bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of Phosphotyrosine Phosphatase-1B (PTP1B) Induces Resistance to the ABL Kinase Inhibitor Imatinib Mesylate (Gleevec®) in BCR-ABL Positive Leukemic Cells.
Authors: Koyama Noriko , Koschmieder Steffen , Tyagi Sandhya , Myloch Silke , Nuernberger Heike , Rossmanith Tanja , Hofmann Wolf Karsten , Hoelzer Dieter , Ottmann Oliver Gerhard
Published: October 2019

BCR-ABL chronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL) have recently received much attention as model diseases for the effectiveness of targeted drug therapy. The ABL kinase inhibitor imatinib mesylate has revolutionized treatment of CML. However, a serious drawback to this specific treatment is the emergence of drug resistance. Protein tyrosine phosphatase-1B (PTP1B) has been demonstrated to block the BCR-ABL mediated transformation of hematopoietic cells in vitro and in vivo. Therefore the impact of PTP1B function on imatinib-response of the BCR-ABL positive cell line K562 and an imatinib-resistant subclone of the cell line SupB15 was investigated.

K562 cells were transfected with wild-type PTP1B (K562/PTP1B), a substrate-trapping dominant-negative mutant PTP1B (K562/D181A), or an empty vector (K562/mock), cultured in the presence or absence of imatinib (0.25–1.0 μM) and analyzed for proliferation, apoptosis and differentiation. K562/PTP1B and K562/D181A cells displayed 1.6-fold higher and 13-fold lower phosphatase activity than K562/mock cells, resp. In both K562/mock and K562/PTP1B cells, 0.5 μM imatinib induced growth arrest, an increase of cells in sub-G1 apoptotic phase (36.9±2.0% and 38.6±3.8%, resp.) and a decrease of cells in S/G2/M phases (18.9±1.1% and 23.7±2.7%, resp.) as determined by FACS analysis of propidium iodide stained cells. Imatinib-induced apoptosis was further confirmed by Annexin V staining and Western blot analysis showing increased active caspase-3 and -8 protein levels and induction of poly ADP-Ribose Polymerase (PARP) cleavage in both cells. Low doses of imatinib (0.25 μM) promoted erythroid differentiation of these cells as evaluated by enhanced expression of glycophorin A. Conversely, K562/D181A cells were significantly less sensitive to imatinib-induced growth arrest (sub-G1: 25±3.9 %, S/G2/M: 34.2±1.4 %) apoptosis, and promotion of erythroid differentiation. Inhibition of PTP1B phosphatase activity by the PTP1B inhibitor bis(N,N-Dimethylhydroxamido)hydroxooxovanadate (DHMV) decreased imatinib-induced apoptosis in wild-type K562 cells (46.7% vs. 28.3% annexin V positive cells; p<0,01). Moreover, the imatinib-resistant subclone of the ALL cell line SupB15, which had been generated in the presence of 1μM imatinib, showed significantly lower PTP1B activity (64.3±5.6%, p<0.05) as compared with imatinib-sensitive wild type cells. In conclusion, our data demonstrate that functional PTP1B is required for imatinib-induced apoptosis, growth arrest, and differentiation. Alterations of PTP1B gene expression and function might contribute to imatinib-resistence and should therefore be assessed in patients with BCR-ABL positive chronic and acute leukaemia who developed restance to imatinib.
Effects of Adaphostin, a Novel Tyrphostin Inhibitor, in Diverse Models of Imatinib Mesylate Resistance.
Authors: Chandra Joya , Tracy Jeannette , Gorre Mercedes , Talpaz Moshe , Griffin James , Verstovsek Srdan , Beran Miloslav , Sawyers Charles , Sausville Edward , Kaufmann Scott
Published: October 2019

The impetus for kinase inhibition as a therapeutic endpoint in leukemia research has been driven largely by the success of imatinib mesylate in chronic myelogenous leukemia. However, while imatinib mesylate has been extremely effective in the treatment of chronic phase CML, resistant disease often emerges in both Philadelphia positive ALL and aggressive phase CML patients. Adaphostin is a tyrphostin inhibitor originally developed to target the bcr/abl kinase and demonstrates selectivity in CML patient isolates. Here we show that in vitro, adaphostin is cytotoxic in numerous models that are resistant to imatinib. Features of adaphostin action in bcr/abl containing cells include apoptosis induction via an oxidant dependent mechanism and degradation of bcr/abl protein. Cell lines containing point mutations in p210 bcr/abl which confer imatinib resistance exhibit intracellular peroxide production and DNA fragmentation in response to adaphostin. Bcr/abl degradation also occurs but can be dissociated from peroxide production and subsequent apoptosis signaling. Similar data has also been obtained in cell lines containing p190 bcr/abl. Studies extending these observations into mononuclear and polymorphonuclear cells from imatinib resistant CML patients and Ph+ ALL patients confirm the same mechanism in primary clinical isolates. Collectively, these data suggest that reactive oxygen species generation by adaphostin bypasses imatinib resistance in CML and Ph+ ALL. Further studies dissecting the mechanism of ROS generation by adaphostin in imatinib resistant cells may lead to the identification of new therapeutic targets.
Efficient Gene Transfer into Primate ES Cells and Stable Transgene Expression during Hematopoietic Differentiation by Sendai Virus Vector.
Authors: Hanazono Yutaka , Sasaki Kyoko , Inoue Makoto , Shibata Hiroaki , Ueda Yasuji , Hasegawa Mamoru , Ozawa Keiya
Published: October 2019

Since human embryonic stem (ES) cell lines have the ability to both proliferate indefinitely and differentiate into multiple tissue cells, they are expected to have clinical applications as well as to serve as models for basic research and drug development. Although efficient and stable gene transfer into primate ES cells would be useful for such purposes, it has been difficult and only lentiviral vectors have been successful in achieving it. We have previously developed Sendai virus (SeV) vector that is capable of self-replication but incapable of transmitting to other cells. The vector replicates in the form of negative-sense single-stranded RNA in the cytoplasm of infected cells and does not go through a DNA phase. SeV vector can efficiently introduce foreign genes without toxicity into various types of cells including hematopoietic progenitor cells. In this study, we have examined the efficacy of SeV vector to introduce a new genetic material into nonhuman primate cynomolgus ES cells.

Cynomolgus ES cells were once exposed to a SeV vector carrying the GFP gene (50 TU/ml). About 60% of cells stably fluoresced at least for a month as assessed by flow cytometry. Fluorescent ES-cell colonies were plucked under a fluorescent microscope once at one month after infection and they were propagated. The GFP gene was then vigorously and stably expressed in about 90% of the cells for a year. The undifferentiated cell fractions were unchanged after the SeV infection of ES cells as assessed by staining with alkaline phosphatase and SSEA-4. In addition, the infected, fluorescent ES cells were able to form fluorescent teratomas when transplanted into immunodeficient mice. Thus, the SeV infection did not hamper pluripotency of ES cells and transgene silencing did not occur during the teratoma formation in vivo. For induction of hematopoietic differentiation, the infected, fluorescent ES cells were seeded onto OP9 stromal cells in the presence of multiple cytokines including BMP-4 and VEGF. Cells at day 18 were subjected to clonogenic hematopoietic colony formation assays. Resulting colonies at day 14 fluoresced and the colonies contained NBT-positive, fluorescent mature granulocytes. Thus, SeV-mediated transgene was stably expressed even after the terminal differentiation to granulocytes. In addition, SeV-mediated GFP expression levels in ES cells could be reduced dose-dependently by the addition of an anti-RNA virus drug, ribavirin, to the culture. Upon discontinuation of ribavirin treatment, the cells regained the original level of GFP expression. Ribavirin is an inhibitor of viral RNA polymerase and used for treatment of hepatitis C infection. In conclusion, SeV vector will be a useful tool for gene transfer into primate ES cells and the method of using anti-viral drugs should allow further investigation for regulated SeV-mediated gene expression.
Prospects for Dendritic Cell Vaccination Against Fungal Infections in Hematopoietic Transplantation.
Authors: Perruccio Katia , Bozza Silvia , Montagnoli Claudia , Bellocchio Silvia , Aversa Franco , Martelli Massimo F. , Bistoni Francesco , Velardi Andrea , Romani Luigina
Published: October 2019

Dendritic cells (DCs) are uniquely able to initiate and control the immune response to fungi. DCs function at three levels in the manipulation of the immune response to these pathogens: 1) they mount an immediate or innate response to them, for example by producing inflammatory mediators upon capture and phagocytosis; 2) through these preceding innate functions, they decode the fungus-associated information and translate it in qualitatively different Th responses; 3) and they are key in containing and dampening inflammatory responses by tolerization through the induction of regulatory T cells (Treg). We demonstrated that both murine and human DCs sense fungi in a morphotype-specific manner, through the engagement of distinct recognition receptors ultimately affecting cytokine production and costimulation (Romani et al. Int Immunol. 2003). In this study we analyzed both myeloid and plasmacytoid murine and human DCs and we observed that they phagocytose fungi and undergo functional maturation in response to them. However, their activation program for cytokine production was different, being IL-12 mainly produced by myeloid DCs and IL-12, IL-10 and IFN-gamma mainly produced by plasmacytoid DCs. This resulted in a distinct ability for T-cell priming, being Th1, Th2 and Treg differently activated by the different DC subsets. The ability of fungus-pulsed DCs to prime for Th1, and Th2 cell activation upon adoptive transfer in vivo correlated with the occurrence of resistance and susceptibility to the infections, respectively. Antifungal protective immunity was also induced upon adoptive transfer of DCs transfected with fungal RNA (Bacci et al. J Immunol. 2002; Bozza et al. Blood. 2003). The efficacy was restricted to DCs transfected with RNA from yeasts or conidia but not with RNA from fungal hyphae. The effect was fungus-specific, as no cross-protection was observed upon adoptive transfer of DCs pulsed with either fungal species. The infusion of fungus-pulsed or RNA-transfected DCs accelerated the recovery of functional antifungal Th1 responses in mice with allogeneic hematopoietic stem cell transplantation (HSCT) without triggering graft-versus-host disease (GvHD) and affected the outcome of the infections. As the ability to respond to fungi was defective in peripheral DCs from patients with HSCT in terms of phagocytosis, cytokine production, and donor T-cell priming, our findings suggest that the adoptive transfer of DCs may restore immunocompetence in HSCT by contributing to the educational program of T-cells. Thus, the remarkable functional plasticity of DCs in response to fungi can be exploited for the deliberate targeting of cells and pathways of cell-mediated immunity in response to fungal vaccines.
Fludarabine (Flu) Based Cytoreductive Regimen and T-Cell Depleted Grafts from Unrelated or Mismatched Related Donors for the Treatment of High Risk Patients with Fanconi Anemia (FA).
Authors: Boulad Farid , Auerbach Arleen D. , Kernan Nancy A. , Small Trudy N. , Prockop Susan E. , Chaudhury Sonali , Castanza Joanne , Casson Anne , Copeland Catherine , Abendroth Heidi , Collins Nancy , O’Reilly Richard J.
Published: October 2019

Between 05/98 and 06/04, 15 consecutive patients with FA received hematopoietic stem cell transplants (SCT) from alternative donors at our Center. There were 7 males and 8 females aged 5 to 24 years (median 11.5). Hematologic diagnoses included aplastic anemia (AA) (N=5), myelodysplastic syndrome (MDS) in RAEB (N=4), RAEBT (N=1) or acute myelogenous leukemia (AML) (N=5). High risk features included: Age > 20 years (n=4), prior multiple transfusions (n=11), prior androgen treatment (n=12), prior infections (n=10), or advanced MDS or AML (n=9). Eight pts had related mismatched donors transplants with respective matching at 3/6 (6/10), 4/6 (6/10), 4/6 (7/10) (n=2)), 5/6 (8/10) (n=3) and 5/6 (9/10) HLA-antigens. Seven pts had unrelated donors transplants with respective matching at 5/6 (7/10), 5/6 (8/10) (n=2), 5/6 (9/10) and 6/6 (10/10) (n=3) HLA-antigens. Cytoreduction included single dose total body irradiation (SDTBI) (450 cGy), fludarabine (Flu) (30 mg/m2 x 5) and cyclophosphamide (Cy) (10 mg/Kg x 4). Immunosuppression included rabbit anti-thymocyte globulin (Thymoglobulin) and tacrolimus for all patients. Grafts were G-CSF mobilized CD34+ and E-rosette negative (E-) peripheral blood stem cell transplants for 12 pts and soybean agglutinin negative (SBA-) and E-rosette negative marrow transplants for 3 pts. Cell doses of the grafts were 1.5 – 29.6 x 106 CD34 cells/Kg and 0 – 26 x 103 CD3 cells/Kg. As evidenced by RFLP or FISH, all 15 evaluable pts were fully engrafted and complete chimeras. Fourteen pts were evaluable for graft-versus-host disease (GvHD). GvHD of the skin and of the gut was suspected in two pts but resolved completely prior to immunosuppressive treatment. With a median follow-up of 2.5 years (range 0.2–6), 13 of 15 pts are alive and 11 of 15 are alive disease-free. There were two deaths: one pt died from sepsis/ARDS at 2 months post SCT and one pt from pneumonitis/ARDS and EBV-infection 6 months post SCT. Three pts relapsed (MDS-RAEB x 1 – AML x 2): One pt relapsed 7 months post transplant, received a 2nd transplant from the same donor following busulfan and Flu and is alive, disease-free 18 months post SCT, while the other two pts are awaiting a second SCT. In summary, this cytoreductive regimen used with T-cell depleted stem cell transplants from unrelated or HLA-mismatched related donors for the treatment of high risk patients with Fanconi anemia, results in rapid hematopoietic engraftment and lymphohematopoietic reconstitution with minimal GVHD and a high disease-free survival.
Alemtuzumab (Campath IH) in Conditioning Therapy with Cyclophosphamide and Total Body Irradiation in Matched and Mismatched Unrelated Donor Transplantation of Children with Acute Lymphoblastic Leukaemia: A Report from 3 U.K. Centres.
Authors: Gharib Maged I. , Greenfield Hayley M. , Wynn Robert F. , Veys Paul , Naik Paru , Will Andrew M. , Cornish Jacqueline M. , Oakhill Anthony , Goulden Nicholas J.
Published: October 2019

Bristol has previously published extensively on the use of Campath antibodies during the unrelated donor cell transplantation of children with relapsed or otherwise high risk Acute Lymphoblastic Leukaemia (A.L.L.). These former reports (Br J Haematol1996;94(3):574–8; Blood1999; 94(7) 2236–46) concerned the use of Campath IG (a rat monoclonal antibody directed at CD52) given pre-transplant to the patients by intravenous infusion during conditioning therapy that also included cyclophosphamide (120 mgs/kg) and fractionated total body irradiation (1440 Gy). During this treatment the unrelated donor marrow was T cell depleted in vitro using Campath IM (also monoclonal and of rat origin). The results of such treatment demonstrated results comparable with HLA-matched sibling BMT, with low levels of acute graft versus host disease. In this communication transplant centres in 3 UK hospitals (Bristol, Great Ormond Street, London and Manchester) report their combined experience of using Campath IH (Alemtuzumab, a humanised monoclonal antibody - also directed at CD52) in the conditioning therapy of children with relapsed or otherwise high risk A.L.L.. The conditioning was otherwise again with Cyclophosphamide and fractionated total body irradiation. In this protocol a T cell replete graft was given and there was no in vitro T cell depletion of the unrelated donor marrow.

We report 34 successive patients where follow up for at least 12 months was available. The median age was 6.5 years and 28 patients received fully matched grafts (matched at HLA-A, -B, -C, -DRB1 and DQB1). The remaining patients received a graft that was mismatched at one of these class I loci. All patients engrafted and the median time to neutrophil count sustained above 0.5x10e9 / l was 20 days (range 12–58 days). Only tow patients experienced grade III acute GvHD and no patient experienced grade IV acute GvHD. 22 patients survive at a median follow up of 23.8 months including 4 of the patients who received a mismatched graft. There were only 3 deaths in the first 100 days following transplant (2 due to disseminated, invasive adenovirus and one to aspergillus). Beyond 100 days 3 further patients have died of adenovirus, 3 have relapsed, 2 have died of chronic GvHD and one died of a pulmonary haemorrhage of uncertain aetiology.

We therefore conclude the combination of Aletuzumab with cyclophosphamide and TBI is an effective and safe conditioning therapy for children with relapsed A.L.L. All patients engraft and there are low rates of acute and chronic GvHD.
Hypoxia-Independent Regulation of Hepatic Erythropoietin Production by Vascular Endothelial Growth Factor.
Authors: Tam Betty Y.Y. , Hoffman Jana , Park Sang-ki , Lee Jeng-Shin , Sundram Uma , Wu Grace , Schrier Stanley L. , Johnson Randall , Mulligan Richard , Kuo Calvin J.
Published: October 2019

Erythropoietin (Epo) is an essential regulator of red blood cell production. While adult Epo synthesis occurs almost exclusively in kidney, extrarenal Epo synthesis has been described in liver and brain in response to hypoxia or anemia. We have used adenoviruses to express soluble ectodomains of the VEGF receptors (sVEGFRs) Flk1 and Flt1 in the circulation of mice, achieving high-level VEGF sequestration, potent conditional ablation of VEGF function in adult animals, and inhibition of tumor growth and angiogenesis (Kuo et al. PNAS (2001). 98: 4605). Intriguingly, adenoviral expression of soluble Flk1 or Flt1 ectodomains, or a recently described soluble receptor VEGF Trap, is associated with profound stimulation of erythropoiesis and polycythemia, with hematocrit as high as 75% from basal levels of ~45%, and elevation of plasma Epo levels. sVEGFR expression results in a profound >100-fold induction of hepatic erythropoietin synthesis while renal Epo production is completely repressed. This effect is specific for adenoviruses expressing soluble VEGF receptors and not other receptor ectodomains, does not require liver transduction or hypoxia, and is not affected by Ad Cre-induced deletion of the hepatocyte HIF-1a gene in HIF-1aflox/flox mice. Selective inhibition of hepatocyte-produced VEGF in Ad Cre-treated VEGFflox/flox mice is sufficient to re-activate hepatocyte Epo production, likely via hepatocyte × endothelium crosstalk. These data suggest VEGF may function physiologically to repress hepatic Epo synthesis, while VEGF blockade leads to de-repression of hepatic Epo production via a novel hypoxia- and HIF-1a-independent pathway.
Sequential Enzymatic Down-Regulation and Degradation of the Lyn Tyrosine Kinase in Erythropoietin-Stimulated Cells.
Authors: Ingley Evan , Schneider Jessica R. , McCarthy David J. , Harder Kenneth W. , Hibbs Margaret L. , Alexander Warren S. , Hilton Douglas J. , Klinken Svend Peter
Published: October 2019

Erythropoietin (Epo) is a key molecule in the development of red blood cells. We have shown previously that the tyrosine kinase Lyn is involved in differentiation signals emanating from an activated Epo-receptor. We have recently identified Cbp (Csk binding protein, a negative regulator of Src) as a novel interactor with Lyn. The proline-rich region of Cbp associates with the SH3 domain of Lyn, which then phosphorylates Cbp on multiple tyrosine residues forming additional binding sites for the Lyn SH2 domain. Phosphorylated Y314 of Cbp, in turn, recruits the negative regulators Csk/Ctk, which phosphorylate Lyn and down-regulate its kinase activity. A phosphotyrosine-specific yeast two-hybrid system we developed, confirmed that the SH2 domain of Csk associated with Y314 of Cbp. Significantly, another well characterized negative regulator, SOCS1, also bound this phosphorylated tyrosine residue, resulting in elevated ubiquitination and degradation of Lyn. Thus, Cbp co-ordinates a two step down-regulation of Lyn. In Epo-responsive J2E cells, Cbp is rapidly phosphorylated by Lyn and the enzymatic activity of Lyn is suppressed by Csk/Ctk after 10 minutes of Epo stimulation. Subsequently, SOCS1 is involved in the turn over of Lyn protein two hours post-Epo induction. These data demonstrate for the first time a two phase process regulated by Cbp for inactivating and degrading Lyn after Epo stimulation.
Janus Kinases Interact with and Phosphorylate Rack-1 (Receptor for Activated C Kinase-1), a WD Motif Containing Protein.
Authors: Haro Takashi , Shimoda Kazuya , Kakumitsu Haruko , Kamezaki Kenjirou , Numata Atsuhiko , Harada Mine
Published: October 2019

We recently reported that Tyk2 was essential for IFN-a-induced B lymphocyte growth inhibition, although Stat1 is not required for this IFN-a-mediated inhibition. This means that other signaling molecules besides Stat1, and which are activated by Tyk2, are thought to transduce the IFN-a signal inhibiting B lymphocyte growth. We performed a yeast two-hybrid screen for proteins that interact with Tyk2, and identified Rack-1, originally described as a receptor for activated C kinase beta, associated with Tyk2. Receptor for activated C kinase (Rack)-1 is a protein kinase C interacting protein, and contains a WD repeat but has no enzymatic activity. In addition to protein kinase C, Rack-1 also binds to Src, phospholipase C gamma, and ras-GTPase-activating proteins. Thus, Rack-1 is thought to function as a scaffold protein that recruits specific signaling elements. In a cytokine signaling cascade, Rack-1 has been reported to interact with the IFN-alpha/beta receptor and Stat1. In addition, we show here that Rack-1 associates with a member of Jak, tyrosine kinase 2 (Tyk2). Rack-1 interacts weakly with the kinase domain and interacts strongly with the pseudo-kinase domain of Tyk2. Rack-1 associates with Tyk2 via two regions, one in the N-terminus and one in the middle portion (a.a.138–203) of Rack-1. In addition, not only Tyk2 but other Jak kinases associate with Rack-1, and each Jak activation causes the phosphorylation of Tyrosine 194 on Rack-1. After phosphorylation, Rack-1 is translocated from cytoplasm or membrane toward the perinuclear region. In addition to functioning as a scaffolding protein, these results raise the possibility that Rack-1 functions as a signaling molecule in cytokine signaling cascades.
Comparison of the Structure, Binding, and Activity of Cell Surface TFPIα and TFPIβ.
Authors: Piro Orlando , Broze George J.
Published: October 2019

TFPI is mainly produced by endothelial cells and alternative mRNA splicing generates two forms, TFPIα and TFPIβ. A small proportion of expressed TFPI remains associated with the cell surface through both direct (TFPIβ) and indirect (TFPIα) glycosylphosphatidylinositol (GPI)-mediated anchorage. TFPIβ lacks the C-terminus and Kunitz-3 (K3) domains of TFPIα and, instead, contains an unrelated C-terminus that directs the attachment of a GPI anchor. TFPIα and TFPIβ migrate with similar MWs (~45,000) on SDS-PAGE due to differing levels of glycosylation; removal of carbohydrate produces protein MWs of 36,000 and 28,000, respectively. Stable clones of ECV304 cells, a human cell line with some endothelial properties, that express reduced levels of TFPIα, TFPIβ, or both were produced using a plasmid-based small interfering RNA gene-silencing technique (siRNA) (Table 1); empty vector transfected clones served as controls. Total TFPI on the cell surface and in the medium of the selected clones was evaluated by flow cytometry (expressed as mean fluorescence intensity) and immunoassay, respectively (Fig. 1). Surface TFPI activity was determined by a two stage chromogenic assay based on the ability of each form to inhibit FX activation by FVIIa on cells with comparable levels of tissue factor (TF). Although TFPIβ accounted for only ~20% of the cell surface TFPI, it was responsible for most of the anti-FVIIa/TF activity (Fig. 2), suggesting a potential alternative role for surface TFPIα. At physiologic concentrations, rTFPIα expressed by ECV304 and mouse C127 cells binds to ECV↓TFPIα cells, but rTFPIα produced by mammalian A-293, HepG2, and CHO cells, and by E. coli does not, implying that post-translational modifications are important for the cell binding of TFPIα. Experiments in C127 cells show that optimal cell binding of TFPIα requires its basic C terminus and the P1 residue (R199) of its K3 domain; mutations within the putative heparin binding region of K3 (K232A, K240A, K241A) do not affect binding. Additional preliminary studies suggest that the cell binding of TFPIα requires N-linked glycosylation and is also reduced by mutation of serine 2, which is phosphorylated in C127 cells. Overall, these results call into question the physiologic relevance of previous cell binding experiments that relied on the use of high concentrations of rTFPIα expressed in E. coli.

table 1

Surface TFPIα (ng/107 cells) Surface TFPIβ (ng/107 cells) Each result represents the average of five independent experiments Cont 2.3 0.5 ECV↓TFPIα 0.4 0.5 ECV↓TFPIβ 2.4 0.04 ECV↓TFPIαβ 0.3 0.05
Influence of Hemoglobin Response to Epoetin Alfa on Quality-of-Life in Anemic Children with Cancer Receiving Myelosuppressive Chemotherapy.
Authors: Razzouk Bassem I. , Hockenberry Marilyn , Hinds Pamela S. , Feusner James , Rackoff Wayne , Hord Jeffrey D.
Published: October 2019

In a double-blind, placebo (PBO)-controlled study of children age 5–18 years (y) with cancer and anemia who received myelosuppressive chemotherapy (CT), the use of epoetin alfa (EPO) increased hemoglobin (Hb) levels (Hb increase ≥1 g/dL from baseline after 4 weeks [wks], independent of RBC transfusion in the previous 28 days; 65.0% for EPO vs 50.0% for PBO; P=.034) and reduced transfusion requirements (transfusion after wk 4 = 51.4% for EPO vs 69.4% for PBO; P=.008). However, no difference in the primary end point, self-reported quality of life (QOL) using the Pediatric Quality of Life Inventory (PedsQL-I™), was observed overall between the 2 treatment arms (ProcASCO 23:abstract 8527, 2004). In adults, increases in Hb during EPO therapy are associated with improved QOL (Cancer95:888, 2002). Given these findings, this post-hoc analysis of the pediatric study was undertaken to determine if a difference in QOL outcomes is observed when the Hb response of children is considered. The study included patients (pts) who were receiving myelosuppressive CT for malignant solid tumors (ST), Hodgkin’s disease (HD), acute lymphocytic leukemia (ALL), or non-Hodgkin’s lymphoma (NHL) and who were anemic (Hb <12 g/dL for boys >12 y, Hb <11 g/dL for girls >12 y, and Hb <10.5 g/dL for children 5–12 y) at study entry. Pts were stratified by tumor type (ST/HD or ALL/NHL) and randomized 1:1 to receive intravenous EPO 600 IU/kg or PBO weekly for 16 wks. The dose was increased to 900 IU/kg weekly after 3–4 wks if Hb increased by <1 g/dL. A total of 222 pts (111 EPO, 111 PBO) were included in primary analysis (98 ST, 75 ALL, 27 HD, and 22 NHL). For this analysis, Hb and PedsQL-I data beyond week 4 were available for 97 EPO pts and 89 PBO pts. PedsQL-I scores improved significantly from baseline among Hb responders but not among Hb nonresponders in both treatment arms (TABLE). The results of this analysis demonstrate that among pediatric cancer pts receiving myelosuppressive CT, Hb response is associated with improvement in QOL. The fact the Hb nonresponders reported higher QOL at baseline in the PBO group than the EPO group may explain, in part, why no difference in final QOL was observed overall in the primary analysis. The significant improvement in QOL among Hb responders suggests that additional studies are warranted to predict and optimize Hb response, QOL, and long-term outcomes with EPO treatment in anemic children with cancer.

Hb Response and PedsQL-I Score

Groups Hb Responder Baseline Final Change P-Value Data presented as mean ± SD (no. of patients) EPO Yes 69.9±17.15 (65) 79.1±15.53 (66) 9.2±20.20 (65) <.001 No 67.1±19.15 (29) 70.0±15.79 (31) 2.6±16.52 (29) .407 PBO Yes 70.0±15.68 (46) 78.0±17.38 (49) 8.5±13.10 (46) <.001 No 72.9±15.76 (40) 75.6±16.51 (40) 2.7±16.96 (40) .315
Lipid Raft Association of a Shared Factor X/Prothrombin Binding Site on Human Platelets Is Mediated by the Gla Domain.
Authors: Ahmad Syed S. , Walsh Peter N.
Published: October 2019

Factor X (FX) initially binds to a high-capacity, low-affinity platelet binding site shared with prothrombin (FII) which then presents FX to a specific, high-affinity site consisting of FVIIIa bound to a high-affinity, low-capacity receptor on activated platelets. We have demonstrated the localization of FX in lipid rafts and shown that FX-raft association requires Ca2+ and is enhanced by saturating concentrations of FVIIIa. Here we investigate FII-raft association and define the domains through which shared FX and FII sites are mediated on the surface of human platelets. Activated (thrombin receptor peptide, SFLLRN, 25 μM) gel-filtered platelets (3.5 x 108/ml) were incubated with 125I-FII or 125I-FX to determine direct platelet binding and then they were lysed with Triton-X100 (0.025-0.25%) followed by sucrose density gradient centrifugation. FII was localized to lipid rafts in SFLLRN stimulated (~25% total binding) but not to unactivated platelets. The optimal associations of FX and FII with lipid rafts required Ca2+ and were not affected by the presence of EGR-FIXa or FIX (45 nM). The association of FII with lipid rafts was completely abolished in the presence of FX (1.5 μM) whereas, Gla (des) FX was unable to compete with raft associated FII. Similarly, 125I-FII fragment 1 association with lipid rafts was inhibited by FX but not by Gla (des) FX. Prothrombin and FII fragment 1 (residues 1-155) were equipotent inhibitors of FX-raft association. FVIIIa (20 nM) had no effect on FII-raft association but significantly increased (~2-fold to ~45%) FX-raft association. In contrast, the presence of FVa (20 nM) had no effect on FX-raft association but significantly (~2-fold to ~45%) increased FII-raft association. The structural integrity of lipid rafts was completely disrupted by 10 mM methyl-β-cyclodextrin (MβCD), a known cholesterol depleting drug, which completely prevented FII or FX association with lipid rafts, and this removal was reversed by cholesterol repletion. Furthermore, MβCD (up to 40 mM) had no effect on the amount of FII or FX bound to activated platelets, thus suggesting that neither platelet activation by SFLLRN nor the exposure of FII receptors was affected by MβCD treatment. These experiments demonstrate the localization of a shared FX/FII site in lipid rafts and support the hypothesis that these interactions are mediated by the Gla-domains of FX and FII and are specific and essential for the assembly of F-X activating complex on the activated platelet membrane.
Molecular Serotype Analysis of Adenoviruses: Clinical Implications.
Authors: Lion Thomas , Ebner Karin , Matthes-Martin Susanne , Watzinger Franz
Published: October 2019

Adenovirus (AdV) infections are a major cause of morbidity and mortality in allogeneic stem cell transplant (SCT) recipients. Human adenoviruses represent a large family, currently including 51 serotypes, which are divided into 6 species (A-F). We have recently demonstrated by a species-specific RQ-PCR approach covering the entire spectrum of human adenoviruses that molecular detection of AdV in peripheral blood precedes the onset of life-threatening virus disease, and provides a basis for early preemptive treatment (Lion et al., Blood102(3):1114–20, 2003). An association of AdV species with clinical manifestation and response to antiviral therapy has been reported, but little is known about the clinical role of individual AdV serotypes. In immunocompromised patients, the use of serological testing for identification of adenovirus serotypes is limited due to the impaired immune response. We have therefore determined the nucleotide sequence of the complete AdV hexon gene in all 51 human serotypes, and identified regions permitting rapid serotyping at the molecular level. Serotypes belonging to the species A,B, C, E, and F, can be determined by fragment length analysis of a single PCR product, respectively. Serotype identification within the largest AdV species D requires sequencing of a single 300bp PCR amplicon. In view of the great predominance of species C in our region, we have also established real-time PCR tests permitting identification of its four serotypes. Analysis of all AdV C positive cases within more than 6.000 clinical specimens investigated at our center over the past years revealed the highest prevalence for serotype 2 (57%), followed by the serotypes 1 (39%) and 5 (4%). In some instances, two different AdV C serotypes were present simultaneously. We have demonstrated that the identification of specific virus strains within individual AdV serotypes, which may be required for investigation of possible transmission of infections within the hospital, can be achieved by sequencing of PCR products derived from an appropriate AdV target region within the hexon gene. The possibility of rapid molecular serotype and strain analysis provides a basis for studies on adenovirus epidemiology, and may in future have implications for the selection of the most appropriate antiviral treatment.
T to Myeloid Lineage Reprogramming by the Synergistic Action of C/EBP and PU.1.
Authors: Laiosa Catherine V. , Xie Huafeng , Ye Min , Graf Thomas
Published: October 2019

During hematopoiesis, transcription factors can not only direct differentiation of committed progenitors but also drive lineage commitment decisions from uncommitted multipotent progenitors. To further study their instructive role, we asked whether critical steps in the hematopoietic differentiation pathway could be reversed, and more specifically, whether myeloid transcription factors, C/EBP alpha and C/EBP beta, could re-direct committed T lymphoid progenitors into a myeloid fate. Previously in our laboratory, it was shown that C/EBP alpha efficiently reprograms CD19+ B cells to Mac1+ Gr1+ functional macrophages (Xie et al. (2004) Cell 117:663). To address lineage conversion of T to myeloid cells, we purified T cell subsets by MACS enrichment or FACS sorting and infected these cells with retroviruses expressing C/EBP alpha or C/EBP beta. Cells were cultured on myeloid supporting stroma with myeloid cytokines and analyzed for expression of antigens and marker genes by flow cytometry and RT-PCR. To rule out the presence of contaminating myeloid progenitors in our cultures, we started with a marked population of T lymphoid progenitors derived from Lck-Cre Rosa-EYFP reporter mice in which T cells are irreversibly marked with EYFP upon T cell commitment. T cell origin was further verified using PCR to detect TCRbeta DJ rearrangements. Using double negative DN3 and DN4 T cells, we show that a large proportion of EYFP+ DN cells infected with C/EBP alpha or C/EBP beta are induced to express surface antigens Mac1 and Gr1, express myeloid cytokine receptors and Fc receptors, and exhibit a myeloid morphology, compared to control infected cells. The reprogrammed cells are also functional as they can bind and phagocytose sheep red blood cells. In addition, the pan-T cell marker Thy1 and other T lineage genes including Lck, Rag2, and preT alpha are downregulated in reprogrammed cells. Furthermore, using a conditional knockout approach developed in Daniel Tenen’s laboratory, we show that endogenous expression of PU.1 is required for activation of Mac1 during this process. In conclusion, C/EBP alpha acts alone to extinguish the T lymphoid program while, in concert with PU.1, it activates the myeloid program leading to reprogramming of the T cell phenotype.
Health-Related Quality of Life (HRQL) in Adult Long-Term Hematopoietic Cell Transplant (HCT) Survivors: A Report from the Bone Marrow Transplant Survivor Study.
Authors: Bhatia Smita , Francisco Liton , Carter Andrea , Baker K. S. , Forman Stephen J. , Gurney James G. , Robison Leslie L. , Weisdorf Daniel J. , Grant Marcia
Published: October 2019

We describe HRQL reported by adult survivors of autologous and allogeneic HCT and the risk factors associated with poor HRQL. Eligible subjects were individuals undergoing HCT at either City of Hope or the University of Minnesota between 1974 and 1998, and surviving two or more years after HCT. We analyzed the data from 1003 HCT survivors older than 21 years enrolled in a retrospective cohort study who had completed a validated City of Hope HCT-QOL tool. Of these, 54.5% were males, 80.2% were Caucasians, and 55% had received allogeneic HCT. The primary diagnoses included CML (n=233), AML (n=241), NHL (n=200), ALL (n=99), HD (n=88), and other (n=142). The median age at HCT was 35.4, median age at study participation: 43.4 years, and the median length of follow-up: 9.4 (2 to 28 years). Survivors’ responses were compared with those of the population norms in Physical, Psychological, Social and Spiritual domains, and overall quality of life (QOL). Poor QOL was defined as QOL scores less than two standard deviations below that reported for the tool norms. Using this definition, 9.2% of this cohort was identified to have poor overall QOL; 13.7% with poor Physical; 10% with poor Psychological; 6.7% with poor Social; and 4.3% with poor Spiritual well-being. Table below shows the results of the risk factors identified for poor QOL by multivariate analysis. Age, sex, type of HCT, and time since HCT were not associated with poor QOL. Thus, overall 9% of the long-term HCT survivors report poor overall QOL. Certain vulnerable sub-populations exist, specifically, low household income, difficulty in obtaining health insurance, inability to return to work, and the presence of pain and anxiety that impact upon the HRQL of this population, and require additional attention and possible intervention.

Predictors of Poor

Risk Factors for Poor QOL Overall QOL Physical Psychological Social Spiritual Relative Risk (p-value) Relative Risk (p-value) Relative Risk (p-value) Relative Risk (p-value) Relative Risk (p-value) Non-white race 2.1 (0.06) 2.4 (0.005) 1.6 (0.1) 2.0 (0.06) 0.5 (0.2) Inability to return to work 3.4 (0.003) 4.9 (<0.001) 3.6 (<0.001) 3.6 (0.002) 0.8 (0.7) Inability to return to school 2.4 (0.2) 4.6 (0.005) 5.2 (0.002) 1.1 (0.8) 0.9 (0.9) Difficulty in obtaining health insurance 1.7 (0.1) 1.9 (0.04) 0.9 (0.9) 3.6 (<0.001) 2.2 (0.05) Absence of health insurance 2.6 (0.05) 1.3 (0.6) 0.7 (0.5) 1.6 (0.4) 4.2 (0.005) Household income <$20k 3.0 (0.04) 0.9 (0.9) 4.4 (0.006) 2.2 (0.1) 3.1 (0.05) CGVHD 2.1 (0.02) 1.8 (0.02) 1.9 (0.04) 1.4 (0.3) 1.5 (0.3) Severe pain 8.8 (<0.001) 12.6 (<0.001) 2.4 (0.07) 5.4 (0.001) 2.8 (0.07) Severe anxiety 240.2 (<0.001) 5.3 (0.001) 60.2 (<0.001) 7.8 (<0.001) 19.7 (<0.001)
In Vivo Drug-Response in Patients with Leukemic Non-Hodgkin’s Lymphomas Is Predicted by In Vitro Chemosensitivity Testing and Gene Expression Profiling.
Authors: Chow Kai Uwe , Nowak Daniel , Kim Soo-Zin , Schneider Bernd , Komor Martina , Boehrer Simone , Mitrou Paris S. , Hoelzer Dieter , Weidmann Eckhart , Hofmann Wolf-Karsten
Published: October 2019

Only a few approaches are available to address the mechanisms of cell death in vivo which are induced by anticancer treatment in patients with malignancies. In this study in vitro chemosensitivity testing of primary peripheral blood leukemic cells of five patients suffering from different leukemic Non-Hodgkin’s lymphomas (atypical CLL, typical CLL, Immunocytoma, Mantle Cell Lymphoma, Prolymphocytic Leukemia (PLL)) was combined with the analysis of the in vivo rate of apoptosis by flow-cytometry (Annexin V and depolarisation of mitochondrial membrane potential (MMP) by JC-1). Furthermore, changes in expression patterns of apoptosis related proteins during chemotherapeutic treatment were detected by Western Blot. Gene expression profiling (HG-U133A, Affymetrix, Santa Clara, CA) was employed to identify common marker genes of in vivo drug response. In vitro chemosensitivity was tested using the cytotoxic agents which the patients were scheduled to receive and was strongly correlated with effective reduction of leukemic lymphoma cells in patients resulting in complete remissions in all five cases. Due to the rapid clearance of apoptotic tumor cells in vivo neither the analysis of the in vivo rate of apoptosis and depolarisation of MMP nor the assessment of expression of regulators of apoptosis showed concordant results concerning the drug response. However, assessment of gene expression during therapy could identify a set of 30 genes to significant discriminate between samples from patients before treatment compared to samples from the same patients after receiving cytotoxic therapy. Among these 30 genes we found a high proportion of genes associated with apoptotic cell death and cell proliferation signalling including complement lysis inhibitor (clusterin, CLU, SP40), beta-catenin interacting protein (ICAT), peroxisome proliferator activated receptor alpha (PPARα), TNF alpha converting enzyme (ADAM 17 / TACE), homeo box A3 (HOXA), inositol polyphosphate 5 phophatase (PPI 5 PIV, SHIP1), FK 506 binding protein (FKBP 38) and inhibitor of p53 induced apoptosis alpha (NME 6). Clusterin is able to mediate apoptosis via p53 and increases drug-induced cell death when overexpressed as detected in our treated samples. The downregulation of NME 6 during chemotherapeutic treatment may enhance this effect. These results indicate that in vitro chemosensitivity testing and gene expression profiling can successfully be utilised to predict in vivo drug response in patients with leukemic NHL’s and can be used to explore new pathway models of drug-induced cell death in vivo which are independent of different lymphoma subtypes and different treatment regimens.
Prognostic Factors and Outcome of Human Herpesvirus 8 (KSHV/HHV-8)-Associated Primary Effusion Lymphoma (PEL) in Patients with AIDS.
Authors: Boulanger Emmanuelle , Gerard Laurence , Gabarre Jean , Molina Jean-Michel , Rapp Christophe , Abino Jean-Francois , Cadranel Jacques , Chevret Sylvie , Oksenhendler Eric
Published: October 2019

Primary effusion lymphoma (PEL) is a rare high-grade B-cell non-Hodgkin lymphoma associated with Kaposi sarcoma-associated Herpesvirus/Human Herpesvirus 8 (KSHV/HHV-8) infection, and mostly observed in the course of HIV infection. The prognosis is poor with reported median survival time shorter than 6 months. To date, no prognostic factor has been identified in this subset of lymphoma. We described here the largest series of HIV-infected patients with PEL, including 30 cases diagnosed in six French centers over a 15-year time period. Prognosis analysis was performed using a Cox proportional hazard regression model. Statistically significant covariates were further analyzed in a forward, stepwise multivariate model. After a median follow-up of 3.2 years (range, 10 months-8.2 years), 9 patients (30%) were still alive, and 8 of them remained progression free. The overall median survival was 5.5 months and the 1-year overall survival rate was 36.7%. Fourteen patients (47%) achieved complete remission, with a 1-year disease-free survival rate at 78.6%. In multivariate analysis, only a performance status > 2 [hazard ratio 6.27, 95% confidence intervals (CI),1.91–20.58] and the absence of highly active antiretroviral therapy (HAART) before PEL diagnosis [hazard ratio 0.28, 95% CI, 0.10–0.77] were found to be independent predictors for shorter survival. Based on a retrospective series of 30 patients, two prognostic factors were identified as being independently associated with impaired clinical outcome in HIV-related PEL, (i) a poor performance status and (ii) the absence of HAART prior to PEL diagnosis.
Expression of VEGF, VEGF Receptors, and Other Angiogenic Markers in Relapsed Aggressive Non-Hodgkin’s Lymphoma : Correlative Sudies from the SWOG S0108 Trial.
Authors: Stopeck Alison , Iannone Maria , Rimsza Lisa , Miller Thomas , Fisher Richard , Bellamy William
Published: October 2019

In the Southwest Oncology Group phase II study S0108, patients with relapsed, aggressive non-Hodgkin’s lymphoma were treated with single agent bevacizumab (Avastin), a recombinant monoclonal antibody directed against VEGF. Pretreatment tissue or bone marrow specimens were examined by immunohistochemistry (IHC) for expression of VEGF and its receptors, VEGFR-1 (Flt-1) and VEGFR-2 (KDR). Circulating endothelial cells (CEC) and plasma levels of VEGF, VCAM, and bFGF were measured at baseline, week 8, and at disease progression. Currently, 45 patients have been enrolled in the study and 18 have had baseline lymphoma specimens submitted and adequate for analysis by IHC for expression of VEGF and its receptors. In 7 of 10 (70%) patients with diffuse large cell lymphoma, the tumors expressed VEGF and either or both VEGF receptors. There was 100% concordance between VEGF and receptor expression in these samples. In 7of 8 (88%) mantle cell specimens examined, VEGF expression was also observed. Expression of both VEGFR-1 and VEGFR-2 was observed in 6 (67%) of these specimens. While VEGF and VEGFR-1 expression was restricted to the malignant cells, VEGFR-2 was predominantly expressed on the endothelial cells in the lymphoma specimens, and in those cases where it was also expressed by the tumor cells it was typically at lower intensity. In 1 of 5 patients with serial lymphoma specimens, VEGF expression increased in the lymphoma cells during the course of bevacizumab therapy. There was a wide range in the plasma levels of VEGF, bFGF, VCAM, and CEC between patients. During the course of treatment, plasma VEGF levels decreased (p = 0.02), VCAM levels increased (p = 0.05), and bFGF and CEC levels remained relatively stable. CEC, VCAM, and bFGF levels did not correlate with VEGF expression in lymphoma samples but there was a trend for lymphomas with higher VEGF expression (+2 or higher) to have higher baseline plasma levels of VEGF (median VEGF level 471 pg/ml compared to 224 pg/ml). Accrual of samples continues and the results will be updated as the analyses are available. The high rate of concordance between VEGF and its receptors in diffuse large cell and mantle cell lymphomas suggests that an autocrine pathway may be involved in lymphoma pathogenesis and/or progression. In addition, these results provide a strong rationale for targeting the VEGF pathway in the treatment of these 2 histologic subtypes of aggressive non-Hodgkin’s lymphoma.