bluuebunny/crossref_metadata_2025 huggingface dataset:
Azacitidine and Lenalidomide Combination In Higher-Risk Myelodysplastic Syndromes-Preliminary Results Of The Vilen-01 Protocol
Authors: Mittelman Moshe , Filanovsky Kalman , Rosenbaum Hanna , Raanani Pia , Braester Andrei , Goldschmidt Neta , Hellman Ilana , Kirgner Ilya , Perri Chava , Oster Howard S
Published: October 2019

Background

Hypomethylating agents are the standard therapy for higher-risk (HR, Int-2 and high-risk) myelodysplastic syndromes (MDS). Lenalidomide is effective in lower-risk (LR) MDS, with or without 5q-, in HR-MDS and in acute leukemia. Many patients remain resistant to a single agent. A potential synergistic effect of both agents has been shown [Sekeres M et al, Am J Hemat 2010; Blood 2012]. Thus, the MDS Israel group has designed a phase II clinical trial (NIH trial #: TASMC-10-MM-0437-09-CTIL).

Protocol

The ViLen-01 protocol consists of 3 phases. The induction phase includes 6 monthly cycles of SC azacitidine (Aza), 75 mg/m daily, days 1-5, and PO lenalidomide (Len) 10 mg daily, days 6-21, followed by a 7-day (days 22-28) respite. The consolidation consists of 6 monthly cycles of Aza only for 5 days each, followed by 12 months of maintenance with Len only. Response is evaluated by the IWG criteria [Cheson B et al, Blood 2006].

Results

As of July 2013, 7 medical centers have enrolled 18 patients. Patients had been diagnosed with either HR-MDS, or LR-MDS with transfusion-dependence, erythropoietin resistance and poor cytogenetics. Adverse events (AE) were as expected in these elderly HR-MDS patients. The common AEs were grade IV transient cytopenias, requiring dose modification in 15 patients. Only 2 patients stopped the protocol due to AE. Thirteen patients completed the induction, 8 continued to consolidation, and 5 patients continued to maintenance. Eight of the 18 enrolled patients (44%, 8 of the 13, 61.5%) who had completed the induction, achieved CR. Three of the 8 patients in CR also attained cytogenetic response. The other 5 had normal karyotype on study initiation. Five other patients obtained erythroid response and became transfusion-free, and another patient achieved platelet response, for a total of 14 (78%) responders. It is still too early to evaluate response duration and survival. One patient is in early induction, and 4 are still being treated. The others have either died or have discontinued for various reasons (patient refusal, progressive disease, transplant).

Conclusions

These preliminary data in a small group of patients with HR-MDS and expected poor prognosis, demonstrate a high response rate and a reasonable safety profile. The study is ongoing. If these results are confirmed in randomized trials, it may set new standards for the treatment of this disease.

Disclosures:

No relevant conflicts of interest to declare.
Erlotinib Is Not Effective In Patients (Pts) With JAK-2 V617F Positive Polycythemia Vera
Authors: Cherry Mohamad , Khawandanah Mohamad , Zhao Zhizhuang Joe , Srour Samer A , Ozer Howard , Selby George , Ghabache Bassam , Al-Kali Aref
Published: October 2019

Introduction

Erlotinib is an epidermal growth factor receptor small-molecule inhibitor and is FDA approved for the treatment of lung and pancreatic cancers. In preclinical study, in vitro colony culture assays revealed that erlotinib at micro-molar concentrations effectively suppressed the growth and expansion of Polycythemia Vera (PV) hematopoietic progenitor cells while having little effect on normal cells. Several JAK inhibitors are being studied for the management of PV, one of which has been approved for the treatment of myelofibrosis (ruxolitinib).

Aim

To study the clinical effect of erlotinib in pts diagnosed with JAK2V617F + PV.

Methods

We conducted a single arm, prospective phase II study at the University of Oklahoma and the Oklahoma City VA hospitals in pts with WHO defined JAK-2 V617F positive PV from June 2010 to August 2012. Appropriate IRB approval was obtained in accordance with Hilsinki declaration. Pts had to be requiring phlebotomy. Toxicity was assessed by treating physicians using NCI version 4. Dose modification for erlotinib was done using label recommendations.

Results

Five Caucasian pts were enrolled (3 (60%) males, with median age at enrollment of 63 years, range 26-79). Pts had pretreatment median hemoglobin14.4 g/dL (10.4 -19.2 g/dL), median platelet count 511 x109 (424-681 x109), median white blood cell (WBC) 14.4 x109(7.8- 18.3 x109). Three pts had splenomegaly prior to treatment. Median number of prior pharmacologic treatments (hydroxyurea, anagralide or interferon) was 1, range 0-2. Pts were given erlotinib 150 mg orally daily for 16 weeks: responders (phlebotomy free or decrease in spleen size) were allowed to continue a total of 1 year of treatment, while non-responders were taken off the study. Three (60%) patients received therapy for 16 weeks and did not achieve hematological response or improvement in spleen size. Two (40%) pts were taken off the study after 2 doses secondary to severe toxicities (grade 3 colitis in 1 case, and grade 3 facial rash in 1case). No therapy continued beyond 16 weeks (due to toxicity or lack of response). All pts in the study developed rash (grade 1 – 3) and diarrhea (grade 1 – 2). Three pts developed mucositis (see Table 1). No death was observed during the study and follow up period (median follow up was 23 months, range 12-37 months). Study was closed due to lack of efficacy.

Conclusions

Despite in vitro efficacy of erlotinib as potent inhibitor of JAK-2 activity, erlotinib is not effective in pts with JAK-2 V617F positive PV with poor toxicity profile. Poor accrual was related to potential toxicity of erlotinib compared to alternative treatments in view of lack of clinical efficacy.

Disclosures:

No relevant conflicts of interest to declare.
Alterations Of The Chemokine Microenvironment In Chronic Lymphocytic Leukemia
Authors: SEILER TILL M , Aydin Roland , Herold Tobias , Holdenrieder Stefan , Hallek Michael , Hiddemann Wolfgang , Dreyling Martin , Bergmann Manuela A.
Published: October 2019

Background

In chronic lymphocytic leukemia (CLL), proliferation of the leukemic cell clone occurs in the bone marrow and lymphatic tissues rather than peripheral blood. In this microenvironment, CLL cells interact with accessory cells, such as T-cells and CD68+ nurselike cells (NLCs). In addition, cytokines and chemokines secreted by leukemic cells, stromal cells and T-cells are essential in forming the disease-specific microenvironment. However, the exact biological role of cytokines and chemokines needs to be defined.

Methods

In order to address this issue, we measured serum levels of 20 chemokines and cytokines in a prospective cohort of 159 previously untreated Binet stage A patients (pts) (GCLLSG CLL1 trial), 50 pts with advanced CLL in need of first line treatment (GCLLSG CLL8 trial) and 27 healthy individuals. For the study cohorts, serum samples had been centrally collected at study entry and stored at -80°C. Sera were analyzed on a Luminex-based multiplex platform, allowing simultaneous screening of multiple serum parameters.

Results

Serum levels of 13 chemokines (EGF, MCP-1, MIP-1alpha, MIP-1beta, sIl2Ralpha, VEGF, MCP-2, MCP-4, SDF-1, 6CKine, CTACK, TRAIL, SCF) differed significantly between healthy controls and CLL patients (p<0.05), indicating that the expression of these cytokines may potentially impact pathways common to CLL and the disease specific microenvironment. In addition, 7 chemokine serum levels were significantly higher in advanced stage CLL compared to patients at primary diagnosis, indicating a pronounced shift of the chemokine homeostasis upon disease progression. (Table)

We identified a strong correlation of inflammatory cytokines (TRAIL, TNFalpha; p=0.039), growth factors (EGF, VEGF, TPO, SCF; p<0.001) and chemokines involved in B- and T-cell migration and homing (MIP-1alpha, MIP-1beta, 6CKine, Il-16; p<0.05). These coordinately regulated cytokines may reflect pathways important to the pathobiology of CLL. Hierarchical cluster analyses revealed distinct chemokine expression patterns in healthy individuals, early CLL and advanced stage disease. Finally, sIl2Ralpha has been previously confirmed as an independent prognostic factor in early stage CLL

Conclusion

Due to the comparison of 27 healthy individuals and 209 CLL pts we were able to demonstrate that the chemokine homeostasis is significantly altered in CLL compared to healthy individuals. Chemokines involved in B- and T-cell migration and homing and recruitment, inflammation and growth factors are coordinately overexpressed, pointing towards pathways crucial to the formation of a disease specific microenvironment. These pathways might serve as potential targets for future therapeutic strategies.

Disclosures:

No relevant conflicts of interest to declare.
Aberrant Megakaryocytic/Erythroid Progenitors Contributes To Transformation Of Cbfb-SMMHC Induced Acute Myeloid Leukemia
Authors: Cai Qi , Jeannet Robin , Liu Hongjun , Kuo Ya-Huei
Published: October 2019

Inv(16)(p13q22) is a recurrent chromosomal rearrangement found in approximately 12% of human acute myeloid leukemia (AML) cases and creates a fusion gene between CBFb and MYH11. The fusion gene encodes a fusion protein CBFß-SMMHC which causes defects in lymphoid and myeloid differentiation. Previous studies also showed that primitive erythropoiesis is impaired by CBFß-SMMHC, however, CBFß-SMMHC knocked-in cells was able to contribute to adult erythropoiesis in chimeric mice. Expressing CBFß-SMMHC in the hematopoietic cells using a conditional knock-in mouse model (Cbfb56M/+/Mx1-Cre; 129SvEv strain) recapitulates inv(16)-associated AML. Previous studies in this model showed that CBFß-SMMHC expression leads to pre-leukemic hematopoietic alterations, and together with additional cooperative mutations, result in spontaneous myeloid leukemia in mice with a 3-6 month latency. We hypothesized that an expanded cell population at the pre-leukemic stage could be the target of additional mutations, and hence the cell of origin of leukemia initiating cells.

To further delineate the pre-leukemic progenitors and leukemia initiating cells, we backcrossed Cbfb56M/+/Mx1-Cre into C57BL/6 for more than 10 generations. Similar to previous studies in the129SvEv strain, expressing CBFß-SMMHC in adult C57BL/6 mice leads to cell number dependent development of AML. Analysis of pre-leukemic bone marrow as early as 2 weeks after induction revealed a 5.7-fold expansion of Pre-Meg/E cells (Pre-Megakaryocyte/Erythrocyte: Lin-cKit+Sca1-CD16-/loCD150+CD105-) compared to similarly treated control mice. While there was a significant increase in Pre-Meg/E population, we did not find significant increase in their proliferation but observed a 4.7 fold decrease of the erythroid progenitor (EP; Lin-cKit+Sca1-CD16-/loCD105hi) subset. Methylcellulose-based colony forming assay showed that pre-leukemic Pre-Meg/E had an impaired differentiation potential for erythroid lineage. In vitro erythroid differentiation assay also showed a partial block of differentiation from pre-leukemic Pre-Meg/E progenitors. These Pre-Meg/E like progenitors were able to induce leukemia in the presence of a known cooperative oncoprotein MPL when transplanted in lethally irradiated congenic recipient mice. In summary, our Results suggest that expression of CBFß-SMMHC impairs adult erythropoiesis at the transition of Pre-Meg/E to EPs, causing an expansion of Pre-Meg/E cells, which can be the target cell of additional mutations contributing to leukemia transformation.

Disclosures:

No relevant conflicts of interest to declare.
Decoding The BH3-Mimetic Pro-Apoptotic Activity Of Quercetin In Jurkat Cells
Authors: Primikyri Alexandra , Hatzimichael Eleftheria , Karali Evdoxia , Kostaras Eleftherios , Manztaris Michalis , Shin Jae-Sun , Chi Seung-Wook , Kolettas Evangelos , Gerothanassis Ioannis , Briasoulis Evangelos , Tzakos Andreas
Published: October 2019

Deregulation of apoptosis contributes largely to the pathogenesis of lymphoid neoplasms allowing the expansion of cell clones that develop resistance to drug-induced cell death. Therefore, subverting inherent resistance to apoptosis is a very challenging therapeutic issue and developing small molecules that can mimic the BH3 domain of the pro-apoptotic Bcl-2 family proteins appears to offer such potentials. Natural products, a rich source of compounds with BH3 mimetic activity, make a sound basis for such an approach. Quercetin, a natural polyphenol, has drawn much attention because it exerts anticancer cytotoxic effects, while sparing normal cells. Pro-apoptotic effects of quercetin have been shown in human lymphoma and leukemic cells and have been associated with Bcl-2 and Mcl-1 downregulation and Bax conformational activation. We undertook a multidisciplinary approach to elucidate the mode of action of quercetin, including cytotoxicity cell assays, biochemical approaches (pull down assays), NMR spectroscopy and docking calculations. We demonstrate that quercetin binds directly to the BH3 domain of the anti-apoptotic Bcl-2 and Bcl-xL proteins, exhibiting BH3-mimetic properties. Specifically, functional cytotoxicity assays of quercetin in Jurkat T-cell leukemic lines, Jurkat Bcl-2 and Jurkat Puro, indicated that quercetin binds to Bcl-2 protein. The direct binding of quercetin to Bcl-2 and to Bcl-xL was biochemically validated using pull down assays. NMR chemical shift perturbation experiments and docking calculations finally revealed that quercetin was bound to the pro-apoptotic BH3-binding site of the anti-apoptotic Bcl-2 family proteins (Figure 1). This property classifies quercetin as a natural flavonoid with BH3 mimetic activity, capable of driving leukemic cells to apoptosis. These results explain the cytotoxic effects of quercetin on Jurkat cells. These data can serve as a basis to consider studying BH3 mimetic natural products as adjuncts in the therapeutic approaches of lymphoid neoplasms in combination with chemotherapy, and can also provide a starting structure for developing novel potent analogues for the treatment of blood cancers.Figure 1Modeling (3D) the complex of quercetin with Bcl-2 (A) and Bcl-xl (B). Quercetin occupies the deep hydrophobic cleft in both proteins making an extended interaction network. The relevant residues are normally occupied by the pro-apoptotic Bak/Bad BH3 binding site in the complexFigure 1. Modeling (3D) the complex of quercetin with Bcl-2 (A) and Bcl-xl (B). Quercetin occupies the deep hydrophobic cleft in both proteins making an extended interaction network. The relevant residues are normally occupied by the pro-apoptotic Bak/Bad BH3 binding site in the complex

Disclosures:

No relevant conflicts of interest to declare.
The ASH Clinical Research Training Institute (CRTI) Positively Impacts The Success Of Early Career Hematologists In Patient-Oriented Clinical Research
Authors: Burns Linda J , Clayton Charles P , George James N. , Mitchell Beverly S. , Gitlin Scott D.
Published: October 2019

There is a recognized need to translate scientific discoveries to patient-oriented clinical research (POCR) in order to improve human health. To overcome obstacles that interfere with the recruitment and retention of physicians for POCR careers, in 2003 the American Society of Hematology (ASH) developed the Clinical Research Training Institute (CRTI), an enhanced and extensive mentored experience for early career POCR investigators. The ASH CRTI is limited to 20 trainees/year (senior fellows or junior faculty) with an equivalent number of faculty mentors. The year-long Institute includes a 1 week workshop with didactic sessions on a variety of POCR and career development topics, disease-specific small group sessions focusing on research protocol development with biostatistician participation, and one-on-one interactions with faculty and representatives with expertise in career-development awards from the U.S. National Institutes of Health. Trainees have additional interactions with their small groups and CRTI mentors throughout the ensuing year to promote career development and research collaborations. Communications between the trainees’ CRTI and home institution mentors are a vital component of the program. Evaluation of the POCR career development successes of the first 7 CRTI classes (140 trainees) was performed by reviewing trainees’ curriculum vitaes and their responses to an electronically distributed survey. Gender, racial and ethnic distributions were similar to that of U.S. Hematology/Oncology fellowship programs. The majority (66%) of trainees were senior fellows or graduates of adult hematology/oncology training programs, 31% of pediatric hematology/oncology programs and the remainder of other hematology-related programs. Eighty-six percent of trainees had self-described success establishing a POCR study and 85% considered themselves clinical investigators. Nearly half of trainees had positions that were primarily research focused. CRTI trainees received at least 144 external grant awards plus additional internal awards, and had published 1035 peer-reviewed manuscripts,173 chapters and 115 review articles. Over 95% of trainees were satisfied with their CRTI experience and testimonials supported their impression that CRTI had a significant impact on their career success. Enhancements introduced into the CRTI program, as a result of ongoing program evaluations, include broadening participant eligibility to include up to 5 international trainees per class to enhance global research collaborations and further enhancements to the trainee-mentor interactions. The outcomes of the ASH CRTI support the hypothesis that enhanced mentoring experiences contribute to the successful career development of physicians pursuing POCR careers.

Disclosures:

Clayton: American Society of Hematology: Employment.
Hematologic Recovery After Liver Transplantation
Authors: Seidler Christopher , Bozorgzadeh Adel , Ferrante-Hersperger Cheryl , Al-Mansour Zeina , George Mridula , Patel Bhavisha , Rawson Amanda
Published: October 2019

Introduction

Chronic liver disease is a national problem with over 3 million American suffering from end stage liver disease. The majority of patients with advanced liver disease have pancytopenia due to multiple factors. Hematology consults generated by the hematologic effects of end stage liver disease comprise 10-20% of consults. The majority of patients undergoing orthotopic liver transplantation achieve some degree of partial or complete hematologic recovery.

Methods

196 sequential records for patients undergoing orthotopic liver transplantation were analyzed. Complete data was available for 92 patients. Hematologic parameters were reviewed 1 month prior to transplantation, 1 month post, 1 year and 2 year post transplantation. A statistical analysis was performed using the two tailed t-test method.

Results

Rapid and sustained hematologic recovery occurred in the majority of patients and was significant except for WBC at 24 months.

Conclusions

Orthotopic liver transplantation results in rapid sustained hematologic response in the majority of patients. Rapid recovery of platelet counts was most significant and does not reflect Ssplenectomy. Co-morbidity and anti-rejection drugs also may lead to delayed marrow suppression. Further sub-set analysis and measure of additional factors will also be presented.

Disclosures:

No relevant conflicts of interest to declare.
Immunosuppression (IST) Can Be Safely Ceased During Chemotherapy For Post-Transplant Lymphoproliferative Disorders (PTLD) In Renal Transplant Patients
Authors: Taylor Emma , Jones Mark , Hourigan Matthew J , Johnson David W , Gill Devinder S , Isbel Nicole , Hawley Carmel M , Marlton Paula , Gandhi Maher K , Campbell Scott B , Mollee Peter
Published: October 2019

Introduction

The optimal reduction of IST in renal transplant patients with PTLD is uncertain. As chemotherapy used to treat PTLD is inherently immunosuppressive, IST may be able to be stopped during this time without compromising graft function. Subsequent long-term reduction of IST is important to reduce PTLD relapse, but may increase long-term risk of graft rejection.

Methods

We performed a retrospective matched cohort study of adult renal transplant patients where IST was ceased during chemotherapy and then resumed at lower dose (calcineurin inhibitor at 50%, prednisolone <6mg daily, no third agent). Outcomes were compared to renal transplant patients without PTLD, matched for creatinine at the equivalent time post-transplant that PTLD was diagnosed in the cases, as well as age, gender, year of transplant and age at transplant. The primary endpoint of time to renal deterioration, defined as the time from PTLD diagnosis (cases) or study entry (controls) to a 25% increase in creatinine, was analysed using competing risk survival analyses. Additional endpoints were time to renal allograft failure requiring dialysis or re-transplant and overall survival.

Results

24 cases were identified: median age at PTLD diagnosis 45yrs; 75% male; median time to onset of PTLD post-transplant was 9 years. 22 cases received CHOP-like chemotherapy and 2 received primary CNS lymphoma protocols, 7 received concurrent rituximab and 4 had consolidation radiation therapy. 88% attained complete remission. Five patients have relapsed. The 5 yr overall survival for the cases was 71% with a median follow-up of survivors of 11.9 years.

The 24 PTLD cases were compared to 84 well matched controls. There were 11 deaths in the cases and 5 in the controls. Three cases recommenced dialysis, compared to 3 controls (HR 2.5, p=0.27). The 5 yr rate of 25% increase in creatinine was 36% in cases and 20% in controls (HR 1.8, p=0.098). Of the 11 cases with ≥25% increase in creatinine, only 1 of these occurred within 6 months of IST cessation in the setting of rapidly progressive PTLD and early death. Only 2 of these 11 cases received rituximab. In contrast 5 of the 7 patients given rituximab did not develop a ≥25% deterioration in renal function.

Conclusions

IST can be safely ceased during chemotherapy for PTLD in renal transplant patients. Whilst long-term reduction in IST is necessary to reduce PTLD relapse, this is associated with a trend to increased risk of chronic allograft nephropathy. Prospective trials are needed to address the ideal reduction of IST in PTLD.

Disclosures:

No relevant conflicts of interest to declare.
Pregnancy Does Not Affect The Thrombotic Thrombocytopenic Purpura (TTP) phenotype Of ADAMTS13-Deficient Mice
Authors: Hayon Yael , Varon David , Kalish Yosef
Published: October 2019

Thrombotic thrombocytopenic purpura (TTP) is a life threatening disease, characterized by sudden onset of the pentad of fever, hemolytic anemia, thrombocytopenia, renal dysfunction and neurological findings. Previous studies in ADAMTS13-deficient mice demonstrated a dependence on both genetic and environmental factors for the expression of the disease. Though homozygous Adamts13-/- mice on a C57BL6/J (B6) background are indistinguishable from wild-type controls, introduction of the CAST/EiJ genetic background results in a spontaneous TTP-like syndrome in a subset of mice.

The aim of this study was to evaluate the effect of pregnancy on TTP expression in this mouse model of TTP.

Adamts13 +/- mice were backcrossed for 20 generations to C57BL/6J to generate N20 B6 Adamts13+/- progeny. These mice were backcrossed for 2 generations into the CAST/EiJ mouse strain to generate N2F1 Adamts13-/- female mice that were tested and compared to N20F1 B6 Adamts13-/- female mice.

Two groups of 20 Adamts13-/- female mice from each strain were tested. Mice from one group were mated with wild type males, and mice from the second group were housed separately from male mice. Pregnant Adamts13-/- mice were compared to control non-pregnant Adamts13-/- female mice. Mice were monitored for a full profile of blood parameters and peripheral blood smears weekly for 10 weeks. Data was collected for mortality, pregnancy outcome and litter size. Von Willebrand factor (VWF) levels were also measured.

All Adamts13-/- mice in the B6 non-pregnant group appeared healthy and exhibited normal blood count parameters, without microangiopathic changes on peripheral blood smears. None of Adamts13-/- mice in the B6 pregnant group developed TTP. Mean platelet count in this group was 844,000±127,000 cells/µl compared to 666,000±56,000 cells/µl in the B6 non-pregnant group (p<0.01)

In contrast to the B6 results, Adamts13-/- CAST/EiJ non-pregnant mice exhibited lower platelet counts 369,000±89,000 cells/µl with spontaneous TTP-like disease in 54% of mice, as expected from previous studies.

Pregnancy did not worsen TTP expression in Adamts13-/- CAST/EiJ mice. Only 45% of mice in the CAST/EiJ pregnant group developed low platelet counts with a higher mean platelet count of 511,000±160,000 cells/µl, compared to non-pregnant group(p<0.01).

The physiological rise in VWF levels of pregnancy had no effect on TTP susceptibility in this model. VWF antigen levels were 167%, 203%, 197% and 245% in B6 non-pregnant, B6 pregnant, CAST/EiJ non-pregnant and pregnant respectively, compared to wild-type B6 levels. Four mice died during follow-up, 2 in the B6 pregnant and 2 in the CAST/EiJ non-pregnant groups.

In summary, TTP expression was unique to Adamts13-/- CAST/EiJ mice. Pregnancy and elevated VWF levels had no deleterious effect on TTP susceptibility in this mouse model.

Disclosures:

No relevant conflicts of interest to declare.
Efficacy, Immunogenicity, Pharmacokinetics, and Safety Of Human-cl rhFVIII – a GCP Study In Children With Severe Haemophilia A
Authors: Liesner Raina , Jansen Martina , Klukowska Anna , Vdovin Vladimir , Szczepanski Tomasz , Knaub Sigurd
Published: October 2019

Introduction

Human-cl rhFVIIIis the first recombinant factor VIII concentrate expressed in a human cell line (Human Embryonic Kidney 293F cells). Studies in adolescent and adult pre-treated patients with severe haemophilia A showed a favorable PK profile with a median half-life of 17.1 hours (one stage assay) and indicated safety and efficacy in preventing and treating bleeding episodes (BE).

Aims

The objectives of this international GCP study were to evaluate the efficacy, safety, immunogenicity and pharmacokinetic (PK) properties of Human-cl rhFVIIIin previously treated children between 2 and 12 years of age.

Methods

All patients started with an in-vivo recovery (IVR) investigation and in a subset of patients, the PK of Human-cl rhFVIII was assessed in comparison to the patient’s previously used FVIII product. After an injection of 50 IU/kg, blood samples were collected up to 48 hours for PK analysis and up to 2 hours for IVR. IVR was repeated in all patients after 3 and 6 months. FVIII plasma levels were measured by chromogenic and one-stage assay in a central laboratory. Patients were treated prophylactically with Human-cl rhFVIII every other day or 3x weekly, injecting 30-40 IU Human-cl rhFVIIIper kg for at least 6 months and > 50 exposure days. Inhibitors were measured before, during and at the end of the study by modified Nijmegen Bethesda assay in a central laboratory. Adverse events were recorded throughout the study.

Results

59 patients (29: 2-5 years; 30: 6-12 years) were enrolled from 15 specialized sites in Europe. 13 children of each age group participated in the comparative PK investigation. Mean PK parameters of Human-cl rhFVIII were similar in both age groups for both the chromogenic and the one-stage assay: AUCnorm 0.23 vs. 0.24 h*IU/mL/[IU/kg]); IVR 1.88 vs. 1.61% per IU/kg; T1/2 9.7 vs. 12.5 h. IVR remained stable throughout the study. A total of 108 BEs in 32/59 patients were treated with Human-cl rhFVIII. The majority of treated BEs were traumatic (60.2%) and minor (56.5%). The mean ± SD monthly bleeding rate of all types of BEs/patient was 0.34±0.43 (spontaneous BEs: 0.12±0.27; traumatic BEs: 0.19±0.29). No inhibitor development was reported; no patient discontinued the study because of an AE.

Conclusion

The data indicate that Human-cl rhFVIII is efficacious and safe in preventing and treating BEs in previously treated children. The PK of Human-cl rhFVIII was comparable in both studied age groups.

Disclosures:

Liesner: Octapharma AG: Consultancy, Investigator Other. Jansen:Octapharma AG: Employment. Klukowska:Octapharma AG: Investigator Other. Vdovin:Octapharma AG: Investigator Other. Szczepanski:Octapharma AG: Investigator Other. Knaub:Octapharma AG: Employment.
A Simple Genetic Thrombosis Score Of Five Single Nucleotide Polymorphisms Is Associated With Risk Of First Venous Thrombosis In Pregnant Women
Authors: Bare Lance A. , de Haan Hugoline G. , Arellano Andre R. , Tong Carmen H. , Devlin James J. , Rosendaal Frits R.
Published: October 2019

Background

Venous thrombosis (VT), a major cause of maternal morbidity and mortality, is increased 4- to 5- fold during pregnancy. A thrombosis score comprising 5 single nucleotide polymorphisms (SNPs) [rs6025 (Factor V Leiden), rs1799963 (Prothrombin 20210 G>A), rs8176719 (ABO 261G>deletion), rs2066865 (FGG 10034 C>T) and rs2036914 (F11 10364T>C)] was previously shown to be associated with VT events in the general population. We asked whether this thrombosis score could predict VT in pregnant and postpartum women in a case-control study on the etiology of thrombosis.

Methods

We studied women during pregnancy and up to three months postpartum (55 controls, 144 cases) selected from over 5000 female cases and controls of the MEGA (the Multiple Environmental and Genetic Assessment of risk factors for venous thrombosis) study. The thrombosis score was calculated for each individual as a sum of risk alleles weighted by the log of the published odds ratio (de Haan et al, Blood 2012). The VT risk per unit of thrombosis score was calculated as a continuous variable in a logistic regression model that adjusted for age, smoking history and family history of VT. The thrombosis scores ranged from 0 to 1.8 in female participants in MEGA with genotypes for the 5 SNPs (n=3,464).

Results

In pregnant and postpartum women, the odds ratio per unit increase in thrombosis score was 10.7 (95%CI 3.2 to 36.2). When the thrombosis score was evaluated in a sub study of pregnant and postpartum women who do not carry factor V Leiden—an important contributant to the risk score— the remaining 4-SNP thrombosis score was also associated with VT: the odds ratio per unit increase in thrombosis score was 10.6 (95%CI 2.3-48.3).The American College of Obstetricians and Gynecologists recommends thromboprophylaxis during pregnancy and postpartum periods in women who are compound carriers of both factor V Leiden and prothrombin 20210 G>A; these compound carriers who do not carry other risk variants would have a thrombosis score of 1.02. Compared with women in the bottom quintile of thrombosis scores, women with thrombosis scores equal to or greater than 1.02 had an odds ratio for VT of 12.3 (95%CI 1.5 to 99.9). About 2% of female controls in MEGA had thrombosis scores equal to or greater than 1.02.

Conclusions

A 5-SNP thrombosis score that combines the VT risk of 5 genetic variants is associated with VT in pregnant women.

Disclosures:

Bare: Celera: Employment. Arellano:Celera: Employment. Tong:Celera: Employment. Devlin:Celera: Employment.
Influence Of Antithrombin Deficiency On Symptomatic Recurrent Venous Thromboembolism (VTE) In Children: A Multicenter Cohort Study
Authors: Kenet Gili , Limperger Verena , Goldenberg Neil A , Heller Christine , Holzhauer Susanne , Vilk Shoshana Revel- , Young Guy , Nowak- Gottl Ulrike
Published: October 2019

Objective

To determine the importance of antithrombin [AT] deficiency as risk factor or predictor for fatal/non-fatal recurrent venous thromboembolism (VTE) in children.

Methods

In the present cohort of 874 consecutively enrolled pediatric patients with VTE aged newborn to <18 years the rate of VTE recurrence and the time to recurrence in relation to AT-deficiency [confirmed by underlying gene mutations; type 1 deficiency n=17; type 2 deficiency n=3], age and sex was determined. Twenty of 874 patients [2.4%] suffered from AT-deficiency. From the same cohort 150 VTE children carrying the heterozygous F5 G1691A mutation [F5: 17.7%] served as controls. Patients were prospectively followed for a median of 84 months. Data were pooled across participating sites to increase power and to enhance the generalisability of the data. Incidence rates were given as events per 1000 person-years.

Results

Of the 170 children enrolled 26 [AT n=9; F5 n=17] had recurrent VTE at a median of 15 months [95%CI: 12-36] following VTE onset: two of nine [AT] and two of 17 [F5] children suffered recurrent VTE while on anticoagulation with warfarin. The overall incidence rate of recurrence was 61.5 in patients with AT-deficiency compared to 23.5 for pediatric F5 carriers [p=0.02]. The recurrent-free survival probability is shown in the figure [logrank p-value: p=0.001]. When comparing AT patients and F5 children multivariate analysis [Cox regression] adjusted for age, sex and duration of anticoagulation treatment showed that AT deficiency [HR/95%CI: 4.1/1.8-9.4] significantly influenced the hazard for recurrent VTE.

Conclusions

Based on multivariate analysis, the presence of AT deficiency was associated with an increased risk of VTE recurrence. AT-deficiency in pediatric patients should be identified at VTE onset and the possible influence of intensified treatment protocols on recurrence should be studied in future prospective international studies.

Condensed abstract

To determine the relative importance of antithrombin deficiency or factor V (FV) mutations as risk factors for fatal/non-fatal recurrence in pediatric thromboembolism (VTE). Antithrombin deficiency influenced the hazard for recurrent VTE.

Disclosures:

No relevant conflicts of interest to declare.
Thrombin Generationand Fibrinolytic Activity in Microparticles In Acute Promyelocytic Leukemia
Authors: Kwaan Hau C. , Rego Eduardo M. , McMachon Brandon , Weiss Ivy
Published: October 2019

Introduction

The coagulopathy in acute promyelocytic leukemia (APL) is unique among the leukemias and accounts for the major complications of hemorrhage and thrombosis. The pathogenesis is in large part the result of increased expression by the APL promyelocytes of the procoagulant TF, and the fibrinolytic components of tPA , PAI-1 and Annexin A2. Recently, microparticles derived from plasma of APL patients and from culture medium of the human APL cell line NB4 have been shown to contain antigens of procoagulant tissue factor (TF), and the fibrinolytic components of tPA , Annexin A2 and PAI-1 (Kwaan HC, Rego EM. Role of microparticles in the hemostatic dysfunction in acute promyelocytic leukemia. Seminars in thrombosis and hemostasis 2010;36:917-24;Kwaan et al:Microparticles in acute promyelocytic leukemia. 53rdASH Annual Meeting; Blood 2011 Abstract No.3346). Results of functional activities of these components and thrombin generation in the microparticles are presented in the present study.

Material and Methods

Blood samples were collected in citrate vacutainers from 23 patients at the time of diagnosis and after induction therapy with ATRA at one month when there was molecular remission, under the approval of the respective institutional review boards. Control samples were collected from 37 healthy subjects.

Preparation of Microparticles

sodium citrated blood is centrifuged at 1500g for 20 minutes. The plasma portion is collected and centrifuged a second time at 3000g for 3 minutes to eliminate any extraneous platelets. 250µl of plasma is then centrifuged at 20,000g for 15 minutes at 4C. The resultant pellet contains the microparticles. [Standardization of platelet-derived microparticle enumeration by flow cytometry with calibrated beads: results of the International Society on Thrombosis and Haemostasis SSC Collaborative workshop. Lacroix R. Robert S. Poncelet P. Kasthuri RS. Key NS. Dignat-George F. ISTH SSC Workshop. Journal of Thrombosis & Haemostasis. 8(11):2571-4, 2010 Nov.

Results

Conclusion

Microparticles bearing TF have been shown to play a thrombogenic role in cancer. We had previously shown that there is an increase of microparticles containing procoagulants and fibrinolytic factors in APL and that these microparticles are mostly derived from CD33 positive (myeloid) cells. Present findings show that procoagulant and fibrinolytic factors are functional. The TF activity, thrombin generation are increased in the APL microparticles. It is noteworthy that thrombin generation is increased in the APL microparticles. In contrast, there is a complete absence of thrombin generation in the control microparticles. This suggests that the TF in microparticles, which are components of cell membranes, are not activated in healthy subjects but in APL.

Disclosures:

No relevant conflicts of interest to declare.
Differential Expression Of Platelet Proteins In Stable Unstable Coronary Artery Disease and Acute Coronary Syndromes
Authors: McRedmond James P , Holohan Brid , Roy Andrew K , Inzitari Rosanna , Alghamdi Kawthar Saad , Quinn Martin , Fitzgerald Desmond J
Published: October 2019

Background

In coronary artery disease (CAD), the risk of platelet-mediated coronary thrombosis varies over time, with the risk of events falling in the months following an episode of acute coronary syndrome (ACS). This study aims to characterize the platelet protein signature that differentiates stable CAD and ACS so as to identify when a patient with stable CAD is at risk for ACS.

Methods

We studied 2 groups of patients (N=10) undergoing PCI for CAD; patients with ACS (NSTEMI, STEMI), and patients with stable CAD undergoing PCI. All patients received a standard antiplatelet regimen of clopidogrel or prasugrel and aspirin. Samples were obtained immediately following PCI (days 1-3) and at follow-up (30-60 days). Total platelet lysate and secreted proteins following TRAP-induced platelet activation were extracted and the proteins trypsin-digested prior to analysis by liquid chromatography coupled to high resolution, high mass accuracy Orbitrap mass spectrometry. Protein identities and relative quantitation were determined in a label-free approach, using the MaxQuant suite of programs.

Results

Lysates: In total 1585 proteins were identified in platelet lysates. Comparing the 2 groups at PCI, there were 266 proteins differentially expressed between stable CAD and ACS; these are enriched in several ontology categories including coagulation and platelet activation. At follow up, 167 proteins were differentially expressed between groups -up, including a cluster of proteins involved in signal transduction.

Secreted proteins

In total 258 proteins were detected in TRAP-induced platelet secretion samples. Clustering analysis of samples from ACS (both PCI and follow up) showed that these patients largely clustered together in that they exhibited a consistent pattern of protein expression. Interestingly, a subgroup of samples from patients with stable CAD was closely related to ACS samples (Figure).

Discussion

Clustering of secreted platelet protein profiles indicates a common pattern of expression in patients with ACS. A subset of stable CAD patients has a similar pattern, and these patients may be at increased risk of thrombosis. Replication of this signature of protein expression in other patient populations and prospective analysis of risk will be required to address its applicability in the assessment of patients with CAD.

Disclosures:

No relevant conflicts of interest to declare.
The Concentration and Separation Of Blood Components Using Acoustic Radiation Force
Authors: Kennedy Daniel R , Gerhardson Tyler , Sporbert Brianna , Mealey Dane , Rust Michael J , Lipkens Bart
Published: October 2019

The concentration of whole blood has a wide spectrum of medical uses, especially in blood transfusions for patients suffering acute blood loss from trauma or surgery. However, current methods rely on centrifugation, which has a tendency to cause fragmentation and deformation of cells. Acoustic standing waves are considered to be a gentler means to concentrate blood cells, but generally suffer from flow rate limitations of >103fold, functioning only on the microscale. To address this issue, we have developed a novel ultrasound technology that works at the macroscale, enabling it to process flow rates that would typically be required to handle medically relevant volumes.

In this study, we have applied the principles of acoustic radiation at the macroscale to re-concentrate 10X diluted porcine blood by trapping the blood cells within a standing wave, resulting in the clumping of cells and platelets, ultimately leading to enhanced gravitational settling. Complete blood counts were measured with a VetScan HM5 hematology analyzer. Using an acoustic force generated by power levels up to 25 Watts, no lysing was observed in any of the experiments. The inlet flow rate through the device was 16 ml/min, and the concentrate flow rate of the blood components was typically 1 ml/min. The transducer was a 2 MHz PZT-8 operating at 10 W, and it was oriented to create an acoustic standing wave perpendicular to the flow direction. Results indicate successful capture of red blood cells, white blood cells, and platelets with separation efficiencies in excess of 90% in a single pass. Furthermore, the blood components were all reconcentrated back to levels similar to those in whole blood.

Subsequent experiments and conditions have allowed for the separation of blood components, suggesting that the future development of acoustic methods to perform blood re-concentration procedures such as erythropheresis, leukapheresis and plateletpheresis in a gentler manner than current methods holds great promise. With almost 15 million transfusions performed each year in the United States, this technology has the potential to make a significant impact on the medical community, particularly for treatment of critically ill patients. Additionally, the novel technology has also demonstrated performance in separating lipid particles from blood, thus indicating its utility for reducing the occurrence of lipid microemboli during re-transfusion of shed blood in cardiopulmonary bypass surgeries, which have been linked with post-operative neurocognitive complications. As a result, the novel acoustophoretic technology may become a general purpose platform for achieving separation in a variety of biomedical applications.

Disclosures:

Mealey: Flo Design Sonics: Employment. Lipkens:Flo Design Sonics: Equity Ownership.
PRL2 Maintains Hematopoietic Stem and Progenitor Cells Through Regulating SCF/KIT Signaling
Authors: Kobayashi Michihiro , Dong Yuanshu , Yu Hao , Bai Yunpeng , Chen Sisi , Gao Rui , Yoder Mervin C. , Kapur Reuben , Zhang Zhong-Yin , Liu Yan
Published: October 2019

The phosphatase of regenerating liver family of phosphatases, consisting of PRL1, PRL2 and PRL3, represents an intriguing group of proteins implicated in cell proliferation and tumorigenesis. However, the role of PRLs in normal and malignant hematopoiesis is largely unknown. While SCF/KIT signaling plays an important role in hematopoietic stem and progenitor cell (HSPC) maintenance, how SCF/KIT signaling is regulated in HSPCs remains poorly understood. We here report that PRL2 regulates HSPC maintenance through regulating SCF/KIT signaling. To define the role of PRL2 in hematopoiesis, we analyzed the hematopoietic stem cell (HSC) behavior in Prl2 deficient mice generated by our group. Prl2 deficiency results in ineffective hematopoiesis and impairs the long-term repopulating ability of HSCs. In addition, Prl2 null HSPCs are less proliferative and show decreased colony formation in response to SCF stimulation. Furthermore, Prl2 null HSPCs show reduced activation of the PI3K/AKT and ERK signaling in steady state and following SCF stimulation. Importantly, we found that PRL2 associates with KIT and the ability of PRL2 to enhance SCF signaling depends on its enzymatic activity, demonstrating that PRL2 mediates SCF/KIT signaling in HSPCs. Thus, PRL2 plays a critical role in hematopoietic stem and progenitor cell maintenance through regulating SCF/KIT signaling. Furthermore, loss of Prl2 decreased the ability of oncogenic KITD814V mutant in promoting hematopoietic progenitor cell proliferation and in activation of signaling pathways. We also checked the expression of PRL2 proteins in human AML cell lines and found increased level of PRL2 proteins in some acute myeloid leukemia (AML) cells compared with normal human bone marrow cells, indicating that PRL2 may play a pathological role in AML. Our results suggest that the PRL2 phosphatase may be a druggable target in myeloproliferative disease (MPD) and acute myeloid leukemia (AML) with oncogenic KIT mutations.

Disclosures:

No relevant conflicts of interest to declare.
NIPA Deficiency Leads To Acceleration Of The Lymphoma Development In EµMyc Mice
Authors: Illert Anna Lena , Klingeberg Cathrin , Albers Corinna , Morris Stephan w. , Peschel Christian , Keller Ulrich , Duyster Justus
Published: October 2019

Timely degradation of proteins that control cell proliferation and apoptosis is an essential mechanism in keeping normal growth from turning into runaway malignancy. We previously reported the cloning of NIPA (Nuclear-Interaction-Partner-of-ALK) and characterized it as a F-Box-protein that defines an oscillating E3-ubiquitin-ligase. Using a conditional-knockout strategy we inactivated Nipa and found Nipa-/- animals to be viable, but sterile due to a block of spermatogenesis. Our studies demonstrate that loss of Nipa has no substantive effect on physiological cell cycle progression of primary MEFs indicating that this cell cycle checkpoint is inactive under optimal proliferation conditions. Interestingly, Nipa checkpoint control can be unmasked by oncogenic c-Myc-transformation. Here we show significant differences in c-Myc-induced transformation: Focus formation ability of c-Myc-infected Nipa-/- MEFs was greatly reduced. Moreover, Nipa-deficiency leads to premature senescence in cultured primary MEFs. Ectopic reexpression of Nipa resulted vice versa in delayed senescence of knockout MEFs. Next, we sought to know, whether increased apoptosis in Nipa-/- c-Myc-transduced MEFs is dependent on a functional p53-Axis. Interestingly, the effect of Nipa deficiency on c-Myc-mediated transformation was totally abolished by p53-knockdown. We observed no differences in focus formation ability or growth behaviour in Nipa-/- MEFs with inactivated p53, suggesting the importance of p53 in Nipa-induced cell death. Looking in more detail on the c-myc-p53 axis we detected a substantial increase in Arf-p19 levels in Nipa-/- cells. Moreover, Nipa-knockdown in Zn-inducible-Arf-NIH/3T3 cells lead to stabilization of Arf p19. To test the impact of these findings in a relevant in-vivo model we intercrossed Nipa-/- animals with a transgene EµMyc-Strain. Nipa-/-EµMycTG/wt animals developed lymphomas within a significantly shorter latency than Nipa+/+EµMycTG/wt animals. Furthermore, lymphomas of knockout animals were more aggressive. FACS- and biochemical-analyses showed no gross differences between Nipa-/- and wt lymphomas except highly elevated Arf-p19 levels in Nipa-/- lymphomas, pointing to an important role of Nipa in Myc-p19-signalling. Taken together our results highlight the functional importance of the Nipa-p53-axis in cell cycle regulation and suggest that deregulation of the protein provides a substantial contribution during the process of tumorigenesis.

Disclosures:

No relevant conflicts of interest to declare.