bluuebunny/crossref_metadata_2025_split huggingface dataset:
Aberrant Expression Of miRNA and mRNA Of Cell Cycle and Adhesion-Related Genes In Bone Marrow Stroma Cells Derived From Patients With Multiple Myeloma
Authors: Berenstein Rimma , Wolfgang Blau Igor , Nogai Axel , Wächter Marlies , Pezzutto Antonio , Dörken Bernd , Blau Olga 
Published: October 2019

Abstract
Multiple myeloma (MM) is a B-cell malignancy characterized by accumulation of malignant plasma cells (PC) within the bone marrow. The bone marrow microenvironment such as bone marrow stroma cells (BMSC) supports MM disease progression, resistance to chemotherapy, protects the tumor cells against apoptosis and causes osteolytic bone disease and angiogenesis.
The aim of this study was to identify constitutive genetic alterations in BMSC derived from patients with MM (MM-BMSC) in comparison to BMSC from healthy donors. For BMSC selection, mononuclear cells were isolated from fresh bone marrow aspirates and were further expanded in cell culture. We studied 25 MM patients and 5 healthy donors. Senescence status was determined in passage 1 of cell cultures. MM-BSMC displayed a considerably higher percentage of senescence cells (p<0,05). We investigated the expression of cell cycle and adhesion-associated genes (CCNE1, CCND1, CDKN1B, VCAM, ICAM, IKK-alpha) in BMSC (passage 4) using SYBR-Green Real-Time PCR and relative quantification by linear regression.
A downregulation of CCNE1 (p=0,05), CDKN1B (p=0,29), and an upregulation of CCND1 (p=0,05), VCAM-1 (p=0,33), ICAM-1 (p=0,33), and IKK-alpha (p=0,05) was demonstrated. Furthermore, the expression profile of miRNAs, targeting the analyzed mRNA genes or correlating with senescence, was studied (miR-16, miR-221, miR-126, miR-223, miR-485-5p and miR-519d). For miRNA detection treatment with Poly(A)-Polymerase and cDNA-Synthesis with a Poly(T)VN-Adaptor primer were carried out following an amplification with an universal reverse primer corresponding to the adaptor sequence and a miRNA-specific primer. miR-16, miR-223, miR-485-5p and miR-519d were significantly upregulated, (p=0,02; p=0,004; p=0,02; and p=0,002, respectively), whereas miR-221 and miR-126 showed no considerable differences to BMSC obtained from healthy donors. Next we investigated incubation of immunmodulatory drug Lenalidomid in BMSC cultures. Cells were treated with 10 µM Lenalidomid over 72 hours and expression was normalized to a 0,01 % DMSO control. Significant difference in gene expression were visible for ICAM-1 (p=0,01). For CDKN1B (p=0,16) and VCAM-1 (p=0,12) we demonstrated changes in gene expression.
Our results indicate aberrant expression of cell cycle and adhesion-related genes, such as CCNE1, CCND1 and CDKN1B VCAM-1, ICAM-1 and IKK-alpha in MM-BMSC as compared with healthy donors. Furthermore, we found significant upregulation of miR-16, miR-223, miR-485-5p and miR-519d. Further investigation are needed to determine molecular mechanisms in MM-BMSC and PC interaction that lead to constitutive changes in BMSC characteristics and gene expression patterns.

Disclosures:
No relevant conflicts of interest to declare.
Monitoring Minimum Residual Disease In Multiple Myeloma Patients By LC-MS/MS
Authors: Bergen H. Robert , Murray David L , Jelinek Diane F. , Tschumper Renee C. , Barnidge David R , Ramirez-Alvarado Marina , Dasari Surendra 
Published: October 2019

Abstract
Proteomics with great sensitivity and specificity effectively analyzes proteins by prior tryptic digestion and subsequent analysis by LC-MS/MS of the tryptic digest. We have utilized this approach in the development of a LC-MS/MS method to characterize minimum residual disease in multiple myeloma. The abundant antibodies produced by multiple myeloma plasma cells are identical and appear as a spike (M-spike) upon protein electrophoresis. The M-spike and histological examination of the bone marrow constitute part of the myeloma diagnostic repertoire. Upon treatment, myeloma cells and the antibodies they produce are reduced in number and amount and become increasingly hard to detect. The bone marrow biopsy serves as the “gold standard” test for clinical remission.
We have developed a sensitive test for the presence of the monoclonal antibody produced by the plasma cells which may serve as a substitute for invasive bone marrow biopsy. We have focused on tryptic peptides comprising the variable CDR regions of the Ig light chains that are unique to each patients antibody clone. Utilizing 2-5 µL of patient plasma/serum from the initial M-spike sample we have utilized SDS-PAGE to yield a crudely purified immunoglobulin light chain. The light chain band is isolated, reduced, alkylated and trypsin digested with subsequent LC-MS/MS analysis to identify CDR specific tryptic peptide(s). These can be identified as variable peaks (elution time and mass) in a base peak ion chromatogram where constant region peptides of either lambda or kappa light chains (clone dependent) serve as “internal standards” for identifying the CDR tryptic peptides. The peptide’s mass and its corresponding MS/MS spectra are unique to this CDR tryptic peptide and this patient’s clone. The unique diagnostic peptide is isolated in subsequent samples by immunoaffinity purification of the target kappa/lambda clone, SDS-PAGE separation and LC-MS/MS analysis of the light chain gel band. An extracted ion chromatogram is generated based upon the CDR peptides identified in the initial analysis of the M-spike sample. In patients in clinical remission the presence of a significant signal from the targeted peptides indicates that targeting a CDR peptide from the M-spike protein is more sensitive than currently available diagnostic tools including immunofixation. Comparison of this test to the gold standard bone marrow biopsy will be examined.

Disclosures:
Barnidge: Mayo Foundation: provisional patent application for technology, provisional patent application for technology Patents & Royalties.
Impact Of Dialysis-Dependent Renal Failure At Time Of Myeloma Diagnosis On Overall Survival - a 15-Year Single Centre Analysis
Authors: Murphy Philip T , Baldeo Cherisse , O'Kelly Patrick , Sargant Jeremy , Thornton Patrick , McCloy Mary , Conlon Peter , Magee Colm , Denton Mark , Quinn John 
Published: October 2019

Abstract
In myeloma, the use of autologous stem cell transplantation in younger patients as well as the introduction of thalidomide, lenalidomide and bortezomib has resulted in improvement in long-term survival of both younger and older patients. Bortezomid and high dose dexamethasone is currently recommended to treat newly diagnosed myeloma patients presenting with renal impairment and may lead to varying degrees of improvement in renal function. We have assessed not only survival trends for all patients diagnosed at our centre over the past 18 years but also the survival of the subset of patients with severe renal impairment who required dialysis at diagnosis.
All patients diagnosed with myeloma at our centre between January 1995 and December 2012 were included. We constructed Kaplan-Meier curves and used the Breslow generalised Wilcoxon test to evaluate overall survival (OS) patterns (diagnosed in three calendar periods: 1995-2000; 2001-2006; 2007-2012) for our total patient population as well as the subset of patients who required dialysis within 4 weeks of diagnostic bone marrow test.
262 patients (60.3% males) were diagnosed between 1995 and 2012. For all patients, median OS significantly increased from 13.2 months in period 1995-2000 to 27 months in period 2001-2006 with median OS not yet reached in period 2007-2012 (p=0.0001). In patients 70 years old or less, median OS significantly increased from 25.4 months in period 1995-2000 to 46.7 months in period 2001-2006 with median OS not yet reached in period 2007-2012 (p=0.0482). Improved median OS was also seen in patients > 70 years old: 4.4 months in period 1995-2000, 17.4 months in period 2001-2006 and 25.1 months in period 2007-2012 (p<0.0001). In contrast, patients requiring dialysis at diagnosis (n = 44) had much worse outcomes: median OS in the period 1995-2000 was 2.8 months and although there was a slight improvement in median OS in the period 2001-2006 (p=0.0318), there has been no further improvement in median OS in the period 2007-2012.
In our overall myeloma patient population, median OS has continued to increase over the time periods 1995-2000, 2001-2006 and 2007-2012, both for younger patients 70 years old or less and older patients >70 years old. Patients requiring dialysis at diagnosis, however, continue to have much poorer median OS, despite the use of bortezomib and dexamethasone containing regimens in recent years. The possible benefit of improved supportive measures and the early use of other emerging novel agents in this poor prognostic subgroup should be explored in the clinical trial setting.

Disclosures:
No relevant conflicts of interest to declare.
Deletion Of Fanca Or Fancd2 Dysregulates Treg Activity and Exacerbates Gvhd In Mice
Authors: Du Wei , Erden Ozlem , Wilson Andrew , Sipple Jared , Schick Jonathan , Mehta Parinda A. , Davies Stella M. , Steinbrecher Kris , Pang Qishen 
Published: October 2019

Abstract
Fanconi anemia (FA) is a genetic disorder associated with bone marrow failure and leukemia. Recent studies demonstrate fundamental immune defects in FA. However, the mechanisms that are critical for FA immunodeficiency are not known. Here we report that deletion of Fanca or Fancd2 dysregulates the suppressive activity of regulatory T cells (Tregs) and exacerbates graft-versus-host disease (GVHD) in mice. Recipient mice of Fanca-/- or Fancd2-/- bone marrow chimeras exhibited severe acute GVHD after allogeneic bone marrow transplantation (BMT). Further study showed that T cells from Fanca-/- or Fancd2-/- mice induced higher GVHD lethality than those from WT littermates. Mechanistically, FA Tregs possessed lower proliferative suppression potential compared to WT Tregs, as demonstrated by in vitro proliferation assay and BMT. Analysis of CD25+Foxp3+ Tregs indicated that loss of Fanca or Fancd2 dysregulated Foxp3 transcriptional activity. Additionally, CD25+Foxp3+ Tregs of Fanca-/- or Fancd2-/- mice were less efficient in suppressing the production of GVHD-associated inflammatory cytokines. Consistently, incremental NF-kB transcriptional activity was observed in infiltrated T cells from FA GVHD mice. Conditional deletion of p65 in FA Tregs desreased GVHD mortality. Our study uncovers an essential role for the FA proteins in maintaining Treg homeostasis and suggests that targeted blocking NF-kB signaling within T cells represents an attractive therapeutic strategy to ameliorate GVHD in FA.

Disclosures:
No relevant conflicts of interest to declare.
Differential Regulation Of Chemokine Receptor Expressions On T Lymphocyte Subsets In Healthy Donors After Mobilization With Rhg-CSF and Its Correlation With Acute GvHD
Authors: Ren Han-Yun , Wang Meng , Ma Xiang-Juan , Dong Yu-Jun , Qiu Zhi-Xiang , Liu Wei 
Published: October 2019

Abstract

Introduction
This study is aimed to investigate chemokine receptors (CCR5, CCR6, CCR7, CCR9, CXCR3 and CCR2) expression on T cell subsets in healthy donors after mobilization with recombinant human granulocyte colony-stimulating factor (rhG-CSF) and analyze its correlation with acute graft-versus-host disease (aGVHD) and to understand the possible mechanisms underlying rhG-CSF-induced immune tolerance.

Methods
Sixty-eight healthy donor and their recipient pairs of family donor allogeneic hematopoietic stem cell transplantation (allo-HSCT) were included in this study. The expressions of chemokine receptors on CD4+ and CD8+ T cells in the peripheral blood (PB) before and after mobilization was detected using flow cytometry (FCM) respectively. Six chemokine receptors (CCR2, CCR5, CCR6, CCR7, CCR9 and CXCR3) were detected on T cell subsets in all the donors, and CCR5 and CCR7 were detected only in eighteen of all the donors. The expressions of chemokine receptor before and after mobilization was compared and its correlation with II-IV aGVHD were analyzed.

Results
After rhG-CSF mobilization, the expression of CCR9 on CD4+ T cells and CCR7 on CD8+ T cells were significantly upregulated compared with that before mobilization (p<0.05). However, the mean value of CCR5, CCR6 and CXCR3 expression on CD4+ and CD8+ T cell subsets in PB after mobilization didn’t differ significantly compared with that before mobilization(p>0.10). However, different individuals showed apparent inconsistencies. According to the changes of chemokine receptor expression on CD4+ and CD8+ T cell subsets, the evaluable donors and their relevant recipients were divided into the down-regulated group and the non-down-regulated (unchanged or up-regulated ) group. The incidence of grade II to IV aGVHD in the two groups were compared in their corresponding recipients. In the univariate analysis, mismatched HLA (p=0.046), down-regulation of CCR7 expression on donor CD4+ T cell subsets (p=0.010), unchangeableness or up-regulation of CCR5 expression on donor CD4+ T cell subsets (p=0.032) and CCR6 down-regulation on donor CD8+ T cells (p=0.045) were risk factors for recipients to develop II-IV aGVHD. In the multivariate analysis, down-regulation of CCR7 expression on donor CD4+ T cells after rhG-CSF was independent risk factor for II-IV aGVHD [RR=3.5, 95% CI (1.3-9.4), p=0.012], while CCR5 down-regulation on CD4+ T cells could reduce the incidence of II-IV aGVHD [RR=0.3, 95% CI (0.1-0.8), p=0.031].

Conclusions
rhG-CSF mobilization could lead to differential regulation of chemokine receptors expression on T cell subsets, which might cause different effects on the migration of T cells in vivo, and decrease T cells trafficking towards GVHD target organs, and thus reduce the incidence of aGVHD after transplantation.

Disclosures:
No relevant conflicts of interest to declare.
Allogeneic Stem Cell Transplantation In Philadelphia Negative Myeloproliferative Or Myelodysplastic/Myeloproliferative Neoplasms: A Retrospective Analysis From The Societe Française De Greffe De Moelle Et De Therapie Cellulaire (SFGM-TC)
Authors: Cahu Xavier , Chevallier Patrice , Clavert Aline , Suarez Felipe , Michallet Mauricette , Fegueux Nathalie , Vigouroux Stephane , Blaise Didier , Socié Gérard , Mariette Clara , Bilger Karin , Yakoub-Agha Ibrahim , Peffault de Latour Regis , Mohty Mohamad 
Published: October 2019

Abstract

Background
Philadelphia negative myeloproliferative or myeloproliferative/myelodysplastic neoplasms may evolve towards secondary acute myeloid leukemia (AML). The prognosis of such secondary leukemia is very poor. At present, there are only a few reports assessing the outcome of adult patients with a philadelphia negative myeloproliferative or myeloproliferative/myelodysplastic neoplasm in blast phase (MPN-BP) who received allogeneic stem cell transplantation (allo-SCT).

Patients and Methods
in this retrospective study, inclusion criteria were: (i) adult patients with a MPN-BP (ii) who received first allo-SCT (iii) between 2000 and 2010 (iv) irrespective of the stem cell source or conditioning regimen. MPN with <20% blasts in blood/bone marrow and AML secondary to myelodysplastic syndromes were excluded from this analysis.

Results
60 patients were included. MPN, AML and allo-SCT characteristics are described in table 1. Median age at allo-SCT was 57 (range, 30-68). Patients received allo-SCT in first complete remission (CR1), CR2 or in advanced disease in 22 (37%), 4 (7%) and 34(57%) of cases, respectively. Engraftment was achieved in 55 cases (92%). With a median follow-up of 31 months (range, 25-44), the 3-year overall survival (OS) and Leukemia-Free-Survival (LFS) were respectively 18% and 9%. The 3-year transplant-related mortality (TRM) was 24% whereas relapse incidence was 68%. The 3-year LFS of patients grafted in CR (n=26) was 18% whereas the 3-year LFS of patients allografted in advanced disease (n=34) was only 3% (p=0.008). In the CR group, the 3-year TRM was 24% whereas relapse incidence was 61%. Intermediate or good AML karyotype (3-year LFS of 33% versus 10% for adverse AML karyotype, p=0.03) and the absence of a previous thrombotic event (3-year LFS of 24% versus 0, p=0.02) were associated with an improved LFS in patients allografted in CR.

Conclusion
These results suggest that the outcome of patients with a MPN-BP is dismal despite allo-SCT due to a high relapse incidence even in patients transplanted in CR. Outside a clinical trial, allo-SCT should be mainly proposed to patients in CR. New strategies are mandatory to improve the outcome of patients in blast phase.

Disclosures:
No relevant conflicts of interest to declare.
Iron Chelation For Diseases Of Regional Siderosis
Authors: Cabantchik Zvi Ioav , Moreau Caroline , Devos David 
Published: October 2019

Abstract
The traditional role of iron chelation therapy has been the reduction of body iron burden via chelation of excess metal from organs and fluids and its excretion via biliary-fecal and/or urinary routes. While that approach is applicable to systemic iron overload, as seen in various forms of hemosiderosis, it might not be suited for treating disorders of iron maldistribution that are characterized by discrete islands of siderosis that appear in a context background of normal or subnormal iron levels (as in sideroblastic anemias, cardiosiderosis in Friedreich ataxia-FRDA and neurosiderosis in Parkinson’s disease-PD).
With the aim of clearing siderotic regions from labile (toxic) metal without interfering with essential local functions or with hematological iron-associated indices, we assessed here a conservative modality of iron chelation based on redeploying tissue chelated metal to physiological transferrin ( Sohn et al Blood 111:1690-9). As test redeployment agent we used deferiprone (DFP), a chelator with clinical proven record for correcting cardiac siderosis in systemic iron overload and in FRDA regional siderosis and for neuroprotection in a translational model of regional (brain) siderosis (that simulate key features of PD)( Moreau et al Am. J. Hematol. 2013;88:E5-E243). As part of a placebo-controlled randomized clinical trial of DFP in early stage PD patients (n=40) already stabilized with dopamine/dopaminergic regimens, we assessed whether a up to 18 month treatment (DFP 30 mg/kg/d in 2 daily syrup servings of Ferriprox) that significantly decreased substantia nigra-pars compacta siderosis (by T2* MRI) and showed neuro-functional benefits (UPDRS score), affected adversely hematological parameters. With the exception of 2 neutropenia patients that were withdrawn early in the trial (and resolved spontaneously) all others on DFP maintained steady plasma iron (80-115 ug/L), Tf- Sat (17-33%) hemoglobin levels (13.8-14.4 g/dL) and hematocrit (40-42 %) throughout the entire study, with just traces of daily sideruria but with serum ferritin that significantly dropped from 95±35 ng/ml to 30 ± 10 ng/ml during the first 12 months but not thereafter. We conclude that to the extent that iron chelation might have beneficial effects for treating neurodegenerative disorders that have a component of neurosiderosis, the modality of the DFP-based therapy provides a paradigm for treating regional siderosis without affecting hematological parameters. Supported by PHRC grants from the French Ministry of Research and the Adelina and Massimo DellaPergola Chair, HUJI, Jerusalem, Israel

Disclosures:
Cabantchik: Aferrix: Consultancy; Apopharma: invited speaker, invited speaker Other; Novartis: Speakers Bureau. Off Label Use: Deferiprone for Parkinson's Disease.
Heme Oxygenase 1 Plays a Role In The Pathophysiology Of β-Thalassemia
Authors: Santos Daniel Garcia , Mikhael Marc , Rivella Stefano , Horvathova Monika , Ponka Prem 
Published: October 2019

Abstract
Thalassemias are a heterogeneous group of red blood cell disorders ranging from a clinically severe phenotype requiring life-saving transfusions (thalassemia major) to a relatively moderate symptomatic disorder, sometimes requiring transfusions (thalassemia intermedia). Thalassemia minor, the least severe form of the disorder, is characterized by minimal to mild symptoms. While thalassemia minor and intermedia are vastly more prevalent than thalassemia major, the latter is often fatal when not treated. Though considered a major cause of morbidity and mortality worldwide, there is still no universally available cure for this severe form of thalassemia. A reason for this is at least in part due to the lack of full understanding of pathophsyiology of thalassemia. The underlying cause of pathology in thalassemia is the premature apoptotic destruction of erythroblasts causing ineffective erythropoeisis. Normally, the assembly of adult hemoglobin (consisting of a tetramer of two α- and two β-globin chains) features a very tight coordination of α- and β-globin chain synthesis. However, in β-thalassemia, β-globin synthesis is decelerated causing α-globin accumulation; while in α-thalassemia the opposite scenario occurs. Unpaired globin chains that accumulate in thalassemic erythroblasts are bound to heme. In addition, in β-thalassemia an erythroid specific protease destroys excess α-globin chains, likely leading to the generation of a pool of “free” heme in erythroblasts. “Free” heme is toxic, but this toxicity will likely be augmented, if heme oxygenase 1 (HO-1) can release iron from heme. To date, virtually no information about the expression of HO-1 in erythroblasts has been produced; however, we have recently provided unequivocal evidence that this enzyme is present in several model erythroid cells1. Based on this novel and important finding, we hypothesize that in β-thalassemic erythroblasts HO-1 mediated release of iron from heme is the major culprit responsible for cellular damage. To test this hypothesis we exploited the mouse model of β-thalassemia, th3/+. Thus far, our data indicates that HO-1 expression is increased in liver, spleen and kidney of β-thalassemic mice compared to wild type mice. Importantly, we observed that Epo-mediated erythroid differentiation of fetal liver (FL) cells isolated from β-thalassemic fetuses, display increased levels of HO-1 as well as decreased phosphorylated eiF2-α. These results indicate that β-thalassemic erythroblasts have inappropriately high levels of unbound heme that is continuously degraded by HO-1. Further research is needed to determine whether HO-1 liberated iron is responsible for the damage of β-thalassemic erythroblasts.
1Garcia Santos D, Schranzhofer M, Bogo Chies JA, Ponka P. Heme Oxygenase 1 plays an unexpected role during erythroid differentiation. Blood (ASH Annual Meeting Abstracts) 118: 344, 2011.

Disclosures:
No relevant conflicts of interest to declare.