bluuebunny/crossref_metadata_2025_split huggingface dataset:
RUNX1 Mutations Are the Most Frequent Leukemia Associated Mutations in Congenital Neutropenia Patients
Authors: Skokowa Julia , Steinemann Doris , Makaryan Vahagn , Klimiankou Maksim , Schnittger Susanne , Kohlmann Alexander , Schlegelberger Brigitte , Zeidler Cornelia , Dale David C. , Welte Karl 
Published: October 2019

Abstract
Abstract 7
Congenital neutropenia is a rare inborn heritable disease with a 20% risk to develop acute myeloid leukemia (AML). One of the most frequently mutated genes in de novo as well as in secondary AML is RUNX1. The aim of our study was to investigate whether patients with congenital neutropenia who developed secondary AML (CN/AML) acquire mutations within the RUNX1 gene prior to the development of AML and whether RUNX1 mutations might be involved in leukemogenesis. Mutation analysis was performed using a sensitive next-generation amplicon deep-sequencing assay (454 Life Sciences, Branford, CT) with minimum coverage of at least 200-fold and confirmed by ABI Sanger sequencing. Using these methods we tested 22 patients with CN/AML for the presence or absence of RUNX1 mutations. Fourteen of the 22 patients (63.6%) demonstrated mutations within the RUNX1 gene. The mutation load was between 8 % and 78 % as assessed by the deep-sequencing read counts. Most of the mutations were detected within the Runt homology/DNA binding domain. Four patients have even two RUNX1 mutations. The RUNX1 mutations consisted of 11 missense mutations, 2 nonsense mutations, 4 frameshift mutations, and one ins/del mutation. Of the 14 patients with RUNX1 mutations 12 were tested positive for inherited congenital neutropenia causing mutations: 10 ELANE mutations, one HAX1 mutation, and one G6PT (SLC37A4) mutation. Cooperating mutations were detected in 8 patients, 7 with CSF3R mutations, one with FLT3-ITD mutation. Cytogenetic analysis of the 14 patients with RUNX1 mutations revealed that 4 patients had monosomy 7 and 3 patients trisomy 21. Sequential analysis of the time prior to overt AML revealed one patient harboring RUNX1 mutation already at birth and a second patient had a RUNX1 mutation 6 months prior to AML. The time course of acquisition of RUNX1 mutations in the remaining 12 patients is presently under investigation. The incidence of RUNX1 and CSF3R mutations in at least 7 patients clearly suggests that these two mutations cooperate in causing leukemia. In conclusion, these data show that patients suffering from congenital neutropenia frequently acquire mutations within the RUNX1 gene prior to development of leukemia.
We thank the Investigators of the Severe Chronic Neutropenia International Registry for supplying us with patient material and clinical data.

Disclosures:
Schnittger: MLL Munich Leukemia Laboratory: Equity Ownership. Dale:AMGEN: Consultancy.
PKCδ Negatively Regulates Primary Megakaryocyte Proliferation and Differentiation
Authors: Kunapuli Satya P. , Kostyak John C 
Published: October 2019

Abstract
Abstract 853
Megakaryocytes are large, polyploid cells that give rise to platelets in the bone marrow and spleen. Megakaryocytes achieve their size and DNA content through a process known as endomitosis via signaling from the cytokine thrombopoietin (Tpo). We have previously determined that the novel Protein Kinase C isoforms theta (PKCθ) and delta (PKCδ) regulate a number of platelet functions including aggregation and secretion. However, the function of these two PKC isoforms in primary megakaryopoiesis has not yet been elucidated. Therefore we chose to utilize primary mouse megakaryocytes from WT, PKCδ−/− and PKCθ−/− mice to characterize the roles of PKCδ and PKCq in megakaryopoiesis. We were first able to determine via western blotting that megakaryocytes express more PKCd than either mononuclear bone marrow cells (p < 0.05) or progenitor cells isolated from bone marrow (p < 0.05). Deletion of PKCδ in mice caused an increase in white blood cell and platelet counts compared to WT mice (p < 005). However, deletion of PKCθ had no effect on murine blood cell counts. Observed increases in platelet counts in PKCδ−/− mice are due to increased platelet production as PKCδ−/− mice contain more thiazole orange positive platelets than WT mice (p < 0.05). Furthermore, we determined via flow cytometry that PKCδ−/− mice had more bone marrow megakaryocytes than WT mice (p < 0.05), although megakaryocyte DNA content was unaltered. Conversely, there was no alteration in megakaryocyte number or DNA content with PKCθ deletion. Interestingly, the increase in bone marrow-derived megakaryocyte count observed in PKCδ−/− mice was heightened following culture of bone marrow cells in 50ng/mL exogenous Tpo (Figure 1). Furthermore, in similar experiments, megakaryocyte DNA content was also enhanced in PKCδ−/− mice compared to WT mice (p < 0.05). PKCδ is an important pro-apoptotic protein, and heightened megakaryocyte number following culture could be due to reduced apoptosis in PKCδ−/− megakaryocytes. However, neither apoptosis nor necrosis was altered with PKCδ deletion as the number of AnnexinV+/7AAD- cells, and the number of AnnexinV+/7AAD+ cells, were not different from WT. Therefore, the observed increases in megakaryocyte number and DNA content could be due to elevated Tpo-induced signaling as ERK1/2 phosphorylation was heightened in PKCδ−/− megakaryocytes compared to WT, in response to exogenous Tpo (Figure 2). These data suggest that PKCδ is an important megakaryopoietic protein, which negatively regulates signaling induced by Tpo in megakaryocytes, while PKCθ is dispensable for primary mouse megakaryopoiesis.

Disclosures:
No relevant conflicts of interest to declare.
Exclusion Criteria In The Dasision and Enestnd Trials: Which Could Their Impact Be On The Front-Line Treatment Of a “Real-life” Patient Population With Chronic Myelogenous Leukemia?
Authors: Latagliata Roberto , Carmosino Ida , Vozella Federico , Volpicelli Paola , Loglisci Giuseppina , Santopietro Michelina , Salaroli Adriano , Romano Angela , Montagna Chiara , Roberto Amanda , Serrao Alessandra , Diverio Daniela , Nanni Mauro , Mancini Marco , Tafuri Agostino , Breccia Massimo , Alimena Giuliana 
Published: October 2019

Abstract

The DASISION and ENESTnd controlled clinical trials have changed the front-line treatment of Chronic Myelogenous Leukemia (CML) leading to the advent of dasatinib and nilotinib in this setting: however, both these company-sponsored trials had many exclusion criteria, with a possible selection bias compared to the real-life CML population. To address the impact of these exclusion criteria on the 1st line treatment in the current clinical practice, we revised 207 unselected newly diagnosed chronic phase CML patients [M/F 108/99, median age 58.8 years, interquartile range (IR) 42.3 – 70.2] treated with front-line imatinib from June 2002 to June 2013 at our Institution and evaluated how many of them would have been excluded from enrolment in the 2 trials. Among these 207 patients, 28 patients should have been excluded by both trials due to polycomorbidities (12 cases), severe cardiopathy (5 cases), age > 80 with frailty (3 cases), drug abuse (2 cases), severe liver impairment, Rendu-Osler disease, active prostatic cancer, chronic obstructive broncopulmonar disease (COPD) + peripheral arterial obstructive disease (PAOD), COPD + arrhythmia, refusal to any marrow examination (1 case each). In addition, 8 patients should have been considered not eligible only for the DASISION due to isolated COPD and 19 patients should have been considered not eligible only for the ENESTnd due to isolated diabetes (10 cases), arrhythmia (4 cases), acute myocardial infarction > 6 months before CML diagnosis (2 cases), chronic pancreatic disease (2 cases), PAOD (1 case). On the whole, 36/207 patients (17.4%) would have been considered not eligible for the DASISION trial and 47/207 (22.7%) for the ENESTnd trial. As expected, these patients potentially not eligible for DASISION and ENESTnd were significantly older and with the imatinib treatment had a worse follow-up in terms of Complete Cytogenetic Response (CCyR), Major Molecular Response (MMolR) and Overall Survival (OS) compared to patients potentially eligible, as shown in the Table.
In conclusion, our data highlight that an important fraction of newly diagnosed patients in a real-life setting would have been excluded by the 2 controlled trials whose results are the current mainstay of the 1st line treatment in CML: thus, an automatic transposition of those results into the current clinical practice should be regarded with caution.

Disclosures:
No relevant conflicts of interest to declare.
Efficacy Analysis With Imatinib Monotherapy Was Superior To Allogeneic Hematopoietic Stem Transplantation In Pediatric Patients With Chronic Phase Of Chronic Myelogenous Leukemia
Authors: Jiang Hao , Liu Daihong , Xu Lanping , Liu Kaiyan , Chen Shanshan , Jiang Bin , Jiang Qian , Chen Huan , Chen Yuhong , Han Wei , Zhang Xiaohui , Qin Yazhen , Lai Yueyun , Liu Yanrong , Huang Xiaojun 
Published: October 2019

Abstract

Introduction
Whether allogeneic hematopoietic stem cell transplantation (allo-HSCT) or imatinib should be first-line therapy for childhood chronic myelogenous leukemia in the chronic phrase (CML-CP) is controversial. This study compared imatinib monotherapy and allo-HSCT for the management of CML-CP in pediatric patients.

Methods
This was a retrospective study of children (aged &lt;18 years) with CML-CP, treated between January 2006 and September 2012 at a hospital in China. Patients were treated with imatinib (260 mg/m2 body surface area) or allo-HSCT. Overall survival (OS), event-free survival (EFS) and adverse events (AEs) were compared.

Results
62 patients (40 males, 22 females) were enrolled: 41 received imatinib, and 21 received allo-HSCT. Median follow-up in the imatinib and allo-HSCT groups was 29 and 56 months, respectively. Imatinib was well tolerated. In the imatinib group, the cumulative complete cytogenetic response (CCyR) and major molecular response (MMR) at 24 months were 96.6% (95%CI, 93.3–99.9%) and 85.6% (95%CI, 78.5–92.7%), respectively; patients achieved CCyR and MMR at a median of 3 and 6 months, respectively. In the allo-HSCT group, allografts were from an HLA-matched sibling (n=3), an HLA mismatched/haploidentical familial donor (n=15) or an unrelated donor (n=3). Twelve patients (57.1%) developed acute graft-versus-host disease (grades 2–6 in 7 patients), and 5 deaths were reported. 5-year OS in the imatinib and allo-HSCT groups was 97.1±2.9% and 73.7±10.3% (P=0.032), respectively, while 5-year EFS was 92.5±4.2% and 63.8±11.1% (P=0.041), respectively.

Conclusion
Treatment with imatinib yielded satisfactory cytogenetic and molecular responses, and superior 5-year OS and EFS to allo-HSCT.

Disclosures:
No relevant conflicts of interest to declare.
Somatic and Germ-Line Molecular Characteristics Of Prefibrotic Myelofibrosis
Authors: Barosi Giovanni , Bonetti Elisa , Catarsi Paolo , Massa Margherita , Campanelli Rita , Poletto Valentina , Viarengo Gianluca , Villani Laura , Fois Gabriela , Biamonte Flavia , Rotunno Giada , Guglielmelli Paola , Magrini Umberto , Vannucchi Alessandro M , Rosti Vittorio 
Published: October 2019

Abstract
Prefibrotic myelofibrosis (pre-MF) is a prodromic variant of primary myelofibrosis (PMF) characterized by prevalence of female sex, young age, high frequency of splanchnic vein thrombosis and indolent disease course (Barosi et al, PLoS One. 2012;7(4):e35631). Here we report the molecular characteristics at diagnosis of 177 consecutive pre-MF patients included in the institutional data-base of PMF patients at the Center for the Study of Myelofibrosis in Pavia from January 2001 through July 2013. As compared with patients with fibrotic type-MF (bone marrow (BM) fibrosis= 1 to 3), the frequency of JAK2V617F mutation in pre-MF patients (BM fibrosis=0) was higher (69.2% vs. 60.9%; P=0.02) but the JAK2V617F allele burden was lower (39% vs. 50.6%; P=<0.001). Patients with pre-MF displayed MPL, EZH2 or ASXL1 mutation at a frequency of 2.6%, 0% and 0% respectively, lower than in fibrotic type-MF (frequency: 7.6%, 4%, 17.4%, respectively). Both 45/1 haplotype of the JAK2 gene (rs12343867), and A3669G polymorphism of the glucocorticoid receptor (GR)- rs6198, known susceptibility alleles for PMF, were over-represented in pre-MF with respect to the normal control population (minor allele frequency, 43.3% vs. 24%, and 22.5% vs. 18.2%, respectively; P<0.001 and P= 0.04, respectively). However, the frequency of the variants were not significantly different in pre-MF with respect to fibrotic type-MF (minor allele frequency, 43.4% vs. 46.9%, and 22.5% vs. 27.1%). In pre-MF JAK2V617F mutated patients, a significant correlation between the burden of the V67F allele and the frequency of 46/1 haplotype (rs 12343867) was documented. By dividing JAK2V617F mutated patients in quartiles of allele burden, the frequency of the minor C allele increased progressively from 0.29% to 0.71% (P<0.001, X2 test for trend). No association was ascertained between the burden of the V617F allele and G allele of CR A3669G polymorphism. In conclusion, pre-MF patients segregated preferentially with JAK2V617F mutated genotype with low allele burden. Pre-MF patients were characterized by an almost complete absence of the acquired somatic mutations (EZH2 and ASXL1) that complement the JAK2V617F mutation in the fibrotic type-MF, and that are known to be associated to poor survival and propensity to blast transformation. Pre-MF patients were associated with susceptibility alleles 46/1 and GR A3669G, and the strength of association was not different from that of the fibrotic type-MF patients.

Disclosures:
No relevant conflicts of interest to declare.
Graft Versus Leukemia With Little Graft Versus Host Disease? Long Term Follow-Up Of a Study Using Transduced Donor T-Cells For Donor Leukocyte Transfusion
Authors: Weissinger Eva M , Borchers Sylvia , Ogonek Justyna , Verma Kriti , Ehrlich Steve , Koenecke Christian , Schweier Patrick , Bonini Chiara , Ciceri Fabio , Dammann Elke , Luther Susanne , Krauter Jürgen , Stadler Michael , Ganser Arnold 
Published: October 2019

Abstract

Fourteen patients with myeloid leukemia (12 with acute and 2 with chronic myelogenous leukemia) were transplanted from their HLA-identical (n = 9) or haploidentical (n = 5) family donors with CD34-enriched stem cells (HSCT) without further immunosuppression. In order to induce a graft versus leukemia (GvL) effect and to investigate the possibility of controlling graft-versus-host disease (GvHD), the standard transplantation protocol was adapted to include transfusion of gene-modified donor T-cells after HSCT in 11 patients, 3 did not receive any T-cells.
Donor-T-cells were transduced with the replication-deficient retrovirus SFCMM-3 which expresses the herpes simplex thymidine kinase (HSV-Tk) as a suicide gene and the truncated low affinity nerve growth factor receptor (ΔLNGFR) for selection purposes. After transfusion, SFCMM-3 transduced T-cells were detectable in all 11 patients by PCR and FACS analyses immediately after transfusion and during the follow up period (range: 1.1-11 years). Two of 9 patients developed acute GvHD: one of the skin, grade 1, 56 days after transfusion of the transduced cells, the other grade II which was successfully treated with ganciclovir. Loss of bcr-abl gene expression was achieved in one patient after expansion of transduced cells. Donor chimerism was stabilized after transfusion of transduced cells in all patients treated. In one patient a cytomegalovirus-reactivation was treated by the transfusion of gene-modified donor T-cells also indicating effectiveness of treatment. To date, 5 patients have relapsed, and died, one after a second HSCT. Two of the patients without transduced T-cells died due to relapse and 7 patients are alive and well and in complete clinical remission.
Thus we have shown that transfusion of transduced T-cells is effective and safe in a long follow-up of 11 years post transfusion.

Disclosures:
No relevant conflicts of interest to declare.
Characterization Of Full-Length, Recombinant Amsh, USP9x, and USP1: Clinically-Relevant Deubiquitinases Involved In Hematological Malignancies
Authors: Russell Nate , Taylor Trevor , Parmar Kalindi , D'Andrea Alan D. , Brasher Bradley B , Melandri Francesco D 
Published: October 2019

Abstract

Attachment of polyubiquitin to substrate proteins generates important biological signaling cues that are inherent to the linkage type of the polyubiquitin chain. For example, K48-linked polyubiquitin chains result in proteasome-mediated degradation of proteins to which they are attached, whereas K63-linked polyubiquitin chains play roles in various intracellular signaling cascades. An important feature of protein ubiquitination is that it is reversible. Substrate-anchored chains may be edited or removed from proteins by highly specialized proteases called deubiquitinating enzymes (DUBs). There are approximately 90 DUBs identified in humans and many have been identified as potential druggable targets because of their involvement in hematological malignancies such as Fanconi Anemia, human follicular lymphomas and diffuse large B-cell lymphomas. DUB activity is regulated by a variety of cues including specificity for a protein substrate(s) to which polyubiquitin chains are conjugated; the presence of protein cofactor(s) that activate or inhibit DUB function; or preference for a specific polyubiquitin linkage type(s). Thus, understanding the mechanisms, regulation, and substrate preferences for deubiquitinases is of great interest, from both academic and clinical viewpoints. To help address these needs, we produced highly purified recombinant deubiquitinase enzymes to facilitate in vitro biochemical studies and drug-discovery efforts. Herein we report the initial characterization of the following, clinically-relevant enzymes:
In conclusion, we have investigated the in vitro substrate preferences and kinetic profiles of three deubiquitinases of clinical relevance. This data will be valuable in the design and analysis of assays used to identify small-molecule inhibitors of these highly specialized proteases.

Disclosures:
Russell: Boston Biochem Inc: Employment. Taylor:Boston Biochem Inc: Employment. Brasher:Boston Biochem Inc: Employment. Melandri:Boston Biochem Inc: Employment.
Genetic Variations In Immune Regulatory Genes and The Risk For Diffuse Large B-Cell Lymphoma
Authors: Kang Jeong-Eun , Kim In-Suk , Kim Nam-Hee , Kim Min Kyoung 
Published: October 2019

Abstract

Introduction
There is growing evidence linking genetic variations of immune regulation genes to non-Hodgkin lymphoma (NHL) etiology. In a large consortial study, it has been recently reported that an increased risk for NHL, especially the major lymphoma subtype diffuse large B-cell lymphoma (DLBCL), with genetic variations in immune regulatory genes that mediate inflammation and autoimmunity. To complement ongoing agnostic approaches for identifying susceptibility genes, we evaluated 57 genetic variations of 7 candidate genes, and their relation to risk for DLBCL in Koreans.

Methods
The case-control series consisted of 192 de novo DLBCL treated at five hospitals throughout Korea from August 2001 through August 2009 and 192 individuals from the population with age and gender matched healthy volunteers. The DNA samples were genotyped using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (Sequenom, Inc., San Diego, CA). We genotyped 11 haplotype-tagging SNPs (htSNPs) of spleen tyrosine kinase (STK), 9 htSNPs of Fc fragment of IgG, low affinity IIa, receptor (FCGR2A), 3 htSNPs of Fc fragment of IgG, low affinity IIIa, receptor (FCGR3A), 15 htSNPs of interferon regulatory factor 4 (IRF4), 9 htSNPs of interleukin 7 receptor (IL7R), 7 htSNPs of interleukin 10 (IL10), and 3 htSNPs of tumor necrosis factor (TNF) genes. We used logistic regression to evaluate the association between genotypes and haplotypes with DLBCL. For DLBCL subtypes, we conducted polytomous multivariate unconditional logistic regression (adjusted for sex, race, age). We calculated P-trends under the co-dominant model for each SNP. The haplotypes were reconstructed according to the genotyping data and the linkage disequilibrium status of these SNPs.

Results
We did not find significant associations between the htSNPs of FCGR2A, FCGR3A, IRF4, IL7R, and TNF genes and the risk of DLBCL. However, the minor allele heterozygotes of the rs3021094 htSNP of IL10 gene (P-trend = 0.028) and the rs2991216 htSNP of the SYK gene (P-trend = 0.035) showed an increased risk of DLBCL. On 10-million permutation testing, the haplotype including rs3021094 variant allele of IL10 gene was significantly associated with an increased risk of DLBCL (P = 0.001), however, the haplotype including rs2991216 variant allele of STK gene was statistically insignificant (P = 0.055).

Conclusions
This study presents several novel aspects of the genetic susceptibility to develop DLBCL. Although this study did not show statistically significant association between STK htSNPs and risk for DLBCL on 10-millinon permutation testing, we demonstrated that genetic variants and haplotypes of IL10 gene showed the significantly increased risk for DLBCL. Larger studies that focus on the role of the STK and IL10 genes will support to confirm causal variants to develop diffuse large B-cell lymphoma.

Disclosures:
No relevant conflicts of interest to declare.
Prognostic Impact Of Beta-2 Microglobulin In Patients With Non-Gastric Marginal Zone Lymphoma
Authors: Yoo Changhoon , Yoon Dok Hyun , Kim Shin , Huh Jooryung , Park Chan-Jeoung , Lee Sang-wook , Suh Cheolwon 
Published: October 2019

Abstract

Background
Although serum Beta-2 microglobulin (B2M) has been suggested as a prognostic factor for several hematologic malignancies, this was rarely investigated in marginal zone lymphoma (MZL).

Materials
Between January, 2000, and May, 2013, a total of 270 patients with non-gastric (NG)-MZL were identified from database of Asan Medical Center, Seoul, Korea. Among them, pretreatment baseline serum B2M was available in 204 patients. Progression-free survival (PFS) and overall survival (OS) were compared according to the level of B2M with cut-off value of 2.5 mg/L.

Results
Median age of study population was 51 year-old (range, 16-81) and 85 (42%) patients were male. Thirty (15%) patients had nodal MZL and 174 (85%) had extranodal MALToma. B2M ≥2.5 mg/L was related with more adverse clinical features, such as poor performance status, ≥2 extranodal sites, advanced stage (III-IV), anemia, elevated LDH, bone marrow invasion, and higher IPI risk group. In univariate analysis, serum B2M (<2.5 mg/L vs ≥2.5 mg/L) was significantly associated with PFS (p<0.001) and OS (p<0.001). In multivariate analysis including B2M and International Prognostic Index (IPI), serum B2M ≥2.5 mg/L was an independent adverse prognostic factor in terms of PFS (hazard ratio [HR]=3.7, 95% CI, 1.5-9.1; p=0.005) and OS (HR=7.5, 95% CI, 1.7-32.7; p=0.008). In analysis including Korean MZL Prognostic Index (MZLPI: nodal MZL, ECOG performance status ≥2 and advanced stage), B2MG ≥2.5 mg/L was also significant for PFS (HR=3.5, 95% CI, 1.5-7.7; p=0.003) and OS (HR=7.3, 95% CI 1.9-28.2; p=0.004).

Conclusion
In patients with NG-MZL, baseline serum B2M is a powerful prognostic factor for PFS and OS, independent of validated prognostic indexes, such as IPI and MZLPI.

Disclosures:
No relevant conflicts of interest to declare.