bluuebunny/crossref_metadata_2025 huggingface dataset:
Analyzing the Stepwise Developmental Pathway From ES/IPS Cells to Functional Mature Erythrocytes.
Authors: Niwa Akira , Fukatsu Tomoki , Umeda Katsutsugu , Kato Itaru , Sakai Hiromi , Morishima Tatsuya , Okita Keisuke , Yamanaka Shinya , Heike Toshio , Nakahata Tatsutoshi
Published: October 2019

Abstract 2534

Poster Board II-511

Induced pluripotent stem (iPS) cells, reprogrammed somatic cells with embryonic stem (ES) cell–like characteristics, are generated by the introduction of combinations of specific transcription factors. Despite the controversy surrounding the gene manipulation, it is expected that iPS cells should contribute to regenerative medicine, disease investigation, drug screening, toxicology, and drug development in future. In the fields of hematology, iPS cells could become used as a new feasible source for transplantation therapy without immunological barrier and for the investigation of various kinds of hematological defects. Previous studies on ES / iPS cells have already demonstrated that they can develop into various lineages of hematopoietic cells including erythrocytes following the similar processes occurred in embryo and fetus. However, it is important to establish the more effective system for developing functional blood cells. Here we present the methods for selectively inducing mature red blood cells from ES / iPS cells in vitro, and show the functional equality of them to natural blood cells. First, Flk1+ mesodermal progenitors were derived from ES / iPS cells on OP9 stromal cells at an efficacy of more than 50% and collected by fluorescence activated cell sorter. Then, those sorted cells were cultured in the presence of exogenous erythropoietin and stem cell factor. They highly selectively developed into erythroid lineages including enucleated red blood cells. Sequential FACS analysis using the antibodies against transferrin receptor CD71 and erythroid specific antigen Ter119 in combination with DNA staining dye Hoechst 33342 demonstrated that ES / iPS cell-derived erythropoiesis in our system follow the normal erythroid developmental pathway occurred in vivo. RT-PCR and Western blot analyses proved the expression of heme biosynthesis enzymes on the produced erythrocytes. Finally, the oxygen dissociation curve showed that ES / iPS cell-derived erythroid cells are functionally virtually equivalent to natural red blood cells as oxygen carriers. Taken together, our system can present the effective methods of investigating the mechanisms of normal erythropoiesis and the deficits in syndromes with disrupted red blood cell production.

Disclosures:

No relevant conflicts of interest to declare.
Interferon-Gamma Is a Critical Regulator of the Hematopoietic Stem Cell Response to Chronic Infection.
Authors: Baldridge Megan T , King Katherine Y , Weksberg David C , Boles Nathan C , Goodell Margaret A
Published: October 2019

Abstract 2549

Poster Board II-526

Hematopoietic stem cells (HSCs) are the progenitors of all blood cells including the effector cells of the immune system. HSCs remain dormant during most of the life of an organism, but can be activated by stress conditions to replenish peripheral blood cells. Infection is a common stress that leads to the consumption of immune cells, and the bone marrow must compensate for increased immune system demands by increasing production of downstream progeny during chronic infection. How this compensation is accomplished is poorly understood.

We investigated the HSC response to chronic infection using an in vivo mouse model of Mycobacterium avium (M. avium) infection. We found that infection stimulates changes in multiple hematopoietic progenitor compartments to maintain peripheral blood homeostasis. Infection activates the HSC itself to differentiate and proliferate, as measured by BrdU incorporation. Since interferon-gamma (IFNg) levels are high during M. avium infection, we hypothesized that this cytokine may be an important mediator of the HSC response. We confirmed that IFNg levels in the bone marrow, where the HSC resides, are elevated in infected mice. Consistent with our hypothesis, we found that the proliferative response to M. avium infection is abrogated in Stat1-deficient and IFNg-receptor-deficient mice, but is unaffected in IFNa-receptor-deficient mice, indicating that the HSC response to M. avium infection is critically mediated by IFNg.

To determine if IFNg alone is sufficient to stimulate the HSC, we tested and confirmed that HSCs express the IFNg receptor, and are thus capable of responding directly to IFNg signaling. We next established that IFNg stimulates expression changes in known IFN regulatory molecules such as Stat1 and Irf9 in the HSC. In agreement with our findings using M. avium, IFNg alone causes HSCs to proliferate both in vitro and in vivo, as assessed by BrdU incorporation. Furthermore, we found that HSCs from an IFNg-deficient mouse model exhibit decreased baseline proliferation and superior engraftment in a competitive transplantation assay. Thus, IFNg participates in maintaining balance between quiescence and proliferation of HSCs even in the absence of infection.

Despite historical data that IFNs are suppressors of bone marrow function, this report signifies the first in vivo evidence that IFNg has a powerful and direct effect on HSC proliferation. Thus it appears that IFN regulation of the bone marrow is a complex, and perhaps context-dependent, phenomenon. These findings inform our understanding of HSC biology as well as our ability to treat patients affected by chronic infections, such as HIV and tuberculosis, illnesses that can be complicated by bone marrow suppression.

Disclosures:

No relevant conflicts of interest to declare.
Short-Term Hematopoietic Stem Cells (ST-HSC) Have Full Long-Term Capacity with Sustained but Reduced Potential Compared with LT-HSC.
Authors: Colvin Gerald A. , Berz David , Quesenberry Peter J. , Papa Elaine , Liu Liansheng
Published: October 2019

Abstract 2550

Poster Board II-527

We evaluated the hypothesis that there was a homing defect between long-term (LT) hematopoietic stem cell (HSC) (KLS-Flk2-) and ST-HSC (KLS-Flk2+) that explained differences in engraftment potential and duration. Short-term HSC by definition have limited self-renewal capacity, generally described as giving rise to lymphohematopoiesis for 4–12 weeks before senescence. We performed three large animal engraftment studies into lethally ablated mice (950cGy split dose) looking at engraftment of both ST and LT-HSC cells delivered via intravenous, intraperitoneal and intra-femoral route. Two-hundred or 500 cells derived from B6/SJL mice were administered to each animal along with 300,000 recipient (C57/BLK) whole bone marrow cells for radioprotection following prior published studied [PNAS:98;14541, Stem Cells 24:1087] with optimization of flourochromes for better discrimination with our Cytopeia sorter. The animals were serially transplanted after eight months or one year to secondary recipients. In our hands, the ST-HSC engrafted animals did not lose chimerism over time. Review of the literature revealed that there were not confirmatory studies from those published from the initial one publication describing the ST-HSC. We found the ST-HSC were not short-term and persisted for one year in primary recipients and at least 3 months in secondary recipients. Engraftment kinetics favored LT-HSC over ST-HSC with engraftment examples at one year of 62% compared with 30% respectively when administered intravenously, 10% verses 4% given intra-femoral and 0.5% verse 0.3% given intraperitoneal. Chimerism was on average 50% better for the LT-HSC when compared with the ST-HSC and was irrespective of route proving that the differences seen are not due to homing deficiency but rather intrinsic differences in the two stem cell pools. Prior studies gave a maximum of 100 cells. Cell number was purposely increased for better differentiate of subtle differences in engraftment kinetics for statistical reasons. To avoid contamination of Flk2+ cells in the Flk2- cohort and vise-versa, discrimination of the gates were enhanced from that which was published prior. Double sorting of the cells confirmed that there was no appreciable cross contamination but obviously we cannot totally rule that out as a potentially confounding factor. In conclusion we found that ST-HSC as described have long-term capacity with intrinsic differences in engraftment potential that is not driven by a homing defect.

Disclosures:

No relevant conflicts of interest to declare.
Myeloid Translocation Gene (MTG)-16 Binds Intracellular Domains of Notch Receptors to Coordinate Notch-Dependent Cell Fate Specification.
Authors: Engel Michael E , Nguyen Hong , Mariotti Jolene , Hunt Aubrey A , Hiebert Scott
Published: October 2019

Abstract 257

The Notch signaling pathway regulates gene expression programs to control the specification of cell fate in diverse tissues. In response to ligand binding, the intracellular domain of Notch receptors is cleaved by the γ-secretase complex and then translocates to the nucleus. There it binds the transcriptional repressor CSL, triggering its conversion to an activator of Notch target gene expression. The events that control this conversion are poorly understood. We show that the transcriptional co-repressor, MTG16, interacts with both CSL and the intracellular domains of Notch receptors. The MTG16 NHR3 domain contributes to CSL binding, while N-ICD binding sites lie within the PST1 and PST2 domains. The Notch intracellular domain disrupts the MTG16—CSL interaction, suggesting a pivotal role in regulating the Notch transcription complex. Using co-culture of Lin-/Sca-1+/c-Kit+ (LSK) cells on OP9-DL1 stromal cells, we show that ex vivo fate specification in response to Notch signal activation is altered in Mtg16 (−/−) hematopoietic progenitors. While Notch signal activation specifies lymphoid fate in Mtg16 (+/+) LSK cells, Mtg16 (−/−) LSK cells display cell surface marker expression reminiscent of myeloid differentiation. We used this lineage allocation assay to assess the contribution of MTG16 to cell fate determination. Lymphoid fate specification is restored by MTG16WT expression in Mtg16 (−/−) LSK cells. However, an MTG16 mutant deficient in N1-ICD binding is defective in this assay, suggesting this region is important to Notch-dependent lineage allocation. These data suggest that MTG family proteins interface with critical components of the Notch transcription complex and intimate a functional relationship between MTG proteins and Notch signaling in normal and malignant hematopoiesis.

Disclosures:

No relevant conflicts of interest to declare.
Measurement of Urine Pyridinoline and Deoxypyridinoline as Markers of Increased Bone Degradation in Sickle Cell Disease.
Authors: Nur Erfan , Mairuhu Willem , Brandjes Dees P. , van Zanten Ton , Biemond Bart J. , Otten Hans-Martin
Published: October 2019

Abstract 2572

Poster Board II-549

Introduction:

Sickle cell disease (SCD) is commonly manifested through skeletal involvement. Besides the characteristic acute musculoskeletal pain, SCD is also associated with chronic skeletal complications such as osteopenia and osteoporosis. During bone resorption, the collagen cross-links pyridinoline (PYD) and deoxypyridinoline (DPD) are released into circulation with subsequent urinary excretion. Measurements of urinary PYD and DPD could serve as valuable tools in detecting osteoporosis in the follow-up of SCD patients but perhaps also in determining the severity of bone infarction during painful crises. Therefore we compared urinary concentrations of PYD and DPD of SCD patients during asymptomatic state and painful crisis with those of race- and age-matched healthy controls.

Methods:

Urinary concentrations of PYD and DPD, adjusted for urine creatinine, were measured in SCD patients both during asymptomatic state (n=38) and painful crisis (n=27) and healthy controls with normal HbA hemoglobin (n=25) using high performance liquid chromatography (HPLC).

Results:

PYD and DPD concentrations were higher in asymptomatic SCD patients compared to controls ((54.8 (41.5–68.6) vs. 44.1 (37.7–49.9),P=0.005 and 11.6 (9.3–15.2) vs. 8.5 (6.8–10.4),P=0.004 respectively), with further increments during painful crisis (63.3 (51.8–76.0),P=0.041 and 15.3(13.0–21.5),P=0.003 respectively). In the asymptomatic patients levels of PYD and DPD were significantly correlated to the degree of hemolysis.

Conclusion:

In sickle cell patients bone resorption is increased and significantly correlated to the degree of hemolysis, compatible with their susceptibility to osteopenia and osteoporosis. Measurement of pyridinoline and deoxypyridinoline could have additional value as biomarkers of osteoporosis in SCD. During painful crises a further increment in bone degradation was observed.

Disclosures:

No relevant conflicts of interest to declare.
Plasma Protein Oxidation and Triglyceride Levels in Sickle Cell Anemia.
Authors: Oztas Yesim , Unal Selma , Ozgunes Nuriman
Published: October 2019

Abstract 2578

Poster Board II-555

Introduction:

A mutant and unstable hemoglobin is characteristic in sickle cell anemia (SCA) with an increased formation of methemoglobin, hemichrome and haemin. Increased oxidative stress and altered lipid balance in the plasmas of SCA patients have been documented previously. Lower total plasma cholesterol (TC) and higher-than-avarage triglyceride (TG) levels have also been reported in SCA patients compared to healthy controls. Plasma phospholipids (PL) were normal or increased according to various studies. However, to our knowledge only a few reports documented the plasma protein oxidation in SCA patients. In this study protein oxidation, lipid levels, bilurubin levels and their probable correlation were investigated in the plasmas of SCA patients, carriers and healthy controls.

Patient and Methods:

15 SCA patients, 10 carriers and 10 healthy controls were included in the study. The patients had not been transfused and hospitalized and had no crisis for the last three months. The oxidation status of plasma and its proteins were detirmined by total sulfhydryl (T-SH) and protein carbonly levels respectively. TC, TG and bilurubin levels were detirmined in the plasma and PL levels were detirmined in the lipid extract from plasma.

Results:

SCA patients had lower T-SH levels compared to controls (p=0.003) and higher protein carbonyl levels compared to carriers (p=0.001) and healthy children (p=0.032). T-SH values were 417.41±77.90 μM, 457.46±106.26 μM, 522.59±77.18 μM and protein carbonyl levels were 0.93±0.15 nmol/mg protein, 0.74±0.08 nmol/mg protein , 0.82±0.05 nmol/mg protein for patients, carriers and controls respectively. Significantly lower mean TC levels than controls (p<0.0001) and higher TG levels than carriers (p=0.001) were observed in SCA patients. TC levels were 116.67±23.60, 127.53±22.40, 151.62±16.53 and TG levels were 89.31±23.50 mg/dl, 60.94±9.18 mg/dl, 82.96±21.06 mg/dl for patients, carriers and controls respectively. Phospholipid levels were not different between the groups. TG levels were positively correlated to protein carbonyl levels (p=0.002) and to bilirubin levels (p=0.033). Bilurubin levels and protein carbonyl levels are also positively correlated (p<0,0001).

Conclusions:

Alteration of lipid balance besides protein oxidation and hemolysis were observed in the plasmas of SCA patients compared to the carriers and healthy group. The positive correlation between total bilurubin, TG and carbonyl levels indicates that oxidative stress may affect the SCA plasma extensively, ie. lipids, proteins and erythrocytes are all affected.

Disclosures:

No relevant conflicts of interest to declare.
Evidence for a Pathological Ratio of CD44s/CD44v6 in Chronic Lymphoctic Leukemia (CLL) Cells.
Authors: Hertweck Magdalena , Erdfelder Felix , Filipovich Alexandra , Uhrmacher Sabrina , Gandhirajan Rajesh Kumar , Gehrke Iris , Paesler Julian , Razavi Regina , Poll-Wolbeck Simon Jonas , Hallek Michael , Kreuzer Karl Anton
Published: October 2019

Abstract 2628

Poster Board II-604

Chronic Lymphocytic Leukemia (CLL) is the most common adult-onset leukemia in the western world. It is characterized by an accumulation of functionally incompetent monoclonal CD5+ B-lymphocytes and an increased resistance to apoptosis. CD44 is a cell surface transmembrane glycoprotein which has more than ten isoforms generated by alternative splicing. It is expressed on different cells as e.g. cells of lymphohematopoietic origin, epithelial cells and keratinocytes. CD44 is involved in lymphocyte activation, recirculation and homing. It acts as an adhesion molecule and plays an important role in angiogenesis, cell proliferation, differentiation and migration. One of these variants, CD44 variant 6 (CD44v6), is shown to be responsible for an increased apoptotic resistance in Jurkat cells. Our aim was to show that i) CD44 is overexpressed in CLL cells, ii) that this overexpression is regulated by the WNT pathway, which is known to be constitutively active in CLL cells, and iii) that this overexpression is a possible cause for the increased resistance to apoptosis in CLL cells. Peripheral blood of CLL patients was incubated with specific antibodies directed against CD5, CD19, CD44 standard (CD44s) and CD44v6 and measured by flow cytometry. We measured the mean fluorescence intensity of CD44s and CD44v6 on CD5/CD19 double positive CLL cells. The patient pool included patients with poor prognosis ( ZAP70+, CD38+) as well as patients having a good prognosis (ZAP70-, CD38-). CD19 positive B-cells from healthy volunteers, aged from 18 to 60, were analyzed for their expression of CD44s and CD44v6 as well. Expression levels of CD44s in 37 CLL samples and of CD44v6 in 29 CLL samples were measured and compared those of 22 healthy volunteers. We found a high expression of CD44s in CLL-cells (mean: 368.02) but an even higher expression in healthy B-cells (mean: 538.8; p<0.05). In contrast, the CD44 variant 6 was expressed five- to six-fold higher in CLL cells compared to healthy B cells (mean CLL cells: 20.0; mean healthy B cells: 3.6; p<0.001). Taken together, we found that there is a significantly altered ratio of CD44s/CD44v6 expression in CLL cells when compared to normal B cells. As CD44v6 is known to be oncogenic this phenomenon may contribute to the malignant phenotype of these cells. Currently, we are investigating the effect of WNT3a, a WNT signaling initiator, on the expression of CD44v6 in CLL cells and the effect of an anti human CD44v6 antibody on the apoptosis rate in CLL-cells.

Disclosures:

Hallek: BayerScheringAG: Honoraria, Research Funding.
Clinical Implementation of a Novel High-Throughput Screen of Primary Leukemia Cells to Customize Therapy for Relapsed/Refractory Acute Myeloid Leukemia (AML).
Authors: O'Dwyer Kristen M , Shum David , Heaney Mark , Brentjens Renier J. , Maslak Peter , Jurcic Joseph G , Djaballah Hakim , Frattini Mark G.
Published: October 2019

Abstract 2657

Poster Board II-633

For most patients with relapsed and refractory acute myeloid leukemia, therapeutic success is unpredictable with current cytotoxic chemotherapeutic regimens. Moreover, multiple courses of therapy have been shown to result in co-morbid conditions, which can often preclude the patient from being able to receive the potentially curative therapy of allogeneic bone marrow transplantation. With the aim of identifying a successful chemotherapy regimen for these patients, based on patient-specific myeloid blast cell chemo-sensitivities, we have developed a high-throughput screening assay that utilizes primary (patient-derived) leukemia cells and tested for chemo-sensitivity against a panel of established chemotherapeutic agents as well as novel agents. In our laboratory, we were able to identify in vitro resistance against the various drugs already clinically administered, including cytarabine, etoposide, and anthracyclines. Importantly, we were able to show in vitro sensitivity to chemotherapy agents that had not been previously administered. The index patient was a 32-year-old woman with primary refractory acute myeloid leukemia who had received six different therapeutic regimens prior to our testing, all of which demonstrated in vitro resistance using our assay. The blasts were sensitive, however, to 6-thioguanine with an inhibitory concentration (IC50) of 70 nM. Based on the in vitro data, the patient began a combination treatment regimen containing oral 6-thioguanine as her white blood cell (WBC) count had increased to greater then 50,000 cells/ml on her previous therapy. Following a maintenance regimen with these agents, her circulating blast count decreased to less then 10,000 cells/ml, and she had partial recovery of the neutrophil count, resulting in a decrease in the overall leukemia burden. This result was not seen with her previous treatment regimens. Importantly, the majority of this therapy was administered in the outpatient setting. Subsequently, we have collected specimens from more than ten patients with AML and used this screening assay to predict their response to various chemotherapeutics. In each case, we have accurately predicted the in vivo clinical response (both sensitivity and resistance) to different chemotherapeutic agents. This demonstrates that the technology currently being used to screen drug therapy for patients with relapsed and refractory AML is clinically useful and has a potential role in designing individualized drug treatment regimens.

Disclosures:

No relevant conflicts of interest to declare.
BACH2 Regulates Foxp3 Expression in Umbilical Cord Blood CD4+/45RA+ T-Cells.
Authors: Lesniewski Mathew Lawrence , Haviernik Peter , Weitzel R. Patrick , Greco Nicholas J , Paul Phil , Laughlin Mary L
Published: October 2019

Abstract 2677

Poster Board II-653

There are clear differences in the response of umbilical cord blood (UCB) T-cells with regards to improved immune tolerance after allogeneic stem cell transplant and lower incidence of acute graft versus host disease (aGvHD) which have not been determined at a cellular level. Activated, UCB CD4+ T-cells express less TNF-α and IFN-γ then their adult complement. At the protein level, NFAT1, a transcription factor known to regulate the expression of FoxP3, TNF-α, and IFN-γ is attenuated in resting UCB CD4+ T-cells. In addition, activated NFAT1 temporal response to stimulation is delayed by ∼6 hours. However our analysis by gene array did not see any attenuation of FoxP3 mRNA compared to normal naïve adult peripherial CD4+/CD45RA+ T-cells. The absence of a key transcription factor activity having little or no effect on subsequent downstream gene expression is normally a strong indicator of compensatory regulatory factor(s). Previous observation demonstrated that the transcription factor BACH2 plays a compensatory regulatory role in the up-regulation of IL-2 in UCB CD4+/CD45RA+ T-cells. Studies of the effect of BACH2 inhibition on FoxP3 expression in UCB CD4+/CD45RA+ T-cells were conducted. Loss of BACH2 expression correlated with the loss of FoxP3 mRNA and protein expression in transient siRNA knockdown experiments as observed by qRT-PCR, and validated by Western Blot. Using cross-linking chromatin immunoprecipitation (ChIP), we show that BACH2 binds to the proximal promoter and within intron 1 of FoxP3 gene in naïve UCB CD4+/CD45RA+ T-cells and within the intron 1 of adult CD4+/CD45RA+ T-cells. This report summarizes studies demonstrating a functional role for BACH2 in UCB CD4+/CD45RA+ T-cells. Transient knockdown of BACH2 by siRNA results in decreased FoxP3 expression at both at mRNA and protein level prior to CD3/CD28 stimulation. There were several putative BACH2 DNA binds sites both associated and unassociated with NFAT1 sites. ChIP analysis demonstrated that BACH2 co-precipitated with 2 separate sites surrounding exon 1 of the FoxP3 gene in unstimulated UCB CD4+/CD45RA+ T-cells, but with only one site in unstimulated AB CD4+/CD45RA+ T-cells. In addition, the bZip protein sMafK was observed co-immunoprecipitated with the same DNA regions in UCB and AB CD4+/CD45RA+ T-cells.

Disclosures:

No relevant conflicts of interest to declare.
MUTATIONAL STATUS of REARRANGED IMMUNOGLOBULIN HEAVY CHAIN VARIABLE Genes in a COHORT of SLOVENIAN PATIENTS with CHRONIC LYMPHOCYTIC LEUKAEMIA.
Authors: Pajič Tadej , Maleŝiĉ Ivan , Zver Samo , Ĉernelĉ Peter
Published: October 2019

Abstract 4399

Introduction

The aims of the study were to determine the mutational status and IGHV gene usage in our cohort of patients with B-CLL and compare the results to those published in the literature.

Patients and Methods

57 B-CLL patients (22 female, 35 male), median age 65 (range from 37 to 88 years) in various Binet stages (47 Binet A, 6 Binet B, 4 Binet C) were included in the study. All the patients were untreated at the time of blood collection. Leukaemia cells were isolated from venous blood samples of patients by Ficoll density gradient centrifugation. Total cellular RNA was isolated from leukaemia cells and used to the prepare cDNA by reverse transcription. After heteroduplex analysis, clonal PCR products were identified with polyacrylamide gel electrophoresis. The PCR products were sequenced and mutational status was determined by comparing the sequence of the IGHV region of the patient sample to the most homologous germ line V sequence. Sequence that was homologous more than 98 % with their corresponding germ line sequence was considered unmutated.

Results

Unmutated and mutated IGHV mutational status in our cohort of patients with B-CLL was found in 42 % (N=24) and 58 % (N=33), respectively.

At the IGH subgroup level, the most frequently used subgroup was IGHV3 (38,55 %), followed by IGHV1 and IGHV4, found in 28,10 % and 22,85 %, respectively. Subgroups IGHV2 and IGHV5 were found out in 2 patients each (3,5 %). The other subgroups, IGHV6 and IGHV2, were determined in one patient each. At the specific gene level, the most frequently used gene IGHV was IGHV1-69, found out in 15,85 %. Genes V4-34, V4-39 in V3-23 were determined in 7,05 %, followed by genes V3-07, V3-11, V3-15, V3-30 and V3-48 identified in 5,25 % each. The others were identified at the lower frequency.

In patients with unmutated IGHV genes, the most frequently observed gene was V1-69 (33,3 %). The most frequently used gene in mutated rearrangements was IGHV4-34 (12,13 %). The gene V3-21 was not found out in our cohort of patients.

Conclusions

In the study performed, we have found out that the portion of unmutated IGHV genes of the clonal B lymphocytes, the gene usage and somatic mutations frequency were similar to those published in the literature. Interestingly, the gene V3-21 was not found out in our cohort of patients, the results that is similar to the Mediterranean-based study. In the future, the geographic comparison and more definitive conclusions could be made on the population based investigations of IGHV gene use in large series of CLL patients in Slovenian region.

Disclosures:

No relevant conflicts of interest to declare.
Resveratrol Plus Simvastatin Are Effective in Decreasing IgM Secretion in Asymptomatic Waldenstrom Macroglobulinemia. One Year Follow-up.
Authors: Iuliano Francesco , Infusino Stefania , Perricelli Alessia , Di Maio Massimo , Pomillo Angelo , Luci Maria , Mingrone Francesco
Published: October 2019

Abstract 4401

Background

Waldenström macroglobulinemia (WM) is a distinct B-cell lymphoproliferative disorder characterized by lymphoplasmacytic bone marrow infiltration along with an immunoglobulin M (IgM) monoclonal gammopathy. Asymptomatic patients with monoclonal IgM and without morphologic evidence of bone marrow infiltration < 10% clonal marrow cells) are classified as having IgM-MGUS.Therapy is postponed for asymptomatic patients, and progressive anemia is the most common indication for initiation of treatment. Resveratrol (3,4',5-tri-hydroxy-trans-stilbene) is an antioxidant constituent of a wide variety of plant species including grapes. It has gained considerable attention because of its anticancer properties, as shown in solid and hematologic malignancies. Published data show that resveratrol has significant antitumor activity in WM cells line. Moreover, simvastatin, a 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitor, induced inhibition of proliferation, cytotoxic effect and apoptosis in IgM secreting cell lines as well as in primary CD19(+) WM cells. With this background we have treated 4 patients with asymptomatic WM with an association of simvastatin and resveratrol to test the efficacy of such of drugs in asymptomatic Waldenstrom macroglobulinemia

Methods

4 pts (3 males and 1 female), median age 42,3 yrs (range, 42–73) and asymptomatic WM were treated with a schedule containing resveratrol 40 mg/die and simvastatin 20 mg/die for at least 90 days. At enrollment patients characteristic were hemoglobin level median, 12.1 g/dL,serum beta(2)-microglobulin level median, 2.4 mg/L, and IgM peaks median, 1.8 g/dL. All patients have taken regularly the drugs and there have been no adverse events.CK and LDH serum levels were kept in the normal range.

Results

In all IgM-MGUS patients a reduction of more than 50% of the IgM peak was observed after 3 months of therapy and it was still maintained at 12 months of follow-up. In SWM patient the reduction was about 25% and it was manteined over time. Striking, another patient with Waldentrom disease resistant to the previous therapy with EDX and anti-CD20 MoAb achieved a CR only after adding resveratrol and simvastatin.

Conclusions

Our data demonstrate clearly that the association between resveratrol with simvastatin decreases IgM secretion in Waldenstrom macroglobulinaemia and can be useful in asymptomatic or low risk patients not having any adverse effects.

Disclosures:

Off Label Use: Simvastatin showed in vitro activity on waldentrom cell lines Resveratrol showed in vitro activity on waldenstro cell lines.
KIR Genes and Their HLA-C Ligands in B-Cell Chronic Lymphocytic Leukemia in a Polish Population.
Authors: Frydecka Irena , Jedynak Anna , Karabon Lidia , Pawlak Edyta , Tomkiewicz Anna , Kielbinski Marek , Woszczyk Dariusz , Wolowiec Dariusz , Kuliczkowski Kazimierz
Published: October 2019

Abstract 4402

Introduction

Dysfunction in cellular and humoral immunity entails an increased risk of B-cell chronic lymphocytic leukemia (B-CLL). It has been suggested that innate immunity, especially natural killers cells play key role in antitumor cytotoxicity regulated by interaction between killer immunoglobulin-like receptors (KIRs) on NK cells and some T-cell subpopulations and their HLA-class I ligands on target cells. KIRs receptors have been divided into two functional groups: with long cytoplasmic tails (DL) - inhibitory and with short cytoplasmic tails (DS) - activating. Two broad haplotypes of KIR genes have been defined: the A haplotype characterized by presence of single activating KIR gene (2DS4) and the B haloptype characterized by two or more activating KIR genes (2DS1, 2DS2, 2DS3, 2DS5 and 3DS1).The 2DL2, 2DL3 and 2DS2 KIRs bind to HLA-C1 allotype (Asp80), while 2DL1 and 2DS1 KIRs bind to HLA-C2 allotype (Lys80). Due to lack of representative data in literature the study was undertaken to determine the association between polymorphism of KIR genes and HLA-C allotypes and susceptibility to B-CLL.

Patients and methods

The following KIR genes were typed: 2DL1, 2DL2, 2DL3, 3DL1, 2DS1, 2DS2, 2DS3, 2DS4fl, 2DS4del, 2DS5, 3DS1, in 200 B-CLL patients and 199 healthy individuals using PCR-SSP method. The HLA-C1 and HLA-C2 were determined using Olerup SSP typing kit in 187 B-CLL patients and 104 controls.

Results

Individual KIR gene content was similar in B-CLL cases and healthy controls although patients with KIR2DS3 gene were more common among cases compared to controls (36% vs. 27%, p=0.06) (Table1). The frequency of AA haplotype did not differ between studied groups (29% vs. 28%). Moreover no differences were found between incidence of HLA-C allotypes (HLA-C1: 74.3% vs. 76.0%, HLA-C2: 67.4% vs.70.2%), genotypes (C1/C1: 32.6% vs 29.8%, C1/C2: 41.7% vs 46,2% and C2/C2: 25.7% vs 24.1) and KIR genes in combination with genes for their HLA-C ligands in B-CLL patients and controls (Table 2).

Conclusions

The data obtain in this study imply that there may be not direct association between KIR and HLA-C gene content in the genome and the presence of B-CLL.

Disclosures:

No relevant conflicts of interest to declare.
Acadesine (Acadra‘) for Patients with Resistant/Relapsed Chronic B-Cell Lymphocytic Leukemia (B-CLL): A Multicentre Phase I/II Study.
Authors: Van Den Neste Eric W , Gonzalez-Barca Eva , Janssens Ann , Cazin Bruno , de Oteyza J. Pérez , Terol Maria Jose , Auton T. , Saunders Andrew , Campas Clara
Published: October 2019

Abstract 4405

Acadesine (Acadra‘) is a nucleoside that induces cell death in B-cell chronic lymphocytic leukemia (B-CLL) cells ex vivo in a dose-dependent manner over the concentration range 50 mM to 1 mM. Acadesine enters B-cells and is phosphorylated to its active metabolite, ZMP, which induces apoptosis independently of ATM or p53. It is active against B-CLL cells from patients who have not responded to prior treatment with fludarabine and/or chlorambucil (Campas et al, 2003).

Based on this pre-clinical data, a Phase I/II clinical study was designed to determine the safety and tolerability of acadesine given intravenously as a 4-hour infusion to patients with B-CLL. This is an open-label dose escalation study of acadesine with two parts. In Part I, the dose escalation part of the study, patients receive a single dose of acadesine on Day 1 and are followed up to Day 22. In Part II, patients will receive up to 5 doses of acadesine at the maximum tolerated dose (MTD) identified in Part I over a period of up to 20 days starting on Day 1. The patient population includes B-CLL patients with refractory or relapsed disease who have received one or more (≥ 1) prior lines of treatment which must have included either a fludarabine or an alkylator based regimen. Primary endpoints of the study evaluate the safety and tolerability of acadesine. Secondary endpoints evaluate the pharmacokinetics of acadesine in plasma, its metabolite ZMP in whole blood, and changes in peripheral blood B-cell and T-cell counts as efficacy biomarkers. Optimal Biological Dose (OBD) is defined as the minimal acadesine dose that gives a maximum Cmax for ZMP in whole blood with a good safety profile.

Twelve patients (6 males and 6 females) have been administered acadesine to date in 3 cohorts in Part I. Acadesine has an excellent safety profile to date. The principal safety finding was asymptomatic acadesine-related hyperuricaemia of short duration in four patients. Prophylactic allopurinol has significantly reduced the incidence of hyperuricaemia.

Pharmacokinetic data showed acadesine rapidly disappears from plasma to be converted into ZMP inside blood cells. The OBD has not been reached yet and maximum dose administered to date is 139.5 mg/kg. The next cohort will receive 210 mg/kg. Additional safety, pharmacokinetics and efficacy data will be presented at the meeting.

Disclosures:

Off Label Use: Acadesine (AcadraTM): nucleoside that induces cell death in B-cell chronic lymphocytic leukemia.. Auton:BTG Limited: Consultancy; Advancell Technologies Inc.: Consultancy. Saunders:BTG limited: Consultancy; Advancell Technologies: Consultancy. Campas:Advancecell Technologies Inc: Employment, Equity Ownership.
Thromboembolic Complications of Intravenous Immunoglobulin (IVIG) in Hypogammaglobulinemic Patients with Chronic Lymphocytic Leukemia (CLL).
Authors: Milani Cannon , Dalia Samir , Colvin Gerald A.
Published: October 2019

Abstract 4416

Infectious complications represent a major cause of morbidity and mortality in patients with chronic lymphocytic leukemia (CLL). The etiology is postulated to be secondary to aberrations in cell-mediated immunity, as well as to therapy-related immunosuppression.

Hypogammaglobulinemia, which occurs in virtually all patients with CLL, may beprofound and correlates with disease duration and stage. In 1981 during treatment of two children with hypogammaglobulinemia and coincidental idiopathic thrombocytopenic purpura (ITP), physicians in Switzerland observed a reproducible increase in the platelet count following IVIG treatment. This clinical observation and follow up systemic investigations further intensified the widespread clinical use of IVIG as a potential immune modulatory agent. Intravenous immunoglobulin (IVIG) therapy has been used successfully to prevent and treat infections in this cohort of patients.

Today, Intravenous immunoglobulin (IVIG) is used in a broad spectrum of autoimmune, inflammatory, and primary and secondary immunodeficiencies. The efficacy has been demonstrated in several control studies. IVIG is a blood product prepared from the serum of between 1,000 and 15,000 donors per batch. The mechanism of action validated by in-vitro models is exerted by a combined effect on autoantibodies, complement activation, cytokines, and saturation of Fc receptors on tissue macrophages.

However IVIG administration and treatment is not benign and should be used with caution given the potential manifestations of thromboembolic complications. High concentration and rapid infusion rate of the IVIG, as well as increased dose and osmolarity of the solution are thought to predispose to thrombotic events. Serum viscosity is the implicated mechanism for compromised blood flow and predisposition of high-risk patients to cardiovascular or cerebrovascular infarction.

In this retrospective review, we detail IVIG related thromboembolic manifestations in CLL patients, to highlight the importance of risk stratifying patients prior to treatment administration. The current consensus surrounding IVIG is that of a relatively safe treatment, with minor adverse effects such as hypertension, fever and chills, nausea, myalgias, or headache.

However our report highlights the importance of proceeding with caution in the application of this therapy, as it's proclivity for thrombotic complications has not been fully elucidated in patients with underlying malignancies. Pre-existing thrombogenic risk factors should be carefully evaluated in patients undergoing treatment with IVIG. Clinical evaluation, with careful attention to vascular history and underlying co-morbidities can potentially unmask the high-risk patient where IVIG could be lethal.

Disclosures:

No relevant conflicts of interest to declare.
Dysregulation of Metabolic Genes May Be Normalized by Epigenetic Drugs (Vorinostat, Decitabine) and Nutritional Compounds (Genistein, Vitamin D) in Leukemic Cells.
Authors: Karlic Heidrun , Varga Franz , Pittermann Elisabeth , Pfeilstocker Michael
Published: October 2019

Abstract 4423

Background

Dysregulation of energy metabolism is a key feature of malignancy and known to be regulated by epigenetic mechanisms. This study explores gene expression in the KG1 cell line and a primary culture of leukemic blasts from a patient, both incubated with SAHA (suberoyl anilide hydroxamic acid, also known as Vorinostat) in comparison to other epigenetic drugs such as Decitabine (DAC) but also nutritional compounds such as genistein and vitamin D3 with a known activity on metabolism where epigenetic mechanisms play a role.

Methods

Following a 3 days incubation with pharmacologic concentrations of the above mentioned compounds, gene expression patterns of the KG1 cell-line were analysed on an Affymetrix Gene Expression Array and specifically evaluated by quantitative real time PCR from KG1 cells and primary leukemic cells of a patient with an AML-M2 which were cultured at the same conditions as the KG1 cells. In order to confirm epigenetic mechanisms induced by targeted drugs, methylation patterns of selected tumor suppressor genes were analyzed using specific primers for bisulfite-treated DNA.

Results

Our experiments show that expression of a large scale of micro RNAs which are known to be responsible for alternative splicing is stimulated by SAHA as well as DAC and to a lesser extent also Vitamin D3 and genistein, but downregulation of mitochondrial RNAs as a consequence of apoptosis was exclusively observable by SAHA.

HDAC-inhibition results in clear repression of metabolic genes involved in glycolysis (5/6 genes) as well as cholesterol-biosynthesis (14/15 genes) and fatty acid synthesis (all 4 genes) which are also known to be regulated by nutrition. Classic tumour suppressor genes, transcription factors and cell-cycle regulators such as P53, P15, P16 and P21 and FOXO were (re-) activated with this treatment while MYC was down-regulated.

Discussion

A downregulation and thus normalization of a large scale of genes including key genes from malignancy-associated stimulation of glycolysis as well as biosynthesis of cholesterol and fatty acids was observed with SAHA and nutritional compounds such as Vitamin D and genistein. We propose that these interactions should be carefully considered in clinical application, and a combination of synergistic drugs and/or dietary strategies may provide a major advantage in future drug development.

Acknowledgment

Supported by Jubilaeumsfonds der Oesterreichischen Nationalbank.

Disclosures:

No relevant conflicts of interest to declare.
Results of a World Wide Survey On the Diagnostic Dilemma Between Type 2B Von Willeband Disease and the PT-VWD.
Authors: Othman Maha , Hamilton Alexander , Lillicrap David
Published: October 2019

Abstract 4436

The Canadian PT-VWD project (www.pt-vwd.org) initiated in 2007 is based on a molecular diagnostic approach to differentiate between type 2B VWD and its closely similar disorder PT-VWD. Recruitment of worldwide samples and data still poses a challenge partly because of the relative rarity of the two bleeding disorders and more importantly because it is dependant on the level of awareness about the requirements for the differential diagnosis and the subsequent impact on the treatment decision. The objective of this survey was to gather an opinion from the worldwide hemostasis community about this diagnostic dilemma and the current utilization of the appropriate diagnostic tests. The survey comprised eight key questions (see link on website) sent individually by email to hematologists from different countries and representing a variety of academic centres. Out of 60 emails we received 37 responses. The highest response was from Europe 16/37 followed by USA 9/37. Other responses were from Africa (3), Canada (2), Asia (2), Middle East (2), South America (1), Mexico (1) and Australia (1). Fifty four percent of specialists have seen or diagnosed 1-5 type 2B VWD cases and 38% have seen or diagnosed 1-5 PT-VWD cases. 82% have reported they have access to laboratory phenotypic tests including RIPA and/or platelet mixing studies with only 59% reporting they have access to genetic testing of the two responsible genes: VWF and platelet GP1BA gene. Genetic analysis was the most discriminative test in the opinion of 54% and the RIPA was chosen to be the discriminative test by 35% while 11% believed the appropriate discrimination should be based on both. 84% agreed to classify PT-VWD under platelet function disorders and 73% have felt that PT-VWD should be investigated when platelet function defects are suspected. On a scale from 1-10 with 10 being most important, 30/37 of participants reported 7-10 to rate the importance of this diagnostic dilemma and 13% have reported 5. Despite the relatively small number of respondents, the data obtained through this survey confirm the rarity of the two disorders, reflects the positive world-wide opinion re the diagnostic challenges of the two closely similar disorders and indicate the significant unavailability of genetic analysis as a useful confirmatory test.

Disclosures:

No relevant conflicts of interest to declare.
Therapeutic Algorithm for Patients with Acquired Von Willebrand Syndrome and Monoclonal Gammopathy.
Authors: Staritz Peter , Krause Manuela , Zimmermann Rainer , Huth-Kuehne Angela
Published: October 2019

Abstract 4445

In contrast to congenital von Willebrand Syndrome therapy of acute bleeding in acquired von Willebrand Syndrome (AWS) with von Willebrand factor containing concentrates (F VIII/vWF) is often ineffective. In patients with IgG monoclonal antibodies therapy with intravenous immunoglobulins (IVIG) is reported to be effective whereas patients with IgM antibodies seem not to respond to IVIG.

Over the last years we diagnosed and treated several patients with AWS either prior to surgery or due to acute bleeding. About half of the patients responded adequately to IVIG as reported in the literature. One patient with no response to IVIG 4 days after the last dose received DDAVP with a significant increase in von Willebrand factor-antigen and ristocetin cofactor and normalized half life. Dental surgery could be performed without any bleeding complications under daily infusions of DDAVP. Another patient with partial response to IVIG and contraindications to DDAVP received F VIII/vWF prior to dental extraction. In contrast to former recoveries with a shortened half life of the infused concentrate (about 2 hours), half life of vWF was significantly prolonged following prior IVIG treatment. Another patient responded sufficiently to single therapy with F VIII/vWF. A further patient with suspected diagnosis of moderate hemophilia A had received DDAVP for severe epistaxis with good response and sufficient half lifes. He was transferred to our centre and we diagnosed AVW with IgM antibodies. We performed another recovery and half life with DDAVP with adequate response.

According to our experience we propose the following therapeutic algorithm: A recovery with DDAVP should be performed first line if treatment is not contraindicated. In case of insufficient half lifes and/or intended major surgery, a recovery and half life with F VIII/vWF concentrate should follow. In case of significantly reduced half life of wWF an attempt with IVIG is necessary. If response to IVIG is not adequate further treatment with DDAVP or F VIII/vWF is indicated.

As treatment with IVIG is not predictable in all patients with IgG antibodies and is very costly a therapeutic attempt with DDAVP in the first line and F VIII/vWF second line is worthwhile. In major surgery requiring prolonged replacement therapy, IVIG treatment, if effective, is less costly than treatment with FVIII/vWF.

Disclosures:

No relevant conflicts of interest to declare.