bluuebunny/crossref_metadata_2025 huggingface dataset:
HSCT PATIENTS with HHV6 Reactivation HAD LOW NUMBERS of CD4+ Effector Memory Lymphocytes IN BLOOD WHAT Associates with Rather POOR SURVIVAL.
Authors: Lange Janusz , Drabczak-Skrzypek Daria , Dlubek Dorota , Jaskula Emilia , Wolowiec Dariusz , Lange Andrzej
Published: October 2019

Abstract 4555

It is known that the pace of immunological recovery influences the outcome of HSCT and herpes viruses reactivation contributes significantly to the post transplant mortality.

The aim of this study was to investigate reconstitution of subpopulations of CD4+ lymphocytes in the context of β-herpes viruses reactivation shortly post HSCT. There were two observation points: (1) at the beginning of lymphocyte recovery (number of lymphocytes > 200 cells/ul) which was seen from 9 to 31 day post HSCT median 14 and (2) 3 weeks later (median day 35 range(31-53)). CD4+ T cell subsets were defined as follow: Naive cells (N) CD4+CCR7+CD45RA+, Central memory (CM) CD4+CCR7+CD45-, Effector memory(EM) CD4+CCR7-CD45+, Terminally differentiated memory cells (TEMRA) CD4+CCR7-CD45RA+ . CMV, EBV and HHV-6 DNA copies were detected in blood with the use of QT-PCR in one week intervals. The study included 44 patients (age 1 to 64 median 45yr., 21 ric/mac, 21/23, sib/mud 21/23 mud, BM/PBPC 4/40.

It was found;

1. Fifteen, 13 and 4 patients were positive for EBV, HHV-6 and CMV DNA copies during the observation period.

(i) CD4+lymphocyte count was lower in patients having CMV copies as compared to those having EBV and/or HHV6 copies (both lymphocytotropic viruses) at the beginning of lymphocytes recovery (45/ul vs 120/ul p=0,047 Mann-Whitney U test), similarly behaved EM cells. 22/ul vs 84/ul EM cells (p= 0,088 Mann-Whitney U test) were seen in pts having CMV reactivation and in those with EBV and/or HHV-6 reactivation, respectively.

(ii) The situation changed three weeks later, it appeared that patients having HHV6 reactivation any time during the observation period had lower CD4+ lymphocyte numbers as compared to patients lacking β-herpes viruses reactivations (CD4+ cells median 93/ul vs190/ul; p=0,096 Mann-Whitney U test) and to those with CMV and EBV reactivation. Again EM cells contributed greatly to these differences. EM CD4+ lymphocytes were 41/ul, 105/ul, 80/ul and 160/ul in patients having HHV-6, CMV or EBV and lacking beta-herpes viruses reactivation, respectively. EM CD4+ lymphocyte numbers for HHV6+ patients as compared to patients lacking β-herpes viruses reactivations was 41/ul vs155/ul.(p=0,012 Mann-Whitney U test).

Conclusions: It appears that HHV6 is the main player in shaping lymphocyte reconstitution post transplant affecting predominantly EM cells what influences the outcome of transplantation. In this group of patients probability of one year survival was 50% vs 65%, in patients having and lacking HHV6, respectively.

Disclosures:

No relevant conflicts of interest to declare.
DynaChip Anti-HLA Antibody Analysis In Sera of Patients Following Allo-HSCT From HLA-Mismatched Unrelated Donors.
Authors: Markiewicz Miroslaw , Siekiera Urszula , Kruzel Tomasz , Dzierzak-Mietla Monika , Zielinska Patrycja , Kyrcz-Krzemien Slawomira
Published: October 2019

Abstract 4575

Introduction:

Anti-HLA antibodies constitute potentially important factor that may influence outcomes of HLA-mismatched allogeneic hematopoietic stem cell transplantation (allo-HSCT). The rationale of this study was to detect presence of anti-HLA antibodies in recipients of allo-HSCT from HLA-mismatched unrelated donors.

Patients and Methods:

Anti-HLA-A,B,C,DR,DQ,DP antibodies were identified in sera collected from 46 recipients of allo-HSCT from HLA-mismatched unrelated donors. Sera were collected between 1 month and 5.5 years after allo-HSCT, and additionally before allo-HSCT in 17 pts. We have used microchips spotted with purified HLA class I and HLA class II antigens to allow binding of anti-HLA antibodies present in tested sera to the surface of the microchip, pre-optimised reagents and DynaChip Processor (Dynal Invitrogen Corporation) for assay processing, data acquisition and analysis.

Results:

Antibodies against HLA class I, II or I and II were detected in 15%, 11% and 35% of pts whereas no antibodies were detected in 39% of patients. Antibodies were directed against HLA-A, B, C, DR and DQ in 37%, 46%, 35%, 48% and 35% of pts, respectively. Pre-transplant anti-HLA antibodies have been detected in 7 pts (41%) out of 17 tested before allo-HSCT. In this group percent of Panel Reactive Antibodies (% PRA) increased following allo-HSCT in 3 pts and decreased in 4. In 5 out of 10 remaining pts without pre-transplant antibodies, %PRA increased post-transplant. DynaChip software allowed to define specificities of HLA-A,B,C,DR and DQ antibodies on low and high resolution levels. The specificity of antigens that masked results of antibody identification has been also defined in 2 pts. At this stage we did not define exactly whether detected anti-HLA antibodies were donor-specific. Cross-reactive groups (CREG's) analysis has been also used to compare antibodies’ reactivity. Anti-HLA-DP antibodies were not detected in the examined group of transplanted patients.

Conclusions:

Presented preliminary study results indicate, that anti-HLA antibodies can appear post-transplant in mismatched allo-HSCT recipients. Further analysis aiming to evaluate their influence on transplant outcomes is ongoing. We intend to extend the search for anti-HLA antibodies with use of Luminex LabScreen method.

Disclosures:

No relevant conflicts of interest to declare.
Survival Impact of Response with Bortezomib Induction on Myeloma Patients Receiving Autologous Stem Cell Transplantation.
Authors: Min Chang-Ki , Eom Hyeon Seok , Cho Byung Sik , Kim Yoo-Jin , Kim Hee-Je , Lee Seok , Cho Seok-Goo , Kim Dong-Wook , Lee Jong Wook , Min Woo-Sung , Park Chong-Won
Published: October 2019

Abstract 4590

Autologous stem cell transplantation (ASCT) is the standard of care for patients with multiple myeloma (MM) aged less than 65 years. Before the era of novel agents the absence of initial response to induction therapy had no impact on outcome after ASCT. With the introduction of novel agents response rate with initial therapy are now between 70% and 100%. This study analyzes the association of progression-free survival (PFS) and overall survival (OS) after ASCT with the response to induction therapy with a regimen that contains bortezomib for patients with MM.

We performed a retrospective analysis of 61 patients with MM who consecutively received bortezomib-containing regimens from 2 institutes before collection of peripheral blood stem cells and ASCT. The median age was 54 (34-65) years and 41 (67.2%) were male. Eight (16.4%) patients received bortezomib as a first-line therapy whereas the others as the second line or more. Melphalan alone was used for conditioning and 29 (47.5%) received thalidomide maintenance therapy.

Fifty four of 61 patients (88.5%) achieved objective response after bortezomib induction treatment. Post-bortezomib complete response (CR), very good partial response (VGPR) and partial response (PR) were present in 33 (54.1%), 6 (9.8%) and 15 (24.6%), respectively. After ASCT CR, VGPR and PR were 41 (67.2%), 3 (4.9%) and 6 (9.8%), respectively. Achieving CR, VGPR or PR after bortezomib therapy failed to show an improvement in PFS and OS after ASCT. In contrast, post-transplant responses markedly influenced the outcomes after ASCT; patients achieving either CR or ≥VGPR had significantly longer PFS (P=.001 and .035, respectively) and OS (P=.002 and .002, respectively) compared to those without CR or ≥VGPR. Post-transplant maintenance treatment was not associated with improved PFS and OS in this cohort.

Our data suggest that the response to bortezomib induction therapy could not predict outcome after ASCT. Achieving post-transplant CR or ≥VGPR is critical for prolongation of PFS and OS.

Disclosures:

No relevant conflicts of interest to declare.
Comparasion of Hyper-CVAD Chemotherapy with Autologous Stem Cell Transplantation In Patients with Peripheral T Cell Lymphomas: A Single Centre Report.
Authors: Xu Yang , Wu Xiaojin , Wang Ying , He Guangsheng , Sun Aining , Qiu Huiying , Tang Xiaowen , Fu Chengcheng , Han Yue , Jin Zhengming , Miao Miao , Depei Wu
Published: October 2019

Abstract 4601

Peripheral T-cell lymphoma (PTCL) is generally characterized by poor prognosis. To determine the role of Hyper-CVAD chemotherapy and autologous stem cell transplantation (ASCT) in PTCL, we retrospectively analyzed the outcomes of 31 patients with PTCL between 1999 and 2009. 15 patients received Hyper-CVAD chemotherapy with 3-year overall survival (OS) of 52.4% and 3-year progression free survival (PFS) of 25.7%. 16 patients received ASCT with 3-year OS of 76.2% and 3-year PFS of 61.3%. There was significant difference in 3-year PFS between the two treatments (P=0.012). Additionally, patients underwent ASCT with elevated LDH, ≥ 2 IPI points and extranodal involvement had a favorable outcome comparing with the ones received Hyper-CVAD chemotherapy. These findings might suggest that ASCT likely offer a durable survival benefit for patients with aggressive peripheral T cell lymphoma.

Disclosures:

No relevant conflicts of interest to declare.
Are Communication Tools Effective in Increasing Reporting Rate of Acute Hemolytic Reactions (AHR) In ITP Patients Treated with Rh (D) Immune Globulin?
Authors: Guirgis Faiz , Muller Carmen , Little Gregg , Sinclair Chris , Genereux Maurice
Published: October 2019

Abstract 4677

BACKGROUND:

Acute Hemolytic Reactions (AHR) associated with administration of Rh(D) Immune Globulin (WinRho® SDF, Cangene Corporation, Winnipeg, MB, Canada) have been infrequently reported during the last 15 years. AHR's as defined were any case reporting hemoglobin decrease, haptoglobin decrease, elevation of bilirubin occurring within hours to number ofdays after WinRho® SDF treatment irrespective of the degree of change from baseline. AHR's where plasma or urinary hemoglobin were positive were categorized as cases of Intravascular Hemolysis (IVH).

AIMS/PURPOSE:

To determine if communication tools such as publication of Case series (Gaines), Dear Doctor Letters and label changes stimulated reporting of AHR's and IVH.

METHODS:

Cangene's WinRho® SDF safety database from USA. was reviewed and analysed for the period 1995–2009. The reporting rate of AHR and IVH were calculated based on cases submitted by year and estimates of number of doses distributed in USA for ITP. These rates were trended to determine if the communication of safety risk was effective in stimulating reporting and decreasing rates of AHR or IVH cases.

RESULTS:

Gaines published 15 cases of IVH after WinRho® SDF in April 2000. Label changes and Dear Healthcare Professional (DHCP) letters were issued in 2000. Gaines reported 6 cases of disseminated intravascular coagulation with IVH in September 2005 and label was changed with Dear Healthcare Professional letters issued in Dec 2005. Reporting rate of IVH was not affected by these warnings. Increase in reporting rate of AHR's was seen after publications and warning letters in 2000 and 2005.

CONCLUSION:

The overall reporting rate of AHR (including IVH) was estimated as infrequent (0.12%) while IVH reporting frequency was rare (0.03%).

Despite significant communication through FDA publications, medical education efforts and DHCP letters, there has been no appreciable change in reporting frequency of hemolytic events following Rh (D) Immune Globulin administration. This suggests that the initial estimate of IVH occurring at approximately 1:1000 doses (Gaines 2000) is still appropriate.

Disclosures:

Guirgis: Cangene Corporation: Employment. Muller:Cangene Corporation: Employment. Little:Cangene Corporation: Employment. Sinclair:Cangene Corporation: Employment. Genereux:Cangene Corporation: Employment.
Design and Construction of Multi-Epitope Fusion Gene Vaccine of Wilm Tumor Gene WT1 and Stimulating Epitope of Mycobacterium Tuberculosis Heat Shock Protein 70
Authors: Tian Weiwei , Qiao Zhenhua , Ye Fang
Published: October 2019

Abstract 4713

Objective

DNA vaccination respresents a novel therapeutic strategy for cancer. Researchs have showed DNA vaccines encoding Wilms tumor gene WT1-Derived HLA *0201-restricted epitopes effectively induce epitope-specific human cytotoxic T-lymphocytes (CTLs) and kill human leukemia cells in vitro. Nevertheless, there is still a need to increase the potency of DNA vaccine and span the HLA diversity of a target population. In this study, we designed polytpoe WT1 DNA vaccine encoding multiple CTL and helper T-lymphocyte (HTL) epitopes and containing HSP70407-426 as immunoadjuvants for immunotherapy.

Methods

On the basis published data, To design a Multi-epitope gene (Multi-WT1) containing three HLA *0201-restricted, one HLA *2402-restricted epitopes, two HTL epitopes and one universal Th Pan- DR epitope (PADRE) sequence. The construct also included IgG K chain leader sequence as signal peptide to facilitate transport into the endoplasmic reticulum and Kozak sequence at the N-terminus to support translation of the gene transcript. DNA-coding sequesces was used computer-based modeling to optimize proteasome mediated epitope processing through the introdcution of amino acid spacer sequences, then cloned into the eukaryotic vector to construct the plasmid PcDNA3.1(+). Mycobacterial HSP70 fragment including stimulatory domain P407-426 as immunotherapy was fused to the C-terminal of Multi-WT1 for enhancing CTL activity and then inserted to PcDNA3.1(+). Transfection HEK-293T cells with the Multi -WT1-HSP70 and identify the expressed product by Western bolt.

Results

Most of Multi -WT1 epitopes can be cleaved by software Net Chop 3.1 and PAPROC± analysis. Western blot showed correct expression of target gene in HEK293T cells.

Conclusion

Multi-WT1-mtHSP70 fusion gene eukaryotic expression vector was successfully designed, which prepared the materials for the research of DNA vaccine.

Disclosures:

No relevant conflicts of interest to declare.
Association of Langerhans Cell Histiocytosis with Erdheim-Chester Disease: How Close Monocyte/Macrophage and Dendritic Cell Lineages Are?
Authors: Hervier Baptiste , Haroche Julien , Hermine Olivier , Donadieu Jean , Charlotte Frederic , Brousse Nicole , Hachulla Eric , Amoura Zahir
Published: October 2019

Abstract 4716

Objectives

Histiocytoses are a heterogeneous group of diseases that can be classified into either Langerhans cell histiocytosis (LCH) or non-Langerhans cell histiocytosis. The latter includes Erdheim-Chester disease (ECD). This study investigated the clinical association between LCH and ECD.

Methods

This retrospective study included 16 patients (10 males, 6 females, median age 41 years) treated at twelve different university hospitals between 1970 and 2010. Inclusion criteria were biopsy-proven LCH in association with two or more diagnostic signs of ECD.

Results

LCH and ECD were diagnosed simultaneously in 4/16 cases, whereas LCH preceded ECD in 12/16 cases. The median time interval was 7.5 years (range 2–22) in these cases. Major organs involved in LCH were the bones (n=12), skin (n=8) and lungs (n=3). ECD mainly affected the large vessels (n=11), bones (n=11) and retroperitoneum (n=9). Non-biopsy proven central nervous system (n=6) and pituitary gland (n=6) involvement also occurred. No specific histologic features were identified in the 65 biopsies studied, including platelet-derived growth factor receptor β expression. Between one and four lines of treatment were required in nine patients diagnosed with LCH. Nine patients were treated with interferon α-2a after the diagnosis of ECD was made. A partial improvement occurred in all assessable patients concerning ECD (n=5) and/or LCH (n=2). These 16 patients were compared with a monocentric cohort of 48 ECD patients; the only difference between the groups was a lower frequency of bone involvement in ECD patients with concomitant LCH (9/13 vs 47/48, p<0.003).

Conclusions

This study suggests that a pathogenic link exists between LCH and ECD. Although the mechanisms responsible for both diseases remain unknown, the present association could argue for transitions between monocyte/macrophage and dendritic cell lineages. The patient characteristics of LCH in association with ECD were similar to those in patients with LCH alone, whereas bone involvement may have been less common in ECD when it was in association with LCH. Clinicians should be aware of this association and should consider the possibility of ECD in patients with LCH, especially in the case of treatment resistance.

Disclosures:

No relevant conflicts of interest to declare.
Clopidogrel, a P2Y12 Receptor Antagonist, Potentiates the Inflammatory Response In An Animal Model of Erosive Arthritis
Authors: Garcia Analia E , Mada Sripal , Rico Mario C , De La Cadena Raul A. , Kunapuli Satya
Published: October 2019

Abstract 4721

The P2Y12 receptor plays a crucial role in the regulation of platelet activation by several agonists. The active metabolite of clopidogrel, a widely used anti-thrombotic drug, irreversibly antagonizes the P2Y12 receptor. However, treatment with clopidogrel has been associated with joint inflammation. In addition, Boilard E. et al identified micro-particles generated by activated platelets in joint fluid from rheumatoid arthritis (RA) patients. In this study we investigated whether platelet activation leads to changes in inflammatory responses using a rat model of erosive arthritis. Here, we evaluated the effect of clopidogrel on inflammation in Lewis rats in a peptidoglycan polysaccharide (PG-PS)-induced arthritis model with four groups of eight rats each: untreated group, animals receiving clopidogrel (clopidogrel group), animals receiving PG-PS (PG-PS group), and animals receiving PG-PS as well as clopidogrel (PG-PS + clopidogrel group). There were significant differences between the PG-PS + clopidogrel treated group when compared to PG-PS treated group including: increased joint diameter [4.24 ± 0.73 vs. 2.70 ± 1.09; p<0.05], clinical manifestations of inflammation, elevated leukocytes count [14.19 ± 0.2 K/uL vs. 9.7 ± 1.4 K/uL; p<0.05] at the expense of neutrophils, increased platelet count [926 ± 195 K/uL vs. 561.25 ± 97 K/uL; p>0.05], and elevated plasma levels of pro-inflammatory cytokines such as IL-1beta, IF gamma, IL-6. Plasma levels of anti-inflammatory cytokine IL-10 were significantly lower in PG-PS + clopidogrel treated group compared to PG-PS group alone. Histological observations indicated an increase in leukocyte infiltration at the inflammatory area of the joint, increased pannus formation and decrease in bone mass upon treatment with clopidogrel in PG-PS-induced arthritis animals. Accordingly to the clinical, histological and cytokine results, we conclude that clopidogrel has a pro-inflammatory effect in PG-PS-induced arthritis animal model and this might implicate that P2Y12 receptor antagonism might enhance inflammatory responses.

Disclosures:

No relevant conflicts of interest to declare.
Clinical, Genetic Characteristics and Geographical Distribution of Gaucher Disease In Iberian Peninsulae and Islands
Authors: Giraldo Pilar , Alfonso Pilar , Irún Pilar , Chabas Amparo , Villageliu Lluisa , Grinberg Daniel , Miranda Clara Sa , Pocovi Miguel
Published: October 2019

Abstract 4729

Background and Objective:

The actual population of Iberian Peninsula (IP) is about 55 millions of inhabitants with a combination of various ethnic groups There are several reports that have analyzed GBA mutations in specific IP population. The aim of this study is to describe the distribution and main clinical characteristics of GD patients in IP.

Patients and Methods:

Data from the National Spanish Gaucher Disease Registry, Genetic Department of Barcelona University and Genetic Medical Institute of Porto since 1970 were jointed. Statistical analysis of demographic data, geographical distribution of GBA mutations and allelic frequency of GBA mutations in IP were analyzed. As well as identified the existence of clusters and the distribution of different types, the type and years on therapy. In addition we have determined the age at diagnosis (aDx) and SSI.

Results:

The prevalence of GD in IP had been 436 diagnosed patients, 421 of them born in IP. The mean aDx was 26.3±19.88 y, there are not differences between aDx before and after the last decade (24.8 vs 26.9 years respectively). Gender: female: 47.74 %, male: 47.98%, unknown: 4.28% type 1: 375 (89.1%), type2: 27(6.4%), type3: 19(4.5%). Mean SSI in type1 GD was 7.7 (range 1–30), and the distribution was 72.7% mild, 25.5% moderate and 1.7% severe. We have identified 70 alleles and 76 different mutations. The most prevalent allelic frequencies were: N370S: 48.8%, L444P: 18.3%, D409H: 3.1%, G3877S: 2.9%, double mutation: L444P+E326K: 2.0%, which account the 75% of total alleles, in 3.3% of alleles the mutation has not been yet identified and the remaining alleles corresponding to private mutations. Nevertheless the most frequent IP genotypes were N370S/L444P, 27.8%, N370S/N370S, 15%, N370S/? 5.2%, G377S/D409H, N370S/G202R, N370S/c.84insG, 2.4% respectively and homozygous for L444P 2.1%. Both Spain and Portugal the most prevalent mutations were N370S and L444P, however N370S is more frequent in Portugal than in Spain (57.1% vs 46.5%) by contrast L444P is more frequent in Spain than Portugal (19.4% vs 14.3%). 247 patients (58.7%) have received ERT for a mean of 10.2±3.89 years and 53 (12.6%) has been treated with miglustat for a mean of 2.1±1.9 years. The 90.4% of type1 and 31.0% of type 3 are alive and all type 2 are dead.

Conclusions:

There are broad spectrums of GBA mutations in GD patients from IP. Five different mutations account for 75% of GBA mutant alleles including N370S, L444P, D409H and G377S. No differences in gender distribution and tendency of aDx were observed. The 98.2% of type1 GD has a mild or moderate grade of severity and 23.0% has never received ERT or SRT.

Disclosures:

No relevant conflicts of interest to declare.
CD11b+gr-1+ myeloid Derived Suppressor Cells (MDSC) In Normal Bone Marrow Suppress T Cell Proliferation and Enhance T-Regulatory Function Via a IL10-, IL4- and IDO-Independent Mechanism
Authors: Forghani Parvin , Harris Wayne , Li jian-Ming , Khorramizadeh M.R. , Waller Edmund
Published: October 2019

Abstract 4801

MDSC have been described as an important negative regulators of autologous anti-cancer immune responses. Considering the important role of MDSC in immune regulation in allogenic stem cell and organ transplantation, we undertook an investigation of the mechanism(s) by which MDSC inhibit T–cell activation and proliferation, and tested the hypothesis that local cytokine secretion or IDO activity is required for suppression of T-cell proliferation.

Two separate populations CD11bhiGr-1hi and CD11bhi Gr-1int were isolated by high-speed FACS from lineage- BM antigen presenting cells (C57 & BALB/c mice). Both MDSC subsets had potent capacity for in–vitro suppression of CD4+ and CD8+ T cells proliferation in response to anti-CD3/anti-CD28 beads and Con A. A ratio of 0.5/1 MDSC: T-cells were sufficient to inhibit >66% control levels of T-cell proliferation. MDSC isolated from transgenic mice that had been “knocked-out” for IFN-γ and IDO had equivalent suppressive activity as MDSC from wild-type donors. Addition of saturating concentrations of anti IL-10 and IL-4 MAb, or in combination with anti- IFN-γ MAb did not abrogate MDSC-suppressive activity. Ex-vivo culture of MDSC with mitogen-activated T-cells generated two—fold more Fox-p3 T-reg compared with cultures of T cell plus mitogen.

Data will be presented regarding the novel role of MDSC involving in the homeostasis regulation of normal T-cell activation and proliferation in non-tumor-bearing mice.

Disclosures:

No relevant conflicts of interest to declare.
Vitamin D Deficiency In Pediatric Patients with Sickle Cell Disease Correlates with Reticulocytosis but Not with Clinical Disease Severity
Authors: Winters Amanda C , Kruse-Jarres Rebecca , Kanter Julie
Published: October 2019

Abstract 4820

Vitamin D deficiency is an established phenomenon in both adult and pediatric patients with sickle cell disease. Between 33% and 78% of children and between 60% and 100% of adults with sickle cell disease (HbSS, HbSC, and HbSβ-thalassemia) have been found to have low serum levels of 25-hydroxyvitamin D, the form most clinically useful for measuring total body levels of vitamin D. Proposed factors contributing to low vitamin D levels in these patients include decreased exposure to sunlight secondary to chronic illness and frequent hospitalizations, malnutrition or malabsorption of vitamin D, higher requirements for vitamin D in sickle cell disease, and impaired metabolism of vitamin D. However, very little data has been published demonstrating a correlation between vitamin D deficiency and burden of disease in patients with sickle cell disease. In addition, there is no published data regarding the effect of vitamin D supplementation on patient-oriented outcomes. We retrospectively reviewed the medical records of pediatric patients with sickle cell disease (ages 0–18 years) to evaluate their serum vitamin D levels as a function of red blood cell turnover, patient's age, and factors relating to burden of disease such as number of emergency room visits and hospital admissions related to vaso-occlusive crises. As expected, we found that essentially all of our patients have insufficient levels of vitamin D at baseline. Although no correlation was seen between vitamin D levels and either age or number of hospital visits related to pain crises, reticulocytosis was significantly correlated (p=0.017) with degree of vitamin D deficiency in our patient population in multiple regression analysis. Based on these preliminary data, vitamin D deficiency in patients with sickle cell disease is more likely the result of the disease process as measured by red blood cell turnover as opposed to the previously hypothesized environmental constraints associated with pain crises and frequent hospitalization. Future analysis will be directed at confirming these findings in adult patients and at evaluating the effects of vitamin D supplementation in both adult and pediatric sickle cell patients. Additional studies should also evaluate the pathogenesis of vitamin D deficiency in states of high red blood cell turnover.

Disclosures:

No relevant conflicts of interest to declare.
Evaluation of TNF Superfamily Molecules Release by Neutrophils and B Leukemic Cells of Patients with Chronic B – Cell Lymphocytic Leukemia
Authors: Sawicka-Powierza Jolanta , Jablonska Ewa , Kloczko Janusz , Piszcz Jaroslaw , Garley Marzena , Ratajczak-Wrona Wioletta
Published: October 2019

Abstract 4855

It was demonstrated that TNF superfamily proteins may affect significantly the time of leukemic cells' survival in the course of B-cell chronic lymphocytic leukemia (B-CLL).

The aim of our study was to evaluate the expression and release of BAFF (B-cell activating factor), APRIL (a proliferation-inducing ligand) and TRAIL (TNF-related apoptosis inducing ligand) molecules belonging to the cytokines of the superfamily of the tumor necrosis factor (TNF) by neutrophils (PMNs) and, for comparison, B cells isolated from the blood of patients with B-CLL vs. their concentration in the blood serum.

40 patients suffering from B-CLL and a control group of 15 healthy subjects were included in the study. Cytoplasmic fractions of PMNs and B cells were analyzed with the use of western blotting for the presence of TRAIL, BAFF and APRIL. Soluble TRAIL, BAFF and APRIL in the culture supernatants and the serum were assessed using ELISA kits.

PMNs and B cells of patients with B-CLL before treatment demonstrated the statistically significantly higher expression of APRIL and BAFF proteins in comparison in relation to the control group of healthy subjects. In contrast, the expression of TRAIL protein in both types of cells of patients was statistically significantly lower than its expression in the control cells.

In the supernatants of PMN and B lymphocytes of patients the decreased concentrations of sBAFF, unchanged of APRIL and increased of sTRAIL molecules were demonstrated.

The results of studies carried out in patients with B-CLL before treatment indicate that the relations demonstrated between APRIL, BAFF and TRAIL molecules, released by neutrophils and B cells and relations between their concentrations in the serum can significantly influence the development of B-CLL.

Disclosures:

No relevant conflicts of interest to declare.
Rituximab Plus Bendamustin (R-B) Treatment In Patients with Aggressive Large B-Cell Lymphoma (LBCL): Response and Tolerability - a Single Institution Experience
Authors: Horn Julia , Kleber Martina , Kohlweyer Ulrike , Hieke Stefanie , Herzog Regina , Schmitt-Gräff Annette , Engelhardt Monika
Published: October 2019

Abstract 4900

Introduction:

Clinical studies have shown that Rituximab plus Bendamustin (R-B) in indolent lymphoma results in favourable responses, progression free survival (PFS) and lower toxicity as compared to R-CHOP. The aim of this analysis was to characterize response and tolerability of R-B in patients with LBCL, who were not qualifying for R-CHOP due to age, comorbidity and/or prior pretreatment (including anthracyclines).

Methods:

We retrospectively identified consecutive patients with LBCL receiving at least two cycles of R-B in our department between 2003 and 2010 using our electronic tumor documentation system. Patient characteristics, response to R-B, and toxicity were assessed.

Results:

We identified 9 caucasian patients (5 females, 4 males) with LBCL; their median age was 71 years (range; 51–82). Two presented with stage I/II, seven with stage III/IV disease at initial diagnosis and before R-B. Six patients had a low or intermediate IPI and three were high risk. Four patients received R-B as first-line therapy, and five were treated for relapsed or refractory disease.

Main determinants for the R-B-selection were contraindications for anthracyclines in five patients and advanced age and/or poor performance status in four patients.

A median of four R-B-cycles were applied (range; 2–6). Response with achievement of CR and PR was observed in 6/9 (CR: 2, PR: 4), two achieved SD. Only one pt showed PD after four R-B cycles. The response of R-B in first-line vs. relapsed appeared similar.

Of note, one female patient with secondary LCBL, after initial Hodgkin's lymphoma and C-MOPP chemotherapy (CTx) and mediastinal irradiation - with excellent response to R-B- failed to successfully mobilize PBSC thereafter. However, she was effectively mobilized with R-Ara-C-thiotepa (peripheral blood CD34+ cells were 5.82 vs. 54/μl, obtaining no vs. 6.72 × 106 CD34+ cells/kg KG via leukapheresis, respectively). Clinical tolerance of R-B in all patients was excellent in a total of 31 R-B-cycles, only two major CTC-events occurred: one infection (CTC grade III) and one thromboembolism (grade IV). Median PFS and overall Survival (OS) were 16 (7- not reached) and 20 (11-21) months.

Conclusions:

If standard R-CHOP cannot be given due to age, comorbidity or CTx-contraindications (e.g. anthracyclines), R-B may represent an effective treatment in LBCL. Larger cohorts and prospective clinical trials are needed to confirm these promising results. Currently, patients with grade III/IV follicular lymphoma are additionally evaluated for response and tolerability under R-B, also being presented at the meeting.

Disclosures:

No relevant conflicts of interest to declare.
PI3KCA and PIM Inhibitors in Peripheral T-Cell Lymphomas
Authors: Martin-Sanchez Esperanza , Rodriguez-Pinilla Socorro M. , Lombardia Luis , Sanchez-Beato Margarita , Dominguez-Gonzalez Beatriz , Romero Diana , Gomez-Abad Cristina I. , Vargiu PierFrancesco , Rodriguez Maria E. , Mollejo Manuela , Alves Javier , Wozniak Magdalena B. , Bischoff James R. , Piris Miguel A.
Published: October 2019

Abstract 4917

T-cell lymphomas (TCL) are a heterogeneous group of aggressive malignancies lacking specific and efficient therapy. Unfortunately, there are neither animal models nor representative cell lines for most TCL types, making functional and pharmacogenomics studies even more difficult. PI3K and PIM are kinases involved in cell proliferation, frequently altered in human cancer that seems to play a critical role in T-cell development and activation. Genomic studies have identified PIK3CD subunit to be significantly associated with in activation of CD40, NF-kB and TCR-pathways. The aim of this project is to determine the efficiency of PI3K inhibitors (PI3Ki) and PIM inhibitors (PIMi) in TCL, looking for biomarkers of their mechanism of action and to identify markers that could identify responders from non-responders.

Twenty PTCL and seven reactive lymph nodes were studied using gene expression microarrays. We performed an in silico analysis using the Connectivity Map program to identify drugs that could potentially reverse PTCL gene expression signature. Among them, several PI3K/mTOR inhibitors were found. A panel of 6 TCL cell lines belonging to different TCL subgroups were treated with 3 PI3Ki (LY294002, ETP-45658, GDC-0941) and one PIMi (ETP-39010). Functional studies were also done to establish the role of each of the targeted genes.

In vitro studies showed that PI3Ki induced G1 cell cycle arrest in all cell lines, and apoptosis in a portion of them, in a time/dose-dependent manner. We also observed a decrease in the levels of pAKT(S473), pGSK3B(S9) and p-p70S6K(T389) after treatment.

In addition, both the analysis of the PTCL gene expression signature as well as western blot studies on TCL cell lines has shown overexpression of PIM family genes, A decrease in cell viability, and a strong induction of apoptosis in all cell lines was seen after PIM inhibition, without cell cycle arrest. Several diagnostic and pharmacodynamic biomarkers of PIMi have been identified at the mRNA and protein level in both cell lines In conclusion, our results indicate that PI3Ki and PIMi are effective therapeutic approaches for TCLs, identifying potential markers for patient's stratification and pharmacodynamic assessment.

Disclosures:

No relevant conflicts of interest to declare.
Combinatorial Effects of CD30 Ligand Stimulation or Inhibitor of Apoptosis (IAP) Antagonist Exposure with Conventional Chemotherapy on CD30 Positive Malignancies
Authors: Walkovich Kelly J , Liu Xuwen , McCollom Anne L , Duckett Colin S
Published: October 2019

Abstract 4926

CD30 is a member of the tumor necrosis factor (TNF) receptor family that is normally found on the cell surface of a small subset of activated lymphocytes but is overexpressed on the surface of anaplastic large cell lymphoma (ALCL) and Hodgkins lymphoma (HL) cells. Although many drugs exist that treat lymphomas by triggering the intrinsic cell death pathway, current chemotherapeutic regimens are limited by unwanted side effects, including secondary malignancies that limit event-free survival. The tumor-restricted overexpression of CD30 makes it an attractive target for therapeutic intervention. Depending on the cellular context, CD30 stimulation has been linked to cell death, cell cycle arrest, or paradoxically, proliferation. In ALCL tumor cell lines, CD30 stimulation activates both the canonical and noncanonical NF-kB pathways while in HL tumor cell lines, CD30 stimulation only slightly enhances NF-kB activity above constitutive levels, implying a role for NF-kB in determining the sensitivity or resistance of lymphoma cells to CD30-induced apoptosis. In addition, IAP antagonists, small synthetic compounds that mimic the structure of the second mitochondrial activator of caspase (Smac) and target IAP molecules that affect the activation of the non-canonical NF-kB pathway, induce apoptosis and/or sensitize cells to death via secondary signals such as TNF. This suggests that the modulation of IAP levels, and consequently regulation of the non-canonical NF-kB pathways, may also have a role in determining tumor cell death. Using representative ALCL and HL tumor cell lines, we have found that CD30 stimulation via its physiologically ligand in combination with standard chemotherapeutic agents results in increased efficacy in tumor cell death in the majority of ALCL cell lines but not HL cell lines. Similarly, IAP antagonists in combination with standard chemotherapeutic agents also resulted in enhanced tumor cell death in most ALCL but not HL cell lines. This augmentation of tumor cell death suggests that CD30-induced apoptosis and IAP antagonist-induced killing may have important consequences in the clinical treatment of CD30 positive malignancies. Currently, we are further investigating the role of both CD30 stimulation via its physiological ligand and IAP antagonists in impacting the activation of the canonical and noncanonical NF-kB pathways alone and in combination with currently utilized chemotherapeutic agents to modulate the apoptotic threshold in CD30 positive lymphoma cells.

Disclosures:

No relevant conflicts of interest to declare.
Heart Function In Systemic and Localized AL Amyloidosis: Role of NT-ProBNP and MPC-1
Authors: Saltarelli Francesca , Moscetti Alessandro , Bruno Guglielmo , Monarca Bruno , Salerno Gerardo , Corsetti Maria Teresa , Cardelli Patrizia , Ferranti Giovanna , La Verde Giacinto
Published: October 2019

Abstract 5006

In AL amyloidosis typical sites of amyloid buildup are heart, skin, gastrointestinal tract, liver, kidneys, and blood vessels. To evaluate the heart involvement in systemic and localized amyloidosis proBNP, peptide (NT-proBNP; 76 amino acids) and MPC-1 were investigated. NT-proBNP have been described as useful marker for the diagnosis heart disease, and its plasma concentrations correlate with the functional classification of patients according to the New York Heart Association (NYHA). MCP-1 is a chemokine that activates mononuclear phagocytes by promoting leukocyte–endothelium binding and migration to sites of inflammation. The MCP-1 levels seem to be related to the severity of cardiac alteration, as demonstrated by the coronary angiogram. NT-proBNP and MPC-1 serum levels were performed in systemic or localized AL amyloidosis to evaluate if there was a difference in the heart involvement. Blood samples were collected from 8 patients with systemic amyloidosis and from 4 patients with localized amyloidosis. To analyze the results of NT-proBNP and MPC-1, Mann-Whitney test was performed. NT-proBNP serum values were significantly (p=0.007) increased in systemic disease. Also, MPC-1 serum levels were significantly (p=0.004) higher in the patients with systemic disease (350.52±58.70 pg/ml) if compared to the group of localized amyloidosis (147.82±26.03 pg/ml). On the basis of our results, the heart seem to be functionally more involved in AL systemic amyloidosis than in localized disease, as demonstrated by the higher NT-proBNP and MPC-1 serum values.

Disclosures:

No relevant conflicts of interest to declare.
Proteasome Inhibitor Bortezomib Disrupts Tumor Necrosis Factor-Related Apoptosis-Inducing ligand (TRAIL) Expression and Natural Killer (NK) Cell Killing of TRAIL Receptor-Positive Multiple Myeloma Cells
Authors: Feng Xiaoli , Yan Jie , Wang Yibiao , Zierath Juleen R. , Nordenskjöld Magnus , Henter Jan-Inge , Fadeel Bengt , Zheng Chengyun
Published: October 2019

Abstract 5021

Bortezomib, a potent 26S proteasome inhibitor, is approved for the treatment of multiple myeloma (MM) and clinical trials are under way to evaluate its efficacy in other malignant diseases. However, cytotoxic effects of bortezomib on immune-competent cells have also been observed. The aim of this study was to investigate the putative effects of Bortezomib on activated human natural killer (NK) cell function. In this project, primary lymphocytes or NK cells were isolated or purified from peripheral blood mononuclear cells of healthy donors. Expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) by interleukin (IL)-2-activated NK cells was quantified by flow cytometry and real time-PCR method. NK cell degranulation activity and cytotoxicity against MM cell lines were evaluated by CD107a surface translocation and 51Cr release assays, respectively. Our results demonstrate that bortezomib markedly downregulated cell surface expression of TRAIL on primary human IL-2-activated NK cells in a dose-dependent manner. Moreover, bortezomib reduced TRAIL mRNA expression in these cells, suggesting that bortezomib regulates TRAIL expression of activated NK cells at the transcriptional level. Interestingly, pharmacological inhibition of the transcription factor NF-κB also profoundly decreased TRAIL expression both at protein and mRNA levels, indicating a novel role of NF-κB in the regulation of TRAIL expression in activated human NK cells. Furthermore, perforin-independent killing of the human MM cell lines was significantly suppressed following bortezomib treatment. In addition, blocking cell surface-bound TRAIL with a TRAIL antibody impaired lymphokine-activated killer (LAK) cell-mediated lysis of the TRAIL-sensitive MM cell line, RPMI8226. Our study demonstrated that bortezomib decreases TRAIL expression by IL-2 activated NK cells in a dose-dependent manner and disrupts TRAIL-mediated killing of TRAIL-sensitive myeloma cells, suggesting that Bortezomib may potentially hamper NK-dependent immunosurveillance against tumors in patients treated with this drug.

Disclosures:

No relevant conflicts of interest to declare.
Inhibition of JAK2V617F Kinase Potently Induce Apoptosis In HEL Cells
Authors: Shen xu-Liang , Wei Wu , Xu Hong-liang , Qin Xiao-qi , Fangping Chen
Published: October 2019

Abstract 5062

Myeloproliferative neoplasms (MPNs), formerly recognized as myeloproliferative disorders(MPDs), are a group of hematological conditions where a molecular defect in a multi-potent hematopoietic stem cell leads to increased production in one or more blood cell lineages. In early 2005, four different groups described an identical JAK2V617F mutation in exon 12 of JAK2 in a large number of patients with MPNs. Whereafter, it has been confirmed that the Gain-of-function mutations of JAK2V617F play crucial roles in the development of MPN. However, how this activated singaling pathway leads to overproduction of blood cells are not fully understood. Our experiment was designed and performed to address one aspect of this issue. Here we report that AG490, a JAK2 kinase inhibitor, is potently inducing apoptosis in HEL cells. HEL cell line, which harbor a homozygous JAK2V617F mutation confirmed by Allele-specific polymerase chain reactions (AS-PCR), restriction enzyme digestion and DNA sequencing, was selected as a cell model. HEL cells were treated with AG490 at different concentrations and for different time length. The Flow cytometry(FCM) with AnnexinV-FITC/PI double staining and genome DNA electrophoresis were performed to determine the apoptosis rate of the HEL cell, respectively; RT-PCR was performed to analyze the effect of AG490 on the expression of Bcl-x mRNA. The FITC-AnnexinV/PI double staining demonstrated that the apoptosis rate of HEL cell increased obviously in a time (F=267.226, P<0.0001)and dose(F=343.77, P<0.0001) dependent manner after treated by AG490. Similar results were observed by DNA ladder. The results of RT-PCR demonstrated that when treated by AG490, the mRNA of Bcl-xL gene was down-regulated obviously(Bcl-xL mRNA was the main species of Bcl-x found), and the down-regulation was strongly time(F=26.402, P<0.0001) and dose(F=39.247, P<0.0001) dependent. Thus, our results suggest that AG490 can induce apoptosis in HEL cells obviously, and JAK2V617F might inhibit cell apoptosis through upregulation of Bcl-xL.

Disclosures:

No relevant conflicts of interest to declare.
T(8;22)(q24;q11) In a Context of Complex Karyotype In Two Patients with B-Cell Lymphoma with Features Intermediate Between Burkitt Lymphoma and Diffuse Large B Cell Lymphoma B (LB/LDCGB)
Authors: Vallespi Teresa , Ortega Margarita , Jaramillo Anny , Lopez-Andreoni Laura , Martínez-Morgado Noemi , Sánchez Carmen , Bosch Francesc
Published: October 2019

Abstract 5093

Background:

In some cases, it is difficult to distinguish between the diagnosis of Burkitt lymphoma (LB) and Diffuse Large Cell Lymphoma B (DLCL-B) with MYC gene rearrangement. The WHO classification distinguishes three clinical entities: LB, LB/DLCL-B, DLCL-B on the basis of morphology, immunophenotype and genetic characteristics of malignant cells. The MYC gene is often rearranged with the gene that encodes the heavy chains of immunoglobulins (IgG 14q32) and, less frequently, with the gene that encodes the light chains kappa (2p12) or lambda (22q11).

Objective:

Describe clinical and biological characteristics of two cases of lymphoma with variant t(8;22)(q24;q11) in a complex karyotype diagnosed BL/DLCL-B according to the WHO classification.

Methods and Patients:

The cytogenetic study was made in the bone marrow (aspirate or biopsy). Leukocytes were incubated during 24h in RPMI without mitogens. Rearrangements of C-MYC, BCL-2, BCL-6 and IGH were studied for FISH techniques. The results were expressed according to ISCN 2009.

Case 1:

A man of 46 years who was admitted with symptoms of spinal cord compression with fever and weight loss. During admission were detected anti-HIV antibodies. In the biopsy were observed large cells with irregular nuclei, with clefts and some multilobulated. Imunohistochemistry: CD10 +, CD20 +, CD79 +, bcl2 + and BCL6 +. The Ki67/MIB1 was 95%. Cytogenetic study of bone cylinder showed a complex karyotype: 49, XY, +1, del (1) (p22), der (2), der (3), +7, t (8;22)(q24;q11), +10, del(10)(q24q26)x2 [3]. The FISH study demonstrated the rearrangement of C-MYC and the absence of BCL2 and BCL6 rearrangement.

Case 2:

A man of 67 year old was admitted for epigastric pain, nausea, vomiting and weight loss. The aspirated cells were of medium and large size, irregularly shaped nuclei, some creases and other poly-lobed with prominent nucleoli. Was detected by immunohistochemistry positivity: CD10, CD20, CD79 and BCL6 Ki67/MIB1 presented a 90%. Cytogenetic study of the bone marrow showed a complex karyotype: 49, XY, t(1;11)(q21;p13),+7, t(7;13) q32;q12), t (8;22)(q24;q11),+12,+19[40]. The FISH study demonstrated presence of the rearrangement of C-MYC and BCL2 and BCL6 rearrangement absence.

Conclusions:

- Both cases are representative of the clinical entity BL// DLCL-B recently recognized by the WHO: anisocitosicas cells with a nucleus of irregular or poly-lobed, with Ki67/MIB1 <90% and C-MYC rearranged with a complex karyotype. - Both cases showed the t(8;22)(q24;q11) rare variant of Burkitt lymphoma and a trisomy 7, trisomy usually observed in DLCL-B.

Disclosures:

No relevant conflicts of interest to declare.