bluuebunny/crossref_metadata_2025 huggingface dataset:
Safety and Efficacy of New Moroctocog Alfa Drug (Octofactor) in Prophylactic Treatment in Adolescent Patients with Severe and Moderate Hemophilia a
Authors: Shiller Ekaterina , Vdovin Vladimir , Petrov Victor , Svirin Pavel , Andreeva Tatiana , Lavrichenko Inna , Bullikh Artem , Koltunov Igor , Davydkin Igor , Kurtov Igor , Shuster Alexandr , Kudlay Dmitry , Lukyanov Sergey , Borozinets Anton , Nikitin Eugene , Klykova Ekaterina
Published: October 2019

Efficacy and safety of a new domestically produced rFVIII-BDD (moroctocog alfa, Octofactor, CJSC "GENERIUM", Russia) was investigated in a controlled, open, prospective, multicenter clinical trial. After screening and a 4-days washout period 12 previously treated patients adolescents (age 12-18 years old) with severe hemophilia A (the activity of FVIII was less than 1%) were included in the clinical trial. The patients were given the moroctocog alfa as prophylactic treatment in a dose of 35±5 ME/kg 3 times per week during 21±1 weeks. Before participation in this clinical trial 2 pts had received treatment with another recombinant FVIII, 2 pts had therapy with pdFVIII and 8 pts had been treated with rFVIII and pdFVIII. The main criterion of drug efficacy was the incidence of spontaneous bleeding occurred within 48 hours after of the moroctocog alfa injection.

The additional criteria were:

· The severity of spontaneous bleeding occurred in 21 ± 1 week.

· Number of injections needed for the one episode of bleeding according of its severity.

· The total amount of drug administered over a period of prophylactic treatment and treatment "on demand".

· The number of patients with severe hemophilia A with a residual activity of FVIII ≥1% in 48 hours after the injection on prophylactic therapy.

During the follow up period (21±1 week) 17 bleeding episodes were registered, 2 of them (11,8%) were severe, 10 (58,8%) were moderate and 5 (29,4%) were mild (Tab.1). Number of bleeding episodes (spontaneous and traumatic) was 17 (1.55 in average). Number of spontaneous bleeding was 3 (17,6%), 1 of them was mild and 2 were moderate. The average number of injections that stop one bleeding episode was 1.7±0.8. The total amount of moroctocog alfa administered over a period was 1.502.000 ÌÅ for prophylactic treatment and 64.750 ÌÅ for "on demand" treatment. The number of patients with severe hemophilia A with a residual activity of FVIII ≥1% in 48 hours after injection on prophylactic therapy was 63,6% on visit 2, 90,09% on visit 3 and 81,1% on visit 4. The safety assessment was performed in 12 patients. There were 8 adverse events and 7 of them were not associated with drugs administration. There was one serious adverse effect, allergic reaction accompanied by arthralgia and cephalalgia. The patient was excluded from the trial without consequences for life and health. There were no infection transmissions and de novo inhibitor incident. The study showed that moroctocog alfa is effective and safe in prophylactic treatment and stopping of bleeding in adolescents with severe hemophilia A.

Table 1. Efficacy evaluation Table 1. Efficacy evaluation

Disclosures

No relevant conflicts of interest to declare.
Short and Long-Term Outcomes in Hemophilia Patients with Inhibitors Undergoing Orthopedic Prosthetic Surgery
Authors: Castaman Giancarlo , Linari Silvia , Carulli Christian , Innocenti Massimo
Published: October 2019

Introduction: Major orthopedic surgery is still a difficult challenge in hemophilic patients with high titer of inhibitor. In addition to possible peri-operative problems, the long-term outcomes are anecdotal.

Aim of the study: to evaluate the short and long term outcomes in prosthetic orthopedic procedures done in hemophilic patients with inhibitor from 1998 to 2014

Results: Sixteen knee replacements have been performed in 12 patients affected by hemophilia A with inhibitors. At the time of surgery 11 out of 12 patients had an inhibitor titer > 5UB (range 5 - 2,500). A preoperative pharmacokinetic with rFVIIa was made in 8 out of 12 patients, with the following mean values of clearance rate: 35 ml/h/kg and half-life: 2.85 hours. The average age was 46 years (range 29-55). All patients were treated with 2-3 doses of rFVIIa (90-120 mg/kg) every two hours during surgery up to suturing of the wound, followed by continuous infusion of rFVIIa at dose of 30-50 mg/kg/h on days 1-3 and 15 mg/kg/h on days 4-14. Plasma levels of FVII:C were maintained > 15U/mL. All but one patients were transfused with 2 units of red blood cells (RBC), according to standard procedure. Two peri-operative complications occurred: 1 pulmonary embolism and 1 major bleeding at the operated site, poorly responsive to rFVIIa and aPCC, treated with 6 units of RBC. Only two patients underwent to prosthesis revision for infection, while another due to rupture of the prosthesis metal cup. After surgery all patients showed a significant improvement in pain and a favorable functional recovery. After a median follow-up of 7 years (1-18 years), a good quality of life as to pain and function of the replaced knee were recorded.

Conclusions: The orthopedic prosthetic surgery in hemophilic patients with inhibitors can be done, with a good outcome. The continuous infusion of rFVIIa provides stable haemostatic coverage, saving on the rFVIIa total dose compared to the bolus standard dose. However, costs remain high and the possible risks of thromboembolic complications should not be overlooked

Disclosures

No relevant conflicts of interest to declare.
Prevalence of Hepatitis C Infection and Virus Clearance in Patients with Hemophilia
Authors: Cisak Kamila Izabela , Pan Jianmin , Rai Shesh Nath , Ashby Patricia , Sharma Vivek R.
Published: October 2019

Introduction

Hemophilia A and B are hereditary genetic disorders caused by deficiency of clotting factor VIII and IX respectively. Infusions of clotting factors constitute the mainstay of treatment. Before availability of recombinant factor VIII and IX, patients with hemophilia received products derived from human plasma and were at risk of blood-borne infections exposure. Since then, ongoing advances have yielded not only safer recombinant clotting factors but also effective treatment of infections. Hepatitis C infection and its complications constitute a common cause of morbidity and mortality in older hemophilia patients. In the last few years, FDA approved various antiviral medications which allow achievement of sustained virologic response and even cure hepatitis C infection. Goal of our study is to show prevalence of hepatitis C infection and virus clearance at our hemophilia treatment center.

Methods

We performed retrospective chart review of patients followed at a single hemophilia treatment center in the United States. We included 59 patients with factor VIII or IX deficiency, age 30 and older, followed in clinic between 2005 and 2014. Patients with acquired hemophilia were excluded from study. Data was collected from physician notes and laboratory tests results which included hepatitis C antibodies and viral load.

Results

39 (66.1%) patients had history of hepatitis C infection. 21 (35.6%) patients had detectable hepatitis C viral load [95% CI 0.24-0.49] while 18 (30.5%) patients cleared the virus [95% CI 0.19-0.44].

Conclusions

Our study showed that 66.1% of hemophilia patients followed at our institution had current or past hepatitis C infection. More than half of them were virus carriers during their last viral load check. Many of them had refused interferon-based treatment due to the requirement of being on it for long duration and concerns about side effects. Others had received the treatment but failed it. This constituted as much as 35.6% of hemophilia patients at our hemophilia treatment center. Availability of the newer antiviral agents that are better tolerated and yield high cure rates provide an opportunity to further reduce the disease burden in these patients.

Disclosures

No relevant conflicts of interest to declare.
Types and Sub Types of Von Willebrand Disease Classified on the Basis of Multimer Analysis
Authors: Naz Arshi , Yadegari Hamideh , Driesen Julia , Oldenburg Johannes , Ahmed Shariq , Tariq Shahla , Amanat Samina Tufail , Razik fazal-E- , Ahmed Nisar , Nadeem Mohammad , Shamsi Tahir
Published: October 2019

Introduction: Von Willebrand disease is a common genetically inherited bleeding disorder. Types of VWD classified on the basis of coagulation factor assays but it cannot characterize further subtypes or verdict the quality and quantity of VWF. Multimer analysis facilitates the classification and prerequisite parameter to accurate diagnosis of VWD.

Objective: To characterize the type and subtypes of VWD type I and II on the basis of laboratory data and analysis of VWF multimer structure.

Setting: NIBD Karachi, University of Bonn Germany, Children Hospital & Chughtai Lab Lahore, PAEC Islamabad and HMC Peshawar.

Material And Method: VWF Ag, Ricof, FVIII, Glycoprotein Ib and multimer analysis of VWD plasma on 1.6% medium resolution SDS-agarose gel electrophoresis was carried out, and then VWF multimer were transferred on nitrocellulose membrane. Incubated nitrocellulose membrane with using polyclonal rabbit anti-human VWF antibody and Goat anti-rabbit IgG HRP conjugate then protein detected by immune reaction after evaluation of the membrane under luminescence.

Results: We studied 57 index patients, 2(3.5%) type I, 4(7.01%) type II, 3(5.26%) unclassified and 48(15.78%) were type III vWD. In type I,II, unclassified and III mean vWF Ag was 52.28, 35.15, 54.5 , 5.7 IU/dl, vWF RCo: 19.5, 18, 38.8, 7.22 IU/dl, vWF Ag/ RCof: 0.655, 0.393, 0.74, 0.55, FVIII 69, 25.7, 61.7, 7.22 IU/dl and GpIb: 27.25, 19.8, 39.8, 4.1. Significant loss of large multimers in type II while multimers were subtle in 2 cases, normal pattern in type I and entirely absence of multimers pattern in case of type III observed.

Conclusion: Types and subtypes of VWD classified on the basis of concentration and distribution of vWF multimer by functional and immunological assays but single test is not enough for appropriate diagnosis. We need molecular genetics for von Willebrand disease for further diagnosis.

Disclosures

Oldenburg: SOBI: Consultancy.
Prinzmetal Angina in Major Hemophilia a Patient: A Case Report
Authors: Massi Mahutondji Romaric , Houssou Bienvenu , Camara Marième , Nisserine Khoubila , Quessar Asmaa AQ , Oukkache Bouchra
Published: October 2019

Introduction: Prinzmetal anginais a special type ofacute coronary syndrome ST+ wich correspond to a transient occlusion of a coronary vessel secondary to spasm. This type of acute coronary syndrome is very rare and is characterized by the presence of signs of myocardial ischemia on electrocardiogram but coronary angiography and coroscaner are frequently normals. Its management in hemophilia patients is difficult because of the use of anticoagulant and antiplatelet drugs wich increase bleeding risk. We report the case of a major hemophilia A patient which presented Prinzmetal angina.

Observation: It is a 64 years old patient, hemophilia A major, chronic smoking (40 pack-year), not diabetic, not hypertensive, which had a retro sternal constrictive pain radiating to the shoulders. At admission he was consciousness. No breath in cardiac auscultation. The electrocardiography showed a heart rate at 61bpm, the axis of the heart was normal. ST-segment elevation was noticed in DIII and AVF: ischemia in the cardiac lower area. Echocardiography was normal. The coroscaner was normal. The troponin I level was at 0.03 µg/L (Normal: 0-0.1µg/L). This patient had a variant Prinzmetal angina. Treatement: Diltiazem Hydrochloride 60 mg 1 tablet / 8 hours. Acetylsalicylic acid 160 mg IV and Clopidogrel 300 mg IV the first day; relay with acetylsalicylic acid 100 mg and clopidogrel 75 mg per day. Transfusion of factor VIII at the dose of 40UI / Kg. Simvastatin 20 mg 1 tablet per day. Perindopril 5 mg 1 tablet the day. The evolution was favorable.

Discussion and conclusion: Coronary syndromes are not frequent in morocco hemophilia patients. Their management is complex and involves the presence of an hematologist.

Disclosures

No relevant conflicts of interest to declare.
Chronic IVC Occlusion Predisposing to Acute Lower Extremity DVT in Children
Authors: Tarango Cristina , Blackmore Anne , Palumbo Joseph S.
Published: October 2019

Congenital atresia of the inferior vena cava (IVC) is a known risk factor for spontaneous lower extremity thrombosis in adults. However, little is known about the incidence of IVC occlusion and its relationship to spontaneous lower extremity thrombosis in children. We reviewed the medical records of consecutive patients with IVC occlusion and lower extremity DVT who presented to our institution between 2008 and 2015. Data were reviewed for risk factors for thrombosis, treatment, and outcomes including recurrent thrombosis, bleeding complications, and post-thrombotic syndrome. This represents the largest reported analysis of previously healthy pediatric patients with occult chronic IVC occlusion presenting with unprovoked acute lower extremity thrombosis. Ten patients aged 13 to 18 years (median age 16.5yrs, 90% male) presenting with acute lower extremity thrombosis were found to have chronic occlusion of the IVC. One patient presented with pulmonary embolism. Seven patients underwent site-directed thrombolysis, and 3 patients received anticoagulation alone. All patients had persistent IVC occlusion regardless of treatment. Two patients had recurrent thrombosis while on anticoagulation. There were no provoking factors identified that preceded the presentation of acute lower extremity thrombosis, and none had predisposing risk factors that could explain the IVC occlusion. Four patients had a negative thrombophilia work up. Five had mild thrombotic risk factors such as heterozygosity for Factor V Leiden or elevated factor VIII levels. Only one patient had a significant thrombophilic risk factor, familial mild protein C deficiency. Five patients had post thrombotic syndrome based on standardized scoring tools. All patients remain on indefinite anticoagulation. This case series suggests that chronic IVC occlusion increases the risk of unprovoked lower extremity thrombosis and pulmonary embolism in healthy children with resultant long-term complications. It also highlights the importance of imaging the IVC in young patients with unprovoked or minimally provoked iliofemoral DVT. Indefinite anticoagulation in patients presenting with unprovoked thrombosis secondary to IVC occlusion should be considered.

Disclosures

No relevant conflicts of interest to declare.
Hyperviscosity Syndrome: A 30-Year Experience in a Tertiary Referral Center in Mexico City
Authors: Armillas-Canseco FM , Hernández-Mata CF , Gómez-Ruiz IA , Aguayo Alvaro , Martínez-Baños D
Published: October 2019

Background: The hyperviscosity syndrome (HVS) is a rare entity which refers to clinical consequences of the high blood viscosity due to abnormal increase in the cellular or acellular blood components. It frequently presents secondary to plasma cell dyscrasias but it also occurs in autoimmune diseases and chronic infections.

Methods: Retrospective and descriptive study. Clinical records of patients with HVS were analyzed between the period of January 1st 1984 and June 30th 2015. We reported etiology, clinical and biochemical features, treatment and outcome.

Results: During this period we evaluated 20 episodes of HVS in 14 patients (50% men). The mean age was 59 years. Ten patients (71%) debuted with HVS as the initial presentation of the underlying disease. Diseases associated to HVS were: five cases of multiple myeloma (MM) (35.7%), 4 Waldenström's macroglobulinemia (WM) (28.5%), 4 Sjögren's syndrome (SS) (28.5%) and 1 case with non-Hodgkin lymphoma (NHL). Seventy percent of the episodes were due plasma cells dyscrasias (IgM 55%, IgG 20%, IgA 10%). Neurological symptoms were present in 100% of the episodes, hemorrhagic manifestations in 70%, visual symptoms in 65% and cardiovascular in 30%. Visual symptoms were more frequent in SS in comparison with MM and WM (p=0.022, p=0.001, respectively). The mean of serum viscosity at diagnosis was 12.6±14.4 (higher in WM and SS, p=0.0216 and p=0.0285, respectively), and after treatment 3.7±2. The mean time elapsed between diagnosis and first session of plasmapheresis and diagnosis and specific treatment of the cause was 3.8±3.8 and 7.95±7.28 days, respectively. In all patients one plasma exchange for session was done and the median of sessions required was 2 (range 1-3). Twenty eight percent of the patients (4/14) had at least one relapse. Seven of the fourteen patients died during follow-up, none due to HVS. All deaths were secondary to infectious complications associated with underlying disease. The median of overall survival was 2071 days (CI95% .000-6201.386). Overall survival divided by diagnosis was different (p=0.002). One and 5 year overall survival were 84 and 58%, respectively. Progression-free survival was 485 days (CI95% .000-1975.58)

Conclusions: In 70% of cases the HVS was the initial presentation of the underlying disease. The main causes were multiple myeloma followed by Waldenström's macroglobulinemia and Sjögren's syndrome. The predominant clinical manifestations were neurological and hemorrhagic symptoms, however visuals symptoms predominated in SS. The highest serum viscosity mean was present in WM and SS. No patient died as a consequence of HVS.

This is the largest study of HVS in Latin America and one of the largest series of HVS worldwide.

Disclosures

No relevant conflicts of interest to declare.
Alteration of HSC Functions in Thalassemia
Authors: Aprile Annamaria , Lidonnici Maria Rosa , Gulino Alessandro , Tripodo Claudio , Mandelli Giacomo , Ferrari Giuliana
Published: October 2019

Beta-thalassemia represents one of the most globally widespread monogenic disorders and is characterized by significantly reduced or absent synthesis of hemoglobin beta-chains. In its severe form the insufficient production of adult hemoglobin results in altered erythropoiesis, hemolytic anemia, bone marrow (BM) hematopoietic hyperplasia and splenomegaly often associated with extramedullary hematopoiesis, requiring regular blood transfusions and iron chelation treatment. Over the last two decades many progresses were made in the field of allogeneic bone marrow (BM) transplantation to definitively cure beta-thalassemia. In parallel, experimental autologous transplantation protocols were developed to correct the disease by gene therapy also in patients lacking a compatible donor. Both in the allogeneic and autologous setting, thalassemic hematopoietic stem cells (HSCs) and the BM niche represent central elements.

Although many aspects of the pathophysiology of thalassemia have been extensively investigated, the HSC and its niche have never been explored. In thalassemia, the BM is a stressed environment, characterized by the compensatory expansion of erythroid progenitors secondary to ineffective erythropoiesis. Whether other hematopoietic subpopulations, such as primitive progenitors and/or HSCs, might be affected by such an altered hematopoietic microenvironment is unknown.

We investigated the frequency of hematopoietic progenitors in a murine model of severe beta-thalassemia intermedia. Immunophenotypic analyses revealed no differences in MEP, GMP, CMP, LMPP and MPP committed precursor subpopulations, whereas a significantly lower frequency of HSCs (Lin- Sca-1+ c-kit+ CD48- CD150+) was observed in thalassemic mice, as compared to age-matched wild-type controls. Competitive transplantation experiments revealed a disadvantage in the engraftment capacity of thalassemic HSCs, which was substantiated by the preliminary results from in vitro and in vivo cell cycle analyses suggesting an accelerated HSC exhaustion. Analyses of other cellular components, such as BM stroma and differentiated hematopoietic cells, revealed that additional elements are altered in the thalassemic BM microenvironment. The cellular and molecular bases of HSC-niche interaction in this pathological condition are under investigation.

Our results uncover a previously ignored defect of HSCs in beta-thalassemia. The investigation of cellular and molecular players that might affect in trans HSC functions in the complexity of this altered microenvironment is ongoing.

Disclosures

No relevant conflicts of interest to declare.
EC-18 (1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol) Exhibits the Immune Regulatory Role through HSC Differentiation of Bone Marrow Cells in Mice
Authors: Lee Ha-Reum , Shin Su-Hyun , Yoo Nina , Yoon Sun Young , Kim Myung-Hwan , Suh Cheolwon , Sohn Ki-Young , Kim Heung-Jae , Han Yong-Hae , Chong Saeho , Kim Jae Wha
Published: October 2019

EC-18 (1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol) was originally isolated as a component of an extract from deer antler is traditionally used as an oriental medicine for hematopoiesis. A trace of MADG (monoacetyldiacylglyceride) could be detected in the deer antler, animal tissue, seed oils and bovine udder, which is identical to its natural source of MADG was chemically synthesized with glycerol, palmitic acid and linoleic acid. Previous study described that EC-18 has effect on the proliferation of hematopoietic stem cells (HSCs) (Biol. Pharm. Bull. 2004, 27(7): 1121-1125). Successively, we investigated the biological role of EC-18 in the differentiation of bone marrow cells into progenitor cell population in mice. EC-18 was administered daily for 1, 5 and 15 days to 6 week-aged C57BL/6 mice. Bone marrow cells of EC-18 administrated mice were collected from femurs and tibiae. Cell population was analyzed by FACs. As results, the total number of bone marrow cells was increased in EC-18 administered mice. To identify hematopoietic lineage cell population, we used lineage cocktail antibody kit including anti-mouse CD3, CD11b, CD45R, Gr-1 and TER-119. The lineages negative population contains HSCs which can self-renew and generate into all lineages of the hematopoietic system. The lineage negative cells significantly increased in time dependent manner in the EC-18-daily administrated mice. Cell population between myeloid progenitor (Lin- Sca1- Kit+) and common lymphoid progenitors (Lin- Sca1+ Kit+ IL-7R-α+) were analyzed using the antibody for Sca-1, c-Kit, IL-7R-α. The data showed that the population of myeloid progenitor cells was markedly increased rather than common lymphoid progenitor cells. In myeloid progenitor cell population, the cell population of megakaryocyte/erythrocyte progenitors (MEP) and granulocyte-monocyte progenitors (GMP) also elevated significantly. Taken together, EC-18 may have a potential role in the HSC differentiation and could be used as a therapeutic agent for anemia and neutropenia.

Disclosures

No relevant conflicts of interest to declare.
Late Clonal Disorders in Patients with Aplastic Anemia
Authors: Kokhno Alina , Mikhaylova Elena A , Fidarova Zalina T. , Ustinova Elena , Troitskaya Vera V. , Sokolov Andrey N. , Mendeleeva Larisa P. , Kuzmina Larisa A. , Tsvetaeva Nina V. , Dvirnyk Valentina N. , Obukhova Tatiana N. , Kovrigina Alla M. , Parovichnikova Elena N. , Savchenko Valeri
Published: October 2019

Long-term survival of AA patients as a result of effective immunosuppressive therapy (IST) is accompanied by an increased risk of MDS / AML development. Clonal evolution of AA in MDS / AML is detected in 2,3-10,6% of cases ( M.G.Afable et al, Hematology ASH Educ Program., 2011; p.90-95).

From 1996 till 2015 385 pts were treated in National Research Center of Hematology, Moscow, Russia. Seventeen (4,4%) pts ( 8 men and 9 women; median age 23 years) with AA (6 severe and 11 moderate AA) were diagnosed with clonal evolution to MDS, AML and CMML.

At the moment of AA diagnostics all those 17 pts had normal cytogenetics and were treated by: ATG+CsA+/-splenectomy (n=8); CsA+/-splenectomy (n=9). G-CSF was not administered in any case. Primary response (hematological response) was registered in all 17 pts with the further development of CR in 13 pts; median response duration till the clonal evolution was 78 (10-360) months. Three pts at the time of progression are conducted IST (CsA).

Clonal disorders developed at a median of 80 (47-408) months from the time of AA diagnosis. The median age at the moment of clonal evolution was 34 (27-60) years. Abnormalities of chromosome 7 were found in 8 pts (44%). In pts with monosomy 7 the median time response duration till the clonal evolution was twice less than in pts with another abnormalities of karyotype (70 (47-209) months vs 145 (70-408) months).

At the moment the clonal expansion was detected all the 17 pts had morphological characteristics of dismyelopoesis, however, bone marrow was hypercellular in 7 cases, hypocellular - in 4; mixed (hyper- or hypoplasia) - in 4 cases. Fibrosis was found in 2 pts, it was difficult to detect karyotype in these cases (not mitosis).

The therapy was conducted in accordance to the type of disease and bone marrow cellularity (IST, hypomethylating agents, chemotherapy). Five pts were transplanted from allogeneic donors. The median life duration since clonal evolution was 22 (2-203) months. Five of the 17 pts are alive ( the median - 64(5 -203) months): after allo- HSST (n=3); IST (n=1); "watch and wait" strategy (n=1).

Conclusion: Our 20 years study of a big cohort of AA pts has demonstrated low frequence of clonal evolution to myeloid disorders - 4,4%. We suggest that the absence of G-CSF in our IST programs may contribute to this fact. Almost half of AL/MDS cases harboured monosomy 7 and the median time to clonal evolution was twice less to this pts (70 vs 145 months). The only curative approach in case of clonal evolution to AL/MDS in pts with AA is allo-HSST.

Disclosures

No relevant conflicts of interest to declare.
No Observation of Chromosomal Instability after Transplantation of RPS14- and TP53-Modified Human HSCs in NSG/NSGS Mice
Authors: Thomay Kathrin , Salari Azam , Vajen Beate , Schienke Andrea , Hagedorn Maike , Ebersold Juliane , Kreipe Hans Heinrich , Krueger Andreas , Schambach Axel , Schlegelberger Brigitte , Göhring Gudrun
Published: October 2019

Chromosomal instability (CIN) is one of the characteristics of myelodysplastic syndromes (MDS) and plays an important role in diagnosis and prognosis as well as in disease progression. Candidate genes are known, but their role in induction of CIN has never been shown due to difficulties in culturing MDS cells long-term, showing the urgent need to have a model to study the fate of cells with typical MDS abnormalities with regard to CIN. The aim of our approach was to establish a xenograft transplantation model, transplanting human hematopoietic stem cells (HSC) with different independent lentivirally-mediated MDS-related modifications into immunodeficient mice. The establishment of appropriate xenograft mouse models is increasing worldwide. Thus, experiences should be shared to contribute towards minimizing loss of mice in this process.

HSCs were intrafemorally transplanted into immunodeficient NSG and/or NSGS mice. Here, we chose 3 different modifications that, in part, were to be combined in later experiments. 1) RPS14-haploinsufficient HSCs via shRNA knockdown, 2) TP53-deficient HSCs via shRNA knockdown and 3) HSCs, with different lentivirally introduced TP53 hotspot mutations (R248W, R175H, R273H, R249S).

We achieved engraftment with HSC in these mice (Figure 1). Cytogenetic analyses of the bone marrow showed human cells with normal karyotypes. However, we were not able to induce chromosomal instability into the cells. NSGS mice transplanted with HSCs carrying a TP53 mutation developed anemia but no histopathological signs of myelodysplasia. In all experiments with NSG mice, mainly control cells as well as the untransduced GFP-negative (GFP-) cells engrafted, not allowing observation of the modified HSCs over a long period. In NSGS mice, the engraftment rate was higher, but mice developed graft-versus-host disease (GVHD).

In summary, engraftment of HSC cells in NSG and NSGS is very promising and could be used to analyze the induction of chromosomal instability. However, the analysis of transduced HSCs with different vectors is limited as yet and further transplantations with the co-transplantation of mesenchymal stroma cells and the inhibition of GVHD are required to improve this model.

Selected examples from the different transplantation experiments representing engraftment success to some extent. Depicted is the median percentage of CD45+ cells equaling engraftment of human cells in target vector- and control vector-transduced cells as well as untransduced mock cells.

Figure 1. Figure 1.

Disclosures

No relevant conflicts of interest to declare.
A Hyperactive Transcriptional State Marks Genome Reactivation during Mitotic Exit
Authors: Blobel Gerd A. , Hsiung Chris C.S. , Huang Peng , Keller Cheryl , Ginart Paul , Jahn Kristen , Bartman Caroline , Stonestrom Aaron James , Evans Perry , Giardine Belinda , Hardison Ross C. , Raj Arjun
Published: October 2019

The tremendous proliferative capacity of erythroid precursor cells underlies the production of over a million red blood cells per second in adult humans. During every mitosis the mammalian nucleus is disassembled and transcriptionally silent. Genome reactivation after mitosis is a key step in the propagation of transcriptional programs through cell generations, yet how this occurs remains largely unexplored. We carried out the first genome wide survey of transcription in cells emerging from mitosis using RNA polymerase II ChIP seq in purified populations at various post-mitotic time points. Using unsupervised approaches, we discover and classify genome reactivation patterns among genes. A surprisingly large fraction of genes (~25%) displays a post-mitotic spike in transcription. This spike represents the first complete round of transcription and accounts for the greatest gene-to-gene variance in temporal patterns of transcription in G1 phase. Another notable and contrasting pattern is gene activation late in the G1 phase.

Single-molecule RNA FISH imaging demonstrates that the post-mitotic transcriptional spike represents the highest activity throughout the cell cycle and results in an increase in mature mRNAs, indicating that the phenomenon has the capacity to alter gene expression. Surprisingly, the post-mitotic transcriptional spike occurs independent of enhancer action and can be recapitulated with promoter sequences ectopically integrated into the genome. In contrast, late-G1 gene reactivation is distinct and requires enhancer function.

Our findings uncover novel modes of transcriptional control during exit from mitosis with implications for our understanding of transitions in gene expression states in dividing cells.

Disclosures

No relevant conflicts of interest to declare.
CREB Controls C/Ebpd to Impair Myeloid Differentiation Process in Acute Myeloid Leukemia
Authors: Tregnago Claudia , Manara Elena , Bisio Valeria , Borga Chiara , Bresolin Silvia , Aveic Sanja , Germano Giuseppe , Basso Giuseppe , Martina Pigazzi
Published: October 2019

Purpose. The cAMP response element binding protein (CREB) is a transcription factor documented to be crucial for normal and neoplastic hematopoiesis. Its overexpression has already been demonstrated to impair myelopoiesis and to aberrantly control cell proliferation, apoptosis and cell cycle progression, both in vitro and in vivo. Its protein overexpression has been found in patients with acute myeloid leukemia (AML) contributing to decrease survival. CREB guides the expression of more than 5000 different targets in a tissue/cell-type specific fashion, and to date its main network towards leukemogenesis remains unknown. Here we aim to identify through which genes CREB triggers AML by using a CREB overexpressing transgenic zebrafish which developed AML in adulthood.

Patients and methods. We performed gene expression profile of RNA from the kidney marrow of 14-months old CREB-zebrafish (n=5) and control zebrafish (n=5). Principal component analysis was performed using Partek Genomic Suite software to integrate zebrafish AML signature with human signature found in pediatric AML to find common CREB target genes. AML cell lines were used to validate CREB targets in vitro.

Results. By GEP analysis and integration data we found 20 differentially expressed genes in both the zebrafish and human leukemia. Several of them were involved in the myeloid differentiation process, such as JUN, FOS and C/EBPδ. We confirmed that C/EBPδ protein levels highy correlated with phosphoCREB levels in zebrafish tumor as well as in a cohort of 66 pediatric patients with AML (r=0.79). We silenced and overexpressed CREB in AML or healthy bone marrow primary cultures respectively, and found that C/EBPδ was controlled by CREB transcriptional activity. We took advantages of a previously created HL60(CREB-) cell line, where CREB protein levels was absent compared to the control cell line called HL60(CREB+), where CREB was overexpressed. HL60(CREB-) cell line expressed lowered C/EBPδ levels compared to HL60(CREB+). To investigate CREB's role on differentiation program, cell lines were treated with all-trans-retinoic acid (ATRA). The evaluation of CD11b expression revealed that HL60(CREB-) differentiated to a higher extent compared to HL60(CREB+).Enhanced myeloid differentiation was also confirmed by cell morphology examination. Then we silenced C/EBPδ gene in HL60(CREB+) cell line and treated with ATRA, confirming that C/EBPδ lowered expression increased CD11b expression, phenocopying the HL60(CREB-) behavior.

Conclusion. These results indicated that C/EBPδ is a CREB target crucial for myeloid differentiation. The new role discovered for CREB proto-oncogene in blocking myeloid differentiation process opens for further opportunities with differentiation agents to cure AML.

Disclosures

No relevant conflicts of interest to declare.
In silico Tissue-Based Expression Analysis of YY1 and Cancer Stem Cell (CSC) Transcription Factors in Hematological Malignancies: Unraveling New Therapeutic Targets
Authors: Kaufhold Samantha , Garban Hermes , Bonavida Benjamin
Published: October 2019

Introduction: Yin Yang 1 (YY1) is a transcription factor, (a 44 kDa protein), ubiquitously expressed in many tissues and cancers. YY1 exerts multiple functions, including transcriptional regulation of many genes involved in cell proliferation, chromatin remodeling, drug and immune resistance, and metastasis [1].Cancer stem cells (CSCs) are a small subset of cancer cells that drive tumorigenesis and metastasis and are considered to be resistant to cytotoxic therapies; they are pluripotent and capable of self-renewal.

Objective: To determine if YY1 is a transcription factor that regulates CSCs.

Hypothesis: We hypothesized that YY1 may be overexpressed in CSCs, and its expression may be coordinated with the expression of CSC transcription factors. The overexpressions and activities of SOX2, OCT4 (POU5F1), BMI1 and NANOG are characteristics of the CSC phenotype in many cancers.

Methods: The above hypothesis was tested by comparing the expression patterns of the four CSC markers and YY1 in hematological malignancies. The data were collected from the Human Protein Atlas proteomics database, and only high and medium expressions were considered positive.

Results: From the data that were collected, the overall percentage of positively stained cells was determined. Our preliminary findings demonstrated that there was a strong correlation between the expression patterns of YY1 and BMI1, some correlation between YY1, OCT4 and SOX2, and no correlation between YY1 and NANOG, which was usually underexpressed. The interactomes of OCT4 and SOX2 are important parts of the regulatory network of hESCs. Further, multiple DNA binding proteins, including YY1, are enriched in both interactomes [2].

Conclusion: The findings suggest strongly that CSCs may overexpress YY1 in coordination with the overexpressions of SOX2, BMI1, and OCT4. They further suggest that YY1 may constitute a CSC biomarker, and they revealed potential therapeutic targets.

[1] Critical Reviews™ in Oncogenesis Volume 16, 2011 [2] Gao F et al., Sci Report 3:1588,2013.

Disclosures

No relevant conflicts of interest to declare.
Downregulation of SATB1 Increase the Invasiveness of Jurkat Cell Via Activation of the WNT/Beta-Catenin Signaling Pathway in Vitro
Authors: Luo Xiaodan , Xu Lihua , Liu Dan , Wang Yaya , Wu Xiaohong , Tan Huo
Published: October 2019

Backgroud: Special AT-rich sequence-binding protein-1 (SATB1) is critical for genome organizer that reprograms chromatin organization and transcription profiles, and associated with tumor growth and metastasis in several cancer types. Many studies suggest that SATB1 overexpression is an indicator of poor prognosis in various cancers, such as breast cancer, malignant cutaneous melanoma, liver cancer, etc. However, their expression patterns and function values for adult T-cell leukemia (ATL) are still largely unknown. Objective: The aim of this study is to examine the levels of SATB1 in ATL and to explore its function and mechanisms in ATL.

METHODS: 20 ATL peripheral blood samples and 20 normal controls were collected. Expressions of SATB1 in both groups were evaluated by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). Cellular proliferation and invasion of SATB1-knockdown Jurkat cells and cells in control group were evaluated by manually count and transwell matrigel invasion assay, respectively.

RESULTS: SATB1 expressions were decreased in ATL peripheral blood mononuclear cells (p<0.001) compared with normal controls. Knockdown of SATB1 gene might increase Jurkat cell invasiveness through the activation of Wnt/β-Catenin signaling pathway.

CONCLUSIONS: SATB1 expression is down-regulated in ATL and decreased expression of SATB1 increase the invasiveness of Jurkat cell through the activation of Wnt/β-Catenin signaling pathway in vitro.

Acknowledgments This study was supported by grants from the National Natural Science Foundation of China (81200399) and Key Clinical Disciplines of Guangdong Province (20111219)

Disclosures

No relevant conflicts of interest to declare.
Identification of Novel Mir-34a Targets in a c-Myc Murine Model
Authors: Contreras Jorge R. , Tran Tiffany M , Palanichamy Jayanth Kumar , Casero David , Rao Dinesh S.
Published: October 2019

MicroRNAs (miRNAs) serve a fundamental function in the post-transcriptional regulation of gene expression. MiRNA-mediated repression is mediated by the RNA-induced silencing complex (RISC) and is generally thought to be predicted by base-pairing in the seed region of the microRNA. In predicting the effects of miRNAs on gene expression, it is important to consider that the transcriptional context will result in differential targeting of transcripts, and necessitates the study of miRNA function in the cell type of interest. Here, to study tumor suppressive functions of two miRNAs, miR-146a and miR-34a, we utilized the Eμ-Myc transgenic mouse bred to targeted knockouts for these two miRNAs. We found that miR-146a deficiency led to more aggressive B-cell neoplasms that showed a more differentiated B-cell phenotype. Transcriptional changes were fairly limited and were not enriched for miR-146a targets as a class. However, the changes included differences that were consistent with regulation of the transcription factor Egr1, which was demonstrated to be a direct target in human cells. In miR-34a deficient mice with the Eμ-Myc transgene, we did not observe changes in survival or in the phenotype of the neoplasms that developed. However, transcriptional profiling revealed changes in many predicted miR-34a targets, as determined by gene set enrichment analyses (GSEA). Novel predicted targets are being analyzed by luciferase assays. These analyses reveal the importance of assessing cell-type and disease-specific miRNA targeting, which can reveal novel and previously unknown miRNA targets and facilitate our understanding of leukemia and lymphoma pathogenesis.

Disclosures

No relevant conflicts of interest to declare.
Potential Crosstalk of Interleukin-6-Heme Oxygenase-1 Dependent Mechanism Involved in Resistance to Lenalidomide in Multiple Myeloma Cells
Authors: Wang Jishi , Wu Weibing , Ma Dan , Wang Ping , Cao Lu , Fang Qin , Zhao Jiangyuan , Lu Tangsheng
Published: October 2019

Interleukin-6 (IL-6), as one of the most important multiple myeloma (MM) survival factors, has been verified to determine poor prognosis of MM. IL-6 mainly originates from paracrine of bone marrow stromal cells and autocrine of MM cells. As an enzyme having cytoprotective effects, heme oxygenase-1 (HO-1) promotes the growth and drug resistance of various malignant tumors. The HO-1 expression levels in bone marrow CD138+ cells of MM patients, which were significantly higher than those of healthy donors, were positively correlated with serum IL-6 levels and intracellular IL-6 mRNA expression levels. Cultivating U266 and CD138+ cells with exogenous IL-6 in vitro induced high HO-1 expressions and allowed them to resist lenalidomide, probably because IL-6 activated the Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) pathway. Without IL-6 co-culture, on the other hand, up-regulating HO-1 expressions in U266 cells and bone marrow CD138+ cells from MM patients significantly up-regulated the mRNA expression level of IL-6 and facilitated autocrine IL-6 production. The findings were associated with high HO-1 expression-enhanced of p38 MAPK phosphorylation. Down-regulating HO-1 expression sensitized U266 and CD138+ cells toward lenalidomide. Therefore, we postulated that HO-1 predominantly controlled IL-6 paracrine and autocrine, and that IL-6 in bone marrow microenvironment of MM patients stimulated MM cells to highly express HO-1 and to resist lenalidomide. High HO-1 expression also stimulated autocrine IL-6 production, thus further augmenting the drug resistance and exacerbating the disease. This study provides valuable experimental evidence for using HO-1 as a possible marker for MM prognosis and drug resistance and as a potential treatment target.

Disclosures

No relevant conflicts of interest to declare.
Histone Deacetylation Inhibitor Sensitizes T-ALLs to Topoisomerase II Inhibitor By Attenuating G2 Cell Cycle Arrest and Blocking Nuclear Entry of Ctip during DNA Repair
Authors: Liu Jin-Hwang , Lin Yu-Hern , Liu Hon-Wen , Gau Jyh-Pyng
Published: October 2019

Double strand DNA repair can be epigenetically modulated to sensitize the malignant cells to chemotherapy. CTV-1 and Molt-3 were segregated as T-acute lymphoblastic cells (T-ALL) according to their expression profiles. We found in these cells doxorubicin, a topoisomerase II (TOP2) inhibitor, induced expression and phosphorylation of cell cycle checkpoint kinase 1(CHK1) associated with G2 cell cycle arrest before achieving apoptosis; while a histone deacetylation inhibitor (HDACi), suberoylanilide hydroxamic acid (SAHA), synergistically enhanced cell death by attenuation of expression and phosphorylation of CHK1, and by attenuated expression and phosphorylation of a double strand DNA repair protein, C-terminal binding protein (CtBP)-interacting protein [CtIP], and was associated with shortened G2 cell cycle arrest. Very often is Doxorubicin as a topoisomerase II inhibitor enrolled in the treatment of T-ALL. We evidenced with isobologram synergistic cell-killing by doxorubicin and SAHA in CTV-1 and Molt-3 T-ALL cell lines of which expression and phosphorylation of CHK1 were negatively affected by SAHA. In the SAHA-treated T-ALL cells, the repair of doxorubicin-induced double strand DNA break (DSB) was associated with increased γH2AX. However, although SAHA increased expression of γH2AX and acetyl H2AX, apoptosis was enhanced with shortened G2/M arrest and the hampered nuclear entry of CtIP revealed by immunofluorescent confocal microscopy. HDACi thus synergistically impaired the topoisomerase II-induced DSB repair and enhanced apoptosis by eliciting DNA repair with γH2AX expression; however, aborted the DNA repair and induced apoptosis by hampering the nuclear entry of CtIP. Attenuation of activated CHK1 may be potentially a biomarkers of synergistic cytotoxicities of HDACi and DSB-inducing chemotherapeutics.

Disclosures

Off Label Use: in vitro study only. suberoylanilide hydroxamic acid in combination with topoisomerase II inhibitor may have synergistic effects. .
Coagulation Disorder after Treatment of Pegaspargase and L-Asparaginase
Authors: Xu Linwei , Wei Xiaolei , Wei Yongqiang , Huang Fen , Hao Xiaoxiao , Zhou Qinjun , Feng Ru
Published: October 2019

Introduction:

Pegaspargase(PEG-ASP) and L-asparaginase(L-ASP) has been widely used in the treatment of acute lymphatic leukemia, changes of coagulation function after treatment are not actually the same. So we performed the present study to analyze coagulation disorder after PEG-ASP and L-ASP treatment of adults with acute lymphatic leukemia.

Methods and materials:

Totally 153 hospitalized patients with acute lymphatic leukemia treated with L-ASP or PEG-ASP were studied from January, 2010 to January, 2015. Of all 153 patients analyzed, they received L-ASP treatment 158 dose times and PEG-ASP treatment 60 dose times respectively.

Results:

There is no difference of the distribution of age, sex, white blood cell count at diagnosis and risk factors of the disease. Agranulocytosis combining with intestinal infection, bleeding or thrombosis and the inducing remission rate between the two groups has no significant difference (p=0.11,0.61,0.33). The total infusion of fresh frozen plasma or cryoprecipitate or fibrinogen after treatment shows no significant difference between the two groups (p=0.11,0.75,0.21).Fibrinogen level decreases slower in the treatment of PEG-ASP(9.37 day vs 7.40 day, p=0.02) than that of L-ASP. What's more, fibrinogen decreases slower when L-ASP used at interval compared with continuous use, however, the incidence rate of bleeding and related complications is higher when used at interval early (p =0.028).

Conslusion:

Because of the preponderance to monitor the changes of fibrinogen and the equal rate of complications and inducing remission rate, it is recommended to use PEG-ASP. L-ASP used at interval can monitor the coagulation function easily than continuous use, but the early use of L-ASP may overlay the drug side effects and related hematology toxicity caused by chemotherapy, then cause an increased incidence of complications.

Disclosures

No relevant conflicts of interest to declare.