bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Application of Growth Factor Stimulants to Improve Ctogenetic Analysis of Patients with Myeloproliferative and Myelodysplastic Syndromes.
Authors: Vaughan Beverley R. , Scott Mike A. , Nachva Elizabeth P. 
Published: October 2019

Abstract
Conventional cytogenetic methods rely on culturing bone marrow aspirates to obtain suitable and sufficient mitotic figures for G banded analysis. Samples from Myeloproliferative (MPD) and Myelodysplastic (MDS) patients often have increased failure rates due to reduced growth and poor morphology all of which hampers the conventional karyotyping investigation . The aim of this study was to investigate whether;
The quality and quantity of metaphase cells from MPD and MDS patients could be improved. To assess whether the applied method(s) led to clonal selection.
Three culture systems using different growth factor stimulants were assessed alongside an un-stimulated control. These were Bone Marrow Growth Supplement (BMGS) containing IL-3, IL-6, G-CSF and CSF, 5637 conditioned medium (5637CM) containing IL-3, IL-6 and G-CSF and myeloid expansion medium (MEM) containing IL-3, IL-6, G-CSF, SCF and Flt-3. Samples were assessed qualitatively and quantitatively for mitotic index (MI = % dividing cells). Clonality was monitored using FISH, when suitable markers were present.
In 39 patients bone marrow samples chromosome morphology of mitotic figures was improved with all stimulated cultures. Variation was observed between patient samples. However the increase in MI was shown to be significant in all stimulated cultures at p<0.05. The most favourable results were obtained with 5637CM, producing increases in MI up to 14 fold (with a mean fold increase of 1.65). 5637CM significantly improved chromosome morphology and consequently banding quality. Examination of 10 patients’ cultures with FISH probes, supports the hypothesis that clonal expansion remains un-altered in stimulated cultures.
Conventionally, peripheral blood (PB) stimulated cultures are not used for cytogenetic analysis of MPD patients. However preliminary studies of six patients have shown that growth factor stimulation successfully produces high quality mitotic figures suitable for analysis by conventional and FISH methods.
Benefits of Early Hemoglobin Response to Epoetin Alfa in Elderly Patients with Chemotherapy-Related Anemia.
Authors: Rosberg James , Cremieux Pierre Y. , McKenzie Scott R. , Fastenau John M. , Piech Catherine T. 
Published: October 2019

Abstract
Background: Previous research has shown early hemoglobin (Hb) response to epoetin alfa (EPO) therapy is associated with reduced transfusion requirements, higher Hb response rates, quality of life score improvements, and decreased EPO drug utilization. This subgroup analysis of elderly (age >/=65) patients (pts) with chemotherapy-related anemia (CRA) assesses the benefit of an early Hb response in this distinct population.
Methods: Data from three large multicenter EPO clinical trials were evaluated. In EPO 1, pts received 10,000 Units TIW with potential escalation to 20,000 Units TIW. In EPO 2 and EPO 3, pts received 40,000 Units QW with escalation to 60,000 Units QW. Pts eligible for this analysis were >/=65 years of age with a non-myeloid malignancy, had a baseline Hb </=11g/dL, and received chemotherapy with or without radiotherapy (EPO 1: N=935: EPO 2: N=1,270; EPO 3: N=269). Early Hb response was defined as >/=1g/dL Hb rise following four weeks of EPO therapy, independent of transfusion in the prior 28 days. Three outcomes of pts who exhibited early Hb response were compared to those of pts who did not: proportion of pts requiring transfusion, subsequent Hb response (Hb rise >/=2 g/dL independent of transfusion), and average weekly EPO dose.
Results: Early Hb response was observed in 54.1%, 47.5%, and 47.2% of pts from EPO 1, 2 and 3, respectively. In all trials, early responders had markedly lower transfusion use (EPO 1: 7.6% v 22.5%, p<.0001; EPO 2: 5.4% v 18.7%, p<.0001; EPO 3: 8.8% v 17.2%, p=.0525), higher subsequent Hb response (EPO 1: 78.7% v, 47.6%; EPO 2: 84.0% v 46.2%, EPO 3: 72.8% v. 47.8%, all studies p<.0001), and lower average weekly EPO dose (EPO 1: 30,467 vs. 40,594 Units, EPO 2: 39,286 vs. 47,310 Units; EPO 3: 38,043 v. 45,835 Units; all studies p<.0001).
Conclusions: Elderly cancer pts achieving an early Hb response to EPO QW or TIW experienced greater clinical benefits (lower proportion of pts transfused, higher subsequent Hb response rates, and less EPO utilization) compared to pts who did not. These findings are consistent with analyses involving pts with younger age groups. As early Hb response is associated with clinical benefits, it should be an important goal of erythropoietic therapy for chemotherapy-induced anemia, especially in the elderly population, as demonstrated here.
Effects of a Leukemia-Associated Gain-of-Function Mutation of SHP-2 Phosphatase on IL-3 Signaling.
Authors: Yu Wen-Mei , Daino Hanako , Chen Jing , Bunting Kevin D. , Qu Cheng-Kui 
Published: October 2019

Abstract
Mutations in SHP-2 phosphatase that cause its hyper-activation have been identified in human leukemias, in particular, juvenile myelomonocytic leukemia (JMML) that is characterized by hypersensitivity of myeloid progenitor cells to granulocyte macrophage colony-stimulating factor and interleukin (IL)-3. However, the molecular mechanisms by which gain-of-function (GOF) mutations of SHP-2 induce leukemia are not fully understood. Our previous studies have shown that SHP-2 plays an essential role in IL-3 signal transduction in catalytic-dependent and -independent manners and that overexpression (5-to-6 fold) of wild-type (WT) SHP-2 attenuates IL-3-mediated hematopoietic cell function through dephosphorylation of STAT5. This raised the possibility that SHP-2-associated JMML was not solely attributed to the increased catalytic activities of SHP-2 GOF mutants. The SHP-2 mutants must have gained other additional functions. To test this possibility, we investigated effects of a GOF mutation (E76K, the most frequent SHP-2 mutation seen in JMML) of SHP-2 on IL-3 signal transduction in great detail by comparing signaling activities of SHP-2 E76K to WT SHP-2. Our results showed that SHP-2 E76K mutation caused myeloproliferative disease in mice, while overexpression of WT SHP-2 decreased hematopoietic potential of the transduced cells in recipient animals. E76K mutation in the N-terminal Src homology 2 domain significantly increased binding of the mutant SHP-2 to Grb2 and Gab2, two adaptor proteins coupling downstream Erk and PI3 kinase pathways to the proximity of the IL-3 receptor. As a result, IL-3-induced Erk and PI3 kinase pathways were highly activated by SHP-2 E76K mutation. In addition, Jak2 kinase activation was also markedly enhanced and due to the E76K mutation the substrate specificity of SHP-2 toward STAT5 was changed. Dephosphorylation of STAT5 by SHP-2 E76K was alleviated. These studies suggest that in addition to elevated catalytic activity, profound changes in protein-protein interaction capacities of GOF mutant SHP-2 play an important role in the pathogenesis of SHP-2-related leukemias.
Microparticle-Associated Tissue Factor Activity: A Link between Cancer and Thrombosis?.
Authors: Tesselaar Margot E.T. , Romijn Fred P.H.T.M. , van der Linden Irma K. , Prins Frans , Bertina Rogier M. , Osanto Susanne 
Published: October 2019

Abstract
Background: Cancer, in particular mucinous adenocarcinomas, is associated with an increased risk of venous thromboembolism (VTE). Tissue factor (TF), a transmembrane-receptor protein and initiator of coagulation, plays a central role in the paradigm that clotting and tumor growth form a vicious circle, in which hypercoagulability facilitates the aggressive biology of cancer and vice versa. Expression of TF is associated with poor differentiation of tumors and poor prognosis. We initiated a study in cancer patients, to find out whether microparticles (MP) derived from malignant epithelial cells, may directly initiate blood coagulation locally or at distant sites via TF expressed on these MP and play a critical role in the development of the hypercoagulability
Patients & Methods: MP were isolated from peripheral blood samples obtained from healthy subjects, and unselected cancer patients, i.e. patients with primary breast carcinoma (before and after surgery), with metastatic breast carcinoma and with pancreatic carcinoma. Tissue factor procoagulant activity associated with isolated MP was studied by factor VIIa dependent generation of factor Xa. MP were examined by flow cytrometric phenotyping, and electron and confocal immunofluorescence laser scanning microscopical examination.
Results: Compared to 37 healthy sujects, significantly increased levels of MP-associated TF activity were found in patients with disseminated breast and pancreatic cancer. Pancreatic (n=5) and breast cancer patients (n=2) who presented with clinically manifest venous thrombosis had a 18-fold increase in MP-associated TF activity as compared to healthy subjects or 6 subjects with idiopathic VTE (P<0.003). In all individuals most of the circulating MP expressed the platelet antigen CD61. MP expressing the epithelial antigen MUC1 -most likely derived from malignant cells- were found in 65% of metastatic breast and 59% of advanced c.q. metastatic pancreatic patients. Co-expression of CD61 and MUC1 on MPs was shown by flow cytometry and confocal immunofluorescence microscopy.
Conclusion: Highly elevated MP-associated TF activity significantly correlated with development of VTE and with the presence of circulating MUC1-positive MP, suggesting a decisive role in the pathogenesis of the prothrombotic state in cancer patients with disseminated mucinous carcinomas. Patients with a low level of TF-activity on MP that also lacked expression of mucin had a higher survival rate at 3–9 months follow-up than those with a high TF-activity and mucin present: the estimated risk of dying was about 0.42 (95% CI 0.19–0.94) for an individual with these 2 predictor variables present, adjusting for the other factors (age cohort, type of cancer, VTE) in the Cox proportional hazards model.
Dynamic Regulation of Src by Csk in Integrin α IIb β 3 Mediated Signalling.
Authors: Vielreicher Martin , Steinmeyer Ralf , Harms Gregory , Walter Ulrich , Obergfell Achim 
Published: October 2019

Abstract
When platelets adhere to fibrinogen, they undergo profound actin rearragements and spreading. These responses require signals triggered by aIIbb3, a process known as outside-in signalling. Although aIIbb3 signaling is required for normal hemostasis, its molecular basis is incompletely understood. Recently we demonstrated by coimmunoprecipitation techniques that several Src family kinases are constitutively associated with aIIbb3 in platelets. Fibrinogen binding to aIIbb3 leads to rapid activation of Src and recruitement and activation of the Syk tyrosine kinase. Theses interactions are significant because murine platelets defiecient in multiple Src kinases or Syk fail to spread on fibrinogen. One of the earliest events upon binding of fibrinogen to the receptor is the assembly of a multimolecular protein complex inculding the tyrosine kinases Src and Syk. Phosphorylation of Src at Tyrosine 418 leads to Src activation, phosphorylation of Tyrosine 529 to inactivation. The inactivation of Src is mediated by Csk. Previous studies have shown that in resting platelets Csk forms a complex with integrin α IIb β3 and Src. After fibrinogen binding Csk leaves this complex.
The aim of this study is to elucidate the dynamics of Src inactivation and the interaction with Csk in this process by using Fluorescence Resonance Energy Transfer (FRET). A5 CHO cells stably expressing integrin α IIb β3 were transfected with Csk-YFP and various Src mutatnts in a GFP vector. After cell adhesion on fibrinogen, a strong FRET signal could be observed at the edges of nascent lamellipodia of the spreading cell. The FRET signal was dynamic since it was transported in waves inside the cell within seconds. After completion of the adhesion process a weak FRET signal remained visible inside the cell. A different pattern could be observed when cells were transfected with Csk and the active Src mutant Y529F. The observed FRET signal was much weaker and less dynamic after cells were allowed to spread on fibrinogen suggesting that the muation of Tyrosine 529 to Phenylalanine inhibited Csk binding and inactivation of Src. This was not due to a spreading defect since the cells appeared to spread on fibrinogen normally. The use of the inactive Src mutant K295R revealed a FRET signal comparable to WT Src. These studies suggest that Src inactivation is dynamically regulated within seconds after cell adhesion on fibrinogen and that Tyrosine 529 is required for the interaction of Csk with Src in this process.
Clinical Tumor Responses Despite Graft Rejection after Nonmyeloablative Conditioning and Transplantation of Partially HLA-Mismatched (Haploidentical) Bone Marrow.
Authors: Levy Moshe Y. , Symons Heather J. , Fuchs Ephraim J. 
Published: October 2019

Abstract
A variety of hematologic malignancies that are resistant to standard chemotherapy can be treated by nonmyelaoblative allogeneic stem cell transplantation, or NST. At our institution, 84 patients have enrolled onto sequential Phase I and II protocols of NST for hematologic malignancies utilizing haploidentical donors. Patients received non-T cell-depleted marrow after conditioning with fludarabine 30 mg/m2 (days −6 to −2), cyclophosphamide (Cy; 14.5 mg/kg/d, days −6 and −5) and 200 cGy total body irradiation on day −1. Novel post-transplantation immunosuppression included high dose Cy (50 mg/kg/d on day 3 only or (n=23) days 3 and 4 (n=40), mycophenolate mofetil from days 5–35 and FK-506 from day 5–180. Sixteen patients (19%) experienced graft rejection, defined as absence of donor chimerism on day 60, including 6/10 patients with myelodysplastic syndrome (MDS), 3/7 patients with chronic myeloid leukemia (CML), 2/3 patients with chronic myelomonocytic leukemia (CMML), 2/21 patients with acute myeloid leukemia (AML), and three patients with lymphoid malignancies. Fourteen patients recovered autologous hematopoiesis, whereas two (1 MDS, 1 NHL) died in aplasia. Interestingly, objective clinical responses were observed in 3/6 of the rejectors with MDS (1 CR, 2 PR), 2/3 rejectors with CML (2 molecular CR), and in both rejectors with CMML (1 CR, 1 PR). Two of the five patients with MDS and blast counts of greater than 20% survived for greater than 2 years with a decrease in blast counts and need for transfusions. Both patients with CML had disease refractory to imatinib mesylate, and both achieved molecular complete responses following transplantation. One of the patients has relapsed, and the other remains in hematologic and cytogenetic complete remission, though in minimal molecular relapse, at 6+ months after transplantation. Of the patients with CMML, one achieved a morphologic and hematologic complete remission at six months after transplantation, and the other achieved improved blood counts and disappearance of marrow blasts before relapsing at six months after transplant. In summary, fatal graft rejection is uncommon following nonmyeloablative BMT with post-transplantation cyclophosphamide. Clinical responses occurred despite graft rejection, particularly among patients with chronic myeloid malignancies. Potential mechanisms of the anti-tumor effect include an effect of conditioning, a transient graft-versus-host reaction by donor T or NK cells, or an abrogation of host tolerance to tumor by either the graft-versus-host or host-versus-graft reaction.
Methylation Profile of B-Chronic Lymphocytic Leukemia Cells.
Authors: Fan Jun , Norio Asou , Matsuoka Masao 
Published: October 2019

Abstract
DNA methylation plays an important role in the development and aging of mammalian cells, and its dysregulation has been frequently observed in cancer cells. The purpose of this study is to investigate the involvement of aberrant DNA methylation in B chronic lymphocytic leukemia (B-CLL) cells. We compared methylation status of B-CLL cells isolated from patients with that of normal CD19+ cells isolated from health donors by methylated CpG island amplification/representative difference analysis method. 5 hypermethylated and 27 hypomethylated DNA regions were identified in B-CLL sample. Among the 27 hypomethylated regions, 5 located on chromosome 9q34, 3 on 10q25-26 and 4 on 19q13. Methylation status was confirmed by sequencing using sodium bisulfite-treated DNA samples. By comparing DNA samples from same patients at different clinical stages, we found that lower methylation density in these regions is linked with disease progression. Expression of 15 genes surrounding hypomethylated regions was studied by RT-PCR. Expression of laminin beta3 gene and melanotransferrin gene was found to be upregulated in all B-CLL cell lines as well as lymphoma cell lines comparing with normal CD19+ peripheral blood mononuclear cells. B-cell CLL/lymphoma 11b gene showed increased expression in only 2 B-CLL cell lines. For other genes, no transcriptional change was found regardless of changed DNA methylation. This study showed the predominance of DNA hypomethylation in B-CLL cells compared with hypermethylation. Hypomethylated regions clustered in a limited number of chromosomes and methylation density appeared to be inversely correlated with disease progress.
Figure Figure
Transgenic Targeting of Human FVIII Expression to Endothelial Cells Corrects the Murine Hemophilia A Phenotype and Re-Establishes a Releasable Pool of FVIII Together with VWF.
Authors: Shi Qizhen , Weiler Hartmut , Haberichter Sandra L. , Cooley Brian C. , Fahs Scot A. , Desai Drashti , Wandersee Nancy J. , Jozwiak Mary A. , Montgomery Robert R. 
Published: October 2019

Abstract
The deficiency of Factor VIII (FVIII) leads to hemophilia A, a severe X-linked bleeding disorder. To date, it is still unknown with certainty where FVIII is synthesized and how the storage pool of desmopressin acetate (DDAVP)-releasable FVIII is established. In vitro studies performed by our laboratory have shown that FVIII will store with VWF in endothelial cells if FVIII expression is induced by transfection or transduction. This FVIII is released by agonist stimulation. Furthermore, our studies have demonstrated that FVIII is not taken up from culture media by cells in culture and transfused FVIII does not re-establish the releasable pool in vivo. In this study, we generated 2 transgenic mouse lines in which human B-domain deleted FVIII (hFVIII) expression is under control of the endothelial-specific Tie 2 promoter (Tie2F8). This transgene was then bred into FVIII-deficient mice so that FVIII expression was restricted to endothelium. Functional FVIII activity (FVIII:C) was quantitated by a chromogenic assay. FVIII antigen (FVIII:Ag) and VWF antigen (VWF:Ag) were quantitated by ELISA. RT-PCR was used to analyze transcription of the transgene. Phenotypic correction was assessed by tail clip survival test, electrically-induced femoral venous thrombus formation, and ferric chloride induced carotid thrombus formation studies. The results demonstrated that the levels of FVIII in our transgenic mice were 559 ± 161 mU/ml in a higher expression line and 186 ± 31 mU/ml in a lower expression line. Tie2F8 transgene transcription was most abundant in lung and heart tissues. All transgenic mice survived tail clipping and achieved hemostasis within 6 hours. Both arterial and venous thrombi formation models demonstrated significant improvement of hemostasis at sites of injury. Confocal microscopy demonstrated recombinant FVIII stored together with VWF in endothelial cells, with especially high expression in the outlet branches of the aorta. Furthermore, this endothelial-specific expressed FVIII can be co-released together with VWF by epinephrine. The ratios of plasma FVIII:C and VWF:Ag post-epinephrine treatment to pre-epinephrine were determined, and the mean ± SEM were 1.68 ± 0.13 and 2.08 ± 0.34, respectively, at 30 minutes post-epinephrine treatment. Our results demonstrate that targeting human FVIII expression to endothelial cells corrects the murine hemophilia A phenotype and re-establishes a releasable pool of FVIII together with VWF.
Low-Dose Recombinante Urate Oxidase (Rasburicase) Is Effective in Treating Hyperuricemia in Patients with Hematologic Malignancies.
Authors: Trifilio S. , Evens A. , Gordon L. , Singhal S. , Tallman M. , Mehta J. 
Published: October 2019

Abstract
Hyperuricemia is a commonly seen in patients with hematologic malignancies and high tumor burden, and is a major feature of tumor lysis syndrome (TLS). Hyperuricemia is conventionally treated with hydration, urine alkalinization, and allopurinol. Allopurinol inhibits the conversion of hypoxanthine to xanthine, and xanthine to uric acid (UA) by inhibiting xanthine oxidase. It has no direct effect on existing UA in the serum. Rasburicase lowers UA rapidly to very low levels at the approved dose of 0.15–0.2 mg/kg daily for 5 days by converting UA to allantoin which is excreted rapidly. Despite this dramatic effect on UA, rasburicase has not been shown to have any beneficial impact on survival in patients with hematologic malignancies. We administered 0.2 mg/kg ideal body weight rasburicase to an obese hyperuricemic patient (actual 120 kg, ideal 60 kg). Serum UA declined from 11 mg/dL to 1.4 mg/dL in 3 h and 0.4 mg/dL in 11 h. The serum UA level remained low for several days and a second dose was not needed. After seeing this dramatic and sustained response, variable low rasburicase doses were used in patients with malignant diseases with close monitoring of biochemical parameters (Trifilio et al, ASH 2004). Here, we present our experince with the use of a single 3 mg dose of rasburicase in 42 adult patients receiving chemotherapy or undergoing hematopoietic stem cell transplantation for hematologic malignancies. In addition to rasburicase, allopurinol and other supportive therapy including hydration were also administered. Serum UA levels (baseline 6.4–16.4 mg/dL, median 9.7) had declined by 12–95% (median 43%) 24 hours after rasburicase administration.
Figure Figure
The baseline serum creatinine was 1.0–8.6 mg/dL (median 2.1), and that 24 hours after rasburicase administration was 0.7–8.0 mg/dL (median 2). The median change in creatinine was a decline of 10%. Except for 3 myeloma patients who were already on chronic hemodialysis, renal impairment did not progress to hemodialysis in any patient - and substantial renal function improvement (at least 50% decline in serum creatinine) was seen in all patients with a baseline serum creatinine of ≥ 3 mg/dL. Based on the Red Book rasburicase cost of $387 for a 1.5 mg vial, the amount of money saved ranged from ~$10,000 to ~$30,000 per patient. Our data suggest that rasburicase is effective at a fraction of the recommended dose. A formal randomized comparison of low- and conventional-dose rasburicase is warranted to see if the recommended higher dose offers any clinical benefit.