrated that combretastatins target acute myeloid leukemia (AML) cells by generating reactive oxygen species within the malignant myeloid cells. We are currently conducting a phase 1 clinical trial of OXi4503 in patients with AML (NCT01085656). However, the vascular targeting mechanisms by which combretastatins regress AML are ill defined. We hypothesized that combretastatins also target bone marrow endothelial cells (BMECs).

Methods:

BMECs were isolated and cultured from bone marrow of healthy subjects and patients with AML. The cells were treated with various doses of the combretastatins CA1 and CA4 for 24 and 48 hours. Cell viability was measured by XTT assay. BMEC function in response to combretastatin treatments was quantified using a modified in vitro scratch assay and 2D capillary formation using Matrigel. Analysis of microtubule cytoskeleton was performed by staining treated BMECs with anti-β-tubulin antibodies and fluorescence microscopy. To ascertain the effects of combretastatin treatment on the ability of BMECs to support and protect AML cells, co-culture experiments were performed.

Results:

The combretastatins CA1 and CA4 did not decrease BMEC viability or metabolic activity. However, scratch assay showed impaired BMEC migration, with only 15.6% and 7.5% of the wound covered by BMECs after CA1 and CA4 respectively, compared to untreated control (80%) (p=0.0173). Matrigel assay also showed impaired BMEC function after combretastatin treatment, with complete lack of capillary tube formation compared to untreated BMECs which generated an average of 27.3 capillary tubes per 15 BMECs (p<0.001). Immunohistochemistry showed a distinct degradation of microtubule cytoskeleton in BMECs treated with CA1 and CA4. Finally, when treated with a vascular targeting combretastatin, AML cells co-cultured with BMECs showed a lower survival rate than AML cells alone (64.1% vs. 75.3%, p = 0.0267).

Conclusion:

Combretastatins directly impair BMEC function and eliminate BMEC protection of AML cells. The dual targeting of combretastatins on BMECs and AML cells leads to enhanced AML regression. These data highlight the role of the leukemia microenvironment as a protective reservoir of disease and provide methods of tracking biologic activity of combretastatins in early phase clinical studies.

Disclosures:

Cogle: OXiGENE: Research Funding; Leukemia & Lymphoma Society: Membership on an entity's Board of Directors or advisory committees, Research Funding.
Glycosylphosphatidylinositol Anchored TFPI As Main Regulator of Factor X Activation On the Surface of Endothelial Cells.
Authors: Dockal Michael , Pachlinger Robert , Baldin-Stoyanova Angelina , Knofl Fabian , Ullrich Nadja , Koehn Jadranka , Ehrlich Hartmut J. , Scheiflinger Friedrich
Published: October 2019

Abstract 2210

Tissue factor pathway inhibitor (TFPI) is a key regulator of factor X (FX) activation in the extrinsic pathway of blood coagulation. TFPI inhibits FXa generation by formation of a quaternary complex consisting of factor VIIa (FVIIa), tissue factor (TF), FXa and TFPI. The main portion (∼80%) of TFPI in humans is reportedly associated with endothelial cells. We used human umbilical vein endothelial cells (HUVECs) as a model to obtain further insight into the function of TFPIα and the glycosylphosphatidylinositol (GPI) anchored TFPI form, which represents TFPIα bound to GPI-anchored surface proteins and/or TFPIβ. In contrast to TFPIα, which consists of 3 Kunitz domains (KD) and a basic C-terminal part, GPI-anchored TFPIβ lacks the third Kunitz domain (KD3) and the basic C–terminal region due to alternative splicing. In TFPIβ these two domains are replaced by a sequence that adds a GPI anchor to the protein linking it to the cell membrane.

Treatment of HUVECs with phosphatidylinositol phospholipase C (PI-PLC) that cleaves GPI-anchors and subsequent fluorescence activated cell sorting (FACS) on living cells showed that GPI-anchored TFPI represents about 70–80% of cell surface TFPI. Staining of TFPI on and in fixed and permeabilized cells (total TFPI) demonstrated that GPI-anchored cell surface TFPI contributes to ∼20% of total cellular TFPI. Enzyme-linked immunosorbent assay (ELISA) showed that PI-PLC treatment released a TFPI protein lacking the KD3 and basic C-terminus. These findings strongly suggest that TFPIβ is the predominant GPI-anchored form of TFPI on HUVECs. FX activation assays performed on the cell surface of PI-PLC treated living HUVECs showed the importance of GPI-anchored TFPI on extrinsic Xase complex activity. PI-PLC treatment resulted in increased FX activation. Although GPI-anchored TFPI displays ∼70–80% of cell surface TFPI, overall FXa generation was increased only by ∼50%. In conclusion, HUVEC surface TFPI is predominantly TFPIβ, and GPI-anchored TFPI is the main but not sole regulator of FX activation on the surface of HUVECs.

Disclosures:

No relevant conflicts of interest to declare.