bluuebunny/crossref_metadata_2025 huggingface dataset:
Thrombopoietin Enhances Expression of Vascular Endothelial Growth Factor (VEGF) in Primitive Hematopoietic Cells through Hif-1α.
Authors: Kirito Keita , Fox Norma , Kaushansky Kenneth
Published: October 2019

The development of hematopoietic stem cells (HSCs) is orchestrated by numerous hematopoietic cytokines, growth factors and chemokines. For the most part these proteins are secreted from bone marrow microenviromental cells, including fibroblasts, endothelial cells and stromal cells, or are displayed on their cell surfaces. HSCs have also been shown to produce numerous cytokines and growth factors. Interestingly, secreted cytokines have also been reported to induce the production of additional cytokines from neighboring cells, suggesting that cytokines drive networks of other cytokines to support hematopoietic cell development. Vascular endothelial growth factor (VEGF), a major regulator of angiogenesis and vasculogenesis, also plays an important role in HSC development, where it acts in an intracellular autocrine fashion to promote cell survival. The secretion of VEGF from endothelial or smooth muscle cells is regulated by extracellular stimuli, inflammatory cytokines and hypoxia. In contrast, it is not clear whether synthesis of VEGF in HSCs is regulated by extracellular signals. Because several early acting cytokines, including TPO, affect VEGF-A expression in hematopoietic cell lines, we hypothesized that TPO could be a regulator of a HSC VEGF autocrine loop. We found that TPO induces VEGF transcripts in primitive marrow derived sca-1+/c-kit+/Gr-1− hematopoietic cells, and that VEGF transcripts are reduced in these cells when derived from Tpo−/− mice. Additional studies determined that TPO induces VEGF expression by increasing the nuclear levels of its primary transcription factor, both in the TPO-dependent primitive hematopoietic cell line UT-7/TPO and in purified sca-1+/c-kit+/Gr-1−marrow cells. Elevation of HIF-1α by TPO is achieved by two different mechanisms; augmented protein synthesis and enhanced stabilization. The latter mechanism is dependent on heat shock protein 90 (Hsp90), as inhibition of Hsp90 function by the specific inhibitor geldanamycin inhibited the TPO-dependent stabilization of HIF-1α. In additional studies we also established that VEGF expression was important for the favorable TPO effect on primitive hematopoietic cells, as blockade of the VEGF receptor with a specific inhibitor, SU5416 (0.1mM), substantially blunted TPO induced growth of sorted single sca-1+/c-kit+/Gr-1−marrow cells in serum-free cultures. Importantly, this inhibitor does not affect TPO induced phosphorylation of Jak2, ERK and AKT in UT-7/TPO cells even at a 100-fold higher concentration. Along with our previous findings that TPO affects Hox transcription factors that regulate HSC proliferation, these data contribute to our growing understanding of the mechanisms by which a hormone can influence HSC development.
A Serine Protease Activity Is Induced by G-CSF and Degrades Stat5 Proteins in Myeloid Cells.
Authors: Qiu Yaling , Zhuang Dazhong , MacRae Alexandra , Dong Fan
Published: October 2019

Granulocytic progenitor cells become progressively less capable of proliferation and survival with terminal differentiation. Granulocyte colony-stimulating factor (G-CSF) is the major regulator of granulopoiesis and stimulates the activation of multiple signaling pathways including the signal transducer and activator of transcription 5 (Stat5) pathway. Little is known about the activation status of G-CSF-stimulated signaling pathways at the distinct stages of granulocytic differentiation. When myeloid 32D cells transfected with the G-CSF receptor were induced to differentiate with G-CSF, Stat5 activation in response to G-CSF was gradually attenuated. Activation of other signaling molecules including Stat1, Stat3, Erk1/2, JNK and p38 was not altered significantly. Stat5 activation was also downregulated in multipotent FDCP-mix cells, which differentiated into mature granulocytes upon induction with G-CSF, but not in pro-B BaF/3 transfected with the G-CSF receptor, which showed no terminal granulocytic differentiation in response to G-CSF, suggesting that the effect of G-CSF is cell type specific. Attenuated activation of Stat5 correlated with reduced Stat5 protein levels, which was associated with expression of a protease activity capable of degrading Stat5 protein in vitro. The Stat5 protease activity was upregulated when myeloid cells were induced to differentiate with G-CSF, but its upregulation by G-CSF was blocked upon expression of leukemogenic proteins Bcr-Abl and Tel-Jak2. The activity of the Stat5 protease was inhibited partially by PMSF and completely by a1-antitrypsin, suggesting that it belongs to the serine family of protease. Our data provide the first evidence that a Stat5 protease activity is upregulated by G-CSF and may have important implications for understanding the molecular mechanism by which G-CSF orchestrates granulopoiesis.
Stabilization of Nmi Protein through Its Interaction with Tip60.
Authors: Zhang Keman , Zheng Gang , Yang Yu-Chung
Published: October 2019

Tip60 (Tat-interactive protein of 60 kDa), a member of MYST family (for its founding members MOZ, YBF2/SAS3, SAS2 and Tip60) histone acetyltransferases, is involved in transcriptional regulation of many genes and diverse signaling transduction. The Tip60 complex consists of multiple subunits, with some implicated in DNA damage and apoptosis. The MYST superfamily members, such as MOZ and MORF, are implicated in the pathogenesis of acute myeloid leukemia. In the present study, Nmi was identified as a novel binding partner for Tip60 by a yeast two-hybrid screen. The association of Tip60 with Nmi was further confirmed by co-immunoprecipitation. Nmi, an interferon and interleukin inducible protein was originally identified as an N-Myc/c-Myc interacting protein. It is expressed in various adult and fetal tissues and in several cancer cell lines with the highest expression in certain myeloid leukemias. Nmi gene is localized on human chromosome 22q13.3 in a region where translocation has been reported in certain human leukemias. The Nmi protein interacts with all the STAT members except STAT2. Although Nmi lacks intrinsic transcriptional activation domain, it functions as an adaptor molecule to enhance STAT-dependent transcription through the recruitment of coactivator CBP/p300. Tip60 interacts with the C-terminus of Nmi in a region essential for Nmi-Nmi homodimerization and Nmi-IFP35 (interferon-inducible protein of 35 kDa) heterodimerization and subcellular localization. Homodimerization of Nmi reduces the interaction of Tip60 with Nmi. Nmi associates with the zinc finger domain of the Tip60, which is important for the transcriptional repression effect of Tip60 on TEL, the translocation E26 transforming-specific (ETS) leukemia. We further showed that Nmi is an unstable protein and is targeted for proteosome-mediated degradation possibly through ubiquitination of its C-terminus. The interaction with Tip60 enhances Nmi stability, which is dependent on the histone acetyltransferase activity of Tip60. Our results suggest that stabilization of Nmi protein through its interaction with Tip60 may be involved in the pathogenesis of certain leukemias.
Patient Preference and Capabilities Are at Least as Important as Clinical Contraindications as Barriers to Prescribing Warfarin in Patients with Atrial Fibrillation.
Authors: Hollowell Jennifer , Ingelgard Anders , Reddy Prabashni , Gold Karen , Fitzmaurice David
Published: October 2019

BACKGROUND: Prior studies have assessed either physicians’ general perceptions of barriers to oral anticoagulation or actual barriers in a sample of patients, but they have not both been evaluated within the same study. We evaluated physicians’ general perception of barriers to warfarin use and actual critical barriers to prescribing warfarin in their own patients, using a newly developed instrument.

METHODS: We identified 45 barriers to warfarin use from a literature review, input from clinical experts, and a physician focus group. Barriers were classified into 4 groups: patient medical characteristics (n = 18), health care system factors (n = 8), patient capability (n = 13), and patient preference (n = 6). A sample of US physicians (n = 30) who treat atrial fibrillation (AF) patients completed the instrument. Physicians ranked on a 1–10 scale their reluctance (10 = very reluctant) in general to prescribe warfarin if a specific barrier was present in a patient (perceived barriers). Physicians then indicated critical barriers to prescribing warfarin in their own specific AF patients not receiving warfarin. The most highly ranked perceived barriers and the most prevalent critical barriers to prescribing warfarin were assessed.

RESULTS: Factors considered most strongly to influence physicians’ decision not to prescribe warfarin (mean score±SD) in general were: severe bleeding <3 months ago (9.2±1.3), inability to rely on a patient to adhere to medication instructions in general (8.5±1.3), inability to rely on a patient to adhere to complex warfarin instructions (8.3±2.2), patient unwilling to undergo repeat laboratory testing (8.3±2.1), and risk of fall (8.2±1.8). The most prevalent critical barriers to prescribing warfarin [n (%)] in specific AF patients (n = 24) were: patient unwilling to undergo repeat testing [7 (29%)], inability to rely on the patient to adhere to medication instructions in general [4 (17%)], inability to rely on the patient to adhere to complex warfarin instructions [4 (17%)], difficulty getting to monitoring appointments [4 (17%)], and bleeding episode >3 months ago [4 (17%)].

CONCLUSION: Patient preference and capabilities are at least as important as clinical contraindications as barriers to prescribing warfarin in AF patients.
An Evaluation of the Impact of Recurrent Venous Thromboembolic Disease and Hemorrhagic Events within the Managed Care Setting.
Authors: Ory Caron , Cyprien Lori , Kim Jennifer , Hauch Ole , Chang Eunice
Published: October 2019

Recurrent venous thromboembolic disease (VTE) has been associated with significant expenditures in health care charges. In this retrospective observational cohort study, managed care enrollees with an initial VTE and treated with warfarin were identified. The rate of recurrent VTE and hemorrhagic events, as well as health care charges incurred for these events were calculated. Study inclusion required a diagnosis of VTE between 01/01/98 through 12/31/00, age 18 or older, a pharmacy claim representing warfarin use 7 through 14 days after diagnosis, no VTE or anticoagulation use during the 6-month period prior to diagnosis and continuously enrolled in the health plan during the 6-month pre-index period through 360 through 720 days after diagnosis. A total of 1,973 patients were identified. Mean age was 69.4 ± 13.3 years and 57.3% were female. Overall, the median length of warfarin therapy was 4.7 months. During the follow-up period, 220 (11.2%) had an acute hospitalization for a subsequent event regardless of warfarin status. Of these, 139 patients (63.2%) experienced a recurrent VTE and 81 patients (36.8%) experienced a hemorrhagic event. Health care charges incurred for the first recurrent VTE or a hemorrhagic event were $15,575 ± $17,209 however, twenty patients experienced a second recurrent event (either VTE or hemorrhagic) and their health care charges increased to $37,356 ± $38,877.

Overall, 11.0% of patients with an initial VTE and who were treated with warfarin experienced a recurrent VTE or hemorrhagic event requiring hospitalization. These data highlight the importance of anticoagulation for secondary prevention of VTE and that multiple events (VTE or hemorrhagic) result in substantial health care expenditures.
Increasing Hemoglobin Levels with Epoetin Alfa in Anemic Hematologic Cancer Patients Receiving Chemotherapy Correlates Significantly with Improved Quality of Life.
Authors: Harousseau Jean-Luc , Fumoleau Pierre , Lange Winand , Welslau Manfred
Published: October 2019

The majority of patients (pts) with hematologic malignancies (HM) are anemic and often have poor performance scores (Ludwig H et al. Blood.2002;100:234a). The correlation between increases in hemoglobin (Hb) with epoetin alfa and improvements in quality of life (QOL) was evaluated in this open-label, multicenter study that enrolled 736 adult pts with various solid tumors and HM (n = 122) who were receiving cytotoxic chemotherapy and had Hb levels <12 g/dL. Epoetin alfa was administered 150 IU/kg or 10,000 IU 3 times weekly (TIW) for a maximum 28 weeks (wks); dose was increased to 300 IU/kg or 20,000 IU TIW if Hb was not increased >1 g/dL above baseline within 4 wks. Results for pts with HM are compared to results from a similar 16-wk trial that included pts with HM (n = 488) and used the 40,000 IU once-weekly (QW) dose, increased to 60,000 IU QW if Hb did not increase ≥1 g/dL within 4 wks (Gabrilove J et al. Int J Hematol.2000;72:55). In the TIW study, the primary efficacy endpoint was change in QOL as measured by the Functional Assessment of Cancer Therapy-General (FACT-G), including subscales for anemia (FACT-An) and fatigue; the FACT was administered at study entry, at 8–9 wks, and at 12 wks. QOL was additionally measured by the Cancer Linear Analog Scale (CLAS; also known as the Linear Analog Scale Assessment [LASA]) administered at every study visit. Dose increases were similar in both studies (33.6% of pts in the TIW study; 36.7% of pts in the QW study). In the TIW study, mean baseline Hb was 9.6 g/dL; mean Hb increased at 4–6 wks (1.35 g/dL), 8–9 wks (2.09 g/dL) and 12 wks (2.46 g/dL) to a mean Hb of 12.0 g/dL. Almost half the HM patients in the TIW study (48.1%) had a complete response (Hb increase ≥2 g/dL without blood transfusion). Hematologic results are similar to those reported in the QW study where mean Hb increased 1.96 g/dL by end of study (P = .0001). QOL improvements were also similar between studies. Mean FACT-An scores for the total population in the TIW study increased 6.7 points after 12 wks, which is clinically significant (Patrick DL et al. Eur J Cancer.2003;39:335–345). The mean increase for overall FACT-G correlated significantly with increased Hb at 12 wks (P <.0001; r = .262). Mean CLAS scores for the total population increased steadily throughout the study. At 12 wks mean increases (based on the 100-mm scale) were clinically significant (Patrick et al): 10.9 mm for Energy, 11.2 mm for Daily Activity, and 10.3 mm for Overall QOL. Mean change for FACT-An for patients from the total population in the QW study (n = 2,230) was 6.0 points (P <.001), which is comparable to the mean changes in the TIW study and the HM population of the QW study (6.59 points, P <.0001). No unexpected adverse events were reported. Epoetin alfa TIW or QW was shown to steadily increase Hb, which significantly correlated with clinically significant improvements in QOL.
Derivation of Hematopoietic Stem Cells from Embryonic Stem Cells.
Authors: Wang Yuan , Yates Frank , Dikovskaia Eugenia , Ernst Patricia , Davidson Alan J. , Zon Leonard I. , Daley George Q.
Published: October 2019

Despite the significant in vitro blood-forming potential of murine embryonic stem cells (ESCs), deriving hematopoietic stem cells (HSCs) that can reconstitute irradiated mice has proven to be challenging. Previously, we successfully engrafted lethally irradiated adult mice with ESCs engineered to ectopically express the homeodomain gene hoxB4. In engrafted animals, blood reconstitution showed a myeloid predominance, likely due to an inability to fully pattern the adult HSC from these embryonic populations. Recently, we have investigated cdx4, a caudal-related homeobox gene whose function has been linked to blood development in the zebrafish. During in vitro differentiation of murine ESCs, cdx4 is expressed during a very narrow time interval on day 3, coincident with the specification of hematopoietic mesoderm. To further characterize the function of cdx4 in mouse hematopoiesis, we have established a tetracycline-inducible murine embryonic stem cell line. When cdx4 expression is conditionally induced over a protracted period from day 2 and 6, we observe a marked enhancement of hemangioblast formation as well as significant increases in primitive and definitive hematopoietic colonies. Cdx4 acts to induce a broad array of hox genes, including a modest elevation in hoxb4. Co-expression of cdx4 and hoxb4 promotes robust expansion of hematopoietic blasts on supportive OP9 stromal cultures. When injected intravenously into lethally-irradiated mice, these cell populations provide robust radio-protection, and reconstitute high-level lymphoid-myeloid donor chimerism. Marrow from engrafted primary animals can be transplanted into irradiated secondary mice. B220+ splenic lymphoid cells and Mac-1/Gr-1+ marrow myeloid cells purified from primary and secondary mice show multiple common sites of retroviral integration, thereby proving the derivation of long-term hematopoietic stem cells from embryonic stem cells in vitro. Our data support a central role for the cdx4-hox gene pathway in specifying murine HSC development, and establish a robust system for hematopoietic reconstitution from ESCs. We have coupled techniques for generating ESCs by nuclear transfer with these methods for blood reconstitution to model the treatment of genetic disorders of the bone marrow.
Low Morbidity but Prolonged Viral Shedding Characterize Parainfluenza Virus 3 Infections in Allogeneic Peripheral Stem Cell Transplant Recipients.
Authors: Aliyu Zakari Y. , Bordner Mary-Ann , Henderson David K. , Sloand Elaine M. , Young Neal S. , Childs Richard , Bennett John , Barrett John
Published: October 2019

Respiratory viruses including human parainfluenza virus 3 (hPIV3) may be significant causes of morbidity and mortality in patients undergoing allogeneic stem cell transplantation. It has been suggested that the acquisition of hPIV3 infections is independent of immunosuppression or transplant type and is driven primarily by patient exposure to the virus during periods of heightened community-wide or nosocomial activity. The incubation period for hPIV3 is 1–3 days and the duration of infectivity/ viral shedding in non-transplant patients is 1–2 weeks. Data regarding the duration of infectivity/ viral shedding and possible predictors of prolong infectivity in peripheral blood stem cell transplant patients (PBSCT) is limited. Of 56 recipients of sibling PBSCT seen at our insitution, 11 (6 inpatients and 5 outpatients) were positive for hPIV3 during a six weeks period, compared with only two positive cases recorded throughout the previous year. At the time of infection patients were between 1 and 7 weeks post transplant. The in-patient attack rate was 11%. Patients in whom respiratory virus infection was suspected were placed on respiratory isolation and followed with weekly nasopharygeal swab/wash (NPW). Care was also taken to limit patient contact of relatives or staff with respiratory symptoms. Most patients developed a flu like illness without major respiratory complications. No mortality was recorded. No case of hPIV 3 was recorded among patients with aplastic anemia receiving ATG in the unit during the same time-period. The median time to conversion from positive to negative hPIV 3 NPW was 2.0 weeks (range 1–6 weeks). The longest duration of viral shedding (six weeks) was in the only haplo-identical SCT patient, who was also the index case. No differences in duration of viral shedding was noted for conditioning regimen type (ablative vs non-ablative), days after transplant of viral acquisition (<15 or >15 days), cyclosporin levels (100–200 ng/l vs 201–400 ng/l), absolute neutrophil counts, absolute lymphocyte counts, CMV reactivation and co-existing peri-hPIV3 infections evidenced by positive blood, urine or sputum cultures. immunosuppression during the outbreak. Our study demonstrates that while hPIV infection in allogenic PBSCT patients has limited morbidity, prolonged duration of viral shedding in this group may contribute to recurrent cycles of epidemics or prolong outbreaks and establish an environmental (community/ nosocomial) contamination or “ping-pong” transmission from patients to asymptomatic health-care workers or family members and back. Our study also emphasize the importance of a universal policy for strict attention to infection-control practices and weekly surveillance NPW until patients are a symptomatic and NPW negative.
Recipient Epstein Barr Virus (EBV) Seropositivity with IgG Antibodies Against Early Antigens (EA) Could Be a Predictive Marker for the Early Onset of Post Transplant Lymphoproliferative Disorders (PTLDs) after Allogeneic Non T Depleted Stem Cell Transplantation.
Authors: Agbalika Felix , Larghero Jerome , Marais Dominique , Esperou Helene , Socie Gerard , Benbunan Marc , Gluckman Eliane , Marolleau Jean Pierre
Published: October 2019

After allogeneic bone marrow (BM) or peripheral blood stem (PBSC)cells transplantation, the occurrence of PTLDs most frequently associated with EBV represent a serious clinical management problem. Identification of risk factors to allow preventive strategies could be useful. In this single center retrospective study from1999 to 2003, we analyzed the EBV virus load in the graft and the EBV serological pretransplant status from 21 recipients with a PTLD and 28 control allografted patients without PTLDs. All patients received a non T cells depleted stem cells transplantation from geno and matched unrelated identical donors for various hematological disorders. The median time from graft to he onset of PTLD was two months (range 1–8 months). All the PTLDs patients received ATG in the conditioning regimen and had an EBV real time-PCR (RT-PCR) highly positive at time of diagnostic.

First, we assessed the EBV viral load in the infused cells by RT-PCR. In the PTLDs group, only 2 cases( one BM and one PBSC) contained infected EBV cells: 1,3x10e7 and 4,33x10e8 per 10e6 cells respectively. In the control group, we did not detect copies (the difference was not significant). The recipient pretransplant EBV infection status (median time: 15 days before transplant) was completed with serology anti-EA (IgA,IgG) and anti -VCA(IgA). Results are in the table 1. We found only a significant difference (p=0,00023) between the recipients with PTLDs and the control group for the early antigens positivity. Anti-EA are associated with a reactivation of EBV.

In conclusion, the EBV viral load in the infused cells is not associated with the onset of PTLD. However, our findings suggest that the detection of IgG antibodies to EA antigens in the recipient pretransplant serology could be a predictive marker of the early onset of a PTLD.

EBV recipient pre transplant status

anti-VCA IgG anti-VCA IgA anti-EBNA IgG anti-EA IgG anti-EA IgA # p=0,00023 PTLDs pts n=21 21/21 (100%) 0/21 (0%) 21/21 (100%) 12/21 (57%)# 0/21 (0%) Control pts n=28 28/28 (100%) 0/28 (0%) 28/28 (100%) 2/28 (7%)# 0/28 (0%)
A Genetic Basis for Adult Onset TTP?.
Authors: Camilleri Raymond S. , Mackie Ian J. , Scully Marie , Machin Samuel J. , Cohen Hannah
Published: October 2019

Thrombotic thrombocytopenic purpura (TTP) presents in childhood or adult life and is caused by a deficiency of von Willebrand factor-cleaving protease (ADAMTS13) activity. Several mutations and polymorphisms have been reported in ADAMTS13 in congenital TTP. The study aim was to elucidate whether a mutation(s) may be linked to adult onset TTP, which may be precipitated by infection, pregnancy or oestrogen-containing preparations and is associated with antibodies to ADAMTS13. We investigated 58 adult onset TTP patients (median age 39 (range 16–74) years) with ADAMTS13 activity levels <5% of pooled normal plasma when tested pre-treatment during the acute phase of their TTP episode. Initial screening using WAVE® technology detected a novel sequence change in exon 24 of ADAMTS13 in 2/14 of these patients (both heterozygous). Gene sequencing identified this nucleotide change as C3178T which results in the significant amino acid substitution arginine to tryptophan (R1060W) in the terminal thrombospondin type 1 (TSP1-8) domain of ADAMTS13. We confirmed this C3178T nucleotide change using a specific PCR and restriction enzyme digest and studied the remaining 44 cases, identifying one homozygote (T3178T) and three additional heterozygous patients, giving a total of 6/58 (10.3%). Fifty-eight healthy normal subjects (median age 33 (range 22–57) years) were also screened for this C3178T nucleotide change and all had the wild-type (C3178C) genotype. There was a significant difference in prevalence of the C3178T missense mutation between the TTP patient and normal control groups (p=0.0072, χ2 test, odds ratio (OR) = 0.00, 95% confidence intervals (CI) = 0.00–0.67). The mutation was only detected in Caucasian patients (n = 6/41). It was not found in any of the Afro-Caribbeans (n = 8), Asians (n = 5) or patients in other ethnic groups (n = 4). The difference in prevalence remained significant amongst the Caucasian contingent (p=0.0024, χ2 test, OR = 0.00, 95% CI = 0.00–0.52). IgG antibodies to ADAMTS13 (found in 37/58 (63.8%) patients) were demonstrated in three of the five heterozygous C3178T TTP patients, but not in the homozygous patient. The C3178T mutation was also detected in the mother of one of these three heterozygous patients and the daughter of the homozygous patient, neither of whom have had TTP. In conclusion, the novel C3178T missense mutation appears to be associated with adult onset TTP and in some instances, antibodies to ADAMTS13. This raises the possibility that adult onset TTP may arise as a result of an interaction between genetic and acquired factors.
Durable Molecular Remission in Two Acute Promyelocytic Leukemia (APL) Patients Treated with Arsenic Trioxide at First Molecular Relapse.
Authors: Douer Dan , Ramezani Laleh , Watkins Kristy , Louie Robert , Tallman Martin S.
Published: October 2019

Background: The recurrent detection of the PML- RARα transcripts in APL patients in complete remission (CR) is predictive of clinical relapse. Intervention at the time of molecular relapse rather than at the time of overt clinical relapse may minimize the risks associated with the disease as well as treatment-related complications. Previous studies have shown that patients treated at molecular relapse had improved overall survival compared to patients treated at clinical relapse; however, the optimal approach to the treatment of molecular relapse has not yet been established. Arsenic trioxide is active as a single agent in APL patients who developed overt clinical relapse after treatment with ATRA and anthracycline chemotherapy. We evaluated the use of single agent arsenic trioxide in APL patients who are still in first hematological CR but have become RT-PCR positive.

Methods: So far, 2 patients were studied, both females with the long form of RAR RARα (bcr1), aged 29 and 39 years. These patients had normal bone marrow morphology but were found to be RT-PCR positive twice in an interval of at least 2 months, while in first CR at 12 and 36 months, respectively. Arsenic trioxide, 0.15 mg/kg/dose, was administered intravenously for 5 days per week for 5 weeks (total of 25 doses) with a 3 to 6 week rest period between cycles up to a maximum of 4 cycles.

Results: Both patients became PCR negative after the first cycle of arsenic trioxide. One patient completed four cycles of therapy. The second patient had a history of diabetes mellitus, received only 20 doses of the first cycle and was removed from the study because of grade III sensory neurotoxicity. The only other side effects were grade II headache in both patients and self-limited grade III liver toxicity in one patient. Both patients remain RT-PCR negative at 30 and 28 months after enrollment, respectively, without further therapy.

Conclusions: Without treatment, these patients could be expected to have developed a clinical relapse within 4–10 months. Arsenic trioxide induced a durable second molecular relapse, extending disease-free survival and obviating the need for more intensive treatment, such as chemotherapy and stem cell transplantation.
All-Trans Retinoic Acid and Gemtuzumab Ozogamicin as Adjunct To Salvage Therapy in Primary Refractory Acute Myeloid Leukemia: Results of Consecutive Phase II Studies of the AMLSG.
Authors: Schlenk Richard F. , Doehner Konstanze , Kerz Martina , Hartmann Frank , del Valle Francesco , Glasmacher Axel , Pralle Hans , Goetze Katharina , Koller Elisabeth , Gastl Guenther , Mergenthaler Hans G. , Salwender Hans , Kirchen Heinz , Haase Detlef , Kremers Stephan , Nerl Christoph , Theobald Matthias , Matzdorff Axel C. , Ganser Arnold , Doehner Hartmut ,   for the German-Austrian, AML Study Group (AMLSG)
Published: October 2019

Introduction: Response to first induction therapy is one of the most important prognostic factors in patients with adult myeloid leukemia (AML). Induction of CR or PR is the primary aim in these patients.

Methods: Between 1993 and 2005 225 consecutive patients (median age: 48.4 yrs, range 16–60 yrs) treated within the AMLHD93 (n=45), AMLHD98A (n=157) and AMLSG 05-04 (n=23, still active) trials were evaluated. All patients had primary refractory AML after one cycle of ICE. The different salvage therapies were as follows: AMLHD93 sequential-HAM (S-HAM) for patients <55 years of age [cytarabine 3g/m2 bid. days 1,2,8,9, mitoxantrone 10mg/m2 days 3,4,10,11], HAM for patients >=55 years of age [cytarabine 3g/m2 bid., days 1–3, mitoxantrone 12mg/m2 days 2,3]; AMLHD98A: A-HAM [HAM with ATRA 45mg/m2 days 3–5, 15mg/m2 days 6–28]; AMLSG 05-04: GO-A-HAM [A-HAM with gemtuzumab ozogamicin 3g/m2 day 1].

Results: The distribution of the different salvage therapies was HAM n=21, S-HAM n=22, A-HAM n=117, GO-A-HAM n=23, other n=31 no further therapy n=11. Response according to salvage therapy was as follows:

response GO-A-HAM A-HAM S-HAM HAM CR 11 (48%) 40 (34%) 5 (23%) 3 (14%) PR 4 (17%) 33 (28%) 5 (23%) 4 (19%) RD 6 (26%) 36 (31%) 12 (54%) 12 (57%) death 2 (9%) 8 (7%) 0 2 (10%)

No CTC-grade 3-5 liver toxicity was seen in patients receiving GO-A-HAM. Multivariable analyses revealed that regimens containing ATRA (odds ratio 2.5, p=0.01) and cytogenetic subgroup [t(11q23) odds ratio 4.2 p=0.04 (n=13), non-complex high risk aberrations odds ratio 4.2 p=0.007 (n=34)] were associated with a significantly better response rate (subsuming CR and PR). 119 of 225 patients have received stem cell transplantation. No case of veno occlusive disease was in 10 so far transplanted pts who have had GO-A-HAM. Median survival was 10.7 months.

Conclusions: Although retrospective in nature our study suggests that ATRA as adjunct to salvage chemotherapy in primary refractory AML patients improves the response rate. The addition of GO in a dosage of 3mg/m2 results in promising response rates without increasing toxicity.
A Phase I Pharmacokinetic Trial of Decitabine Administered as a 3-Hour Infusion to Patients with Acute Myelogenous Leukemia (AML) or Myelodysplastic Syndrome (MDS).
Authors: Cashen Amanda , Shah Ajit , Helget Angela , Todt Laura , Fisher Nicholas , DiPersio John
Published: October 2019

Background: Decitabine (DacogenTM) is a cytosine analogue that promotes hypomethylation of DNA. Although decitabine has been extensively studied in Phase II and III trials in patients with MDS, relatively limited pharmacokinetic (PK) data is available in this patient population. Recently, a Phase III trial of decitabine in advanced MDS demonstrated that decitabine administered as a 3-hour infusion of 15 mg/m2 every 8 hours for 3 days (cycles repeated every 6 weeks) results in a significantly higher response rate and improves time to AML or death compared to best supportive care (J Clin Oncol. 2005;23(suppl 16S):570s).

Methods: The PK of this dosing regimen was evaluated in twelve patients (8M/4F, age 50–87 yr) with MDS (n=6, IPSS ≥ 1.5) or relapsed/refractory AML (n=6). Patients were treated with the same dose and schedule as the Phase III trial, with a second cycle repeated six weeks after the first. Plasma samples were obtained pre-dose and during the first 8-hour dosing interval on each dosing day during cycle 1, and at pre-dose and just prior to the end of infusion during cycle 2. PK samples were assayed for decitabine by a sensitive and specific LC/MS/MS method.

Results: Day 1 cycle 1 PK results (mean±SD) from ten patients are shown in the table below (data from two patients excluded due to dosing error).

In plasma, decitabine did not accumulate during three days of dosing, and CLp remained unchanged. For the 5 patients who have received decitabine for two cycles, Cmax values for cycle 1 (49.0±22.2 ng/mL) and cycle 2 (62.7±45.2) were comparable, indicating unchanged PK during both cycles. This regimen of decitabine was generally well-tolerated. Serious adverse events included neutropenia and fever (2 patients) and infection (one patient with gram positive bacteremia and pneumonia). In addition, one patient died of sepsis, and two died of progressive AML. Of four patients who have been assessed for response after 2 cycles of decitabine, one patient with MDS had a complete response, one patient with MDS had hematologic improvement, and two patients (one MDS, one AML) had no response.

Conclusions: We conclude that repeated administration of decitabine does not result in systemic accumulation of the drug, and PK remains unchanged from cycle to cycle. It will be of interest in future studies to correlate pharmacokinetics of decitabine with pharmacodynamic changes in CpG island methylation and clinical responses.

Cmax (ng/ml) Tmax (hr) AUC(0-∞) (ng•hr/ml) T1/2 (hr) CLp (L/hr/m2) Vdss (L/m2) AUC Ratio (Day3/Day1) *Geometric mean 79±54.1 2.67±0.55 170±105 0.65±0.30 122±73.0 75.8±70.1 0.92±0.15 65.1* 144*
Dual Inhibition of Blood Coagulation Factors Xa and Iia Synergize To Reduce Thrombus Weight and Thrombin Generation in a Rabbit Arteriovenous Shunt Model of Thrombosis.
Authors: McClanahan Thomas B. , Baxi Sangita M. , Chi Liguo , Dahring Tawny , Gould Weston R. , Johnson Ted , Hicks Gary , Welch Kathy , Leadley Robert J.
Published: October 2019

Several compounds currently in development for the treatment of thrombotic disorders demonstrate high levels of specificity for single targets of the blood coagulation cascade such as factor Xa and thrombin. However, development of a single molecule dual inhibitor against factor Xa and thrombin may expand the efficacy to safety ratio of treatment options for arterial and venous thrombosis. The objective of this study was to determine if simultaneous administration of PD 0313052, a selective Xa inhibitor and argatroban, a direct thrombin inhibitor, would lead to a synergistic antithrombotic effect in a rabbit AV shunt model of thrombosis. Intravenous administration of PD 0313052 alone at doses of 0.1, 0.3, and 1.0 mg/kg/min resulted in thrombus weight (TW) reductions of 11±3, 25±10 and 67±7 % compared to the vehicle group. Argatroban at 1, 3 and 10 mg/kg/min reduced TW 16±13, 47±10 and 75±6 %. When PD 0313052 was administered at 0.1 mg/kg/min in combination with argatroban at 1, 3 or 10 mg/kg/min TW was reduced 50±7, 60±7 and 82±9 %. Likewise, argatroban at 1 mg/kg/min combined with 0.1, 0.3 or 1mg/kg/min of PD 0313052 resulted in TW reductions of 56±9, 60±9 and 84±5 %, respectively. At the lowest combined doses of PD 0313052 and argatroban there was no change in bleeding time relative to the additive fold-increases from each drug alone. The EC50 of intravenously administered PD 0313052 and argatroban was 67±23 and 178±58 ng/ml, respectively. When the drugs were combined the EC50 was reduced to 12±6 ng/ml with the PD 0313052/argatroban combination and to 83±29 ng/ml with the argatroban/PD 0313052 combination. A synergistic effect was also observed in an ex vivo assay of thrombin generation (TG). Predicted additive inhibition of TG based on the individual effects of each compound was −9±7, 9±2 and 29±7 % compared to 10±5, 32±5 and 55±3 % with the 313052/argatroban combination. The predicted effects of the argatroban/PD 0313052 combination was −9±7, 1±7 and 16±9 % compared to the actual inhibition of 5±3, 14±5 and 31±7 %. These results demonstrate a significant synergistic antithrombotic effect by combining low doses of a factor Xa and a thrombin inhibitor and support the hypothesis that development of a single molecule inhibitor against different hemostatic targets may offer greater efficacy in the prevention and treatment of venous and arterial thrombosis.
Anti-Thrombotic Action of DZ-697b, a Novel Anti-Platelet Agent, on Photochemically Induced Thrombosis with Lower Bleeding Risk in Guinea Pigs.
Authors: Shibutani Tomoko , Iijima Takako , Kaneda Yuki , Muramatsu Sumie , Ogihara Yoshiyasu , Shibano Toshiro
Published: October 2019

Introduction: DZ-697b, a novel anti-platelet compound, selectively inhibits ristocetin- and collagen-induced platelet aggregation, whose pharmacological profile is different from any current medicines. In order to clarify the benefit and risk balance as an anti-thrombotic agent, effects of DZ-697b and aspirin were compared on thrombus formation and gastric bleeding model in guinea pigs.

Materials and Methods: Fasted male Hartly guinea pigs under anesthesia were used. DZ-697b (3 - 200 mg/kg) or aspirin (100–300 mg/kg) was orally administered 60 min prior to evaluation.

PIT model: Left carotid artery was exposed. Photochemically induced thrombosis (PIT) was initiated by intravenous injection of rose-bengal (10 mg/kg) and exposure of green light (540 nm) to the carotid artery. The time to occlusion by thrombus formation was measured by pulse Doppler blood flow meter.

Gastric bleeding model: The stomach was exposed, and esophagus and pylorus were ligated. A cannula was inserted into stomach and gastric hemorrhagic erosion was induced by intraluminal application of 0.7N HCl in a volume of 5mL. Gastric contents were collected every 10 min and measured hemoglobin (Hb) leakage as indices of bleeding.

Results: DZ-697b at 3 mg/kg to 100 mg/kg dose-dependently prolonged the time to occlusion in the PIT model, and significant anti-thrombotic effects were observed from 10 mg/kg. However, gastric bleeding induced by 0.7N HCl in gastric lavage was not affected by DZ-697b up to 100 mg/kg. Statistically significant increase in gastric bleeding was observed when DZ-697b was administered at 200 mg/kg. Aspirin at doses of 100–300 mg/kg tended to lengthen the time to thrombotic occlusion in a dose-dependent manner, but there was no statistically significant difference. In contrast, aspirin dose-dependently increased gastric bleeding in the lavage and significant enhancement was found at doses of 200 and 300 mg/kg. Both DZ-697b and aspirin did not give any influences on the area of hemorrhagic erosion induced by gastric application of 0.7N HCl. Thus, the balance between anti-thrombotic effect and bleeding risk of DZ-697b seemed more favorable than that of aspirin.

Conclusion: DZ-697b showed anti-thrombotic effects at doses much lower than those inducing gastric bleeding in comparison with aspirin. These results suggest that inhibitors of vWF and collagen related signaling pathway are promising target in the development of new anti-platelet drugs with lower bleeding risk.
Coupling Tissue Type Plasminogen Activator to Carrier Erythrocyte Protects Against Plasma Inhibitors.
Authors: Ganguly Kumkum , Cines Douglas B. , Muzykantov Vladimir R.
Published: October 2019

Conjugating tissue plasminogen activator (tPA) to carrier red blood cells (RBC) restricts its permeation into tissues and pre-existing hemostatic clots, minimizing side effects, and prolongs its circulation, which permits it to be incorporated within nascent clot which it lyses from inside. We now report that RBC/tPA dissolves clots formed from mouse blood more effectively than free tPA, while they were equipotent against clots formed in vitro from PAI-1 KO mouse blood. To test whether tPA acquires resistance to plasma inhibitors when conjugated to RBC, we compared the activity of RBC/tPA vs free tPA in the presence of purified PAI-1, α2 macroglobulin (α2M) and α1anti-trypsin (α1AT). At equimolar concentrations, PAI-1 completely inhibited the activity of soluble tPA in vitro, whereas RBC/tPA retained 100% and 75% of its amidolytic and fibrinolytic activity, respectively. RBC/tPA, but not free tPA, was also resistant to equimolar concentrations of α2M and α1AT. However, tPA coupled to RBC pre-incubated with a mixture of hyaluronidase, heparinase and neuraminidase was as susceptible to inactivation by PAI-1, α2M and α1AT as free tPA. Further, tPA coupled to glycocalyx-stripped RBC bound two-fold more 125I-labeled PAI-1 than tPA coupled to naïve RBC. We conclude that the RBC glycocalyx protects tPA from inactivation by PA inhibitors, likely by steric hindrance. This unexpected benefit may enhance the utility of RBC/tPA in thromboprophylaxis and suggests a previous under-appreciated role for the glycocalyx in modulating PA activity on the vasculature and other cell surfaces.
Incidence of HCV and HIV NAT Positive in Cord Blood Donors.
Authors: Rhamy Jennifer , Beltz Mark , John Karla , Feria David , Waxman Dan , Dracker Robert
Published: October 2019

Introduction:

Donors of cord blood (CBD) are screened similarly to whole blood donors (WBD). It has been reported that CBD have a higher incidence of both true positive and false positive disease markers tested by EIA[1]. This abstract compares the rate of positive results for viral RNA (NAT) in CBD vs. WBD in a large cohort of donors.

Methods:

NAT was performed using the Chiron Procleix MPX or discriminatory assays. EIA testing was performed using Abbott bead methodology following manufacturer’s instructions. Confirmatory results for EIA were performed with Chiron RIBA. Results from 12/17/04 to 6/30/05 were collated.

Results

EIA HIV EIA HCV NAT HIV/HCV HIV EIA POS(%) HCV EIA POS (%) HIV NAT POS HCV NAT POS WBD 74299 74299 74299 75(0.10) 107(0.14) 1 (0) 31(0.04) CBD 11996 12001 12007 50(0.42) 46(0.38) 0 13 (0.11)

A higher percentage of CBD were positive for HIV and HCV EIA as previously reported1. HIV NAT positive donors were 0.00% for both populations. The ratio of HCV EIA positive to HCV NAT positive donors was 28.97% for WBD and 28.26% in CBD. The ratio of CBD HCV EIA positives to WBD HCV EIA positives is 2.71 and the ratio of WBD HCV NAT positives to WBD HCV NAT positives is 2.75. The ratio of HIV CBD EIA positives to HIV WBD EIA positives is 4.10, which is significantly higher (p=<0.01).

Conclusions

Both CBD and WBD have a low rate (0%) of HIV NAT positive donors, but CBD have a 4x higher rate of HIV EIA. This suggests a 4x higher donor loss to false positive antibody results. The CBD donors have a 2.7x higher rate for both EIA and NAT positive results, showing a higher rate of viremic donors in CBD. The similar ratios for EIA positive to NAT positive donors in the two populations suggest that the false positive rate is not elevated in CBD versus WBD.

The higher incidence of HCV infection in CBD could be geographic or ethnic in origin. More study is needed to determine the reasons and further predict donor loss. The HIV EIA results are not predictive of actual infection in this population. A more aggressive logarithm for reentry than used for WBD may be merited.
Molecular and Clinical Features of Diffuse Large B-Cell Lymphoma with MYC Alterations: Inverse Correlation with p53 Pathway Disruption.
Authors: Moller Michael B. , Nielsen Ole , Christensen Bjarne E. , Pedersen Niels T.
Published: October 2019

Diffuse large B-cell lymphoma (DLBCL) is a clinically and genetically heterogeneous disease. Frequent genetic lesions include alterations of BCL2 and BCL6, as well as aberrations of the RB pathway and the p53 pathway resulting in deregulation of cell cycle progression and apoptosis. MYC translocations and amplifications have been detected in a subset of cases. Recently, MYC was shown to control a major regulatory network in B-cells (Nature Genet. 37; 382–90: 2005). We thus hypothesized that DLBCLs with constitutively active MYC are associated with distinctive genetic and clinical features when compared to cases with wild-type MYC. Thirty seven de novo DLBCLs were analyzed using immunohistochemistry for CD10, bcl6, MUM1, and bcl2 expression, as well as fluorescence in situ hybridization for MYC, BCL2, BCL6, IGH, IGK, and IGL breakpoints and amplifications. The lymphomas were previously extensively characterized for aberrations at the genetic, epigenetic, or protein level of components of the RB pathway (pRb, p16, cyclin D3, cdk4) and the p53 pathway (p53, p14ARF, MDM2, E2F1). Five cases (14%) had MYC alterations (4 breakpoints and 1 amplification). Of the MYC+ DLBCL cases, 2 showed GCB immunophenotype and 3 were of non-GCB type. Perturbation of the p53 pathway components p53, p14ARF, MDM2, and E2F1 were identified in 28%, 16%, 53%, and 20% of the MYC− cases, respectively, and 83% had aberrations of 1 or more p53 pathway component. In contrast, only 1 (20%) of the MYC+ cases had a p53 pathway aberration (MDM2 overexpression; p=0.013). Thus, MYC alterations and p53 pathway aberrations were inversely correlated. RB pathway aberrations were equally frequent among MYC+ (40%) and MYC− (43%) DLBCLs. However, the pattern of aberrations was different. Lack of pRb expression was the only alteration of the RB pathway in the MYC+ DLBCLs, whereas 30 of 31 (97%) MYC− lymphomas had normal pRb expression (p=0.045). p16 alterations (point mutation, deletion, hypermethylation, lack of expression) and/or cyclin D3 overexpression were identified in 12 (38%) MYC− cases but in none of the MYC+ cases (p=0.038). MYC status was also related to cytogenetic BCL2 alterations. Four of 5 (80%) MYC+ DLBCLs showed BCL2 breakpoints or amplification in contrast to 4 of 32 (13%) of MYC− DLBCLs (p=0.005). Furthermore, all MYC+ DLBCLs were bcl2 positive (p=0.05) and MUM1 negative (p=0.022), and the proliferation index was lower in the MYC+ DLBCLs (p=0.038). Clinically, MYC+ DLBCL patients were characterized by advanced stage disease (p=0.037) and high tumor burden (p=0.037) but favorable treatment response (p=0.05). Taken together, our data indicate that DLBCL with MYC alterations constitute a subgroup of DLBCL with distinct clinical and molecular features. The patterns of aberrations of the p53 pathway, the RB pathway, and BCL2 indicate profound differences in the molecular pathogenesis between MYC+ and MYC− DLBCL.