bluuebunny/crossref_metadata_2025 huggingface dataset:
Relapsed Multiple Myeloma: Who Benefits From Salvage Autografts?
Authors: Chow Annie W.S. , Lee Cindy H.S. , Hiwase Devendra K , To Luen Bik , Horvath Noemi
Published: October 2019

Abstract 3059

Aim

Despite novel agents, multiple myeloma (MM) remains incurable. The majority of patients with MM will relapse at some stage after induction therapy and high dose chemotherapy with autologous stem cell transplant (ASCT). Published studies have recommended salvage ASCT as an effective option in patients with relapsed MM. However, the patient cohort who will derive maximum benefit from salvage ASCT is still undefined and is likely to evolve with increasing therapeutic options.

We aimed to evaluate the role of salvage ASCT in relapsed MM patients.

Methods

We performed a retrospective analysis of patients who underwent salvage ASCT for relapsed MM in South Australia between 1992 and 2011.

Results

During this period, autologous stem cell transplants were performed for 457 patients with newly diagnosed MM. Thirty-nine patients subsequently underwent salvage ASCT for relapsed MM. The median age of patients at salvage ASCT was 59 (34–73) years, and 85% were ISS I and II at diagnosis. The majority of patients (90%, n=35) had a progression free interval (PFI) of at least 12 months after initial ASCT, consistent with recommendations by current literature. Salvage ASCT was performed for four patients with PFI shorter than 12 months, due either to suboptimal response to novel agents (thalidomide, lenalidomide, bortezomib), or the lack of novel agent availability in the earlier years.

The median progression-free survival (PFS) and median overall survival (OS) following salvage ASCT were 22 months (95% CI: 11–32) and 52 months (95% CI: 30–74) respectively. Non-relapsed mortality at 2 years was 7%. Following salvage ASCT, the median PFS (28 vs. 12 months, p = 0.006) and OS (83 vs. 26 months, p = 0.02) were significantly longer in patients whose progression free interval after the initial ASCT was > 24 months (Figures 1 and 2). Use of novel agents in salvage induction and maintenance therapy post salvage ASCT did not appear to influence outcome.

Conclusion

Our results suggest that there remains a role for salvage ASCT, even in the era of novel therapies. Salvage ASCT may result in durable responses, particularly in patients with longer progression free interval (> 24 months) following initial ASCT. This interval could be included as a selection criterion for patients with relapsed myeloma who remain transplant eligible.

Disclosures:

No relevant conflicts of interest to declare.
12-HETrE, An Endogenous Inhibitor of Platelet Activation,
Authors: Yeung Jennifer , Ikei Kenneth , Vesci Joanne , Arnouk Alex , Holman Theodore R , Holinstat Michael
Published: October 2019

Abstract 3254

Platelet activation plays a pivotal role in thrombosis and hemostasis. Understanding the signaling events mediating this process is essential in preventing unwanted clot formation, which can lead to the development of heart attack, stroke, and venous thromboembolism. Fatty acids may play an important role in determining the level of platelet reactivity, however the mechanism(s) by which this occurs are not entirely clear. 12-lipoxygenase (12-LOX) has been shown to oxidize the fatty acid, arachidonic acid (AA), in order to produce the bioactive eicosanoid 12-HETE, which has recently been shown to play a role in tissue factor activation and subsequent thrombin generation in the platelet (Thomas et al., 2010, J Biol Chem; 285:6891–903). 12-HETE has also been shown to signal to cells, in part, through the G protein-coupled receptor GPR31 (Guo et al, 2011, J Biol Chem; epub). While research on 12-LOX-mediated eicosanoid regulation of platelets has primarily focused on 12-HETE, the potential for regulation of platelets by eicosanoids derived from other fatty acids has been overlooked. As the fatty acid content on the platelet membrane is extremely dynamic and fatty acid supplementation is correlated with a reduced risk for cardiovascular disease, we hypothesized that 12-LOX oxidation of another fatty acid may play a direct role in regulating platelet function. To test this hypothesis, the Ω-6 fatty acid dihomo-γ-linolenic acid (DGLA) as well as its eicosanoid derived from 12-LOX oxidation, 12-hydroxyeicosatrienoic acid (12-HETrE), were applied to washed platelets followed by stimulation with thrombin, PAR1-AP, or PAR4-AP. Platelets treated with either DGLA or 12-HETrE showed significant attenuation in platelet aggregation following stimulation with thrombin or PAR-AP. A number of biochemical intermediates were also tested in the presence of 12-HETrE or DGLA including Rap1 activation, αIIbβ3 integrin activation, α-granule secretion, and dense granule secretion. All endpoints tested were attenuated in the presence of DGLA or 12-HETrE relative to control. To confirm the regulation was unique to DGLA and its metabolite, the same endpoints were measured in the presence of AA or 12-HETE. Pre-treatment with either AA or 12-HETE did not attenuate any of the agonist-mediated platelet activation endpoints. Thus, our data supports a unique role for 12-HETE and 12-HETrE and that fatty acid regulation of platelet function may be highly dependent on the lipid content of the platelet. Shifting the ratios of fatty acids in the platelet through dietary supplementation or pharmacological intervention may be sufficient to induce a cardio-protective state, in part, through increasing 12-HETrE formation and subsequently inhibiting platelet activation.

Disclosures:

No relevant conflicts of interest to declare.
A Genome Wide Association Study Identifies Novel Susceptibility Genes for Pediatric Venous Thrombosis,
Authors: Arning Astrid , Hiersche Milan , Bidlingmaier Christoph , Stoll Monika , Nowak-Gottl Ulrike
Published: October 2019

Abstract 3336

Background:

Genome wide association studies (GWAS) are the current method of choice to dissect the genetic basis of common complex diseases. Up-to-date, studies in families with a known first onset of symptomatic arterial or venous thrombosis (VT) in early childhood are lacking. Methods: Here, we performed a GWAS in a large family-based study sample comprising 241 nuclear families with pediatric VT using the Illumina 660W Infinium SNP array. The average genotype call rate was >99.5%, and the genomic inflation factor was a= 1.012. Single point and haplotype association was assessed using the Transmission Disequilibrium Test (TDT) as implemented in PLINK and FBAT, respectively, and corrected for multiple testing using permutation testing. In addition, associations were corrected for age, gender, and in a subsequent analysis for Factor VLeiden. Results and Conclusion: Four SNPs exceeded the threshold for genome wide significance in this dataset as determined by permutation testing using 100.000 bootstrap permutations (p<10−5), and are likely true associations. Among these, 2 SNPs reside in a region on chromosome 6q13 comprising the gene for beta-1,3-glucoronyltransferase 2 (B3GAT2), a member of the human natural killer 1 (HNK1) carbohydrate pathway are associated with pediatric VT with p-values for rs1304029 (p=3.42×10−6) and rs2748331 (p=6.92×10−6). The corresponding haplotype association test resulted in a p=5.37×10−6 for the GA-haplotype, further underlining the robustness of the association. The SNPs rs636434 on chromosome 6q12 and rs1565242 on chromosome 15 both reside within hypothetical genes and are associated with VT with a p=2.70×10−6 and p=8.24×10−6, respectively. Additional SNPs exceeding a p<10−5 are included in subsequent analyses looking at gene networks and replication in independent study samples including our second GWAS on pediatric thromboembolic stroke (TS). Future studies using larger study samples are warranted to validate these findings and to characterize the genetic architecture underlying VT and TS in children.

Disclosures:

No relevant conflicts of interest to declare.
Non-Steroidal Anti-Inflammatory Drugs and Imatinib; Drug Interactions That May Impact Efficacy,
Authors: Wang Jueqiong , Hughes Timothy P , Kok Chung Hoow , Saunders Verity A , Frede Amity , GrootObbink Kelvin , Osborn Michael Philip , Somogyi Andrew A , D'Andrea Richard J , White Deborah L.
Published: October 2019

Abstract 3501

Aim:

The human organic cation transporter-1 (OCT-1) is the primary active influx protein for imatinib (IM) into target BCR-ABL-positive cells. The functional activity of the OCT-1 protein (OCT-1 activity; OA) is predictive of molecular response and event-free survival in de-novo chronic phase chronic myeloid leukemia (CP-CML) patients. Due to the common use of non-steroidal anti-inflammatory drugs (NSAIDs) among CML patients on IM to treat musculoskeletal pain, it is of particular interest to investigate the impact of NSAIDs on OCT-1 mediated IM uptake.

Methods:

12 NSAIDs were firstly examined for their impact on OA using the intracellular uptake and retention (IUR) assay and [14-C]-IM. The degree of in vitro kinase inhibition achieved was assessed using p-Crkl in the IC50imatinib assay. Viable cell numbers were determined by trypan blue exclusion method after 72 hours incubation with the co-administration of NSAIDs and IM. NSAIDs concentration was adjusted to median blood levels on standard doses.

Results:

The majority of NSAIDs (10 of 12) selected did not induce any significant change in OA in CML cell lines. Compared to control cells, increased OA were observed when K562 or KU812 cells were treated with diclofenac (10μM), and this resulted in a concomitant decrease in the IC50imatinib. In keeping with this, after 72 hours co-incubation with IM, diclofenac resulted in a significantly lower viable cell number compared with IM alone. In contrast, ibuprofen (130μM) led to a significant decrease in OA, resulting in an increased IC50imatinib and thus reduced the inhibition of IM on cell growth number (Table 1).

The effects of diclofenac and ibuprofen on IM efficacy were then further investigated using primary samples. In keeping with the results observed in cell lines, diclofenac resulted in significant increases in OA, which translated to a significant decrease in IC50imatinib. Ibuprofen induced significant decreases in OA in all patients and healthy donors (Table 1).

Conclusion:

These finding provide strong evidence for interactions between some NSAIDs and IM which may substantially impact the response of CML patients to IM. While drugs such as diclofenac are likely to have a positive influence on IM efficacy, the findings observed with ibuprofen suggest caution when administrating NSAIDs to CML patients on IM treatment.

Disclosures:

Hughes: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; ARIAD: Honoraria, Membership on an entity's Board of Directors or advisory committees. Osborn:Novartis: Research Funding. White:Novartis Pharmaceuticals: Research Funding; BMS: Research Funding.
Chronic Lymphocytic Leukemia Associated with Autoimmune Hemolytic Anemia Is Characterized by a Distinctive miRNA Signature,
Authors: Ferrer Gerardo , Navarro Alfons , Hodgson Kate E , Colomer Dolors , Aymerich Marta , Baumann Tycho , Pereira Arturo , Monzó Mariano , Moreno Carol , Montserrat Emili
Published: October 2019

Abstract 3897

Chronic lymphocytic leukemia (CLL) is frequently associated with autoimmune hemolytic anemia (AIHA). However, the mechanisms governing the association between CLL and AIHA are poorly understood. MicroRNAs (miRNAs) are small 18–22 nucleotide long RNA molecules that regulate gene expression and play a key role in several biological processes. Importantly, deregulated miRNA expression has been implicated both in CLL and autoimmunity. This led us to speculate that patients with CLL who develop AIHA might have a different miRNA expression pattern as compared to those in whom this complication is not observed. We report here the first results of this study. We have evaluated the miRNA expression in purified CLL cells (CD19+, CD5+) from 14 patients who developed AIHA over the course of their disease and 19 sex-, age-, and clinical stage-matched controls who, after a comparable follow up time, did not develop this complication. The expression of 377 mature miRNAs was analyzed using TaqMan Human MicroRNA Arrays A v2.0 (Applied Biosystems) in an ABI 7900 HT sequence detection system. miRNA expression data was analyzed by the 2–ΔΔCt method, using RNU48 as endogenous control. Statistical analyses were performed with TiGR MultiExperiment Viewer, BRB-ArrayTools and R software. The unsupervised hierarchical cluster analysis identified two groups (CLL AIHA+ and CLL AIHA-). The supervised analysis revealed 7 miRNAs that were down-regulated in CLL AIHA+ patients compared to CLL AIHA- patients: miR-19a, miR-20a, miR-29c, miR-146b-5p, miR-324-3p, miR-340, miR-660. Interestingly, miR-146, which has previously been related to autoimmunity, regulates IL-1 receptor-associated kinase 1 and TNF receptor-associated factor 6, two key adapter molecules downstream of Toll-like and cytokine receptors. The prediction analysis of microarrays (PAM) was used to determine a set of miRNAs able to classify the samples into CLL AIHA+ and CLL AIHA-. We obtained a 122-miRNA model that classified CLL AIHA+ patients with a sensitivity of 69.4% and specificity of 68.4%. This model correctly classified 67% of the analyzed samples. In summary, CLL AIHA+ samples were characterized by a distinctive signature of 7 down-regulated miRNAs, one of which (miR-146) has previously been related to autoimmunity. Moreover, we identified 122 miRNAs that are able to predict AIHA in CLL patients. This is the first study establishing a relationship with CLL associated with AIHA and a distinctive miRNA signature, which should be useful for further studies aimed at unfolding the biologic complexity of AIHA in CLL.

Disclosures:

No relevant conflicts of interest to declare.
Association of Disparities in Known Minor Histocompatibility Antigens with Relapse-Free Survival and Graft-Versus-Host-Disease Upon Allogeneic Stem Cell Transplantation,
Authors: Hobo Willemijn , Broen Kelly , van der Velden Walter J.F.M. , Greupink-Draaisma Annelies , Adisty Niken , Wouters Yannick , Kester Michel G.D. , Fredrix Hanny , Jansen Joop H. , van der Reijden Bert A. , Falkenburg J.H. Frederik , de Witte Theo , Preijers Frank W.M.B. , Schattenberg Anton , Feuth Ton , Blijlevens Nicole N.A. , Schaap Nicolaas , Dolstra Harry
Published: October 2019

Abstract 4136

Purpose:

Allogeneic stem cell transplantation (allo-SCT) can induce remission in patients with hematological malignancies due to graft-versus-tumor (GVT) responses. This, however, is often accompanied by graft-versus-host disease (GVHD). Both the GVT effect and GVHD are mediated by minor histocompatibility antigen (MiHA)-specific T cells recognizing peptide products from polymorphic genes that differ between recipient and donor. Here, we evaluated whether MiHA mismatches are associated with clinical outcome after partial T cell depleted allo-SCT.

Patients and Methods:

We retrospectively analyzed the impact of MiHA mismatches in a cohort of 327 patients who received a partially T cell-depleted allo-SCT because of a hematological malignancy. MiHA allele genotyping was performed by fluorescence-based competitive allele-specific PCR. Subsequently, a multivariable statistical analysis of immunogenic MiHA disparity rates and association with clinical outcome was performed. In addition, development of MiHA-specific T cell responses was assessed by dual-color tetramer staining.

Results:

Statistical analysis revealed that an autosomal MiHA disparity on DNA level associates with increased relapse-free survival in sibling transplants, especially in patients transplanted for multiple myeloma. In addition, mismatches for the ubiquitous Y chromosome-encoded MiHA resulted in more acute GVHD (grade 3–4), while other MiHA mismatches, either ubiquitous or restricted to hematopoietic cells, were not associated with GVHD. Finally, we demonstrated considerable differences between MiHA in the capability to induce in vivo T cell responses post-transplantation.

Conclusion:

These data support that autosomal MiHA contribute to the induction of GVT immunity providing a rationale for MiHA-based post-transplantation immunotherapy to prevent and treat persistent and recurrent cancer following allo-SCT.

Disclosures:

No relevant conflicts of interest to declare.
Favorable Responses to Novel Agents for Multiple Myeloma in African American Patients,
Authors: Saraf Santosh , Patel Pritesh R. , Ozer Howard , Peace David , Nimmagadda Sangeetha , Quigley John G. , Rondelli Damiano
Published: October 2019

Abstract 4213

Multiple myeloma (MM) accounts for 10% of hematologic malignancies in the U.S. African Americans (AA) have twice the risk of developing multiple myeloma and, in previous studies, a higher mortality rate when compared to non-African Americans (non-AA). In recent years, clinical outcomes for patients with MM have improved as a result of new agents, such as immunomodulatory drugs (IMiDs) and bortezomib. However, the therapeutic impact of these new therapies in AA vs. non-AA patients has not been evaluated. In this study, 53 consecutive patients (23 AA, 30 non-AA) with newly diagnosed MM were retrospectively analyzed after induction treatment with thalidomide/dexamethasone (n=22), lenalidomide/dexamethasone (n=8), bortezomib/dexamethasone (n=5), bortezomib/thalidomide/dexamethasone (n=3), lenalidomide/bortezomib/dexamethasone (n=12), or an IMiD plus melphalan (n=3). AA and non-AA patients were comparable for age, immunoglobulin isotype, MM stage of disease, serum β2microglobulin and albumin levels, and cytogenetic abnormalities including del13 (27% vs. 38%, respectively, p=NS). When measured according to international uniform criteria, the response rate to induction therapy was not statistically different between AA and non-AA patients: complete remission (CR, 22% vs. 21.4%), very good partial remission (VGPR, 26% vs. 21.4%), or partial remission (PR, 43.4% vs. 32.1%). However, the rate of stable/progressive disease following induction therapy was significantly higher in non-AA patients (p = 0.03). Of 53 patients, 34 received high-dose chemotherapy followed by autologous stem cell transplant (ASCT). In this group, the CR + VGPR rate following ASCT was significantly higher in AA compared with non-AA patients (59% vs. 35%, p=0.0007). At a median follow up of 47 months, the relapse rate was 59% in AA and 46% in non-AA patients (p=NS) and the median time to progression (TTP) was 9.1 months in both groups. Five patients (9%) died. All deaths occurred in the non-AA cohort and in 4/5 cases death was due to disease progression. In conclusion, our findings demonstrate that despite their increased disease risk, AA patients with MM have a favorable outcome that is equivalent to that of non-AA patients when treated with IMiDs and/or bortezomib.

Disclosures:

No relevant conflicts of interest to declare.
A Retrospective Evaluation of the Effects of Severe Aplastic Anemia Treatment in Children with Horse and Rabbit ATG.Polish Pediatric Hematology Group
Authors: Pawelec Katarzyna , Matysiak Michal , Salamonowicz Malgorzata , Kowalczyk Jerzy , Balwierz Walentyna , Zaleska-Czepko Ewa , Chybicka Alicja , Szmyd Krzysztof , Szczepanski Tomasz , Bubala Halina , Wysocki Mariusz , Kurylak Andrzej , Wachowiak Jacek , Szprecht Dawid , Mlynarski Wojciech , Bulas Monika , Krawczuk-Rybak Maryna , Leszczynska Elzbieta , Urasinski Tomasz , Peregud-Pogorzelski Jaroslaw , Balcerska Anna , Kaczorowska Barbara
Published: October 2019

Abstract 4375

Antithymocyte horse globulin (hATG) or rabbit (rATG) is used to treat children with severe aplastic anemia (SAA) in the absence of a compatible bone marrow family donor’s. We presenting (viewing) retrospective evaluation of the effects of SAA therapy using hATG and rATG.

49 children (20 girls and 29 boys, aged 1.2–17) were treated between 1993 and 2000 - hATG (15mg/kg/day for 5 days with cyclosporine 5mg/kg/day for 12–24 mths). On day 180 day of treatment there was a complete response (CR) in 22 of 49 children (44.5%) and a partial response (PR) in 12 children (24.4%). 34 of 49 children (69,4%) responded to treatment. There was no remission (NR) in 15 of 49 children (30.6%). In the whole group there were 7 deaths during the first 6 months. There were no relapses within 5 years of observation. The 5-year probability of survival in this group was 84%.

55 children (22 girls and 33 boys, aged 3,5–17,7 years) were treated in the years 1996–2009 using rATG (3.75 mg/kg/day for 5 days and cyclosporine 5mg/kg/day for 12–24 mths). On day 180 of treatment 5 patients (9%) achieved a CR, 23 children (41.8%) had a PR. 28 of 55 children (50.9%) responded to treatment. NR was found in 27 children (49%).In the rATG group 15 deaths (27%) were recorded, 4 patients (7%) had relapses. In one child paroxysmal nocturnal hemoglobinuria was noted.The 5-year survival in the rATG.group was 63.54%.

Our results show the greater effectiveness of treatment with horse globulin than rabbit globulin in terms of hematological response and overall survival.

Disclosures:

No relevant conflicts of interest to declare.
Unexpected 5 Year Survival Benefit in Patients Given Oral Cryotherapy During Conditioning for Stem Cell Transplantation. A Prospective Randomized Study
Authors: Svanberg Anncarin , Öhrn Kerstin , Birgegard Gunnar
Published: October 2019

Abstract 4559

Patients receiving high dose chemotherapy (HDC) in connection with bone marrow transplantation (BMT) often are afflicted with severe oral mucositis (OM). OM may affect 75–99% of patients. Oral cryotherapy has been shown to alleviate symptoms of mucositis alleviating oral pain and inability to maintain nutritional status. In a randomised controlled trial we have shown that patients receiving oral cryotherapy had less mucositis, less use of i.v. opioides and fewer doses of total parenteral nutrition (TPN) than a control group receiving routine oral care. Adult patients scheduled for bone marrow transplantation were randomly assigned to experimental (EXP) or control (CTR) group. A stratified randomisation was used with regard to type of transplantation (autologous vs allogeneic/unrelated donor (URD)). Randomisation was performed between January 2002 and August 2004. The final sample consisted of 78 patients, (31 autologous BMT and 8 allogeneic/URD BMT), and 39 constituted the CTR group and received standard treatment (31 autologous BMT and allogeneic/URD BMT). Concern has been raised for a possible protection of tumor cells by cryotherapy which could increase the risk of relapse and reduce survival. Thee aim of the present study was to investigate any difference in survival and relapse rates 5 years post-BMT for the two treatment groups from the randomised study.

After 5 years, 25/39 (64%) of the cryotherapy patients were alive compare to 15/39 (38%) of the control group (odds ratio 0,35, 95 % CI 0,14–0,88, p = 0,025)(Figure 1). No significant difference could be found with regard to the relapse rate between the groups. Most of the deaths were due to relapse.

The study offers no support for the speculation about tumor protection from cryotherapy during high dose chemotherapy conditioning for stem cell transplantation. These data indicate that oral cryotherapy is a safe prophylactic treatment for mucositis during chemotherapy.

Unexpectedly, the cryotherapy group showed a significantly better 5-year survival. Further analyses are needed to explore the difference in survival rate.

Disclosures:

No relevant conflicts of interest to declare.
DNMT3A Mutations Are Connected with Lower DNA Methylation Levels and Numbers of Concurrently Hypermethylated Genes in AML Patients
Authors: Hajkova Hana , Markova Jana , Haskovec Cedrik , Cermak Jaroslav , Petrbokova Radka , Michalova Kyra , Schwarz Jiri
Published: October 2019

Abstract 4629

Recently, mutations in DNA methyltransferase 3A (DNMT3A) have been found in patients with acute myeloid leukemia (AML) and have been confirmed to be connected with adverse clinical outcome. As DNMT3A plays direct role in the process of DNA methylation by adding methyl group to the cytosine residue of CpG dinucleotides, the question is obvious: What is the impact of DNMT3A mutations on DNA methylation levels?

We examined 79 AML patients at diagnosis for the presence of aberrant DNA methylation of 12 tumor suppressor genes (TSG) (CDKN2B, CALCA, CDH1, ESR1, SOCS1, MYOD1, DAPK1, TIMP3, ICAM1, TERT, CTNNA1, EGR1) by methylation specific real-time PCR (MethyLight) and for mutations in the gene DNMT3A by direct sequencing. Next we studied methylation status of 24 HOX genes from all four clusters A-D using methylation-restriction endonucleases followed by RQ-PCR arrays in 10 AML samples compared to 4 healthy donor samples.

Sequencing of cDNA between amino acids 300 and 930 revealed that 32 of 79 AML patients had DNMT3A mutation. The reason for higher DNMT3A mutation incidence in our patients’ cohort consists in preferential selection of AML patients with a higher percentage of probability of DNMT3A mutation (it means normal karyotype and mutations in NPM1, FLT3 and/or IDH1/2 genes). MethyLight assessment of 12 TSG showed subsequent frequencies of hypermethylation: CDKN2B (47%), CALCA (43%), CDH1 (22%), SOCS1 (24%), MYOD1 (18%), ESR1 (14%). The remaining 6 genes were weakly methylated in less than 10 % AML patients at diagnosis and were therefore excluded from further analysis. DNA methylation arrays revealed a set of differentially methylated HOX genes (n=12): 11 HOX genes were hypermethylated (HOXA4, HOXA6, HOXB13, HOXB3, HOXB4, HOXB7, HOXB8, HOXC8, HOXD10, HOXD11, HOXD3) and HOXA5 was hypomethylated compared to healthy donor samples. Comparing overall cumulative DNA methylation levels and numbers of simultaneously hypermethylated genes to mutational status of DNMT3A gene, we did observe lower levels of DNA methylation (P<0.0001) as well as displaying lower numbers of concurrently hypermethylated genes (P<0.0001) in patients with DNMT3a mutations. We observed the same trend also in DNA methylation levels of HOX genes when compared mutant (n=4) versus wild-type (n=6) DNMT3A patients.

These results clearly show that numbers of simultaneously hypermethylated genes and DNA methylation levels of chosen tumor suppressor genes (TSG) as well as HOX genes differs between AML patients with wild type and mutant DNMT3A.

This study is part of the COST Action BM0801 (EuGESMA) and is supported by NS10632-3/2009, OC10042 and IHBT00023736.

Disclosures:

No relevant conflicts of interest to declare.
Pregnancy-Induced Thrombocytopenia in Pseudo-Von Willebrand Disease
Authors: George Simi , Donahue Laura L. , Funk Dorothy M (Adcock) , Allen Steven L.
Published: October 2019

Abstract 4658

Platelet-type von Willebrand disease, or pseudo-VWD, is a rare congenital platelet disorder due to a mutation in the glycoprotein 1b alpha receptor. This results in increased binding of normal VWF to platelets, leading to platelet aggregation and intermittent thrombocytopenia. Although the mechanism is similar to type IIb VWD, this gain of function mutation affects the platelet, rather than plasma VWF.

We report a patient with pseudo-VWD who developed progressive thrombocytopenia during pregnancy. She was initially seen at 10 weeks gestation, when the platelet count was 143 × 109 /L (figure 1). As the pregnancy progressed, the platelet count decreased, accompanied by an inverse rise in VWF antigen and activity, factor VIII activity, and an increase in high molecular weight (HMW) multimers (figure 2). The VWF activity to antigen ratio remained abnormal at all times. At the time of delivery, the platelet count reached a nadir of 36 × 109/L. Post-partum, the platelet count normalized to 185 × 109/L with a concomitant fall in VWF antigen and activity levels. VWF multimer analysis after delivery showed an absence of high molecular weight multimers.FIGURE 1FIGURE 1. FIGURE 2Weeks GestationPlatelet count (×109 /L)VWF antigenVWF activityFactor VIIIPlatelet aggregationVWF multimers1014381%35%88%↑ ristocetin 0.6normal3654144%57%205%not donenormal+15 post-partum18555%19%49%not doneHMW multimers absent

The likely etiology of the progressive decrease in the platelet count is secondary to the physiologically stimulated rise in VWF levels during pregnancy. With rising VWF levels, the abnormal platelet GP1b alpha receptor binds VWF, resulting in platelet-VWF aggregates with subsequent clearance. Transfusion of normal platelets would be the only way to correct the abnormality. Our patient, a Jehovah's Witness, would not accept platelet transfusion if it became necessary. She underwent an uncomplicated cesarean section for obstetric indications.

Our patient's family was the subject of the landmark article initially describing this condition (Weiss HJ, Meyer D, Rabinowitz R, et al. Pseudo- von Willebrand's disease: An intrinsic platelet defect with aggregation by unmodified human factor VIII/von Willebrand factor and enhanced adsorption of its high-molecular-weight multimers. N Engl J Med 1982; 306:326-333). This patient is the 2-year old who was not tested when the family was initially evaluated. To our knowledge, this is the first reported case of pregnancy-associated thrombocytopenia in a patient with pseudo-VWD.

Disclosures:

No relevant conflicts of interest to declare.
Anti-Leukemic Activity Is Increased with Low Dose Busulfan and Irradiation: A Preclinical Model
Authors: Patel Pritesh R. , Mahmud Dolores , Zhang Wei , Zhou Tong , Mahmud Nadim , Weichselbaum Ralph R , Garcia Joe GN , Rondelli Damiano
Published: October 2019

Abstract 4696

Combinations of busulfan (Bu) and low dose total body irradiation are commonly used in conditioning regimens for allogeneic stem cell transplant in acute myeloid leukemia (AML). In this study we tested the in vitro effect of physiologic doses of Bu and ionizing radiation (IR) to address the question whether additive or synergistic antileukemic activity can be demonstrated in two leukemic cell lines, one sensitive (HL60) and one resistant (K562) to radiation.

Cells were treated for 24 hours with Bu at 0, 12.5, 25, 50, 100 or 200 μ g/ml, or IR at 1.5, 3 or 6 Gy, or a combination of both with 12.5 or 25 μ g/ml for 24 hours, followed by IR at 1.5 or 3Gy. Cells were then tested for proliferation by thymidine uptake, apoptosis by surface expression of annexin V and caspase 3, and in-vitro colony formation assays. Exposure to Bu or IR for 24, 48 and 72 hours induced a dose dependent inhibition of proliferation and colony formation in both cell lines. However, 3 or 6 Gy of IR were both very effective against HL60 cells whereas a significant increase in cytotoxicity in K562 cells was observed only by using 6Gy (p=0.008).

In-vitro treatment with either Bu or IR caused apoptotic cell death mainly in HL60 cells, but in a very limited amount in K562 cells. To test the effect of the combination of Bu and IR we utilized a low dose of Bu for 24 hours with low doses of radiation. HL60 cells were strongly inhibited by both agents when separate or combined. In contrast, treatment of the relatively radio-resistant K562 cells with low dose IR (3Gy) alone inhibited colony formation by 28%. The inhibitory effect increased to 84% when combined with low dose Bu.

In order to identify genes associated with a response to Bu, we formed a linear regression model controlled for cancer type and also leukemic cell lines using GI50 and Stanford cDNA array data from the NCI-60. Among 7 genes (p<0.001 and FDR ∼50%) that were identified for all cancers, CLK-2 was also shown to correlate with response to Bu in leukemic cell lines (p=0.047). We then tested how these significant genes performed in the Affymetric U133 plus 2.0 platform. Six other genes (ERC2, HCLS1, CD74, KCNH2, HLADQB2, CD53) were significantly associated with response to Bu (p<0.05).

In conclusion, we demonstrated an additive effect of Bu and low dose IR even on radioresistant leukemic cells. These findings prompted the design of a clinical trial with IV Bu and marrow irradiation in the conditioning regimen prior to allogeneic stem cell transplant for acute leukemia. Expression of genes correlating with response to Bu may represent a novel biomarker to predict the outcome of leukemic patients undergoing stem cell transplant and could lead to a personalized anti-leukemic therapy with Bu.

Disclosures:

No relevant conflicts of interest to declare.
Examining Knowledge and Information Seeking Behaviors towards Blood Transfusion Among Individuals with Cancer
Authors: Naim Ahmad B , Walls Danielle , Gollins Jan , Reynolds Chuck
Published: October 2019

Abstract 4762

Objective:

Examine knowledge and information seeking behaviors towards blood transfusions among individuals with metastatic or recurrent cancer.

Methods:

An online survey was conducted from a nationally representative patient panel in 1Q2011. All respondents were ≥18 years and diagnosed with cancer by a physician. Participants were asked about their blood transfusion history, information seeking behaviors, and knowledge about blood transfusion.

Results:

206 individuals responded to survey. 65% (n=133) were female and 25% (n=52) were over 55 years. 55% (n=114) were anemic and 45% (n=92) not anemic. 62% (n=128) had received blood transfusion, whereas, 38% (n=78) had no transfusions. Top two sources of information are doctor (85%) and Internet (78%). Among those previously transfused, 74% received right amount of information, whereas, 11% received too little information, and 15% received too much information. More than 90% of transfused indicated they knew the reasons for and benefits of getting a blood transfusion. Less than two-thirds received information about the costs. Over 65% of not transfused said that it is extremely important to know risks of infections, right blood type and screening techniques. Among previously transfused, about 70% agreed that they made an informed choice about receiving blood transfusions. Among the previously transfused, 78% agree that they knew the benefits compared with 68% not transfused. About 68% of transfused and not transfused agree they knew the risks of blood transfusion.

Conclusions:

Doctor's office and internet are primary sources of information about blood transfusions. Gaps in knowledge exist about benefits, risks, and costs of blood transfusions. A significant number feel that they need more information about blood transfusion to make an informed choice. Providers should consider adopting shared-decision making with their patients.

Disclosures:

Naim: Janssen Scientific Affairs, LLC: Employment. Walls:Janssen Scientific Affairs, LLC: Consultancy. Gollins:Janssen Scientific Affairs, LLC: Consultancy. Reynolds:Janssen Scientific Affairs, LLC: Consultancy.
EYA3 May Be Required for Globin Gene Expression in Erythroid Differentiation
Authors: Branco Diana Santos , Brugnerotto Ana Flavia , Lanaro Carolina , Fertrin Kleber Yotsumoto , Cunha Anderson Ferreira , Costa Fernando Ferreira
Published: October 2019

Abstract 4797

Background:

Extensive studies have led to a considerable understanding of the cellular and molecular control of haemoglobin production during red blood cell differentiation, however, identification of the genes expressed as part of the erythroid differentiation programme remains an important goal because of the insights that these data will bring to erythrocyte biology and disease. Previous results using SAGE identified 93 differentially-expressed genes during erythroid development. One of these genes, EYA3, a homologue gene of Eyes Absent 3 in Drosophila, is a transcription cofactor with intrinsic phosphatase activity and its expression was observed to be high at the end of CD34+ cell differentiation and in human bone marrow.

Aim:

To evaluate globin gene, fetal hemoglobin (HbF) expressions and apoptosis levels in the erythroleukemic K562 cell line after EYA3 gene silencing and induction with hemin.

Methods:

Four different cultures from human K562 cells (1×105cells/mL in DMEM, 10% FBS, penicillin/streptomycin, 5% CO2, 37°C) were transfected with control or EYA3 knockdown lentiviruses (MOI=2.5). After proliferation and selection of successfully transfected cells with puromicin (2.0 ug/mL), cells were treated with 30μM hemin and collected after 0, 24, 48, 72 and 96h for gene expression and flow cytometry analyses. EYA3, LXN, α, and g-gene expression was measured by qRT-PCR and normalized using the Genorm program. HbF expression and apoptosis were evaluated by flow cytometry.

Results:

Analysis of globin gene expression showed that α-globin gene was downregulated in EYA3 silenced K562 culture cells compared with the control culture in 72h after hemin addition (1.791±0.1735; 0.7404±0.1709, respectively, **P<0.001, n=4). g-globin gene expression was found to be downregulated in K562 EYA3 silenced culture after 24h (1.350±0.1296; 0.5285±0.1736, respectively, *P<0.05, n=4) and 72h (1.554±0.1042; 0.6889±0.1535, respectively, **P<0.001, n=4). HbF expression was found to be downregulated in the same culture compared to the control culture at 72h after hemin addition (3568±41.00; 1947±206.50, respectively, *P<0.05, n=4). Apoptosis levels were found increased in EYA3 silenced K562 culture cells compared with the control culture in 72h after hemin addition (4.7±0.10; 8.55±0.55, respectively, *P<0.001, n=4).

Conclusions:

Our results show that silencing EYA causes modifications in the expression pattern of α- and g-globin gene expression as well as in HbF expression pattern and apoptosis levels in a model of erythroid differentiation. Further studies should be performed in primary erythroid cell cultures using siRNA-based gene silencing and overexpression of these genes to determine how these genes are involved in the mechanisms of globin gene regulation.

Support by FAPESP, CNPq and INCTS

Disclosures:

No relevant conflicts of interest to declare.
Cell Fate Modulation by Microvesicles: Transcriptionally-Mediated and Long Term in Nature
Authors: Quesenberry Peter J
Published: October 2019

Abstract 4801

Cell-derived membrane enclosed vesicles containing mRNA, protein, microRNA, and DNA, can enter cells and effect a phenotype change. We have shown that lung-derived microvesicles enter marrow cells inducing them to express pulmonary epithelial cell-specific protein and mRNA, a variety of microRNA and to enhance their capacity to engraft in irradiated mice and express the phenotype of type II pneumocytes (Aliotta et al, Exp Hematol 38,2010). In the present studies using rat/mouse hybrid cultures and measuring species-specific mRNA, we have shown that immediately after co-culture of rat lung across from mouse marrow, mouse marrow cells express both rat and mouse specific surfactants B and C mRNA. However, when these cells are cultured in steel factor supported long-term culture, rat-specific mRNA disappears rapidly, while mouse-specific mRNA persists out to 12 weeks in liquid culture. Identical studies with rat liver cultured across from mouse marrow have shown early expression in mouse marrow of both rat and mouse albumin mRNA, but in long term in vitro culture, expression of albumin mRNA was mouse-specific. Thus, the major long-term persistent event is an alteration of transcription in the target marrow cells. In a similar fashion, marrow modulated by lung microvesicles in vitro and engrafted into lethally irradiated (950 cGy split dose) mice, evidences expression of pulmonary epithelial cell-specific mRNA or protein (surfactants) in host lung, marrow, thymus, spleen and liver 6 weeks after engraftment – the furthest time tested. These results indicate that microvesicle cell fate modulation is biologically meaningful and represents an important new mechanism for cell phenotype determination.

Disclosures:

No relevant conflicts of interest to declare.
A Wharton's Jelly Mesenchymal Stromal Cell Derived 3D Osteogenic Niche Allows for Cord Blood Stem Cell Expansion Using Cytokine-Free Culture Media
Authors: Aljitawi Omar S. , Keefe Peggy , Henderson Christianna , Akel Salem , Abhyankar Sunil , McGuirk Joseph , Detamore Michael , Garimella Rama
Published: October 2019

Abstract 4832

Introduction:

In prior experiments, a 3 dimensional (3D) osteogenic niche was developed using Wharton's jelly mesenchymal stromal cells (WJMSC) that allowed for successful attachment of CD34 + umbilical cord blood (UCB) stem cells. The 3D osteogenic scaffold resulted in 10 times expansion of total nucleated cells (TNCs), however, most of the expanded cells were CD34 negative possibly secondary to their differentiation attributed to the cytokine-rich media. Accordingly, it was proposed to use a cytokine-free media in expansion of CD34+UCB using the same 3D osteogenic structure.

Aims:

To assess the ex vivo expansion of CD34+UCB stem cells in a 3D osteogenic niche using cytokine-free culture media. The expansion in 3D conditions was compared to 2D and liquid-culture conditions.

Methods:

CD34+ selected UCB stem cells were expanded in 3D osteogenic scaffold using cytokine-free media for 2 weeks. In 2D conditions, CD34+ UCB stem cells were expanded over a monolayer of osteogenic differentiated WJMSCs using the same cytokine-free media. In liquid culture conditions, the CD34+ UCB stem cells were expanded in culture flask. Pre- and post-expanded TNC was determined and post expansion CD34 expression was assessed using flow cytometry. Colony forming unit (CFU) assays were used to compare the expanded population in the three culture conditions.

Results:

TNCs were expanded 26X in 3D, 265X in 2D, and only 2X in liquid culture conditions. CD34 expressing cells increased 2.4X in 3D, 10X in 2D, and decreased in liquid cultures. Relative to 3D, CFU expansion in 2D was increased 2.6X. In 3D, CFUs were almost exclusively CFU-GM (>93%), compared to 2D (74%). Absence of CFU-E was noticed in the expanded population that remained attached to the 3D osteogenic scaffold. On the other hand, low CFU-E percentage was noticed in the population of expanded cells that dettached from the 3D osteogenic scaffold.

Conclusions:

The experimental conditions lead to a modest but significant expansion of CD34+UCB stem cells in 3D as well as 2D conditions. Though the majority of the expanded population did not express CD34, they retained their colonogenic potential. Contrary to the expanded cells that detached from the 3D osteogenic scaffold, the cells that remained attached did not form CFU-E.

Disclosures:

No relevant conflicts of interest to declare.
Early Events of Innate Immune Response to Influenza Virus in a Complex Cellular Environment Revisited with Multi-Parametric Flow Cytometry
Authors: Olive Daniel , gondois-Rey Françoise , Le Thi Kieu Suong , Herrera Diana , Hirsch Ivan
Published: October 2019

Abstract 4931

The development of an innate immune response against viruses involves multiple cellular interactions implying infected cells and the cells endowed with innate immune functions. These cells have to act in unison to develop a potent response. Although multiple interactions sustained the development of the innate response, much of the knowledge on the function of human innate immunity comes from investigations of isolated cells or dual partner coculture. Our aim was to investigate the early interactions of influenza virus with innate immune cells in a complex cellular environment in order to deepen our knowledge on the dynamics of the innate immune response. Polychromatic flow cytometry has been the appropriate method of exploration. We designed panels of 12 markers to explore PBMC stimulated with live-influenza virus or a mix of TLR agonists mimicking the interactions of influenza virus with TLR7/8 and TLR4. The DC populations were identified as lineage−HLA-DR+ cells and we took advantage of the polychromatic cytometry to split the lineage markers into separate channels to analyze NK, T cells and monocytes simultaneously to DC. Intra-cellular expression of IFN-a, TNF-a, IL-12, IL-6, IFN-g, and CD107 were analyzed in a time-dependent fashion. Our results demonstrate the benefits of using complex cell culture for the understanding of innate immune response to virus. We show a new field of application for multi-parametric flow cytometry as a powerful tool of investigation of innate immune responses in complex cell systems, with a sensibility to detect subtle modifications comparable to isolated cell systems. The multiple time-dependent results allowed to approach innate immune activation as correlated dynamic events. Dynamic studies lead to re-visit the relative roles of infection and TLR induced-DC response in the early specific features of Flu induced innate activation.

Disclosures:

No relevant conflicts of interest to declare.
A Study of Outcomes of Diffuse Large B Cell Lymphoma in the Elderly
Authors: Hoffmann Marc , Ahmadi Tahamtan , Cobain Erin , Loren Alison , Frey Noelle V , Porter David , Stadtmauer Edward A. , Schuster Stephen J. , Nasta Sunita D.
Published: October 2019

Abstract 4950

Introduction:

Despite improvements in outcome using chemoimmunotherapy, treating elderly patients with diffuse large B cell lymphoma (DLBCL) remains challenging. Reliable risk stratification beyond age is lacking and consequently elderly patients are often treated with reduced-dose therapeutic regimen with palliative intent. We retrospectively analyzed outcomes of DLBCL patients aged 65 and older treated at the University of Pennsylvania.

Patients and method:

We identified 41 patients (pts) with diagnosis of DLBCL and age >65 years. Median age was 74 years (range: 65–86). Eight pts (20%) were age 80 or older. There were no differences in IPI, elevated LDH, or bcl-2 expression, whereas bcl-6 expression was more common in patients >80 years with 75% (6/8) vs. 27% (24/33) (p=0.01).

Overall, 31 pts were treated with R-CHOP, 2 pts with R-HyperCVAD and one pt with R-CVP. Seven patients with a median age of 77 years were considered too frail for standard chemotherapy and were treated with a “split R-CHOP” regimen consisting of: rituximab 375 mg/m2 day 1, cyclophosphamide 375 mg/m2 day 1 and 15, adriamycin 25 mg/m2 day 1 and 15, vincristine 1 mg day 1 and 15 and Prednisone 50 mg day 1–5 and day 15–19.

Results:

Overall, the complete remission (CR) rate was 56% with seven treatment related deaths (17%). For patients between 65 to 80 years of age and deemed fit for standard chemotherapy (n=28), CR rate was 57% with four treatment related deaths (14%). Among patients over 80 and deemed fit for standard chemotherapy (n=6), the CR rate was 50% with 2 treatment related deaths (33%). Frail patients treated with the “split R-CHOP regimen” (n=7) had a CR rate of 57% with one (14%) treatment related death.

For all patients, the median progression-free survival (PFS) was 1 year with a median overall survival (OS) of 2 years. The median PFS for pts between 65 and 80 years of age treated with standard chemotherapy was 16 months. Median PFS in pts >80 years of age treated with standard chemotherapy was 7 months, whereas median PFS in frail pts treated with “split R-CHOP regimen” was 11.7 months.

Conclusions:

Our data reveal interesting findings about elderly pts treated for DLBCL. PFS and OS in general are poor as has been reported by others. In our data set, pts>80 years considered fit for standard chemotherapy, had a shorter PFS then fit patients between 65 to 80 years.

Intriguingly, PFS among frail elderly patients treated with a “split R-CHOP” regimen appears to be superior to that of elderly deemed more robust who were treated with standard dose RCHOP-21. Though limited by small numbers, our institutional data suggest that frail patients can tolerate a modified R-CHOP regimen although survival remains short.

Disclosures:

No relevant conflicts of interest to declare.