bluuebunny/crossref_metadata_2025 huggingface dataset:
Blockade of Fas-dependent apoptosis by soluble Fas in LGL leukemia
Authors: Liu Jin Hong , Wei Sheng , Lamy Thierry , Li Yongxiang , Epling-Burnette P.K. , Djeu Julie Y. , Loughran Thomas P.
Published: October 2019

Altered expression of the Fas-Fas ligand apoptotic pathway leads to lymphoproliferative and autoimmune diseases. Inlpr/lpr mice and children with autoimmune lymphoproliferative syndrome, defective apoptosis is due to Fas mutations. Large granular lymphocyte (LGL) leukemia is a clonal lymphoproliferative disorder associated with rheumatoid arthritis. Leukemic LGLs are resistant to Fas-dependent apoptosis despite expressing high levels of Fas. Such resistance can be overcome by activating leukemic LGLs in vitro, suggesting inhibition of Fas signaling in leukemic cells. We report that sera from patients with LGL leukemia contain high levels of soluble Fas. Ten of these 33 patients with LGL leukemia also had rheumatoid arthritis. Cloning and sequencing revealed expression of multiple Fas messenger RNA variants in leukemic LGL. These Fas variants, including 3 newly described here, encode soluble Fas molecules. Supernatants from cells transfected with these Fas variants blocked Fas-dependent apoptosis of leukemic LGLs. These results suggest that blockade of Fas-signaling by soluble Fas may be a mechanism leading to apoptosis resistance in leukemic LGLs.
Retrospective analysis of HHV-8 viremia and cellular viral load in HIV-seropositive patients receiving interleukin 2 in combination with antiretroviral therapy
Authors: Malnati Mauro , Broccolo Francesco , Nozza Silvia , Sarmati Loredana , Ghezzi Silvia , Locatelli Giuseppe , Delfanti Fanny , Capiluppi Brunella , Careddu Anna , Andreoni Massimo , Monini Paolo , Poli Guido , Lazzarin Adriano , Lusso Paolo , Tambussi Giuseppe
Published: October 2019

The combination of interleukin 2 (IL-2) and antiretroviral therapy (ART) represents an emerging strategy in the treatment of patients infected with HIV. Aside from its immunomodulatory role, however, IL-2 may induce replication of human herpesvirus 8 (HHV-8)/Kaposi sarcoma (KS)–associated herpesvirus. We retrospectively evaluated HHV-8 plasma viremia and cellular load, as well as anti–HHV-8 antibody titers, in sequential samples from 84 patients receiving ART alone or in combination with IL-2. At baseline, HHV-8 plasma viremia was present only in 2 HHV-8–seropositive patients in whom KS subsequently developed during or immediately after termination of IL-2 therapy. The level of viremia increased during follow-up and peaked at the time of the clinical manifestation of KS. Moreover, transient peaks of HHV-8 viremia were temporally associated with administration of IL-2. HHV-8 plasma viremia was never detected in the other 47 patients receiving IL-2 nor in 35 controls treated only with ART. Thus, IL-2 therapy seems safe in most patients infected with both HIV and HHV-8, except for those with detectable HHV-8 viremia, who may not be eligible for IL-2 treatment.
The TRQQKRP motif located near the C-terminus of Rac2 is essential for Rac2 biologic functions and intracellular localization
Authors: Tao Wen , Filippi Marie-Dominique , Bailey Jeffrey R. , Atkinson Simon J. , Connors Bret , Evan Andrew , Williams David A.
Published: October 2019

Rac GTPases regulate a wide variety of cellular processes including actin cytoskeleton organization, gene expression, cell-cycle progression, and apoptosis. Here we report that the TRQQKRP motif of Rac2 located near the C-terminus, a region of sequence disparity among Rac proteins, is essential for complementation of Rac2 function in Rac2-deficient cells. Deletion of this sequence can also intragenically suppress the dominant-negative Rac2D57Nmutation in a variety of functional assays. In Rac2-deficient cells, expression of TRQQKRP-deleted Rac2 protein is unable to completely rescue migration and nicotinamide adenine dinucleotide phosphate oxidase deficiencies previously described in these cells. In fibroblasts, the Rac2D57N mutant phenotypes of abnormal proliferation, cell morphology, and membrane ruffling are suppressed by the TRQQKRP motif deletion. In myeloid hematopoietic cells, the deletion of the TRQQKRP motif eliminates a Rac2D57N-induced block in in vitro differentiation of neutrophils not previously described with this mutant. Mechanistically, deletion of the TRQQKRP motif results in diminished geranylgeranylation and delocalization of intracellular Rac2 protein. Taken together, these results indicate that the TRQQKRP motif in Rac2 protein is required for efficient prenylation and correct intracellular localization of Rac2 protein and is essential for Rac2 to mediate a variety of its biologic functions. These data suggest that precise localization of Rac2 protein in intracellular compartments and/or with other proteins/lipids is a prerequisite for its diverse functions.
Peripheral blood but not tissue dendritic cells express CD52 and are depleted by treatment with alemtuzumab
Authors: Buggins Andrea G. S. , Mufti Ghulam J. , Salisbury Jonathan , Codd Jane , Westwood Nigel , Arno Matthew , Fishlock Keith , Pagliuca Antonio , Devereux Stephen
Published: October 2019

CAMPATH antibodies recognize CD52, a phosphatidylinositol-linked membrane protein expressed by mature lymphocytes and monocytes. Since some antigen-presenting dendritic cells (DCs) differentiate from a monocytic progenitor, we investigated the expression of CD52 on dendritic cell subsets. Four-color staining for lineage markers (CD3, 14, 16, 19, 20, 34, and 56), HLA-DR, CD52, and CD123 or CD11c demonstrated that myeloid peripheral blood (PB) DCs, defined as lineage−HLA-DR+CD11c+, express CD52, while expression by CD123+ lymphoid DCs was variable. Depletion of CD52+ cells from normal PB strongly inhibited their stimulatory activity in an allogeneic mixed lymphocyte reaction and also reduced the primary autologous response to the potent neoantigen keyhole limpet hemocyanin. CD52 is thus expressed by a myeloid subset of PBDCs that is strongly allostimulatory and capable of initiating a primary immune response to soluble antigen. Administration of alemtuzumab, a humanized monoclonal antibody against CD52, to patients with lymphoproliferative disorders or as conditioning for hematopoietic stem cell transplantation resulted in a marked reduction in circulating lineage−HLA-DR+ DCs (mean 31-fold reduction,P = .043). Analysis of monocyte-derived DCs in vitro revealed a reduction in CD52 expression during culture in granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4, with complete loss following activation-induced maturation with lipopolysaccharide. In contrast to the findings in PB, epidermal and small-intestine DCs did not express CD52, suggesting either that transit from blood to epidermis and gut is associated with loss of CD52 or that DCs in these tissues originate from another population of cells.
Cyclosporine inhibition of P-glycoprotein in chronic myeloid leukemia blast phase
Authors: List Alan F. , Kopecky Kenneth J. , Willman Cheryl L. , Head David R. , Slovak Marilyn L. , Douer Dan , Dakhil Shaker R. , Appelbaum Frederick R.
Published: October 2019

Chronic myeloid leukemia blast phase (CML-BP) cells commonly express the multidrug transporter, P-glycoprotein (Pgp). To determine whether Pgp inhibition improves treatment outcome in CML-BP, the Southwest Oncology Group performed a randomized, controlled trial testing the benefit of the Pgp modulator, cyclosporin A (CsA). Seventy-three eligible patients were assigned to treatment with cytarabine and infusional daunorubicin with or without intravenous CsA. Treatment with CsA yielded no improvement in treatment outcome as measured by the frequency of induction resistance (68% vs 53%), rate of complete remission or restored chronic phase (CR/CP, 8% vs 30%), and survival (3 vs 5 months). Blast expression of Pgp (63%) and LRP (71%) was common, whereas only Pgp adversely impacted the rate of CR/CP (P = .025). We conclude that Pgp has prognostic relevance in CML-BP but that the modulation of Pgp function with CsA as applied in this trial is ineffective.
Evidence for a Single Step Mechanism in the Origin of Hyperdiploid Childhood Acute Lymphoblastic Leukemia.
Authors: Paulsson Kajsa , Morse Helena , Fioretos Thoas , Behrendtz Mikael , Strombeck Bodil , Mitelman Felix , Johansson Bertil
Published: October 2019

High hyperdiploidy (>50 chromosomes) in childhood acute lymphoblastic leukemia (ALL) is characterized by non-random multiple trisomies and tetrasomies, involving in particular chromosomes X, 4, 6, 8, 10, 14, 17, 18, and 21. It is the most common cytogenetic subgroup in pediatric ALL, but in spite of this, the mechanisms behind its formation remain elusive. Four different pathways are possible: (1) initial near-haploidy followed by doubling of the chromosomes, (2) prior polyploidization with subsequent losses of chromosomes, (3) sequential gains of chromosomes in consecutive cell divisions, and (4) a simultaneous gain of all additional chromosomes in a single abnormal mitosis. Although these alternatives are difficult to distinguish experimentally, investigations of possible uniparental disomies (UPDs) and of the allelic ratios of loci located on tetrasomic chromosomes may provide some clues. In a previous study of 10 cases of hyperdiploid childhood ALL, we could exclude the first pathway as a general mechanism. Furthermore, the results did not favor the second alternative. Finally, our findings of equal allele dosage for chromosome 21 loci in cases with tetrasomy 21 suggested that the hyperdiploidy arose by a simultaneous gain of chromosomes. However, because all disomies and tetrasomies were not investigated, formation via a polyploid state or by a sequential gain could not be definitely excluded. In the present study, we have addressed this issue further by investigating 15 new cases of hyperdiploid childhood ALL using a total of 57 polymorphic microsatellite markers mapped to 23 of the 24 human chromosomes. Ten of the cases were analyzed with multicolor fluorescence in situ hybridization (M-FISH) and the remaining five with interphase FISH using probes for X, 4, 6, 8, 10, 14, 17, 18, and 21. Markers localized to all disomic and all tetrasomic chromosomes were then applied. One case displayed multiple UPDs and a non-typical pattern with only tetrasomies; this ALL probably arose via a near-haploid pathway. Two other cases had UPDs for 4/9 and 2/14 disomic chromosomes, respectively, and may have originated via a polyploid state. However, in the vast majority of the cases (12/15) there was no evidence for any UPDs, excluding that the hyperdiploidy originated by the near-haploid or polyploid mechanisms. Investigating the tetrasomies, 25 of a total of 27 tetrasomies present among the 15 cases displayed equal allele dosages, indicating a simultaneous gain of chromosomes as opposed to sequential gains. The two tetrasomies with unequal allele dosages were present in the same case, suggesting that this ALL constituted an exception. In conclusion, the results of the present study strongly suggest that hyperdiploidy in childhood ALL generally arises by a simultaneous gain of all additional chromosomes in a single abnormal mitosis.
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

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

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

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

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

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

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

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

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

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.
Notch Activation Converts B Cells into a T Cell Fate at the Earliest Stages of B Cell Committed Progenitors.
Authors: Taneera Jalal , Smith Emma , Sigvardsson Mikael , Hansson Emil , Lindahl Urban , Martensson Inga-Lill , Jacobsen Sten Eirik
Published: October 2019

Notch activation has been suggested to promote T cell development at the expense of B cell commitment at the level of a common lymphoid progenitor prior to B cell commitment. Here, we explored the possibility that Notch activation might be able to switch the fate of already committed B cell progenitors towards T cell development upon Notch activation. To address this we overexpressed constitutively activated Notch-3 (N3IC) in B cell progenitors purified from transgenic mice in which human CD25 is expressed under control of the λ5 promoter. Strikingly, whereas untransduced and control transduced B220+λ5+CD3− B cell progenitors gave rise exclusively to B cells, CD4+ and CD8+ T cells but no B cells were derived from N3IC-transduced cells when transplanted into sublethally irradiated NOD-SCID mice. Gene expression profiling demonstrated that untransduced B220+ λ5+CD3− B cell progenitors expressed λ5 and CD19 but not the T cell specific genes GATA-3, lck and pTα, whereas CD3+ T cells derived from N3IC-transduced B220+λ5+CD3−cells failed to express λ5 and CD19, but were positive for GATA-3, lck and pTα expression as well as a and b T cell rearrangement. Furthermore, DJ rearrangements were detected at very low levels in CD3+ cells isolated from normal non-transduced BM, but were more abundant in the N3IC-transduced CD3+ BM cells. Noteworthy, N3IC-transduced B220+λ5+CD3−CD19+ proB cell progenitors failed to generate B as well as T cells, whereas N3IC-transduced B220+λ5+CD3−CD19− pre-proB cells produced exclusively T cells, even when evaluated at low cell numbers. In conclusion Notch activation can switch committed B cell progenitors from a B cell to a T cell fate, but this plasticity is lost at the Pro-B cell stage, upon upregulation of CD19 expression.
The Clinical Impact of Mild Hemophilia A, Report of a Cohort Study.
Authors: Scully Mary-Frances , MacGregor David , Walsh Meghan , Barrett Brendan , Kawaja Marc , Sheppard Charlotte
Published: October 2019

Due to a founder affect certain regions of the Canadian province of Newfoundland and Labrador have a very high prevalence of mild Hemophilia A. To accurately plan for future health related needs of these patients, a base-line cross-sectional study of a large cohort segregating a known founder mutation (Val2016Ala) was undertaken.

Characteristics of Study Participants Characteristic Affected Males Control Males P-Values (Affected vs Control) Carrier Females Control Females P-Value (Carrier vs Control) Values are expressed as number, mean*, median(interquartile range)†, or percentage Overall N 61(5–76yrs) 44(4–81yrs) 0.883 88(8–88yrs) 65(16–78yrs) 0.935 Factor VIII* 0.15(0.05) 1.29(0.37) <0.001 0.78(0.31) 1.34(0.41) <0.001 Hepatitis Bc Antibodies 15.8%(9/57) 0%(0/28) 0.012 0%(0/75) 0%(0/58) - Hepatitis C Positive 19.7%(12/61) 0%(0/33) 0.001 0%(0/88) 0%(0/65) - HIV Positive 1.6%(1/61) 0%(0/33) 0.420 0%(0/88) 0%(0/65) - Diabetes 24.4%(11/45) 6.1%(2/33) 0.031 - - - BMI†(Adults Only) 30.0(27.0–33.0) 27.0(26.0–30.0) 0.028 29.0(20–57) 28.0(19–46) .446 Statistically Significant Difference SF-36 & Colorado PE-05 Mean Scores for Affected and Unaffected Males Affected Males Affected Males Affected Males Unaffected Siblings Unaffected Siblings Unaffected Siblings t-test Difference vs References Dimension n Mean SD n Mean SD P-Value P<0.05)* Affected Males(age 23–76 years) Unaffected Siblings(age 20–81 years) SF-36 Scores General Health Scale 44 58.1 28.7 32 71.4 21.4 0.024* Role Emotional 44 89.2 19.9 33 98.0 5.5 0.007* Colorado PE-05 Musculoskeletal Scores Axial Deformity 47 0.9 1.4 33 0.2 0.8 0.008* Range of Motion 47 4.2 2.0 33 2.9 1.8 0.005* Gait 47 2.4 3.5 33 0.4 1.4 0.002* Left Ankle 47 3.3 5.0 33 1.0 2.9 0.015* Right Ankle 47 3.2 4.6 33 0.7 1.3 0.002*

There was a positive association between the SF-36 General Health Scale scores and the following values of the Colorado PE-05 Scores. Gait (R -.45, p<.001), Range of Motion (R-.08, P=.508), Axial Deformity (R -.35, P<.001), Right Ankle (R -.42, p<.001), and Left Ankle (R-.49, p<.001). The association between the SF-36 Role Emotional Scales for affected males and the values of the Colorado PE-05 was also positive. Gait (R -.52, P<.001), Range of Motion (R - .30, P = .01), Axial Deformity (R -.54, P<.001), Right Ankle (R -.06, P<.001) and Left Ankle (R-.52, p<.001). No patients studied have acquired an inhibitor to Factor VIII. Obese males (BMI >30) had significantly greater impairment in their Range of Motion than non-obese males (4.59 vs (3.34), p<0.05) independent of whether or not they had hemophilia. These results are consistent with our clinical experience that mild Hemophilia A causes episodic bleeding, lack of recognition and delayed treatment leads to significant morbidity. The association with diabetes was unexpected. Future interventions will now focus on prevention and early treatment of ankle bleeds and will include strategies to reduce the risk of obesity.
A Prospective, Observational Study of the Effectiveness and Safety of a Weekly Fixed Dose of Darbepoetin alfa with Current Practice Iron Management for the Treatment of Chemotherapy-Induced Anemia (CIA): A Subanalysis in Patients (pts) with Lymphoproliferative Malignancies (LPM).
Authors: Alegre A. , Salar A. , Roig I. , Bermejo J.C. , Palacios A. , de Paz R. , Arranz R. , Fernández A. , Calzada R. , Iturralde P. ,   On Behalf of the AMG-DAR-2002-01 Study Group
Published: October 2019

Background: Darbepoetin alfa (Aranesp®) is a unique erythropoiesis-stimulating protein effective for the treatment of CIA when administered weekly (QW), every 2 weeks (wks) or every 3 wks. Fixed dosing of darbepoetin alfa is routinely used in clinical practice, but little data are available on its patterns of use and the impact of iron status on its effectiveness.

Methods: This was a subanalysis of LPM pts included in a prospective, single-arm, multicenter study conducted in Spain. Pts were ≥ 18 yrs old, anemic (hemoglobin [Hb] ≤11 g/dL), scheduled to receive ≥ 12 wks of chemotherapy, and without iron, vitamin B12, or folate deficiencies. Pts were treated with darbepoetin alfa 150 mcg QW; the dose was to be doubled to 300 mcg QW if Hb increased <1 g/dL after 4 wks. Oral iron (200 mg QD) was recommended if transferrin saturation was ≤ 30%, transferrin was ≥ 400 mcg/dL, and/or ferritin was ≤ 300 mcg/L at any time during the study. The primary endpoint was hematopoietic response (Hb increase ≥ 2 g/dL or Hb ≥ 12g/dL in the absence of transfusions in the previous 28 days). Secondary endpoints included the percentage of pts with functional or absolute iron deficiency and its correlation with response rates, change in FACT-Fatigue score from baseline to end of treatment (EOT), and safety.

Results: Of a total of 293 patients enrolled, 129 had LPM and were included in this analysis. Of the LPM patients, 55% were women, median age was 67.3 yrs (range: 20.6–88.4), median weight was 67 kg (range: 40.5–115.0). Eleven percent had Hodgkin’s disease, 33.9% had non-Hodgkin’s lymphoma. 5.5% had chronic lymphocytic leukemia, and 49.6% had multiple myeloma. Most pts (80.5%) had baseline Hb between 9–11 g/dL; 19.5 % had Hb < 9 g/dL. Darbepoetin alfa was administered for a median of 16 wks (range:1–16). At wk 5, 25.6 % of pts had their dose doubled to 300 mcg QW. The observed hematopoietic response was 70.73% (95%CI: 62.69, 78.77). The proportion of pts transfused from wk 5 to EOT was 12.7%. During the study, 31.5% pts received oral iron, 10.3% developed absolute iron deficiency, and 8% had iron blockade. Mean (95% CI) FACT-Fatigue score increased from 34.1 (32.2, 35.9) to 40.0 (38.2, 41.8) (p<0.0001). Darbepoetin alfa was well tolerated with 2.4% of pts experiencing adverse events considered related to the study drug.

Conclusions: darbepoetin alfa, given as a fixed dose, is an effective and well-tolerated treatment for CIA in pts with LPM. Darbepoetin alfa significantly improved health-related quality of life in this routine clinical practice setting.
Erythropoietin Response to Anemia Is Impaired in Patients with Hematologic Malignancies.
Authors: Shen Ti , Shi Yuankai , Zhu Jun , Han Bing
Published: October 2019

Anemia is a common complication in patients with hematologic malignancies, either before or after chemotherapy. To find out whether anemic patients with hematologic malignancies have a lower erythropoietin (Epo) response, we analyzed serum Epo levels in these patients. Serum samples for measurement of Epo levels were obtained from 80 patients from three medical centers. Patients had multiple myeloma (MM, n=26), chronic lymphocytic leukemia (CLL, n=6) or non-Hodgkin’s lymphoma (NHL, n=48). Thirty patients had anemia (mean hemoglobin [Hb] level 8.4±1.6 g/dl) and 50 patients were not classed as anemic, with mean Hb levels of 12.7±1.4 g/dl. To provide control values, serum Epo levels were also determined in patients with iron-deficiency anemia (IDA; n=20) and in healthy individuals (n=20). Serum Epo levels were measured by enzyme-linked immunosorbent assay (ELISA). Patients with severe liver or renal dysfunction, low platelet counts or blood loss were excluded. The complete blood cell count (CBC), reticulocyte count, serum lactate dehydrogenase (LDH), liver and renal function, serum iron (SI), total iron-binding capacity (TIBC), transferrin saturation (TSAT), and serum ferritin (SF) levels were evaluated concomitantly. There were no significant differences in CBC, LDH, liver and renal function, SI, TIBC, TSAT and SF values between malignant patients with or without anemia (P>0.05). In patients with hematologic malignancy, those with anemia had higher Epo levels (mean 97.8±183.9 mIU/ml) than those without anaemia (mean 27.8±85.4 mIU/ml; P<0.001). In patients with IDA, the Epo response was inversely correlated with Hb level (r= −0.5, P<0.05), whereas the expected inverse linear relationship between serum levels of Epo and Hb was absent in the group with hematologic malignancies (r= −0.14). Anemic patients with malignancy, and IDA patients both had elevated Epo levels compared with normal controls (means, 97.8±183.9 mIU/ml, 158.3±308.6 mIU/ml and 9.2±4.0 mIU/ml, respectively; both P<0.001 vs controls). However, after correcting for Hb level, anemic patients with malignancy were found to have significantly lower Epo levels than patients with IDA (P=0.032) indicating a decreased Epo response in the former group. These findings indicate that anemia associated with hematologic malignancy may result from an inappropriately low Epo response. Epo treatment should benefit this group of patients.
The Role of Lactadherin in the Phagocytosis of Phosphatidylserine-Expressing Sickle Red Blood Cell by Macrophages.
Authors: Dasgupta Swapan K. , Thiagarajan Perumal
Published: October 2019

Sickle cell anemia, the most common serious hemoglobinopathy, is associated with a markedly reduced life span of red blood cells due to their preferential clearance by macrophages. During polymerization of sickle hemoglobin, phosphatidylserine, an anionic phospholipid normally present exclusively on the inner leaflet of the membrane bilayer is exteriorized to outer leaflet. This exposure of phosphatidylserine is thought to be a tag for macrophage recognition. Lactadherin, also known as milk fat globule-EGF factor 8, is a phosphatidylserine-binding glycoprotein secreted by macrophages that promotes the engulfment of apoptotic cells. Here, we investigated the role of lactadherin in the phagocytosis of sickle red blood cells.

The binding of fluorescein-lactadherin to normal and sickle red blood cells was studied by flow cytometry. We quantified the effect of lactadherin on phagocytosis of red blood cells by monocyte-derived macrophage.

In normal individuals, less than 0.5% of red blood cells showed any binding to lactadherin when analyzed by flow cytometry. However, in sickle cell patients, circulating red blood cells showed 2 to 10- fold increase in lactadherin binding (P<0.0002). Lactadherin stimulated the phagocytosis of resting sickle red blood cells by macrophages but had no significant effect on the phagocytosis of normal red blood cells. Deoxygenation of sickle red blood cells further increased the lactadherin binding and phagocytosis. Antibodies to integrin αVβ3 also inhibited macrophage binding and phagocytosis.

These results show lactadherin may play a major role in sickle red cell clearance by anchoring the phosphatidylserine-expressing sickle red blood cells to integrins on tissue macrophages.