bluuebunny/crossref_metadata_2025_split huggingface dataset:
Transdifferentiation Of Malignant B-Cells Into Macrophages In a Murine Model Of Burkitt´s Lymphoma
Authors: Bruns Heiko , Mueller Sebastian , Pasemann Shirin , Mackensen Andreas , Gerbitz Armin 
Published: October 2019

Abstract

Background
The presence of abundant macrophages is characteristic morphological hallmark for the diagnosis of Burkitt´s lymphoma. They are considered to be the removers of cellular debris which occurs due to the rapid proliferation of such lymphomas. However, although macrophages represent the main non-malignant population within such lymphomas and therefore can be considered a substantial part of the stroma, little is known about how these cells are being recruited and how they influence malignant growth. Since malignant B–cells show high plasticity and have capacity to differentiate into various phenotypes we analysed the origin of macrophages in a murine model of Burkitt´s lymphoma. In this transgenic model a human c-myc gene is overexpressed in a B-cell specific fashion.

Results
When long term cultured lymphoma cells from transgenic animals were transferred into wild type animals expressing the congenic marker CD45.1 lymphomas grew out within 14 days post transfer. Lymphomas were minced into single cell suspension and macrophages were sorted by flow cytometry based on the expression of CD11b and F4/80. Approximately 6% of the cells expressed F4/80 and CD11b as macrophage markers and displayed an M2 phenotype. Surprisingly approximately 50% of these macrophages also expressed CD45.2 and therefore were not of host origin but derived from the transferred lymphoma cells. These cells were positive for the c-myc transgene, but displayed reduced expression of c-myc as shown by RT PCR in comparsion to the lymphoma cell line in culture. Furthermore, lymphoma derived macrophages displayed reduced PAX5 expression and were negative for CD19 surface expression. When lymphoma cells were retrovirally transduced to express bcl2 to block apoptosis, and were treated with chemotherapy in vivo, we observed a massive increase in macrophage numbers in remaining tissues suggesting transdifferenziation as an alternative exit strategy in case of cellular stress.

Conclusion
These results demonstrate that BL cells are able to differentiate into a macrophage phenotype to form their own supporting stroma. In addition, differentiation after DNA damage such as chemotherapy is increased suggesting a new exit strategy when classic apoptotic pathways are blocked.

Disclosures:
Mackensen: Eli Lilly: Consultancy.
Polyclonal Gamma Globulin Suppression and Risk Of MGUS Progression Into Multiple Myeloma
Authors: Sheqwara Jawad Z. , Alhyari Mohammad , Keating Shannon , Kuriakose Philip 
Published: October 2019

Abstract
Monoclonal gammopathy of undetermined significance (MGUS) is the most common form of plasma cell dyscrasia, with a prevalence of 3% in the general population above age of fifty. MGUS has a malignant evolution rate of 1% per year. Large longitudinal studies have suggested that virtually all patients diagnosed with multiple myeloma (MM) had a preceding MGUS, with 75 % having detectible Monoclonal (M) protein ≥8 years prior to diagnosis. It is important to identify the features at diagnosis that can predict neoplastic transformation to MM.

Purpose
We identified 239 patients at our institute in whom MGUS was diagnosed between 2000 and 2010. The presenting clinico-hematologic features were correlated with the frequency of evolution into MM to identify early predictors of evolution. The primary end point was progression to MM.

Results
The patients' mean age was 70.7 years. The Male/Female ratio was 0.7. The mean concentration of the M component (MC) was 0.7 g/dL. IgG was the most frequent MC (77%), followed by IgA (13%). The median ratio of MC protein to total protein was 0.5. Single or multiple background polyclonal (PC) suppression was noted in 36% of patients. PC suppression of 50% or more was noted in 20.1% of patients, 49.8% had < 50% and 30.1% had no suppression. Mean bone marrow plasma cell percentage was 4.5 percent and mean hemoglobin was 12.4 g/dL.
Eighteen of the 239 patients with MGUS progressed into MM over ten years of follow up.
Univariate comparisons of all variables between those who progressed and those who did not, showed that the initial concentration of the serum M protein, ratio of M protein to the total protein, number of PC gamma globulins suppressed, degree of PC suppression and IgM gamma globulin suppression were statistically significant risk factors that correlated with progression into MM. Fourteen out of eighteen patients with progressive disease had either PC suppression or background IgM suppression.

Conclusions
Monoclonal protein concentration, ratio of M protein to the total protein and abnormal serum free light chain ratio are simple variables that have been shown in multiple previous studies to predict the progression of MGUS into MM. In our study, we additionally found that number of PC suppressed, degree of suppression and IgM suppression are also key risk factors that can predict progression.
We believe that these variables can be potentially applied into an approach that uses a detailed risk stratification system to predict which cases of MGUS will progress into MM and to provide more intensive monitoring for patients more likely to progress.

Disclosures:
No relevant conflicts of interest to declare.
CD3 Antibody Treatment Prevents Gvhd and Allows For Rapid, Long Term, Multilineage Engraftment Of Unfractionated UCB Into Immunodeficient Mice
Authors: Wunderlich Mark , Brooks Ryan , Rhyasen Garrett , Mulloy James C. 
Published: October 2019

Abstract
NSG mice are currently the best hosts for human hematopoietic stem cell xenografts, due in large part to their ability to initiate robust grafts comprised of diverse human blood cell lineages. Unfortunately, NSG mice are susceptible to GVHD induced by contaminating T cells. Therefore, stem and progenitor cells must be selected with a high degree of purity. This not only complicates the procedure, but also prevents modeling of whole umbilical cord blood (WUCB) and bone marrow transplantation. Here, we have explored both in vivo and in vitro methods to prevent xenogenic GVHD utilizing the anti-T cell antibodies ATG and OKT3. IP injection of either antibody within the first 72 hours of transplant proved effective at preventing T cell expansion. Alternatively, a simple short-term incubation of the cell suspension with antibody was equally effective. ATG, but not OKT3, had significant non-specific activity resulting in reduction and even prevention of the non-T cell portion of the graft, including SCID repopulating cells, using either the in vivo or in vitro approach. However, using the OKT3 in vitro incubation protocol, we were able to initiate stable, long term, multi-lineage engraftment from whole, unselected UCB without GVHD. Additionally, WUCB transplanted mice engrafted more readily, with a wider array of human cell types in the peripheral blood as early as 2 weeks, relative to CD34+ engrafted mice. Stem cell engraftment was confirmed by successful transplant of the human graft to secondary recipients. Limiting dilution experiments showed that significant human grafts could be generated with as few as 1 million total WBCs per mouse, indicating that very large cohorts of mice can be generated with a single UCB sample. This protocol not only has the potential to significantly streamline hematopoietic xenograft studies while greatly reducing the cost and time commitments involved, but also to allow for whole cord blood transplantation modeling and generation of unique subsets of human cells in vivo.

Disclosures:
No relevant conflicts of interest to declare.
Towards a Reference Human Platelet Transcriptome: Evaluation Of Inter-Individual Correlations and Its Relationship With a Platelet Proteome
Authors: Londin Eric R. , Hatzimichael Eleftheria , Loher Phillipe , Edelstein Leonard C. , Shaw Chad , Delgrosso Kathleen , Fortina Paolo M. , Bray Paul F , McKenzie Steven E. , Rigoutsos Isidore 
Published: October 2019

Abstract
Next generation sequencing of RNA (RNA-seq) is an emerging technology that has so far been used successfully to profile the transcriptomes of several cell types and cell states. For the platelet transcriptome, RNA-seq descriptions exist for only a few subjects. Additionally, there have been no studies of the same individual’s transcriptome using two different technologies. As such, it has been unclear how well platelet transcriptomes correlate among different donors or across different RNA platforms, and what the transcriptomes’ relationship is with the platelet proteome.
We generated RNA-seq profiles of the long RNA transcriptomes from the platelets of 10 healthy young males (5 white and 5 black). In addition to RNA-seq, we profiled the platelet messenger RNAs of the same 10 individuals using the Affymetrix GeneChip System. We observed that the abundance of platelet mRNA transcripts was highly correlated across the 10 individuals, a finding that was independent of race and of the employed technology. Additionally, our RNA-seq data showed that these high inter-individual correlations extend beyond mRNAs to several categories of non-coding RNAs. However, there was a notable exception: the category of pseudogenes exhibited a clear difference in expression by race.
Comparison of our mRNA signatures with the only publicly available quantitative platelet proteome data showed that most (87.5%) identified platelet proteins had a detectable corresponding mRNA. Interestingly, there was also a high number of mRNAs that were present in the transcriptomes of all 10 individuals but had no representation in the proteome. Spearman correlation of the relative abundances for those platelet genes that were represented by both an mRNA and a protein, revealed an unexpectedly weak correlation between the transcriptome and the proteome. Further analysis of the overlapping and non-overlapping platelet mRNAs and proteins identified groups of genes with very distinct characteristics. Gene Ontology analysis of the respective gene identifiers revealed that the gene groups corresponded to distinct cellular processes, an interesting finding that provides novel insights for platelet biology.
The very high inter-individual correlations of the transcriptome signatures across 10 different subjects representing two races together with the results of our analyses indicate that it is feasible to assemble a platelet mRNA-ome that can serve as a reference for future platelet transcriptomic studies of human health and disease.

Disclosures:
No relevant conflicts of interest to declare.
Targeting Oncogenic Interleukein-7 Receptor Signaling With N-Acetylcysteine In T-Cell Acute Lymphoblastic Leukemia
Authors: Mansour Marc R. , Reed Casie , Eisenberg Amy , Tseng Jen-Chieh , Yoda Akinori , Rodig Scott J , DeAngelo Daniel J. , Sallan Stephen E. , Weinstock David M. , Izraeli Shai , Kung Andrew L , Kentsis Alex , Look Thomas 
Published: October 2019

Abstract
Activating mutations of the interleukin-7 receptor (IL7R) occur in approximately 10% of patients with T-cell acute lymphoblastic leukemia. Most mutations generate a cysteine at the transmembrane domain leading to receptor homodimerization through disulfide bond formation and ligand-independent activation of STAT5. We hypothesized that the reducing agent N-acetylcysteine (NAC), a well-tolerated drug used widely in clinical practice to treat acetaminophen overdose, would disrupt disulfide bond formation, and inhibit mutant IL7R-mediated oncogenic signaling.
To first identify a suitable cell model to study mutant IL7R signaling, we sequenced exon 6 of IL7R in 21 T-ALL cell lines. We identified a 4-amino-acid insertion (p.L242_L243insLSRC) in DND-41 cells which is predicted to form IL7R homodimers through disulfide bond formation with the unpaired cysteine of neighboring mutant IL7Rs. We found that treatment with NAC at clinically achievable concentrations disrupted IL7R homodimerization in IL7R-mutant DND-41 cells in vitro (IC50 approximately 150 micromolar) and led to STAT5 dephosphorylation and cell apoptosis. These effects could be rescued in part by a constitutively active allele of STAT5, indicating the mechanism of NAC is mediated predominantly through disruption of IL7R-STAT5 signaling in these cells. In a murine xenograft model of T-ALL, intraperitoneal NAC treatment led to significant inhibition of tumor progression, indicating NAC has activity in vivo. Previous studies of NAC pharmacokinetics in humans have shown steady state plasma levels range from 200 to 900 micromolar when given on standard treatment regimens for acetaminophen overdose, well within the therapeutic range required to kill DND-41 cells in vitro. Targeting leukemogenic IL7R homodimerization with NAC offers a potentially effective, cheap and feasible therapeutic strategy that warrants testing in clinical trials.

Disclosures:
Rodig: Daiichi-Sankyo/Arqule Inc., Ventana/Roche Inc., Shape Pharmaceuticals Inc.: Consultancy; Ventana/Roche Inc.: Research Funding.
Hereditary Thrombocytosis Caused By a Novel Germ-Line Mutation In The Gelsolin Gene
Authors: Pianta Annalisa , Liu Kun , Lundberg Pontus , Shimizu Takafumi , Hao-Shen Hui , Dirnhofer Stephan , Kralovics Robert , Shapiro Amy D. , Skoda Radek C. 
Published: October 2019

Abstract
Hereditary thrombocytosis (HT) is a familial myeloproliferative disorder with clinical features resembling sporadic essential thrombocythemia. In some families germline mutations causing HT have been identified in the genes for thrombopoietin (THPO) and its receptor, MPL. However, in many HT families the disease-causing genetic lesion remains unknown. Here we studied a HT pedigree with 15 affected family members in 5 generations. Thrombocytosis is transmitted as an autosomal dominant trait with high penetrance (Figure 1). Genome-wide linkage analysis was performed on DNA from 21 family members using microsatellites and single nucleotide polymorphism arrays. A single co-segregating region with a LOD score of 4.3 was found on chromosome 9. All exons and splice junctions of genes within the co-segregating region were sequenced by classical DNA sequencing and by Next Generation Sequencing (Illumina). We found a candidate mutation in the gelsolin gene (GSN). This C/T transversion was not reported in any SNP database. Computational analysis predicts that the resulting Gly to Cys amino acid change will be damaging to the protein structure. Platelet biogenesis in vitro assays in DAMI cell line stably transfected with the mutant GSN showed that the candidate alteration increased the release of platelets-like particles. To study the in vivo role of the candidate mutation, we generated transgenic mice expressing the mutant GSN gene. These mice developed thrombocytosis and showed increased numbers of megakaryocytes in bone marrow (Figure 2). Thus, our genetic and functional data strongly suggest that GSN mutation is causing thrombocytosis in this family. The exact mechanism of how this newly identified genetic alteration leads to increased megakaryopoiesis needs further investigation.

Disclosures:
No relevant conflicts of interest to declare.
Is There An Expiration Date For Cord Blood Units In Storage?
Authors: Parmar Simrit , de Lima Marcos J , Lee Dean A. , Worth Laura , Kongtim Piyanuch , Petropoulos Demetrios , McNiece Ian K. , Shpall Elizabeth J. , Rondon Gabriela , Champlin Richard E 
Published: October 2019

Abstract

Little is known about the impact of storage duration of an umbilical cord blood unit (CBU) on its’ long term engraftment potency in a clinical setting. We hypothesized that the longevity of storage would not affect CBU engraftment kinetics, when stored under standard conditions. A total of 86 patients undergoing single CB transplants were identified and impact of CBU age was analyzed on transplant outcomes including engraftment and survival. Based on age of the CBU, 4 quartiles were identified: (Q1: Age < 0.7 years (median: 0.07; n=21); Q2: 0.71-1.96 years (median: 1.3 years; n=22); Q3: 1.97-3.87 years (median: 2.8 years; n=22) and Q4: 3.88-12.2 years (median: 5.3 years; n=21)) (Table 1). We also performed analysis on dichotomous distribution of Old CBU (age ≥ 5 years; n=15) vs. New CBU (age < 5 years; n=71). No significant differences in the baseline patient characteristics were seen in any of the subgroups (Table 2). Engraftment was comparable across all quartiles. When comparing quartile Q1, Q2, Q3, Q4, the distribution of outcomes was: median time to neutrophil engraftment: 24, 24, 21, 22 days and median time to platelet engraftment: 42, 54, 41, 42 days, respectively (Figure1 A, B). No differences were observed in the duration of hospitalization: median 43, 48, 43 and 44 days, respectively for the 4 quartiles. For the dichotomous comparison, median time to neutrophil engraftment was 19 vs. 22 days; median time to platelet engraftment was 41 vs. 44 days; and median duration of hospitalization was 41 vs. 46 days, respectively, for Old CBU vs. New CBU. Acute GVHD rates and overall survival rates were also similar. We conclude that old CBUs remain a viable source for stem cell transplant.

Disclosures:
No relevant conflicts of interest to declare.
Rituximab Maintenance Therapy After Autologous Stem Cell Transplantation Prolongs Progression Free Survival In Patients With Mantle Cell Lymphoma
Authors: Dietrich Sascha , Weidle Julia , Meissner Julia , Radujkovic Aleksandar , Ho Anthony D. , Dreger Peter , Witzens-Harig Mathias 
Published: October 2019

Abstract

Background
It was recently shown that rituximab maintenance therapy prolongs progression free (PFS) and overall survival (OS) in patients with mantle cell lymphoma (MCL) after conventional chemotherapy treatment. However, it is not clear if rituximab maintenance can further improve outcome of MCL patients after autologous stem cell transplantation (autoSCT).

Methods
All patients with MCL who underwent an autoSCT at the University Hospital of Heidelberg from 2000 onwards were selected for this analysis. MCL patients who have received rituximab maintenance therapy after autoSCT in a prospective phase-2 trial were compared with regard to PFS and OS with MCL patients who were transplanted during the same time period but did not receive rituximab maintenance therapy by multivariate cox regression analysis correcting for known confounders. For the comparison of rituximab maintenance therapy with the control group, rituximab maintenance therapy was considered as a time dependent event in order to avoid that early progression before start of rituximab maintenance therapy may compromise results.

Results
A total of 72 patients met the inclusion criteria. Twenty-two patients participated in a phase-2 trial and received rituximab maintenance therapy for two years. Median age was 60 years (30-74). Prior to autoSCT all patients received rituximab and 45 patients were treated with high dose cytarabine (HD-ARA-C). AutoSCT was performed after first line treatment in 51 patients. A complete response (CR) before autoSCT was achieved by 27 patients. Patients with and without rituximab maintenance therapy did not significantly differ with regard to age, upfront autoSCT, CR rates and HD-ARA-C treatment prior to autoSCT. However, there was a trend that patients who received rituximab maintenance therapy were transplanted more recently (p=0.06).
Median observation time after autoSCT was 56 months. Two-year PFS and OS of the control group were 65% and 80%, respectively, as compared to 90% and 88%, respectively, of the rituximab maintenance group. By univariate analysis rituximab maintenance therapy was associated with significantly better PFS (HR 0.21, p=0.014; Figure 1A) but so far not OS (Figure 1B). Multivariate adjustment for age (HR per year 0.98; p=0.79), year of transplant (HR per calendar year 1.0; p=0.96), achievement of CR prior autoSCT (HR 1.59; p=0.26), upfront autoSCT (HR 0.81; p=0.80) and high dose ARA-C treatment (HR 0.69; p=0.63) confirmed the beneficial impact of rituximab maintenance therapy (p=0.02 HR 0.23).

Summary
Rituximab may prolong PFS in MCL patients who have undergone autoSCT. Prospective studies are highly warranted to confirm our preliminary finding.

Disclosures:
Ho: Sanofi-Genzyme: Research Funding, travel grant Other.
Dysregulation Of Cyclin D1 Or Activation Of KRAS Accelerate The Development Of Monoclonal Gammopathy and Cooperate To Cause Its Progression
Authors: Chesi Marta , Garbitt Victoria , Palmer Stephen , Bergsagel P. Leif 
Published: October 2019

Abstract
We have previously proposed the dysregulation of the D type cyclins by chromosome translocations to the IgH locus or by not fully characterized mechanisms in hyperdyploid patients as a unifying genetic event in the pathogenesis of Monoclonal Gammopathy of Uncertain Significance (MGUS) and multiple myeloma (MM). However, no experimental data are available to evaluate the direct consequences of cyclin Ds overexpression in plasma cells. Similarly, activating mutations of KRAS have been identified in 15% of MM patients, but never in MGUS, and have been proposed to contribute to the progression of MGUS to MM, but this hypothesis has never been experimentally validated. We evaluated the contribution of CyclinD1 and KRAS to the development and progression of monoclonal gammopathy (MG) that develops spontaneously in C57BL/6 mice. We aged a cohort of EuCCND1 mice, and lslKRASV12 mice crossed to three different CRE expressing mice: IgG1CRE, CD19CRE or Vk* CRE (KRAS/CRE). A cohort of Vk* MYC mice, Vk* MYC x EuCCND1 (MYC/CD1) and triple transgenic mice (CD1/KRAS/CRE) was also analyzed. Mice were bled every 10 weeks beginning at 30 weeks, and the presence of MG was evaluated by serum protein electrophoresis. The median Time To M-Spike (mTTS) detection was 97 weeks in C57Bl/6, 63w in EuCCND1, 54w in KRAS/CRE, 54w in Vk* MYC, 38w in CD1/KRAS/CRE and 36w in MYC/CD1 mice. This indicates that acceleration of the development of MG occurs in all these strain of mice. However, we noticed that the spontaneous MG in C57BL/6 wt, EuCCND1 or KRAS/CRE rarely progresses (as determined by detection of a M-spike >7 g/L), while in the other mouse strains it frequently progresses, with a mTTS >7 g/L around 65w. A clear progression of MGUS to MM was observed only in Vk* MYC mice, and was further accelerated in MYC/CD1 mice, which are characterized by extra-medullary disease and shorter overall survival.
In conclusion, these data support our hypothesis that Cyclin Ds dysregulation in plasma cells is the initial transformation step leading to MGUS. Further oncogenic events, like activating mutations of KRAS are required to expand the monoclonal plasma cells. Among the oncogenes examined MYC provides the greatest acceleration of the MG in wildtype and EuCCND1 mice, and results in a disease with the malignant features of MM.

Disclosures:
No relevant conflicts of interest to declare.