bluuebunny/crossref_metadata_2025 huggingface dataset:
The Clinical Outcome of Newly Diagnosed Patients >60 Years of Age with Diffuse Large B-Cell Lymphoma (DLBCL) Treated with Standard or Liposomal Chemotherapies
Authors: Ahmed Mohamed Amin , Oki Yasuhiro , Noorani Mansoor , Nastoupil Loretta , Fayad Luis E. , Hagemeister Fredrick B. , Rodriguez Alma , Davis Richard Eric , Westin Jason R
Published: October 2019

Background:

Effective and safe treatment approaches are needed for diffuse large B-cell lymphoma (DLBCL) in the elderly. One approach is to substitute a chemotherapeutic agent with liposomal formulation, expecting greater anti-tumor activity and less toxicity. We have previously conducted two phase II studies using this approach: DRCOP (Clin Lymphoma Myeloma Leuk 2015), utilizing pegylated liposomal doxorubicin in place of conventional doxorubicin in RCHOP, and RCHMP (Brit J Haematol 2013), utilizing liposomal vincristine in place of conventional vincristine in RCHOP. We sought to compare the outcome of patients who are > 60 years of age with newly diagnosed DLBCL, and received DRCOP, RCHMP and RCHOP. This retrospective analysis was in part supported by Spectrum.

Methods:

The outcome of patients with DLBCL who were > 60 years old were analyzed from the study of DRCOP (DLBCL, n=79) and RCHMP (DLBCL elderly, n=32). In addition, we collected the outcome of patients > 60 years old who received standard RCHOP therapy in 2012 (n=39) from our Lymphoma Outcome Database. The baseline clinical characteristics including age adjusted IPI score (aaIPI), determined by stage, LDH and performance status were collected, along with progression free survival (PFS) and overall survival (OS). The retrospective study of patients with this population has been approved by the institutional review board.

Results:

The baseline characteristics were slightly different in all three groups, though statistically not significant; patients with aaIPI 2/3 were 40/79 (51%), 13/32 (40%) and 12/39 (31%) in DRCOP, RCHMP and RCHOP group, respectively (p=0.13). The 3-year PFS rates in aaIPI 0/1 group were 74%, 83% and 81%, respectively. The 3-year PFS rates in aaIPI 2/3 group were 52%, 67% and 48%, respectively. The 3-year OS rates in aaIPI 0/1 were 84%, 94% and 80%, respectively. The 3-year OS rates in aaIPI 2/3 were 63%, 77% and 65%, respectively. None of the survival differences were statistically significant. Kaplan-Meyer curves are shown in Figure.

Conclusion:

In summary, our data showed that liposomal formulation of chemotherapeutic agents produced similar survival outcome to standard therapy. The number of patients analyzed here is small, but these data support the idea of a randomized evaluation of RCHMP against standard RCHOP therapy in a prospective study in patients with DLBCL.

Figure 1. Figure 1.

Disclosures

Off Label Use: Doxil and Marquibo are both not approved for diffuse large B-cell lymphoma but are discussed.. Nastoupil:Celgene: Honoraria; Genentech: Honoraria; TG Therapeutics: Research Funding; Janssen: Research Funding; AbbVie: Research Funding. Rodriguez:Orthobiotech: Research Funding. Westin:Spectrum: Research Funding.
Bispecific T-Cell Engager Antibody Construct Blinatumomab Shows Durable Response in a Long-Term Follow-up Analysis of 38 NHL Patients Treated in a Phase I Trial
Authors: Dufner Vera , Goebeler Maria-Elisabeth , Sayheli Cyrus , Bargou Ralf C.
Published: October 2019

Introduction: Treatment of relapsed/refractory (rr) Non-Hodgkinxs lymphoma of B-cell type (B-NHL) is still challenging and new and more effective therapies are urgently warranted. Blinatumomab, a bispecific T-cell engager (BiTE®) antibody construct, engages CD3+ cytotoxic T cells, resulting in T-cell expansion and lysis of CD19+ B cells. In a prior phase I study, blinatumomab treatment resulted in an overall response rate (ORR) of 69% and 55% in diffuse large B-cell lymphoma (DLBCL), respectively for patients treated at 60 μg/m2/d. Based on this study we here present a single-center analysis on long-term outcome of patients with B-NHL after treatment with blinatumomab. We here report first preliminary data on long-term follow-up of patients with rr NHL after blinatumomab treatment.

Methods: Patients with rr B-NHL who achieved an objective response (PR or CR) upon blinatumomab treatment were eligible for long-term follow-up analysis. Kaplan-Meier estimates for progression free survival (PFS) probability were calculated from first infusion to relapse or death. Patients without an event were censored at last follow-up. This study was conducted in accordance with the Declaration of Helsinki.

Results: Out of 38 treated patients (17 follicular lymphoma (FL), 14 mantle cell lymphoma (MCL), 4 DLBCL, 3 others) 22 received the effective target dose of 60/90 μg/m²/d. 47,4% (CR/PR=28,9%/18,4%, n=38) and 68,2% (CR/PR=40,9%/27,3%, n=22) of the patients experienced response, respectively, whereas only 18,8% of the patients who were not treated with the effective target dose responded to blinatumomab (CR/PR=12,5%/6,3%, n=16). 6 patients are still in ongoing remission.

Median overall survival (OS) of all 38 patients was 1560 days, median PFS 204 days and treatment free survival (TFS) 233 days. Median OS in the 22 patients treated with the target dose was 1793 days, median PFS 492 and TFS 752 days, compared to only 412 days median OS, 46 days PFS and 82 days TFS in the patients, who did not receive blinatumomab in an effective dosage.

Conclusion: Blinatumomab has the potential to induce durable remissions in patients with rr NHL. Our data suggests that treatment at target dose of 60 μg/m²/d is an important prerequisite to achieve long-term remission.

Figure 1. Kaplan Meier analysis shows significantly longer PFS in patients on target dose Figure 1. Kaplan Meier analysis shows significantly longer PFS in patients on target dose

Disclosures

No relevant conflicts of interest to declare.
Pharmacokinetics and Pharmacodynamics after Subcutaneous Dosing of the iNKT Cell Depleting Antibody NKTT120
Authors: Schaub Robert , Thariath Abraham , Scheuplein Felix , Mashal Robert
Published: October 2019

The iNKT cell (invariant Natural Killer T cell) is a unique immune cell that has been shown to be associated with chronic inflammation in sickle cell disease. One approach to reducing iNKT mediated inflammation would be to reduce the number of iNKT cells in the tissue of SCD patients. NKTT120 is a humanized monoclonal antibody directed to the unique T cell receptor of invariant NKT cells that can deplete iNKT cells by antibody dependent cellular cytotoxicity. It recently completed a first in man Phase 1 study that demonstrated that intravenous single doses in adults with SCD specifically reduced iNKT cells without dose limiting toxicity in the therapeutic range of 0.3 and 1.0 mg/kg.

Although intravenous dosing was found to be effective and safe, the ability to dose patients by subcutaneous administration would be an asset to a chronic treatment strategy and patient compliance. The purpose of this study was to assess the bioavailability and the efficacy of NKTT120 administered subcutaneously (SC) to cynomolgus monkeys. Female adult cynomolgus monkeys were administered NKTT120 as a single subcutaneous dose of 0.03, 0.1, 0.3 mg/kg (n=3 each) or as a single intravenous (IV) dose of 0.3 mg/kg (n=3). Animals were monitored for iNKT cell depletion of peripheral blood by FACS analysis and NKTT120 concentration was monitored by ELISA.

All doses resulted in complete depletion of blood iNKT cells without any change in other blood cells. The return of measureable peripheral blood iNKT cells occurred in each group when the plasma concentration was ≤ 100 ng/mL. The time to recovery was dose dependent with measureable iNKT cells found at 4-6 weeks with the 0.03 mg/kg dose and 8-14 weeks with the 0.3 mg/kg IV and SC doses. NKTT120 was dose proportional with time to maximum plasma concentration of 48 hours following SC dosing. Bioavailability of NKTT120 after SC dosing was ~60% and T1/2 was 15 days for the IV dose group and ranged from 11-19 days for the SC dose groups. The duration of iNKT cell depletion and time to recovery for the 0.3 mg/kg SC dose was similar to that observed for the 0.3 mg/kg IV dose group.

The results support a SC dosing strategy for NKTT120 and suggest that similar pharmacodynamic responses could be expected when a therapeutic intravenous dose (0.3 mg/kg) is administered by the subcutaneous route in SCD patients every 3-4 months.

Plasma Concentration vs. Time for NKTT120 Following IV or SC doses to Cynomolgus Monkeys

Figure 1. Figure 1. Figure 2. Figure 2.

Disclosures

Schaub: NKT Therapeutics: Employment, Equity Ownership. Thariath:NKT Therapeutics: Employment, Equity Ownership. Scheuplein:NKT Therapeutics: Equity Ownership. Mashal:NKT Therapeutics: Employment, Equity Ownership.
Alterative Expression Pattern of T Cell Immunosuppressive Receptors in Peripheral Blood of Patients with ITP
Authors: Zhong Jun , Chen Shuaohua , Lai Jing , Xu Ling , Zhang Tao , Yu Zhi , Lu Yuhong , Yang Lijian , Wu Xiuli , Li Bo , Li Yangqiu
Published: October 2019

Immune Thrombocytopenia (ITP) is an autoimmune disorder characterized by production of auto-antibodies against platelet antigens. Dysregulation of T cells is a major role involved in this disease, while anti-platelet antibodies induce platelets destruction due to an imbalanced immune response. Increasing data showed that abnormal expression of T cell immunosuppressive receptors such as Programmed death 1 (PD-1) and Cytotoxic T lymphocyte antigen-4 (CTLA-4) may relate to autoimmune disease pathogenesis. In this study, we analyzed the expression levels of T cell immunosuppressive receptors including PD-1, PD-L1, CTLA-4, T cell immunoglobulin mucin 3 (Tim-3), lymphocyte activation gene-3 (LAG-3) and B and T lymphocyte attenuator (BTLA) in RNA from peripheral blood mononuclear cells (PBMCs) of ITP patients (18 cases, male: 12, female: 6, median age: 38.5 years, range 24-68 years), PBMCs from 20 healthy individuals were served as control group, by real-time PCR. The results showed that significant lower expression levels of PD-1 (median: 0.0015) and PD-L1 (median: 0.0572) were showed in ITP group in comparison with healthy group (PD-1, median: 0.0117, p < 0.0001; PD-L1, median: 0.5428, p < 0.0001), while the expression levels of Tim-3 (median: 0.1352), LAG-3 (median: 0.1403) and BTLA (median: 0.1403) were significant higher than that from healthy group (Tim-3, median: 0.0751, p < 0.007; Lag-3, median: 0.0155, p < 0.0001; BTLA, median: 0.0315, p < 0.0001). There was not significant different on the CTLA-4 expression level between ITP group (median: 0.0818) and healthy group (median: 0.1667) (p = 0.219). In conclusion, we firstly described the alterative expression pattern of T cell immunosuppressive receptors in ITP, which may have a role in ITP pathogenesis.

Disclosures

Li: The National Natural Science Foundation of China (81370605): Research Funding.
Bone Marrow Findings in Veterans (V) with Hepatitis C Infection (HCV) at a VA Medical Center
Authors: Srinivas Shanthi , Kim Katherine , Gonzalez Melanie , Zhong Fengming , Crump Barbara , Victor Chang
Published: October 2019

Background: Hepatitis C Virus (HCV) is a major public health problem, with an increased prevalence in Veterans. HCV has been associated with B cell Lymphoproliferative disorders and Type II and III Cryoglobulinemias.Prior studies also found that patients with HCV are more likely to develop cytopenias compared normal. Little is known about other possible hematologic manifestations of HCV.We present a series of 54 Veterans seen in a hematology oncology clinic with HCV and their hematologic and bone marrow findings

Methods: In an IRB-approved protocol, we reviewed the records of V with HCV who underwent bone marrow biopsy from January 1993 to July 2015. V with hematological malignancies, coinfection with human immunodeficiency virus (HIV), hepatitis A or B, were excluded. Demographics, Vietnam era status, clinical and pathological data, indications for bone marrow biopsies, and results were abstracted. Statistical analysis was performed using SAS 9.4.

Results: Of the 54 V, 30 Caucasians, 22 African Americans and 2 Hispanics met the eligibility criteria. 37 served in the Vietnam era. The median (M) age was 59 years (range 39-90). M for WBC 6.9 x109/L (2.9-36.3), hemoglobin 14.3 gm/dL (6.9-19), MCV 93.1 fL (68.8-110..6), platelets 172 x109/L (39-493) , total bilirubin 0.75 mg/dl (0.1-5.3), alkaline phosphatase 96.5 U/L (56-341), albumin 3.9 g/dl (1.6-4.9), lactate dehydrogenase 191 IU/L (91-867), AST U/L (19-426), ALT 56.5.U/L (4-480), INR 1.09 (0.84-2.04). 12 V had splenomegaly by imaging. 10 V at the time of bone marrow biopsy had cirrhosis. The most common indication for bone marrow biopsy was cytopenia (n=38) in at least one of the lineages. The remaining (n=16) were for paraproteinemia, lytic lesion, leukocytosis or lymphocytosis. 48 V had abnormal bone marrow biopsy findings (summarized in Table 1) and 6 V had normal biopsy findings.Table 1.Bone Marrow Findings (n=48)Dysplasia15Hyperplasia14Benign Lymphoid Aggregate4Plasma Cells3Leukemia, Plasma cell dyscrasia4Combination of hyperplasia, lymphoid aggregates or plasma cells8

Conclusions: Veterans with HCV develop cytopenias and hematological malignancies. While Cytopenias can result from splenomegaly, autoimmune mechanism, and medications, some of the cytopenias are unexplained Bone marrow changes from chronic HCV infection may be a contributing factor. Understanding these bone marrow changes from HCV may help in determining the etiology and further management of hematological problems related to HCV

Disclosures

No relevant conflicts of interest to declare.
The Comparison of Hemostatic Effects of Recombinant Activated Factor VII (rFVIIa) in Thrombocytopenic Patients (pts) and Hemophilia Pts with Inhibitors
Authors: Galstian Gennadii M. , Kolosova Irina V. , Gemdzhian Eduard , Zorenko Vladimir Yu. , Polyanskaya Tatyana A. , Orel Elena B. , Savchenko Valeri G.
Published: October 2019

Background: Administration of rFVIIa is used as a method of choice in the treatment of hemophilia pts with inhibitors. Besides, rFVIIa may be useful in thrombocytopenic pts with inefficiency or impossibility of platelet transfusions due to alloimmunization or religious beliefs.

Aim: The aim of the present study was to compare the effects of rFVIIa on hemostasis in thrombocytopenic pts and hemophilia pts with inhibitors.

Patients and Methods: rFVIIa was used in 20 pts with thrombocytopenia (I group) and in 20 hemophilia pts with inhibitors (II group). Indications for use of rFVIIa in thrombocytopenic pts were: gastrointestinal and intra-abdominal bleeding, intracranial hemorrhage, insertion of the central venous catheter, lumbar puncture, bleeding from the femoral artery puncture site, epistaxix and pulmonary hemorrhage. In thrombocytopenic pts blood platelet count varied from 1*109/l to 72*109/l (median 15 *109/l). The media dose of rFVIIa in thrombocytopenic pts was 79 μg/kg (ranges from 50 to 144 μg/kg). All hemophilia pts received a single dose of rFVIIa (90 μg/kg). All pts received rFVIIa (Generium, Russia). APTT, Quick prothrombin test (QPT), FVII:C level, endogen thrombin potential (ETP) and Thromboelastography (TEG) parameters (R, MA) were evaluated before and after rFVIIa administration.

The median and interquartile range (IQR) were calculated as descriptive statistics. Mann-Whitney U test and Mood's median test were used to evaluate group differences. Statistical analyses were performed with SAS 9.1 (using the Npar1way procedure).

Results: rFVIIa was effective in all hemophilia pts. Administration of rFVIIa in 15 min shortened but did not correct APTT, increased QPT and ETP, FVII:C (Table 1). Prior to treatment the hemophilia pts have no clotting determined by TEG, but after rFVIIa administration TEG demonstrated close to normal pattern. In thrombocytopenic pts after rFVIIa administration bleeding stopped in 51% pts, decreased in 44% and did not change in 4% pts. In thrombocytopenic pts maximal hemostatic effect was achieved within 1 hour after rFVIIa administration (Table 1). 1 case of ischemic stroke was obtained. The maximum effect of rFVIIa in thrombocytopenic pts coincided with decrease of plasma activity of FVII perhaps due to its consumption.

Conclusion: In the most hemophilia pts with inhibitors the response was achieved within 15 min of administration of rFVIIa. In thrombocytopenic pts haemostatic effect of rFVIIa was delayed and reached within 1 hr, good response was achieved in 51% pts.

Figure 1. Parameters of hemostasis before and after rFVIIa administration expressed as a median (IQR) Figure 1. Parameters of hemostasis before and after rFVIIa administration expressed as a median (IQR)

Disclosures

No relevant conflicts of interest to declare.
Identification of Molecular Defects in ITGA2B and ITGB3 Genes and Phenotypic Correlation in Pakistani Patients with Glanzmann Thrombasthenia
Authors: Jamal Yonus , Ahmed Shariq , Najmuddin Akbar , Imran Ayisha , Khan Tehmina nafees sonia , Shamsi Tahir , Naz Arshi
Published: October 2019

Background:

Glanzmann thrombasthenia (GT) is most common inherited platelet functional defect. It is an autosomal recessive disorder, characterized by a bleeding diathesis. Incidence is increased in locations where consanguineous marriages are common. The defect is caused by mutations in the genes encoding ITGA2B or ITGB3. This results in qualitative or quantitative abnormalities of the platelet receptor, αIIb-β3 integrin.

Objectives:

The aim of this study was to identify and correlate the mutations in GT patients with phenotype of the patient.

Subjects and methods:

20 patients with GT were enrolled in the study to identify the molecular defects and to correlate their phenotype with their genotype. CBC with peripheral film, PT, APTT and Fibrinogen levels were done initially. Platelet aggregation studies, flow cytometry, and mutation analysis was done by Sanger sequencing. Genomic DNA was extracted from peripheral blood by QIAamp DNA Blood mini kit (Qiagen) and Exon specific PCR was done for GT gene and Direct gene sequenced on automated ABI-3130 Genetic Analyzer (Applied Biosystems). For any variation wild type was matched on HGMD (Human Gene Mutation Database http://www.hgmd.cf.ac.uk/ac/index.php) and wild type color fasta sequence (http://pga.gs.washington.edu/). Pathogenecity score was evaluated by using software tools including :

Polyphen-2(http://genetics.bwh.harvard.edu/pph2/) ,SNP&GO(http://snps.biofold.org/snps-and-go/index.html), MUpro (http://mupro.proteomics.ics.uci.edu/) ,SIFT (http://sift.jcvi.org), Provean (http://provean.jcvi.org/about.php) .

All samples were sequenced at the Gene Sequencing Lab of NIBD (National Institute of Blood diseases and Bone Marrow Transplantation) Karachi.

Results:

Mutations were identified in all patients. Missense mutations were seen in most of the GT patients. The remaining mutations were heterogeneous and were distributed throughout the length of the gene.

Conclusions:

The severe type I GT was the most common subtype found in this study.Carrier detection and genetic counseling in these families is a potentially effective alternative for decreasing the burden of severe type of GT.

Disclosures

No relevant conflicts of interest to declare.
A Predominant Mutation in Regulatory Region of SERPINC1 Gene and Venous Thrombosis
Authors: Hu Bei , Wang Qingyun , Tang Liang , Hu Yu , Liu Han
Published: October 2019

Background

Antithrombin (AT) is a critical physiological anticoagulant in haemostasis. SERPINC1, the gene encoding antithrombin, is 13.5kb in length and located on chromosome 1q23-25. Mutations in the SERPINC1 gene may cause antithrombin deficiency and are supposed to confer a high risk for venous thrombosis. However, most mutations reported so far are located in the coding or splicing region of this gene. Therefore, we investigate SERPINC1 gene to identify possible regulatory variants that would increase susceptibility of thrombosis in Chinese people.

Methods and Results

The potential promoter and 5'-untranslated region of SERPINC1 was screened by direct DNA sequencing in 100 individuals diagnosed with unprovoked venous thrombosis. Further genotyping was performed by Restriction Fragment Length Polymorphism (RFLP) analysis in case-control populations (1750 vs 1900). By DNA sequencing, we identified a c.-11.G>A transition, located 11bp upstream from the transcription initiation codon. Next case-control study showed that this mutation was present in ten individuals of thrombosis group and two of control group. All the mutation carriers we identified were heterozygotes. Therefore, individuals carrying the mutant A allele conferred a 5.42-fold increased risk for venous thrombosis (p<0.05) .

Conclusion

The c.-11G>A mutation of SERPINC1 gene is a risk factor for venous thrombosis in the Chinese population. It's the first time that a predominant mutation in the regulatory region of SERPINC1 gene reported to be associated with venous thrombosis in a large population.

Disclosures

No relevant conflicts of interest to declare.
Atherosclerotic Cardiovascular Risk in Adult Patients with Hemophilia: What We Can Do to Reduce Risk of Cardiovascular Events? Implementation of 2013 ACC/AHA Guideline on the Treatment of Blood Cholesterol 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 genetic disorders characterized by deficiency of clotting factors resulting in delayed bleeding. Despite hypocoagulable state, patients with hemophilia are prone to developing coronary artery disease or its equivalents. It is known that proper treatment of dyslipidemia has relevant impact of atherosclerotic cardiovascular events reduction. The goal of our study was to determine implementation of newest 2013 ACC/AHA Guideline on the Treatment of Blood Cholesterol in our patients with hemophilia and assess how many more patients currently may require lipid-lowering therapy.

Methods

We performed retrospective chart review of patients followed at single hemophilia treatment center in United States. We included 30 patients with factor VIII or IX deficiency, age 30 and older, followed in clinic between 2005 and 2014 with available lipid profile results. Patients with acquired hemophilia were excluded from study. We used stepwise approach proposed by above guidelines and divided patients into four groups.

Results

4 patients among 30 were already on lipid lowering therapy. 1 (3.3%) additional patient [95% CI 0.001-0.17] required lipid lowering therapy due to presence of clinical atherosclerotic cardiovascular disease (group 1), 0 patients had LDL-C at least 190 mg/dl (group 2), 2 (6.7%) additional patients [95% CI 0.008-0.21] required therapy due to presence of diabetes mellitus and 40 to 75 year of age and LDL-C levels of 70 to 189 mg/dl (group 3); 9 (30%) additional patients [95% CI 0.17-0.51] should receive therapy due to age 40 to 75 and estimated 10-year ASCVD risk above 7.5%. We had total 12 (40%) additional patients among 30 with known lipid profile who were not on lipid lowering therapy but who require such therapy based on the latest 2013 ACC/AHA Guideline on the Treatment of Blood Cholesterol.

Conclusion

Aggressive cardiovascular risk factor modifications play a significant role in prevention of coronary artery disease, stroke and peripheral vascular disease. This may be even more relevant in patients with hemophilia who have an increased baseline risk of bleeding and may therefore be at greater risk of complications from anti-thrombotic therapies used for treating cardiovascular disease. Above results suggest that according to actual 2013 ACC/AHA Guideline on the Treatment of Blood Cholesterol, a significant number of patients with hemophilia may require lipid lowering therapy. It is important for hemophilia treatment centers to screen their patients with regard to this since many of them may either not have primary care physicians or may not be perceived as having high risk for cardiovascular disease due to their bleeding disorder.

Disclosures

No relevant conflicts of interest to declare.
Baseline Thrombin Generation Markers and Functional Antithrombin Levels in Sepsis Associated Coagulopathies Are Predictive of the Severity of Pathogenesis
Authors: Hoppensteadt Debra , Williams Mark , Syed Daneyal , Guler Nil , Mosier Michael , Fareed Jawed
Published: October 2019

Introduction: Severe sepsis remains the most common cause of death in critically ill patients. Thrombin plays a crucial role in the pathogenesis of sepsis associated disseminated intravascular coagulation (DIC). Thrombin generation markers and functional AT levels provide useful prognostic information in the management of these patients. The purpose of this study was to compare the baseline values of such thrombin generation markers of prothrombin fragment (F1.2), thrombin antithrombin complex (TAT) and functional antithrombin levels in patients enrolled in the Phase 2B and a Phase III study to investigate the therapeutic effect of recombinant thrombomodulin (ART-123) in sepsis associated coagulopathy.

Materials and Methods: Plasma samples from seven hundred and fifty eight patients enrolled in the ART-123 study, a phase 2b, international, multicenter, randomized controlled trial, and patients enrolled in an ongoing (n=200) phase III clinical trial were profiled for F1.2 and TAT using commercially available ELISA methods (Siemens, Deerfield, IL, USA). Functional AT activity was measured using a chromogenic substrate method. All of these samples included blood drawn prior to the first of ART-123 or placebo treatment.

Results: The results are tabulated in the following tables.

Phase 2b Study (n=750) Table 1.ParameterMeanStd ErrorMinMaxMedianQ1Q3F1.2 (pmol/L)515.541.7109,239305182571.5TAT (ng/ml)26.74.50.31,213.87.54.215.1AT (% NHP)84.91.383.020672.882.3101.3

Phase 3 Study (n=176) Table 2.ParameterMeanStd ErrorMinMaxMedianQ1Q3F1.2 (pmol/L)604.259.418.88,618401.8207.6695TAT (ng/ml)28.94.21.86524.812.46.1227.6AT (% NHP)51.424.82.1122.650.332.567.3

Discussion: The inclusion criteria for the phase 2b study was based on the revised ISTH criteria and the phase III study used an INR>1.4, 30K=<PLT<150K, shock, and/or respiratory organ dysfunction as criteria at baseline. The median values were TAT (7.5 ng/ml Phase 2b study; 12.4 ng/ml Phase III), F1.2 ( 305 pmol/L Phase 2b; 401.8 pmol/L Phase III) and AT ( 72.8 %, Phase 2b; 50.3% ,Phase III). There was a difference noted in the markers of thrombin generation between the two studies. The TAT and F1.2 were higher and the AT was lower in the Phase III study suggesting a higher generation of thrombin in this study. Therefore the strict criteria used in the Phase III study shows that this patient group had more activation of coagulation compared to the Phase 2b patients. Furthermore the modified criterion used for the recruitment of patients in the phase III study provides a defined project population to investigate the therapeutic effects of ART-123. These observations also underscore the importance of inclusion criterion for sepsis associated coagulopathy patients to validate the therapeutic effects of newer drugs such as ART-123.

Disclosures

Williams: Asahi Kasei Pharma America: Employment.
A Generic Version of Recombinant FVIIa Is Similar to the Branded Product (NovoSeven)
Authors: Sadeghi Nasir , O'Malley Paul , Kahn Daniel , Hoppensteadt Debra , Fareed Jawed
Published: October 2019

Background: Commercially available recombinant factor VIIa (Novoseven) is widely used in the management of hemophilia patients with inhibitors. Recently several generic versions of recombinant VIIa (rFVIIa) have become available. The generic versions of rFVIIa are claimed to be biosimilar to the barnded Novoseven (Novo Nordisk, Copenhagen, Denmark). The purpose of this study is to compare the US and European Novoseven products with a generic version of rFVIIa namely, Aryoseven (Aryogen, Tehran, Iran).

Methods: Four commercially available random lots of Novoseven were obtained from the US and European sources. Four different batches of Aryoseven were obtained from Aryogen. All individual rFVIIa preparations were diluted to obtain working concentrations of 100, 10, 1 and 0.1 ug/ml. Protein content (Lowry's method), molecular profile using surface enhanced laser desorption ionization (SELDI), gel electrophoretic profile (GEP), factor VII related antigen level (FVII:Ag), factor VII correction studies in depleted plasma and thrombin generation (TG) studies were carried out. In addition, VIIa/tissue factor mediated thrombin generation studies were carried out in various prothrombin complex concentrates such as Beriplex and Prothromplex.

Results: The protein content and SELDI mass spectrophotometric profile of all 4 rFVIIa preparations were comparable. There was no differences in the Novoseven obtained from the US and European sources. The GEP of the two groups of agents showed a comparable profile with distinct peaks at 50 KDa and 25 KDa. The FVII related antigen levels were also comparable in the Novoseven and Aryoseven preparations. Supplementation of both the Novoseven and Aryoseven preparations at 10 and 100 ug/ml resulted in a comparable correction of the factor VII deficient plasma as measured by PT(INR). Thrombin generation was comparable in the branded and generic product.

Conclusions: These results demonstrate that the US and European Aryoseven are comparable. Four batches of Novoseven and 4 individual clinical batches of Aryoseven were found to be comparable. When the US purchased Novoseven preparation was compared with the European Novoseven product, no differences were noted. Thus, the generic Aryoseven is biosimilar to barnded Novoseven and warrant in vivo validation studies.

Disclosures

No relevant conflicts of interest to declare.
Transformed Phenotype of Bone Marrow Stromal Cell Lines Derived from K14E7 Fancd2-/- mice
Authors: Sivanathan Aranee , Zhang Xichen , Franicola Darcy , Cao Shaonan , Shields Donna , Epperly Michael W , Greenberger Joel S.
Published: October 2019

To determine whether cytokeratin 14 promoter linked expression of the Human Papilloma Virus (HPV) oncogene detectably influenced biologic parameters of cell phenotypes other than squamous epithelium, continuous bone marrow cultures were derived from K14E7 Fancd2-/- mice (Park, et al., Cancer Research, 70(23): 9959-9968, 2010). Long-term bone marrow cultures derived from K14E7 Fancd2-/-, control Fancd2-/- (129/Sv), K14E7 (FVB/N), and wild type 129/Sv X FVB/N F1 mice were evaluated for longevity of hematopoiesis in long-term cultures and stromal cell lines were derived from each. Similar to Fancd2-/- mouse, long-term marrow cultures, K14E7 Fancd2-/- marrow cultures demonstrated decreased longevity of hematopoiesis with cessation of production of multi-lineage colony forming progenitor cells after 14 weeks. In contrast, wild type F1 and K14E7 long-term marrow cultures continued to produce hematopoietic cells for a significantly longer duration 25 weeks (p=0.0257). Bone marrow stromal and IL-3 dependent hematopoietic cell lines were derived from each genotype marrow culture. K14E7 Fancd2-/- hematopoietic cells showed reversal of the radiation resistance of Fancd2-/- IL-3 dependent cell lines (D0 of 1.34 ± 0.197Gy, ñ 4.0 ± 0.9 compared to D02.213 ± 0.124 Gy (p = 0.0284), ñ 3.3 ± 0.8. Thus, one phenotypic difference associated with K14E7 oncogene expression was reversal of radioresistance of Fancd2-/- hematopoietic cells. In contrast, bone marrow stromal cell lines from K14E7 Fancd2-/- remained radiosensitive similar to those from Fancd2-/- mice. K14E7 and wild type F1 marrow stromal cell lines showed intermediate radioresistance (p= 0.1759). To determine whether the E6/E7 oncogene had a biological effect in tissues other than squamous epithelium, tissue analysis for cytokeratins 13, 14, 6, and 10 was carried out. Cytokeratin 14 was detected only in squamous cells of the esophagus and oral cavity, not in bone marrow. E6/E7 oncogene was detected only in squamous cell lines expressing cytokeratin 14. However, bone marrow stromal cell lines from K14E7 Fancd2-/- marrow cultures demonstrated a unique phenomenon of cellular density, piling up and formation of tumors in vitro. Each of 22 single cell derived clonal sub-lines of K14E7 Fancd2-/- stromal cell lines demonstrated the same transformed phenotype. These data provide support for indirect effects of the E7 oncogene linked to the K14 promoter in Fancd2-/- hematopoietic and mesenchymal stem cell tissues.

Supported by research grant NIAID/NIH, U19A168021.

Disclosures

No relevant conflicts of interest to declare.
CDKN2 Gene Deletion As Poor Prognosis Predictor Involving in the Progression of Adult B-Lineage Acute Lymphoblastic Leukemia Patients
Authors: Li Yuling , Xu Na , Zhou Xuan , Huang Jixian , Li Lin , Lu Qisi , Lu Ziyuan , He Bolin , Pan Chengyun , Liu Qifa , Xiaoli Liu
Published: October 2019

Background : Genes CDKN2A (MTS1) and CDKN2B (MTS2), which encode for p16INK4a/p14ARF and p15INK4b respectively from the 9p21.3 locus, are thought to be an important growth suppressor gene. INK4a/ARF regulates p53 tumor suppressor function by interacting with MDM2. P15/INK4b works as a CDK inhibitor, which inhibits phosphorylation of Rb by inactivating cyclin/ CDK-4/6 complexes and thereby controls cell-cycle G1 progression. Deletion of CDKN2A/B was well known in many hematologic malignancies, but only few report investigated this deletion effect on clinical prognosis.

Methods: This study performed analysis of the CDKN2 deletion in 215 adult B-ALL patients, and relation with cytogenetic prognostic factors (BCR/ABL; E2A/PBXl; TEL/AML1; MLL rearrangement; MYC, IGH translocation).

Results: The prevalence of CDKN2 deletions in all study population was 28.4%. There is no difference between patients with CDKN2 deletion and wild-type patients in sex, age, white blood cells(WBC) count, BM blast percentage,extra infiltration and induction complete remission(CR) rate. Analysis at relapse patients revealed increased rate of CDKN2 loss (44.6%) compared with the rate in all patients (28.4%) (p=0.006). Deletions of CDKN2 were significantly associated with poor outcomes including decreased overall survival (OS) (p<0.001), lower disease free-survival (DFS) (p<0.001), and increased cumulative incidence of relapse (p=0.002); Also, CDKN2 deletions were strongly associated with IGH translocation (p=0.021); and had an adverse effect on patients who with BCR-ABL fusion gene or MLL rearranged. Among of patients with the CDKN2 deletions , the subgroup of received Allo-HSCT treatment had a better OS and DFS compared with the subgroup received chemotherapy only (OS: p<0.000; DFS: p<0.000).

Conclusion: Patients with CDKN2 gene deletion were benefited from allogeneic hematopoietic stem cell transplantation (Allo-HSCT). Deletion of CDKN2 gene was commonly observed through leukemia progression and was a possible poor prognostic marker in long-term outcomes.

Disclosures

No relevant conflicts of interest to declare.
Tumor Vs Normal Gene Expression Profiles Identify Deregulated Pathways in ETV6/RUNX1 Positive Acute Lymphoblastic Leukemia: A Microarray-Based Meta-Analysis
Authors: Heiden Thomas , Nickel Janette , Preissner Robert , Seeger Karl
Published: October 2019

Acute lymphoblastic leukemia (ALL), a clinically and biologically heterogeneous disease, is the most common type of childhood cancer. Approximately 25% of B-cell precursor ALL (BCP-ALL) carry the cryptic chromosomal translocation t(12;21)(p13;q22), the most frequent genetic aberration in pediatric ALL. This translocation combines two transcription factors and essential regulators of normal hematopoiesis, ETV6 and RUNX1, generating the fusion oncogene ETV6/RUNX1 (synonym TEL/AML1). Recent studies in various animal models have strengthened the view that ETV6/RUNX1 positive cells give rise to preleukemic clones with a differentiation block in the pro/pre-B stage of B cell development that, after acquisition of additional mutations, may transform into full malignancy. However, the effects triggered by the expression of ETV6/RUNX1 and the associated additional mutated genes are not well understood.

We compared global gene expression data from ETV6/RUNX1 positive ALL and normal hematopoietic progenitor cells to identify expression signatures underlying the malignant phenotype. Our meta-analysis comprised Affymetrix HG-U133A und HG-U133 Plus 2.0 data from a total of 321 patients with ETV6/RUNX1 positive ALL, different ETV6/RUNX1 positive cell models, leukemias with 11q23 rearrangements and hyperdiploid ALL, as well as 251 samples from various normal controls including normal bone marrow, hematopoietic stem cells (HSCs) and normal B-(progenitor) cells (multipotent progenitor, pro-, pre-, immature-, naive-, centrocyte-, centroblast-, memory-, plasmablast-B cells). To eliminate potential lab batch effects, we performed RMA normalization of all microarray CEL files and next applied the batch effect removal algorithm ComBat. For determination of differentially expressed genes and pathway analyses, the R/Bioconductor packages limma and SPIA were used, respectively.

We generated a gene expression landscape of the normal B-cell development and were able to show that pre-B cells are the closest normal counterpart of ETV6/RUNX1 positive ALL. Furthermore, in comparisons with normal HSCs the p53-, mTOR-, PI3K-AKT-, Apoptosis-, and JAK-STAT signaling pathways were found to be deregulated in those ALL. The combination of multiple global gene expression data sets from ETV6/RUNX1 positive ALL offers the possibility of an integrated large-scale meta-analysis and reveals novel insights into deregulated gene networks in this type of leukemia.

Disclosures

No relevant conflicts of interest to declare.
Targeting Heme Oxygenase-1 Improves Poor Therapeutical Outcomes of BCR-ABL-Positive Adult B-Allwithikaros Isoform 6
Authors: Wang Jishi , Wang Ziming , Lin Xiaojing , Fang Qin , Chen Shuya , Huang Jun , Zhe Nana , Yu Meisheng , Zhang Yaming , Zhou Bingfeng
Published: October 2019

Genomic profiling studies have demonstrated that the dominant-negative Ikaros isoform 6 (IK6) is overexpressed in BCR-ABL1-positive B-lineage acute lymphoblastic leukemia (BCR-ABL1-positive B-ALL), and it is strongly associated with a poor outcome. In addition, IK6 expression leads to BCR-ABL-positive B-ALL patients insensitive to tyrosine kinase inhibitors (TKIs). Moreover, a study has revealed that Heme oxygenase-1 (HO-1), an essential survival factor, could be a potential target in treating ALL. Therefore, we were wondering whether targeting HO-1 could improve poor therapeutical outcomes of BCR-ABL-positive adult B-ALL with IK6. Firstly, we detected that IK6 existed in 20 of 42 (47.6%) adult BCR-ABL1-positive B-ALL by using reverse transcribed polymerase chain reaction (PCR) and nucleotide sequencing. We also found that early clinical response was poor in BCR-ABL1-positive B-ALL patients with IK6, and they were usually high WBC counts at diagnosis and a high relapse rate. Compared to healthy donors' CD34+ cells, the levels of HO-1 expression were higher in primary CD34+ cells derived from adult BCR-ABL1-positive B-ALL patients with IK6 by qRT-PCR and western blot studies. And there was a strong correlation between the expression of IK6 and HO-1. In primary CD34+ leukemic cells derived from our IK6-positive patients' pool, silencing HO-1 resulted in the reduce of proliferation and a substantial proapoptotic effect, including a significantly enriched subG1 population, an increase of Annexin V-positive cells and of caspase3 cleavage. Furthermore, silencing HO-1 also increased their sensitivity to TKIs. This phenomenon was also supported by the observation that ZnPP IX substantially augmented the growth-inhibitory effects of imatinib on primary leukemic cells. Finally, we found that IK6 led to STAT5 activiation, and HO-1 was one of the downstream target genes of STAT5. In conclusion, the existence of extra IK6 seems to exacerbate poor prognosis and risks of relapse in adult BCR-ABL1-positive B-ALL. HO-1 plays an important role as a survival factor in BCR-ABL1-positive B-ALL with IK6, and targeting HO-1 can probably improve the clinical outcomes.

Disclosures

No relevant conflicts of interest to declare.
Dynamic Telomere Shortening and Chromosomal Instability in Irradiated CD34+ Cells Transduced with TP53 Hotspot Mutations R175H, R248W and R249S
Authors: Salari Azam , Thomay Kathrin , Schienke Andrea , Hagedorn Maike , Ebersold Juliane , Krueger Andreas , Schambach Axel , Schlegelberger Brigitte , Göhring Gudrun
Published: October 2019

Patients with MDS and a complex karyotype have a very short median survival and a high risk of transformation into AML. We showed earlier that TP53 mutations are associated with complex karyotype and disease progression. However, it is poorly understood how TP53 mutations contribute to the induction of chromosomal instability in hematopoietic stem and progenitor cells.

We therefore established a long-term cell culture (LTC) model and investigated the role of TP53 in human CD34+ hematopoietic stem and progenitor cells (HSCs) isolated from cord blood and in HT1080 cells over 6 weeks. We chose 3 different modifications: 1) TP53-deficient HSCs via shRNA knockdown, 2) HSCs with different lentivirally introduced TP53 hotspot mutations (R248W, R175H, R273H, R249S) and 3) HT1080 cells with different lentivirally introduced TP53 hotspot mutations (R248W, R175H, R273H, R249S). We performed each LTC at least three times. In order to stress the cells and induce chromosomal instability, we irradiated half of the cells. Besides functional assays in the first week, we performed detailed cytogenetic analysis including telomere length measurement at weeks 1, 3 and 6.

TP53 mutations and the downregulation of TP53 led to impaired hematopoiesis with decreased erythroid differentiation, increased apoptosis and decreased proliferation. None of the modifications induced chromosomal instability in cells without irradiation. In the irradiated cells, all cells carrying a TP53 mutation or a TP53 downregulation developed a chromosomal instability in comparison to the cells transduced with control vectors. However, no stable complex clones developed. Telomeres shortened during follow-up in HSCs carrying the mutations R175H, R248W and R249S. No other cells showed a dynamic response in telomere length. In order to analyze the DNA repair capacity, we performed yH2AX foci assay. Surprisingly, the same cells which showed a telomere shortening showed a lower amount of foci. This could be due to a lower amount of double-strand breaks or to a lower ability to form foci.

In summary, TP53 mutations and the downregulation of TP53 led to an increased chromosomal instability in irradiated cells only. Modifications alone did not lead to the development of complex karyotypes. Furthermore, only the mutations R175H, R248W and R249S led to a telomere shortening. In conclusion, a TP53 mutation seems to require additional passenger mutations in order to lead to MDS or AML with complex karyotype.

Disclosures

No relevant conflicts of interest to declare.
Peg-PLGA-PLL Nanoparticles in Combination with Gambogic Acid for Reversing Multidrug Resistance of K562/A02 Cells to Daunorubicin
Authors: Xu Peipei , Wang Ruju , Ouyang Jian , Chen Bing
Published: October 2019

Purpose: The present chemotherapy in malignancy treatment, including leukaemia, is plagued by one main problem: multidrug resistance (MDR) which is often related to excessive expression of adenosine triphosphate-dependent efflux pump. In recent years, attempts have been focused on searching competitive inhibitors. However, high toxicity and severe side effects were the main impediments for the application of inhibitors.

Methods: this study investigated a new strategy to utilise polyethylene glycol-polylactic-co-glycolic acid-poly-L-lysine (PEG-PLGA-PLL) as a delivery system to combine with gambogic acid (GA) with the purpose of delivering daunorubicin (DNR) to the target location to reserve MDR with maximised therapeutic efficacy and minimised side effects. The anti-tumour ability of DNR in the presence of GA-PEG-PLGA-PLL was evaluated by methyl thiazolyl tetrazolium (MTT) assay, flow cytometry and 4',6-diamidino-2-phenylindole (DAPI) staining. The possible signalling pathway was demonstrated by quantitative real-time polymerase chain reaction (QPCR) and Western blot assay.

Results: The intracellular concentration of DNR can evidently increase with the presence of GA-PEG-PLGA-PLL, and the resistance of K562/A02 cells to DNR can be reversed when combined with GA-PEG-PLGA-PLL, which acted as a delivery system. The anti-tumour efficiency was promoted.

Conclusion: GA-PEG-PLGA-PLL can be a promising agent in reversing MDR of K562/A02 to DNR.

Disclosures

No relevant conflicts of interest to declare.
Drug Induced MLL Fusion Degradation Via Hsp90 Inhibition
Authors: Cantilena Sandra , Goulden Nicholas , Williams Owen , de Boer Jasper
Published: October 2019

The survival rate for infants is less than 50%. The fast majority of infant acute leukemias are characterized cytogenetically by balanced chromosomal translocations involving the mixed lineage leukemia (MLL) gene. Leukemic therapies that degrade the driver oncogene are associated with loss of cancer cell self-renewal and excellent cure rates. Therefore, therapy that degrades the MLL fusion gene would offer new hope to these patients.

Recently it was shown that the Drosophila Trithorax gene, an analogue of the human MLL, is degraded by Radicicol. Radicicol is a natural compound and a well-known Hsp90 inhibitor.

Here, we show that Radicicol is able to induce a dose dependent degradation of the MLL-fusion protein in a panel of human MLL rearranged cell lines and in human cord blood-derived MLL-AF9 immortalised myeloid cells. This drug induced degradation of the MLL-fusion gene results in down-regulation of the expression of MLL target genes, including HOXA9, MEIS1 and c-MYB. Functionally, this results in a loss of self-renewal of the leukemic stem cells, as shown by methylcellulose colony forming assays.

Radicicol proved ineffective and too toxic for in vivo use. One of the best tolerated Hsp90 inhibitors is Ganetespib. It is currently in phase II/III clinical trials.

We extended our Radicicol data to Ganetespib. Like Radicicol, Ganetespib induces MLL-fusion protein degradation and downregulation of MLL target genes. Treatment of MLL rearranged leukemia with Ganetespib results in a loss of leukemic stem cell activity.

In conclusion, we show how the inactivation of the MLL-fusion and down-regulation of MLL target genes results in a block of leukemic stem cell self- renewal. We will validate these findings in a pre-clinical in vivo model in the near future.

Disclosures

No relevant conflicts of interest to declare.