bluuebunny/crossref_metadata_2025 huggingface dataset:
Does Vitamin D Deficiency Play a Role in Sickle Cell Nephropathy?
Authors: Hamideh Dima A , Raj Vimal , Zilleruelo Gaston , Alvarez Ofelia A.
Published: October 2019

Abstract 4755

Background:

Although improvements in the management of sickle cell disease (SCD) have increased patient survival into adulthood, morbidity and mortality from end-organ damage remain major concerns. One of the most serious complications of SCD is renal failure, affecting about 20% of patients. Secondary prevention of the progression of SCD nephropathy is hindered by a lack of easily modifiable risk factors. Based on animal and observational human studies, vitamin D deficiency is potentially a novel, modifiable risk factor that may interrupt or delay the progression of kidney disease by suppressing renin gene expression and inflammation. Studies have reported varying prevalence rates of vitamin D deficiency in patients with SCD patients but their clinical significance is uncertain. The purpose of this exploratory study was to determine the association of vitamin D insufficiency with albuminuria among a cohort of patients with SCD.

Methods:

This cross-sectional study was performed using a cohort of patients with SCD. Serum creatinine, serum cystatin C, spot urine for albumin/creatinine ratio, 25-hydroxyvitamin D [25(OH) D] levels, measures of hemolysis, inflammation and other laboratory studies were obtained. Vitamin D insufficiency was defined as 25(OH) D levels ?30 ng/ml.

Results:

27 patients with SS disease were evaluated. The mean age was 18 +/− 4.9 years; 40% were males and 60 % females. Mean GFR by serum creatinine was 167 +/− 40 ml/min/1.73 m2 and by cystatin C was 150 +/− 29.4 ml/min/1.73 m2. Increased protein/creatinine ratio (ratio ? 0.2) occurred in 29 % of patients (n= 9). Increased albumin/creatinine ratio (ACR ?30 mg/g of creatinine, mean 216 mg/g of creatinine) occurred in 37% of patients (n= 10). 88% of subjects showed vitamin D insufficiency [n=24, mean 17+/− 7 ng/ml]. Mean 25 OH vitamin D level was 14.8 +/− 8.9 ng/ml (5–28) and 18.2 +/− 5 ng/ml (12–30) in subjects with and without albuminuria, respectively. Urinary ACR inversely correlated with 25(OH) D levels (r=-.4, P=.02). No correlations were observed between urinary ACR and other clinical or laboratory parameters (age, hemoglobin levels, percentage of reticulocytes and lactate dehydrogenase levels). Proteinuria correlated with age (r=-.4, P=.02) and reticulocyte counts (r=-.4, P=.02) in SS disease but not with other parameters.

Conclusion:

In conclusion, vitamin D insufficiency was common and significantly associated with albuminuria among patients with SCD. Additional studies are needed to clarify the causal relationship of vitamin D insufficiency with albuminuria and to determine the role of vitamin D supplementation to attenuate the development of SCD nephropathy.

Disclosures:

No relevant conflicts of interest to declare.
Validation of a Non-Invasive Hemoglobin Estimation in Patients with Sickle Cell Disease
Authors: Al-Khabori Murtadha K. , Al-Hashim Abdulhakeem , Jabeen Zeba , Farsi Khalil Al , Al-Huneini Mohammed , Al-Riyami Arwa Z. , Al-Kemyani Nasser , Daar Shahina
Published: October 2019

Abstract 4770

Introduction:

The care of patients with Sickle Cell Disease (SCD) is frequently complicated by difficult venous access complicating blood sampling for laboratory investigations including hemoglobin (Hb) measurement. Non-invasive hemoglobin concentration monitoring is a potential solution, albeit not validated yet in patients with SCD. The primary objective of this study was to validate non-invasive pulse CO-oximetry based hemoglobin estimation in this patient population.

Methods:

We conducted a prospective observational study on patients with SCD admitted to the inpatient wards over 4 weeks in a tertiary care hospital. We estimated a spot Hemoglobin (Sp Hb) measurement using Masimo Pronto-7 Pulse CO-oximetry device (two measurements per patient) and compared it to a venous sample Hb (Reference Hemoglobin; Ref Hb) measured using Abbott CELL-DYN Sapphire hematology analyzer. We calculated Pearson correlation coefficient and coefficient of determination (R2). The multivariable linear regression model of predicting the estimation differences included age, gender, weight, height, blood pressure and reference hemoglobin.

Results:

We enrolled 98 patients (45 males, 53 females) with a mean age of 26 years (SD 8.8; 14–75) and a mean Ref Hb of 9.2 g/dL (SD 1.5; 5.3–13). The mean Sp Hb was 10.1 g/dL (SD 2.0; 5.3–14.5). The correlation coefficient between the Sp Hb and Ref Hb was 0.54 (R2 = 29%) with a mean difference of 0.9 g/dL (SD 1.7; −4.8 to 4.5). In the multivariable model, gender (p =0.042) and Ref Hb level (p=0.001) were statistically significant predictors for the difference in measurement. A strong correlation between the two CO-oximetry Hb measurements was obtained (correlation coefficient = 0.81, R2 = 65%).

Conclusions:

Our study demonstrated the validity of the CO-oximetry Hb measurement in adult patients with SCD. Larger prospective studies are needed to confirm our results.

Disclosures:

No relevant conflicts of interest to declare.
Study On the Expression of 14-3-3ζ in Acute Myeloid Leukemic Cell Lines
Authors: Liang Rong , Xiong Hua , Wang Zhe , Chen Qun xie , Gao Xun guang , Dong Xia bao , Zhang Liang
Published: October 2019

Abstract 4820

Objectives:

It is known that one of main reasons of refractory and relapsed acute myeloid leukemia (AML) is multidrug resistant (MDR). Despite it was showed that 14-3-3ζ was over- expressed in HL-60/VCR MDR cells than in HL-60 sensitive AML cells by examining the difference of gene expression profile with Affymetrix GeneChip Human Genome U133 set A. Yet, the understanding on role of 14-3-3ζ in the survival of AML cells remains poor.

Methods:

Semi-quantitative RT-PCR method was used to examine the expression of mdr1 mRNA in AML cell lines to validate the results of microarray. Western blot was performed to investigate Pgp#x2610;A14-3-3ζ#x2610;ABCL-2#x2610;AMCL-1 proteins expression. Immunofluorescence assay was used to detect the subcellular location of 14-3-3ζ protein in HL-60 and HL-60/VCR cells by laser scanning confocal microscope. 14-3-3ζ knockdown cells were obstained by transduction with lentivirus-mediated shRNA to silence 14-3-3ζ in AML cell lines. MTT and cell count method were used to analyze the changes of growth of AML cells.

Results:

mdr1 mRNA and Pgp was not expressed in HL-60 cells, but significantly overexpressed in HL-60/VCR cells(P<0.01). Except 14-3-3σ, the expression of other subtypes of 14-3-3 was higher in HL-60/VCR cells than in HL-60 cells, especially 14-3-3ζ(P<0.01). The higher increased expression of 14-3-3ζ, BCL-2, MCL-1 protein was observed in HL-60/VCR cells than in HL-60 cells. These results were same as gene chip. It was also noticed that14-3-3ζ was located in the cytoplasma and nuclear of AML cell lines, especially over-expressed in HL-60/VCR cells(P<0.05). Furthermore, suppression of14-3-3ζ by RNA interference resulted in inhibition of the proliferation of AML cells with BCL-2 and MCL-1 decreased protein expression, especially in HL-60/VCR cells(P<0.01).

Conclusion:

It was implied that14-3-3ζ played an important role in AML MDR and was associated with BCL-2 and MCL-1 expression. These results suggested that development of therapy targeting 14-3-3ζ may provide novel, effective strategies for refractory and relapsed AML.

Disclosures:

No relevant conflicts of interest to declare.
MiR-146a Expression Level As a Molecular Marker in Acute Promyleocytic Leukemia
Authors: Zhong Hua , Wang Chun , Fangyuan Chen , Xu Lan , Zhong Jihua , Jiong HU
Published: October 2019

Abstract 4821

MiR-146a which is located on chromosomes 5 may be involved in normal hemtopoisis and the pathogenesis of several hematopoietic diseases through inhibiting the expression of its targets. Acute promyelocytic leukemia (APL) cell line NB4 was taken as research model to find out whether miR-146a expression was associated to cell differentiation and proliferation. The miR-146a expression level of NB4 cells decreased after ATRA treatment, meanwhile the expression level of its predicted target gene Smad4 increased simultaneously.

MiR-146a expression plasmid, no related negative control sequence plasmid and recombined luciferase reporter gene vector containing the miR-146a complementary combined site on Smad4 plasmid were constructed by DNA recombination technical. Both recombined plasmids were transiently co-transfected into 293T cells with control plasmid pRL-TK. The interaction between miR-146a and its predicted target gene Smad4 was confirmed by the relative activities of dual luciferase detection.

This interaction relationship was further confirmed by liposome entrapment transfection of miR-146a and its antisense molecular in NB4 cells. The expression level of miR-146a in NB4 cells did no influence on Smad4 gene expression but Smad4 protein expression levels (fig 1).

The changes of NB4 cells on biological behaviors were observed by cell morphology, proliferation curve and flow cytometry methods under different miR-146a expression status. The results showed that cell proliferation ratio decreased and cell apoptosis ratio increased after miR-146a expression suppression in NB4 cells.

The investigation of miR-146a expression levels in mononuclear cells of APL patients' bone marrow and clinical data were carried out. It was showed that the expression levels of miR-146a were higher in APL patients than normal controls. The expression level of miR-146a was positive correlated with peripheral blood white cell count (fig 2). MiR-146a expression level was negative correlated with Th/Ts ratio among these patients (fig 3).

MiR-146a/Smad4 signal pathway is expressed in acute promyelocytic leukemia. The proliferation and apoptosis of the cells are modified by the Smad4 protein expression regulated by miR-146a expression. MiR-146a expression is somewhat associated with acknowledged clinical prognosis factors. It has potential possibility to take miR-146a as prognosis factor.

Disclosures:

No relevant conflicts of interest to declare.
Efficacy and Safety of Linezolid in Immunocompromised Patients with Hematological Malignancy
Authors: Qian Shenxian , Song Hua , Zhang Xuejin , Zhang Xuejin , Zhang Xiaohong , Luo Wenji , Luo Wenji , Huang Qian
Published: October 2019

Abstract 4838

BACKGROUND:

The aim of this study was to determine the safety, tolerance and efficacy of linezolid for the treatment of infections from Gram-positive bacteria in immunocompromised patients with Hematological Malignancy.

METHODS:

This was a prospective non-comparative unblinded and multi-center study in the Hematology Unit over a one-year period, administering combined use of linezolid in patients with Hematological Malignancy.

RESULTS:

Fifth-four adult patients received linezolid (600 mg, iv, q12h per day). Thirty-four children received linezolid (30 mg/kgr: 3 i.v. per day).Mean duration of linezolid administration was 12.8 days (range, 10–23days). Among the evaluable patients, fifty four were adults, the median age was 67(range, 30–101years); thirty four were children (range, 11 months-12.6 years), while the median age was 6 years. Primary diagnosis was acute lymphoblastic leukemia (forty-five patients), lymphoma (twenty-three patients), multiple myeloma (eleven patients), Aplastic anemia(four patients), myelodysplasia syndrome(one patients), leucopenia and hemophagocytic syndrome (two patient each). All patients were immunocompromised, 90% of them in the midst of chemotherapy cycles. The mean base number of platelet was 41×109/L(range, 30 – 138×109/L), platelet count less than 30×109/L patients were 28 cases (33%), among them, less than 10×109/L were 13 cases (14.8%). 27 out of 88 patients had positive blood cultures, methicillin-resistant Staphylococcus aureus, eighteen patients; Enterococcus, seven patients; Methicillin Resistant Staphylococcus Epidermidis and hemolytic streptococcus (one patient each).All patients were considered clinically cured after the end of the linezolid regimen (100% efficacy). The main adverse events were thrombocytopenia. There were 11 cases happened transient thrombocytopenia, Among them, four cases were happened within 4 days, seven cases happened four to seven days after received linezolid. Eliminate the possibility caused by reasons such as severe infection which can also result in thrombocytopenia, Linezolid related thrombocytopenia were only two cases (2.3%). Chemotherapy-induced myelotoxicity (90% patients) was not worsened during linezolid therapy. The platelet recovery time was also not lengthened during linezolid therapy. No bleeding episodes were presented, no Self-limited diarrhea were happened. The total adverse event rate was 2.3%; however, there was no premature cessation of linezolid in any patient.

CONCLUSIONS:

Linezolid may be another effective and safe therapy to treat infections from resistant Gram-positive bacteria in immunocompromised patients, even in children.

Disclosures:

No relevant conflicts of interest to declare.
Brain Lymphoma Treatment with Chemotherapy or Chemoradiotherapy from an Institution
Authors: Jalaeikhoo Hassan , Shafaeean Bahman , Keyhani Manoutchehr
Published: October 2019

Abstract 4859

Background:

Primary Brain Lymphoma (PBL) is an aggressive malignancy arising from the brain parenchyma. It is potentially curable brain tumor, or at least one in which durable remission can be achieved. We examined survival among patients with PBL treated with combination chemotherapy and radiotherapy and/or chemotherapy alone.

Methods:

We conducted a retrospective study of 38 patient diagnosed with PBL and examined long-term survival and morbidity among patients treated with either combination chemotherapy and radiotherapy or chemotherapy alone between July 1, 2004 to July 1, 2012. Patients were eligible for the study if they were between 16 and 80 years of age, HIV negative and had positive pathology and immune- histochemical staining for non Hodgkin's lymphoma of the brain. All patients had CT scan of chest, abdomen and pelvic, total body bone scan and bone marrow aspiration as part of their initial work up.

Enrolled patients were treated with combination of chemotherapy and radiotherapy if they were younger than 60 and with chemotherapy only if they were older than 60 years to reduce cognitive complications related to radiotherapy in older adults. The chemotherapy regimen included Methotrexate 2500 mgs per M2 with Leucovorin rescue (started day two) and Vincristine 1.4 mg per m2 (day two) which was administered every other week for 6 weeks. Chemotherapy in younger patients (<60) was followed by daily radiotherapy for 3 weeks to a total of 4500 rads. Both groups received Cytarabine 3000 mgs per m2 for 2 successive days (3 weeks apart). Intrathecal injection of Methotrexate 15 to 20 mg plus Cytarabine 100 mg were given in all patients in whom spinal fluid was drown on suspicion of meningeal involvement.

Result:

Among the 38 patients 77 % had diffuse large B cell lymphoma and 56% presented with multiple lesions and 44% with a single lesion on MRI. The median age at diagnosis was 53 years (range19 to 75). Two thirds (65.7%) were men. Mean duration of follow-up was 12 months (range 1 to 100 months). Overall (21%) of all patients died from early relapse, 2 patients had disease outside of brain found after relapse one in the liver and another one in mediastinum. The vast majority of deaths were among patients younger than 60 (87.5%). Significant morbidity included memory loss, hemi paresis, blindness, abnormal gate, psychiatric problems and incontinence which persisted or developed in 30% of patients. Among all patients 50% are alive and without significant morbidity. Disease recurrence was noted in 30% of all patients. Relapsed patients have been treated indefinitely with high dose Methotrexate.

Discussion:

Current management of PBL either with chemotherapy alone and/ or combination chemotherapy and radiotherapy has improved long-term survival in these patients. The most troublesome complication of treatment has been memory loss.

Disclosures:

No relevant conflicts of interest to declare.
Hepatosplenic T Cell Lymphoma Mimicking Hemophagocytic Lymphomhistiocytosis: A Case Report
Authors: Ahn Inhye E , Rice Lawrence
Published: October 2019

Abstract 4864

Background.

Hepatosplenic T cell lymphoma (HSTCL) is rare and aggressive extranodal lymphoma characteristically involves young males in third or fourth decade of life. It frequently manifests as pancytopenia with severe thrombocytopenia, hepatosplenomegaly, and much rarely, hemophagocytic lymphohistiocytosis (HLH).

Methods.

Medical records of a patient who presented with HLH as a presenting signs of HSTCL were reviewed. Literature search was performed to identify characteristic demographics and natural course of HSTCL reported to date.

Results.

A 30-year-old male presented after 6 months of constitutional symptoms. Remarkable findings were pancytopenia, mildly elevated LFT, and high ferritin level (89,000). Extensive work up for autoimmune and infectious etiology was negative. Worsening anemia and thrombocytopenia prompted the third bone marrow biopsy, which revealed the first evidence of hemophagocytosis. Despite of Cyclosporine A and Etoposide, pancytopenia worsened which prompted splenectomy and core needle liver biopsy. Sinusoids of spleen and liver were densely infiltrated with atypical lymphocytes consistent with T cell phenotype. Diagnosis of HSTCL was confirmed after PCR detection of gamma delta T cell receptor rearrangement. Previous bone marrow biopsies were retrospectively reviewed, which revealed small clusters of cells staining positive for CD3. The patient underwent three courses of chemotherapy that included high-dose Cytarabine, Etoposide and adriamycin. Post-chemotherapy course was complicated with disseminated Candidiasis complicated with mycortic aneurysm and worsening pancytopenia. The patient expired due to overwhelming septic shock 6 months after the pathologic diagnoses of HSTCL.

Conclusion.

HSTCL causes aberrant expansion of gamma delta T cells and defective innate immunity, and is an important secondary etiology for HLH. Splenectomy has diagnostic significance but little therapeutic benefit. Longer survival was reported in patients who underwent cytarabine-based chemotherapy; however median survivals in anecdotal case series all fall within 2 years regardless of regimen.

Disclosures:

No relevant conflicts of interest to declare.
ACY-1215, a First-In-Class Selective Inhibitor Of HDAC6, Demonstrates Significant Synergy With Immunomodulatory Drugs (IMiDs) In Preclinical Models Of Multiple Myeloma (MM)
Authors: Quayle Steven N , Jones Simon S
Published: October 2019

Histone deacetylase (HDAC) enzymes represent attractive therapeutic targets in multiple myeloma, but unfortunately non-selective HDAC inhibitors have led to dose-limiting toxicities in patients. ACY-1215 is a first generation, orally available HDAC inhibitor that is 11-fold selective for HDAC6, and synergizes in vitro and in vivo with bortezomib in preclinical models of MM without inducing unfavorable toxicities (Blood, 20[210]: 4061). Ongoing Phase Ib clinical trials with ACY-1215 have thus far confirmed an exceptional safety and tolerability profile (Raje, et al, EHA, 2013).

The IMiD class of drugs, including lenalidomide and pomalidomide, exhibit striking anti-myeloma properties in a variety of MM models, and have demonstrated significant clinical activity in MM patients. Prior studies have shown clinical activity of a combination of the non-selective HDAC inhibitor vorinostat with lenalidomide and dexamethasone in myeloma patients (Richter, et al, ASH, 2011). However, many patients experienced significant toxicities with this regimen that significantly limits its clinical utility. In support of our ongoing clinical development program for ACY-1215 in MM, we show here that combining ACY-1215 with either lenalidomide or pomalidomide leads to synergistic decreases in the viability of MM cells in vitro. The relevance of inhibition of HDAC6 to this synergistic effect was validated by demonstrating synergistic interactions of either IMiD molecule with ACY-775, which is more than 300-fold selective for HDAC6 over class I HDAC’s. Further, the combination of ACY-1215, lenalidomide, and dexamethasone was well tolerated in vivo with no overt evidence of toxicity, and combination efficacy studies with this combination are now ongoing in models of MM. By demonstrating that a selective inhibitor of HDAC6 synergizes with IMiD’s while maintaining an improved safety profile, these results provided a rational basis for the clinical development of the orally available combination of ACY-1215 and lenalidomide plus dexamethasone in an ongoing Phase Ib clinical trial (NCT01583283) for the treatment of MM.

Disclosures:

Quayle: Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership. Jones:Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership.
Common Gamma Chain Cytokines Contribute To Acute GvHD Via Perforin/Granzyme B-Mediated Cytotoxicity Of CD8 T Cells
Authors: Schwarze Vincent , Hechinger Anne-Kathrin , Leonhardt Franziska , Prinz Gabriele , Schmitt-Gräff Annette , Kovarik Jiri , Zeiser Robert
Published: October 2019

Allogeneic hematopoietic cell transplantation (allo-HCT) is an important treatment option for different hematological malignancies, but also for some nonmalignant hematological disorders such as sickle cell anemia, aplastic anemia or thalassemia(1). In the ladder group the graft-versus-leukemia (GvL) effect mediated by donor T cells is less important and prevention of graft-versus-host disease (GvHD), which occurs in 40-50% of allo-HCT patients, is a major priority. The common gamma chain (CD132) is a cytokine receptor sub-unit that is common to the interleukin (IL) receptors of IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21. Since several of these cytokines were shown to be increased in the serum of patients developing acute GvHD, we reasoned that inhibition of CD132 could have a profound effect on acute GvHD by inhibiting the bioactivity of multiple proinflammatory cytokines.

We observed that anti-CD132 treatment reduced GvHD potently with respect to survival, production of TNF, IFN-γ, IL-6, MCP-1 and GvHD histopathology. Protection was only seen when anti-CD132 was applied in a CD8 T cell-dependent GvHD model while no protection was seen when only CD4 T cells were given. Mechanistically, we could show that CD8 T cells isolated from mice treated with anti-CD132 had reduced levels of Granzyme B and that GvHD induced by Perforin-deficient T cells was resistant towards blockade by anti-CD132 treatment. These data indicated a role of the common gamma chain cytokines for the induction of Perforin/Granzyme B in CD8 T cells during GvHD. Compatible with this notion, exposure of CD8 T cells towards IL-2, IL-7, IL-15 and IL-21 alone or in combination induced increased levels of Granzyme B.

Based on these findings, we concluded that CD8 T cells that are activated by common gamma chain cytokines during GvHD produce then Granzyme B which can be blocked by anti-CD132 treatment. This therapeutic approach has particular clinical potential for patients undergoing allogeneic transplantation for nonmalignant indications, since graft-versus-tumor activity is not required.

Disclosures:

No relevant conflicts of interest to declare.
Inhibition Of S100A6 Induces Graft Versus Leukemia Effects In MLL/AF4-Positive ALL In Human PBMC-SCID Mice
Authors: Tamai Hayato , Yamaguchi Hiroki , Dan Kazuo , Inokuchi Koiti
Published: October 2019

Mixed-lineage leukemia (MLL)/AF4-positive acute lymphoblastic leukemia (ALL) is associated with poor prognosis even after allogeneic hematopoietic stem cell transplantation (allo-HSCT). The resistance to graft-versus-leukemia (GVL) effects may be responsible for the poor effect of allo-HSCT on MLL-AF4-positive ALL. Cytotoxic effector mechanisms mediated by tumor necrosis factor-alpha (TNF-a) was reported to contribute to the GVL effect. We reported previously that MLL/AF4-positive ALL shows resistance to TNF-a, which is the main factor in GVL effect, by upregulation of S100A6 expression followed by interference with the p53-caspase 8-caspase 3 pathway in vitro. We examined whether inhibition of S100A6 can induce an effective GVL effect on MLL/AF4-positive ALL in a mouse model. To examine the long-term effects of inhibition of S100A6,MLL/AF4-positive ALL cell lines (SEM) transduced with lentiviral vectors expressing both S100A6 siRNA and luciferase (SEM-Luc-S100A6 siRNA) were produced. SEM-Luc-S100A6 siRNA cells and SEM-Luc-control siRNA cells were injected into groups of five SCID mice (1×107/body). After confirmation of engraftment of SEM cells by in vivo imaging, the mice in each group were injected with 4.8×107 human peripheral blood mononuclear cells (PBMCs). Although there were no significant differences between the serum concentrations of human-TNF-a after injection of human PBMCs of SEM-Luc-S100A6 siRNA injected mice and those of SEM-Luc-control siRNA injected mice (P=0.95), SEM-Luc-S100A6 siRNA-injected mice showed significantly longer survival periods than SEM-Luc-control siRNA-injected mice (P = 0.002). SEM-Luc-S100A6 siRNA-injected mice showed significantly slower tumor growth than those injected with SEM-Luc-control siRNA (P < 0.0001). These results suggested that inhibition of S100A6 may be a promising therapeutic target for MLL/AF4-positive ALL in combination with allo-HSCT because it induce effective GVL effectson MLL/AF4-positive ALL which is resistant to GVL effects.

Disclosures:

No relevant conflicts of interest to declare.
Hematopoietic Progenitor Cell Counts Is a Useful Indicator To Determine Appropriate Time For Peripheral Blood Stem Cell Collection
Authors: Kong Sun-Young , Shim Hyoeun , Lee Se-Na , Kong Jung-Hee , Eom Hyeon-Seok , Park Byung-Kiu , Park Hyeon Jin , Yun Tak , Lee Hyewon , Song Yoon Kyung
Published: October 2019

Background

The optimal peripheral blood stem cell (PBSC) collection is a key step for successful outcome in hematopoietic stem cell transplantation (HSCT). Many indicators including preharvest white blood cell (WBC), mononuclear cell (MNC), and CD34 positive cell counts have been used for deciding the adequate time for collection of PBSCs, but each indicator has limitations. Here we investigated hematopoietic progenitor cell (HPC) count as an indicator for PBSC collection. Methods: Data from 851 autologous PBSC collections from 233 patients at the National Cancer Center, Korea, were analyzed. The correlations between harvested CD34 cell counts with preharvest WBC, MNC, CD34 cell counts, and HPC were analyzed, as were correlations by disease and mobilizing agent. Also how the outcome for engraftment can be predicted based on HPC count was studied. Results: The median age of patients was 41 years (range 0.1-72 years). The most frequent diseases were multiple myeloma (n=64) and non-Hodgkin lymphoma (n=56). The correlation coefficient between collected CD34 cells and preharvest CD34 count was (r=0.669, p<0.001), followed by preharvest HPC count (r=0.419, p<0.001), preharvest MNC (r=0.190, p<0.001) and preharvest WBC (r=0.014, p=0.679). The most adequate cut-off value for obtaining >1x106 CD34+ cells/kg at first time of PBSC was 24.0 HPCs/μL with sensitivity and specificity of 67.7% and 74.3% respectively. The cutoff as 28.0 HPCs/μL was adequate for obtaining 2.0 x106 CD34+ cells/kg with sensitivity and specificity of 73.7% and 72.2% respectively. HPC was well correlated with CD34 in PBSC of patients with multiple myeloma (r=0.326, p=0.009), non-Hodgkin lymphoma (r=0.353, p=0.008), especially diffuse large B-cell lymphoma (r=0.810, p<0.001) and acute leukemia (r=0.998, p<0.001). HPC was a better indicator for non-cyclophosphamide (r=0.337, p<0.001) than cyclophosphamide-based chemomobilization (r=0.572, p=0.052). Infused number of HPCs did not affect the times to engraftment of platelets (p=0.896) and neutrophils (p=0.953), though CD34 count of infusion had positive effect on platelet engraftment (p=0.017). Conclusion: HPC count represented good correlation with CD34+ and high area under the curve. Considering advantages of ease for use and cost-effectiveness than those of CD34 count, HPC is a good surrogate marker to determine appropriate timing for PBSC.

Disclosures:

No relevant conflicts of interest to declare.
Post-Transplant Cyclophosphamide Following a Myeloablative Conditioning For Hematologic Malignancies Receiving An Unmanipulated Haploidentical Bone Marrow Transplant
Authors: Raiola Anna Maria , Ghiso Anna , Dominietto Alida , Di Grazia Carmen , Gualandi Francesca , Varaldo Riccardo , Galaverna Federica , Van Lint Maria Teresa , Lamparelli Teresa , Bregante Stefania , Miglino Maurizio , Ballerini Filippo , Bacigalupo Andrea
Published: October 2019

Background

Unmanipulated family haploidentical transplants are becoming increasingly used in patients lacking an HLA identical family donor, or a well matched unrelated donor.

Aim of the study

This is an updated analysis of our program of haploidentical bone marrow transplant (hBMT) , followed by post-transplant high dose cyclophosphamide (PT-CY) in 95 patients with hematoloigic malignancies receiving a myeloablative conditioning.

Methods

The conditioning regimen consisted of thiotepa, busulfan, fludarabine (TBF) (n=47) , thiotepa melphalan fludarabine (n=5) , or 9.9-12 Gy TBI + fludarabine (n=43). The patients’ median age was 49 years (17-74); the disease status at the time of transplant, was first complete remission (CR1, n=29) , CR2 (n=23), or active disease (n=38,40%). The diagnosis was AML (35%), ALL (24%), MDS (12%) myeloproliferative disorders (12%), other malignancies (17%). The total nucleated cell dose infused was 3.37 (1.4-9.17). The median follow up for surviving patients is 440 days (90-930). Graft versus host disease (GvHD) prophylaxis consisted in PT-CY on day+3 and +5 , cyclosporine (from day 0) , and mycophenolate (from day +1).

Results

The cumulative incidence of neutrophil (PMN) engraftment was (90%) and all surviving patients on day +30 had full donor chimerism. The median day for neutrophil engraftment was day +17 (11-32). The cumulative incidence (CI) of grade II-III acute GvHD was 14%, and of moderate chronic GvHD 8%, severe cGvHD 9%. The CI of transplant related mortality (TRM) is respectively 7%, 17%, 26% for patients in CR1, CR2, or with active disease. The CI of relapse is respectively 17%, 30%, 37% for patients in CR1, CR2, or with active disease. The overall actuarial survival is respectively 75%, 44%, 32% for patients in CR1, CR2, or with active disease (p=0.002) (Fig.1). The most frequent cause of death was leukemia (26%) followed by infections (9%).

Conclusions

These data confirm our first report on 50 patients and support the use of myeloablative conditioning regimens, followed by h-BMT with PT-CY. The risk of acute and chronic GvHD seems relatively low , as well as the overall transplant related mortality. We have not seen the excess relapse rate reported when patients are given a non myeloablative regimen, and disease control has been satisfactorily both for patients in remission, as well as for patients with active disease at the time of transplants.

Disclosures:

No relevant conflicts of interest to declare.
Hypothyroidism and Cardiac Complications In Thalassemia Major Patients
Authors: Gamberini Maria Rita , Meloni Antonella , Rossi Giuseppe , Secchi Giuseppina , D'Ambrosio Alfonso , Macchi Silvia , Pulini Stefano , De Franceschi Lucia , Vallone Antonino , Lombardi Massimo , Pepe Alessia
Published: October 2019

Introduction

In the non-thalassemic population hypothyroidism has been associated with an increased risk of cardiac disease while the link thyroid-heart disease has been little explored in thalassemia major (TM). This retrospective cohort study aimed to systematically evaluate in a large historical cohort of TM in the cardiovascular magnetic resonance (CMR) era whether hypothyroidism was associated with a higher risk of heart complications (heart failure, arrhythmias and pulmonary hypertension).

Methods

From a cohort of 957 TM patients who underwent CMR for myocardial iron overload (MIO) assessment, quantification of biventricular function and detection of myocardial fibrosis within the MIOT network (Myocardial Iron Overload in Thalassemia), we identified 115 (12%) hypothyroid patients. Each hypothyroid patient was matched by sex and age (at the time of the CMR) with two non-hypothyroid patients, creating 115 triples. A cardiac event was considered valid if diagnosed at an age older than the hypothyroidism’s onset age for the hypothyroid patient in the belonging triple.

Results

Hypothyroid and non-hypothyroid patients had comparable MIO, but hypothyroid patients showed significantly lower biventricular stroke volume index, ejection fraction and left ventricular cardiac index. Accordingly, the prevalence of overall heart dysfunction (LV, RV or both) was higher in hypothyroid patients (43.5% vs 33.5%, P=0.0314). Hypothyroid patients had a significant higher frequency of heart failure (19.1% vs 9.1%, P=0.003) and arrhythmias (11.3% vs 4.3%; P=0.003).

Figure1 shows odds ratios (OR) estimating the relationship between hypothyroidism and cardiac involvement. Hypothyroid patients had a significant higher risk of heart dysfunction, heart failure and arrhythmias, also adjusting for the endocrine co-morbidity.

Conclusions

Hypothyroidism seems to increase the risk for heart failure, arrhythmias and heart dysfunction in TM patients. Our data confirm the link thyroid-heart disease also in TM patients and they stress the need to prevent hypothyroidism in this population.

Disclosures:

No relevant conflicts of interest to declare.
Spi-C Negatively Regulates Murine Eosinophil Differentiation
Authors: Schollaert Kaila L , Stephens Michael R , Mingler Melissa K. , Rothenberg Marc E. , Fulkerson Patricia C.
Published: October 2019

Eosinophils are bone marrow-derived granulocytes that normally comprise less than 5% of leukocytes in the blood, but can be found in higher numbers in tissues such as the bone marrow and gastrointestinal tract. Eosinophilia is associated with numerous clinical disorders including atopic diseases, parasitic infections, hypereosinophilic syndrome, and cancer. Expression of Spi-C was originally reported to be limited to B cell and macrophage lineages, but we have reported that upregulation of Spi-C expression within the lung was dependent on the presence of eosinophils in an experimental chronic asthma model. In this study we investigated the role of Spi-C during eosinophil differentiation. At baseline, we observed increased numbers of bone marrow and splenic eosinophils in Spi-C-deficient (Spi-CKO) mice. Stimulation of low-density bone marrow (LDBM) cells with the eosinophil-promoting cytokine IL-5 resulted in 100-fold increase in Spi-C mRNA expression. Cultured Spi-CKO LDBM cells had an accelerated rate of eosinophil progenitor (EoP) differentiation which yielded higher numbers of eosinophils with increased effector functions, including enhanced chemotaxis, granule protein production and release compared to wild-type mice. In addition, induction of asthma resulted in amplified airway eosinophilia in Spi-CKO mice compared to wild-type controls. Together our data indicate that Spi-C negatively regulates eosinophil differentiation during homeostasis and disease. Defining the molecular regulators of eosinophil differentiation and function will undoubtedly provide key information with clinical applications.

Disclosures:

Rothenberg: Teva Pharmaceutical: Consultancy, Ownership Interest receiving Other; Immune Pharmaceutical: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Ownership Interest receiving, Ownership Interest receiving Other.
Platelet Glycoprotein VI Haplotypes and The Generation Of Microparticles In Health and Disease
Authors: Ware Jerry , Dent Judith
Published: October 2019

Platelets are well known for their role in hemostasis, thrombosis, and wound repair. However, there is growing evidence for established platelet paradigms being relevant to other biological processes, such as inflammation and cancer. As one example, the platelet-specific collagen receptor, glycoprotein (GP) VI, has been shown to exacerbate rheumatoid arthritis in mouse models of the disease and also support experimental metastasis. GPVI exists as a membrane surface receptor that once engaged is capable of eliciting activation-dependent intraplatelet signals. An outcome of GPVI engagement is the release of platelet-derived microparticles (arbitrarily defined as less than 1 μm) and, in the case of rheumatoid arthritis, the microparticle further augments the inflammatory cascade. Beyond this example, platelet-derived microparticles are being recognized as relevant in a wide range of proinflammatory and prothrombotic pathophysiologic states. Characterization of the human GPVI gene has revealed genetic haplotypes corresponding to altered in vitro platelet response. A growing body of evidence has also linked these haplotypes to pathologic responses in the acute setting, such as myocardial infarction and stroke. The GPVI haplotypes, referred to as GP6a and GP6b, differ in 5 codons resulting in 3 polymorphic amino acids in the extracellular portion of the GPVI (S219P, K237E, and T249A) and 2 polymorphic amino acids in the cytoplasmic tail (Q317L and H322N). While GPVI haplotypes have been linked to cardiovascular disease outcome, a gap exists in determining whether the same polymorphisms influence platelet microparticle production and whether microparticle levels might vary in disease states. We have performed a detailed flow cytometry analysis of GPVI-dependent platelet-derived microparticles both in vivo and following agonist stimulation in vitro using blood from normal donors. A correlation exists between GPVI haplotypes and circulating levels of platelet-specific (αIIb) microparticles. Comparing homozygous GP6a (n=34) and heterozygous GP6a/b (n=17) individuals reveals an approximate 2-fold difference in the number of platelet microparticles that is statistically significant (p= 0.005). The GPVI dependent in vitro release is also haplotype specific following addition of type I fibrillar collagen. However, in a human condition of chronic inflammation, such as rheumatoid arthritis, haplotype-dependent changes in platelet-derived microparticle are not observed and reveal a maximum level of microparticle release occurring in the chronic inflammatory state. The results demonstrate that common polymorphisms within the human GPVI gene impact the level of circulating platelet-specific microparticles in normal individuals and in a state of chronic inflammation. The physiologic contribution of GPVI in disease states must consider the representative haplotype and levels of GPVI-dependent microparticles.

Disclosures:

No relevant conflicts of interest to declare.
Effects Of Helicobacter Pylori Eradication In Adult Patients With Acute Idiopathic Thrombocytopenic Purpura (ITP)
Authors: Moghimi Minoosh , Dizaji Saeid Razavi , Mortazavi Yousef , Mazloomzadeh Saeideh , Keyhanian Shaharbano , Alamsamimi Mozhgan
Published: October 2019

Background

Different studies have investigated the link between helicobacter pylori and extra digestive diseases suchas ITP.

Aims

In most studies chronic ITP has been investigated, therefore in the present study, we focused on the effect of H.pylori eradication on chronicity of ITP in adult patients with acute ITP.

Methods

After obtaining informed consent, eighty five patients with acute ITP whose platelet count were less than 30 ×103 /μl were enrolled to the study. Urea breath test (UBT) was carried out for all the patients. Based on the UBT results,the patients were divided into 3 groups: 1- H.pylori positive patients who underwent standard triple therapy. 2- ITPpatients negative for H.pylori and 3- ITP patients positive for H.pylori, but without eradication therapy. Evaluation of the H.pylori eradication was done 4 weeks after the treatment and also at the end of month 6.

Results

52(61.2%) of patients were female and 33(38.8%) were male with mean age of 34.8±12.2 years. There was nosignificant difference between the mean age and sex in different groups. No significant difference was seen in mean platelet count at the baseline among the groups, but there was a significant statistical difference in mean platelet betweenthe groups at the end of the first month. However, a significant difference was not seen in mean platelet count in months 2to 6. Chronicity in the first group was less than the 3rd group which statistically was significant. Also, the chronicity rate in non-infected ITP patients was lower than the H.pylori positive patients (P=0.03).

Summary / Conclusion

Our results show that eradication of H.pylori can reduce the chronicity rate in adult patients with acute ITP. Further studies on larger number of patients with longer follow-up are recommended.

Disclosures:

No relevant conflicts of interest to declare.
The Dynamic Motion and Delicate Control Of Vkor Catalysis
Authors: Li Weikai
Published: October 2019

Vitamin K epoxide reductase (VKOR) is an intramembrane enzyme required for blood coagulation and is the target of the anticoagulant warfarin. The reduction of vitamin K epoxide is coupled with disulfide-bond formation at the active site of VKOR. To regenerate the active site, VKOR is reduced by protein partners that transfer electrons to VKOR. Here we report two crystal structures of a bacterial VKOR homolog with its reducing partner captured in different conformational states. These structures reveal a short helix at the hydrophobic active site of VKOR that undergoes stretching and compressing motions. Motions of this “horizontal helix” promote electron transfer to the VKOR active site by regulating the positions of two cysteines in an adjacent loop. Compression of the helix also separates the “loop cysteines” to prevent backward electron transfer. During these motions, hydrophobicity at the active site is maintained to facilitate vitamin K reduction. Biochemical experiments support the structural model and suggest that several warfarin-resistant mutations act by changing the conformation of the horizontal helix. Taken together, these structures provide a comprehensive understanding of electron transfer and vitamin K reduction catalyzed by VKOR.

Disclosures:

No relevant conflicts of interest to declare.
Trib2 Pseudokinase Marks Early CMP Progenitors, and Promotes Granulocytic Plus Erythroid Progenitor Cell Formation
Authors: Li Lei , Kuhrt David , Dev Arvind , Jachimowicz Edward , Akashi Koichi , Wojchowski Don M.
Published: October 2019

Altered expression of Trib2 pseudokinase has been associated with AML (M2, M4 subtypes) (Cancer Cell, 10: 401), and ectopic expression of Trib2 in hematopoietic progenitors drives myeloid leukemogenesis (Blood, 116: 1321). In addition, inhibition of Trib2 expression by EPO in erythroid progenitors has been demonstrated (Blood, 111: 5390). Beyond this, little is known about Trib2’s expression profiles and roles during normal myelopoiesis. To critically address functional roles, we have generated and characterized a novel Trib2-KO mouse model. Within bone marrow CFU-GM formation faltered, and Trib2-KO Linneg progenitors exhibited deficit granulocyte production (with a relative increase in monocytes). This was not associated with significant differences in survival or cell cycle features among developing granulomonocytic cells. Instead, Trib2 levels proved to be heightened among Linneg progenitors (and inhibited in GM cells following GMCSF, GCSF or ATRA exposure). This prompted analyses of CMP and early-CMP (eCMP) populations in which RNAseq demonstrated peak Trib2 expression. Furthermore, eCMP and CMP pools were elevated in Trib2-KO mice. Within progenitors of the erythroid lineage, Trib2 expression levels also selectively increased, and in Trib2-KO mice BFUe and CFUe levels were decreased (and RBC plus HGB were diminished). Intriguingly, when Trib2-KO mice were challenged with Flt3L plus GMCSF, BFUe (but not CFU-GM or GEMM) were mobilized to peripheral blood at levels 13.3-fold above wild-type controls. This indicates candidate effects of Trib2 on a proposed BFUe niche. Overall, novel multi-lineage roles for Trib2 are revealed in regulating eCMP pools, GM-CSF populations, balanced granulocytic vs monocytic cell formation, and the production of erythroid progenitor cells.

Disclosures:

No relevant conflicts of interest to declare.
Loss Of NIPA Leads To Defects In The Repair Of DNA Double Strand Breaks In Mice
Authors: Illert Anna Lena , Antinozzi Cristina , Kawaguchi Hiroyuki , Kulinski Michal , Klitzing Christine , Peschel Christian , de Rooji Dirk , Morris Stephan w. , Barchi Marco , Duyster Justus
Published: October 2019

Regulated oscillation of protein expression is an essential mechanism of cell cycle control. The SCF class of E3 ubiquitin ligases is involved in this process by targeting cell cycle regulatory proteins for degradation by the proteasome. We previously reported the cloning of NIPA (Nuclear Interaction Partner of ALK) in complex with constitutively active oncogenic fusions of ALK, which contributes to the development of lymphomas and sarcomas. Subsequently we characterized NIPA as a F-Box protein that defines an oscillating ubiquitin E3 ligase targeting nuclear cyclin B1 in interphase thus contributing to the timing of mitotic entry.

Using a conditional knockout strategy we inactivated the gene encoding Nipa. Nipa-deficient animals are viable, but show a lower birth rate and a reduced body weight. Furthermore, Nipa-deficient males were sterile due to a block of spermatogenesis during meiotic prophase. Virtually no spermatocytes progress beyond a late-zygotene to early-pachytene stage and reach an aberrant stage, with synaptonemal complex disassembly and incomplete synapsis. Nipa-/- females are sub-fertile with an early and severe meiotic defect during embryogenesis with extensive apoptosis in early prophase (E13.5-E14.5).

Here we report, that Nipa-/- meiocytes exhibit persistent cytological markers for DNA double strand break repair proteins (like DMC1, RAD51) in meiotic prophase with more than twice as many DMC1 foci as control animals. Kinetic analysis of the first wave of spermatogenesis showed increased DMC1/RAD51 foci in Nipa-/- cells as soon as early-pachynema cells appear (13-14 days post partum). Moreover, we show that Nipa deficiency does not lead to a defect in meiotic sex chromosome inactivation despite epithelial stage IV apoptosis. Nipa-deficient spermatocytes exhibit numerous abnormalities in staining of chromosome axis associated proteins (like SYCP3 and STAG3) indicating that chromosome axis defects were associated with compromised chromosome axis integrity leading to overt chromosome fragmentation.

Further in vitro analyses with bleomycin treated MEFs displayed high pH2AX levels in cells lacking NIPA. Repair of DNA DSB seemed to be abolished in these cells as the pH2AX-level were sustained and still visible after 90 min of timecourse, where wildtype cells already repaired sides of DNA Damage. Consistent with these findings NIPA-deficient spleen cells showed compromised DNA Damage repair measured in a comet assay with a significantly longer olive tail moment in NIPA knockout cells under repair conditions.

Taken together, the phenotype of Nipa-knockout mice is a definitive proof of the meiotic significance of NIPA and our results show a new, unsuspected role of NIPA in chromosome stability and the repair of DNA double strand breaks.

Disclosures:

No relevant conflicts of interest to declare.