bluuebunny/crossref_metadata_2025 huggingface dataset:
T Cell Metabolism and Memory
Authors: Rathmell Jeffrey C
Published: October 2019

Lymphocyte activation leads to rapid proliferation and differentiation and we have shown that CD4 T cell subsets are metabolically distinct. These specific metabolic programs may allow new understanding and approaches to manipulate immunity. Using metabolic network analysis of metabolomics and proteomics we defined several metabolic nodes differentially utilized by CD4 T cell subsets, including glutamine metabolism. By genetically deleting the glucose transporter Glut1 or the glutaminolysis enzyme, Glutaminase (GLS), we have shown that glycolysis and glutaminolysis are both used by activated T cells. All effector T cells require glycolysis, but we found that Th17 cells preferentially require GLS while Th1 cells are actively impaired by this enzyme. Thus, inhibition of GLS both reduces Th17 responses and promotes differentiation of Th1 cells and can lead to signs of T cell exhaustion. We show that GLS-deficiency can protect against Th17-mediated inflammatory models and also can augment effector function of Th1 CAR-T cells against B cell targets. Understanding mechanisms that regulate T cell metabolism may provide new tools to modulate immunity the balance of T cell effector populations to both suppress inflammation or promote effector function.

Disclosures

Rathmell: Calithera: Research Funding.
Analysis of Elderly Patients with Chronic Myeloid Leukemia
Authors: Kelkitli Engin , Goker Hakan , Turgut Mehmet , Atay Memis Hilmi
Published: October 2019

Aims

We aimed to evaluate in clinical results >65 years with chronic myeloid leukemia (CML).

Methods

191 CML patients treated with imatinib frontline were evaluated retrospectively. 56 patients who aged >65 years were included our study. The clinical characteristics and treatment outcomes of these patients were evaluated.

Results

A total of 56 patients (24 [57.1%] men, 32 [42.9%] women) were evaluated retrospectively. The median age of the patients was 72 years (range 66-92years). Of the patients, 56 (98.2%) were in the chronic phase and one (1.8%) were in the accelerated phase when diagnosed. All of patients (100%) were receiving imatinib as the first-line therapy.

Imatinib treatment induced 51 (91.8%) complete hematological responses at 3 months, 29 (51.8 %) complete cytogenetic responses at 6 months and 37 (66.6%) major molecular responses at 12months.

As a result, nilotinib and dasatinib were used in 28 (50%) patients as second-line therapy because of intolerance 5 (8.9%), primary and soconder unresponsiveness 14 (25%), 9 (16.1%) respectively. Second-line Tyrosine kinase inhibitors (TKI) induced 23 complete hematological responses (82.1%) at 3 months, and 20 major molecular responses (71.4%) at 12 months. Fifty-two of 56 patients (92.9%) are still alive. The 5 year median overall survival and progression free survival rates were 92% and 85% respectively.

Conclusion

The prognosis of CML patients has changed with Tyrosine kinase inhibitors (TKI). In our study, we found that the response rate and survival rates elderly patients were similar to the younger patients in the literature.

Disclosures

No relevant conflicts of interest to declare.
Normal and Aberrant NKL-Code in B-Cell Development and Lymphoma
Authors: Nagel Stefan , Roderick MacLeod A.F. , Meyer Corinna , Kaufmann Maren , Drexler Hans G.
Published: October 2019

Homeobox genes encode transcription factors which regulate basic processes in development and cell differentiation. Several members of the NKL subclass are deregulated in T-cell progenitors and support leukemogenesis. We have recently described particular expression patterns of nine NKL homeobox genes in early hematopoiesis and T-cell development. Here, we screened NKL homeobox gene activities in normal B-cell development and extended the NKL-code to include this lymphoid lineage. Analysis of public expression profiling datasets revealed that HHEX and NKX6-3 were the only members differentially active in naïve B-cells, germinal center B-cells, plasma cells and memory B-cells. Subsequent examination of different types of B-cell malignancies showed both aberrant overexpression of NKL-code members and ectopic activation of subclass members physiologically silent in lymphopoiesis including BARX2, DLX1, EMX2, NKX2-1, NKX2-2 and NKX3-2. Based on these findings we performed detailed studies of the B-cell specific NKL homeobox gene NKX6-3 which showed enhanced activity in patient subsets of follicular lymphoma, mantle cell lymphoma and diffuse large B-cell lymphoma (DLBCL), and in three DLBCL cell lines to serve as in vitro models. While excluding genomic and chromosomal rearrangements at the locus of NKX6-3 (8p11) promoter studies demonstrated that B-cell factors MYB and PAX5 activated NKX6-3 transcription. Furthermore, aberrant BMP7/SMAD1-signalling and deregulated expression of chromatin complex components AUTS2 and PCGF5 promoted NKX6-3 activation. Finally, NKL homeobox genes HHEX, HLX, MSX1 and NKX6-3 were expressed in B-cell progenitors and generated a regulatory gene network in cell lines which we propose may provide physiological support for NKL-code formation in early B-cell development. Together, we identified an NKL-code in B-cell development whose violation may deregulate differentiation and promote malignant transformation.

Disclosures

No relevant conflicts of interest to declare.
Flow Cytometric Analysis of Platelet Function in Patients on Antiplatelet Therapy and Suspected Thrombocytopathy
Authors: Huskens Dana , Roest Mark , Florin Lisa , De Laat Bas , Devreese Katrien
Published: October 2019

Background: Light transmission aggregometry (LTA) is still the most used test for the identification and diagnosis of platelet function defects (PFD). Disadvantages of LTA include its laborious nature, the large volumes of blood required and the relative insensitivity to small changes in platelet function. We investigated if the flow cytometry-based whole blood platelet activation test (WB-PACT) correlates with LTA or could be used as a complimentary test.

Methods: WB-PACT quantifies αIIbβ3 activation and P-selectin expression in response to 3 agonists and VWF binding to platelets in response to ristocetin. In total, 212 patients (51 on dual antiplatelet therapy (acetylsalicylic acid and ADP receptor antagonist), 161 suspected for bleeding diathesis) were tested. For WB-PACT parameters, the 2.5th, 10th and 25th percentiles were determined in 56 healthy donors to score the patient samples. For LTA, maximal aggregation, disaggregation and prolongation of the lagtime in response to 4 agonists and ristocetin was scored. Patients with at least one parameter lower than the 10th percentile measured with WB-PACT in healthy donors or at least one value deviating from the normal range measured with LTA were diagnosed with PFD. A bleeding score (BS) was calculated with the ISTH/SSC bleeding assessment tool (Rodeghiero et al., JTH, 2010).

Results: A moderate correlation between LTA versus WB-PACT was found with a R of 0.60 (Figure 1). In total, 95 patients were diagnosed with PFD by both tests (κ=0.40, Table 1) and BS were recorded for 28/95 patients. Of these 28 patients, 25% had an elevated BS (adapted according to gender and age: >5 in women, >3 in men and >2 in children) and 36% had a BS>3. BS were recorded for 30/38 and 22/24 patients who were diagnosed with PFD according to WB-PACT only and LTA only, respectively. Interestingly, 27% of patients with PFD according to WB-PACT had an elevated BS and 40% had a BS>3. For LTA, only 13% of patients with PFD had an elevated BS and 13% had a BS>3.

Conclusions: Flow cytometric analysis of platelet function by WB-PACT gives additional value to LTA to determine PFD.

Disclosures

No relevant conflicts of interest to declare.
How KEAP1 Deletion in Single Cell Mitigates Sickle Cell Pathology
Authors: Lukwete Nadine Keleku , Suzuki Mikiko , Panda Harit , Otsuki Akihito , Katsuoka Fumiki , Saitou Ritsumi , Saigusa Daisuke , Uruno Akira , Yamamoto Masayuki
Published: October 2019

Sickle cell disease (SCD) is one of the most frequent blood disorders caused by a point mutation of β-globin gene. In the pathology of SCD, we often encounter contributions of vascular and cellular components. The activation of vascular endothelial cells promotes adhesion of inflammatory cells and vaso-occlusion responsible for low blood flow, hypoxemia and organ damages. Hemolysis triggers activation and recruitment of myeloid cells to the inflammation site, further sustaining the inflammatory process. Recent progress has created the need to examine targeted induction of the KEAP1-NRF2 system as a potential therapeutic tool for the SCD. We have demonstrated that genetic as well as pharmacological induction of NRF2 in SCD prevent organ damages and suppress inflammation, although it still remains unclear how the NRF2 induction modifies the pathology or which type of cells contribute to the NRF2-mediated improvement of SCD. To address these questions, we conducted targeted deletion of Keap1 gene and induced expression of Nrf2 gene in endothelial cells and macrophages/monocytes using Tie1-Cre and LysM-Cre system, respectively. We found that SCD::Keap1F/F::Tie1-Cre mice (NRF2-increase in endothelial cells) showed low hematocrit and low platelet counts. While severe infiltration of monocytes, lymphocytes and macrophages was observed in the lungs of SCD mice, such phenotype was diminished in the SCD::Keap1F/F::Tie1-Cre mice. In the compound mutant mice, livers showed fewer necrotic zones and less to none accumulation of free iron. On the contrary, imaging MS (iMScope) analyses revealed decrease of free heme in livers and spleens in SCD::Keap1F/F::LysM-Cre mice (NRF2-increase in macrophages/monocytes). Furthermore, upregulations of most known Nrf2-dependent pathways and other yet-to-be-defined pathways are found in an RNA-sequencing analysis of the mice. Importantly, NRF2 induction enhanced heme catabolism and decreases the plasma level of free heme and the quantity of accumulated free heme in the SCD::Keap1F/F::LysM-Cre mouse tissues, whereas the change is not obvious in SCD::Keap1F/F::Tie1-Cre mice. On the contrary, vascular leakage of fluids was decreased in the SCD::Keap1F/F::Tie1-Cre mice. Thus, our results demonstrate that cell-lineage-specific activation of Nrf2 does inhibit the inflammation and organ injury with some particularities to each genotype.

Disclosures

No relevant conflicts of interest to declare.
Grb10 Is a Tumor Suppressor in Human Acute Myeloid Leukemia
Authors: Chai-Ho Wanxing , Roos Martina M , Li Michelle , Amara Pang , Zhang Yurun , Fang Tiancheng , Termini Christina , Chute John P
Published: October 2019

Acute myeloid leukemia (AML) is a common and potentially fatal hematologic malignancy. Allogeneic stem cell transplantation is the only curative therapy for most subtypes of AML, but carries a significant risk of transplant-related mortality. The development of novel therapies to eradicate AML remains a substantial area of unmet medical need. Growth factor receptor bound protein 10 (Grb10) is a member of the family of imprinted genes. Our laboratory demonstrated that in normal hematopoietic system, deletion of the maternal allele of Grb10 significantly increased hematopoietic stem cell long-term repopulating capacity (Yan et al. Cell Rep 2016). Grb10 has been shown to bind the intracellular domain of various tyrosine kinase receptors, e.g. KIT, FLT3 and RET, as well as low-density lipoprotein receptor-related protein 6, a negative regulator of the Wnt/β-catenin pathway. Analyzing RNAseq data from the Leucegene Project, we found that Grb10 is expressed on the majority of patient AML samples regardless of leukemia mutation profile. Silencing of Grb10 expression via Grb10 shRNA increased the proliferation and colony forming capacity of human AML cell lines, Kasumi-1, THP-1 and OCI-AML3 in vitro (p<0.0001 and p<0.01). Conversely, overexpression of Grb10 suppressed human AML cell growth (p<0.05). In order to determine the role of Grb10 in regulating AML growth in vivo, we transduced bone marrow lineage negative cells from mice with Grb10 maternal allele deletion (Grb10 m/+) and wild type (Grb10 +/+) mice with HoxA9-Meis1-neo-MSCV (gift from G. Savageau) and transplanted the progeny into congenic mice. Primary and secondary mice transplanted with Grb10m/+ HoxA9-Meis1 leukemia cells displayed significantly decreased survival compared to mice transplanted with Grb10+/+ HoxA9-Meis1 cells (p<0.001 and p<0.05). Furthermore, leukemia cells with Grb10 maternal allele deletion displayed an increase cell cycle progression and increased leukemia colony forming capacity. RNAseq analysis of Grb10 m/+ leukemia cells from diseased mice revealed significant dysregulation of the canonical Wnt/β catenin signaling pathway compared to Grb10 +/+ mice. RT-qPCR analysis confirmed that Wnt/β-catenin target genes, including MYC, CCND1, and SOX2 were significantly up-regulated in Grb10 knockdown human AML cells lines. Taken together, these data suggest that Grb10 is a powerful tumor suppressor in human AML, and represents a novel mechanistic target for the development of new therapies for human AML.

Disclosures

No relevant conflicts of interest to declare.
Intravesicular Cidofovir in the Treatment of BK Virus-Associated Hemorrhagic Cystitis (BKV-HC) Following Allogeneic Hematopoietic Stem Cell Transplant (HSCT)
Authors: Tooker Graham , Badros Ashraf Z. , Nishioka Jennifer , Riedel David
Published: October 2019

Background: BKV HC is a well-known complication following allo-SCT. Supportive care with bladder irrigation and blood transfusions were the only available treatment. Since our initial report (Bridges B et al. Am J Hematol 2006;81:535), several studies confirmed that intravesicular cidofovir is a potential effective treatment for BKV HC. In this study, we report a large series of consecutive patients who developed BKV HC following allo SCT and received intravesicular cidofovir.

Methods: We conducted a retrospective review of allo SCT patients who developed BKV HC and were prescribed intravesicular cidofovir from 2012 to 2017.

Results: 33 patients were diagnosed with BKV HC. The median age was 50 years (range=23-73), and 18 (55%) were male. Acute myeloid leukemia (n=12, 35%) was the most common diagnosis followed by non-Hodgkin lymphoma (n=7, 21%) and B cell acute lymphoblastic leukemia (n=4, 12%). Conditioning regimens were myeloablative (n=19, 58%) or reduced-intensity (n=14, 42%); 15 (45%) patients received cyclophosphamide, and 22 (67%) received total body irradiation. The median time to onset of HC symptoms following SCT was 37 days (range: 8-178); 17 (52%) patients had acute graft vs. host disease. HC symptom severity ranged from grade 0-4 (median=2). The median BK urine viral load pre-treatment was 100,000,000 IU/ml. Patients received a median of 2 intravesicular treatments (range=1-7) at a dose of 5 mg/kg. Four patients (12%) were also treated concurrently with intravenous cidofovir. 19 (59%) patients demonstrated complete clinical resolution of symptoms, 9 (28%) demonstrated partial response to treatment, and 4 (13%) had no change in symptoms following treatment. These improvements in clinical status were independent of viral load, though most had reductions in the viral load. The median time to symptom resolution was 17 days (range=7-53; n=28). 82% of patients had no recurrent symptoms of HC. The main side effect of intra-vesicular instillation was increased discomfort and bladder spasms; severe in 3 patients (9%). No patient had impaired renal function directly attributable to intra-vesicular cidofovir. At 12 months after BKV HC diagnosis, 26 (79%) patients were alive.

Conclusions: To our knowledge this is the largest study of intravesicular cidofovir for BKV HC reported to date; 77% of patients with BVK HC achieved clinical improvement of symptoms with minimal side effects. Clinical trials of intravesicular cidofovir could provide further evidence for adding intravesicular cidofovir as a standard tool for the treatment of BKV HC.

Disclosures

Badros: Celgene: Consultancy, Research Funding; Karyopharm: Research Funding; GSK: Research Funding.
Socioeconomic Disparities Affect Survival of Classical Hodgkin Lymphoma in a Statewide Cancer Registry Analysis
Authors: Myint Zin W. , Shrestha Runa , Chen Quan , Huang Bin , Goldberg Amit , Ramlal Reshma , Monohan Gregory , Fleischman Roger A. , Herzig Roger H. , Hildebrandt Gerhard Carl , Saeed Hayder
Published: October 2019

Introduction:

Low socioeconomic status (SES) has been shown to shorten survival in classical Hodgkin lymphoma (cHL). We sought to determine if differences in outcome of cHL varied according to SES and geographic setting in a population-based analysis.

Methods:

All adult patients diagnosed in 2000-2014 with cHL were collected through Kentucky Cancer Registry (KCR). Baseline SES and clinical variables were collected. Pearson Chi-square, log-rank, and cox regression tests were utilized. To minimize selection bias, cases with less than 6 months survival after cancer diagnosis were excluded.

Results:

A total of 1075 cHL patients were included, of which 605 (56.3%) were early stage vs. 405 (37.7%) were advanced stage. Most patients were under age 50 (63.2%) with a male predominance (55.4%). Nodular sclerosis was the most common histology (57.6%). Therapy consisted of chemotherapy alone in 729 (67.8%) patients, while 222 (20.7%) had received both chemotherapy and radiation. 633 (58.8%) lived in metro areas. Metro-area residents were more likely to come from non-Appalachian counties (93.7%), have more advanced education (76.8%) and have private insurance (60.1%) than non-metro-area residents (41.2%) (p<0.0001). There were no differences in age, gender, stage and histology at presentation according to geographic setting. Patients living in metro-area had a better OS by bivariate analysis (p=0.01) (Figure 1). A multivariate analysis identified age <50, early stage, privately insured, non-lymphocyte-depleted histology, and treatment with combined chemo-radiation as independent factors favoring an improved OS.

Conclusion:

Our study shows that despite the absence of differences in presenting clinical variables based on SES or geographic setting, significant discrepancies in outcome are observed. This disparity in outcomes may have been affected by differences in access to or quality of care delivery and may widen with the introduction of more costly novel interventions.

Disclosures

No relevant conflicts of interest to declare.
The Middle-Aged Bone Marrow Microenvironment Drives Hematopoietic Stem Cell Aging through Altered Production of Insulin-like Growth Factor-1 (IGF1)
Authors: Young Kira , Loberg Matthew , Eudy Elizabeth , Bell Rebecca , Trowbridge Jennifer J.
Published: October 2019

Globally, lifespan continues to increase. A growing challenge is how to maintain a healthy aging population. In hematopoiesis, healthspan is limited by a decline in regenerative capacity and overproduction of myeloid cell types at the expense of lymphoid cell types. These alterations contribute to decreased immune function, poor response to vaccination and increased susceptibility to myeloid neoplasms. Methods to extend hematopoietic healthspan are unclear, in part due to our relatively poor understanding of the factors controlling aging.

To address this gap in knowledge, we utilized mouse models to examine at what age did impaired regenerative capacity and myeloid-biased hematopoiesis begin to occur. We find that these phenotypes become significant by 9-12 months of age, representing middle age in mice. Through reciprocal transplantation studies of purified long-term hematopoietic stem cells (LT-HSCs) between young and middle-aged mice, we find that the middle-aged bone marrow (BM) microenvironment is sufficient to cause impaired regenerative capacity and myeloid-biased hematopoiesis from young LT-HSCs. These studies clearly implicate components of the BM microenvironment at middle age as causing hematopoietic aging.

To uncover the factors altered in the middle-aged BM microenvironment that cause hematopoietic stem and progenitor cell aging, we utilized single cell RNA-Seq and ELISA analysis. Together, these identified a reduction in Insulin-Like Growth Factor-1 (IGF1) in the BM microenvironment as the top candidate regulator of hematopoietic stem and progenitor cell aging. Expression of the receptor for IGF1, IGF1R, was dynamically altered on hematopoietic stem and progenitor cell populations with aging, suggesting that these respond to altered local levels of IGF1. In vitro colony-forming unit studies demonstrate that stimulating hematopoietic stem and progenitor cells isolated from middle-aged mice with exogenous IGF-1 ligand resulted in a dose-dependent suppression of myeloid cell overproduction, rescuing the aging phenotype. Finally, direct restoration of IGF1 levels in the BM microenvironment by short-term intrafemoral injection, at middle age, suppressed myeloid cell overproduction and restored B cell production in the BM and peripheral blood.

In summary, this study reveals a novel role for IGF1 in hematopoietic aging. We show that decline in local levels of IGF1 in the BM microenvironment at middle age cause hematopoietic aging. In addition, we demonstrate that short-term restoration of IGF1 at middle age can rescue hematopoietic aging. IGF1 thus represents an attractive candidate molecule to restore hematopoietic regenerative capacity and balanced lineage cell production to extend hematopoietic healthspan into older age.

Disclosures

No relevant conflicts of interest to declare.
Extravesicular Mir-7977 Released from Leukemic Cells Inhibites the PCBP1-Mediated mRNA Stabilization and Hippo-YAP Signaling Pathway in Bone Marrow Mesenchymal Stromal Cells
Authors: Yoshida Masahiro , Iyama Satoshi , Horiguchi Hiroto , Goto Akari , Kikuchi Shohei , Murase Kazuyuki , Takada Kohichi , Miyanishi Koji , Ikeda Hiroshi , Maeda Masahiro , Kato Junji , Kobune Masayoshi
Published: October 2019

[Background] We and others have revealed that various abnormalities of the bone marrow (BM) environment such as aberrant cytokine expression and impaired microRNA (miRNA) biogenesis are observed in patients with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). However, underlying mechanisms to induce BM stromal dysfunction have not yet been clarified. Recently, we have found that abundant extracellular vesicles including exosomes, were detected in the BM interstitial fluid. Furthermore, we have shown that exosomal miR-7977 was highly released from AML cells (GSE64029) and was taken up by BM mesenchymal stem cells (MSCs). Based on these findings, we hypothesized that exosomal miR-7977 could be involved in the alteration of BM stromal function of MDS/AML.

[Materials and Methods] To gain an insight into the function of exosomal miR-7977 in BM, we firstly performed transduction of a miR-7977 mimic into BM MSCs and compared transcriptomes between control-transduced and miR-7977-transduced MSCs (GSE108186). Subsequently, we selected the differentially expressed genes (DEGs) and conduced gene set enrichment analysis (GSEA).

[Results] Regarding DEGs, we focused on poly(rC) binding protein 1 (PCBP1), since PCBP1 was predicted to be a target gene of miR-7977 by an online database for miRNA target prediction using miRDB. The reductions in PCBP1 mRNA and protein were confirmed in MSCs after miR-7977 transfer by real time PCR. Because PCBP1 is known to be involved in splicing, and the expression levels of several genes, we analyzed the expression of several hematopoietic factors and found that expression level of stem cell factor (SCF), angiopoietin-1 (ANGPT1) and Jagged-1 (JAG1) were remarkably decreased. Regarding the pathway analysis, GSEA showed that the gene sets of Yes-associated protein 1 (YAP1)_up were significantly enriched (p<0.001, q<0.25), suggesting that miR-7977 modulates the Hippo-YAP signaling pathway. Visualization of pathway and network showed that miR-7977 significantly reduced the expression of Hippo core kinase, STK4. The transfer of miR-7977 mimic inactivated the Hippo-YAP signaling pathway as proven by GFP-tagged YAP nuclear trans localization and TEAD reporter assay. The miR-7977-transduced MSCs showed elevated saturation density and enhanced entry into the cell cycle.

[Conclusion] Collectively, these results indicated that miR-7977 is an important factor that inhibits normal hematopoiesis via PCBP1 reduction and up-regulate the growth of functionally-disturbed MSCs via inactivation of Hippo-YAP signaling pathway in MDS/AML.

Disclosures

No relevant conflicts of interest to declare.
Direct Oral Anticoagulants and Mortality in Atrial Fibrillation
Authors: Ellis Martin , Ruslan Sergienko , Ariel Hammerman , Greenberg-Dotan Sari , Battat Erez , Hamburger-Avnery Orly , Greenberg Dan , Ronen Arbel
Published: October 2019

Background

Direct oral anticoagulants (DOACs) are at least as effective as vitamin K antagonists (VKAs) for stroke prevention in patients with non-valvular atrial fibrillation (NVAF). DOACs are associated with less intracranial hemorrhage and are easier to administer and therfore have become standard of care for stroke prevention in NVAF patients. Nevertheless, many eligible patients still receive no anticoagulation. The effect on overall mortality in routine practice of DOAC therapy compared to no anticoagulation in NVAF patients is unknown.

Methods

We identified all newly diagnosed, anticoagulant naive NVAF patients eligible for DOAC therapy from 2011 to 2016 in Clalit Health Services, the largest HMO in Israel. We created a matched cohort, using 1:1 propensity score matching of DOAC -treated versus non-anticoagulant treated NVAF patients. The primary outcome was overall mortality rate in the two cohorts. Secondary outcomes were rates of stroke, major cardiac and bleeding events.

Results

28,195 newly diagnosed CHADS2 ≥2 NVAF patients were identified. Of these 8 298 received a DOAC and 10 603 received no anticaogulation. The remainder received a VKA and were not included in this study. Patients recieving DOAC therapy were younger (77.4 vs 78.0 years), had a higher socioeconomic status (5.6 vs. 5.3), had a female predominance (53.7% vs 52.0%), had a higher BMI (29.6 vs 28.3) and had a greater prevalance of accompanying cardiovascular morbidities namely congestive heart failure (CHF) (29% vs 27%) and stroke (33% vs 30%) (P<0.001 for all variables) . 5,657 patients who received DOAC therapy were matched with 5,657 patients not receiving an anticoagulant using propensity score matching. DOAC therapy was associated with significantly lower risk for death (hazard ratio 0.66, 95% CI 0.60-0.71, P<0.001). Rates of stroke and major cardiac were significantly lower in the DOAC-treated than in the non-anticoagulated patients. DOAC therapy was associated with a significant survival benefit across all patient subgroups.

Conclusions

In this cohort of newly diagnosed NVAF patients treated in routine clinical practice, DOAC therapy was associated with a lower risk for death compared to no oral anticoagulation. Our findings provide further evidence for the importance of DOAC therapy in NVAF patients.

Disclosures

No relevant conflicts of interest to declare.
D-Dimer Levels and Risk of Recurrence Following Provoked Venous Thromboembolism: Findings from the Riete Registry
Authors: Ellis Martin , Mar Martin , Manuel Monreal , Hamburger-Avnery Orly , Bura-Riviere Alessandra , Barillari Giovanni , Mazzolai Lucia , Mahe Isabelle , Marchena Pablo Javier , Verhamme Peter
Published: October 2019

Background. Patients with venous thromboembolism (VTE) secondary to transient risk factors or cancer may develop VTE recurrences after discontinuing anticoagulant therapy. Identifying at-risk patients could help to guide the ideal duration of anticoagulant therapy in these patients.

Methods. We used the RIETE database to assess the prognostic value of d-dimer testing after discontinuing anticoagulation to identify patients at increased risk for recurrences. The proportion of patients with raised d-dimer levels was determined and the hazard ratio (HR) for VTE recurrences compared to those with normal levels was calculated. Univariate and multivariate analyses of factors associated with VTE recurrence were performed.

Results. 3 606 patients were identified in the database in April 2018: 2 590 had VTE after a transient risk factor and 1016 had a cancer. D-dimer levels were measured after discontinuing anticoagulation in 1 732 (67%) patients with transient risk factors and 732 (72%) patients with cancer-associated VTE and these patients formed the cohort in which recurrent VTE rate was calculated. D-dimers and were elevated in 551 (31.8%) of patients with a transient risk factor and were normal in 1181 (68.2%). In the cancer-associated group, d-dimers were elevated in 398 (54.3%) and normal in 334 (45.7%) patients. The adjusted hazard ratio for recurrent VTE was: 2.32 (95%CI: 1.55-3.49) in patients with transient risk factors and 2.23 (95%CI: 1.50-3.39) in those with cancer.

Conclusions. Patients with raised d-dimer levels after discontinuing anticoagulant therapy for provoked or cancer-associated VTE are at increased risk for recurrent VTE and death. Future studies could target these patients for extended anticoagulation.

Disclosures

No relevant conflicts of interest to declare.
Sickle Cell Education for Medical Professionals: A Pre and Post Test Analysis
Authors: Simmons Diana D , Lucia Robert , Saving Kay Linn , Alwan Nicole
Published: October 2019

Background and Objectives: Sickle cell disease is a severe inherited form of anemia caused by a genetic mutation. Polymerization of hemoglobin leads to a cascade of effects decreasing blood flow. This causes tissue hypoxia leading to acute and chronic damage to the organs and endothelial lining. This disease requires complex management that relies on comprehensive training and knowledge regarding the disease process. Often accurate knowledge of sickle cell disease and how to provide appropriate care in the general medical population is limited. The purpose of this project was to develop a sickle cell educational training module for medical professionals. Such a module could be used to guide the provision of accurate education regarding sickle cell disease and best practice when caring for this patient population.

Methods: Goals and learning objectives were created and current medical literature about caring for sickle cell disease was reviewed. A comprehensive PowerPoint presentation was produced along with a provider tip sheet and a pre and posttest. The presentation, tip sheet, and tests were reviewed by a board certified pediatric hematologist/oncologist along with the hospital's educational review committee in the Department of Professional Regulation. Once approved, the PowerPoint, tip sheet, and tests were combined into a learning module and uploaded onto an online learning system utilized by the hospital system. The module was sent to over 2,400 outpatient providers and staff and to all inpatient staff on units where sickle cell patients stay when admitted. The module consisted of the participant completing a 10 question pretest, then reviewing the PowerPoint presentation and tip sheet. Following the review of the PowerPoint and tip sheet, the participant completed a 10 question posttest and completed an evaluation of the module.

Analysis: There were 223 people who completed the Sickle Cell Disease Learning Module. A paired t-test was conducted to compare pre-test scores to post-test scores. There was a significant difference in the pre-test scores (M = 5.98, SD = 1.66) and post-test scores (M = 9.17, SD = 1.36); p = <0.0001.

Conclusion: The goal of this module was to increase baseline medical knowledge of sickle cell disease. The results indicate there was statistically significant improvement in baseline knowledge, based on pre and post data (p = <0.0001). While the results indicate statistically significant increases in performance, it would be important to see if improvements are sustained over time. Reassessment of participants one year after completion of module can be beneficial to see if learned knowledge has been retained.

Disclosures

No relevant conflicts of interest to declare.
Invariant Natural Killer T Cells from Donor Lymphocyte Infusions (DLI-iNKTs) Contribute to Anti-Tumor Immunity after Allogeneic Hematopoietic Cell Transplantation
Authors: Jahnke Simona , Schmid Hannes , Secker Kathy-Ann , Duerr-Stoerzer Silke , Keppeler Hildegard , Baur Rebecca , Schumm Michael , Handgretinger Rupert , Bethge Wolfgang , Kanz Lothar , Schneidawind Corina , Schneidawind Dominik
Published: October 2019

Allogeneic hematopoietic cell transplantation (HCT) is a curative treatment option for hematologic malignancies but relapse remains the most common cause of death. Infusion of donor lymphocytes (DLIs) can induce remission and prolong survival by exerting potent graft-versus-leukemia (GVL) effects. However, sufficient tumor control cannot be established in all patients. We showed previously that invariant natural killer T (iNKT) cells promote anti-tumor immunity in murine models of allogeneic HCT without exacerbating graft-versus-host disease (GVHD).

We therefore studied iNKT cells in DLIs and investigated how culture-expanded iNKT cells from such DLIs (DLI-iNKTs) could lyse leukemia cells. We analyzed 63 cryopreserved DLI samples by flow cytometry. iNKT cells were identified using the PBS57-loaded CD1d tetramer. Under steady state conditions, iNKT cells represent 0.04% (range, 0.01-0.6) of live donor lymphocytes and need to undergo ex vivo expansion for further experiments and clinical application. We established a two-week protocol resulting in a 300-fold expansion of functional DLI-iNKTs with a purity of 94%. Interestingly, we observed a preferential expansion of CD4+ DLI-iNKTs. This subset turned out to be critical for tolerance induction and prevention of GVHD after allogeneic HCT. For degranulation and leukemia cell lysis assays, culture-expanded DLI-iNKTs were co-cultured with leukemia cells. CD107a as a marker of degranulation and iNKT-cell activity was upregulated on DLI-iNKTs upon engagement with leukemia cells that were subsequently lysed in a dose-dependent manner. We also observed an increased release of cytokines like IFN-γ (85 vs. 7 pg/ml, p=0.04). Moreover, the cytotoxic effects of culture-expanded DLI-iNKTs were CD1d-dependent: blocking the interaction between the MHC-I-like molecule CD1d and the T-cell receptor of DLI-iNKTs abrogated iNKT-cell degranulation and efficient leukemia cell lysis.

Our results suggest that iNKT cells from DLIs are involved in anti-tumor immunity after allogeneic HCT and therefore may reduce the risk of relapse and improve progression-free and overall survival. Prior expansion of iNKT cells could promote beneficial effects on tumor control, immune tolerance and overall survival.

Disclosures

Handgretinger: Miltenyi Biotec: Patents & Royalties: Co-patent holder of TcR alpha/beta depletion technologies, Research Funding. Bethge:Miltenyi Biotec GmbH: Consultancy, Honoraria, Research Funding; Neovii GmbH: Honoraria, Research Funding.
Effect of Renal Impairment on the Pharmacokinetics and Safety of Rivipansel in Subjects with Renal Impairment and in Healthy Subjects
Authors: Tammara Brinda , Ryan Kelly , Plotka Anna , Shafer Frank E. , Wei Hua , Readett David , Fang Annie , Korth-Bradley Joan
Published: October 2019

Background: Rivipansel is a pan-selectin inhibitor in phase 3 development for treatment of sickle cell disease vaso-occlusive crises. Previous studies have shown almost complete elimination of unchanged drug in urine following an intravenous (IV) infusion. The objective of this study was to evaluate the effect of varying degrees of renal impairment on the pharmacokinetics (PK), safety, and tolerability of rivipansel.

Methods: A single 840-mg dose of open-label rivipansel was administered IV over 20 minutes to 7 subjects with mild, 7 with moderate, and 7 with severe renal impairment, and to 7 healthy subjects with normal renal function. Classification of renal impairment groups was based on the Cockroft-Gault estimated glomerular filtration rate (CGeGFR): 60-89 mL/min (mild), 30-59 mL/min (moderate), and <30 mL/min (severe). Normal renal function was CGeGFR ≥90 mL/min. Plasma and urine samples were collected for 96 hours postdose and analyzed by validated LC-MS/MS methods. Pharmacokinetic parameters were estimated using noncompartmental modeling. ANOVA was used to assess the effect of renal impairment on PK parameters.

Results: All 28 subjects completed the study. A summary of PK parameters is presented in Table 1. Overall rivipansel exposure was greater in subjects with mild, moderate, and severe renal impairment, with values 1.4×, 2.3×, and 5.5× that of subjects with normal renal function, respectively. Renal clearance decreased with decreasing renal function. Total clearance was lower by 31%, 56%, and 82% in the mild, moderate, and severe renal impairment groups, respectively, compared with the normal renal function group. Five treatment-emergent adverse events (TEAEs) were reported in 3 subjects in the mild renal impairment group, and 3 TEAEs were reported in 2 subjects in the severe renal impairment group. None of the TEAEs reported was considered to be treatment-related.

Conclusions: Greater rivipansel exposure and decreased clearance were observed in subjects with renal impairment compared with subjects with normal renal function. A single 840-mg IV dose of rivipansel was well tolerated in all groups.

Disclosures

Tammara: Pfizer Inc.: Employment. Ryan:Pfizer Inc.: Employment. Plotka:Pfizer Inc.: Employment. Shafer:Pfizer Inc.: Employment. Wei:Pfizer Inc.: Employment. Readett:Pfizer Inc.: Employment. Fang:Pfizer Inc.: Employment. Korth-Bradley:Pfizer Inc.: Employment.
Altering Glycosphingolipid Composition to Improve Multiple Myeloma Bone Complication
Authors: Leng Houfu , Ersek Adel , Morris Emma , Molina Beatriz Gamez , Edwards Claire M. , Horwood Nicole
Published: October 2019

Multiple myeloma (MM) is an incurable cancer of plasma cells (PC), with a median survival of 5-7 years. Osteolytic bone disease and skeletal complications occur in more than 80% of MM patients and significantly contribute to the morbidity and mortality of these patients. Glycosphingolipid (GSL), an essential constituent of the outer leaflet of the cellular membrane, is altered in MM and other hematological cancers. We previously reported that GM3, a subtype of GSL promotes osteoclastogenesis. On the other hand, the GSL synthase inhibitor N-butyl-deoxynojirimycin (NB-DNJ) reduces myeloma bone disease in the 5TGM1 mouse model of MM. Mechanistically, NB-DNJ prevents osteoclast (OC) development and activation by disrupting RANKL-induced localization of TRAF6 and c-SRC into lipid rafts and preventing nuclear accumulation of the transcriptional activator NFATc1.

Although NB-DNJ is an FDA-approved drug treating Gaucher's disease, it has many undesired off-target effects, such as inhibiting lysosomal and plasma membrane Beta-glucocerebrosidase and interfering with intestinal glucosidases which leads to gastrointestinal toxicities and severe weight loss. Therefore, more specific GSL inhibitors are required to minimize the side effects.

Here we report a novel GSL inhibitor called Genz112638 with comparable effects as NB-DNJ but reduced side effects. Genz112638 inhibits both OC formation (p < 0.01) and MM cell growth (p < 0.0001) in vitro in a dose-dependent manner. Moreover, compared to NB-DNJ, Genz112638 more significantly improved bone condition and potentially reduced MM burden, as evidenced by the amelioration of bone loss in the 5TGM1 model of myeloma, and a reduction in the proportion of MM within bone marrow and spleen without obvious adverse effects (n=6) (p < 0.01).

As excessive malignant PC in MM normally arise from germinal centre, we also checked the effects of Genz112638 on germinal centre reactions in wildtype mice. We found that Genz112638 suppresses the formation of germinal centre B cells in mouse spleen induced by sheep red blood cells (n=7). Thus, Genz112638 may affect the pathogenesis of MM disease at the initial stage.

Taken together, our data elucidate a novel specific GSL inhibitor as a promising candidate drug relieving two main features of MM: bone destruction and tumour burden with negligible side effects. In vitro, it decreases OC differentiation and proliferation, and meanwhile decreases MM viability and proliferation. In vivo, it may suppress B cell formation in germinal centre, ameliorate bone destruction, and potentially interfere with the vicious cycle between increased OC and susceptibility to MM. In short, we provide a preclinical platform for GSL inhibition as a new tool against MM and its related complications.

Figure. Figure.

Disclosures

Horwood: Genzyme: Research Funding.
Safety and Efficacy of Dose-Escalation Hydroxyurea Therapy in Infants and Young Children: A Retrospective Cohort Study
Authors: George Alex , Tran Jennifer Nguyen
Published: October 2019

The BABY HUG clinical trial of hydroxyurea in infants and young children with sickle cell disease (SCD) established the safety and clinical benefit of hydroxyurea therapy in this age group. The study did not establish, however, if escalation to maximum tolerated dose (MTD) could be safely accomplished and resulted in increased benefit in this age group.

We sought to determine the safety and clinical effectiveness of dose-escalation hydroxyurea therapy in children with SCD under the age of two years. We conducted a retrospective chart review of children with HbSS or HbS β0Thalassemia who initiated hydroxyurea therapy before the age of two years between 2011 and 2017 at the Texas Children's Hematology Center. We assessed the clinical and laboratory response to dose-escalation therapy as well as any adverse events associated with hydroxyurea therapy.

We identified 95 patients ranging from 5 to 24 months in age who were initiated on hydroxyurea therapy during the study period. MTD was achieved in 75 patients (76.5%), of whom 51 were still at MTD at end of monitoring. There were significant increases in hemoglobin concentration (Hb) and hemoglobin F (HbF) levels and decline in absolute reticulocyte count (ARC) on hydroxyurea therapy (Table 1). Additionally, the magnitude of increases in Hb and HbF were greater than those seen in the BABY HUG hydroxyurea treatment group both at the time of MTD and at end of assessment. The proportion of patients experiencing neutropenia or thrombocytopenia as well as the number of events per patient-year (PPY) over 193.9 patient-years were similar to those in the BABY HUG hydroxyurea treatment group (Table 2). The incidence of acute chest syndrome (ACS) and transfusions was similar between the two groups, while episodes of splenic sequestration were more common in patients advanced to MTD.

In summary, hydroxyurea therapy with dose escalation to MTD appears to be safe in infants and young children and results in more significant and sustained increases in hemoglobin concentration and %HbF than were seen with fixed dosing in the BABY HUG study. In the context of this retrospective study, though, hydroxyurea dose escalation did not clearly result in increased clinical benefit relative to fixed-dose treatment.

Disclosures

No relevant conflicts of interest to declare.
Attempt to Prove the Existence of Abnormal B Lymphocyte As Myeloma-Initiating Cells from B Cell-Derived Induced Pluripotent Stem Cells
Authors: Yanagi Aki , Tsuyama Naohiro , Yanai Yukari , Abe Yu , Sugai Misaki , Ota Akinobu , Sivasundaram Karnan , Shigemura Tomonari , Sasatani Megumi , Kamiya Kenji , Hanamura Ichiro , Ikezoe Takayuki , Onodera Masafumi , Sakai Akira
Published: October 2019

We previously established normal B cell-derived induced pluripotent stem cells (BiPSCs; BiPSC13 and MIB2-6). BiPSCs are known to maintain VDJ rearrangement of the IgH gene, and they can be induced by the tet-off system to express activation-induced cytidine deaminase (AID; BiPSC13-AID and MIB2-6-AID) and differentiate into hematopoietic progenitor cells (HPCs) (Scientific Rep, 2017). Using these BiPSCs, we attempted to prove the existence of abnormal B cells, which are thought to be myeloma-initiating cells, originating from mature B cells transformed by reprogramming. We speculated that BiPSCs could develop into myeloma-initiating cells that undergo chromosomal translocation or gain genetic abnormalities during redifferentiation into mature B cells. First, using the comet assay, we confirmed the DNA-damaging effect of AID in BiPSCs-AID. Secondly, we differentiated BiPSC13-AID into CD34+/CD38-/CD43-/CD45- cells by co-culture with stromal cells (mouse embryo cell line: 10T1/2), and we subsequently transplanted the cells into the bone marrow (BM) of immunodeficient NRG mice. The presence of CD34+ cells was still observed in mouse BM 4 months after transplantation; however, no differentiation into B cells was detected. Next, using the CRISPR/Cas9 system, we attempted to make BiPSCs with chromosomal translocation t(11;14); we succeeded in establishing a 293T cell line with t(11;14), then confirmed t(11;14) in MIB2-6-AID, and the clone is now being established. Furthermore, we established BiPSC13-Pax5, which can be induced by the tet-off system to express Pax5, and we then differentiated BiPSC13-Pax5 into CD34+/Pax5+/CD38-/CD43-/CD45- cells by co-culture with stromal cells (10T1/2). We expect that the HPCs or hematopoietic stem cells (HSCs) derived from BiPSCs will further differentiate into B cells due to the expression of Pax5 in the BM of NRG mouse. We also established BiPSC13-AID-p53-/-, in which p53 was deleted using the CRISPR/Cas9 system, and the cells differentiated into HPCs. Interestingly, we detected some CD43+/CD45+ cells among CD34+/CD38- cells after the co-culture of BiPSC13-AID with aorta-gonad-mesonephros-derived stromal cell (AGM-S3) instead of 10T1/2. Therefore, AGM-S3 may promote the differentiation of BiPSCs into cells that are more similar to HSCs. These CD34+ cells differentiated from BiPSCs will be transplanted into the BM of NRG mouse.

Disclosures

Hanamura: CHUGAI PHARMACEUTICAL CO., LTD.: Research Funding; Kyowa Hakko Kirin Company, Limited: Research Funding; Fujimoto Pharmaceutical Corporation: Research Funding; Takeda Pharmaceutical Company Limited.: Other: Lecture fee; Bristol-Myers Squibb: Other: Lecture fee, Research Funding; Celgene: Other: Lecture fee.