bluuebunny/crossref_metadata_2025_split huggingface dataset:
Modulation of Leukotriene Signaling Affecting Cell Growth in Chronic Myeloid Leukemia: A Key Pathway to Cure?
Authors: Yektaei Elham , Nilsson Anders , Näsman-Glaser Barbro , Ekblom Marja , Qian Hong , Rådmark Olof , Zovko Ana , Kanter Lena , Wallvik Jonas , Stenke Leif 
Published: November 2019

Abstract

Background: Recent data suggest that tyrosine kinase inhibitor (TKI)-insensitive leukemic stem cells often prevail in chronic myeloid leukemia (CML) patients subjected to long-term TKI treatment. To achieve cure additional aberrant pathways in these patients, such as leukotriene (LT) signaling, may need to be targeted. We have previously shown that human CML cells have an increased capacity to produce LT and that LT can stimulate normal myelopoiesis (Tornhamre ExpHem 2003). Chen later noted a striking up-regulation of ALOX5/5LO (catalyzing an initial step in LT formation) in CML mice, and that inhibition of this enzyme improved survival of these animals by a magnitude similar to that induced by imatinib (Chen NatureGen 2009). Here wexve assessed the effect of LT modulating agents on the growth of human CML cells in vitro.
Materials and methods: Human CML cells, derived from the three cell lines K562, Kcl22 and KU812, were grown in microtitre plate cultures, in the presence of RPMI 1640. The MTT technique was used to evaluate the number of viable cells at 72 hours. Protein expression was assessed by Western blot and immunocytochemistry.
Results: Several specific modulators of LT-signaling were capable of inducing dose-dependent growth inhibitory effects on CML cells. Thus, the cysteinyl-LT receptor antagonist montelukast, a drug with approved clinical use in human asthma, was shown to reduce the growth of all three tested CML cell lines. In Fig. 1 this is examplified by K562 cells, where also additive effects between montelukast and imatinib are indicated. All cell lines expressed the cysLT1-receptor. Furthermore, the LTB4 receptor antagonist etalocib, as well as the 5-LO activating protein (FLAP) inhibitors licofelone and Bay-X-1005, also executed inhibitory effects at concentrations considered as physiological. As expected, addition of the TKIs imatinib, nilotinib or dasatinib to the cultures also generated dose-dependent inhibitory effects on the growth of all tested CML cell lines. An LD50 of approximately 0.5 µM, 10 nM and 0.1 nM was noted for imatinib, nilotinib and dasatinib, respectively, for K562 cells at 72 hours.
Conclusions: We demonstrate that several compounds, known to specifically inhibit leukotriene signaling by different mechanisms, were at clinically relevant concentrations capable of significantly suppressing the growth of human CML cells. Some of these compounds are already in clinical practice for non-hematological disorders. They could provide an additional therapeutic possibility in the quest to cure CML.
Figure 1. Figure 1.

Disclosures
No relevant conflicts of interest to declare.
BCR-ABL Fluctuation Analysis in Chronic Phase Chronic Myeloid Leukemia (CP-CML) Patients with MR 4.5
Authors: Pereira Mariana Pinto , Vanelli Tito , Gomes da Silva Eduardo , Dias Américo André , Burin Mariana M , Soares Tahiane , Silla Lucia , Daudt Liane , Fogliatto Laura 
Published: November 2019

Abstract

Introduction: Imatinibe, a target tyrosine kinase inhibitor, have revolutionized CP-CML treatment. Considering that it is being discussed imatinibe treatment interruption in patients with MR ≥ 4.5 lasting two years or more, we believe to be important to establish predictors of major molecular response (MMR) loss. Fluctuations in BCR-ABL transcripts are being advocated to be able to predict MMR loss, although the real impact of small fluctuations among patients with in MR ≥ 4.5 is uncertain.
Objective: Correlate BCR-ABL fluctuations and MMR loss in patients considered to be in MR ≥ 4.5, as well as to recognize possible factors associated with these small fluctuations.
Methods: We conducted a retrospective analysis in CP-CML patients receiving imatinibe (as first or second treatment line) that achieved MR ≥ 4.5 (defined as a 4.5 log reduction on an international scale) and surveyed for BCR-ABL fluctuations. We considered a fluctuation to be a rise of at least 0.5 log of BCR-ABL transcripts. Treatment interruptions were considered only when imatinibe was not taken for 15 or more consecutive days. The presence of comorbities was evaluated by applying the Charlson Index.
Results: Fifty-eight patients were evaluated between with a median follow-up 7 years. Fifty-five percent were men and 28 were women, with a median age of 46 (18 - 93) years-old, most of the patients were older than 60 yo (75,9%). Among 32 patients evaluated by the Sokal risk score 97,5% were estimated to be low or intermediate. Twenty six patients presented at least one fluctuation. Regarding the number of fluctuations per patient, 18 presented only 1 fluctuation, 5 presented 2 fluctuations and 3 patients presented 3 fluctuations. Among those patients with at least one fluctuation 3 (11%) presented MMR loss, while there was none among patients without documented fluctuations (p=0,15). Among these three patients considered to have MMR loss, two presented fluctuations that reached MR 3.0, while the other patient presented a history of long treatment suspension. More patients that interrupted treatment presented BCR-ABL fluctuations, although this difference was not statically significant (78% vs 50% p=0,11).
Conclusion: Small fluctuations among patients with MR ≥ 4.5 didn't seem to correlate with MMR loss, however we do believe that our population is underpowered to rule out this difference. We considered that it very interesting that patients which reached MR 3.0 presented MMR loss (considering that the other patient had to abandon treatment during pregnancy).

Disclosures
No relevant conflicts of interest to declare.
Predictive Factors of Treatment Response to Hypomethylating Agents in Patients with Myelodysplastic Syndrome
Authors: Wu Ping , Du Xin , Weng Jianyu , Deng Chengxin , Geng Suxia , Lu Zesheng , Lai Peilong , Huang Xin , Luo Chengwei 
Published: November 2019

Abstract
Background: Hypomethylating agents (HMAs), including decitabine and azacitidine, have improved outcomes for patients with myelodysplastic syndrome (MDS). However, not all patients benefit from this therapy, and no reliable prognostic tool can predict differential likelihood of benefit from HMAs.
Methods: To investigate predictive factors of treatment response to HMAs in patients with MDS, we analyzed the baseline biomarkers, clinical variables and TP53 mutations of 52 newly diagnosed Chinese patients with MDS who received decitabine or azacitidine (1-23 cycles, median number cycles: 7)
Results: In the 46 assessable patients who received more than 2 cycles of standard treatment, a total of 22 patients had an overall response after treatment, and 24 patents showed no response. The results showed that there was no significant difference in response by treatment regimen (P=0.147) or source of sample (P=0.413). Furthermore, age (P=0.382), sex (P=0.595), IPSS risk groups (P=0.117) and cytogenetic abnormalities (P=0.935) were not associated with response rate. However, in univariate analysis of the overall cohort (n=40) who received more than 3 cycles of standard treatment, the response rate was significantly higher for those patients who had platelet count doubling after 2 cycle of treatment (P=0.013), but there was no statistically difference after 1 cycle(P=0.376). Moreover, a total of 5 patients (11%) had TP53 mutations, overall response rate to HMAs was 100% in patients with TP53 mutation, which is significantly higher than that of patients with wild type cases (p = 0.013).
Conclusions: These findings indicate that patients with TP53 mutation or achieving platelet count doubling after 2 cycle of HMAs respond well.

Disclosures
No relevant conflicts of interest to declare.
A Rapid, Sensitive and Specific Method for Quantifying Calr Mutant Allele Burden in Persons with Myeloproliferative Neoplasms
Authors: Zhou Jiao , Yao Qiumei , Gale Robert Peter , Li Jinlan , Li Lingdi , Li Ning , Chen Shanshan , Ruan Guorui 
Published: November 2019

Abstract
Background: CALR mutations were recently identified in a substantial proportion of persons with essential thrombocythemia (ET) and with primary myelofibrosis (PMF) without JAK2V617F. Consequently rapid, sensitive and specific methods to detect and quantify these mutations are needed.
Methods: We studied samples from 1088 persons with myeloproliferative neoplasms (MPNs) including 421 JAK2V617F negative subjects with ET, PMF, polycythemia vera (PV), chronic myeloid leukemia (CML) and hyper-eosinophilic syndrome (HES). Detection of CALR exon 9 mutations was done by PCR amplification followed by fragment length analysis and direct sequencing. Dilution assays were used to determine CALR mutant allele burden.
Results: We detected CALR mutations in blood and bone marrow samples from 152 subjects with ET and with PMF but not in samples from normal or persons with PV, CML or HES. CALR mutant peaks were distinct from wild-type peaks and dilution experiments indicated a sensitivity level of 0.5-5% for a CALR mutant allele in a wild-type background. Diverse types of mutations were detected including deletions, insertions and complex indels. All mutations were confirmed by direct sequencing. We also used dilution experiments to quantify mutant allele burden. We were able to reproducibly detect mutant allele levels as low 5% (0.5-5%) in a wild-type background.
Conclusions: PCR amplification followed by fragment length analysis is a rapid, sensitive and specific method for screening persons with MPNs for CALR mutations, especially those with ET and PV with JAK2V617F and for estimating mutant allele burden.
Figure 1. Standard curve for the detection of mutant allele burden. Figure 1. Standard curve for the detection of mutant allele burden. Figure 2. Titration analyses of sensitivity of CALR mutation screening by sequencing and fragment analyses. Figure 2. Titration analyses of sensitivity of CALR mutation screening by sequencing and fragment analyses. Figure 3. Figure 3 Sequencing traces show heterozygous mutation of CALR. Gene scan electropherogram from PCR method and partial sequence of CALR exon 9 from sequencing method (numbering according to GenBank access number: NC_000019.9). A-P: Detected a wild type and 15 CALR mutation types by sequencing and fragment analysis methods. A: wild type, B-I: deletions, H-L: insertions, M-P: complex indels. Figure 3. Figure 3 Sequencing traces show heterozygous mutation of CALR. Gene scan electropherogram from PCR method and partial sequence of CALR exon 9 from sequencing method (numbering according to GenBank access number: NC_000019.9). A-P: Detected a wild type and 15 CALR mutation types by sequencing and fragment analysis methods. A: wild type, B-I: deletions, H-L: insertions, M-P: complex indels.

Disclosures
No relevant conflicts of interest to declare.
GDF11 Increased in Patients with Myelodysplastic Syndrome
Authors: Han Yu , Wang Huaquan , Shao Zonghong 
Published: November 2019

Abstract
Objective To analyze the concentration of growth differentiation factor 11(GDF11) in peripheral blood of patients with myelodysplastic syndrome (MDS), so as to evaluate the relationships between these changes and erythropoiesis functions and to explore the role of GDF11 in the pathogenesis of MDS.
Methods The concentration of GDF 11 in peripheral blood was detected by enzyme-linked immuno sorbent assay in 44 MDS patients and 10 normal controls from September 2014 to June 2015 at our hospital. The percentage of nucleated erythrocyte (CD235a) in bone marrow was detected by flow cytometry. The correlation between these changes and erythropoiesis functions, including red blood cell count, hemoglobin, reticulocyte (RET%), hematokrit (Hct), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular-hemoglobin concentration (MCHC) and late erythroblast in bone marrow were evaluated.
Results (1)The concentration of GDF11(128.67±47.62)in high-risk MDS patients was significantly higher than that of low-risk MDS patients (65.96±36.55,p<0.01)and higher than that of normal controls (29.76±10.10,p<0.01); The concentration of GDF11 in low-risk MDS patients was significantly higher than that of normal controls (p<0.05). (2) The expression of CD235a in high-risk group(38.49±5.42)was not different with that in low-risk group(42.64±7.36, p>0.05). (3)In high-risk MDS patients, the expression of GDF11 was negatively correlated with Hb, RET%, RBC, MCHC, Hct in peripheral blood and late erythroblast, CD235a+ cells in bone marrow(r=-0.437,r=-0.428,r=-0.444,r=-0.553,r=-0.661,r=-0.436,r=-0.52,all p<0.05),and the expression of GDF11 was positively correlated with MCV(r=0.52, p <0.05),but it was not correlated with MCH (p >0.05).(4) In low-risk MDS patients, the expression of GDF11 was negatively correlated with Hb, RET% (r=-0.491Ar=-0.606,both p<0.05),it was not correlated with RBC, MCHC, MCV, MCH, Hct, late erythroblast and CD235a+ cells (all p>0.05).
Conclusion GDF11 increased in patients with MDS and it was negatively correlated with late erythropoiesis.

Disclosures
No relevant conflicts of interest to declare.
IGF2BP1 Protein Binding of Erythroblast Transcription Factor mRNA: A Cytoplasm-Localized Mechanism for Globin Gene Regulation
Authors: de Vasconcellos Jaira F. , Lee Y. Terry , Byrnes Colleen , Clarke Benjamin A. , Xu Pauline C. , Rabel Antoinette , Proia Richard L. , Miller Jeffery L. 
Published: November 2019

Abstract
Pharmacologic and genetic regulation of fetal hemoglobin (HbF) remains a major goal for treatment of the beta-thalassemias and sickle cell disease. Recently, the let-7 microRNA heterochronic pathway of RNA-binding factors was shown to be developmentally correlated with HbF expression in humans. Experimental manipulation of the pathway components (LIN28, let-7, and the IGF2 binding proteins) demonstrated moderate increases in fetal hemoglobin (around 15-35% HbF) with the exception of IGF2BP1, which caused a nearly complete reversal in gamma- and beta-globin gene expression resulting in HbF levels of 60-70% among cultured adult human erythrocytes. To further explore IGF2BP1 effects on HbF and erythropoiesis, lentiviral transduction of CD34(+) cells was performed with let-7 resistant expression of IGF2BP1 driven by the human SPTA1 gene (IGF2BP1-OE). Donor-matched control transductions were studied for comparison. For protein localization studies, confocal imaging of sorted cells was utilized. IGF2BP1-OE caused IGF2BP1 protein expression throughout the cytoplasm and localized to small granules. Granules were unevenly distributed and more abundantly observed in the perinuclear regions. Nuclear detection of IGF2BP1 protein remained at background control levels. We thus hypothesized that IGF2BP1 protein in these cytoplasmic granules may regulate globin gene expression by binding RNAs that encode epigenetic modifiers of the beta-globin locus. To test this hypothesis, RNA-immunoprecipitation (RIP) was performed using a RIP-certified anti-IGF2BP1 antibody. Next generation sequencing and qRT-PCR were used to determine the mRNA species bound by IGF2BP1. RIP-enrichment of BCL11A mRNA was identified (4.6 ± 1.2 fold compared to input sample), as well as mRNA encoding other modifiers of globin gene expression including KLF1 and ZBTB7A. Further analyses of BCL11A showed no significant changes in BCL11A mRNA levels among IGF2BP1-OE cells (control: 6.5.E+03 ± 3.8.E+03, IGF2BP1-OE: 4.1.E+03 ± 1.2.E+02, p=0.403). However, BCL11A protein detection was reduced to background levels by Western analysis compared to transduction control cell lysates. These studies suggest that RNA-binding and cytoplasmic compartmentalization provide IGF2BP1 with a mechanism for increasing fetal hemoglobin via post-transcriptional regulation of globin gene transcription factors.

Disclosures
No relevant conflicts of interest to declare.
Intravascular Large B Cell Lymphoma in the United States (US): A Population- Based Study Using Surveillance, Epidemiology, and End Results Program and National Cancer Data Base
Authors: Rajyaguru Devalkumar J , Bhaskar Chaithanya , Borgert Andrew J , Smith Angela , Parsons Benjamin 
Published: November 2019

Abstract
Background: Intravascular large B cell lymphoma (IVLBCL) is a rare and aggressive non-Hodgkin lymphoma lacking any prior US based population studies. We aimed to conduct a population based study of IVLBCL in the US and compare characteristics and survival to diffuse large B cell lymphoma, not otherwise specified (DLBCL, NOS).
Methods: We used Surveillance, Epidemiology, and End Results (SEER) registries from 2000 to 2013 to calculate incidence rates (case/1,000,000) and age-adjusted to the US 2000 standard population. We collected patient level data from the NCDB Participant User Files using ICD-O-3 histology code 9680 with ICD-O-3 primary site modifier C499 and analyzed for demographic and clinical characteristics as well as overall survival (2004-2013). Chi-Square test was used to compare the characteristics of patients with IVLBCL to that of DLBCL, NOS. Overall survival analysis was performed using Kaplan- Meier method and compared by log rank. Overall survival (OS) of IVLBCL was compared to propensity-matched cohort of DLBCL, NOS. Multivariate analysis was conducted using weighted Cox Proportional Hazard regression model.
Results: In the years 2000-2013, the overall crude incidence rate of IVLBCL was 0.093 (case/1,000,000) and age-adjusted incidence rate of IVLBCL was 0.095 (case/1,000,000). A total of 388 IVLBCL patients were identified in NCDB database. Patients with IVLBCL (n=388), compared to DLBCL, NOS (n=155,436), were more likely to be of age ≥ 65 (64.2% vs 56.9%, p=0.0039), female (51.8% vs 46.5, p=0.036), diagnosed at advanced stage (stage IV, 51.5% vs 33.4%, p<0.0001) and less likely to receive chemotherapy (70.4% vs 78.9%, p = 0.0001).The median 1, 3 and 5-year OS rates for IVLBCL were 66.2%, 51.8% and 46.3% respectively. After propensity matching, the 5-year overall survival of IVLBCL was comparable to DLBCL, NOS (46.6% vs 45.4%, p=0.61). On multivariate analysis, advanced age (HR 1.03, 95% CI 1.032-1.034; p<0.0001), advanced stage (Stage III: HR 1.34, 95% CI 1.31-1.38; <0.0001, Stage IV: HR 1.81, 95% CI 1.77-1.85; p<0.0001), having Medicaid (HR 1.72, 95% CI 1.66-1.79; p<0.001) or Medicare (HR 1.24, 95% CI 1.20-1.26; p<0.0001) predicted worse OS, whereas female gender (HR 0.86, 95% CI 0.85-0.88; p<0.0001) and use of radiation therapy (HR 0.76, 95% CI 0.75-0.78; p<0.0001) predicted better OS.
Conclusions: This is the largest series of IVLBCL reported to date. Our study provides characteristics and OS of a large cohort of this rare entity, which can be used for patient education and may provide background information for future research. In the Rituximab era, the OS of patients with IVLBCL is comparable to DLBCL, NOS. In addition, our study also reports predictors of OS in patients with IVLBCL.

Disclosures
No relevant conflicts of interest to declare.
An Essential Erythroid-Specific Enhancer of ATP2B4 Associated with Red Blood Cell Traits and Malaria Susceptibility
Authors: Stern Emily N , Lessard Samuel , Schupp Patrick G. , Sher Falak , Lettre Guillaume , Bauer Daniel E. 
Published: November 2019

Abstract
Genome-wide association studies (GWASs) have identified thousands of common genetic variants associated with human traits and disease susceptibility. Given that the majority of GWAS-identified SNPs are located within non-coding regions of the genome, the mechanisms of these trait associations are frequently unknown. Here we investigate the biological underpinnings of common trait-associated genetic variation at ATP2B4, a gene on chromosome 1q32 that encodes a major plasma membrane calcium ATPase (also known as PMCA4). Genetic variation within this locus is associated with several phenotypes including various red blood cell traits (mean corpuscular hemoglobin concentration and red cell distribution width) as well as susceptibility to severe malaria infection. We conducted an expression quantitative trait loci (eQTL) mapping analysis in human erythroblasts and identified a set of SNPs associated with ATP2B4 gene expression in cis. These included the same genetic variants found by GWAS to be associated with RBC traits and malaria susceptibility. Furthermore, these SNPs overlap an intronic erythroid DNase I hypersensitive site at ATP2B4. An analysis of the ENCODE database showed that this element was erythroid specific in that it lacked DNase I hypersensitivity in >30 queried non-erythroid cell types. We used the CRISPR/Cas9 genome editing system to delete this element in HUDEP-2 immortalized human erythroid precursor cells. We observed that cells bearing a 927 bp biallelic deletion of this noncoding element displayed near complete loss of ATP2B4 expression (3% residual gene expression), while cells bearing heterozygous deletions showed an intermediate gene expression phenotype. We identified a core element that encompassed 3 of the highly trait associated SNPs and 5 GATA1-binding motifs. Biallelic deletion of this 98 bp core led to 83% reduction in ATP2B4 expression. Disruption of individual GATA1-binding motifs resulted in partial reduction of gene expression, suggesting the contribution of multiple binding sites to appropriate gene expression. Overall, this study suggests that variation within an essential erythroid-specific enhancer of ATP2B4 underlies the association of ATP2B4 with RBC traits and malaria susceptibility. Furthermore these results encourage combined analyses of gene expression, chromatin state, and prospective genetic perturbation as a means to explore the variants, elements and genes responsible for heritable blood phenotypes.

Disclosures
No relevant conflicts of interest to declare.