bluuebunny/crossref_metadata_2025_split huggingface dataset:
Minimal Residual Disease Monitoring By Quantitative RT-PCR In t (8; 21) AML Early After Allogeneic HSCT Can Predict Clinical Outcomes and Allow Further Risk Stratification
Authors: Wang Yu , Liu Yanrong , Qin Yazhen , Huang Xiao-Jun 
Published: October 2019

Abstract

Background
Recent results from our prospective multi-center study identified patients with t (8; 21) AML as high-risk according to their MRD status after the second consolidation chemotherapy and allo-HSCT can benefit this part of patients; however, the relapse rate was reported to be 22% even after allo-HSCT for those high-risk patients. To date, there have been no studies to answer the question of whether the specific fusion gene, RUNX1/RUNX1T1 can be used to further distinguish between patients with low and high risks of relapse in allo-HSCT setting, just like the already established standard for MRD measurement in CML and APL.

Methods
Sixty consecutive AML patients with t (8; 21) and identified as high-risk according to the criteria from our recently published report patients who received allo-HSCT were enrolled between January 2006 and January 2013. Serial MRD monitoring by RQ-PCR post HSCT was done. The impact of MRD monitoring on transplant outcomes was assessed.

Results
A > 3 log reduction at 1 month after HSCT in RUNX1/RUNX1T1 transcripts from diagnosis, is associated with 2-year CIR of 17% in the 88% of patients achieving it, compared with 43% in the remaining (p=.02). A > 3 log reduction at 2 months after HSCT in transcripts from diagnosis, is associated with a CIR of 9% and LFS of 66% in the 86% of patients achieving it, compared with CIR of 100% and LFS of 0% in the remaining (both p<.001). A > 3 log reduction at 3 months after HSCT in RUNX1/RUNX1T1 transcripts from diagnosis, is associated with a CIR of 11% and LFS of 73% in the 78% of patients achieving it, compared with CIR of 44% and LFS of 0% in the remaining (both p<.001). Of the 60 patients analyzed, 28 patients had positive MRD, occurring at a median of 110 days (30-540 days) after transplant. The predictive value of sequential monitoring could be demonstrated in 8 patients culminating in clinical relapse (representing 73% of all relapsing patients). The median time from MRD positivity in BM to morphological relapse was 95 days (range, 33-140 days).

Conclusions
A > 3 log reduction at the first 3 months after HSCT in RUNX1/RUNX1T1 transcripts from diagnosis is highly prognostic. We conclude that MRD monitoring by RQ-PCR at regular early time points post HSCT in t (8; 21) AML allows further identification of patients at high risk of relapse even after allo-HSCT and could now be incorporated in clinical trials to evaluate the role of risk directed prophylactic/preemptive therapy.

This work was partly supported by The Key Program of National Natural Science Foundation of China £¨Grant No. 81230013£©, Beijing Municipal Science & Technology Commission (No.Z121107002812033) and Bejing Municipal Science & Technology Commission£¨No.Z111107067311070).

Disclosures:
No relevant conflicts of interest to declare.
High-Dose Chemotherapy and Autologous Stem Cell Transplantation In Patients With High-Risk Neuroblastoma : Results Of Turkish Pediatric Oncology Group- Neuroblastoma 2003 (TPOG-NBL 2003) Protocol
Authors: Aksoylar Serap , Varan Ali , Vergin Canan , Hazar Volkan , Akici Ferhan , Dagdeviren Ayhan , Unal Emel , Oniz Haldun , Anak Sema , Koseoglu Vedat , Ince Dilek , Olgun Nur 
Published: October 2019

Abstract

Introduction
TPOG-NBL 2003 national protocol was designed to improve treatment results of the high risk patients by adjunction of stem cell rescue with intensive multimodal therapy.

Material and Methods
High risk stratification was made according to COG criteria. Just before the third cycle of chemotherapy, patients without progression were allocated into two treatment groups non-randomly by physicians’ and/or parent’s choices guided to the center’s facility, toxicity and social-economical facility to attain the megatherapy. After an induction of 6 chemotherapy cycles, the protocol was divided into two arms which were designed to continue the intensive conventional chemotherapy (CCT), or initiate myeloablative therapy with autologous stem-cell rescue (ASCR). All patients were also given 13-cis-retinoic acid as maintenance therapy.

Results
Fifty-six percent (272 patients) of all neuroblastoma patients was evaluated as high risk. Response rate to induction chemotherapy was 81% (CR/VGPR: 32%, PR: 49%) in patients at the end of induction chemotherapy. Overall EFS and OS at 3-years were 36% and 45%, respectively. Intention-to-treat analysis documented post-induction (after the six cycles of induction chemotherapy) EFS of 46% in CCT arm (137 patients) and 37% in ASCR group (55 patients) (p= 0.037); whereas, OS was 59% and 43%, respectively (p=0.052). Thirty-one patients (11%) died of treatment-related complications.

Conclusion
Survival rates of high-risk neuroblastoma have improved over the last decade in Turkey. The main problems when managing these patients were an effective local control, early progression and death. Megatherapy has not augmented the therapeutic end point in our country’s circumstances. However; the better the supportive care and the higher the patients’ compliance is attained, the higher the survival rates might be obtained in Turkish neuroblastoma patients.

Disclosures:
No relevant conflicts of interest to declare.
Sideroflexin 4 Deficiency Results In An Erythroid Differentiation Defect
Authors: Nwanaji-Enwerem Jamaji C. , Hildick-Smith Gordon J. , Bauer Daniel E. , Huston Nicholas , Paw Barry H. 
Published: October 2019

Abstract
Megaloblastic anemia characterizes molecularly heterogeneous disorders, most commonly marked by macrocytosis, red blood cell enlargement, and abnormal nuclear maturation. Aside from vitamin B12 and folate deficiencies, genetic defects may be responsible for hereditary forms of megaloblastic anemia, though some remain unknown (Ghersim C, et al. 2013 J. Biol. Chem.). Loss of function of the gene Sideroflexin-4 (SFXN4) has been implicated as a cause for combined macrocytic anemia and mitochondriopathy. We have previously shown a defect in SFXN4 in humans and successfully modeled the erythroid and mitochondrial defects in the zebrafish (Hildick-Smith GJ, et al. 2013 AJHG). Here, we expand on prior studies involving SFXN4 in zebrafish and human fibroblasts by generating a murine cultured cell model deficient in SFXN4.
Clustered regularly interspaced short palindromic repeats (CRISPR)-cas targeting technology (Cong L, et al. 2013 Science) was utilized to generate SFXN4 knockout Friend mouse erythroid leukemia (MEL) clones. We conducted a screen of clones using qRT-PCR to select MEL clones with the lowest expression of Sfxn4 mRNA. The CRISPR knockout clone shows significantly reduced Sfxn4 mRNA relative to wild type (Panel A). Furthermore, the CRISPR knockout's reduced Sfxn4 mRNA is comparable to Sfxn4 mRNA levels in clones silenced by shRNA hairpin constructs (data now shown).
We also investigated the differentiation and maturation potential of the CRISPR-derived SFXN4 knockout clone. Initially, the MEL clone was differentiated for 3-5 days with DMSO treatment. o-dianisidine stains revealed that the CRISPR knockout clone had a reduction in hemoglobinization. Furthermore, Wright-Giemsa staining demonstrated that the same clone possessed megaloblastic features with immature maturation and a high nuclear to cytoplasmic ratio (Panel B). Both sets of results from experiments support the hypothesis for an essential role of SFXN4 in vertebrate red cell development.
In summary, our data: (1) supports the hypothesis of SFXN4's involvement in erythropoiesis and candidacy as a gene for combined macrocytic anemia and mitochondriopathy, and (2) demonstrates the promise of shRNA knockdown and now CRISPR SFXN4 knockout clones as tools for further studying the biochemical relationship between SFXN4 and megaloblastic anemia.

Disclosures:
No relevant conflicts of interest to declare.
Barth Syndrome and Neutropenia
Authors: Dale David C. , Bolyard Audrey Anna , Marrero Tracy M. , Bonilla Mary Ann , Phan Lan , Steward Colin 
Published: October 2019

Abstract
Barth syndrome is an X-linked, hereditary cause for neutropenia, cardiomyopathy, muscle weakness and growth retardation. It is attributable to mutations of TAZ, a gene encoding a highly conserved acyltransferase necessary for the maintenance of the phospholipids of the inner layers of mitochrondrial membranes. There is a wide diversity in the TAZ mutations but as yet no recognized genotype-phenotype correlations.
We collected and analyzed data for 83 patients (all males, Severe Chronic Neutropenia International Registry, Barth Syndrome Foundation and NHS Barth Syndrome Service in the UK). Fifty-four are currently less than 18 years old (median age 13.6 years, range 0.2-31.6 years). Absolute neutrophil counts (ANC) varied remarkably (median 1.1 x 109/L, range 0.14-5.4 x 109/L). A few patients have cyclic variations in the ANCs, but most have not had a sufficient series to determine if the variations show periodicity. Mouth ulcers, bleeding gums, skin infections, and upper respiratory infections were the most common complications, but pneumonia, sepsis, and more severe infections occurred sporadically. Comparisons of reported events for the population suggest that patients with baseline neutrophil counts< 0.5 x 109/L have more infections than patients with higher counts.
Data were available for 42 patients with neutropenia sufficiently severe to be treated with G-CSF by their physicians. On average, G-CSF was begun at 5.8 years (median age 4.3, range 0.05-21.5 years), with the median dose of 1.67 mcg/kg/d, mean 2.78 +/- 0.78 (SEM) mcg/kg/d (range 0.45-12.8 mcg/kg/d), with an average exposure of 7.3 years.
With longitudinal follow-up, 15 patients have had cardiac transplants and no patients have developed AML or received bone marrow transplantation and treatment responses to G-CSF are maintained long term. Bone marrow examinations were generally not performed, either before or on G-CSF treatment. Longitudinal observation suggests that the hematological profiles of individual patients are largely unchanged from childhood into adulthood, without evidence of worsening or amelioration. Additional clinical and laboratory data are need to define the genetic determinants and hematological features of Barth syndrome.

Disclosures:
Dale: Amgen: Consultancy, Research Funding.
Making “Sense” Of Persistent Polyclonal B Cell Lymphocytosis
Authors: Tsui Hubert , Paltser Geoff , Adissu Hibret , Dosch Michael , Keating Armand 
Published: October 2019

Abstract
Neuronal regulation of the hematopoietic stem cell niche includes elements of the sympathetic nervous system however, a role for sensory neurons has not been explored. While investigating sensory nerves in hematopoiesis, we unexpectedly found mice deficient in the neurotransmitter substance P (Tac-1-/-) to exhibit peripheral blood lymphocytosis that is persistent from 8 weeks to at least 35 weeks of age. Flow cytometry showed the increased population to be comprised of polyclonal B-lymphocytes. Hematopathology review demonstrated abnormal peripheral blood bi-nucleated lymphocytes and enlarged splenic marginal zones. Lymph nodes were not affected. To confirm that sensory nerve disruption and not hematopoietically derived substance P was the cause, wildtype C57BL/6 mice were chemically denervated and found to exhibit similar peripheral blood lymphocytosis. By immunohistochemistry, sensory nerves synapsed at splenic marginal zones, consistent with previous reports and providing a neuro-anatomical link for the observed lymphoid hyperplasia. A human correlate to this hematological phenotype featuring long-term peripheral polyclonal B cell lymphocytosis, bi-nucleated lymphocytes and expanded splenic marginal zones is Persistent Polyclonal B-cell Lymphocytosis (PPBL), a rare disorder affecting predominantly female chronic smokers. The pathoetiology of this disorder is unknown but its strong association with smoking supports a novel hypothesis where chronic smoking leads to respiratory tract sensory nerve desensitization with possibly more systemic effects, including on the spleen, thereby mimicking the substance P deficient environment of Tac-1-/- mice. Indeed, pulmonary function tests on chronic smokers have shown a sub-population of patients with hyporeactivity to respiratory sensory nerve stimulation, explained molecularly by increased activation thresholds. It will be interesting to determine if PPBL patients fall into this category. In Tac-1-/- mice, further characterization of the expanded B cell population, immunoglobulin profiles, VDJ utilization and effect of neurokinin-1 receptor modulators is proposed. These results support a neuroimmune regulatory network involving sensory nerves with potential relevance in both lymphoid homeostasis and lymphoproliferative disease.

Disclosures:
No relevant conflicts of interest to declare.
Thrombin Induces Specific Signaling Pathways Distinct From Simultaneous Stimulation With PAR1 and PAR4 Agonist Peptides In Human Platelets
Authors: Arachiche Amal , Nieman Marvin T. 
Published: October 2019

Abstract
Platelet activation by thrombin plays an important role in hemostasis and thrombosis. Protease activated receptor 1 (PAR1) and PAR4 are G- protein coupled receptors expressed in human platelets and activated by the proteolytic cleavage of their N-terminus with thrombin. We have previously shown by Bioluminescence Resonance Energy Transfer (BRET) that thrombin induces the formation of PAR1-PAR4 heterodimers in HEK293 cells. In contrast, simultaneous stimulation with the PAR1 and PAR4 agonist peptides, TFLLRN and AYPGKF, respectively, does not induce the formation of heterodimers. Signaling events that are specific to PAR1-PAR4 heterodimers in platelets are not known. In the current study we compared signaling in human platelets activated with thrombin or a mixture of TFLLRN and AYPGKF agonist peptides by examining the phosphoproteome using mass spectrometry. A global analysis of proteins phosphorylated on tyrosine, serine, or threonine residues was performed using nanoflow liquid chromatography-tandem mass spectrometry (MS/MS). Human platelets were pretreated with tirofiban, a GPIIb/IIIa integrin inhibitor, in order to block the outside in signaling and limit the analysis to the initial signaling events prior to integrin activation. Platelets were then activated with 3 nM thrombin or both TFLLRN (50 μM) and AYPGKF (500 μM) for 3 min and compared to unstimulated platelets. The results showed the expected signaling pathways activated in both thrombin-stimulated and agonist peptide-stimulated platelets. Importantly we identified 51 proteins that were specifically phosphorylated in thrombin-stimulated platelets. Next we wanted to know if the specific phosphorylation sites identified play a role in the function of these proteins. We identified a single phosphorylation residue on Ser552 of β-catenin, a signaling component of the Wnt pathway, after stimulation with thrombin but not with TFLLRN+ AYPGKF. This phosphorylation is important for the stabilization of β-catenin. We also identified that Disabled-2 (Dab2) and protein kinase C beta (PKC-β) are phosphorylated after stimulation with TFLLRN+ AYPGKF, but not thrombin. Dab2 is a cargo-specific adaptor protein involved in endocytosis and cell signaling. The phosphorylation on Ser401 regulates function and localization of Dab2 whereas the phosphorylation on Thr500 and Thr642 regulates PKC-β function. Taken together, we have identified specific signaling pathways initiated by thrombin stimulation, since thrombin induces the formation of PAR1-PAR4 heterodimers, the signaling events identified in our study may be specific to PAR1-PAR4 heterodimers. In conclusion, the understanding of the molecular arrangement of PAR1 and PAR4 in platelets will provide insight for the development of specific antiplatelet therapies.

Disclosures:
No relevant conflicts of interest to declare.
Histone 3 Methyltransferase (EZH2) Inhibition Enhances TRAIL-Induced Apoptosis In Mantle Cell Lymphoma Cells By Accelerated cFLIP Degradation
Authors: Braun Frank K , Mathur Rohit , Sehgal Lalit , Berkova Zuzana , Samaniego Felipe 
Published: October 2019

Introduction
Mantle cell lymphoma (MCL) is an aggressive form of non-Hodgkin lymphoma that is characterized by the t(11:14)(q13:p32) translocation. MCL cells have altered cyclinD1 levels, impaired cell cycle regulation, DNA damage response, and likely defects in apoptosis signaling. Furthermore, up-regulated anti-apoptotic mediators such as the target of NF-κB c-FLIP were correlated with decreased apoptosis signaling. Also many cancer cells and malignant tumors show a prevalent resistance to apoptosis induction by TRAIL. Thus, by understanding the underpinnings of apoptosis resistance, we will be in a better position to develop strategies that improve TRAIL-induced killing of lymphoma cells.

Methods/Results
MCL cell lines (Mino, JeKo-1, JVM-2 and Z-138) were treated with DZNep (3-Deazaneplanocin A; 0.2-5µM) for 24 h followed by incubation with TRAIL (10-20ng/ml, 6-16h). Cell death, DNA fragmentation, and mitochondrial membrane potential (Δψm) were determined by calcein staining, subG1 analysis, and TMRM staining, respectively. Neither DZnep alone nor in combination with TRAIL showed a significant induction of necrosis as determined by LDH-release levels, but DZNep alone showed strong antiproliferative properties at higher concentrations (Promega CellTiter 96 assay). Activation of the caspase signaling cascade (caspase-8, -9, -3, Bid, and PARP cleavage) was analyzed by Western blotting. TRAIL-induced signaling was significantly increased and caspase-8 processing enhanced in DZNep pretreated cells indicating a regulation at the TRAIL/DISC. Although a reduced expression of DR5 in total cell lysates of DZNep treated cell was observed, the surface receptor levels were not altered. Interestingly, downregulation of the well-known caspase inhibitor, cFLIP, correlated with the DZNep-induced increased TRAIL sensitivity in MCL cell lines. However, it appears that cFLIP levels are not reduced due to blocked NF-kB signaling but rather by an accelerated ubiquitin-mediated degradation.

Conclusions
This study reveals that inhibition of histone methyltransferase (EZH2) activity by DZNep has a profound positive impact on TRAIL signaling; it enhances TRAIL sensitivity by promoting processing of caspase-8 through enhanced cFLIP degradation. The capacity of DZNep to target stability of cFLIP, which represents a center piece in DISC regulation underscores its potential for enhancing efficacy of TRAIL-based cancer therapies.

Disclosures:
No relevant conflicts of interest to declare.
Control Of Allograft Rejection With Mitomycin C Incubated Blood Cells
Authors: Greil Johann , Kleist Christian , Mohr Elisabeth , Sandra-Petrescu Flavius , Jiga Lucian , Dittmar Laura , Satagopam Venkata , Demirdizen Haydar , Hundemer Michael , Becker Luis , Lang Peter , Opelz Gerhard , Terness Peter 
Published: October 2019

Immunosuppressive treatment in allogeneic transplantation is an effective way to control allograft rejection. But unfortunately it can cause serious side effects, such as infections or malignancies. Cell therapeutic approaches for induction of specific tolerance are on the rise and may have fewer side effects. We observed that monocytes or peripheral blood mononuclear cells (PBMC) of healthy persons after culture with dendritic cell-inducing cytokines and differentiation blockade with the chemotherapeutic drug mitomycin C (MMC) show the ability to suppress allogeneic T cell responses. Interestingly these cells resemble morphologically as well as phenotypically myeloid-derived suppressor cells (MDSC) which occur in cancer patients impairing the anti-tumor immunity. FACS- and gene expression analyses of these MMC incubated blood cells revealed downregulation of the immunostimulatory cell surface molecules CD80, CD83, CD86 and HLA-DR, and upregulated transcription of immunosuppressive and pro-apoptotic genes such as iNOS, arginase-1, IL-10, COX2, TGF-β, placental growth factor, immunoglobulin-like transcript 3, adrenomedullin, SMAD5, LRDD and of the transcription factor C/EBPβ. For use in preclinical transplantation models in vitro culture of MMC treated donor-derived blood cells with dendritic-cell inducing cytokines was not applicable and was therefore omitted. The tolerance inducing properties of donor-derived MMC incubated blood cells were investigated in two different animal models of solid organ transplantation. When injected into rats, donor-derived PBMC incubated with MMC strongly prolonged heart allograft survival. Suppression of rejection was donor-specific. Recipients showed an increased number of CD4+CD25+FoxP3+ regulatory T-cells and tolerance could be transferred to syngeneic recipients with blood or spleen cells. Donor-derived MMC incubated blood cells also prolonged kidney allograft survival in pigs. Furthermore, in a first clinical use a 6-year-old girl with relapse of acute lymphoblastic leukemia and imminent rejection of a third haploidentical stem cell transplant received donor-derived CD3/CD19-depleted peripheral blood stem cells, which have been treated in vitro with MMC, in order to prohibit graft rejection. We describe a simple method for in vitro generation of tolerance inducing donor-derived blood cells for potential use in clinical organ transplantation.

Disclosures:
No relevant conflicts of interest to declare.
Allogeneic Compact Bone-Derived Mesenchymal Stem Cell Transplantation Attenuates The Severity Of Idiopathic Pneumonia Syndrome In a Murine Bone Marrow Transplantation Model
Authors: Qiao Shukai , Ren Hanyun , Shi Yongjin , Liu Wei , Yuan Jing 
Published: October 2019

Introduction
Idiopathic pneumonia syndrome (IPS) is a serious and life-threatening lung complication following allogeneic hematopoietic stem cell transplantation (allo- HSCT) and currently no effective therapies exist. The present study was designed to determine whether transplantation of allogeneic murine compact bone derived- mesenchymal stem cells (CB-MSCs) could prevent the development of IPS.

Methods
We tested the effects of allogeneic CB-MSCs transplantation on IPS using an established murine model of IPS, wherein lethally irradiated male BALB/c (H-2d) recipient mice received bone marrow and spleen cells from female C57BL/6(H-2b) donors. Survival rates, body weight change, clinical GVHD scores, lung histological changes and wet-dry lung ratios were assessed after IPS induction. Mechanistically, concentrations of cytokines (TNF-alpha, IFN-gamma and IL-4) and chemokines (CCL5, CXCL9 and CXCL10) in bronchoalveolar lavage fluid (BALF) from the recipient mice were measured at different time point post-transplantation. CD4+CD25+Foxp3+ regulatory T cell (Treg) percentage, CCR5, CXCR3 and CCR7 expression on CD3+ T cells, and lung CXCR3, CCR5, CCR7, T-bet and GATA-3 mRNA levels were also evaluated at different time point post-transplantation.

Results
In the IPS model, co-transplantation of CB-MSCs significantly attenuated the severity of lung injuries and increased the survival rate of mice compared to non-cotransplanted mice. Furthermore, a higher Treg percentages, reduced TNF-alpha, IFN-gamma, CCL5, CXCL9 and CXCL10 levels, CXCR3 and CCR5 down-regulation, as well as CCR7 up-regulation, were observed in MSCs co-transplantation mice. Additionally, the prophylactic effect of CB-MSCs was also associated with a shift of Th1/Th2 balance toward anti-inflammatory Th2 polarization.

Conclusions
Our present data suggested that allogeneic CB-MSCs attenuated the severity of IPS due to its profound immunomodulatory capacity in our murine model. This may offer a novel prophylactic approach for IPS patients after allo-HSCT.

Disclosures:
No relevant conflicts of interest to declare.