bluuebunny/crossref_metadata_2025 huggingface dataset:
PML Mutation in PML-Rarα Alters PML Nuclear Body Organization and Induces ATRA Resistance in Acute Promyelocytic Leukemia
Authors: Zhu Hong-Hu , Wu Congying , Sun Yujie , Hu Jiong
Published: October 2019

Acute promyelocytic leukemia (APL) has become a highly curable disease using target drugs including all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) with or without chemotherapy. However, APL patients with acquiredPML mutationsin PML-RARα had dismal outcomes and showed resistance not only to ATO but also to ATRA.In this study, we confirmed in the clinic that APL patients with mutations in the PML moiety of PML-RARα showed different resistanceto ATRA (cross-resistance). We demonstrated that critical mutations are responsible for this "cross-resistance". Super-resolution microscopy and electron microcopy were employed to examine the fine structural changes of PML/PML-RARα nuclear bodies (NBs) in response to therapy. We observed drug-induced acceleration of NB movement, which was absent in the mutant cells. Furthermore, NB doublets were revealed, and fusion events were captured upon drug treatment. We proposed that SUMOylation contributed to increased NB dynamics, leading to more fusion incidents and thustaking functional NBs for oncoprotein degradation. Different protein density in the mutant NBswas identified by single molecule quantification, indicating reduced permeability and recruitment of clients. Taken together, our results revealed that critical sites in the PML moiety of the PML-RARα fusion gene disrupted NB scaffold organization and perturbed fusion protein degradation in response to ATRA.Our work will largely improve the mechanistic understandings of APL pathology and therapy and will shed new light on unveiling the structure and function of PML NBs.

Disclosures

No relevant conflicts of interest to declare.
The Lysine Acetyltransferase Tip60 Is Required for Hematopoietic Stem Cell Maintenance
Authors: Numata Akihiko , Kwok Hui Si , Zhou Qi-Ling , Li Jia , Hannah Rebecca , Park Jihye , Angarcia Vladimir Espinosa , Gottgens Berthold , Yang Henry , Lough John , Bararia Deepak , Tenen Daniel G.
Published: October 2019

Hematopoietic stem cells (HSCs) are maintained for their two defining properties, a self-renewal and a multi-lineage differentiation ability, under various epigenetic processes. One of the epigenetic processes essential for HSC is a lysine acetylation, which was demonstrated in several previous studies utilizing knockout mice of a gene encoding a lysine acetyltransferase (KAT), such as Cbp/p300, Moz, or Mof. The lysine acetyl transferase 5, Kat5 (also called Tip60), is a member of MYST KAT family, defined by a protein containing a C2HC-type zinc finger and an acetyl-CoA binding domain, consisting of five members: Tip60, Moz, Morf, Hbo1, and Mof. Tip60 regulates gene transcription, genome stability, cell growth and apoptosis and has been demonstrated to be essential for embryonic development.

To assess the role of Tip60 in murine HSC, we generated Tip60 conditional knockout mice in two different Cre strains (Mx1-Cre and Vav-iCre), wherein Tip60 gene was successfully deleted in HSCs. Tip60 abrogation induced embryonic and adult lethality due to hematopoietic failure. Remarkably, HSCs were rapidly depleted upon Tip60 disruption, exhibiting robust apoptosis, aberrant cell cycle progression, and accumulated DNA damages. Tip60Δ/Δ fetal LSK cells transduced with retroviruses expressing wild-type Tip60 recovered a long-term hematopoiesis in transplantation experiments, whereas acetyl transferase defective Tip60 did not rescue the reconstitution to any detectable level, suggesting that the Tip60 acetyltransferase activity is essential for HSC function. Gene set enrichment analysis in RNA-seq demonstrated that Tip60 was significantly associated with genes involved in cell-cycle and DNA repair process, and Myc transcriptional factors target gene sets. A genome-wide chromatin profiling corroborated these findings, uncovering a strong similarity between Tip60 and c-Myc binding genomic regions. Considering similarities of HSC phenotype between our Tip60 conditional knockout mice and c-myc and N-myc double knockout mice (Laurenti, E. et al., Cell Stem Cell 2008), Tip60 maintains HSC by regulating Myc target genes.

We next evaluated alteration in histone modifications evoked by Tip60 deletion, performing ChIP-seq analysis in Tip60f/f; Rosa26-CreERT2 and Tip60f/f fetal c-Kit+ cells that were treated with 4-OHT. Intriguingly, we found that acetylated H2A.Z was enriched at the Tip60-bound active chromatin and Tip60 deletion reduced the acetylation level of H2A.Z at Myc target genes promoters, whereas neither H3K27 acetylation level, active promoter / enhancer mark, nor H3K27 tri-methylation level, repressive mark, were altered. Collectively, our results demonstrate that Tip60 - H2A.Z could be an epigenetic axis critical for active transcription of Myc target genes to maintain murine HSC.

Disclosures

No relevant conflicts of interest to declare.
Decreased Factor V Ameliorates Bleeding Diathesis in Patients with Combined Deficiency of Factor V and Factor VIII
Authors: Shao Yanyan , WU Wenman , Xu Guanqun , Xuefeng Wang , Qiulan Ding
Published: October 2019

Combined factor V (FV) and factor VIII (FVIII) deficiency (F5F8D) is a rare autosomal recessive bleeding disorder caused by mutations in LMAN1 and MCFD2 gene. Though characterized with deficiencies of both FV and FVIII, it failed to lead to more severe bleeding than single defect of FVIII in similar degree. Our study on F5F8D patients revealed that mild decrease of FV and FVIII had counteractive effect on blood coagulation. Out of 5 subjects investigated, three showed potent thrombin generation as observed in thrombin generation assay (TGA), with peak heights ranging from 138 nM to 166 nM, compared with 81 nM in normal plasma when coagulation was initiated with 1pM TF. The same trend was seen when the TF concentration was increased to 5 pM.

Supplement of FV and FVIII, however, demonstrated distinct impact in TGA, with FVIII addition inducing more thrombin generation whereas higher FV level failed to further boost thrombin generation despite the shortened lag time.

FV is a Janus-faced protein and suppresses blood coagulation by protecting TFPI from degradation.Similar to patients with severe FV deficiency, both total and free TFPI levels also decreased slightly in F5F8D patients. Total TFPI levels decreased from healthy donors (65.95±8.35 ng/mL) to F5F8D patients (65.79±12.99 ng/mL), to severe FV-deficient patients (51.30±11.15 ng/mL, P=0.037). A more pronounced trend was observed for free TFPI levels, as free TFPI in healthy controls were 12.68±2.91 ng/mL, higher than those in F5F8D (4.73±1.31 ng/mL, P=0.004), and in severe FV-deficient patients (3.81±1.03 ng/mL, P=0.004).The lowered TFPI in patients with F5F8D may neutralize the impact of FV and FVIII deficiency on blood coagulation.

Desmopressin (DDAVP) increases endogenous FVIII and is used in the management of mild hemophilia A patients. In current study, 4 patients with F5F8D were treated with DDAVP. The administration of DDAVP saw no elevation of FV yet the increase of FVIII. The mean basal FV and FVIII levels were 11.5±3.4% and 15.1±4.8% respectively. The peak value of FVIII levels rose to 66.1±20.5% 30 minutes after the administration, and was sustained at two-folds of the baseline four hours post administration.

The distinctive changes brought by combined FV and FVIII deficiencies on blood coagulation functions indicates the lone FVIII replacement without FV supplement might be sufficient in bleeding management of patients with F5F8D. DDAVP can be considered as a potential substitute for FVIII concentrate in the treatment of F5F8D patients.

Disclosures

No relevant conflicts of interest to declare.
Prophylaxis of Trimethoprim-Sulfamethoxazole for Pneumocystis Pneumonia in Diffuse Large B-Cell Lymphoma Treated with R-CHOP Therapy: A Single Center Experience
Authors: Ha Joo Young , Lee Kyoungmin , Hong Jung Yong , Yoon Dok Hyun , Kim Sung-Han , Jang Seongsoo , Park Chan-Sik , Huh Jooryung , Ryu Jin-Sook , Lee Sang-wook , Suh Cheolwon
Published: October 2019

Background: Recent studies reported increased risk of Pneumocystis pneumonia (PCP) in patients treated with rituximab-containing chemotherapy and its incidence ranged from 2% up to 13%. However, there are no standard guidelines for PCP prophylaxis in patients with diffuse large B-cell lymphoma (DLBCL). Herein, we investigated the efficacy and safety of trimethoprim/sulfamethoxazole (TMP/SMX) as primary prophylaxis for PCP in patients with DLBCL treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone (R-CHOP).

Method: We retrospectively review the patients with DLBCL who received R-CHOP every 21 days (R-CHOP-21) and received PCP prophylaxis with daily single-strength TMP/SMX (80mg/400mg) in Asan Medical Cencer, Seoul, Korea. We included patients only who completed at least 4 cycles of R-CHOP-21 and PCP prophylaxis. At first, we searched for patients with a positive test result for an immunofluorescence assay for PCP using bronchoalveolar lavage (BAL) samples. As a confirmative test of PCP, a direct immunofluorescence assay to detect P.jirovecii was performed with a commercially available kit (Light Diagnostics TM Pneumocystisjirovecii DFA Kit, Millipore, Billerica, MA, USA). Secondly, we searched for patients who received therapeutic doses of TMP/SMX for following clinical conditions; 1) symptoms such as fever, cough, or dyspnea, 2) radiologic findings compatible to PCP on chest X-ray or chest computed tomography, 3) no other definite cause of pneumonia.

Result: We identified 176 patients with DLBCL who received at least 4 cycles of R-CHOP-21 and concurrent PCP prophylaxis between June 2016 and December 2017. The median age was 59 (range, 22-84), 79 patients (44.8%) had advanced stage (stage III/IV) and 163 patients (92.6%) received 6 or 8 cycles of R-CHOP. The median follow-up duration of 13.6 months (range, 4.4 - 24), and TMP/SMX prophylaxis median cycles was 6 (range, 4-8). Among 176 patient, we identified 2 patients who had undergone bronchoscopy and the immunofluorescence assay for PCP using BAL samples in suspicious of PCP but no patients revealed positivity for the test. We also found that no patients received therapeutic doses of TMP/SMX during the chemotherapy. Grade 3/4 neutropenia and thrombocytopenia occurred in 30.6% and 8.1% of patients, respectively.

Conclusion: There was no PCP event in patients with DLBCL treated with R-CHOP-21 and TMP/SMX prophylaxis with daily single-strength TMP/SMX (80mg/400mg). Side effect profiles seems to be comparable with R-CHOP-21 alone.

Disclosures

No relevant conflicts of interest to declare.
Risk Factors Predisposing Development of Priapism in Sickle Cell Disease Patients
Authors: Alkindi Salam , AlMufargi Said , Pathare Anil
Published: October 2019

Background and Purpose: Penile erection and detumescence are complex physiologic processes, which require delicate neurohormonal and cardiovascular response. Priapism is defined as a persistent and painful erection lasting longer than four hours without sexual stimulation. Stuttering priapism is characterized by a self-limited, recurrent, and intermittent erection, frequently occurring in patients with sickle cell disease(SCD). The aim of this study was to identify the clinical and laboratory features in SCD patients with priapism in Oman.

Methodology: In this retrospective case-control study, medical records of all patients with SCD who developed priapism were retrieved from the hospital information system and compared in a 1: 1 ratio with age and gender matched SCD patients who did not ever had priapism. Addition information extracted include hematological and laboratory parameters, treatment, and complications. Data obtained were analyzed using IBM SPSS version 23. The study was conducted following approval from the hospital medical research and ethics committee.

Results & Discussion: Amongst the forty-one SCD patients evaluated, in the 21 patients with priapism (mean age 24.7 yrs), there was a significantly higher WBC, platelet, retic counts, LDH and serum total bilirubin as compared to controls (p<0.05, student's t test). However, there was no significant differences in the incidence of vaso-occlusive crisis, acute chest syndrome, pulmonary hypertension or cerebrovascular accident (p >0.05, Chi Square test). Patients with priapism were managed with exchange transfusion (100%), aspiration (52%) and shunting (5%). Interestingly, none of these patients showed impotence, although 10% manifested with infertility.

Conclusions: Priapism in this SCD cohort is a disease of the young, and seen predominantly in the hemolytic phenotype of the disease. Patients with priapism showed high WBC's, Platelets, Bilirubin, LDH and retics with a lower hemoglobin as compared to the controls. Favorable outcome is dependent on early intervention and almost half of these patients needed surgical intervention.

Disclosures

No relevant conflicts of interest to declare.
Detection of the BCR-ABL Gene By the Real-Time PCR Method in Patients with Chronic Myeloid Leukemia in Rio Grande Do Norte, Brazil
Authors: Paiva Aldair Sousa , Paiva Hugo Diogenes De Oliveira , Cavalcanti Geraldo Barroso , Leão Gioconda DR , Leão Marcos Dias , Vasconcelos Roberto C , Fernandes Andrea LAC , Bahia Frank , Soares Victor lima , Gil Erica Aires , Januario Geisa , Santos Karla Cristina , Lemos Telma Araujo , Jardim Alessandra Suelen
Published: October 2019

Background: The Philadelphia chromosome is a cytogenetic change resulting from a reciprocal translocation of genetic material between ABL genes from chromosome 9 and BCR from chromosome 22 or t(9; 22) (q34; 11), forming the chimeric gene BCR- ABL, being associated with chronic myeloid leukemia (CML), acute lymphoid leukemia (ALL) and acute myeloid leukemia (AML). The p190 variant is usually associated with acute forms of leukemia, including AML and ALL, whereas the p210 variant is associated with the chronic phases of CML. Due to the high sensitivity and specificity, nucleic acid amplification techniques by real-time PCR have replaced the conventional cytogenetic techniques for the identification of the Philadelphia chromosome and its p190 and p210 variants. Molecular analysis has been indicated in the initial diagnostic phase and also for the therapeutic monitoring defining the percentage of neoplastic cells present in the patients during the different phases of the treatment (Minimum Residual Disease or MRD).The aim of this study was the transcript BCR-ABL identification in patients with suspected of CML and evaluation of the gene frequency in these patients. Methods: The presence of BCR-ABL gene was investigated in blood samples from 42 patients with suspected CML. The RNA extraction was performed by phenol/chloroform method. The cDNA was submitted to PCR, using specific primers for and BCR-ABL genes by Real time PCR. Results: From all studied patients, 16 (38.10%) were negative, and 26 (59.09%) positive for one of rearrangements: p210 b3a2 and b2a2 in 18 cases (40.91%) and p190 a1a2 in 2 cases (4,76%) and double positive p120/190 in 6 cases (14,28%). We observed that the most common rearrangement was the p210 b3a2, and the molecular results were compatible with clinical and hematologic suspicion. Conclusions: The Real-timePCR, because of its specificity and sensitivity, can be considered the most used technique in routine diagnosis and investigation of MRD of CML patients.

Disclosures

No relevant conflicts of interest to declare.
Recurrent Stroke: The Role of Thrombophilia in a Large International Pediatric Stroke Population
Authors: Deveber Gabrielle , Kirkham Fenella Jane , Shannon Kelsey , Brandao Leonardo R. , Sträter Ronald , Kenet Gili , Clausnizer Hartmut , Moharir Mahendranath , Kausch Martina , Askalan Rand , MacGregor Daune , Stoll Monika , Torge Antje , Dlamini Nomazulu , Ganesan Vijeya , Prengler Mara , Singh Jaspal , Nowak Goettl Ulrike
Published: October 2019

OBJECTIVE:

Risk factors for arterial ischemic stroke (AIS) in children are multiple and include cardiac disease, vasculopathy, and prothrombotic risk factors (PR). The relevance of these factors to a second AIS event is incompletely understood.

METHODS:

We conducted a multicenter cohort study to assess the rate of symptomatic stroke recurrence following initial AIS, pooling data on recurrent AIS from the databases held in Canada, Germany, and UK. We followed 894 patients aged 1 month to 18 years (median 6 years) at initial AIS for median 35 months.

RESULTS:

160 of 894 patients (17.9%) had recurrence from 1 day to 136 months (median 3.1 months) after first AIS. Recurrence was significantly more common in children with (hazard ratio (HR) 2.5, 95% confidence intervals (CI) 1.92-3.5, p<0.001) compared to children without vasculopathy. After adjusting for vasculopathy, antithrombin deficiency, elevated lipoprotein (a), and the presence of any combined PR were independently associated with recurrence. Recurrence rates calculated per 100 person-years were 10 (95%CI: 3-24) for antithrombin deficiency, 6 (95%CI 4-9) for elevated Lp(a), and 13 (95%CI 7-20) for combined PR.

CONCLUSIONS:

Identifying children at increased for recurrent AIS events is important in intensifying preventative measures. Among 894 Canadian, English and German pediatric stroke patients, 17.9% experienced recurrent AIS at a median of 3.1 months after the index stroke. The presence of more than one prothrombotic risk factor is associated with AIS recurrence in children

Disclosures

No relevant conflicts of interest to declare.
Prostaglandin E2 Glyceryl Ester Contributes to Hyperalgesia in a Humanized Model Pf Sickle Cell Disease through P2Y6 Receptors
Authors: Simone Donald , Gupta Kalpna
Published: October 2019

Sickle Cell Disease (SCD) is the most common monogenic red blood cell disorder characterized by a remarkable phenotypic complexity. Common complications of SCD are unpredictable episodic and chronic pain. Transgenic homozygous HbSS-BERK mice expressing >99% human sickle hemoglobin and exhibiting both pain and neurochemical changes similar to humans, and HbAA control mice expressing normal human hemoglobin, were used to determine the contribution of the prostaglandin E2 glyceryl ester (PGE2-G), to mechanical and heat hyperalgesia and the mechanism of its pro-nociceptive effect. Mechanical hyperalgesia was defined by a decrease in paw withdrawal threshold, and heat hyperalgesia was defined by a decrease in paw withdrawal latency to radiant heat applied to the plantar hind paw. Considering that prostaglandin E2-G (PGE2-G) is the product of oxidation of 2-arachidonylglycerol (2-AG) by cycloxygenase-2 (COX-2), the expression of COX-2 protein and COX-2 activity was measured in dorsal root ganglia (DRG). The levels of 2-AG and PGE2-G in DRG were determined by HPLC-MS. Mechanical and heat hyperalgesia in HbSS mice were associated with a decreased level of 2-AG and increased level of PGE2-G in DRGs of HbSS mice due to elevated level of COX-2 protein and COX-2 activity. In HbAA control mice, intraplantar (i.pl.) injection of PGE2-G (1 µg/10 µl) produced mechanical hyperalgesia in the injected paw independently from PGE2 receptors. Also, PGE2-G (1 µg/10 µl, i.pl.) produced mechanical and heat hyperalgesia in the injected paw in C3H mice. The hyperalgesia was blocked by co-injection of PGE2-G with MRS 2578, the purinergic P2Y6 receptor antagonist (1.4 mg/10 ml, i.pl.). Importantly, MRS 2578 (4.7 mg/50 ml, i.p.) also attenuated hyperalgesia in HbSS mice. Thus, increased expression of COX-2 in DRG neurons of HbSS mice results in the oxidation of 2-AG and production of PGE2-G, a pronociceptive mediator. PGE2-G produced mechanical and heat hyperalgesia in HbAA and C3H mice, and these effects were mediated by P2Y6 receptors.

Acknowledgments: Supported by the National Heart, Lung and Blood Institute (HL135895-16).

Disclosures

Gupta: Novartis: Honoraria; Tau tona: Consultancy.
The Role of Thrombin Deficiency on Skeletal Health
Authors: Taylor Hillary J , Goldscheitter Galen , Taylor Jason A
Published: October 2019

Introduction

Persons with hemophilia have decreased bone mineral density (BMD) and increased risk of fracture. Our group has demonstrated that mice with factor VIII deficiency have decreased BMD and decreased biomechanical strength. The underlying mechanism for the decreased skeletal health remains undetermined. This study uses small interfering(si) RNA to knockdown prothrombin gene expression to test the hypothesis that thrombin deficiency results in decreased skeletal health.

Method

Seventeen 3-week old male C57BL/6J wild-type mice were given subcutaneous injections of siRNA against α-prothrombin (1 mg/Kg) and compared to an equal number of mice injected with saline. Mice were injected at 3, 4, 5, 7, 9, 11, 13, 15, 17, and 19 weeks of age with a volume of 200 ul and measurements taken at peak bone mass at 20 weeks of age. In initial dose response studies, this regimen results in >85% knockdown of prothrombin gene expression (data not shown). Studies included whole body and femoral BMD, thromboelastography (TEG) of plasma, micro computed tomography (µCT), and three-point breaking.

Results

TEG studies show significant differences between mice treated with siRNA (n=17) and mice treated with normal saline (n=6) with R, K and α-angle being p < 0.02, p < 0.014, and p < 0.005 respectively. These results confirm that the thrombin is knocked down by the siRNA against α-prothrombin. Comparative analysis of the whole body and femoral BMD between the treatment and saline groups demonstrate no significant differences (p = 0.64 and p = 0.74). There was no significant difference seen in µCT scans of the right femur, including femoral cortical area (p=0.19), cortical thickness (p= 0.63), and cancellus area (p= 0.25). Three-point break data show no significant difference in ultimate force (p = 0.268), strength (p = 0.546), or modulus (p = 0.07).

Conclusion

This study seeks to understand the underlying mechanism for bone disease in hemophilia. The results indicate that >85% prothrombin knockdown in wild-type mice starting at 3 weeks of age does not significantly alter skeletal health. This suggests that either 1) thrombin deficiency is not involved in the mechanism of decreased skeletal health in hemophilia or; 2) the role of thrombin deficiency occurs early in bone development (prior to 3 weeks of age). To evaluate these two potential explanations, thrombin deficiency needs to be evaluated earlier in development. Thrombin deficient mice are embryonically lethal. Therefore our laboratory is currently using a series of transgenic mice to characterize the role of thrombin receptors present osteoblasts and their role in bone metabolism.

Disclosures

Taylor: Alnylam: Research Funding; Shire: Research Funding; CSL: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bioverativ: Membership on an entity's Board of Directors or advisory committees.
The 3.2 Angstrom Crystal Structure of a Bioengineered Factor VIII Construct Illustrates Two Conformations of the C2 Domain
Authors: Smith Ian W , d'Aquino Anne , Parker Ernest T. , Doering Christopher B , Spiegel Paul C
Published: October 2019

Treatment for hemophilia A consists of repeated intravenous infusions of blood coagulation factor VIII (fVIII), either recombinant or derived from pooled plasma cryoprecipitate. While several recombinant versions are clinically available, all existing fVIII replacement products express at low levels, harbor short half-lives, and are cost intensive to produce for prophylaxis and therapeutic intervention. Here, we report the 3.2 Å structure of a bioengineered construct of fVIII, termed ET3i, which is a human/porcine chimeric B domain-deleted heterodimer with an improved expression profile and slower A2 domain dissociation following proteolytic activation of fVIII (Figure 1A). Previous structural knowledge of fVIII is at 3.7 Å resolution or lower, and these structures display inconsistencies in their atomic details. The structure reported herein shows remarkable conservation amongst the core folds and domain arrangement of the fVIII protein as well as provides a more detailed view of the interface between the A2 domain and the remaining fVIII structure. With two fVIII molecules in the asymmetric unit of the crystal, we independently observe two distinct conformations of the C2 domain relative to the remaining fVIII structure. While one model (chain A) superimposes with previously-determined fVIII structures without significant deviation, the second model (chain B) indicates a 'barrel tilt and roll' conformational change of the C2 domain that may have implications for how proteolytically activated fVIII binds to activated platelet surfaces (Figure 1B). The improved model and stereochemistry of the ET3i A chain was subsequently used as a scaffold to generate an improved, refined model of human fVIII. In the case of both original datasets to 3.7 Å (pdb#: 2R7E) and 3.98 Å (pdb#: 3CDZ), our new model based on the ET3i structure resulted in improved refinement statistics for human fVIII. These improved structures further illustrate the conserved nature of the ET3i protein as well as provide a more confident model of fVIII for next generation engineering efforts to develop fVIII therapeutics with longer half-lives, higher expression levels and lower immunogenicity.

Figure 1 Figure 1.

Disclosures

Doering: Expression Therapeutics, LLC: Equity Ownership, Research Funding.
An In Vivo Genome-Wide CRISPR Screen Identifies Novel Dependencies for Blast Crisis Chronic Myelogenous Leukemia
Authors: Bajaj Jeevisha , Scott-Browne James , Spinler Kyle , Reya Tannishtha
Published: October 2019

Poorly differentiated aggressive myeloid diseases such as Acute Myelogenous Leukemia (AML) and blast crisis Chronic Myelogenous Leukemia (bcCML) are often resistant to standard therapy and associated with significantly poor survival in both children and adults. There is thus a significant need for a better understanding of the mechanisms that drive disease progression and for finding novel therapeutic targets. Thus, to determine the molecular effectors of myeloid leukemia growth in vivo, we carried out a genome-wide CRISPR/Cas9 dropout screen using the lentiviral Brie gRNA library. This library targets 19,674 genes, and has on average 3 gRNAs for each gene, and 1000 control non-targeting gRNAs. We carried out this whole-genome screen in a mouse model of Cas9+ blast crisis CML (bcCML) driven by BCR-ABL/ NUP98-HOXA9 since this represents a very aggressive phase of myeloid cancer where 90% of the leukemic blasts are undifferentiated and cancer stem cell-like. This in vivo screen led to the identification of 3636 genes essential for leukemic growth and propagation in the bone marrow of recipient mice, constituting pathways such as metabolism, protein translation and DNA replication. The genes that were significantly depleted included known drivers of myeloid cancer progression and regulators of myeloid cancer stem cells (for example, Brd4, Kdm1a, Pafah1b1/Lis1, Rptor), indicating that our screening strategy can successfully identify functional drivers of cancer growth.

While intrinsic signals that drive myeloid cancer progression are well described, little is known about how interactions with the surrounding microenvironment can control leukemic growth and propagation. Our whole-genome screen identified ~130 cell surface genes that are significantly depleted in the bcCML stem cells transplanted in vivo. Since environmental factors commonly signal through receptors on the surface of leukemic cells, this subset is likely to include most, if not all, genetic effectors of niche driven signals required for in vivo growth and propagation of aggressive myeloid leukemia cells. Of these 130 genes, several have earlier been shown by us and others to be essential for myeloid cancer progression including Itgb1, Cxcr4 and Cd44. We are currently testing the functional contribution of novel candidate cell surface molecules, which can integrate signals from the environment, on the in vivo growth and progression of myeloid malignancies. We anticipate that these studies will provide a basis for testing antibody-mediated therapeutic inhibition of specific microenvironmental signals on myeloid leukemia growth and propagation.

Disclosures

No relevant conflicts of interest to declare.
The Effect of Health Care Disparities on Complications and Mortality in Sickle Cell Disease
Authors: Perimbeti Stuthi Pavani , Hou Kevin Ye , Ramanathan Sabarina , Woodard Adonas , Kyung Daniel , Wang Qian , Crilley Pamela Ann , Ward Kristine , Styler Michael
Published: October 2019

Introduction:

Sickle cell disease (SCD) is a chronic and debilitating disorder that affects approximately 100,000 Americans and results in the development of significant complications, leading to high numbers of hospitalizations, healthcare cost and mortality. Despite the advent of newer therapies, the overall rate of complications has continued to rise. We aimed to study the prevalence of complications in SCD as well as its relation to differing insurance status.

Methods:

Patients with SCD were identified using ICD-9 codes 2826, 28260, 28261, 28262, 28263, 28264, 28268 and 28269 from the Healthcare Cost and Utilization Project's Nationwide Inpatient Sample from 1999 to 2014. Admission with acute chest syndrome, acute myocardial infarction (AMI), avascular necrosis of the hip (AVN), end stage renal disease (ESRD), pneumococcal infections, splenic sequestration and stroke. Univariate and bivariate analyses were performed using the Chi square test. Cox proportional hazard regression was used to control for multiple confounders in calculating the hazard ratios of an event occurrence and mortality.

Results:

A total of 216,438 (Weighted=1,066,536) observations were identified from the years 1999 to 2014. The median age for male patients was 25 years and that for females was 27. Observing the trends from 1999 to 2014, the prevalence of acute chest syndrome increased from 1.22% to 8.82% (p=0.002), splenic sequestration from to 0.08 % to 1% (p=0.01) and AVN from 1 % to 8.8% (p=0.001). The prevalence of stroke and ESRD were unchanged over the interval studied. After controlling for confounding factors such as race, age, sex, income, comorbidities and insurance status, the hazard ratio of mortality for various complications is significantly elevated. Also, after controlling for multiple confounders, the patient's insurance status plays a significant role in the risk of developing a complication and subsequent mortality (Table 1).

Discussion:

The data indicates that the rate of complications from SCD have risen since 1999. With newer therapies and better understanding, the life expectancy of SCD patients has risen over time, nearly doubling from 1951 to 2018. The increased frequency of complications may be attributed to better survivorship and a rising number of older SCDs patients. However, our data also suggests that insurance status plays a significant role in the complication rate of SCD. The uninsured and patients with Medicaid have significantly increased risk of developing disease complications and resultant mortality. This could be the result of reduced access to care and health disparities due to race, socioeconomic status and insurance status.

Disclosures

No relevant conflicts of interest to declare.
Single-Cell RNA-Seq Analysis of Native Murine Megakaryocytes from Young and Old Mice Reveals Significant Metabolic and Mitochondrial Differences throughout Megakaryocyte Development
Authors: Davizon-Castillo Pavel , Jones Kenneth L. , Trahan George D , Di Paola Jorge
Published: October 2019

Traditional transcriptome analysis of megakaryocytes relies on ex-vivo culture and expansion of hematopoietic cells in thrombopoietin-rich media for several days prior to RNA isolation for bulk RNA-seq analysis. This approach has been widely used, however, it is not clear as to what extend this ex vivo expansion of megakaryocytes represents the transcriptome landscape of megakaryocytes at the time of harvest from the bone marrow. In order to get a more detailed megakaryocytic transcriptome landscape immediately after bone marrow harvest, we have optimized a method to perform single cell RNA-seq analysis of native freshly isolated bone marrow megakaryocytes. This was accomplished throughout a series of steps to enrich murine bone marrow for megakaryocytes followed by cell capture using the 10X Genomics platform. In order to assess the transcriptome of native murine megakaryocytes and the effect of age on transcriptional signatures we performed single cell RNA seq analysis of 3 young mice (age 2-3 months old) and 3 old mice (>18 months old). Bioinformatics analyses identified seven transcriptionally different clusters of cells that represent the megakaryocyte ploidy status (Figures 1 and 2). Within these seven regions, 3 of them appear to represent late states of maturation (regions 5, 6 and 7) and possibly, the pro-platelet forming groups of cells due to the elevated expression of megakaryocyte transcripts such as Vwf, Pf4, Itga2b, Itgb3, Gata-1 and Mpl. Ingenuity pathway analysis (IPA) shows that the top three differentially regulated pathways between megakaryocytes from young and old mice are: a) protein ubiquitination; b) mitochondrial dysfunction and; c) oxidative phosphorylation. Furthermore, we validated the RNA-seq analysis at the protein level by measuring ALDHA1 from platelet lysates, the top differentially expressed transcript between groups. Finally, platelets from old mice have a distinctive mitochondrial phenotype characterized by elevated mitochondrial mass and significantly elevated oxygen consumption upon activation by thrombin, features that might be directly contributing to platelet hyperreactivity of aging. In summary, we present a novel methodology to study the transcriptional profile of native megakaryocytes. This initial approach highlights that metabolic and mitochondrial pathways appear to be important modulators of megakaryocyte and platelet development and function.

Disclosures

No relevant conflicts of interest to declare.
Chronic Myeloid Leukemia in Adolescents and Young Adults Treated with Generic Form of Imatinib in Resource Limited Setting: A Single Center Experience from Myanmar
Authors: Han Yin Nwe , Gyi Aye Aye , Htut Khin Thida
Published: October 2019

Chronic myeloid leukemia (CML) is an acquired myelo-proliferative disorder characterized by the presence of BCR-ABL1 fusion transcript with deregulated tyrosine kinase activity. Leukemias are one of the leading causes of cancer related deaths in adolescents and young adults (AYA) particularly in resource limited countries. Recent democratic government in Myanmar holds supportive policies for treatment of cancers in younger population and subsidizes some therapies including imatinib mesylate, a selective BCR-ABL 1 tyrosine kinase inhibitor, although there is still limitation for cytogenetic and molecular monitoring of therapy. This study on response to imatinib in newly diagnosed chronic phase CML was carried out during January 2016 to February 2018 at the North Okkalapa General and Teaching Hospital of Yangon, Myanmar where molecularly confirmed new CML in chronic phase were treated with generic form of imatinib bought by hospital tender system, Unitinib (United Biotech (P) ltd. India) followed by cytogenetic analysis of bone marrow and molecular detection of BCR-ABL1 transcript from peripheral blood by in house real time PCR machine at one year. They are divided into AYA (aged 15-39 years) and adult age groups (40 years and older) and clinical parameters and response to treatment with imatinib 400mg per day were compared. Among 56 cases (median age of 39.5 years, male to female ratio of 1.5:1), half were AYA (28 of 56) with median age of 28.5 years compared to 50 years in adult group with higher male to female ratio of 3.6:1 in AYA group. Patients in AYA had larger spleen size (11 vs 5.5 cm) and higher median white cell count compared to adults (366 x 109/l vs 224 x 109/l) although initial platelet count, peripheral blood eosinophil, basophil and blast percent and Sokal score were comparable. Additional chromosomal abnormalities were detected in 2 AYA and 3 adults with CML. There was no difference in complete haematologic response at 3 and 12 months between AYA and adults (96.4% vs 96.4% and 92.9% vs 89.3% respectively). At 12 months, complete cytogenetic response of AYA at 67.9% was less than 75.0% of adults, it was statistically not significant. Using in house molecular method although not standardized, 14.3% of AYA and 32.1% of adults were molecularly undetectable at one year. The response of AYA to imatinib in this study was comparable to adults despite having adverse prognostic features and receiving only generic forms of treatment in resource limited setting and it would further help support from authorities for leukemia in younger population.

Disclosures

No relevant conflicts of interest to declare.
Comparison of Rivaroxaban with Argatroban in Heparin Induced Thrombocytopenia Using Historical Controls : A Case-Control Study
Authors: Modi Vivek Suresh , Jatwani Karan , Rajeeve Sridevi , Kakked Gaurav , Patel Hardikumar , Bandyopadhyay Dhrubajyoti , Mannem Siva Krishna
Published: October 2019

Introduction

Heparin-induced thrombocytopenia (HIT) is an uncommon and a serious complication of heparin therapy. Currently, only two intravenous Direct Thrombin Inhibitors (DTI) are approved for its therapy. However, their cost and frequent monitoring make the Direct-Acting Oral Anticoagulants (DOACs) an attractive option for its treatment. Several case series and systematic reviews have been published with positive results for use of NOACs in HIT. No head to head trials comparing NOACs and DTI have been published as infrequency of cases make such a study prohibitive. The landmark multicenter trials establishing the role of Argatroban in the treatment of HIT used historical controls to establish it's efficacy. In this study, we aim to compare a cohort of patients treated with Rivaroxaban as recruited from prior case series with a historical cohort treated with Argatroban to establish it's safety and efficacy compared to Argatroban.

Methods:

A literature review was done using Embase and Pubmed to find HIT patients treated with Rivaroxaban. A total of 14 publications were found including case studies, case series, and systematic reviews. A database was created of a total of 51 HIT patients treated with Rivaroxaban alone or argatroban followed by Rivaroxaban using all the publications. All the cases were confirmed HIT cases based on Serotonin release assay and/or HIT antibody. Platelet nadir and Platelet count at the start of therapy as noted wherever possible. Patients with known bleeding diathesis and bleeding sites were excluded from both the cohorts. Outcomes were thrombosis after initiation of therapy and bleeding after initiation of therapy. The cohort was compared with a historical cohort of patients treated with Argatroban alone in the paper published by Lewis et al. Chi-square test was done to compare new thrombosis and bleeding during hospitalization.

Results:

No significant difference was found in the rates of new thrombosis (OR: 0.18, CI: 0.02-1.36, p: 0.06). 5% of patients had major bleeding in the argatroban cohort while none of the patients had major bleeding in Rivaroxaban cohort.

Conclusion:

The study indicates that Argatroban is not significantly better in the treatment of HIT in preventing new thrombosis. However, the bleeding complications might be lower with Rivaroxaban therapy compared to Argatroban therapy. The study is limited by less than ideal comparability of cohorts and low power. However, it provides only such evidence of it's kind.

Table. Table.

Disclosures

No relevant conflicts of interest to declare.
Study of Bone Marrow Response to Platelet Transfusion in Severe Traumatic Brain Injury Patients
Authors: Subramanian Arulselvi , Albert Venencia , Agrawal Deepak , Pati Hara Prasad
Published: October 2019

Introduction: SevereTraumatic Brain Injury (TBI) patients are predisposed to secondary brain insults due to thrombocytopenia and coagulopathy, warranting platelet transfusion. Thrombopoietin (Tpo) has been shown to be a physiological regulator of thrombopoiesis. An inverse correlation between Tpo and the circulating platelet has been previously reported for several clinical conditions. Response pattern of Tpo to circulating platelets in TBI patients has not been studied yet. We hypothesised that in severe TBI patients thrombopoiesis response to TPO will be impaired

Methodology: Plasma Tpo levels were measured by enzyme-linked immunosorbent assay was measured 3 times i.e. Pre-platelet transfusion of platelets,24h and 48h post-transfusion in 100 severe (GSC≤8)TBI patients. Platelet transfusion response was evaluated and defined as a ≥20% increase in platelet count from baseline and patients were categorized as responders and non-responders to platelet transfusion.

Results: Platelet count was low at admission (89.4±31.1*103 per cumm). Less than 2 units of random donor platelets(RDP) was given to 14% patients,67% patients required2-4 units of RDP and 19% required >4 units of RDP. TPO levels decreased 24h post transfusion compared to pre transfusion (Mean±SD)(127.8±90.2 vs. 142.2±82.6 pg/ml; P 0.07), but elevated at 48h (209.1±74.8 pg/ml; p Pre vs. 48h 0.05). On Average the platelet count improved after transfusion at 24h to 112.7±74.6pg/ml (p Pre vs. 24h 0.003) and continued to rise at 48 h (123.7±82.1 pg/ml; p Pre vs. 48h 0.02). Platelet count and TPO levels were found to be independent (rho -0.12, p 0.38). 62% showed a ≥20% increase in platelet count from baseline (Responders), 38% were non-responders. As significant increase in 24 h post transfusion Tpo levels were seen in responders [median (IQR)] [173.6(0-202.4) pg/ml vs. 164.3(137.1-203.7) pg/ml; p 0.04] compared to pre transfusion levels. Tpo levels in Non-responders did not vary post platelet transfusion [156(0-198.11) pg/ml vs. 158.88(0-198.11) pg/ml; p 0.97]. 28% of Non-responders had low (<81.25 pg/ml) and 2.8% had high (>237.7 pg/ml) pre transfusion Tpo levels. At 24 h post transfusion 26.6% had low and zero had High Tpo levels.

Conclusion: In the present study platelet transfusion response inversely correlates with Tpo levels, as 24h post platelet transfusion Tpo levels were transiently low but insignificant and elevated at 48h post transfusion. In non-responders, the post transfusion TPO levels were not sufficient to mitigate the low platelet counts. Hence, the possibility of transient bone marrow depression in severe TBI patients is suggested, which requires further analysis in a larger study cohort.

Disclosures

No relevant conflicts of interest to declare.
In vitro phenotypic correction of hematopoietic progenitors from Fanconi anemia group A knockout mice
Authors: Rı́o Paula , Segovia José Carlos , Hanenberg Helmut , Casado JoséAntonio , Martı́nez Jesús , Göttsche Kerstin , Cheng Ngan Ching , Van de Vrugt Henri J. , Arwert Fré , Joenje Hans , Bueren Juan A.
Published: October 2019

Fanconi anemia (FA) is a rare autosomal recessive disease, characterized by bone marrow failure and cancer predisposition. So far, 8 complementation groups have been identified, although mutations in FANCA account for the disease in the majority of FA patients. In this study we characterized the hematopoietic phenotype of a Fanca knockout mouse model and corrected the main phenotypic characteristics of the bone marrow (BM) progenitors using retroviral vectors. The hematopoiesis of these animals was characterized by a modest though significant thrombocytopenia, consistent with reduced numbers of BM megakaryocyte progenitors. As observed in other FA models, the hematopoietic progenitors from Fanca−/− mice were highly sensitive to mitomycin C (MMC). In addition, we observed for the first time in a FA mouse model a marked in vitro growth defect ofFanca−/−progenitors, either when total BM or when purified Lin−Sca-1+ cells were subjected to in vitro stimulation. Liquid cultures ofFanca−/−BM that were stimulated with stem cell factor plus interleukin-11 produced low numbers of granulocyte macrophage colony-forming units, contained a high proportion of apoptotic cells, and generated a decreased proportion of granulocyte versus macrophage cells, compared to normal BM cultures. Aiming to correct the phenotype of Fanca−/−progenitors, purified Lin−Sca-1+ cells were transduced with retroviral vectors encoding the enhanced green fluorescent protein (EGFP) gene and human FANCAgenes. Lin−Sca-1+ cells fromFanca−/−mice were transduced with an efficiency similar to that of samples from wild-type mice. More significantly, transductions with FANCA vectors corrected both the MMC hypersensitivity as well as the impaired ex vivo expansion ability that characterized the BM progenitors ofFanca−/−mice.
The cytoplasmic domain of Mpl receptor transduces exclusive signals in embryonic and fetal hematopoietic cells
Authors: Challier Cécile , Cocault Laurence , Berthier Rolande , Binart Nadine , Dusanter-Fourt Isabelle , Uzan Georges , Souyri Michèle
Published: October 2019

The Mpl receptor plays an important role at the level of adult hematopoietic stem cells, but little is known of its function in embryonic and fetal hematopoiesis. We investigated the signals sent by the MPL cytoplasmic domain in fetal liver hematopoietic progenitors and during embryonic stem (ES) cell hematopoietic commitment. Mpl was found to be expressed only from day 6 of ES cell differentiation into embryoid bodies. Therefore, we expressed Mpl in undifferentiated ES cells or in fetal progenitors and studied the effects on hematopoietic differentiation. To avoid the inadvertent effect of thrombopoietin, we used a chimeric receptor, PM-R, composed of the extracellular domain of the prolactin receptor (PRL-R) and the transmembrane and cytoplasmic domains of Mpl. This allowed activation of the receptor with a hormone that is not involved in hematopoietic differentiation and assessment of the specificity of responses to Mpl by comparing PM-R with another PRL-R chimeric receptor that includes the cytoplasmic domain of the erythropoietin receptor (EPO-R) ([PE-R]). We have shown that the cytoplasmic domain of the Mpl receptor transduces exclusive signals in fetal liver hematopoietic progenitors as compared with that of EPO-R and that it promotes hematopoietic commitment of ES cells. Our findings demonstrate for the first time the specific role of Mpl in early embryonic or fetal hematopoietic progenitors and stem cells.
Rituximab treatment results in impaired secondary humoral immune responsiveness
Authors: van der Kolk Lizet E. , Baars Joke W. , Prins Martin H. , van Oers Marinus H. J.
Published: October 2019

In lymphoma patients, treatment with chimeric CD20 monoclonal antibodies (rituximab) results in a depletion of normal and malignant B cells, persisting for 6 to 9 months. This B-cell depletion leads neither to a decrease in immunoglobulin levels nor an increase in the number of infectious complications. However, the effect of rituximab treatment on the immune responsiveness is unknown. In 11 patients with relapsed, low-grade lymphoma, we investigated the effect of rituximab treatment on the humoral immune response to 2 primary antigens and 2 recall antigens. After rituximab treatment, the humoral immune response to the recall antigens was significantly decreased when compared with the response before treatment. Already before rituximab treatment, none of these patients was able to mount a response to the primary antigens. These findings are relevant regarding the feasibility of rituximab in maintenance treatment and may also offer a rationale for the treatment of antibody-mediated autoimmune diseases with rituximab.