bluuebunny/crossref_metadata_2025_split huggingface dataset:
Targeted Next Generation Sequencing of the Five Hemochromatosis Genes in Italian Patients with Iron Overload and Non-Diagnostic First Level Genetic Test: A Pilot Study
Authors: Badar Sadaf , Busti Fabiana , Zamperin Giampiero , Ferrarini Alberto , Bozzini Paolo , Manna Daniele , Campostrini Natascia , De Matteis Giovanna , Delledonne Massimo , Girelli Domenico 
Published: October 2019

Abstract

Background and Aim: Molecular diagnosis of HFE-related hereditary hemochromatosis (HH) is typically made by searching for the C282Y and H63D mutations (first level genetic test). However, in the Mediterranean area up to one third of patients with HH phenotype do not have the “diagnostic” genotypes (C282Y homozygosity, or C282Y/H63D compound heterozygosity). This pilot study was designed to develop a “second level” next generation sequencing (NGS)-based test for rapid and simultaneous analysis of the five HH genes (HFE, HFE2, HAMP, TFR2 and SLC40A1).
Methodology: we studied 61 patients with relevant biochemical signs of iron overload (IO) and non-diagnostic first level genetic test suggesting a possible “non-HFE” HH. The five HH genes were captured by Halo-Plex™ technology, and then sequenced using aNGS platform (Illumina HiSeq 1000). Sequenced reads were aligned against human reference HG19 and analyzed by GoldenHelix™ software to annotate all the variants possibly involved in the disease.
Results: In IO patients a large number of new non-synonymous variants (according to bioinformatics tools based on publicly available databases including the 1000-genomes project) were found. Many of them were relatively frequent and detected also in controls, thus being considered likely “non-pathogenic”, unless clearly enriched in patients. On the other hand, some rare variants (i.e. limited to a single or very few individuals), particularly in SCL40A1, TFR2, and HFE, were found exclusively in patients, and could be considered “potentially pathogenic”.
Conclusions: The combination of the Halo-Plex™ approach with NGS platform and GoldenHelix™ algorithm appears a suitable approach for a better molecular characterization of patients with unexplained HH phenotype, and could represent a good option for second level genetic testing in referral centers. However, establishing the clinical relevance of NGS-detected “novel” genetic variants in a prevalently autosomal recessive disorder like HH remains a difficult task, requiring further functional studies and national/international collaborative efforts.

Disclosures
No relevant conflicts of interest to declare.
Clinical Impact of CD8+ Cells in Hodgkin Lymphoma Lymphadenopaties. Contribution of Flow Cytometry
Authors: Alonso Sara , Vidriales Maria Belen , Caballero Maria Dolores , Blanco Oscar , Galende J , Peñarrubia Maria Jesús , Zato Esther , Barez Abelardo , Davila Julio , Caballero Juan Carlos , Orfao Alberto , González Marcos , Garcia-Sanz Ramon 
Published: October 2019

Abstract
CONTEXT: Microenvironment of Hodgkin and Reed Sternberg(H-RS) cells is a current focus of interest to define risk and predict evolution of Hodgkin′s Lymphoma.
OBJECTIVES: To determine the role of infiltrating CD8+ cells, using flow cytometry (FCM), in the evolution of patients with HL.
DESIGN: Cell suspensions obtained after mechanical disintegration of ganglion biopsies of patients with newly diagnosed HL were analyzed using FCM. Definitive diagnosis was made by conventional histology and immunohistochemistry. Clinical data were collected from medical records. Statistical analysis was performed using SPSS 20.0 software.
SETTING: In the University Hospital of Salamanca, we consecutively analyzed by FCM all lymph nodes with suspected lymphoma at diagnosis. There was no selection bias when collecting patients, except for some cases with inadequate quality(insufficient cells).
PATIENTS OR OTHER PARTICIPANTS: From 1996(earliest available FCM data) to 2014, 104 of that samples had a definitive diagnosis of HL. Treatment depended on stage: a)early diagnosis received ABVD(x3) & local radiotherapy(RT) (20-30 Gy), b)advanced: ABVD(x6 to 8), plus RT in selected cases. Median follow-up was 10 years.
INTERVENTIONS :This was a retrospective observational study.
MAIN OUTCOMES MEASURES: Primary end points were overall survival(OS) and freedom from treatment failure(FFTF), considered from diagnosis to progression or relapse.
RESULTS: Most cells obtained were lymphocytes (Median17.1±86.9%) with a T/B/NK distribution of 72%/27%/1.6%(median) and predominance of CD4+ (median CD4:51%-CD8:12%). Median CD8+ cells(12%) was used to divide patients into 2 groups.FFTF was longer in patients with more than 12% of CD8+cells(93% vs. 71%, p=0.01). When analyzed separately patients with early/advanced disease, the clinical benefit remained in the group with advanced disease (p=0.006), whereas the statistical significance was lost in the group with early disease, possibly due to the excellent prognosis for those patients(FFTF 10 years > 95%). No differences were observed in the OS, because second line therapy was highly effective. In the multivariate analysis using Cox regression, advanced stage (HR=9.6 with 95% CI 1.2 to 73.9) and>12% CD8+ tumor infiltrating T-cells were independent variables(HR=0.26, 95% CI 0.07 to 0.9).
CONCLUSIONS: Increased number of CD8+ in the H-RSC microenvironment of HL is associated with better FFTF, particularly in the advanced-disease group. This should be considered as a new biomarker to identify high-risk patients.
Figure 1. Figure 1.

Disclosures
No relevant conflicts of interest to declare.
Glasgow Prognostic Score Is Superior to Other Inflammation-Based Scores in Predicting Survival of Diffuse Large B-Cell Lymphoma
Authors: Hao Xiaoxiao , Wei Yongqiang , Huang Fen , Wei Xiaolei , Zhang Yuankun , Feng Ru 
Published: October 2019

Abstract
Inflammation-based prognostic scores, such as the glasgow prognostic score (GPS), prognostic index(PI), prognostic nutritional index(PNI), neutrophil lymphocyte ratio(NLR), platelet lymphocyte ratio(PLR) was related to survival in many solid tumors. Recent study showed that GPS can be used to predict outcome in diffuse large B-cell lymphoma(DLBCL). However other inflammation related scores had not been reported in DLBCL, and it also remained unknown which of them was more useful to evaluate the survival in DLBCL. In this retrospective study, a number of 252 newly diagnosed and histologically proven DLBCL patients from January 2003 to December 2014 were included. An elevated GPS, PI, NLR, PNI and PLR were all associated with decreased overall survival(p=0.000, p=0.000, p=0.006, p=0.001 and p=0.001, respectively) and event-free survival (p=0.000, p=0.000, p=0.011, p=0.001 and p=0.009, respectively) in univariate analysis. Multivariate analysis indicated that GPS(RR=1.768, 95%CI=1.043-3.000, p =0.034) remained an independent prognostic predictor in DLBCL. The area under the curve of GPS (0.735, 95%CI=0.645-0.824) was greater than that of PI(0.710, 95%CI=0.621-0.799), PNI(0.600, 95%CI=0.517-0.683), NLR(0.572, 95%CI=0.503-0.642), and PLR(0.599, 95%CI=0.510-0.689) by Harrell's C-statistics. Especially in the DLBCL patients treated with R-CHOP, GPS also remained the most powerful inflammation-based prognostic score when comparing with PI, NLR, PNI and PLR (p=0.004, p=0.000, respectively for OS and EFS). In conclusion, these results indicate that Inflammation-based prognostic scores such as GPS, PI, NLR, PNI and PLR can be used to evaluate the outcome in DLBCL patients. Among them, GPS is the most powerful tool in predicting survival in DLBCL patients, even in the rituximab era.

Disclosures
No relevant conflicts of interest to declare.
Switch to Subsequent Line of Treatment in Children and Adolescents with Chronic Myeloid Leukemia (CML) Treated with Imatinib: Experience of the International Registry for Chronic Myeloid Leukemia in Children and Adolescents (I-CML-Ped Study)
Authors: Millot Frédéric , Guilhot Joelle , Suttorp Meinolf , Meunier Anne Sophie , Adalet Meral Gunes , Sedlacek Petr , de Bont Eveline , Li Chi Kong , Kalwak Krzysztof , Lausen Birgitte , Culic Srdjana , De Moerloose Barbara , Biondi Andrea , Baruchel Andre 
Published: October 2019

Abstract
Aims: To determine in children and adolescents with chronic myeloid leukemia (CML) in chronic phase (CP) treated with imatinib front line, (i) the probability of switch to a second line therapy, (ii) to characterize the reasons and the type of switch and (iii) to determine the impact of the switch on outcome.
Methods: Children and adolescents (<18 years) with CML diagnosed later than the year 2000 and enrolled in the international registry for childhood CML (I-CML-Ped Study) were the subjects of the study.
Results: The I-CML-Ped study enrolled 301 children and adolescents with CML in CP treated with imatinib front line. Among them 112 patients subsequently switched to a second line therapy (median duration of imatinib treatment before the switch: 16 months [range: 1-111]).The probability of switch at 38 months was 50% (95% CI: 29-60). Primary resistance was the cause of switch in 47% of the patients: failure to achieve complete hematologic response (CHR, 1%), complete cytogenetic response (CCR, 20%) or major molecular response (MMR, 24%); not detailed (2%). A loss of response (CHR loss [2%] or CCR loss [4%] or MMR loss [13%]) or progression were the cause of switch in 19% and 4% of the patients, respectively. Occurrence of non hematologic toxicity (mainly muscle-skeleton pain) was the cause of switch in 10% of the patients. The reason of switch was the physician's choice in 20% of the patients (switch to hematopoietic stem cell transplantation [HSCT] while the patients were in MMR or deeper molecular response). The second line therapy consisted of second generation tyrosine kinase inhibitors (63%), chemotherapy (4%) or HSCT (33%). With a median follow up of 38 months (range: 2-150), overall, 8 deaths were recorded among switching patients: all were patients transplanted for acute phase (4 patients), hematologic resistance (1 patient), loss of hematologic response (1 patient) or physician's choice (2 patients). The causes of death were treatment related mortality (7 patients) and relapse (1 patient). One death only was recorded among the non switching patients. The probability of overall survival at 48 months was 90% (95% CI: 81% - 95%) for switching patients and 98% (95% CI: 89% - 100%) (p=0.005) for the non switching patients.
Conclusion: Treatment failure is the main reason for a switch to a second line therapy in children and adolescents treated with imatinib front line. Efficacy of second line therapy still needs improvement.

Disclosures
No relevant conflicts of interest to declare.
Evaluating Frequency of Anti-Xa Monitoring in Treatment of Pediatric Venous Thromboembolism
Authors: Roberts Laura E , Fan Jennifer L , Lee-Kim YoungNa J , Shah Mona D 
Published: October 2019

Abstract
BACKGROUND/INTRODUCTION: With the rising incidence of pediatric venous thromboembolism (VTE), it is imperative that anticoagulation treatment protocols are standardized. While unfractionated heparin and warfarin were considered the standards of care, enoxaparin has now become the drug of choice in many pediatric institutions, given improved efficacy, fewer bleeding complications, and a more favorable pharmacokinetic profile. The increased bioavailability, predictable dose response, and less frequent drug monitoring have made enoxaparin popular in the adult population. In pediatrics, there are insufficient guidelines regarding frequency of enoxaparin monitoring. At Texas Children's Hospital (TCH), we titrate the enoxaparin dose during initial hospitalization to a therapeutic Anti-Xa level of 0.5 - 1.0 units/mL. Patients are instructed to return for outpatient follow-up levels within 9 days of discharge, and monthly thereafter. It is unclear whether this frequency of monitoring is optimal.
OBJECTIVES: In an effort to reduce overall patient morbidity and healthcare costs, we evaluated current Anti-Xa level monitoring practices in children with VTEs at TCH to develop recommendations for standardizing Anti-Xa level monitoring.
METHODS: Using the TCH DVT Registry, we retrospectively identified 107 subjects diagnosed with uncomplicated VTE and treated with enoxaparin. Demographic information, Anti-Xa levels, dose modifications, and outcomes were abstracted using the electronic medical record (Epic®) and analyzed.
RESULTS: 103 patients had therapeutic Anti-Xa levels at the time of hospital discharge. As instructed, 82 (80%) patients returned for follow-up within 9 days of discharge, with only 59 (72%) patients having therapeutic Anti-Xa levels. Of these 59 subjects, only 34 patients returned to clinic for their 1-month follow-up visit, of which 27 (80%) had therapeutic levels requiring no dose adjustments. Of the original 59 subjects, 29 patients returned for their 2-month follow-up, of which 26 (90%) patients continued to have therapeutic levels.
CONCLUSIONS: Initial therapeutic levels immediately post-discharge are critical to ensuring therapeutic dosing throughout the course of therapy. Once confirmed, the majority of subjects continued to have therapeutic levels at their 1-month (80%) and 2-month follow-ups (90%). Given the discomfort of laboratory testing, costs, and time to families, it may be reasonable to decrease the frequency of laboratory monitoring at these monthly visits.

Disclosures
Off Label Use: Enoxaparin for anticoagulation of pediatric patients with venous thromboembolism.
Enhancer Landscape Dynamics and the Role of IRF8 in Mononuclear Phagocyte Development
Authors: Kurotaki Daisuke , Nakabayashi Jun , Nishiyama Akira , Sasaki Haruka , Kaneko Naofumi , Koizumi Shin-ichi , Ozato Keiko , Suzuki Yutaka , Tamura Tomohiko 
Published: October 2019

Abstract
Monocytes and dendritic cells (DCs) are critical mononuclear phagocytes that regulate innate and adaptive immune responses. Hematopoietic stem cells give rise to monocytes and DCs via intermediate myeloid progenitor populations, such as granulocyte-monocyte progenitors (GMPs), monocyte-DC progenitors (MDPs), and common monocyte progenitors (cMoPs) or common DC progenitors (CDPs). However, the molecular mechanism underlying their lineage determination is poorly understood. Recently, promoter-distal enhancers have been found to be key for cell fate decision. In this study, we performed chromatin immunoprecipitation-sequencing (ChIP-seq) analysis of three histone modifications (H3K4me1, H3K27ac, and H3K4me3, representing primed enhancers, active enhancers, and transcriptional intiation, respectively) in seven myeloid populations (GMPs, MDPs, cMoPs, CDPs, monocytes, DCs, and neutrophils), and identified approximately sixty thousand putative enhancer regions. We found that a majority of monocyte- and DC-specific active enhancers were gradually established at progenitor stages prior to gene expression and terminal differentiation. Computational DNA motif analysis implicated that these enhancers were regulated by combinations of lineage-determining transcription factors such as PU.1, RUNX, C/EBP, and IRF. Indeed, ChIP-seq of PU.1 confirmed that the myeloid master regulator PU.1 was the common transcription factors bound to enhancers at all the stages examined. IRF binding motifs were enriched at the enhancer regions in MDPs, cMoPs and CDPs, but not GMPs. Among IRFs, IRF8, a partner of PU.1, has been shown to be highly expressed in MDPs and their descendants, and to be required for MDP-to-CDP and cMoP-to-monocyte transitions. Indeed, ChIP-seq analysis in Irf8-/- progenitors demonstrated that the enhancer landscapes of Irf8-/- GMPs, MDPs, and cMoPs all remained similar to that of wild-type GMPs. Moreover, ChIP-seq for IRF8 binding revealed that IRF8 directly promoted the priming and activation of many enhancers in MDPs and cMoPs. These results contribute to a comprehensive understanding of how transcription factors govern the enhancer landscape dynamics during mononuclear phagocyte development.

Disclosures
No relevant conflicts of interest to declare.
Molecular Classification of Bone Marrow Failure Syndromes: Protein Signatures As Surrogate Biomarker for Accurate Diagnosis of Severe Aplastic Anemia, Hypoplastic Myelodysplastic Syndrome and Paroxysmal Nocturnal Hemoglobinuria Patients
Authors: Alaiya Ayodele , Alzahrani Hazza A , Shinwari Zakia , Owaidah Tarek , Al Mohareb Fahad , Al Sharif Fahad , Al Nounou Randa , Chaudhri Naeem A. , Aldawsari Ghuzayel , Hanbali Amr , Shaheen Marwan , Alfraih Feras , Ahmed Syed Osman , Aljurf Mahmoud 
Published: October 2019

Abstract
Background/Purpose: Bone marrow failure syndrome is an example of disease entity where accurate diagnosis of Severe Aplastic Anemia (SAA), Paroxysmal Nocturnal Hemoglobinuria (PNH) and Hypoplastic Myelodysplastic Syndrome (MDS) is very challenging. The aim of this study was to identify panels of disease-specific /disease-associated proteins biomarkers to be used for more objective diagnosis and better prediction of disease prognosis of patients presenting with features of bone marrow failure syndromes.
Methodology:
Bone marrow plasma (MBP) and peripheral blood plasma (PBP) samples from 20 patients with bone marrow hypoplasia; including AA/MDS/PNH were subjected to expression proteome analysis using label-free quantitative liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS).
Results: Approximately 300 unique protein species were identified of which 107 and 218 were significantly differentially expressed (> 2- ∞- fold change & p < 0.05) in BMP and PBP respectively. These protein fingerprints independently discriminates patients into three distinct clusters; AA/MDS/PNH. Furthermore, only approx. 25% of the proteins were common between the two datasets from BMP and PBP. Some of the identified proteins were filtered and mapped using Ingenuity Pathway Analysis, and were associated with five different networks. The top two of these networks involved cell-to-cell signaling interaction, hematological system development and function, and immune cell trafficking. Only three of the differentially expressed proteins were uniquely expressed in SAA and MDS but absent in PNH, thus making these proteins potential biomarkers.
The probable diagnostic utility of these proteins would be validated in large archival clinical samples. Our data indicates the utility of multivariate analysis of quantitative proteome data as a means of discovery of disease related or disease specific biomarkers for bone marrow syndromes.
Conclusions:
We have identified protein signatures capable of objective classification of bone marrow failure syndromes patients. Our expression proteomics strategy is very promising for identification of clinically useful biomarkers. These proteins once validated, on a larger cohort of patients, might be valuable to complement the currently existing parameters for reliable and objective disease diagnosis, monitoring treatment response and clinical outcome of bone marrow failure syndrome patients.

Disclosures
Owaidah: King abdulaziz city for science, Novo Nordisk, Bayer: Honoraria, Research Funding.
PKR Inhibits Hematopoietic Stem Cell Differentiation
Authors: Bennett Richard L , Byrne Michael T. , Cheng Xiaodong , Patel Mohini , Patel Zeel , Asous Amalin , May W. Stratford 
Published: October 2019

Abstract
Recent work from our laboratory has demonstrated that increased expression and activation of the interferon-inducible, dsRNA-activated kinase (PKR) is associated with inferior survival for many hematologic malignancies including AML. Mice expressing a PKR transgene (TgPKR) specifically in hematopoietic cells develop a preleukemic/myelodysplastic syndrome (MDS)-like disease consisting of peripheral blood (PB) cytopenias and bone marrow (BM) dysplasia with an increased frequency of BM blasts. In addition, TgPKR mice have a reduced number of BM Lin-, Sca+, cKit+ hematopoietic stem and progenitor cells (HSPCs) and an increased percentage of these cells are quiescent (G0) compared to WT mice. New data now reveals that BM of TgPKR mice have a significantly reduced frequency of multipotent progenitor cells (MPPs) compared to wild type (WT) mice. Furthermore, MPPs isolated from BM of TgPKR mice display a partial block of the cell cycle in G0/G1 compared to MPPs from WT mice. In contrast, PKR knockout mice (PKRKO) have an increased number of HSPCs that are more frequently proliferating (S+G2/M) than WT mice. While the BM of PKRKO mice have a significantly increased frequency of MPPs there is a decreased frequency of both phenotypic LT- and ST-HSCs compared to either WT or TgPKR mice. Thus, in long term culture initiating cell (LTC-IC) assays BM of TgPKR mice has nearly double the colony forming capacity after >4 weeks in culture compared to BM of PKRKO mice, indicating that the TgPKR expressing mice have increased numbers of HSCs that are functional in vitro but not in vivo. Significantly, K562 cells with reduced PKR expression by siRNA knockdown display an increased rate and extent of PMA-induced megakaryocyte differentiation compared to control cells. Taken together these results suggest that increased PKR expression/activation as observed in high-risk MDS, AML and other hematologic malignancies may have a previously unrecognized function to block hematopoietic cell differentiation. This may account, at least in part, for why TgPKR mice accumulate quiescent HSPCs that are unable to terminally differentiate in vivo. Since we recently discovered that nuclear PKR inhibits DNA damage response signaling that results in an increased frequency of somatic mutations, we propose that increased PKR may contribute to leukemogenesis by promoting both genomic instability and a differentiation block in the HSPC populations.

Disclosures
No relevant conflicts of interest to declare.