bluuebunny/crossref_metadata_2025 huggingface dataset:
Gene Frequencies of the HNA-4a and -5a Neutrophil Antigens in Brazilians and a New Polymerase Chain Reaction-Restriction Fragment Length Polymorfism Method for HNA-5a Genotyping.
Authors: Cardone José D.B. , Chiba Akemi K. , Norcia Angela M.M.I. , Vieira-Filho João P.B. , Bordin José O.
Published: October 2019

The HNA-4 and HNA-5 systems are located on the b2-integrin, that share a common b subunit (b 2 or CD18) noncovalently associated with four different a subunits. The HNA-4a (Mart) and HNA-5a (Ond) antigens are polymorfic variants of a M (CD11b) and a L (CD11a) subunits, respectively, and both are associated with single nucleotide polymorphisms (SNP) leading to amino-acid dimorphisms. HNA-4a has been recently linked to alloimmune neonatal neutropenia while the HNA-5a clinical significance is not clear yet. Using a PCR with sequence-specific primers (SSP) the frequency of allelic polymorphism of HNA-4a among 121 Brazilian blood donors and 114 Amazon Indians was determined. A novel PCR-restriction fragment length polymorphism (RFLP) method digesting the PCR product with the enzyme Bsp1286 I for HNA-5a genotyping was developed, and the gene frequencies of this antigen were determined among 123 Brazilian blood donors and 114 Amazon Indians. In order to validate this proposed new genotyping method, the amplified genomic DNA from 6 previously genotyped samples (2 of each genotype) was purified using a commercial kit (GFX PCR DNA and Gel Band Purification Kit, Amersham Bioscience, Piscataway, NJ, USA), sequenced using a cycle-sequencing kit (BigDye Terminator, PE Applied Biosystems, Foster City, CA, USA) and analyzed on a genetic analyzer (ABI Prism 3100, PE Applied Biosystems). The calculated HNA-4a (+/+), HNA-4a (+/−) and HNA-4a (−/−) genotype frequencies found in blood donors (0.686, 0.273 and 0.041) and Amazon Indians (1.000, 0.000 and 0.000) presented statistically significant differences (p<0.01). The frequencies of HNA-5a (+/+), HNA-5a (+/−) and HNA-5a (−/−) genotypes among blood donors (0.512, 0.399 and 0.089) and Amazon Indians (0.746, 0.219 and 0.035) also differed significantly (p<0.01). Sequencing studies demonstrated absolute concordance with PCR-RFLP genotyping results in all 6 analyzed samples. Overall, the present data indicate that comparing to other population studies, the Brazilian population presents a higher frequency of the HNA-4a-negative allele, suggesting that Brazilians would be more susceptible to HNA-4a alloimmunization by pregnancy or blood transfusion. Moreover, the distribution of the HNA-4 system alleles observed in Amazon Indians is quite similar to that already reported among Korean individuals. Besides that, a new effective and efficient HNA-5a genotyping technique is now available for population studies.
Platelet-Derived Microparticles Promote Clot Stability.
Authors: Loncar Robert , Dzepina Daniel , Stoldt Volker , Zotz Reiner B. , Scharf Rudiger E.
Published: October 2019

Microparticles (MP) are the plasma membrane fragments. They are formed along with membrane remodelling processes of most stimulated eukaryotic cells or generated upon cellular stimulation including platelets. After having been considered “inert cell debris” previously, recent findings suggested that MP can modulate distinct cellular responses in the related microenvironment. For example, the concentration of circulating platelet-derived MP is increased in acute myocardial infarction and stroke. It is hypothesized that platelet-derived MP promote hemostasis and thrombosis. However, the precise role of MP is still unknown. In this study, we evaluated the influence of platelet-derived MP on clot stability. Anticoagulated blood (3.8% sodium citrate) was obtained from healthy blood donors. Platelet-rich plasma was centrifuged at 1500g (10 min, 22°C). The pelleted platelets were washed three times in PBS (pH 7.4), resuspended in 1 ml of the same buffer and activated with human collagen of type I (10 min, 35°C) at a final concentration of 10 μg/ml. The supernatant (1500g 10 min, 22°C) containing activated platelet MP was centrifuged at 13,000g (30 min, 4°C). The pellet of MP was resuspended in 450 μl of PBS. MP were identified by scanning electron microscopy and flow cytometry following immunolabelling with an anti-GPIbα FITC-monoclonal antibody. The influence of platelet MP onto clot stability, determined as platelet contractile force (PCF) and clot elastic modulus (CEM), was evaluated with a Hemodyne haemostasis analyzer (Hemodyne, Richmond, USA). Mean PCF and CEM in blood of healthy donors (n=7) were 6.5±3 Kdynes and 9.6±6 Kdynes/cm2, respectively. Addition of 100 μl of platelet-derived MP increased PCF (forces generated by platelets within a clot) without reaching statistical significance (mean increase of 11% as compared to controls without MP). By contrast, addition of platelet MP significantly enhanced CEM as measure of clot stability from 9.6±6 Kdynes/cm2 to 94 ± 62 Kdynes/cm2, (p<0.05). In experiments conducted with platelet-rich plasma or platelet-poor plasma instead of anticoagulated whole blood, no influence of added platelet-derived MP on clot stability was observed. In a patient with thrombocytopenia (70,000/μl) supplementation of whole blood with platelet MP increased CEM by 70%. Our ex vivo experiments demonstrate that collagen-induced platelet-derived MP can modulate clot stability. However, this effect is restricted to anticoagulated whole blood and not observed in platelet-rich plasma or plasma alone. Therefore, interaction of platelet-derived MP with other cellular elements than platelets, e.g. monocytes, may be relevant to promote clot stability. In general, microparticles may be a pharmacological target in the management of hemostatic disorders.
Impact of Joint Bleeding on Joint Function in a Murine Model of Hemophilia A.
Authors: Mejia-Carvajal Carmen R. , Valentino Leonard A. , Hakobyan Narine , Kazarian Tamara
Published: October 2019

Hemophilia is a congenital bleeding disorder that commonly results in musculoskeletal complications due to recurrent bleeding into joints. After an acute joint bleed, there is pain, swelling and limited motion due to the increase in intra-articular pressure and inflammation. Blood in the joint space provokes a proliferative and inflammatory disorder known as hemophilic synovitis (HS), which causes mechanical dysfunction and eventually destruction of the articular surface and bone. Among persons in the USA with severe hemophilia, nearly half have some restriction in activity (Evatt 2004) and it is estimated that 73% of boys enter adolescence with arthropathy and between 13–16 years of age, two or more major joints are affected (Brackmann,1992). Detail examination of the earliest changes in joint function following 1–3 joint hemorrhages is difficult to perform in patients. In this project, a murine model of HS (Valentino,2004) was used to examine the impact of a minimum number of hemarthrosis on joint function. The hypothesis tested was that there is a relationship between hemarthrosis and joint dysfunction. It was expected that joint dysfunction would be observed following few bleeds. Also, it was expected that a single hemarthrosis will result in permanent joint dysfunction. To test these hypotheses, 25 mice deficient in factor VIII coagulant activity were injured, times three, at weekly intervals to induce hemarthrosis then their ability to ambulate monitored using a Rotorod: Economex apparatus, which is intended for testing coordination and impairment of locomotor agility of laboratory animals. Mice were trained to ambulate on the rotating rod then injured and retested 2, 4 and 7 days after each hemarthrosis. A 2nd group of 25 mice was not injured and served as controls. At the conclusion of the experiment, mice were sacrificed and the knee joints examined by gross and histological methods. Correlations were drawn between the ambulatory dysfunction and the pathological changes observed. Preliminary data showed:

mice can be trained to ambulate on the Rotarod, acute hemarthrosis temporarily impairs their ability to ambulate, and after mice recover from acute injury they regain their lost ability to ambulate to the point that their performance is better than previous.
Isolated Alpha-2 Antiplasmin Deficiency Presenting as a Spontaneous Bleeding Disorder in a 63 Year Old Man.
Authors: Harish Vallathucherry C. , Lawson Heather L. , Zhang Lin , Huff Jason D. , Owen John
Published: October 2019

Spontaneous disruption of hemostasis in adults is a common clinical presentation. However, in a few patients, no cause is found. We were faced with such a situation when a 63 year old Caucasian male presented with a large spontaneous hematoma of his left thigh. There was no prior history of bleeding disorders in our patient or amongst his family members. Initial investigations did not show any conclusive coagulation factor abnormalities. Fibrinogen, D-dimer, liver function tests and platelet aggregation studies were normal. Subsequent tests showed that while his activatable plasminogen and euglobin clot lysis were normal, the function of his alpha-2 antiplasmin was at 35% of normal. Laurell immunoassay demonstrated a type 1 deficiency with antigen levels proportionately decreased at 30% of normal. Treatment with low dose epsilon amino caproic acid resulted in resolution of the hematoma and control of bleeding.

We therefore sought to determine the cause of his isolated alpha-2 antiplasmin deficiency. Alpha-2 antiplasmin is a serpin which targets plasmin. It is synthesized in the liver and deficiency results in a bleeding disorder.Congenital deficiencies of alpha-2 antiplasmin are uncommon bleeding disorders. However, since heterozygotic mutations of alpha-2 antiplasmin can present with initial bleeds late in life, we sequenced his alpha-2 antiplasmin gene. This gene is located on chromosome 17, spanning 16 Kb. It has 10 exons which we amplified by PCR in 13 segments and sequenced by the di-deoxy method. All splice junctions and the 70 base pairs upstream of exon 1 were normal. Three heterozygous polymorphisms were found, ruling out major gene deletions. The polymorphisms are as follows: Exon 2, C to T causing substitution of Alanine by Valine in the signal peptide, Exon 3, C to T causing substitution of Arginine by Tryptophan, Exon 10, G to A causing substitution of Arginine by Lysine. Each of these 3 polymorphisms have been found in blood donors.

This raises important issues about his deficiency. First, lack of production could be due to defective translational factors or epigenetic factors regulating gene transcription. It could also be due to the Alanine to Valine change in the signal peptide which we are currently investigating. Secondly, non-inhibitory antibodies to alpha-2 antiplasmin could explain his type 1 deficiency. Finally, age-related vascular and connective tissue defects may unmask a dormant inherited bleeding disorder. Unfortunately, his demise due to the development of nephrotic syndrome and sepsis may leave us without answers to these questions. There is no case linking nephrotic syndrome with the use of epsilon amino caproic acid.

In conclusion, since response to anti- fibrinolytics can be dramatic, deficiencies of alpha-2 antiplasmin must be considered in patients presenting at any age with a spontaneous bleeding disorder.
Hyperconcentrated (Dry) Versus Standard Platelet Apheresis: An In Vitro Quality Study.
Authors: Perotti Cesare , Bergamaschi Paola , Viarengo Gianluca , Del Fante Claudia , Bellotti Laura , Bressan Daniela , Balduini Carlo , Salvaneschi Laura
Published: October 2019

Introduction: Platelet concentrates (PC) collected by apheresis are effective in supporting deeply thrombocytopenic patients. The reduced risk of multiple allogeneic exposure and transmissible infectious diseases together with the high WBC depletion and diminished transfusion reactions are the main advantages offered by PC transfusion. At moment, the availability of several synthetic solutions for platelets storage permits to prepare hyperconcentrate(dry) apheresis platelets with the advantage of reducing febrile non-hemolytic transfusion reactions and, in low body weight patients the citrate toxicity, without the necessity of further manipulations. The aim of this study was to test the quality of 20 dry platelets (DP) in comparison to 20 standard plateletpheresis (SP) concentrates.

Materials and methods: A total of 40 apheresis procedures were performed by the single-needle Cobe Trima separation device (Gambro BCT, Lakewood, CO, USA) collecting either DP or SP concentrates. Within 1h after collection, the bag containing DP was added with the appropriate amount (70% of DP final volume) of synthetic solution for platelets storage (SSP, MacoPharma). Both DP and SP concentrates were stored at room temperature with gentle agitation for 4 days. For both concentrates, platelet yield was calculated and in vitro studies of membrane glycoproteins expression and aggregation at day +1 and day +4 were carried out.

Results: The comparison between 20 DP and 20 SP concentrates in terms of ability to aggregate in vitro and membrane glycoproteins expression at day +1 and day +4 of storage is reported in table A and B respectively.

Conclusions: The in vitro tests documented a major activation of dry platelets. In particular, the ability to aggregate was reduced in the 20 DP concentrates analised and this phenomenon was more evident at day +4 of storage. The alteration of membrane glycoproteins expression (markers of storage lesion) confirms the lower in vitro quality of DP concentrates. The effectiveness of this new blood component in vivo should be evaluated in a controlled clinical trial.

Table A. At collection Day +1 Day +4 SP DP SP DP SP DP Collagen μg/ml 4 93 88 97 82 80 53 ADP 10 μM 32 11 24 16 16 5 Ristocetin 1.5 mg/ml 91 97 77 81 71 52 Collagen 10 μg/ml + Adrenaline 10 μM 98 95 93 94 93 80 ADP 10 μM + Adrenaline 10 μM 87 72 76 61 57 30 Table B. At collection Day +1 Day +4 SP DP SP DP SP DP GPIb alfa (MFI) 5.06 5.75 6.31 6.13 5.26 4.54 GPIIb-IIIa (MFI) 35.47 36.71 34.61 37.7 31.76 40.9 GP IV (MFI) 11.49 11.4 11.67 10.28 11.65 11.38 GP 53 24.23 27.11 21.74 25.91 21.46 31.84 GMP 140 21.79 29.29 22.65 30.38 20.58 34.89
Safety of Central Venous Catheter Removal in the Setting of Thrombocytopenia and Peripheral Blood Stem Cell Collection.
Authors: Mantha Simon , Wu Yanyun
Published: October 2019

Thrombocytopenia is not uncommonly seen in patients undergoing peripheral blood stem cell collection. It is most likely secondary to the administration of chemotherapy and G-CSF, as well as the apheresis process itself. Peripheral blood stem cell collection causes a variable loss of platelets, which are collected along with the mononuclear cells. The ensuing thrombocytopenia can be at times severe, but the bleeding risk in this setting is unclear, as is the need to administer platelets prophylactically when the central catheter used for collection is removed. In this study we retrospectively analyzed data on 40 patients who had a central venous line removed immediately after their last autologous stem cell collection and had a platelet count of less than 50000/μL before the apheresis procedure. Patients were divided into two groups, according to whether or not they had received a platelet transfusion. The expected pre-catheter removal platelet count was calculated, according to the patient’s blood volume, the number of platelets in the stem cell product and the amount of platelets transfused in random donor platelet units (RDP) equivalents. Each chart was reviewed for the occurrence of catheter removal-associated bleeding. The results are shown in table 1. Post-apheresis platelet measurement is not routinely performed, due to its inconvenience to the patient and delay in catheter removal. Most clinicians use the pre-apheresis value as a guide to transfuse platelets before taking out the central line. As shown in table 1, in spite of significantly lower measured platelet counts before the harvest procedure in the transfused group, the calculated final counts were similar, due to the transfusion of allogeneic platelets. The patients’ weight was significantly lower in the transfused group, and this might actually have contributed to their lower platelet counts after repeated peripheral stem cell collections. There was no bleeding associated with catheter removal in either group. One patient had persistent oozing at the catheter insertion site, likely secondary to an unusually large incision. Removal was uneventful however. In this study, removing the central venous catheter was performed safely without platelet transfusion in patients who had a pre-apheresis platelet count above 30000/μL. It might be safer to administer prophylactic platelet transfusions to patients with lower counts.

Table 1 Untransfused group Transfused group P N 34 6 Patient weight in kg (median, range) 93 (52–125) 76 (63–89) 0.03 Measured platelet count pre-apheresis X 1000/μL (median, range) 40 (28–49) 24 (19–30) <0.01 Number of platelets removed X 1011 (median, range) 0.51 (0.05–1.44) 0.48 (0.14–2.34) 0.58 Number of transfused RDP equivalents per patient (median, range) 0 5 (4–6) - Calculated platelet count post-apheresis (median, range) 32 (19–44) 39 (8–45) 0.56 Number of bleeding episodes 0 0 -
Role of TPO in the Differentiation of Embryonic Stem Cells into Hematopoetic Cells.
Authors: Wang Zheng , Andri Pramono , Julia Skokowa , Karl Welte
Published: October 2019

Thrombopoetin (TPO) is a primary regulator of megakaryocyte and platelet production. However, studies in c-mpl-deficient mice and in congenital amegakaryocytic thrombocytopenia-patients with non-sense c-mpl mutation who develop pancytopenia during the first years of life suggest that TPO also play an important role on early hematopoesis. We demonstrated that TPO enhances FLK-1 (VEGF-receptor) expression on hemangioblasts during murine embryonic stem cell differentiation in embryoid body-liquid cultures (up to 73%). To extend our studies, we investigated the TPO signaling in FLK-1 positive cells. ES cells at different time point of differentiation showed that TPO enhances c-mpl-, BMP4-, Notch-, HOXB4-, HOXB9-, HOXA10-, Runx1-and CD133- mRNA expression. To investigate mesoderm formation, we also analyzed GATA-4 and T-brachyury mRNA level expression. Interestingly, we found that TPO alone did not increase GATA-4- and T-brachyury- mRNA expression, suggesting that TPO requires other cytokines to form the mesoderm. We also found that TPO could maintain VEGF-A mRNA expression level during differentiation of ES-cells. We hypothesize that VEGF expression together with c-mpl expression is required in hematopoetic differentiation of ES cell. This activity of Tpo was also observed during Rhesus monkey embryonic stem cell differentiation into hematopoetic cell. Only combinations of TPO and VEGF were capable of increasing CD34 positive hematopoietic progenitor cells (up to 8%), but TPO alone failed to induce high levels of CD34+ cell. In addition, analysis of gene expression during hemangioblast development demonstrated that TPO was capable of increasing the expression of VEGF receptors (FLK-1) and TPO receptors (c-mpl) in mice and primates. The in-vitro differentiation of mouse and rhesus monkey ES cells provides an opportunity to better understand the role of TPO in the early stage of hematopoietic development from ES cells to mature hematopoietic cells.
Pharmacokinetics and Pharmacodynamics of CNTO 528, a Novel Erythropoietin Receptor Agonist in Normal and Anemic Rats.
Authors: Bugelski Peter , Nesspor T. , Spinka-Doms T. , Makropoulos D. , Eirikis E. , Fisher J. , Volk A. , Shamberger K. , James I. , Fisher P. , Pool C. , Jang H. , Miller B. , Huang C. , Heavner G. , Knight D. , Ghrayeb J. , Scallon B.
Published: October 2019

CNTO 528 is a member of a group of novel stimulants of erythropoiesis that has recently entered clinical trials. Although CNTO 528 bears no sequence homology to erythropoietin (EPO), it is an EPO receptor agonist, capable of rescuing EPO-dependent cells from apoptosis in vitro and stimulating erythropoiesis in vivo. The purpose of these experiments was to explore the pharmacodynamics and pharmacokinetics of CNTO 528 in normal rats and to evaluate its efficacy in rat models of anemia. In vitro, CNTO 528 was approximately 10-fold less potent on a molar basis than recombinant human EPO (rhEPO) in stimulating the growth of UT-7EPO cells. Despite the lower in vitro potency, when compared to rhEPO and darbepoetin in normal rats, a single subcutaneous dose of CNTO 528 caused a longer-lived reticulocytosis and a longer-lived increase in hemoglobin. Also, CNTO 528 caused only minor changes in red cell distribution width (RDW) or mean cell volume (MCV) led to the release of mature reticulocytes and had no effect on mean platelet volume (MPV). We have also shown that CNTO 528 was efficacious in rat models of anemia and in a rat model of pure red cell aplasia. Taken together, our data show that CNTO 528 is a novel stimulant of erythropoiesis in rats.
Pegfilgrastim and Daily Granulocyte Colony-Stimulating Factor (G-CSF) Patterns of Use and Neutropenia-Related Outcomes in Cancer Patients in Spain: Results of the Learn Study.
Authors: Almenar D. , Mayans J. , Juan O. , Bueno J.M. García , Jalón Ji , Frau A. , Guinot M. , Cerezuela P. , Buscallà E. García , Sánchez J. ,   On Behalf of the LEARN Study Group
Published: October 2019

Background: Daily G-CSFs(Filgrastim, lenograstim) are widely used to reduce duration of chemotherapy-induced neutropenia(CIN) and incidence of febrile neutropenia(FN) in cancer patients(pts). Data on their patterns of use and effectiveness in routine clinical practice are however limited. The introduction of the once-per-cycle G-CSF pegfilgrastim(Neulasta®) in Spain in 2003 may have changed patterns of use of daily G-CSFs and CIN-related outcomes.

Methods: Multicentre, retrospective, observational study of daily G-CSF and pegfilgrastim patterns of use and outcomes in adult subjects with non-myeloid malignancies receiving myelosuppressive chemotherapy(CT). Consecutive patient medical records with documented use of daily G-CSF or pegfilgrastim were abstracted from 10 Spanish centers during 2003.

Endpoints: percentage of proactive(primary prophylaxis) vs reactive(secondary prophylaxis/treatment) use of G-CSFs, duration of treatment with G-CSF, and CIN-related outcomes(dose delay, dose reduction, incidence of FN, hospitalization and antibiotic consumption).

Results: 248 charts documented pegfilgrastim or daily G-CSF use; 75 pts received pegfilgrastim only; 111 pts received daily G-CSF only(99 Filgrastim, 12 lenograstim); 62 pts received both daily G-CSF and pegfilgrastim during their CT(data not shown). Most common tumor types were lung(25%), breast(20%), malignant lymphomas(20%). Pattern of use (% pts on primary or secondary prophylaxis, or treatment at any time during CT) was:pegfilgrastim(39%,48%,17%, respectively) vs daily G-CSF(40%,48%,30%, respectively). Median number of injections/cycle in the daily G-CSF group was 6 (range 1–13) in primary prophylaxis, and 5 (range 1–11) in secondary prophylaxis and treatment. CIN-related outcomes are shown in the table below.

Conclusions: Patterns of use of daily G-CSFs and pegfilgrastim were similar for primary and secondary prophylaxis, but a potential trend to less frequent treatment use in the pegfilgrastim group was observed. CIN-related complications, including incidence of FN, were observed to be lower in pts receiving pegfilgrastim.

CT-related complications% pts (95% CI) Pegfilgrastim(n=75) Daily G-CSF(n=111) Dose Delay 44% (33; 55) 46% (36; 55) Dose Reduction 14.7% (8.2; 24.6) 20.7% (14.2; 29.2) Dose Reduction due to Neutropenia 6.7% (2.5; 15.0) 20.7% (14.1; 29.2) Febrile Neutropenia (FN) 10.7% (5.3; 19.9) 24.3% (17.2; 33.1) Hospitalization due to FN 9.3% (4.3; 18.3) 19.8% (13.4; 28.3) Antibiotic Consumption due to FN 8.0% (3.4; 16.7) 17.1% (11.2; 25.3)
Expression of Hes-1 (Notch-1 Effector) during “In Vitro” Normal Erythroid Differentiation.
Authors: Cappellini Maria D. , Libani Ilaria V. , Calzavara Elisabetta , Ronzoni Luisa , Chiaramonte Raffaella , Comi Paola
Published: October 2019

Hematopoiesis involves highly regulated proliferation and differentiation during which a small number of multipotent stem cells give rise to differentiated progenies. In several developmental systems stem cells fate is influenced by soluble molecules acting via cell-cell interaction, including those mediate by the Notch receptor family. Members of Notch family transmembrane receptors are found on primitive hematopoietic precursors, suggesting a role for Notch signaling in mammalian blood cells development. Notch signaling regulates cell fate controlling asymmetric cell division during stem/progenitor cell differentiation. A previous study on K562 cell line showed that Notch signaling inhibits erythroid/megakaryocytic development by suppressing GATA-1 activity. Furthermore there are evidences that Notch is expressed in early murine erythroid precursors. Probably Notch signaling in these uncommittted precursors may lead to enhanced survival, preserving multilineage potential. The role of Notch pathway during human adult erytropiesis has not been described. The aim of this study is to investigate the modulation of Notch activity during “in vitro” human erythropiesis. Human CD34+ from perpheral blood of normal adult subjects were differentiate “in vitro” for two weeks by the addiction of IL-3, SCF and Epo. This method of colture reproduces all stages of adult erythropoiesis. We analized the modulation of the expression of Notch-1, of its effector Hes-1 and of several erythoid specific genes, at different stages of differentiation using real time PCR. Our analysis shows that Hes-1 expression, which indicates the activation of Notch-1 pathway, is very high in the early steps of differentiation (BFU-E, CFU-E) while in the late stages rapidly decreases to undetectable levels. The Notch-1 gene expression doesn’t seem to be modulated way, but we didn’t invstigate the protein levels yet. These data suggest that Notch pathway is involved in the early stages of erythroid differentiation where it may enhance erythroid progenitors survival up to CFU-E, as hypothisized in mouse model, preventing them from apoptotic stimuli and promoting their proliferation. Involvement of Notch-1 signaling in preventing erythroid progenitors from apoptosis during erythroid differentiation could be important in some erythropoietic disorders such as b-Thalassemia syndromes or diserythropoietic anemias.
Mechanisms of Increased Reactive Oxygen Species (ROS) Generation Induced by Organic Arsenic Derivative S-Dimethylarsino-Glutathione (SGLU; ZIO-101).
Authors: Cheng Xiaodong , Taghi Manshouri , Huang Peng , Golemovic Mirna , Zingaro Ralph , Freireich Emil J. , Andreeff Michael , Kantarjian Hagop M. , Verstovsek Srdan
Published: October 2019

ZIO-101 is organic arsenic with similar anti-leukemia activity to arsenic trioxide (ATO) but much less toxicity. Consequently, ZIO-101 can be given at substantially higher doses than ATO and may be active in more diverse cancers than ATO. The precise anti-cancer mechanism of ZIO-101 is unknown: ZIO-101 increases intracellular production of reactive oxygen species (ROS) in dose dependent manner, resulting in apoptosis of leukemic cells. Modifying intracellular ROS levels alters the ability of ZIO-101 to induce apoptosis. We focused on the 2 most important intracellular ROS generators, mitochondria and NADPH oxidase. Cells were treated with ZIO-101 and NADPH oxidase expression levels, membrane translocation and interactions of subunits were studied. Effects of modified NADPH oxidase levels on ZIO-101-induced ROS production were studied (diphenyleneiodonium [DPI], a specific inhibitor of NADPH oxidase, and SiRNA technique were used to inhibit enzyme function, while bryostatin-1, a specific activator of NADPH oxidase, was used to increase enzyme function). Mitochondrial DNA-depleted HL60 Rho-0 cells were also used to evaluate mitochondrial input to ROS-generation. Our data show that ZIO-101 activates NADPH oxidase. A low concentration of ZIO-101 (1uM) requires 14 h to significantly increase intracellular ROS levels and kill leukemic cell. This effect is strongly inhibited by DPI-pretreatment and is also seen in P47-SiRNA transfected HL60 cells. On the other hand, this A higher dose of ZIO-101 (4uM) increases ROS levels more rapidly (2–6 h). This early increase is not inhibited by DPI-pretreatment or SiRNA transfection. A 14 h but not 2–6 h increase in ROS is detectable in Rho-0 HL60 cells. These data indicate dose-dependent mechanisms of ZIO-101 induction of ROS:

1uM ZIO-101 induces NADPH oxidase activity which results in ROS production detectable at 14 h; there is no mitochondrial component; 4uM ZIO-101 disrupts mitochondrial function resulting in an earlier increase in ROS levels and subsequent apoptosis. effect is significantly enhanced in cells that have increased enzyme function. ROS-production in Rho-0 HL60 cells is unaffected by these interventions.
A Biologically Distinctive Subgroup of Acute Myelogenous Leukemia with Characteristic Nuclear Morphology and a High Frequency of FLT3 Mutation.
Authors: Chen Weina , Rassidakis George , Routbort Mark , Jones Dan , Medeiros L. Jeffrey , Bueso-Ramos Carlos
Published: October 2019

Acute myelogeneous leukemia (AML) is a heterogeneous group of diseases. Mutations of the fms-like tyrosine kinase-3 (FLT3) gene of internal tandem duplication (ITD) type have been identified as one of the most frequent molecular defects in AML. However, even within FLT3 mutated/ITD positive cases, there is heterogeneity in clinical outcomes. In this study, we describe a biologically distinctive subgroup of AML in which blasts exhibit prominent nuclear invagination with frequent fishmouth-like nuclear indentation and had tendency of high frequency of FLT3/ITD. We identified 23 AML, FAB-M1 cases with fishmouth morphologic feature (study series, SS) diagnosed between 1998 and mid-2005 at our institution, which represents approximately 20% of AML, M1 cases and 1% of the entire AML cases. The age-matched control series (CS) of 20 AML, M1 cases without this nuclear feature were established to compare the incidence of FLT3 mutations, cytogenetics (CG), immunophenotype by multiparameter flow cytometry and clinical outcome. All the laboratory tests were performed on the bone marrow (BM) samples except one case using peripheral blood.

Characteristics of AML cases with and without fishmouth-like nuclear morphology age (y) sex M:F WBC x109/L Normal CG FLT3/ITD lack of CD34 lack of HLA-DR The values of age and WBC are the range and median. SS (n=23) 18–81 62 5:17 1.4–335, 48 15/22 (68%) 6/7 (86%) 16/23 (70%) 10/23 (43%) CS (n=20) 20–80 65 13:7 0.7–550, 35 9/20 (45%) 7/14 (50%) 2/20 (10%) 2/20 (10%) p value > 0.05 0.006 > 0.05 0.21 0.17 0.0004 0.017

As illustrated in the table, AML cases with fishmouth-like nuclear morphology are significantly associated with female gender and lack of expression of CD34 and HLA-DR. There is statistical trend toward high frequencies of normal cytogenetics (68% diploid by conventional karyotyping) and FLT3 mutation of ITD type (86%) in the study series. The white cell count, bone marrow blast percentage (82–98%, median 90% in SS vs. 76–97%, median 85% in CS) and expression of CD13, CD33 and CD117 are not significantly different. Electron microscopic examination demonstrated condensed collections of mitochondriae within the indented/invaginated nuclear pockets. Complete remission rate was higher in AML cases with fishmouth-like nuclear morphology (73% vs. 40%, p=0.032). However, there was no difference in the overall survival rate between these two groups (1-year survival of 38% in SS vs. 39% in CS). In conclusion, AML cases with fishmouth-like nuclear morphology may represent a rare but distinctive AML subgroup with specific clinical, immunophenotypic and genetic features. Further prospective studies of larger cohorts of patients are required to confirm the prognosis of this entity.
Study of Sequential Treatment of Newly Diagnosed De Novo AML Patients with DA and CAG Regimens as Remission Induction Therapy.
Authors: ping Sun Hui , Liu Wei Hong , Li Jun min , Chen Qiu sheng , Chen Yu
Published: October 2019

Objectives

To evaluate the efficacy and safety of sequential treatment of newly diagnosed de novo AML patients with DA and CAG regimens as induction therapy.

Methods

Those who were newly diagnosed as de novo AML (FAB classification criteria) were enrolled and DA regimen chemotherapy were administered. Bone marrow aspirates were performed and BM smears were examined at 48 hours since the end of chemotherapy. If severe hypocellularities were not achieved, the percentage of blasts in BM was between 20%–60% and peripheral WBC was in the range of (0.5–10) x109/L, the patients would receive CAG regimen therapy since 72 hours. Patients’ general status and the important parameters, such as peripheral blood count, liver function, renal function, thrombosis and hemostasis parameters were monitored throughout the course of the treatment and thereafter. When the clinical symptoms were relieved and peripheral blood counts returned to normal, or it was the end of the second or third week since the end of the CAG regimen, Bone marrow were examined again to evaluate the efficacy of the sequential therapy.

Results

14 patients consisted of 9 male and 5 female patients were enrolled. Out of them, 2 were M1, 5 M2, 4 M4 and 3 M5 according to FAB classification criteria. Median of blasts in BM were 38.5%(20%–60%) before CAG regimen. Of the 14 patients, 10 reached CR, 2 PR and 2 NR. CR rate was 71.4% (10/14) and total response rate was 85.7%(12/14). Time to achieve CR was on 15th(14th–29th)day medianly since the end of the treatment. During the CAG therapy courses, the nadir of peripheral blood cell counts and the time when it occurred were as follows: WBC 1.0(0.2–3.5)(x109/L),10(1–23)(d); Hb 57.5(44–69) (g/L), 10(1–27)(d)and PLT 11.5(10–65)(x109/L), 12(3–23)(d), respectively. Neutropenia (WBC<1.0x109/L) and thrombocytopenia (PLT<20.0x109/L) were lasted for 0(0–24) and 11(0–21)days, respectively. Median units of transfusions of platelets and red blood cells required by each patient were 3(0–10)(u) and 4(0–12)(u), respectively. The most commonly observed side effect of the regimen was bone marrow proliferation inhibition. Infections, usually respiratoy tract infections, were the second. However, sepsis was rare, which appeared in 1 out of 14 patients.

Conclusions DA and CAG regimens sequential treatment as remission induction chemotherapy in patients with newly diagnose de novo AML was highly effective and well tolerated. It would be beneficial for those who might not be sensitive enough to DA regimen chemotherapy only.
An Initial Analysis of an Allele-Specific Q-PCR Assay Which Detects Minimal Residual Disease in the Peripheral Blood of Follicular Lymphoma Patients.
Authors: Kautz Naomi , Ingola Diane , Denney Dan , Theriault Thomas
Published: October 2019

The monitoring of minimal residual disease (MRD) in peripheral blood cells of follicular non-Hodgkin’s lymphoma patients may provide important information regarding disease prognosis and response to therapy. The traditional method of assessing MRD in follicular lymphoma is by amplification of the t(14;18) translocation at the major breakpoint region (MBR/JH) locus. We are developing an allele-specific (ASO) quantitative PCR (Q-PCR) approach which has the ability to monitor tumor cells in the peripheral blood of patients who do not have a t(14;18) breakpoint within the MBR sequence. The patient-specific ASO assay amplifies immunoglobulin heavy chain sequence defined by unique CDR2/CDR3 (complementarity-determining region) primers. Both the generic MBR/JH and the patient-specific ASO Q-PCR assays were performed on peripheral blood lymphocytes collected before and after chemotherapy and following treatment with MyVax® personalized immunotherapy. MyVax® personalized immunotherapy (Id-KLH conjugate) is an active idiotype immunotherapy manufactured by Genitope Corporation that targets each patient’s unique tumor-associated immunoglobulin and is co-administered with GM-CSF. Preliminary Q-PCR data generated from these analyses could be grouped into MBR positive and MBR negative sets. Initial results have shown MBR values can track ASO values at sequential time points during therapy. In the MBR negative group, we were able to monitor tumor-specific cells present in peripheral blood by the ASO assay. In terms of MRD assessment in follicular lymphoma patients, the allele-specific Q-PCR assay may provide a valuable extension of analysis beyond the standard approach of solely monitoring MBR/JH values. Genitope Corporation is focused on the development of novel means for both treating follicular lymphoma and monitoring the patient’s MRD in a patient-specific manner.
Isophosphoramide Mustard-Lysine (ZIO-201): Active Moiety of Ifosfamide (IFOS) without Kidney Toxicity.
Authors: Taub Mary , Springate James E. , Zaki Emed , Struck Robert
Published: October 2019

Ifosfamide (IFOS) and cyclophosphamide (CPA) are widely used anti-cancer drugs. Both are pro-drugs which must be metabolized before their metabolites cross-link DNA. Use of these drugs, especially high-dose IFOS, is complicated by substantial adverse effects of metabolites not directly involved in DNA-cross-linking including kidney, bladder and CNS toxicities. Recently, a lysine-stabilized form of isophosphoramide mustard (IPM-lysine; ZIO-201), an IFOS-metabolite, was developed. ZIO-201 directly cross-links DNA and is active against human cancer cell lines and in mice with human cancer xenografts. To evaluate nephrotoxicity of ZIO-201 we used an in vitro primary rabbit kidney proximal tubule (RPT) cell culture system, which retains many of the characteristics of renal proximal tubule cells including a polarized morphology, a Na+/glucose cotransport system and a p-aminohippurate transport system, glutathione status, and hormone responses (including a parathyroid hormone sensitive adenylate cyclase). We studied effects of ZIO-201 and other IFOS metabolites on primary RPT cells. IFOS is activated by the cytochrome P450 system producing a mixture of 2- and 3-dechloroethylifosfamide and resulting in formation of chloroacetaldehyde (CAA). Also, metabolism of IFOS by the cytochrome P450 system produces 4-hydroxyifosfamide, ultimately resulting in the production of acrolein (ACR). CAA and ACR are IFOS metabolites implicated in kidney toxicity in humans. Effects of ZIO-201 and CAA on the viability of confluent monolayers of primary rabbit kidney proximal tubule (RPT) cells were tested at 15–100 μM. Viability was determined by neutral red dye uptake by the primary cultures after 8 h incubation. ZIO-201 did not significantly reduce viability of primary RPT cells; the LD50 for CAA was about 40 μM with complete inhibition at 75 μM. Effects of 4-hydroperoxyifosfamide (HIFA), a synthetic prodrug of 4-hydroxyifosfamide and ACR were also studied. Although ACR had an LD50 of 80 μM HIFA did not significantly affect viability. The IFOS metabolite 4-hydroxyifosfamide, was prepared from 4-hydroperoxyifosfamide (1). At 200 μM, 4-hydroxyifosfamide resulted in extensive cell death after 4 h incubation. These data show that ZIO-201 avoids the kidney toxicity caused by metabolites of pro-drugs like IFOS. Whereas 2 IFOS metabolites, CAA and ACR were cytotoxic to kidney cells, ZIO-201 was not. An explanation for the toxic effects of 200 μM 4-hydroxyifosfamide t might be metabolism to ACR in vitro.
Disruption of Leukemia/Stroma Cell Interactions by CXCR4 Antagonist AMD3465 Enhances Chemotherapy-Induced Apoptosis in AML.
Authors: Zeng Zhihong , Konopleva Marina , Nowak Billie J. , Plunkett William , Borthakur Gautam , Estey Elihu , Champlin Richard , Bridger Gary , Andreeff Michael
Published: October 2019

Chemokine receptor CXCR4 is critically involved in the migration of hematopoietic cells to the stromal derived factor (SDF-1α)-producing bone marrow microenvironment. We and others have previously demonstrated that stroma/leukemia interactions mediate protection of leukemic cells from chemotherapy-induced apoptosis (Konopleva, Leukemia2002:1713; Burger Blood2000: 2655). Using AMD3465, the second-generation small-molecule CXCR4 inhibitor with a greater potency than AMD3100, we tested the hypothesis that CXCR4 inhibition interferes with stromal/leukemia cell interactions resulting in increased sensitivity to chemotherapy. Our results showed that AMD3465 inhibited surface expression of CXCR4 on AML cell lines in a dose dependent manner. AMD3465 (1μM) significantly inhibited SDF-1α and stromal (MS-5)-induced migration of OCI-AML2 cells (78% and 54% inhibition, respectively), U937 cells (71% and 41.3%) and diminished SDF-1α- or stromal-induced migration of leukemic blasts from four primary AML samples tested (SDF-1α, 43.4 ± 8.6%, MS-5, 38.4 ± 8.5% inhibition). In in vitro co-culture systems, stromal cells significantly protected leukemic cell lines and primary AML cells from spontaneous and chemotherapy induced apoptosis (p<0.01; p<0.001). Measurements of intracellular Ara-CTP levels determined by HPLC showed that stromal cells diminished incorporation of Ara-C into leukemic cells by 20%. AMD3465 enhanced AraC- and Busulfan-induced apoptosis by 44% and 69%, respectively. Western blot revealed that AMD3465 downregulated AKT signaling in AML cells. Most importantly, it decreased stroma-mediated protection from AraC-induced apoptosis in five out of ten primary AML samples with surface expression of functional CXCR4 (mean increase, 29.9±19.5% compared to chemotherapy alone). Curiously, the highest sensitization was observed in a sample from AML patient harboring Flt3/ITD mutation (Ara-C, 30.3% annexinV(+); Ara-C+AMD, 62.8%), confirming recently documented role for Flt3/ITD in modulation of CXCR4 signaling (Fukuda, Blood2005:3117). Taken together, our data suggest that SDF-1α/ CXCR4 interactions contribute to the resistance of leukemic cells to chemotherapy-induced apoptosis. Disruption of these interactions by the potent CXCR4 inhibitor AMD3465 represents a novel strategy for targeting leukemia cell/bone marrow microenvironment interactions. A clinical trial testing this concept in patients with AML is in preparation.
Novel Combination of Gallium Nitrate, Rituximab and Dexamethasone for the Treatment of Relapsed or Refractory Diffuse Large B-Cell Lymphoma, Mantle Cell Lymphoma or Transformed Follicular Lymphoma.
Authors: Smith Scott E. , Stiff Patrick J. , Rodriguez Tulio , Toor Amir , Klein Jared
Published: October 2019

More than 56,000 new cases of non-Hodgkin’s lymphoma (NHL) will be diagnosed this year in the United States. Despite advances in treatment modalities such as radiation, biologic agents, and cytotoxic chemotherapeutic regimens, only 25–30% of these patients will be cured of their disease. Standard salvage regimens such as DHAP, ESHAP, ICE and EPOCH have proven efficacy at the cost of increasing toxicity and hospitalization costs. The reported response rates for these are on the order of 50–75% as first-line salvage regimens. However, as our aging patient population develops worsening performance status and co-morbidities, it seems appropriate to develop effective lymphoma treatments, which have fewer toxicities and lower costs for administration. One approach is to combine non-myelosuppressive therapies. One non-myelosuppressive agent, which has efficacy in lymphoma, is gallium nitrate. Investigation of gallium nitrate for cancer treatment dates back to the 1970’s. While it is currently approved for the treatment of bisphosphonate resistant hypercalcemia of malignancy, it has also been shown to inhibit ribonucleotide reductase and bind transferrin and potentially complex with the transferrin receptor, which is highly expressed in intermediate and aggressive histology lymphomas. It appears that the binding of the transferrin receptor on the lymphocyte as well as its inhibition of ribonucleotide reductase, eventually impairs iron metabolism, which is a necessary component of the intracellular cytochrome systems/mitochondrial function and ultimately oxidative phosphorylation.

The current study is a phase II clinical trial investigating the combination of gallium nitrate, rituximab and dexamethasone (GaRD) for relapsed or refractory DLBCL, MCL or transformed follicular lymphomas. The gallium nitrate is given at 200mg/m2 CIV days 1–7, rituximab 375mg/m2 IVPB day 1 and dexamethasone 40 mg po days 1–4. Eligible patients must have proven relapsed or refractory disease and have a SWOG PS <3. Patients may have failed prior ASCT or allogeneic SCT. The accrual goal is 37 patients. We have enrolled 14 patients on study to date and have 12 evaluable patients; as part of the ongoing safety evaluation for this study, we have the following results: ORR 9/12 (75%); CR 2/12 (17%); PR 7/12 (58%); SD 2/12 (17%); and PD 1/12 (8%). Most of these patients were refractory to prior salvage regimens 8/12 (67%), including ESHAP, DHAP or high-dose cyclophosphamide. No patients developed grade 3 or 4 toxicities, with the exception of grade 4 lymphopenia and grade 3 anemia (most likely due to transferrin receptor binding).

Conclusions: gallium nitrate, rituximab and dexamethasone (GaRD) appears to be an effective and relatively non-toxic salvage regimen for patients with relapsed DLBCL, MCL or transformed FL.
A Phase II Trial of CHOP Chemotherapy Followed by Yttrium 90 (90Y) Ibritumomab Tiuxetan (Zevalin) for Previously Untreated Elderly Diffuse Large B-Cell Lymphoma (DLBCL) Patients.
Authors: Zinzani Pier Luigi , Tani Monica , Stefoni Vittorio , Alinari Lapo , Marchi Enrica , Fina Mariapaola , Baccarani Michele
Published: October 2019

In the last 20 years, the major improvement over the use of CHOP has been the addition of anti-CD20 immunotherapy (Rituximab). This advancement was first demonstrated in a randomized trial in elderly patients with diffuse large B-cell lymphoma (DLBCL). Single-agent radioimmunotherapy activity, in particular Yttrium 90 (90Y) Ibritumomab Tiuxetan (Zevalin), has been demonstrated in heavily pretreated DLBCL patients. Recently, the preliminary data of a phase II trial have showed that 90Y Ibritumomab Tiuxetan have useful activity in the treatment of relapsed/refractory elderly DLBCL (Morschhauser et al, Blood 2004, 104: 41a), with no unexpected toxicities observed. The results of this study support a further evaluation of 90Y Ibritumomab Tiuxetan in combination with chemotherapy earlier in the time course of elderly DLBCL. We conducted a prospective, single-arm, open-label, non-randomized, phase II to evaluate the efficacy and safety of 90Y Ibritumomab Tiuxetan of a novel new approach combining induction chemotherapy with CHOP followed by consolidation with 90Y Ibritumomab Tiuxetan for patients with previously untreated elderly DLBCL. Patient eligibility was represented by: patients older than 60 years with biopsy-proven, untreated, bidimensionally measurable stage II, stage III, or stage IV DLBCL expressing the CD20 antigen; performance status of 0 to 2, a pretreatment granulocyte cell count of 1500/μL or greater, and a platelet count of 100.000/μL or greater. All patients were notified of the investigational nature of this study and signed a written informed consent approved in accordance with institutional guidelines, including the Declaration of Helsinki. The study was approved by the institutional review board. Patients were treated with standard CHOP chemotherapy every 21 days for 6 cycles. Patients were restaged 4 to 8 weeks after completion of the sixth cycle of CHOP chemotherapy. Patients achieving at least a partial response after 6 cycles of CHOP chemotherapy were eligible for consolidation with 90Y Ibritumomab Tiuxetan provided the granulocyte count was greater than 1500/μL, the platelet count exceeded 100.000/μL, and the bone marrow examination at the completion of CHOP chemotherapy demonstrated no more than 25% involvement with lymphoma. All patients were to receive a single dose of 90Y Ibritumomab Tiuxetan 14.8 MBq/kg (0.4 mCi/kg) up to a maximum dose of 1184 MBq (32 mCi). A total of 20 patients have been enrolled and preliminary results of this trial will be presented.