bluuebunny/crossref_metadata_2025 huggingface dataset:
Non-Invasive Urinary Markers of Renal Dysfunction in Sickle Cell Disease.
Authors: Mohtat Davoud , Moulton Thomas , Driscoll M. Catherine , Woroniecki Robert P
Published: October 2019

Abstract 4621

Renal failure is one of the major complications of sickle cell disease (SCD), affecting 5-18% of the patients. Microalbuminuria (MA), which has a prevalence of 26.5% in all children with SCD, is used as a marker of preclinical glomerular damage and renal disease progression. However, measuring MA alone may miss renal damage involving tubular proteins. We hypothesize that sickle cell nephropathy subjects will have increased urinary excretion of Transforming Growth Factor Beta-1 (TGF β1) and Neutrophil Gelatinase–Associated Lipocalin (NGAL) as compared to age-matched controls (CTR). We also hypothesize that urinary excretion of these proteins will be associated with severity of anemia in SCD. Enzyme-Linked Immunosorbent Assay (ELISA) kits were used to detect urinary excretion of TGF β1/NGAL, and corrected to urine creatinine. Mean age, gender, and race were not statistically different among the groups. Urinary excretion of TGF β1 was significantly higher in SCD subjects (26.4 pg/mg Cr +/-1.5) vs. CTR (14.9 pg/mg Cr +/-2.4), (p<0.00001). There was no difference in urinary NGAL between the SCD subjects vs. CTR. SCD patients with hemoglobin less than 9gm/dl had higher urinary TGF β1 than SCD patients with milder anemia (p=0.002). Urinary TGF β1 was lower in SCD patients on hydroxyurea (23.61+/-2.6), vs. those who were (27.69+/-1.8), p=0.08. Surprisingly, there were no correlation between urinary TGF β1 and urinary microalbumin or estimated glomerular function, indicating that microalbuminuria and serum creatinine are poor measures of renal injury in SCD patients.

Our data suggests that urinary TGF β1 may serve as a urinary biomarker to monitor the progression of renal disease in SCD. This assumption needs to be tested prospectively.

Disclosures:

No relevant conflicts of interest to declare.
Cognitive Dysfunction in Children and Adolescents with Sickle Cell Disease without Evidence of Stroke: Preliminary Results.
Authors: Silva Janete , Leite Ana , Maia Heber , Thomé Ursula , Moura Patricia , Lobo Clarisse Lopes
Published: October 2019

Abstract 4623

INTRODUCTION

Sickle cell disease (SCD) is the most frequent cause of stroke in children. It is possible that cognitive dysfunction occurs in the absence of identifiable stroke due to silent cerebrovascular disorders.

OBJECTIVE

To evaluate SCD compromises cognitive functions of patients with no radiological nor clinical evidence of stroke.

METHODS

Thirty eutrophic children with SCD, with normal neurological examination and imaging (brain CT) and without past cerebrovascular disease were submitted to neuropsychological assessment between 2002 and 2008. The neuropsychological battery included the following tests: WISC-III, BTN (battery of neuropsychological tests, assessing laterality), Rey complex figure (visual perception and memory), and behavioral screening for ADHD, learning disorders and antisocial behavior.

RESULTS

66.7% of patients were male. The average age of patients was 9.6 years (6 - 15 years). The mean total IQ was 78.8 (50 - 105) and the executive IQ was 77.9 (47 - 108), both at the borderline limit. The verbal IQ mean was 84.9 (55 - 113), on lower average. Verbal comprehension verbal was on average 87.4 (58 - 115). The results of visual memory was at the lower limit of normal (mean percentile 16 to 18). There was no correlation between the rates of behavior disorders (inattention, hyperactivity, learning difficulties and conduct disturbance) and IQ results. There was no statistically significant difference between the results of males and females or between age groups. 40% of the sample had not developed manual preference.

CONCLUSION

The subjects of this sample showed significant cognitive impairment in the absence of clinical or radiological evidence of cerebrovascular disease. The study has limitations as the sample size, the imaging technique used and neuropsychological battery comprehensiveness, but the results are quite suggestive of a relationship between SCD and cognitive impairment. The research group is developing a study with a larger sample (100 patients) to clarify the correlations between these findings. This will be important to outline earlier neuroprotective measures, which will provide improvements in neurodevelopment, learning, quality of life and social inclusion.

Disclosures:

No relevant conflicts of interest to declare.
The Eveluation of Pulmonary Functions in Children with Sickle Cell Anemia.
Authors: Kurt Cem , Sasmaz Ilgen , Antmen Bulent , Kilinc Yurdanur , Kurdak Sadi , Kuleci Sedat
Published: October 2019

Abstract 4628

Aim

In this study we evaluated to pulmonary functions and determined relations of these findings with clinical parameters in children with sickle cell anemia (SCA) who were at follow up in our pediatric heamatology clinic.

Materials and Methods

24 children with sickle cell anemia and 9 healthy children as control group include to the study. Complete blood count, hemoglobin electrophoresis and biochemical values were eveluated for both groups. At pulmonology department, the carbonmonoxide diffusion test performing for both groups. At the same day spirometric respiratory function evaluation and exercise test performed both groups at department of sports physiology. The data recieved are compared statistics.

Results

HbS, HbF, SGPT, ferritin, total bilirubine, direkt bilirubine and Fe++ values were high at patient group (p<0.05). Hemoglobin and hematocrit values were low at patient group according to control group as expected (p<0.05). The number of SCA patient who had one-three venoocclusive crises (VOC) were 14 (58.3%), patient who had three or more VOC were 7 (29.2%) and patient who had no VOC were 3 (12.5%). The number of patient who had acute chest syndrome (ACS) were 5 (20.9%) and 19 patients had no ACS (79.1%). Ýmpaired isole carbonmonoxide diffusion test was established at the 62.5% of the patient's. At patient group, spirometric FEV1 and MEF25 measurement were found lower than the control group (p<0.05). At exercise test VO2/HR rate were lower for patient group (p<0.05). VE/VO2 rate (p=0.023) and R (p=0.016) measurement were found higher.

Conclusion

Pulmonary gas transfer was found difficult in patients with SCA. Respiratory airways established obstructed in spirometric evaluation. Obstructive defficiensies have to be follow up. Oxygen pulse and respiratory exchange rates were determined low and more oxygen usage was observed for aerobic metabolic activity. With these results, ýt can be say that chronic inflamation process at lung due to oxygen radicals and hipoksemia in sickle cell patients, the aerobic respiratory load was increased.

Disclosures:

No relevant conflicts of interest to declare.
Modified Vaccinia Ankara Virus (MVA) Induces a Th1-Polarized Immune Response in Antigen-Presenting Cells (APCs).
Authors: Flechsig Christin , Suezer Yasemin , Kapp Markus , Sutter Gerd , Einsele Hermann , Grigoleit Götz Ulrich
Published: October 2019

Abstract 4650

Introduction

MVA is one of the most promising vaccine candidates for infectious diseases as well as for malignancies. Astonishingly, little information exists about the mechanism by which immune responses to MVA are generated. It was shown that among leukocytes - which are essential for the generation of cellular and humoral immune responses - APCs like dendritic cells, monocytes and B cells are preferentially infected. But little is known about the effects on APCs. Therefore we wanted to analyze in more detail the general effects of MVA infections on different immune cells.

Methods

Primary human peripheral blood mononuclear cells (PBMCs) and isolated leukocyte populations in particular monocyte derived DCs, monocytes and B cells were infected with (wildtype) wtMVA or MVA-gfp (green fluorescent protein) to verify the infection. Subsequently changes in surface markers and cytokine expression were assessed.

Results

Among PBMCs and specific isolated leukocyte populations, monocytes, DCs and B cells were most susceptible to MVA infection. NK cells showed a lower and T cells a very low infection rate. Surprisingly, selected monocytes were less susceptible to MVA as compared to unselected ones. This phenomenon is due to phagocytosis of other infected PBMCs by monocytes. Furthermore we could show that MVA causes a downregulation of CD14 on iDCs and monocytes as well as of CD25, CD80, and CD86 on B cells. Furthermore, there was a slight downregulation of CD1a on iDCs and mDCs and of CD80 on iDCs. On the other hand MVA caused an upregulation of HLA-DR on monocytes and additionally a slight upregulation of CD40 on iDCs. Moreover, MVA evoked a slight upregulation of CD83 on iDCs but a slight downregulation on mDCs. Above all, we could demonstrate that MVA induces an upregulation of CXCL10 in iDCs, mDCs, monocytes, and B cells, and an upregulation of TNFα, IL-6, and IL-12p70 in iDCs, mDCs, and monocytes. In addition, we revealed a downregulation of CXCL8 in monocytes as well as of IL-β in B cells.

Conclusions

These results suggest that MVA induces a Th1-polarized immune response in APCs. Thus, MVA seems to be an appropriate vaccine vector for antiviral immunotherapy of stem cell transplant recipients.

Disclosures:

No relevant conflicts of interest to declare.
Analysis of Risk Factors for Overall Survival at 5 Years in 96 Patients after Allogeneic Hematopoietic Cell Transplantation.
Authors: Ding Jiahua , Ding Jiahua
Published: October 2019

Abstract 4669

The aim was to analyze the risk factors for overall surrival at 5 years in 96 patients undergoing allogeneic hematopoietic stem cell transplantation by retrospec- tive analysis. 11 clinical parameters were selected for univariate analysis by using a Cox regression: age, sex, morbid rate, HLA locus, donor type, donor-recipient blood type, conditioning regimen, aGVHD, HC, VOD and IP. Factors significant at the 0.1 level on univariate analysis were evaluated by multivariate analysis by a Cox regres- sion. The cumulative incidence of aGVHD and patients survival rate were calculated by the method of Kaplan and Meier. 95 patients achieved sustained donor engraftment except one patients not. The median time of leukocyte engraftment was 13 days. The incidence rates of grades I∼IV acute GVHD was 43.75%.one of grades I aGVHD was 11.46%,gradesIIaGVHD was 19.79%,grades III∼‡WaGVHD was 12.50%. Ten patients relapsed and 38 dead,the overall survival at 5 years was 60.42%. The COX method analysis showed that aGVHD and disease states at transplant significantly im- proved OS. In conclusions, The key to improve the outcome of allo-HSCT is to red- uce the incidence and severity of aGVHD, meanwhile selecting the suitable disease state of CR1 at transplantation to treat the patients in advanced refractory and recure- nt situation.

Disclosures:

No relevant conflicts of interest to declare.
Mesenchimal Stroma Cells From Patients with Myelodysplastic Syndrome and Acute Myeloid Leukemia Show Distinct Cytogenetic and DNA-Mutation Data as Compared with Bone Marrow Leukemic Blasts.
Authors: Blau Olga , Hofmann Wolf-Karsten , Baldus Claudia D , Thiel Gundula , Nolte Florian , Schuemann Elke , Benlasfer Ouidad , Sindram Annette , Thiel Eckhard , Blau Igor W.
Published: October 2019

Abstract 4697

Bone marrow mesenchymal stroma cells (BMSC) are key components of the hematopoietic microenvironment. BMSC from patients with acute myeloid leukemia (AML) and myelodisplasic syndrome (MDS) display functional and quantitative alterations. To gain insight into these questions, we carried out cytogenetic analyses, FISH, FLT3 and NPM1 mutation examinations of both hematopoietic (HC) and BMSC derived from 53 AML and 54 MDS patients and 35 healthy donors after in vitro culture expansion. Clonal chromosomal aberrations were detectable in BMSC of 12% of patients. Using FISH we have assume that cytogenetic markers in BMSC were always distinct as the aberrations in HC from the same individual. 17% and 12% of AML patients showed FLT3 and NPM1 mutations in HC, respectively. In BMSC, we could not detect mutations of NPM1 and FLT3, independent from the mutation status of HC. For control analysis, BMSC cultures from 35 healthy donors were prepared under the same conditions. BMSC from healthy donors did show normal diploid karyotypes and absence of specific DNA-mutations of NPM1 and FLT3.

Our data indicate that BMSC from MDS and AML patients are not a part of malignant clone and characterized by genetic aberrations. Lack of aberrations as detected in HC and appearance of novel clonal rearrangements in BMSC may suggest enhanced genetic susceptibility and potential involvement of BMSC in the pathogenesis of MDS and AML.

Disclosures:

No relevant conflicts of interest to declare.
High Progenitor Cell Frequency at Diagnosis Predicts High Minimal residual Disease Level in Childhood ALL.
Authors: Ebinger Martin , Witte Kai , Lang Peter J. , Handgretinger Rupert
Published: October 2019

Abstract 4705

Acute lymphoblastic leukemia (ALL) as the most frequent childhood malignancy exhibits a favorable prognosis, yet a significant proportion of patients suffer a relapse. Comparable to acute myeloblastic leukemia, immature leukemic cells may be resistant to therapy and initiate a new population of leukemic cells. 42 patients with childhood ALL treated according to the ALL-BFM 2000 protocol were included in the present study. We determined an aberrant immunophenotype of the leukemic population at diagnosis and investigated the expression of CD34, CD38 and CD45 in leukemic blasts. The fraction of immature leukemic cells defined by CD34+/CD38-/CD45low at time of diagnosis as well as level of minimal residual disease (MRD) at day 33 and day 80 was determined. This is the first study to show a significant correlation of the initial fraction of immature leukemic cells with minimal residual disease levels at day 33 and day 80 in childhood ALL. Thus the initial level of this CD34+/CD38-/CD45low population may serve as marker for adverse prognosis in pediatric ALL.

Disclosures:

No relevant conflicts of interest to declare.
The Serum Fas and Fas Ligand levels in Childhood Acute Leukemias.
Authors: Kiziltas Alev , Antmen Bulent , Sasmaz Ilgen , Kilinc Yurdanur , Yilmaz Mustafa , Yigin Akin
Published: October 2019

Abstract 4724

Aim

Abnormalities and alterations in apoptosis mechanism may lead to cancer development. Cystean proteases enzymes, called caspases, appear to be involved in both the initial signaling events. There are many proteins that trigger intrinsic and extrinsic pathway and induce apoptosis signals. Fas and its specific ligand that known as Fas Ligand are the best defined dead receptors and have functions in apoptosis regulation with many tumor types. Fas binds the ligand on the cytotoxic T cells and start apoptosis. Objectives of this study were to determine serum levels of Fas and Fas Ligand at the time of diagnosis in childhood acute leukemias that may be play important role in apoptosis mechanism.

Patients and Methods

In this study, we investigated serum Fas and Fas Ligand levels by using ELISA method in childhood acute leukemias. Twenty-nine cases with acute lymphoblastic leukemia and twenty-three cases with acute myeloblastic leukemia at the ages of 1-18 years are included this study. The age distrubition of the control group varied 1-15 years consisted of twenty-seven children. We investigated serum Fas and Fas Ligand levels at the time of diagnosis from peripheral blood samples.

Results

The comparison of the mean values of Fas and Fas Ligand levels in acute leukemia patients groups and control group have shown important difference as statistically (p<0,05). The mean values of Fas and Fas Ligand levels were higher in ALL and AML patients. The comparison of the mean values of Fas and Fas ligand levels in ALL and AML patients have shown no difference (p>0,05). The comparison of the Fas levels in ALL patients according to immunophenotypes; CALLA(+) B-ALL have higher mean level than T-ALL and shown important difference as statistically (p<0,05). The comparison of the mean values of Fas level at the diagnosis in ALL patients who had relapsed and patients who had remission have shown important difference (p<0,05). The mean values of Fas level were found higher in relapsed ALL patients. In these results showed that Fas and Fas ligand may play important role in apoptosis mechanism.

Disclosures:

No relevant conflicts of interest to declare.
MGN1703, a Novel TLR9-Agonist - Results of a Phase 1 Clinical Trial in Patients with Metastatic Malignancies.
Authors: Weihrauch Martin R , Schmidt Manuel , Tschaika Marina , Richly Heike , von Bergwelt-Baildon Michael S , Hacker Ulrich , Shimabokuro-Vornhagen Alexander , Wittig Burghardt , Scheulen Max E
Published: October 2019

Abstract 4730

Background

The DNA-based immunomodulator MGN1703 stimulates the innate and cellular immune system mainly via the TLR9-receptor. The results of the recent in vivo experiments showed potent anti-tumor efficacy of MGN1703 in several mouse tumor models in prophylactic and therapeutic settings as well as a good safety profile in various animals. Two investigator-initiated pilot trials of MGN1703 as adjuvant in patients with metastatic solid tumors also showed good safety and tolerability of the drug as well as a positive effect on the response rate in patients treated with MGN1703.

Patients/Methods

In this Phase 1 clinical trial MGN1703 is administered subcutaneously in escalating doses (0.25 mg, 2 mg, 10 mg, 30 mg, and 60 mg; 3-6 patients per group) either in a single or in a multiple (2x / week over 6 weeks) dose regimen. Patients with metastatic tumors of the following entities are recruited for the study, if no other standard treatment options are available: Colorectal cancer, breast cancer, lung cancer, renal cell carcinoma and melanoma. Primary endpoints are evaluation of the safety and tolerability of escalating single doses and of escalating multiple doses of s.c. administered MGN1703, determination of the maximum tolerated dose (MTD) and dose limiting toxicity (DLT), and recommendation of a dose for a Phase 2 trial in patients.

Results

Currently, 12 patients have been treated and evaluated in the single dose groups of 0.25 mg, 2 mg, 10 mg and 30 mg (3 patients each). In the multiple dose group, 4 patients have been treated with 0.25 mg, 3 patients with 2 mg, 3 patients with 10 mg and 3 patients with 30 mg MGN1703, so far. Therapy was well tolerated except for sporadic transient symptoms as mild redness and induration of injection sites in two patients, increase of temperature to 38 °C in one patient, and fatigue in two patients. In the 0.25 mg group, one patient showed a stable disease (SD, according to RECIST) after 6 weeks of treatment, and in the 2 mg group, 3 of 3 patients showed a SD after 6 weeks. Treatment results of the last 2 dosing groups are pending. The four patients, who responded to the treatment with MGN1703, were treated with MGN1703 for further 6 weeks within an extension phase of this clinical trial. Two of them still had a SD after 12 weeks of treatment.

Conclusions

MGN1703 showed safety and tolerability at dosages up to 30 mg so far. The detailed evaluation of clinical and immunological responses is still ongoing. There has been no DLT at this point of the Phase 1 trial.

Disclosures:

Weihrauch: MOLOGEN AG: Membership on an entity's Board of Directors or advisory committees. Schmidt:MOLOGEN AG: Employment. Tschaika:MOLOGEN AG: Employment. Wittig:MOLOGEN AG: Membership on an entity's Board of Directors or advisory committees.
Catastrophic NAD+ Depletion in Activated T Lymphocytes through Nampt Inhibition Reduces Demyelination and Disability in EAE.
Authors: Bruzzone Santina , Fruscione Floriana , Morando Sara , Poggi Alessandro , Garuti Anna , Selmo Martina , Ferrando Tiziana , Benvenuto Federica , Cea Michele , Soncini Debora , Moran Eva , Rocco Ilaria , Cirmena Gabriella , Zoppoli Gabriele , Ballestrero Alberto , Sordat Bernard , Patrone Franco , Uccelli Antonio , Nencioni Alessio
Published: October 2019

Abstract 4732

Nicotinamide phosphoribosyltransferase (Nampt) inhibitors such as FK866 are potent inhibitors of NAD+ synthesis that show promise for the treatment of different forms of cancer. Based on Nampt upregulation in activated T lymphocytes and on preliminary reports of lymphopenia in FK866 treated patients, we have investigated FK866 for its capacity to interfere with T lymphocyte function and survival. Intracellular pyridine nucleotides, ATP, mitochondrial function, viability, proliferation, activation markers and cytokine secretion were assessed in resting and in activated human T lymphocytes. In addition, we used experimental autoimmune encephalomyelitis (EAE) as a model of T-cell mediated autoimmune disease to assess FK866 efficacy in vivo. We show that activated, but not resting, T lymphocytes undergo massive NAD+ depletion upon FK866 mediated Nampt inhibition. As a consequence, impaired proliferation, reduced TNF-a production, ATP shortage, and, finally, autophagic cell demise result. We demonstrate that upregulation of the of the NAD+-degrading enzyme poly-(ADP-ribose)-polymerase (PARP) by activated T cells enhances their susceptibility to NAD+ depletion. We relate defective TNF-a production in response to FK866 to impaired SIRT6 activity. Finally, in line with its capacity to interfere with T lymphocyte function and survival, we show that FK866 strikingly reduces the neurological damage and the clinical manifestations of EAE. In conclusion, Nampt inhibitors (and possibly sirtuin inhibitors) could be used to modulate T cell-mediated immune responses and thereby be beneficial in immune-mediated disorders.

Disclosures:

No relevant conflicts of interest to declare.
Risk Factors for Candidemia in Pediatric Oncology Patients.
Authors: Zaoutis Theoklis E , Prasad Priya A , Localio A. Russell , Reilly Anne , Bell Louis M , Walsh Thomas J , Gross Robert
Published: October 2019

Abstract 4735

Background

Candida species are among the most common causes of bloodstream infection and are associated with significant morbidity and mortality, particularly in oncology patients. Few studies have identified the risk factors for candidemia in pediatric oncology patients.

Methods

We conducted a nested case-control study within a cohort of pediatric patients admitted to the oncology unit at a children's hospital between 2001 and 2004. A case was defined as a patient with a positive blood culture for Candida spp. As time at risk is an important confounding variable in nosocomial studies assessing antibiotic risk factors, we selected two controls per case based on incidence-density sampling. Demographic and clinical data were collected by medical record review. Assessment of exposures was focused on the 2 weeks prior to the development of infection (case)/study entry (control). Conditional multivariate analyses were performed to determine independent risk factors for candidemia.

Results

We identified 45 oncology patients with candidemia during the study period. The most commonly isolated species were C. albicans (49%), C. parapsilosis (18%), C. glabrata (9%), and C. lusitaniae (9%). The median age of cases was 6.2 years (Interquartile Range (IQR): 2.9– 13.8 years). Median time to candidemia was 12 days (IQR: 4-22 days). There was no significant difference in mortality between cases (7%) and controls (7%). Independent risk factors for candidemia included receipt of aminoglycosides for >3 days (OR: 10.51, CI: 2.96, 64.43), receipt of total parenteral nutrition (OR: 13.82, CI: 2.48, 44.49), and Graft versus Host Disease (GVHD) (OR: 15.09, CI: 0.91, 250.24).

Conclusion

The receipt of aminoglycosides for >3 days, use of TPN, and GVHD were independently associated with candidemia in pediatric oncology patients. These results may inform targeted interventions to reduce the risk of candidemia in pediatric oncology patients.

Disclosures:

Zaoutis: Merck & Co.: Research Funding; Cephalon: Research Funding; Enzon: Research Funding.
Combined Phosphoepitope Specific Flow Cytometry and MHC-Multimer Staining - A New Tool to Monitor Antigen-Specific Signaling in CMV-Specific T-Cells.
Authors: Kapp Markus , Thiele Rainer , Baumeister Elke , Fick Kerstin , Stuhler Gernot , Opitz Andreas , Einsele Hermann , Grigoleit Götz Ulrich
Published: October 2019

Abstract 4737

Flow cytometry has become a routine method in both clinical and basic immunological research. Its ability to differentiate between distinct populations of cells by surface staining of various parameters is a main advantage since we have the possibility to identify antigen-specific T-cells by flow cytometry through the development of soluble multimeric peptide–MHC complexes. Nevertheless, surface staining does not provide information about the functionality of the analyzed cell populations. Hence, further methods have been described to define cells by detection of intracellular epitopes. These assays include the intracellular staining of distinct cytokines or phosporylated signaling molecules (Phosflow). MHC-multimer approaches combined with intracellular cytokine staining are routinely used, whereas the detection of intracellular p-kinases under MHC-multimer staining applying the Phosflow-protocols has not been realized so far. The use of phosphoepitope analysis in antigen-specific T-cells is of high interest in infections or especially during immunosuppressive drug treatment. Therefore, we aimed to establish a dual multimer-phospho-staining protocol to provide a method to get insight into the biochemical signaling processes in antigen-specific T-cells. We chose CTL responses against CMV as model system due to well established epitopes and high frequency in healthy donors. The original Phosflow-protocols did not turn out to be suitable for a combination with MHC-multimer staining. The very harsh fixation and permeabilization procedures largely or completely abrogated the antigen-specific staining. We have been able to stain both the CMV-specific T-cell-receptor and phosphorylated kinases following polyclonal stimuli (e.g. PMA, IL-2 etc.) using different protocols for some p-kinases (ERK, STAT5, NfKB, p38). These protocols allow a combination of specific T-cell-receptor staining with that of intranuclear phosphoepitopes after polyclonal stimulation. In preliminary experiments, we have also been able to show a specific phosphorylation of the ERK molecule after stimulation with CMV-specific artificial antigen-presenting cells or antibody-coated plates. As mentioned above, the use of phosphoepitope analysis in antigen-specific T-cells may offer the possibility to correlate immunological anergy with distinct signaling processes in defined clinical situations, e.g. in immunosuppressed patients post alloSCT.

Disclosures:

No relevant conflicts of interest to declare.
Multicentric prospective Study of Differential Treatment for Elderly Patients Affected by Aggressive Non Hodgkin Lymphoma Based On a Geriatric Multidimensional Analysis.
Authors: Gini Guido , Olivieri Attilio , Bocci Caterina , Catarini Massimo , Visani Giuseppe , Montanari Mauro , Guiducci Barbara , Isidori Alessandro , Brunori Marino , Alesiani Francesco , Giuliodori Luciano , Marcellini Massimo , Offidani Massimo , Caraffa Patrizia , Stronati Andrea , Capelli Debora , Trappolini Silvia , Leoni Pietro
Published: October 2019

Abstract 4775

From November 2002 to March 2006 in the centres of the region Marche participating in the study have been diagnosed 91 new cases of lymphoma B diffuse large cells in patients aged 65 years or more.

All the 91 patients enrolled prospectively in our study were assessed on the basis of multidimensional geriatric evaluation (MGE).

MGE analysed in detail the following aspects of elderly: Functional status, Socio-economic conditions, State cognitive, State emotional, Therapy pharmacology, State nutritional and every geriatric syndromes.

On the basis of MGE we have identified 3 groups. The first group was composed of the patients who had no disability and that they could receive a standard chemotherapy with CHOP-Rituximab scheme.

The second group of patients had at most 2 co-morbidities of moderate grade in the absence of geriatric syndromes. These patients have received a therapy chop-like with anthracycline liposomal peghilated (Caelyx).

The third group composed of “frail patients” showed over the 85 years, 3 or more co-morbidities ADL <6 and one or more geriatric syndromes. These patients run therapy with chop scheme with reducing dose of 50%.

54 patients (59%) were allocated to the first group, 22 (25%) in the second and 15 (16%) in the third. In the first group, after the treatment, 82% has obtained the complete remission, and in the second and third group is obtained respectively in 64% and 60%. It was observed 1 death toxic to the first and third group and 2 in the second. Currently with a median follow-up of 55 months are live in complete remission 31 patients (57%) of the group 1, 7 patients (32%) of the group 2 and 3 patients (20%) of the group 3. The MGE has proven a useful instrument for an objective evaluation of elderly patients and has been possible to guide to the choice of therapy more appropriate also in patients that showed relevant co-morbidities.

Disclosures:

Offidani: Celgene: Honoraria; Janssen Cilag: Honoraria.
Histone Deacetylase Inhibitors Demonstrate Significant Preclinical Activity as Single Agents, and in Combination with Bortezomib in Waldenstrom's Macroglobulinemia.
Authors: Sun Jenny , Xu Lian , Tseng Hsiuyi , Ciccarelli Bryan , Fulciniti Mariateresa , Hunter Zachary , Maghsoudi Kaveh , Hatjiharissi Evdoxia , Zhou Yangsheng , Yang Guang , Zhu B. , Liu Xia , Gong Ping , Ioakimidis Thea , Sheehy Patricia , Manning Robert , Hanzis Christina , Lewicki Megan , Patterson Christopher J , Chauhan Dharminder , Rodig Scott J. , Munshi Nikhil C. , Anderson Kenneth. C , O'Connor Owen O , Treon Steven
Published: October 2019

Abstract 4785

Waldenstrom's Macroglobulinemia (WM) is a B-cell lymphoproliferative disorder characterized by bone marrow infiltration of CD19+ cells and production of a monoclonal IgM protein. Despite advances in treatment, WM remains incurable. As part of these efforts we sought to define the role of HDAC-inhibitors in WM. Gene expression profiling of bone marrow CD19+ cells from 30 WM patients and 10 healthy donors showed over-expression of HDAC4, HDAC9, and Sirt5 in WM patients. Evaluation of the HDAC inhibitors suberoylanilide hydroxamic acid (SAHA or Vorinostat), Trichostatin A (TSA), LBH-589 (Panobinostat), and sirtinol demonstrated dose dependent killing of BCWM.1 cells with IC50 of 3.5 uM, 70 nM, 0.8 uM, and 30 uM, respectively, whilst the combination of these agents with bortezomib resulted in at least additive tumor cell killing. TSA is more potent than bortezomib in inducing apoptosis in primary WM tumor cells in patients with prior treatment. TSA and bortezomib showed synergistic effect in 25% of the patients samples tested. We also observed that TSA and bortezomib-induced apoptosis of BCWM.1 cells depended on the activation of a similar set of caspases. Conversely, changes in cell cycle regulators were distinctly different between TSA and bortezomib treated BCWM.1 cells. The results of these studies demonstrate over-expression of distinct members of HDAC in WM cells, and provide a framework for the examination of HDAC-inhibitors as monotherapy, as well as combination therapy with bortezomib in the treatment of WM.

Disclosures:

No relevant conflicts of interest to declare.
Low-Dose Bexarotene Alone or in Combination for the Treatment of Relapsed or Refractory Cutaneous T-Cell Lymphoma.
Authors: Beltran Brady , Paredes Antonio , Moises Celia , Sanchez Gadwin , Sokol Lubomir , Castillo Jorge
Published: October 2019

Abstract 4790

Introduction

Selective ligands of retinoid X receptor (i.e. bexarotene) are used in the treatment of early and advanced stages of cutaneous T-cell lymphoma (CTCL). The objective of this study is to report a Peruvian single-institution experience on 13 cases of relapsed or refractory CTCL who were treated with low-dose bexarotene alone or in combination with phototherapy or interferon alfa-2a.

Methods

From 2002 to 2008, thirteen patients with relapsed or refractory CTCL were treated with bexarotene at low dose (75-300 mg/day) as monotherapy (BEX; 3 cases) or in combination with interferon alfa-2a (INF) at 3-9 million units subcutaneously three times a week (5 cases) or phototherapy (PT; 4 cases). Therapy was continuously administered until progression of disease or unacceptable toxicity. Eight patients with mycosis fungoides (MF), two patients with aggressive epidermotropic CD8+ cytotoxic lymphoma (AECL), two patients with Sezary Syndrome (SS) and one with smoldering adult T-cell leukemia/lymphoma (ATLL) were included in this study.

Results

Mean age was 57 years (range 26 to 74 years). In average, patients were exposed to three prior lines of therapy (range 1 to 5). From the 8 cases of MF, 3 cases were treated with BEX monotherapy, achieving 1 complete response (CR), 1 partial response (PR) and 1 progressive disease (PD); 3 cases were treated with BEX and PT, obtaining 1 CR and 2 PR; and 2 cases were treated with BEX and IFN, both showing PD. From the 2 cases with AECL, 1 case received BEX/PT achieving a PR and 1 case received BEX/IFN showing PD. From the 2 cases with SS, both were treated with BEX/IFN achieving a PR. The case of ATLL achieved a PR with BEX/PT. The overall response for the entire group was 69.2% (9/13) with a mean duration of response of 15 months (range 2 to 67 months). From the therapy point of view, BEX alone achieved a 66% overall response rate (ORR), BEX/IFN showed a 40% ORR and BEX/PT showed a 100% ORR with a 20% CR. Most common adverse events were mild, seven patients experienced grade 1–2 hypercholesterolemia, and hypertriglyceridemia grade 1–2 was seen in seven patients and grade 3 in two cases. Four patients developed grade 1–2 hypothyroidism.

Conclusions

Low-dose BEX alone or in combination with INF or PT is effective in the treatment of patients with relapsed or refractory CTCL and is associated with mild toxicity. Prospective studies, if possible, are needed to further investigate novel therapeutic approaches in these hard-to-treat patients.

Disclosures:

No relevant conflicts of interest to declare.
Artesunate,a Antimalarial Agent,Exhibits Potent Anti-Leukemia Activities in Vitro and Inbibit Wnt/b-Catenin Pathway.
Authors: Xu Na , Xiaoli Liu , Du Qingfeng , Liu Zhi , Li Rong , Yang Jun , Wang Shuang , Zhong Min , Wang Yu
Published: October 2019

Abstract 4822

Artesunate (ART), a remarkable antimalarial agent, its antitumor effect was widely studied recently. But whether the antitumor mechanisms of ART involves in Wnt/b-catenin signalling, a major oncogenic pathway have yet been poorly understood. In address this question,we treated human leukemia cell lines,such as K562 cells, HL-60 cells,U937cells and KG1a cells with 12.5ug/ml,50ug/ml, 100 ug/ml and 200ug/ml ART for 24h, 48h and 72h respectively. Determined by the Cell Counting Kit-8 and flow cytometry analysis on apoptosis, we found that ART suppressed the leukemia cells proliferation and promoted the apoptosis of leukemia cells in a time- and concentration-dependent manner. Furthermore, we detected the mRNA level and protein expression of b-catenin and Wnt/b-catenin target genes c-myc and cyclinD1 by QRT-PCR and western blotting when the above leukemia cell lines treated with ART for 48h. The results demonstrated that the the mRNA level and protein expression of c-myc and cyclinD1 was lower in ART groups than in the contorl groups,and in concentration-dependent manner. on the other hand, there was no significant difference in the mRNA level and protein expression of b-catenin in the ART groups compare to the contorl groups. Moreover, we observed b-catenin by immunofluorescence technology, the Bioluminescent imaging demonstrated that ART translocated b-catenin from nucleus to adherent junctions of membrane,and binding to E-cadherin on the cell membrane. Our results provide the vitro evidence for the anti-luekemia mechanism of ART correlated to the inhibition of hyperactive Wnt/b-catenin signaling pathway. Combination With the present studies, the antitumor mechanism of ART is invoved in multiple signaling pathways, and the well known low toxicity. We thus speculate that ART might be a promising candidate drug for the treatment of leukemia.

Disclosures:

No relevant conflicts of interest to declare.
Obesity Adversely Affects Survival of Pediatric Patients with Severe Aplastic Anemia After Hematopoietic Cell Transplantation.
Authors: Barker Collin C. , Agovi Manza- A , Logan Brent , Gupta Vikas , Lazarus Hillard M. , Ballen Karen K , Hale Gregory A. , Frangoul Haydar , Rizzo J. Douglas , Pasquini Marcelo C.
Published: October 2019

Abstract 652

Prevalence of obesity in children has been rapidly rising in the last decade in the US and other industrialized countries (US prevalence, ages 2-19, 16%, NHANES, 2008) and it represents a serious health concern. The impact of recipient's weight on mortality after hematopoietic cell transplantation (HCT) for non-malignant diseases is not well characterized. We studied the effect of body mass (BMI) on transplant outcomes in 1,281 patients aged 2-19 years who underwent HCT for severe aplastic anemia from 1990-2005. The study population was divided in five groups based on age-adjusted BMI percentiles. Patients in the <5th, 5-25th, 26-75th, 76-95th and >95th percentile BMI were classified as underweight, at risk for underweight, normal weight, at risk for overweight, and overweight respectively. Cox proportional hazards regression models for survival and acute graft-versus-host disease grade III-IV (aGVHD) were performed using BMI groups as the main effect and the normal weight (26-75th percentile) as the baseline comparison. Two-year overall survival was lower for overweight children (p<0.001, table). The risk of death, adjusted for significant covariates was higher for overweight compared to normal weight children (p=0.001, table). BMI groups were not associated with excess risk for aGVHD. Other significant factors associated with mortality include race and region of US, donor type, performance score at HCT, recipient age, and year of transplant. Risk of death was higher in African American patients (1.84, p=0.009) compared to Caucasians in the US, donors other than HLA-matched sibling and patients who received HCT before 2002. Among recipients of HLA-matched sibling grafts, use of busulfan-containing regimens (3.13, p<0.001) or cyclophosphamide without antithymocyte globulin (ATG) (1.92, p=0.03) had a higher risk of death compared to recipients of cyclophosphamide and ATG. Conditioning regimens in recipients of unrelated donor HCT were not associated with mortality. Among the reported causes of death, organ failure and infections occurred more frequently in overweight patients compared to the other BMI groups. In conclusion, overweight children with severe aplastic anemia have a higher risk of death after HCT independent of other factors analyzed in the study.BMI percentileN100 day mortality Probability (95% CI)2-y OS Probability (95% CI)RR of death (95% CI)p26-75th152515 (10-20)76 (70-82)1.00-<5th10812 ( 7-19)77 (68-84)1.01 (0.65-1.57)0.975-25th19611 ( 9-14)77 (74-81)1.38 (0.99-1.91)0.0676-95th30417 (13-21)73 (68-78)1.22 (0.93-1.61)0.14>95th14229 (22-36)59 (51-67)1.71 (1.24-2.35)0.0011Baseline group

Disclosures:

No relevant conflicts of interest to declare.
Differential MLL Interaction and H3K4me3 Mark Maintenance at PU.1 Regulatory Region by AML Translocations-Associated Oncoproteins AML1-ETO and CBFβ-SMMHC.
Authors: Huang Gang , Yan Xiaomei , Zhao Xinghui , Rao Yalan , Sashida Goro , Bowie Rajeana
Published: October 2019

Abstract 674

The interplay between genetic and epigenetic regulators is a fundamental mechanism to ensure gene regulation and cell fate decision. Hematopoiesis and leukemia are excellent systems in which to study this process. The Mixed-Lineage Leukemia (MLL) protein, a Set1-like H3K4 methyltransferase, and the heterodimeric transcription factor RUNX1 (AML1)/CBFβ are critical for definitive and adult hematopoiesis. They are required for the generation of all hematopoietic lineages and act as tumor suppressors in human leukemia. PU.1 is a critical downstream target gene for AML1 in adult hematopoiesis. AML1 regulated PU.1 through 3 AML1 binding sites in the PU.1 URE region (1). AML1 is responsible for the H3K4me3 mark at PU.1 URE and promoter region. AML1-ETO represses PU.1 expression through PU.1 URE region (2). Dysregulation of PU.1 levels cause leukemia in both mouse and human. PU.1 is absolutely required for the normal development of monocytic and B cell lineages, in which the core binding factor (CBF) fusions (CBFβ-SMMHC or TEL-AML1) or MLL fusions (MLL-AF9 or MLL-AF4) have been high frequently identified in human acute leukemias. These suggested that AML1/CBFβ and MLL might regulate PU.1 expression and AML1/CBFβ or MLL fusions might dysregulate PU.1 expression at genetic and epigenetic levels and eventually develop similar leukemias. We found that the AML1-ETO/CBFβ complex interacts with MLL in a similar manner as AML1/CBFβ (3), while the AML1/CBFβ-SMMHC complex interacts with MLL more strongly. Surprisingly, the CBFβ-SMMHC by itself interacts with MLL through the junction region of the fusion protein. The interactions with MLL by these fusion proteins complexes, AML1-ETO/CBFβ and AML1/CBFβ-SMMHC, correlate with their ability to maintain H3K4me3 levels at PU.1 URE and promoter regions in 416B cell lines stable expressing AML1-ETO or CBFβ-SMMHC. Taken together, our data indicate that AML1-ETO/CBFβ complex preserves the interaction with MLL, while the AML1/CBFβ-SMMHC complex enhances it. This suggests that these two leukemogenic fusions downregulate PU.1 expression through different epigenetic mechanisms in the presence of H3K4me3 mark at PU.1 regulatory region. The effects of these differential interactions on the H3K4me3 mark maintenance and on target gene expression, particularly PU.1, may be critical for the aberrant regulation that underlies the etiology of M2 and M4Eo acute myelogenous leukemia (AML).

1. Huang G, et al. Nat. Genet. 2008; 40: 51-60

2. Zhang P, et al. Blood 2008; 112: 594.

3. Huang G, et al. Blood 2008; 112: 282.
Combined Modality Treatment with Intensified Chemotherapy and Dose-Reduced Involved Field Radiotherapy in Patients with Early Unfavourable Hodgkin Lymphoma (HL): Final Analysis of the German Hodgkin Study Group (GHSG) HD11 Trial.
Authors: Borchmann Peter , Diehl Volker , Goergen Helen , Mueller Horst , Mueller Rolf Peter , Eich Hans T. , Mueller-Hermelink Hans Konrad , Herrmann Richard , Markova Jana , Ho Anthony D. , Hiddemann Wolfgang , Doerken Bernd , Greil Richard , Engert Andreas
Published: October 2019

Abstract 717

Purpose:

Combined modality treatment consisting of 4 cycles of chemotherapy (CT) followed by involved field radiotherapy (IF-RT) is the standard treatment for early unfavourable HL. In our prior trial for this group of patients (HD8), overall survival (OS) and freedom from treatment failure (FFTF) at 5 years were 91% and 83%, respectively. The HD11 trial thus addressed two major questions: (1) improving outcome by intensifying CT (4xABVD vs. 4xBEACOPPbaseline; Bbas) and (2) defining the best radiation dose (30Gy vs. 20Gy IF-RT).

Patients and methods:

Between May 1998 and January 2003, 1395 eligible patients aged 16–75 years with untreated early unfavourable stage HL (CS I, IIA with at least one of the risk factors large mediastinal mass (a), extranodal disease (b), elevated ESR (c) or ≥ 3 nodal areas (d); IIB with risk factors c and/or d) were randomized into one of the following 4 treatment arms: 4xABVD + 30Gy (A), 4xABVD + 20Gy (B), 4x Bbas + 30Gy (C) or 4x Bbas + 20Gy (D). Since there are strong indications for an interaction between CT- and RT-doses, a comparison of pooled treatment arms (A+B vs. C+D for comparison of 4×ABVD vs. 4× Bbas and A+C vs. B+D for comparison of 30Gy IF-RT vs. 20Gy IF-RT) would be misleading. Therefore all treatment arms were analysed separately.

Results:

Patient characteristics were well balanced between the 4 arms (median age 33 years, 49% male, 6% stage I, 29% B-symptoms). CT- and RT-related acute toxicity occurred significantly more often in the arms with the more intensive therapy (CT: 74.1% vs. 51.8%; RT: 12.3% vs. 5.5%). The complete remission rate 3 months after end of therapy was 94.1% for the whole group and did not differ significantly between the 4 arms. The 5-year estimate of FFTF (primary endpoint) is 85.0% (OS 94.5%, PFS 86.0%).

Bbas is more effective than ABVD if followed by 20Gy IF-RT (5y-FFTF difference 5.7%, 95%-CI [0.1%; 11.3%]). This effect does not exist in combination with 30Gy IF-RT (5y-FFTF difference 1.6% [-3.6%; 6.9%]). Similar results are observed for the RT-question: After 4 cycles of Bbas, 20Gy is not inferior to 30Gy (5y-FFTF difference -0.1%, 95%-CI [-5.1%; 4.9%]), whereas after 4xABVD, a relevant inferiority of 20Gy cannot be excluded (-4.0% [-9.5%; 1.4%]).

Conclusion:

A reduction of RT dose from 30Gy to 20Gy IF-RT seems to be justified only in combination with Bbas, but not with a less effective chemotherapy such as 4xABVD. Patients will benefit from an intensified CT such as Bbas only in combination with 20Gy IF-RT but not with 30Gy IF-RT.

Disclosures:

No relevant conflicts of interest to declare.
Mapping the Protease Activated Receptor 4 (PAR4) Homodimer Interface.
Authors: Nieman Marvin T
Published: October 2019

Abstract 773

Thrombin activates platelets by binding and cleaving protease activated receptors 1 and 4 (PAR1 and PAR4). PAR1 and PAR4 communicate with each other to lower the concentration of thrombin required for PAR4 activation (Nieman Biochemistry, 2008). In addition, PAR1 and PAR4 form homo and heterodimers. However, where these receptors interact has not been defined and it is not known if dimerization influences receptor activation, downstream signaling, or both. Since PAR4 activation is important on human and mouse platelets, we sought to characterize the interaction site between PAR4 homodimers. Using bioluminescence resonance energy transfer (BRET), we mapped the PAR4 homodimer interface. The PAR4 homodimers show a specific interaction as indicated by a hyperbolic BRET signal in response to increasing PAR4-GFP expression with a fixed concentration of PAR4-Rluc. The threshold maximum BRET signal was disrupted in a concentration-dependent manner by unlabeled PAR4. In contrast, the unrelated G-protein coupled receptor, rhodopsin, was unable to disrupt the BRET signal indicating that the disruption of the PAR4 homodimer is a specific interaction. We have mapped the region required for PAR4 homodimer formation using chimeras between rhodopsin and PAR4. PAR4 does not interact with rhodopsin in BRET assays. Using a library of rho-PAR4 chimeras that have the junction at the beginning of transmembrane (TM) 2, 3, 4, 5, 6 or 7, we determined where dimer formation is restored. When the junction is placed at the beginning of TM4 or TM5, the chimera does not interact with PAR4-WT. In contrast, when the junction is moved to the end of TM2, the BRET signal is restored. These results indicate that the region on PAR4 required for homodimer formation encompasses a 63 amino acid region that includes the first extracellular loop, TM3 and the second intracellular loop. These studies establish techniques that may be used to define the interactions between other GPCRs found on the platelet surface. These receptor-receptor interactions may be another level of regulation of agonist activity and platelet function in vivo and may provide novel targets for anti-platelet therapies.

Disclosures:

No relevant conflicts of interest to declare.