bluuebunny/crossref_metadata_2025 huggingface dataset:
Pattern of Mir-155 and PU.1 Expression in CLL/SLL and Aggressive Lymphomas
Authors: Stopka Tomas , Vargova Karin , Huskova Hana , Burda Pavel , Curik Nikola , Vlckova Petra , Kulvait Vojtech , Pospisil Vit , Savvulidi Filipp , Necas Emanuel , Mraz Marek , Pospisilova Sarka , Mayer Jiri , Karban Josef , Obrtlikova Petra , Trneny Marek
Published: October 2019

Abstract 4600

MicroRNA miR-155 represents a candidate pathogenic factor in chronic lymphocytic leukemia (CLL) and lymphomas (Eis PS, et al. 2005, Fulci V, et al. 2007, Calin GA, et al. 2005, Kluiver J, et al. 2005, Metzler M, et al. 2004, van den Berg A, et al. 2003., Vargova K. et al 2011). In diffuse large B-cell lymphoma (DLBCL) expression of miR-155 was associated with NFkB pathway (Rai et al., 2008). In addition, increased miR-155 levels were associated with more aggressive (Activated B-cell like) DLBCL (Eis et al. 2005). The targets of up-regulated miR-155 apparently include a key hematopoietic transcription factor PU.1. Down-regulation of PU.1 represents an important and critical step for both myeloid as well as lymphoid tumorigenesis. We herein found the increased levels of miR-155 coupled with downregulation of its target PU.1 are frequent events in B cells of a vast majority of CLL patients (N=169). To advance understanding of miR-155 and PU.1 in lymphoid malignancies we studied lymph node biopsies derived from patients with lymphomas. We show that increased miR-155 levels are also found in DLBCL (N=31, P < 0.0001), Hodgkin lymphoma (HL, N=22, P < 0.0001), follicular lymphoma (FL, N=23, P < 0.001), small lymphocytic lymphoma (SLL, N=12, P < 0.01), and marginal zone lymphoma (MZL, N=11, P < 0.01). The miR-155-overexpressing tumors display downregulation of PU.1. In contrast, peripheral T cell lymphoma (T-NHL, N=6) and mantle cell lymphoma (MCL, N=7) expressed miR-155 and PU.1 comparably as control lymph nodes. Our data indicate that expression pattern of miR-155 and its target PU.1 represents a hallmark of some but not all lymphoid malignancies. Interestingly, the miR-155/PU1 levels vary substantially within each diagnosis, therefore, our aim is to further analyze the occurrence of this relationship in different prognostic groups and analyze the clinical outcome as well. (Grants: NT10310-3/2009 & NT11218-6/2010, MPO FR-TI2/509, GAUK251135 82210 & 251070 45410, SVV-2011-262507)

Disclosures:

No relevant conflicts of interest to declare.
The Expression of BAG3 Gene in Chronic Lymphocytic Leukemia and Its Clinical Significance
Authors: Zhu Huayuan , Liu Peng , Li Jianyong
Published: October 2019

Abstract 4605

Objective:

BAG3 (BCL2-associated athanogene 3) is a member of BAG family. Previously, many reports indicated that BAG3 was over-expressed in different human cancer tissues, such as thyroid carcinoma, pancreatic cancer, prostate cancer, and leukemic cells. However, there is no or low expression of BAG3 in normal tissues. These imply that BAG3 maybe have some important roles in the human cancers. This study was aimed to investigate the expression level of BAG3 mRNA in chronic lymphocytic leukemia (CLL) and its significance in evaluation of CLL prognosis.

Methods:

100 CLL human patients and 20 healthy controls were enrolled in this study. The BAG3 mRNA expressions were measured by real-time PCR with fluorescent dye SYBR Green I, the β-actin was used as internal control. The relative quantitative value of BAG3 expression was calculated by means of 2 (–ΔCt). We analyzed the results with established CLL prognostic factors, overall survival (OS) and treatment-free survival (TFS).

Results:

The expression of BAG3 mRNA in 100 CLL patients was significantly higher than 20 normal control (p = 0.038). Furthermore, The BAG3 expression was obviously increased in CD38 positive patients CD38 (n = 31) than CD38 negetive patients it (n = 61, p = 0.0238). We also found that the expression of BAG3 in the patients with unmutated immunoglobulin heavy-chain (n = 29) was markedly higher than those with mutated immunoglobulin heavy-chain (IgHV) (n = 62, p = 0.0326). It is also found that the patients with enlarged lymph nodes (n = 24) have higher BAG3 expression, compared with those without enlarged lymph nodes (n = 76, p = 0.0004). The increased BAG3 level can also be found in the patients with splenomegaly (n = 32),compared with those without splenomegaly (n=68, p = 0.0393). No association was found between BAG3 expression and patient clinical baseline information (gender and age), as well as other established prognostic factors (lymphocyte count, cytogenetics analysis and p53 mutation status). 25 of these patients were treated with fludarabine-based therapy, interestingly, BAG3 expression level was significantly increased in patients who were relapse and/or refractory to fludarabine-based treatment (n = 10, p = 0.019). However, there are no differences in OS and TFS between patients with low BAG3 expressions and high expression, which indicated BAG3 didn’t affect the patients’ OS and TFS.

Conclusion:

These results showed that the BAG3 expression in CLL patients is markedly higher than normal controls. CD38, mutation of IgHV, lymph nodes and splenomegaly are related to the expression of BAG3 in CLL. Furthermore, the patients, who were relapse and/or refractory to fludarabine, have a higher expression of BAG3. These imply that BAG3 may be involved in the pathogenesis of CLL and the roles of BAG3 in CLL need further investigation.

Disclosures:

No relevant conflicts of interest to declare.
Usability Testing of An Online Transition Program for Adolescents with Hemophilia
Authors: Breakey Vicky R. , Warias Ashley , Ignas Danial M , Blanchette Victor S. , Stinson Jennifer
Published: October 2019

Abstract 4777

Purpose:

This study explored the usability of a new Internet-based educational program of disease-specific information, self-management strategies and social support for adolescents with hemophilia.

Methods:

A comprehensive eight-module educational website was developed, based on results of an in-depth needs assessment. The website was tested for usability, using qualitative methods that included semi-structured, audio taped interviews and observation by a trained observer. To determine the usability and intuitiveness of the user interface of the “Teens Taking Charge: Managing Hemophilia Online” intervention, testing occurred in three cycles (4 participants per cycle). Participants were asked to work through standardized parts/features of the program, with changes to the prototype made after each cycle. Thematic analysis using a collaborative and iterative process was used to organize data into categories that reflected the emerging themes.

Results:

A purposive sample of twelve adolescents (range 12–18 years, mean 15.4 years) was recruited from a Canadian tertiary care center. All of the participants had access to a computer at home and felt comfortable using the Internet. Teens responded positively to the content, appearance and theme of the website (promoting self-management in youth with hemophilia). Subjects thought that it was easy to navigate, use and understand. Overall, they felt the content was appropriate and geared to meet the unique needs of adolescents with hemophilia. The multi-media components (videos, animations and quizzes) were thought to enrich the experience and make the program appealing. Adolescents provided ideas on how the website user-interface could be improved in terms of its usability (navigation, format and layout). Minor changes to the website user-interface were made and tested after the first and second cycles of testing. No further problems were identified in final cycle of testing. At the teens' suggestion, additional social media elements were added (discussion board, “ask the expert” section) to build in elements of support and increase interactivity. Most participants felt this program would be helpful prior to transition of care and beyond.

Conclusions:

Usability testing was the crucial first step in ensuring the acceptability and ease of use of “Teens Taking Charge: Managing Hemophilia Online”. Findings from this study were used to refine the website prototype. A pilot study is underway to determine the feasibility of using a randomized controlled trial to study the Internet-based self-management program.

Disclosures:

Breakey: Baxter Bioscience: Research Funding, Salary Support. Ignas:Baxter Bioscience: Salary Support.
γ-IFN May Act As a Positive Regulator to Hematogenesis Through PI3K/Akt and JAK2/STAT5 Pathways
Authors: Liu Hong , Zhou Lu , Wang Han
Published: October 2019

Abstract 4785

It is known to us all that γ-IFN is a growth inhibitory to hematogenesis and plays a critical role in the pathophysiology of aplastic anemia(AA). However, γ-IFN actually is a bilateral regulatory factor. In certain experimental situations, it can facilitate the proliferation and growth of hematopoitic stem cells. Interferon regulatory factor-1 (IRF-1) and IRF-2 are the key regulators of IFN system and IRF-1 has been shown to participate in development of CD8+ T cells, Th1 differentiation, and NK cell development. Thus, IRF-1 may has crucial functions against hematopoietic stem and/or progenitors cells. To investigate the function of IRF-1and the molecular mechanism of proliferative effect of γ-IFN, IRF-1-siRNA was designed to knocked out IRF-1 gene in 32D cells, mouse multipotent progenitor cells, and the apoptosis was assayed by flow cytometry 48 hr after incubation with γ-IFN in three different concentrations. It showed that after IRF-1 gene was knocked out, γ-IFN reversed its action on hematogenesis and apoptosis,it potentiated the proliferation and growth of 32D cells. Interestingly, at this situations, the inhibitory effect of γ-IFN on Akt activation was abolished completely and reversed to enhanced Akt phosphorylation significantly. The expression of pStat5 also changed when γ-IFN only exert proliferative effect.Nevertheless,The negative effect of γ-IFN on Erk1/2 activation was not blocked after IRF-1 gene was knocked out. Taken together, γ-IFN can enhance or antagonize cell proliferation depending on the function of IRF-1 and may act as a positive regulator to hematogenesis through PI3K/Akt and JAK2/STAT5 signaling pathways. Our findings may contribute to understanding the decreased number of stem cells characteristic of aplastic anemia and will provide novel therapeutic strategies for this disease.

Disclosures:

No relevant conflicts of interest to declare.
Expression of PTH in Bone Marrow Stem Cells in Adult SD Rats
Authors: Zou Weiying , Yang Bei , Wu Lei , Chen Ting , Zhang Dalei , Xiong Mingdi , Chen Jiaxiang , Zou Ting
Published: October 2019

Abstract 4817

Introduction:

Parathyroid hormone (PTH) is a major regulator of calcium and phosphate metabolism and is secreted by the cells of the parathyroid gland. Recent research showed that PTH treatment may regulate the hematopoietic stem cell niche, leading to beneficial effects on the HSC pool, and increasing stem cell number and the number of stem cells mobilized into the bloodstream. Many studies demonstrated that the serum PTH was decreased but maintained at the lower level suggesting persisting PTH secretion after total parathyroidectomy without autotransplantation. Some research showed that the thymus is another source of PTH. Can the bone marrow cells express PTH mRNA and release PTH and be a third source of PTH production? The aim of this study was to explore whether PTH could be expressed in bone marrow stem cells(BMSCs).

Method:

BMSCs were separated from adult male SD rat bone marrow and were cultured in DMEM supplemented with 10% fetal bovine serum. The total RNA was extracted using Trizol one-step method. RT-PCR was performed to examine the expression of PTH and GAPDH in BMSCs. The optical densities (ODs) value of RT-PCR product was measured using the Gel Imageware System. The signal intensity of PTH bands was normalized by corresponding GAPDH bands. The ODs in each sample were expressed as a ratio PTH over GAPDH densitometric intensities. The resultant PCR product of PTH was sequenced in both directions. Immunocytochemistry staining was utilized to examine the expression of PTH in the BMSCs from adherent cell fraction of the 2nd passage.

Results:

The expressions of PTH mRNA in BMSCs were detected in all 11 rats by RT-PCR. The PCR product was 193bp for PTH (Fig 1). The ratio of PTH over GAPDH ODs value ranged from 0.4407 to 3.1506, and the average ODs value (m ± SD) was 1.2617 ± 0.8953. The sequence of the PTH PCR products matched 100% with the PTH sequence in Genebank (Gene ID: 24694 Pth; gb K01268.1 RATPTH3, Rat parathyroid hormone gene, exons II and III). Some of the BMSCs were PTH positive by immunocytochemistry. The PTH positive cells were middle in size and spindle. PTH staining was localized in the cytoplasma and cell membrane of BMSCs cultured (Fig 2).

Conclusion:

The PTH mRNA and protein were detected in BMSCs. The results suggest that the BMSCs may be a third cell source responsible for the synthesis and secretion of PTH. It would be interesting to know what type of cells in BMSCs is secreating the PTH and to explore the role of PTH in bone marrow stem cell niche.

Disclosures:

No relevant conflicts of interest to declare.
Chromosomal Instability in Cultured Mouse Hematopoietic Cells Associated with Oxidative Stress
Authors: Liu Alice M. , Qu William W. , Liu Xia
Published: October 2019

Abstract 4819

Hematopoietic stem cells (HSCs) that give rise to all blood cell types are important vehicles for cell-based and gene therapies. After isolation from the bone marrow, HSCs are often cultured in laboratory settings for purposes of ex vivo expansion, gene transduction, and bone marrow transplantation for the treatment of various disorders of the blood and immune systems. While undergoing proliferation and differentiation in vitro, test tube and dish culturing can potentially induce genomic instability in HSCs due to prolonged culturing periods or the exposure to increased levels of oxygen. Here we demonstrate that in vitro culturing outside their bone marrow niches, HSCs may change even under very short durations of time. Lineage− Scal-1+ c-Kit+ (LSK) cells that are enriched with HSCs revealed significant levels of genomic instability in culture, as evidenced by the emergence of aneuploidy cells. To further determine the effects of in vitro culturing conditions, whole bone marrow cells were cultured in a hypoxic environment of 2–3% oxygen, mimicking conditions inside the body's bone marrow. In this case, cells proved to undergo less genetic alterations. Proper dosages of the antioxidant N-Acetyl-Cysteine (NAC) similarly decreased occurrences of chromosomal changes. Furthermore, in vitro normoxic culture-induced chromosomal instability was enhanced in aged hematopoietic cells compared to that in young hematopoietic cells due to noted increased oxidative stress in aged cells. These results reveal that in vitro cell culturing does indeed cause genomic instability in hematopoietic cells. Reduced oxygen levels and additions of antioxidants can be employed as a possible agent to lower oxidative stress and decrease chances of transformation. Additionally, since hematopoietic cells are commonly developed in laboratory settings before transplantation for patient treatment, our findings raise a concern for using cultured hematopoietic cells for therapeutic purposes.

Note: Alice Liu and William Qu contributed equally to this work.

Disclosures:

No relevant conflicts of interest to declare.
TIF1γ Knockdown Enhances Hematopoietic Stem Cell Self Renewal with Preferential Myeloid Differentiation and Delayed Erythropoiesis
Authors: Dorn David C , He Wei , Massague Joan , Moore Malcolm A.S.
Published: October 2019

Abstract 4829

The role of TIF1γ in hematopoiesis is still incompletely understood. We previously identified TIF1γ as a novel binding factor for Smad2/3 in the Transforming Growth Factor-β (TFGβ)-inducible signaling pathway implicated in the enhancement of erythropoiesis. To investigate the function of TIF1γ in regulation of hematopoietic stem cells we abrogated TIF1γ signaling by shRNA gamma-retroviral gene transfer in human umbilical cord blood-derived CD34+ hematopoietic stem/ progenitor cells (HCS/ HPCs). Upon blocking TIF1γ the self-renewal capacity of HSCs was enhanced two-fold in vitro as measured by week 5 CAFC assay and three-fold in vivo as measured by competitive engraftment in NOD/ SCID mice over controls. This was associated with a delay in erythroid differentiation and enhanced myelopoiesis. These changes were predominantly observed after TIF1γ knockdown and only mildly after Smad2 depletion but not after Smad3 or 4 reduction. Our data

reveal a role for TIF1γ-mediated signaling in the regulation of HSC self-renewal and differentiation.

Disclosures:

No relevant conflicts of interest to declare.
Optimizing Hydroxyurea Use in Children with Sickle Cell Disease: Least Tolerated Dose Is Effective
Authors: Sharef Sharef Waadallah , Al-Hajri Maya , Beshlawi Ismail , Al-Shahrabally Amer Qais , Wali Yasser
Published: October 2019

Abstract 4851

Background:

Hydroxyurea (HU) is an antimetabolite that effectively ameliorates the course of sickle cell disease (SCD). Starting dose is usually 20 mg/kg/day and the dose is escalated up to 35 mg/kg/day in most of the centers. In Oman, due to high level of consanguinity, ethnic neutropenia affects around 10% of population rendering higher doses difficult to maintain in most of the patients.

Materials and Methods:

161 patients (age 2–16 years, 61% male and 39% female) with SCD at Sultan Qaboos University Hospital, Muscat, Oman, were started on HU for clinically severe course (defined as: annual vaso occlusive crisis admissions more than 3, and/or occurrence of acute chest syndrome) with starting dose of 15 mg/kg and a mean dose of 18 mg/kg/day. All patients were included in the study except for 19 patients who were excluded for various reasons (8 lost follow up, 6 stopped HU by parents, and 5 stopped due to side effects: dizziness, abdominal pain, and myelosuppression). Remaining 142 patients were followed up for median treatment duration of 4 years (range, 1.5–10 years). Patients were assessed for clinical and laboratory response to HU.

Results:

There was significant reduction in the annual number of admissions due to vaso occlusive crisis (P <0.001, Wilcoxon Signed Ranks Test) with a mean of 4.7 and 1.5 before and after HU use respectively.

There was also observed clinical improvement regarding the incidence of acute chest syndrome. Only 12 out of 39 patients initiated on HU for acute chest syndrome had a recurrent attack during a follow up period of 3–7 years.

The laboratory parameters were consistent with previous reports: significant increase in hemoglobin level, fetal hemoglobin (HbF) level, Mean Corpuscular Volume, whereas significant decrease in platelet, absolute neutrophil, and absolute reticulocytic counts.

All 142 patients tolerated the treatment well. Observed side effects included abdominal pain, dizziness, rash, tremor in one patient each. Of note, 21 patients (11.1%) developed neutropenia (ANC<1000 × 10̂9/l) while 7 patients (3.7%) had thrombocytopenia (<100 × 10̂9/l) which interrupted the treatment for period ranging from 3 weeks to 3 months only.

Conclusion:

In SCD patients with background of ethnic neutropenia, lower starting dose and limited range of dose escalation of HU ensured safety and yet did not affect efficacy.

Disclosures:

No relevant conflicts of interest to declare.
Inflammatory Bone Loss Drives Skeletal Deterioration in a Murine Model of Sickle Cell Disease
Authors: Archer David R. , Mulupuri Prasad , Chappa Prasanthi , Vikulina Tatyana , Roser-Page Suzanne , Yamaguchi Masayoshi , Capers Patrice L , Hyacinth Hyacinth I , Hibbert Jacqueline , Weitzmann Neale
Published: October 2019

Abstract 4855

Sickle cell disease (SCD) results from a point mutation in the beta globin gene that leads to the replacement of glutamic acid with a valine at the sixth position of the beta globin chain of hemoglobin. Children with SCD experience delayed skeletal maturation and more than 70% of adult SCD patients' exhibit osteopenia or osteoporosis. The underlying mechanisms responsible are unclear; however in a number of disorders inflammation is established to drive bone loss. Importantly, persistent immune-activation in SCD leads to a chronic inflammatory state that may be pertinent. In this study we characterized the skeletons of four month old Berkeley SCD mice, an animal model that recapitulates key features of SCD including inflammation. We show that as in children with SCD, the skeletons of SCD mice have significantly reduced bone mineral density (BMD). Micro-computed tomography shows that homozygous sickle cell mice were also significantly denuded in cancellous and cortical bone volumes, a consequence of an enhanced pro-osteoclastogenic bone microenvironment. Similarly to animal models of postmenopausal osteoporosis we found a significant increase in T cell activation state and TNFa production in SCD mice, and an elevated expression of receptor activator of NF-kB ligand (RANKL), the key osteoclastogenic cytokine in the bone marrow. Finally, we show a significantly elevated number of monocytes (early osteoclast precursors) that likely further amplifies osteoclastogenesis in the context of elevated RANKL in SCD. Taken together our data validate the Berkeley sickle mouse as a suitable model to explore the mechanisms of bone loss in SCD, and suggest a disruption in the immuno-skeletal interface resulting from chronic inflammation as key to bone loss in SCD.

Disclosures:

No relevant conflicts of interest to declare.
Addition of Hemoglobin Level Into the Prognostic Scoring System Together with White Blood Cell and Platelet Count Provides Better Prognostic Stratification for Overall Survival, Complete Remission Rate and Early Death, but Not for the Relapse Risk in Adult Patients with Acute Promyelocytic Leukemia
Authors: Park Silvia , Jung Chul Won
Published: October 2019

Abstract 4898

Background:

Although the introduction of all-trans retinoic acid (ATRA) and combined chemotherapy improved clinical outcomes of acute promyelocytic leukemia (APL), treatment failure still occur due to early death or disease relapse. The PETHEMA study suggested that the risk adapted stratification of disease into 3 groups (low, intermediate and high risk) according to the white blood cell (WBC; 10×109/L) and platelet counts (40×109/L) could improve treatment outcomes of APL patients. However, this stratification system needs to be validated in an independent cohort of patients. The current study attempted to validate the prognostic significance of WBC count, platelet counts, hemoglobin level and PETHEMA risk stratification system in Asian population and to propose new clinical prognostic system consisting of WBC counts, platelet counts and hemoglobin level.

Methods and patients:

Between 2000 and 2009, total of 156 consecutive patients with APL from 7 institutes in Republic of Korea were retrospectively reviewed. The patients received Idarubicin/ATRA based combination chemotherapy during remission induction, and consolidation therapy was given with idarubicin monotherapy or Idarubicin plus cytarabine. In the new 3 score prognostic model, WBC more than 10×109/L, platelet less than 40×109/L as well as hemoglobin less than 8g/dL were separately considered as an one risk factor, and patients having “0”, “1” and “2 or 3” risk factors fall into a score “0”, “1” and “2”, respectively.

Results:

With median follow-up of 3.8 years, the CR rate following remission induction treatment was 81.4% in overall patients (127 out of 156), and 24 (15.4%) were considered as failures due to early death (ED) within 30 days of APL diagnosis. The 4-year OS, LFS and CIR rate was 73.8±4.1%, 85.7±3.7, and 18.7±3.8%, respectively. Compared to the single variables such as WBC count, platelet count or hemoglobin level, the combined prognostic system such as PETHEMA stratification or CBC score provided better stratification for the prognosis in APL patients. The risk stratification system by PETHEMA could predict OS (p=0.014) and ED (p=0.024), but not CR rates or relapse. However, the proposed 3 score prognostic system could provide better stratification of APL patients in term of the CR rates (p=0.004), OS (p=0.004) and ED (p=0.008), but not of relapse risk.

Conclusions:

This retrospective study suggested that the proposed 3 score prognostic system could provide better stratification of APL patients in term of the CR rates, OS and ED, but not of the relapse risk. Prospective study with a single protocol will be needed to reach a clean conclusion of better clinical prognostic stratification of APL patients.

Disclosures:

No relevant conflicts of interest to declare.
Immunophenotyping Features in Acute Myeloid Leukemia (AML) with NPM1+ and/or FLT3+
Authors: Topcuoglu Pervin , Dalva Klara , Bozdag Sinem Civriz , Arslan Onder , Ozcan Muhit , Ilhan Osman , Akan Hamdi , Beksac Meral , Gurman Gunhan
Published: October 2019

Abstract 4908

Immunophenotyping Features in Acute Myeloid Leukemia (AML) with NPM1 and/or FLT-3 Positive

Pervin Topçuoglu, Klara Dalva, Sinem Civriz Bozdag, Önder Arslan, Muhit Özcan, Osman Ýlhan, Hamdi Akan, Meral Beksaç, Günhan Gürman

Aim:

We aimed to evaluate immunophenotypical (IP) features in AML pts with NPM1+ and/or FLT3+ except on acute promyelocytic leukemia.

Patients&Method:

Between Nov 2009 and Feb 2011, we retrospectively analyzed IP features by flow cytometry (FCM) in 51 pts (46M;17F) with new diagnosed AML. Median age was 46 years (range: 14–71 ys). The mutations of NMP1 and FLT-3 TKD&ITD were determined by the methods of RQ-PCR or RFLP, respectively in the samples of bone marrow (n=31) or periheral blood (n=20) at the diagnosis. Antigenic expression of leukemic cells was analyzed by four-color FCM (FITC, PE, PerCP&APC) based by Nomdedeu et al researh (Leuk res 2011; 35:163) (Table-1).

Results:

We detected NMP1+ mutation in 16 patients. Of these, three were associated with mutations of FLT3-ITD (n=2) or -TKD (n=1). Twelve patients had FLT3+ (9 ITD or 3 TKD). More than half of the patients without any mutation were CD15+/CD34+/HLA-DR+ and 11.5% for CD34 negative. Similarly, the patients with FLT-3 positive were mostly CD34+ as the pts w/o any mutations. Contrary, most of the pts with NMP1+ were CD34 negative (56.3%) (Table 1). When evaluated the complete IP in leukemic cells, the expression of CD123 was significantly marked in the patients with NPM1+ and/or FLT3+ than those w/o mutations (p=0.008). While the co-expression of CD7 and CD117 was found in 67% of the pts w/o any mutations, 30% of the pts with NMP1 and/or FLT-3 ITD (p=0.01). CD56 expression was detected in more pts with NMP1+ than those with FLT-3+ (40% vs 8%, p=0.04). Besides, CD36 expression was positive in the all pts with FLT3-ITD than TKD+ (p=0.005). More intensive CD33 expression was seen in NMP1+ pts. The expression of CD64 was similar in all three mutations.

Conclusion:

Though NMP1 mutation was associated more CD34+ cells, more FLT3+ pts had CD34 positivity. The expression of CD123 was especially associated with the mutations. Aberrant expression of CD56 was in more pts with NPM1+, but CD36 for FLT3-ITD. These data might be a step for a study aiming to show a correlation between the type of mutations combined with IP features of leukemic cells and clinical characteristics or disease course of AML pts.

Disclosures:

No relevant conflicts of interest to declare.
Allogeneic Hematopoietic Stem Cell Transplantation for Blastic Plasmacytoid Dendritic Cell Neoplasm
Authors: Zhou Hongsheng , Zhang Xian , Yu Guopan , Deng Yongjian , Jiang Qianli , Fan Zhiping , Sun Jing , Liu Qifa
Published: October 2019

Abstract 4938

Background

Blastic plasmacytoid dendritic cell neoplasm is a rare malignancy that typically follows a highly aggressive clinical course,formerly known as blastic natural killer (NK)-cell lymphoma and CD4+/CD56+ hematodermic neoplasm. Here we report 2 cases of blastic plasmacytoid dendritic cell neoplasm that underwent allogeneic stem cell transplantation.

Patients and Methods

Case one was recognized as acute lymphocyte leukemia and then was revised to BPDC at recurrence, and another initially presented with typical cutaneous involvement and was promptly diagnosed by biopsy. After induction/re-induction and subsequent consolidation chemotherapy, 2 cases of BPDC received HLA identical-sibling donor allogeneic stem cell transplantation (Allo-HSCT) after BU/Cy, TBI/Cy+VP-16 conditioning regimen, respectively. Both achieved rapid hematopietic recovery in 2 weeks. Multi-color labeling flow cytometry was performed to monitor minimal residual disease (MRD) and the patient received donor lymphocyte infusion (DLI) to control primary malignancy as MRD-triggered or preventive strategy.

Results

Case 1 survived refractory acyclovir-resistant Epstein-Barr virus viremia after rituximab treatment, but failed in DLI-related grade IV graft versus host disease (GVHD) mixed with thrombotic microangiopathy (TMA) on 1 year post-transplantation. Case 2 died from relapse.

Conclusion:

Allo-HSCT seems to be a promising treatment for aggressive BPDC aided with MRD monitoring and MRD-triggered DLI. GVHD mixed with TMA deserves more attention for treatment of refractory GVHD.

Disclosures:

No relevant conflicts of interest to declare.
Lymphomatoid Granulomatosis: A Clinicopathologic Study of a Series of Cases in a Single Institution
Authors: Chavez Julio C , Zhang Ling , Sagatys Elizabeth M. , Sokol Lubomir , Moscinski Lynn , Sotomayor Eduardo M. , Shah Bijal D.
Published: October 2019

Abstract 4960

Introduction:

Lymphomatoid granulomatosis is a rare lymphoproliferative disorder with a proclivity for extranodal involvement, including the lungs, brain, kidney, liver, and skin. It usually affects middle-age adults, with male predominance (M:F ratio >2:1). Its association with EBV (Epstein Barr Virus) infection has been described.

Objectives:

To present our experience of 8 cases of lymphomatoid granulomatosis at Moffitt Cancer Center (MCC) in the last 10 years. Results: We collected the data of 8 cases of lymphomatoid granulomatosis that were diagnosed and treated at MCC from January 2000 to January 2011. The median follow up was 7.5 months. Five out 8 (63%) cases were males. The median age was 65. 3/8 (12.5%) had a performance status (PS) more than 2. 4/8 (50%) of cases had elevated LDH. All patients (100%) had an Ann Arbor Stage IVE. The IPI (International Prognostic Index) was more than high-intermediate in 6/8 (75%) cases. 4/8 (50%) of cases were positive for EBV. 6/8 (75%) have received chemotherapy to date, most commonly R+CHOP. The median time to progression was 8.6 months (1-8 months) following the first treatment and was shorter after the second treatment (4.8 months). 2/8 (25%) are disease free after chemotherapy, all remaining patients have active disease. 2/8 (25%) have died at 7 and 13 months after diagnosis.

Conclusions:

Lymphomatoid granulomatosis is a rare lymphoproliferative disease. In our experience it presents as an advanced and high risk disease. Interestingly, EBV was found in only 50% of cases. The overall response to standard chemotherapy is suboptimal and further strategies are needed for optimal management. These might include aggressive chemotherapy and up front stem cell transplantation.

Disclosures:

No relevant conflicts of interest to declare.
A PML–PPAR-δ Pathway for Fatty Acid Oxidation Regulates Hematopoietic Stem Cell Maintenance Through the Control of Asymmetric Division.
Authors: Ito Keisuke , Carracedo Arkaitz , Arai Fumio , Ala Ugo , Avigan David , Schafer Zachary , Evans Ron M. , Suda Toshio , Lee Chih-Hao , Pandolfi Pier Paolo
Published: October 2019

Abstract 2327

Stem-cell function is an exquisitely regulated process. Thus far, the contribution of metabolic cues to stem-cell function has not been well understood. Here we have established a new assay for the assessment of asymmetric division in hematopoietic stem cells (HSCs), and identify a previously unknown promyelocytic leukemia (PML)–peroxisome proliferator-activated receptor δ (PPAR-δ)–fatty-acid oxidation (FAO) pathway for the maintenance of HSCs and the control of asymmetric cell division.

HSCs exist in a quiescent state in the bone marrow niche and are the source of all hematological progenitors and differentiated cells throughout the lifespan of an organism. One of the central tasks of stem-cell biology is to define the modes and mechanisms that regulate the self-renewal and commitment of stem cells, as alterations in this equilibrium have a substantial effect on hematopoietic homeostasis and maintenance. It has been suggested that asymmetric division of HSCs ensures that a fraction of daughter cells retain features of stem cells while replenishing the committed compartment of hematopoietic progenitors. Therefore, identifying the factors that regulate this process would be of great biological and therapeutic relevance.

The PML tumor-suppressor gene, originally cloned at the break point of the t(15;17) chromosomal translocation of acute promyelocytic leukemia, has a key role in the maintenance of HSCs. However, how PML exerts its crucial function in the biology and maintenance of HSCs has not been established. In this study, we will present a new metabolic pathway downstream of PML, which can be regarded as a fine-tuning rheostat essential for HSC maintenance and their asymmetric division.

Specifically, we find that loss of PPARδ or inhibition of mitochondrial FAO induces loss of HSC maintenance both in vitro and in vivo, whereas treatment with PPARδ agonists improved HSC maintenance. We demonstrate that PML exerts its essential role in HSC maintenance through regulation of PPAR signaling and FAO. Mechanistically, we show that the PML–PPAR δ–FAO pathway controls the asymmetric division of HSCs. Deletion of the Ppard or Pml genes as well as inhibition of FAO results in the symmetric commitment of HSC daughter cells, whereas PPARδ activation increases asymmetric cell division, and rescues the defect exhibited by Pml-deficient HSCs, therefore ensuring the correct maintenance of the HSC population.

Thus, our findings identify a metabolic switch for the control of HSC cell fate with potential therapeutic implications.

Disclosures:

Avigan: Curetech: Research Funding; Celgene: Research Funding; Genzyme: Membership on an entity's Board of Directors or advisory committees.
The Role of Bone Marrow T Cells in Regulating Hematopoietic Stem Cell Function.
Authors: Brandao Claudia , de Bruin Alexander M. , Nolte Martijn A.
Published: October 2019

Abstract 2349

After immune activation, effector/memory T cells, including virus-specific CD8 T cells, are known to migrate to the bone marrow (BM), where they can be maintained by the production of IL-15 by the stroma; however, it is not yet known whether these T cells also have a function at this site. Since depletion of T cells from allogenic BM grafts compromises HSC engraftment, we hypothesize that T cells can directly influence the balance between differentiation and self-renewal of hematopoietic stem cells (HSCs). To test the ability of T cells to affect hematopoiesis, we performed co-cultures of HSCs and T cells isolated from murine BM. We found that T cells localized in the BM are able to enhance HSC differentiation as well as their self-renewal capacity. This feature is specific for BM central memory (CM) CD8 T cells, since other T cell subsets are not able to affect HSCs to the same extent. Moreover, depletion of CM CD8 T cells from the total BM T cell pool abrogates the impact on HSC differentiation and self-renewal, indicating that this particular T cell population is both sufficient and required for the observed effects. BM CM CD8 T cells do not affect quiescence of HSCs, but do enhance their proliferative capacity, and we found that supernatant from CM CD8 T cells is sufficient for this effect. Interestingly, competitive transplantation assays showed that HSCs cultured with CM CD8 T cells-derived supernatant contribute much better to leukocyte formation than medium-treated HSCs. This effect is seen in both the myeloid and lymphoid compartment, indicating that CM CD8 T cells are able to release soluble factors that support and enhance the multilineage reconstitution capacity of HSCs. Functional studies with blocking antibodies or knock-out mice showed that the supernatant-mediated effect is not caused by the hematopoietic cytokines IL3, IL6, IL21, GM-CSF, RANTES, TNFα or IFNγ. Preliminary data indicate that this feedback mechanism of the immune system on the hematopoietic process in the bone marrow is also present in the human situation, since autologous BM T cells increase the numbers of human HSCs, as well as their differentiation capacity.

Overall, these findings demonstrate that T cells have an important function in the BM and that especially CD8 TCM cells can directly influence HSC homeostasis. We postulate that this feedback mechanism of the immune system on the hematopoietic process in the BM is particularly relevant during viral infection, as the efficient migration of virus-specific CD8 T cells to the BM could well benefit the replenishment of the HSC/progenitor cell compartment and restoration of blood cell numbers that got lost upon infection.

Disclosures:

No relevant conflicts of interest to declare.
DNMT3A Mutation Leads to Hematopoietic Dysregulation.
Authors: Xu Jie , Zhang Wei-na , Zhen Tao , Li Yang , Shi Jing-yi , Mi Jian-qing , Wang Yue-ying , Chen Sai-juan , Chen Zhu
Published: October 2019

Abstract 2382

Epigenetic modification process is required for the development of hematopoietic cells. DNA methyltransferase DNMT3A, responsible for de novo DNA methylation, was newly reported to have a high frequency of mutations in hematopoietic malignancies. Conditional knock-out of DNMT3A promoted self-renewal activity of murine hematopoietic stem cells (HSCs). However, the role of mutated DNMT3A in hematopoiesis and its regulative mechanism of epigenetic network mostly remain unknown. Here we showed that the Arg882His (R882H) hotspot locus on DNMT3A impaired the normal function of this enzyme and resulted in an abnormal increase of primitive hematopoietic cells. In both controlled in vivo and in vitro assays, we found that the cells transfected by R882H mutant promoted cell proliferation, while decreased the differentiation of myeloid lineage compared to those with wild type. Analysis of bone marrow (BM) cells from mice transduced by R882H reveals an expansion of Lin−Sca-1+C-kit+ populations and a reduction of mature myeloid cells. Meanwhile, a cluster of upregulated genes and downregulated lineage-specific differentiation genes associated with hematopoiesis were discovered in mice BM cells with R882H mutation. We further evaluated the association of mutated DNMT3A and HOXB4 which was previously detected to be highly expressed in clinical samples carrying R882 mutation. Compared with wildtype DNMT3A, R882H mutation disrupted the repression of HOXB4 by largely recruiting tri-methylated histone 3 lysine 4 (H3K4). Taken together, our results showed that R882H mutation disturbed HSC activity through H3K4 tri-methylation, and transcriptional activation of HSC-related genes.

Disclosures:

No relevant conflicts of interest to declare.
GLI1 Directly Regulates the Transcription of AKT Genes in Diffuse Large B-Cell Lymphoma.
Authors: Agarwal Nitin K , Qu Changju , Kunkalla Kranthi , Liu Yadong , Vega Francisco
Published: October 2019

Abstract 2399

Activation of the Hedgehog (Hh)/glioma-associated oncogene (GLI) pathway has been found in a growing number of malignancies. We have provided evidence that canonical Hh signaling is required for cell survival and proliferation of DLBCL cell lines. To confirm the pathogenic role of GLI1 in DLBCL, we established GLI1 knock down DLBCL cell lines (OCI-Ly19, HBL-1 and BJAB) using a lentiviral shRNA system and performed cell viability and apoptosis assays. Cell viability assays demonstrated that GLI1 knockdown DLBCL cells experienced a statistically significantly decrease in the number of viable cells in comparison with control cells harboring scramble shRNA. To examine whether decreases number of cell viability in GLI1 knock down cells were due to apoptosis, we performed annexin V and PI assays. We observed marked increase of apoptosis in GLI1 knock down DLBCL cells versus controls (2.5 fold increase for OCI-Ly10, and 5 fold for HBL1 and BJAB). To investigate the mechanism by which GLI1 regulates tumorigenesis and cell survival, we searched for whole genome GLI1-target genes in DLBCL cells using CHIP sequencing technique and identified AKT genes as potential targets of GLI1. Using pharmacological and silencing approaches, we observed that Hh signaling modulates the expression of AKT genes in DLBCL cells. We further identified two putative binding sites for GLI1 in the AKT1 promoter region and confirmed their functionality using chromatin immunoprecipitation, luciferase reporter and site-directed mutagenesis assays. To investigate whether there is any correlation between AKT1 and GLI1 mRNA expression in human DLBCL tumors, we performed quantitative real-time PCR analyses in 17 frozen DLBCL specimens including apharesis samples from pleural effusions. The real time PCR analysis revealed a strong Spearmen correlation coefficient (R2=0.9) between GLI1 and AKT1 mRNA expression. In summary, we provide evidence of the role of GLI1 in the pathobiology of DLBCL and demonstrated a cross talk, at the transcriptional level, between Hh signaling and AKT in DLBCL. A link between these 2 pathways at the trasncriptional level was not previoulsy documented. This finding is of clinical interest as AKT has a key role in lymphoma cell survival and constitutive activation of AKT has been described in DLBCL.

Disclosures:

No relevant conflicts of interest to declare.
Fap1 Controls Beta-Catenin Activity in a Gsk3-Beta Dependent Manner in Chronic Myeloid Leukemia.
Authors: Huang Weiqi , Bei Ling , Eklund Elizabeth A.
Published: October 2019

Abstract 2404

Fap1 (Fas-associated phosphatase 1) interacts with the C-terminus of multiple proteins via a PDZ-type protein-protein interaction domain. Interaction of Fap1 with Fas results in Fas-de-phosphorylation and inhibits Fas-induced apoptosis. In previous studies, we determined that PTPN13, the gene encoding Fap1, is a target gene for the interferon consensus sequence binding protein (referred to as Icsbp or Irf8). Icsbp functions as a leukemia suppressor in chronic myeloid leukemia (CML), and we previously demonstrated Icsbp/Fap1-dependent apoptosis resistance in cells expressing the CML-associated oncoprotein, Bcr-abl. Another known interaction partner for Fap1 is the adenomatous polyposis coli protein (Apc). However, the functional significance of this interaction has not been previously explored. In the current stuides, we investigate whether interaction between Fap1 and Apc impacts the pathogenesis of CML. Apc is involved in assembly of a multi-protein complex that includes Apc, Axin, Gsk3-beta and beta-catenin. Association of these proteins results in serine/threonine phosphorylation (pS/T) of beta-catenin by Gsk3-beta. pS/T beta-catenin is released from the complex and degraded by the proteasome. Previous studies demonstrated that pS/T of beta-catenin, and consequent beta-catenin degradation, is impaired in CML, but the mechanism for this impairment is unknown in most cases. We find that Icsbp-dependent increase in Fap1 results in increased interaction between Fap1 and Apc in Bcr-abl+ myeloid progenitor cells. We also find that increased Fap1/Apc interaction is associated with inactivation (de-phosphorylation) of Gsk3-beta and a decrease in inhibitory pS/T of βcatenin. We find that this results in increased beta-catenin protein and activity. We also find that Fap1 co-immuno-precipitates with Apc, Axin, Gsk3-beta and beta-catenin. This result suggests the possibility that Gsk3-beta is a substrate for Fap1 protein tyrosine phosphatase activity. Consistent with this hypothesis, we find that Fap1 is able to dephosphorylate Gsk3-beta in vitro. We tested the functional significance of this observation in experiments using an activated form of Gsk3-beta that cannot be dephosphorylated at a key residue (Y216D-Gsk3-beta). We find that expression of this Gsk3-beta mutant partly reverses the effect of Bcr-abl and Fap1 on beta-catenin protein stability and activity. Increased beta-catenin activity is a characteristic of leukemia stem cells (LSC) in CML, and increasing beta-catenin activity in the bone marrow is associated with poor prognosis in CML. Therefore, these studies identify a novel pathway that regulates events significant to CML pathogenesis. These studies also identify a role for Apc in the pathogenesis of leukemia, and another mechanism for the leukemia suppressor effect of Icsbp.

Disclosures:

No relevant conflicts of interest to declare.
Sleeping Beauty Transposon Insertional Mutagenesis Identifies Drivers of Erythroid Leukemia in Mice.
Authors: Loeb Keith R. , Hughes Bridget T , Fissel Brian M. , Choi Yongiae E. , Pogosov Galina , Knoblaugh Sue E , Wood Brent L. , Clurman Bruce E.
Published: October 2019

Abstract 2407

Background and Methods:

Cyclin E-Cdk2 is a cell cycle regulator that promotes the G1-S transition by phosphorylating proteins involved in cell division and genome duplication. Cyclin E activity is frequently increased in many cancers, usually as a result of deregulation of mechanisms that control cyclin E protein levels and activity. Cyclin E T74A/T393A mice, in which cyclin E has been rendered resistant to degradation by the Fbw7 ubiquitin ligase by ablating two phosphorylation sites, exhibit enhanced epithelial proliferation and erythroid hyperplasia with inefficient erythropoiesis and macrocytic anemia. However, despite this widespread hyperproliferation, cyclin E T74A/T393A mice do not develop spontaneous tumors. To accelerate tumorigenesis and identify cooperating oncogenes in cyclin E T74A/T393A mice that complement the block in erythroid differentiation, we performed an insertional mutagenesis screen using the Sleeping Beauty (SB) transposon system.

Results:

Expression of SB by the inducible Mx-Cre caused rapid leukemiagenesis within 9–15 weeks post poly (I/C) injection in both control and cyclin E T74AT393A mice. The majority of tumors were T-cell lymphomas/leukemias and erythroid leukemias. Surprisingly, both wild type and cyclin E T74AT393A mice developed erythroid leukemias at similar frequencies. The erythroleukemia were composed of homogeneous populations of immature proerythroblasts akin to pure erythroleukemia in humans. Immunophenotypic profiling showed dim CD45 with variable CD117 (c-kit) and CD71 (transferrin receptor) expression. Preliminary sequencing of 29 erythroid leukemias identified 27 common insertion sites (CIS), including several genes associated with AML (Flt3, Erg and Ets1) as well as erythropoiesis (Fli-1 and Epo), highlighting the specificity of the SB-induced mutations for myeloid and erythroid leukemias. Preliminary analyses have also revealed preferential gCISs in erythroid leukemias isolated from cyclin E T74AT393A mice.

Conclusion:

We conclude that the Mx-Cre SB mutagenesis system provides a unique and rapid murine model of pure erythroid leukemia. We have identified a set of genes that promote erythroleukemia in mice may represent potential therapeutic targets for human erythroid and myeloid leukemias.

Disclosures:

No relevant conflicts of interest to declare.