bluuebunny/crossref_metadata_2025_split huggingface dataset:
B Cell Receptor Stimulation of CLL Cells Leads to Upregulation of IRF4 Proteinexpression Influenced by SNP Expression,
Authors: Patz Michaela , Linde Nina-Felizitas , Frenzel Lukas P. , Pallasch Christian P. , Brinker Reinhild , Claasen Julia , Hallek Michael , Wendtner Clemens 
Published: October 2019

Abstract
Abstract 3886

Introduction:
As previously published by Di Bernado et al. (2008) the single nucleotide polymorphism (SNP) RS872071 located in the 3'UTR of IRF4 (Interleukin Regulatory Factor 4) has influence on the risk for developing chronic lymphocytic leukemia (CLL). IRF4 is a key player in the development of B lymphocytes and multiple myelomas. The SNP is either expressed as Adenine (A) or Guanine (G). Expression of G/G increases the risk to develop CLL. MicroRNAs (miRNAs) adhere posttranscriptional to the 3'untranslated region (UTR) of mRNAs and can influence the expression of mRNA and proteins. The SNP lies within the 3'UTR of IRF4 and offers some putative binding sites for miRNAs. The aim of this project is to proof that this SNP has influence on the binding behavior of miRNAs, which are influencing the IRF4 expression.
Methods and results: To identify miRNAs binding to this region we cloned the neighboring region of the SNP into a luciferase expressing vector and generated the SNP by mutagenesis. In luciferase assays 15 miRNAs were checked for differences in binding affinity dependent on SNP expression. Three of them showed significant SNP dependent binding behavior. In all cases expression of SNP G leads to reduced luciferase expression, indicating suppression of IRF4. To elucidate the consequences on SNP expression on cellular level in CLL we collected DNA derived from B cells of CLL patients (n=104) and sequenced the SNP region. All together our pool of patients expressed 25% A/A, 31% A/G and 44% G/G. MRNA and protein expression of IRF4 was considered SNP-dependently and compared to healthy B cells. On mRNA and protein level CLL cells have a significant higher IRF4 expression compared to healthy B cells. SNP-dependent comparison between CLL cells on mRNA-level shows a tendency of less IRF4 expression in B cells of patients expressing the G/G SNP compared to patients expressing A/A SNP. However on protein level this tendency was not detected. As IRF4 is known as a key regulator for extracellular stimuli, we focused on SNP dependent IRF4 regulation after different stimuli. Whereas CD40 and IL-4 stimulation did not show SNP dependent regulation, stimulation of the B cell receptor (BCR) leads to a higher IRF4 induction in patients carrying A/A (n=6) compared to patients carrying G/G (n=5) and A/G (n=8) (p<0.05).

Conclusion:
For the first time connections between the SNP RS872071 and molecular mechanisms explaining his influence on the pathogenesis on CLL were drawn. Dependent on expressed SNP miRNAs bind with different affinities to the 3'UTR. In further experiments the connection between IgM stimulation and miRNA expression has to be tightened.

Disclosures:
No relevant conflicts of interest to declare.
Characterization of Clonotypic B Cell Progenitors in Bone Marrow of Multiple Myeloma Patients,
Authors: Boucher Kelly , Parquet Nancy , Shain Kenneth H. , Baz Rachid , Alsina Melissa , Koomen John , Durand Ashley , Anasetti Claudio , Dalton William , Perez Lia 
Published: October 2019

Abstract
Abstract 4002
The lack of specific molecules to define malignant B progenitor cells in multiple myeloma (MM) has hampered the evaluation of minimal residual disease (MRD). We have identified a bone marrow (BM) CD138- subset that co-express CD19+ with identical κ or λ light chain (LC) restriction as the abnormal plasma cell (PC), as previously shown by others. The majority of LC restricted (LCR) B progenitors are CD19+/CD34- and <0.5% of whole BM (WBM) cells exhibit an immature phenotype: CD19+/CD34+ with aberrant CD27 expression. Immature B cell precursors are undetectable in peripheral blood (PB). LCR CD138-/CD19+ cells represent only 0.72± 0.5% of WBM in newly diagnosed patients (n=23) and do not increase (0.47± 0.51%) in patients with relapsed disease (n=21). The κ/λ LC ratio is 1.46±0.6 regardless of disease stage suggesting that conventional LC ratios for PCs (> 4 or <0.5) may not apply in B progenitors. LCR B progenitors (CD19+/34+ or CD19+/34-) are CD117+, Notch+ and Survivin+ as MM patient's hematopoietic stem cells (HSC). ALDH enzymatic activity is 3.1% (0.1-–7.26%) in LCR B cells. Flow sorted CD138+ did not grow in a colony formation assay (methylcellulose with PHA-LCM), whereas CD19+/CD34- or CD19+/CD34+ grew colonies with efficiency of 1:25,000 or 1:10000 respectively. Cells harvested from colonies have a lympho-plasmacytoid appearance and LCR B progenitors differentiated into CD138+ PC (80±5%) compared to HSC (10±5%). Colony efficiency was optimized (3 fold) using conditioned medium (CM) from HS5-stroma. Isolated CD138-/CD19+ cells were relatively bortezomib and melphalan resistant compared to CD138+ PC. We hypothesize that CD138-/CD19+/CD34+ cells contains earlier progenitor B cells that differentiate into the malignant PC. Surrogate assays for stem cell activity and xenotransplant models should determine cancer stem cell activity of immature B cell precursors. Research studies of MM putative progenitor cells will allow developing novel treatments to eradicate potential MM MRD reservoir.

Disclosures:
Baz: Millenium: Research Funding, Speakers Bureau; Celgene: Research Funding.
The Effect of KIRs Expression Profile in Donor/Recipient Pairs in HLA-Identical Sibling Hematopoietic Stem Cell Transplantation,
Authors: Wei Xiaoai , Tang XiaoWen , Feng Yufeng , Zhu Ziling , He Jun , Xu Yang , Sun Aining , Depei Wu 
Published: October 2019

Abstract
Abstract 4017

Objective:
We investigated the distribution characteristics of KIRs expression profile in donor/recipient pairs with acute leukemia (AL) receiving HLA-identical sibling hematopoietic stem cell transplantation (sib-HSCT). We further explored the effect of KIRs expression profile in donor/recipient pairs on clinical outcome, including dynamics of donor T cell and NK cell engraftment.

Methods:
The genotypes of donor/recipient KIRs were determinated by polymerase chain reaction- sequence specific primer (PCR-SSP) for 80 pairs of donor/recipient receiving HLA-identical sibling hematopoietic stem cell transplantation. The multiple short tandem repeat (STR) PCR was used to evaluate the status of engraftment of donor T cells and NK cells at +14 days, 21 days, 28 days, 60 days and 90 days after transplantation in 24 cases.

Results: 1.
In 80 pairs of donor/recipient: (i) the KIRs were completely identical in 57.5% of donor/recipient pairs; (ii) the donors' KIRs contained the recipients' in 13.75% pairs; (iii) the recipients' KIRs contained the donors' in 17.5% of pairs; (iv) the KIRs were completely different in 11.25% pairs. The graft versus host (GVH) direction KIR-matched group was 75%. The percentage of group donor B/X and group donor A/A was 50%, respectively. 2. Comparing the patients from GVH direction KIR-matched and mismatched group, the incidence of acute (a) GVHD was 60% and 30%, respectively (P =0.0222), and 2-year OS was 62.96% and 94.12%, respectively (P =0.0492). Particularly, grade III-IV aGVHD rate of KIR-matched group was higher than that of non-KIR matched group(15% vs 0%). 3. Donor B/X group had a higher 2-year OS and 2-year relapse-free survival (RFS) compared with donor A/A group (89.23% vs 49.57%, P =0.0159, and 90% vs 59.71%, P =0.0239, respectively). Patients with three or less aKIRs had a lower 2-year OS (58.9% vs 92.44%, P =0.0338) and a lower RFS (65.14% vs 92.59%, P =0.0398), compared with patients with more aKIR. 4. Sequential monitoring of chimerism status of donor NK-cells in 24 cases revealed that on day+14, the percentage of full donor NK cells chimerism was higher in non-KIR matched patients than that of KIR matched patients (85.7% vs 52.9%, P =0.0456).

Conclusions:
Donor KIR genotype appears to have a direct impact on aGVHD, OS and RFS. Therefore, donor KIR genotype should be evaluated as an outcome predictor of the HLA-identical sib-HSCT.

Disclosures:
No relevant conflicts of interest to declare.
Prevention of Graft-Versus-Host Disease by HY-Specific Induced Tregs Through Activation-Dependent Manner,
Authors: Li Jun , Semple Kenrick , Koasaard Kane , Haarberg Kelley M.K. , Chen Fang-ping , Anasetti Claudio , Yu Xue-Zhong 
Published: October 2019

Abstract
Abstract 4023
Naturally occurring regulatory T cells (nTregs) may prevent graft-versus-host disease (GVHD) while preserving graft-versus-leukemia (GVL) activity. However, clinical application of nTregs has been severely hampered by their scarce availability and non-selective suppression. To overcome these limitations, we took the alternative approach to induce antigen-specific Tregs (iTregs) and tested their efficacy and selectivity in the prevention of GVHD in a pre-clinical model of bone marrow transplantation (BMT). We selected HY as target antigen, because it is a naturally processed and ubiquitously expressed minor histocompatibility antigen (miHAg) with a proven role in GVHD and GVL effect. To generate HY-specific iTregs, naïve CD4+CD25− cells were isolated from MHC II-restricted, HY-specific transgenic and Foxp3/GFP knock-in mice, and were stimulated with HY peptide and APC, in the presence of TGFb and retinoic acid. Marrow plus polyclonal CD4 T effector cells (Teffs) from B6 donor splenocytes induced GVHD when transplanted into lethally irradiated (B6 x bm12)F1 recipients, and adoptive transfer of HY-specific iTregs (CD4+CD25+GFP+) significantly decreased GVHD mortality in male (HY+) but not female (HY−) recipients. On a per cell basis, HY-specific iTregs were significantly more potent than polyclonal Tregs in the prevention of GVHD. These data indicate that HY-specific iTregs were effective in controlling GVHD in an activation-dependent manner. Because polyclonal Teffs cells recognize bm12 alloantigen whereas iTregs recognize HY miHAg, these data suggest that Tregs may control GVHD through a linked-suppression on Teffs in vivo. Mechanistically, by measuring iTregs and Teffs in spleen and liver of the recipients, we found that HY-specific iTregs expanded extensively and significantly suppressed expansion and infiltration of Teffs in male but not female recipients. We finally generated alloreactive iTregs from polyclonal CD4 precursors, and found that these iTregs highly suppressed the Teffs alloresponse in vitro and in vivo. These results show that Ag-specific iTregs are promising cell therapy for effective GVHD prevention in human allogeneic hematopoietic cell transplantation.

Disclosures:
No relevant conflicts of interest to declare.
IL-15 Administration Increases Graft Versus Leukemia Activity in Recipients of Haploidentical Hematopoietic Stem Cell Transplantation,
Authors: Sauter Christopher , Biswas Chandra , Bailey Cavan , Panis Michelle , Budak-Alpdogan Tulin , Alpdogan Onder 
Published: October 2019

Abstract
Abstract 4027
The success of haploidentical (HI) hematopoietic stem cell transplantation (HSCT), suggests that graft-versus-leukemia (GVL) effect might have a substantial role in this transplant modality. Rigorous T-cell depletion (TCD) of the graft decreases the occurrence of graft-versus-host disease (GVHD) in HI-HSCT, however this results in immunodeficiency and high disease relapse rate, especially in patients with resistant or residual leukemia. Therefore, enhancing GVL activity of HSCT without increasing GVHD is crucial for improving the outcome of haploidentical transplant. Post-transplant IL-15 administration is shown to enhance immune reconstitution, particularly donor-derived NK and CD8+ T cell populations in murine models. We evaluated the efficacy of IL-15 for enhancing GVL effect in recipients of HI-HSCT.
For developing clinically relevant haploidentical transplant models, different hybrid mice with B6 background that share the same haplotype (H2Kb) are used for our murine haploindentical transplant experiments. Lethally irradiated B6D2F1/J (H2Kb/d) mice are transplanted with B6CBAF1/J (H2Kb/k) TCD bone marrow (BM) and T cells at varying doses. Some animals were also given P815 tumor cells on the day of transplant. Administration of IL-15 significantly increased the numbers of CD8+ T and NK cells in the spleen and BM in the T cell depleted model at post-transplant day 28. Infusion of very low dose haploidentical T cells (1×104) with TCD-BM resulted in a conflicting effect on immune reconstitution, i.e. increased T cell numbers, and decreased NK cell population. Post-transplant IL-15 administration also changed this immune reconstitution pattern and significantly increased both T and NK cell numbers in recipients of HI-HSCT. In P815 challenged mice that were transplanted with very low dose T cell added TCD-BM, IL-15 administration significantly increased anti-tumor activity of the graft and improved survival (Figure 1) without increasing GVHD. This effect was observed when IL-15 administration was given at a later time point rather than immediately following transplantation, possibly allowing for more donor cell engraftment and T cell proliferation to take place. IL-15 administration without T cell infusion did not result in any survival improvement. We conclude that in our experimental HI transplant models, IL-15 administration augments anti-tumor effect of the HI-HSCT without increasing GVHD risk, and this effect requires presence of donor derived T cells.

Disclosures:
No relevant conflicts of interest to declare.
Influence of Genetic Polymorphisms in CYP2C8 and ABCB1 on the Mobilization Ability and Toxicity of Paclitaxel,
Authors: Lozano María L. , Niño Elkin A. , Anton Ana Isabel , Padilla Jose , Rivera Jose , Cerezo Juan José , Castilla-Llorente Cristina , Garcia Vicente 
Published: October 2019

Abstract
Abstract 4050

Introduction.
We and others have described the efficacy of paclitaxel-based chemotherapy in mobilizing large amounts of hematopoietic progenitors (HP) both in patients with solid tumors,(Bone Marrow Transplant 2000;25:231–5), and with hematological malignancies (Haematologica 2008; 93:161–3). Like most drugs, the taxanes are not controlled by the actions of one gene, but believed to be dependent on several polymorphic proteins. Single nucleotide polymorphisms (SNPs) in the ABCB1 gene, which encodes the transport protein P-glycoprotein, or in in metabolic enzymes, such as CYP2C8, have been suggested to influence the pharmacokinetics and clinical response to paclitaxel. Aim. To retrospectively evaluate the effects of five known allelic variants in the CYP2C8, and ABCB1genes on the mobilization ability and toxicity of the anticancer agent paclitaxel. Patients and Methods. 107 patients with hematological malignancies (43 lymphoma, 41 myeloma, 23 acute leukemia) received paclitaxel 170 mg/m2 i.v. by continuous infusion for 24 hours (day 1) followed by 8 mg/kg s.c G-CSF daily until the last apheresis. 77% received this treatment after failure of mobilization with G-CSF, and the rest as first line therapy because of risk factors for failure to achieve successful mobilization. The genetic variants (ABCB1 rs1045642 A >G, ABCB1 rs2032582 C>A, ABCB1 rs2032582 C>T, CYP2C8 rs10509681 C>T, and CYP2C8 rs11572080 A>G), were genotyped by allelic discrimination polymerase chain reaction (PCR) assays using TaqMan®Genotyping Assays (Applied Biosystems). The effect of genotypes on mobilization efficacy and on maximal hematological toxicity (according to the NCI version 3) was retrospectively assessed. Results. Allelic frequencies for rs1045642 A>G, rs2032582 C>A, rs2032582 C>T, rs10509681 C>T, and rs11572080 A>G variants, were 0.46, 0.40, 0.8, 0.13, and 0.13, respectively. Successful mobilization (>2 106/kg CD34+ cells) was achieved in 57% of patients. No reproducible significant associations between genotype and outcome (evaluated as number of CD34+ cells/kg in the first and total apheresis, number of collections performed, and on the mobilization success [p>0.05]), nor on myeloid or platelet toxicity (p>0.05) were found for any of the SNPs analyzed. Discussion. This study on a well-defined patient population suggests that the presently evaluated variant alleles in CYP2C8 and ABCB1 genes do not explain the substantial interindividual variability in the outcome or hematological toxicity of the mobilization schedule using paclitaxel and G-CSF. Funding. This study was supported in part by a research grants 04515/GERM/06; RECAVA RD06/0014/0039, and FIS 10/02594.

Disclosures:
No relevant conflicts of interest to declare.
High Level Secretion of HLA-G5 Might Inhibit the Incidence of Gvhd,
Authors: Wu Xiuli , Chen Yinkui , Liu Qifa , Xuan Li , Ling Yiwen , Zhang Yu , Fan Zhiping 
Published: October 2019

Abstract
Abstract 4068
Human leucocyte antigen G (HLA-G) is a nonclassical HLA class I molecule, the tolerogenic role of HLA-G is highly supported in pregnancy immunization, tumor immune escape and organ transplant. HLA-G molecules also act as an inhibitor of the T-lymphocytes, NK cells and antigen presenting cells. HLA-G can be expressed as 7 different isoforms including 4 membrane-bound (HLA-G1 to -G4) and 3 soluble (HLA-G5 to-G7) proteins. The soluble HLA-G5 isoform encoded by intron-4 retaining spliced transcript has been previously detected in vivo in sera and grafts from solid transplanted patients who had significantly better graft acceptance. In the present study, we analyzed the expression levels of HLA-G in patients after hematopoietic stem cell transplantation (HSCT) and investigated the relationship between HLA-G and GVHD. The levels of soluble HLA-G (sHLA-G) in the plasma of peripheral blood from 90 patients underwent HSCT were determined by enzyme-linked immunosorbent assay (ELISA). Flow cytometry was used to detect the expression of membrane bound HLA-G (mHLA-G). The levels of mHLA-G of patients at pre-HSCT, +15d post-HSCT and +30d post-HSCT were similar with the normal individuals. High levels of sHLA-G in the plasma of recipients were found after allogeneic HSCT (allo-HSCT). The soluble HLA-G5 and HLA-G6 levels in the plasma of recipients at +15d post-HSCT and +30d post-HSCT were significantly higher than that at pre-HSCT, but the soluble HLA-G7 levels in the plasma of recipients at +15d post-HSCT or +30d post-HSCT were similar with that at pre-HSCT. In contrast, the levels of sHLA-G in the plasma of recipients had no significant change after autologous HSCT (auto-HSCT). High level secretion of HLA-G5 after allo-HSCT (the levels of sHLA-G increased more than 1.0 fold compared to the pre-HSCT group) significantly reduced the incidence of acute GVHD and chronic GVHD; High level secretion of HLA-G6 after allo-HSCT also significantly reduced the incidence of chronic GVHD, but had no influence to the incidence of acute GVHD; High level secretion of HLA-G7 after allo-HSCT had no influence to the incidence of acute GVHD or chronic GVHD. The increased levels of HLA-G5 after allo-HSCT which compared with the pre-HSCT group had a significant negative correlation with the severity of GVHD. In conclusion, the high level secretion of HLA-G5 might inhibit the incidence of acute and chronic GVHD. Detected the secretion level of HLA-G5 after HSCT might benefit to predict and evaluate the GVHD.

Disclosures:
Liu: National Natural Science Foundation of China (No.30971300), Science and Technology Planning Project of Guangdong Province of China (No.2009A030200007): Research Funding.
First and Second High Dose Treatment Supported by Autologous Hematopoetic Cell Transplantation Rescue in Multiple Myeloma
Authors: Bozdag Sinem Civriz , Topcuoglu Pervin , Beksac Meral , Ilhan Osman , Ozcan Muhit , Gurman Gunhan , Arslan Onder 
Published: October 2019

Abstract
Abstract 4528

Purpose:
We aimed to evaluate the outcome of multiple myeloma patients underwent double autologus hematopoietic cell transplantation in our center.

Patient&Method:
We retrospectively analyzed 31 multiple myeloma patients (22F;9M) receiving two times high dose therapy supported by auto-HCT in Adult BMT Unit of Ankara University between 2005–2010. Median age was 52 years (34–66ys) prior to first auto-HCT. We evaluated the response rates of progression free survival (PFS) of both transplantation, respectively and also overall survival (OS) from the diagnosis.

Results:
The median time from diagnosis to the first transplantation was 11 months (5–68ms). The response rates were 87% before the first auto-HSCT [2 Complete response (CR),4 near-CR, 7 very good partial remission (VGPR), 14 PR) but four patients (11%) had refractory disease. The response of first auto-HCT was complete remission or partial response in 50% of the pts and 36% for stable disease. Only 4 pts was progressed at early period of 1st auto-HCT. The median time from the 1st to 2nd auto-HSCT was 16 months (3–64 ms). Seventy-seven percent patients sue to progressive disease responded completely or partially to one or two step treatment regimens before the second auto-HSCT. When evaluated the responses after the second auto-HCT, 14 (46%) patients were in CR or near-CR, 12 (38%) for stabile disease and 5 for refractory. PFS was estimated similar after both first and second auto-HCT (median 9 months vs 10 months, p=0.3) (Graphic 1). Five- year OS from the diagnosis was 75±10%.

Conclusion:
PFS of the myeloma pts was estimated similar median time after 1st&2nd auto-HSCT. Collection of adequate hematopoietic stem cells enough for more than one auto-HCT in myeloma patients can be appropriate. Although the majority of responses after the first AHSCT were partial responses, complete or near complete responses were dominant after the second AHSCT.Improvement in responses and the similarity of progression free survival can be explained by the contribution of the new antimyelom treatment options

Disclosures:
No relevant conflicts of interest to declare.
The Involvement of NF-κB Subunits in the Interaction Between CLL-B Cells and Bone Marrow Stromal Cells
Authors: Sun Aining , Feng Guo , Xu Jingjing , Wang Wenjuan , Depei Wu 
Published: October 2019

Abstract
Abstract 4590

Objective:
To investigate the function of NF-κB signaling pathway in the interaction between chronic lymphocytic leukemia B cells and bone marrow stromal cells.

Method:
The expression of NF-κB family members at mRNA and protein levels were examined by quantitative RT-PCR and Western bloting analyses individualy, and the differences between CLL-human bone marrow stromal cell (hBMSC) and non-CLL-hBMSC were investigated with the methods above. Cell death was measured by flow cytometry analysis after B-CLL cells were co-cultured with hBMSC and treated with proteasome inhibitors. The changes of NF-κB expression at the protein levels were examined by Western bloting after co-culture experiment.

Result:
The expression of NF-κB family members turned out to be heterogeneous at both mRNA and protein levels in B-CLL cells, and the members demonstrated a different κB-DNA binding activities. The mRNA expression of NF-κB family members in B-CLL cells was shown at a remarkable higher level than that of the controls. The relative mRNA expression of relA was 0.0214±0.012, whereas it was 0.0130±0.012 for the controls of CD19 positive cells. The relative mRNA expression of p50 and p52 were 66.0860±21.649 and 0.0208±0.011 respectively, and the corresponding expression of CD19 positive cells were 24.8440±9.749 and 0.0065±0.002. The differences were statistically significant. There was no notable difference between CLL-hBMSC and Non-CLL-hBMSC. hBMSC protected B-CLL cells against the proteasome inhibitors and facilitated the survival of B-CLL cells.

Conclusion:
NF-κB expression is heterogeneous in bone marrow B-CLL cells. There is no significant difference between CLL-hBMSC and non-CLL-hBMSC. hBMSC can protect the survival of B-CLL cells dependending on the endogenous NF-κB activity. hBMSC can increase the drug resistance of B-CLL cells to proteasome inhibitors.

Disclosures:
No relevant conflicts of interest to declare.