bluuebunny/crossref_metadata_2025 huggingface dataset:
Low Incidence of Cytogenetically-Defined MDS/AML among Newly Diagnosed Patients Treated According to Total Therapy 1 (TT 1) or Total Therapy 2 (TT 2).
Authors: Thertulien Raymond , Barlogie Bart , Sawyer Jeffrey , Bailey Clyde , Reiner Maureen , Rasmussen Erik , Fassas Athanasios , Zangari Maurizio , Tricot Guido
Published: October 2019

The leukemogenic potential of hematopoietic stem cell-toxic alkylating agents (melphalan, nitrosoureas) and of topoisomerase inhibitors are well established. We have previously reported, among 1,326 patients treated at our institution with melphalan-based tandem autotransplants, that the incidence of cytogenetically-defined MDS (CG-MDS) was directly related to the duration of prior alkylating agent therapy, patient age, and could be anticipated in those with poor stem cell mobilization. Using appropriate FISH probes applied to interphase nuclei, we have also reported that the MDS cytogenetic abnormality (MDS-CA) was usually already present in the cryopreserved PBSC. We have now examined the post-transplant incidence of MDS-CA among 195 patients receiving a first autotransplant as part of TT 1 and among 569 patients as part of TT 2. MDS-CA included del 20q11 in 4, t(1;7) (q10; p10) in 4, del 7q22/-7 in 4, del 13q in 3, del 5q22 in 3 and del 16q22 in 1 patient. 16 total events were observed, 10 on TT 1 and 6 on TT 2, with a low 5-yr estimate of MDS-CA of 1% with TT 2 and 4% with TT 1. Of these 16 patients, 3 have developed overt AML. Subsequent management included anthracycline/ara-C combination chemotherapy, as well as further autotransplant in 2 patients. 8 of the 16 patients have died, 2 due to AML, 3 due to progressive MM, and 3 to other causes. These findings indicate that limited exposure to chemotherapy and the use of non-genotoxic agents as induction treatment for myeloma reduce the risks of the potential development of t-MDS after autologous transplantation. Given the observed survival benefit for patients treated on these 2 protocols, these findings also support the notion of an aggressive approach to myeloma treatment using intensive, hematopoietic stem cell sparing induction and post-transplant consolidation therapy as administered in TT2.
Molecular Transfer of Human CD40 and OX40 Ligands to Leukemic Human B-CLL Cells Extensively Expands Tumor-Reactive Cytotoxic T-Lymphocytes.
Authors: Biagi Ettore , Dotti Giampietro , Yvon Eric , Rousseau Raphael , Lee Edward , Gotschalk Stephen , Brenner Malcolm
Published: October 2019

CD40 ligand is an accessory signal for T-cell activation and can overcome T-cell anergy. The OX40-OX40 ligand pathway is involved in the subsequent expansion of memory T cells. We expressed both human CD40L and OX40L on B-Chronic Lymphocytic Leukemia (B-CLL) cells, by exploiting the phenomenon of molecular transfer from fibroblasts engineered to over-express both of these TNF-receptor superfamily members. We analyzed the effects of the modified B-CLL cells on the number, phenotype and cytotoxic function of autologous T cells in seven B-CLL patients. Transfer of CD40L and OX40L to B-CLL cells was observed in all patients (mean value from 1% pre to 76% post for CD40L; from 0.7% pre to 88% post for OX40L). Subsequent up-regulation of the costimulatory molecules CD80 (B7-1) and CD86 (B7-2) was obtained after engagement of the endogenous CD40 receptor on B-CLL by the transferred CD40L molecules (mean value from 8% pre to 64% post for CD80; from 36% pre to 95% post for CD86). Co-culture of modified and unmodified B-CLL cells with autologous T cells revealed profound differences in the immune responses they induced. With unmodified B-CLL cells, or cells expressing either CD40L or OX40L individually, less than a 10-fold expansion of autologous T cells was observed, with a <100-fold expansion in tumor reactive T cells (measured by IFN-gamma Elispot with autologous B-CLL cells as stimulators, and allogeneic B-CLL cells as controls). By contrast, co-culture with B-CLL cells expressing both CD40L and OX40L induced a >40 fold expansion of autologous T cells - including both CD8+ T cells and CD4+ T cells with a Th1 pattern of cytokine release - and a >2500-fold increase in leukemia-reactive T cells. These expanded T cells were also directly cytotoxic to B-CLL targets, producing a mean 48% B-CLL killing at an E:T ratio of 10:1. A proportion of these tumor-reactive CD8+ T cells were specific for survivin, a B-CLL associated tumor antigen. Hence the combination of CD40L and OX40L expression by B-CLL cells allows generation of potent immune responses to B-CLL, which may be exploitable either by using active immunization with CD40L/OX40L-modified tumor cells or by adoptive immunotherapy with CD40L/OX40L generated tumor-reactive T cells.
CD38 Ligation in B-Chronic Lymphocytic Leukemia Cells Induces Sequential Tyrosine Phosphorylation of ZAP70, PLC- γ2 and ERK1/2 Proteins.
Authors: Deaglio Silvia , Vaisitti Tiziana , Malavasi Fabio
Published: October 2019

CD38 is a negative prognostic marker in B-chronic lymphocytic leukemia (B-CLL). In a previous study we observed that CD38 and IL-2 cooperate in inducing proliferation and survival of B-CLL cells. Now attention is given to the signaling pathway ruled by CD38 in B-CLL cells. We show that CD38 ligation triggers a prominent tyrosine phosphorylation of the ZAP70 kinase in 6 out of 9 patients studied (2 representative shown in figure 1). ZAP70 activation is evident 1 minute after receptor cross-linking and tends to diminish within 5 minutes. Tyrosine phosphorylation of syk is undetectable in these conditions. A functional relationship between CD38 and ZAP70 was confirmed by the analysis of a panel of B cell lines. Indeed, CD38 ligation in Nalm-6, a pre-B cell line identified as ZAP70+, wais followed by ZAP70 tyrosine phosphorylation, with a kinetics overlapping the one observed with the B-CLL samples.

Downstream events include the sequential activation of BTK and PLC-γ2, the latter likely responsible for the cytoplasmic Ca2+ mobilization, observed both in B-CLL and in the Nalm-6 cells. The signaling events culminate with the activation of ERK1/2, but not of SAP/JNK and p38 proteins. The results obtained with purified B-CLL cells from 2 representative patients are shown in figure 1.

Together, these data i) highlight for the first time a functional relationship between CD38 and ZAP70 in B cells and ii) identify crucial cytoplasmic steps in the signaling cascade promoted by CD38, thus providing a mechanistic base to explain why B-CLL patients with a CD38+/ZAP70+ clone are characterized by the most unfavourable clinical course. Selected bibliography

Figure. Figure.
Nurselike Cells in Chronic Lymphocytic Leukemia (CLL) Express Both BAFF and APRIL that Protect CLL B Cells from Spontaneous Apoptosis by Paracrine Manner.
Authors: Nishio Mitsufumi , Tsukada Nobuhiro , Ohata Junko , Zvaifler Nathan J. , Kipps Thomas J.
Published: October 2019

Large, adherent nurse-like cells (NLC) can differentiate from the blood mononuclear cells of patients with chronic lymphocytic leukemia (CLL) that, in turn, can support leukemia cell survival in vitro, and presumably in vivo. These cells differentiate in the context of CLL B cells and can be found in the secondary lymphoid tissues of patients with this disease. NLC express high levels of B cell activating factor belonging to the tumor necrosis factor (TNF) family (BAFF), a factor that can support normal and leukemia B cell survival in vitro. BAFF binds to three distinct members of TNF-receptor superfamily : TACI (transmembrane activator and CAML interactor), BCMA (B cell maturation antigen) and BAFF-R, the latter two of which are expressed by CLL B cells. We found that addition of saturating amounts of a recombinant BAFF-receptor-Fc fusion protein (BAFF-R:Fc) to cultures of NLC and CLL B cells failed to inhibit the protective effect(s) of NLC on CLL B cell survival in vitro. This suggested that another factor(s) made by NLC can contribute to the survival effect(s) of BAFF on CLL B cells. A candidate for this is a proliferation-inducing ligand (APRIL), another member of the TNF family that can interact with TACI and BCMA, but not BAFF-R. We found that, in contrast to CLL B cells or blood mononuclear cells, NLC express high-levels of APRIL mRNA and protein, as assessed by quantitative RT-PCR and immunoblot analyses. Furthermore, recombinant human APRIL also could support CLL B cell survival via mechanism similar to that used by recombinant BAFF. Consistent with the fact that APRIL and BAFF each can function to support CLL B cell survival, we found that a recombinant BCMA-Fc fusion protein could significantly inhibit the protective effects of NLC on CLL B cells in vitro. These data suggest that BCMA-Fc might be better suited than BAFF-R:Fc to inhibit the interactions between NLC and CLL B cells in vivo.
Tcl-1 Inhibits Nuclear Export of TR3 and Is Highly Expressed in ZAP70-Positive CLL B-Cells.
Authors: Pedersen Irene M. , Luciano Frederic , Zhang Xiao-kun , Croce Carlo M. , Kipps Thomas J. , Carson Dennis A. , Reed John C.
Published: October 2019

Chronic Lymphocytic Leukemia (CLL) is the most frequent leukemia afflicting the Western world. Unlike other more aggressive leukemias and lymphomas, CLL B-cells do not divide at a high rate and expansion of the malignant clone appears to be due to underlying defect in apoptosis.

Elevated levels of anti-apoptotic Bcl-2 are thought to be involved in the pathogenesis of CLL. The activity of Bcl-2 can be modulated my interactions with other cellular proteins. Recently, orphan nuclear receptor TR3 (nur77) was shown to exit the nucleus, bind Bcl-2, and convert Bcl-2 into a killer protein (Lin, et al., CELL, 116:527, 2004). We evaluated the expression of TR3 by immunoblotting in CLL B-cells specimens. High levels of TR3 protein were present in 8 of 8 CLL patient specimens. Subcellular fractionation studies showed predominately nuclear localization of TR3 in 2 of 2 CLL specimens examined. Because Akt is known to regulate TR3 activity in the nucleus (Pekarsky, et al., Proc. Natl. Acad. Sci.98:3690, 2001), we also examined the expression of the Akt regulator, Tcl-1. The Tcl-1 protein was expressed in the majority of CLL samples and at high levels in 8 of 16 CLL specimens, correlating with ZAP70-positivity. In gene transfection experiments using tumor cell lines, over-expression of Tcl-1 inhibited nuclear export of TR3, as determined by immunofluorescence confocal microscopy. We propose a model in which expression of the oncogene Tcl-1 in CLL B-cells (perhaps related to ZAP70 expression) modulates nuclear Akt activity, in turn suppressing TR3 function, and thus reducing activity of the TR3-mediated pathway for conversion of Bcl-2 from protector to killer. This hypothesis requires further experimental testing.
Gene Expression in CLL Cells Associated with Lymphadenopathy and Chromosome 11q23 Deletion.
Authors: Dickinson John D. , Joshi Avadhut , Gilmore Jamie , Bierman Philip J. , Warren Sanger , Joshi Shantaram S.
Published: October 2019

Previously we have demonstrated that peripheral blood samples with B-cell Chronic Lymphocytic Leukemia (CLL) have different gene expression profiles associated with chromosome aberrations detected by fluorescence in situ hybridization (FISH). In particular, the vast majority of differentially expressed genes were related to the presence of the 11q23 deletion. The 11q23 deletion has previously been shown to be correlated with shortened over-all survival and extensive/bulky lymphadenopathy. In this study we sought to identify genes whose expression may play role in the progression of CLL in patients that carry the 11q23 deletion. Gene expression from 10,700 human gene specific 50-mer oligos (MWG Biotech, Ebersberg, Germany) was compared between two groups of peripheral blood CLL samples. The first group consisted of CLL patients with the 11q23 deletion detected by FISH as well as with the presence of abdominal/mediastinal lymphadenopathy. The second group consisted of CLL patients without the 11q23 deletion and without the presence of known abdominal/mediastinal lymphadenopathy. The non-11q deletion group included CLL patients with the 13q14 deletion, trisomy 12, 17p13 deletion, as well as several without any detectable abnormality. Immunoglobulin heavy chain variable region (IgVH) mutational status was compared in CLL samples in both groups to ensure that resulting expression differences were not the result of this known prognostic marker. Median of ratios was compared between the two groups. Eighty-eight (87) genes had a p-value < 0.01. Gene function was classified at the European Molecular Biology Laboratory (EMBL) Bioinformatic Harvester website. Twenty of these differentially expressed genes were cell cycle/cell signaling related genes that were over-expressed in the 11q23 deletion group. Examples include: activating transcription factor 4, rho-associated coiled-coil containing protein kinase 2, fibroblast growth factor 21 precursor, signal transducing adaptor molecule 1, mad2-like 1, interferon receptor 1, pim-2 oncogene, and zw10 interactor. In comparison, using the same peripheral blood CLL samples, 78 genes were differentially expressed (p < 0.01) between those samples that had a mutated IgVH versus those that had an unmutated IgVH. Therefore, the presence of both the 11q23 deletion and bulky abdominal/mediastinal lymphadenopathy significantly alters the gene expression profile of peripheral blood CLL cells, particularly genes related to the cell cycle and cell signaling related processes. Biological roles of some of these genes may help further elucidate the basis of the clinical behavior of CLL patients
CD6-Depleted Mobilized Stem Cells for Modification of HVG and GVH Reactions after HLA-Haploidentical Marrow Transplantation.
Authors: Kolb Hans-Jochem , Bigalke Iris , Simoes Belinda , Falk Christine , Tischer Johanna , Hill Wolfgang , Ledderose Georg
Published: October 2019

Allogeneic stem cell transplantation is limited to patients with a histocompatible donor, but for patients with advanced acute leukemia and high-grade lymphoma HLA-haploidentical transplantation may be considered. Rejection of the transplant and graft-versus-host disease are major obstacles and T-cell depletion eliminates the graft-versus-leukemia effect and causes prolonged immune deficiency. Here we studied the use of CD6-depleted G-CSF mobilized blood cells (mbc) 6 days after transplantation of unmodified marrow in order to suppress host-versus-graft and graft-versus-host reactions (GVHD) retaining a graft-versus-leukemia effect and the capacity to reconstitute the immune system. CD6-depleted mbc contain a large proportion of NK and NK-T cells. 63 patients with advanced disease (AML 32, ALL 15, NHL 11, CLL 2, CML 2, SAA 1) were transplanted with marrow from family donors sharing one HLA-haplotype and differing in 0 – 4 HLA-antigens of the second haplotype. Conditioning consisted of total body irradiation (TBI), antithymocyte globulin (ATG) and cyclophosphamide (CY), post-grafting immunosuppression of cyclosporin A (CSA) and a short course of methotrexate (sMTX). A transfusion of donor leukocytes was given prior to CY. Complete engraftment was observed in 34 evaluable patients given 12 Gy TBI. The dose of TBI could be reduced to 4 Gy without rejection in 25 evaluable patients. GVHD was severe (grade III and IV) in 12 of 48 evaluable patients. An improved method of CD6-depletion was administered to mbc in 9 patients and severe GVHD did not develop. GVHD responded to corticosteroids in most patients. 15 patients survive disease free up to 6 years (median 784 days). Recurrent infections including PTLD were the major cause of transplant-related mortality. Absolute counts of lymphocytes, CD4 and CD8 subpopulations were not different from those of a contemporary group of 46 patients in advanced disease given HLA-identical sibling transplants. However naïve CD4 cells and TRECs were low. Rejection, GVHD and GVL have been controlled by this regimen, but immune reconstitution remains a problem that may be solved by early discontinuation of immunosuppression in this regimen.
Hemostatic Alterations in Patients Undergoing Hematopoietic Stem Cell Transplantation.
Authors: Buchholz Stefanie , Woywodt Alexander , Scheer Johanna , Hambach Lothar , Kamal Haytham , Haller Hermann , Haubitz Marion , Ganser Arnold , Hertenstein Bernd , von Depka Mario
Published: October 2019

Damage of endothelial cells (EC) is known to be involved in pathogenesis of microangiopathy, hepatic veno-occlusive disease, sepsis, capillary leak syndrome, and acute or chronic graft-versus-host disease (GvHD) as major causes of morbidity and mortality in patients after hematopoietic stem-cell transplantation (HSCT). We recently demonstrated in 39 patients undergoing allogeneic stem cell transplantation that numbers of circulating EC (CEC) increased significantly after conditioning regimens and that patients treated with reduced intensity conditioning (RIC) showed significantly lower cell numbers (Woywodt et al., Blood, 2004 May 1;103(9):3603-5).

Here we report on the measurement of plasma levels of von Willebrand factor (vWF), thrombomodulin (TM), PAI-I and TAFI in the course of HSCT.

Measurement of vWF, TM, PAI-I and TAFI was performed in the 39 patients (20 male, 19 female; n = 21: HLA-matched unrelated donor; n = 18: related donors). Blood samples were collected before starting conditioning regimen (day −7), the day before transplantation (day −1) and at day +7, +14 and +21 after transplantation. 28 patients received a conventional regimen with cyclophosphamide (120 mg/kg) and either total body irradiation with 12 Gy (n = 14) or busulfan (16 mg/kg, n = 14). 11 patients undergoing stem-cell transplantation were treated with reduced intensity conditioning with fludarabine (150 mg/m²) and busulfan (8 mg/kg body) or melphalan (120 mg/m²).

Median baseline vWF was elevated (262%, range 68–612, normal range: 50–150). Median baseline PAI-I (11.3 U/l; range 1.8–34.4; normal range: < 20) and median baseline TAFI (90%; range 46–126; normal range: 70–120) were within normal limits. TM level was lower than normal values (median 3.95 ng/ml; range 2.69–9.36; normal range: 6.6–10.6). Levels of vWF increased after conditioning regimen and remained elevated until day +21 (day −1: median 262; day+7: 268; day +14: 327; day +21: 374; p < 0.01). Median TM remained low at all time points (day −1: median 4.26; day +7: 3.86; day +14: 3.97; day +21: 4.52).

Levels of TAFI remained unchanged (day −1: median 82; day +7: median 91, day +14: median 88; day +21: median 89). There were also no differences in levels of PAI-I before or after conditioning regimen or after transplantation (day −1: median 11.4, day +7: median 10.8, day + 14: median 14, day + 21: median 11.8). There was no significant difference in vWF in patients undergoing reduced intensity conditioning compared to patients treated with conventional regimens.

Interestingly, there was no correlation between the endothelial markers as vWF and TM and numbers of CEC.

Our data indicate that significant alterations of the hemostatic system occur in patients undergoing HSCT. Further studies are warranted to define the clinical role of both, hemostatic alterations and CEC in the course of HSCT.
Molecular Targets of Glucocorticoids in Context of Apoptosis Induction in Childhood Precursor T-Cell ALL (T-ALL).
Authors: Karawajew Leonid , Rhein Peter , Kostka Dennis , Hoffmann Rosemarie , Schrappe Martin , Spang Rainer , Ludwig Wolf-Dieter
Published: October 2019

Glucocorticoids (GCs) are principal therapeutic agents in ALL. In spite of their prominent clinical significance, molecular targets of GCs and, consequently, the mechanisms of GC-resistance are still unclear. In the present study, we focussed on molecular mechanisms of GC cytotoxicity in T-ALL, and investigated GC-specific response in leukemic cells at transcriptional and cellular levels. To this end, T-ALL samples (n=14) were investigated for GC-specific gene expression changes before onset of apoptosis (10 μM dexamethasone, 6 hr of incubation). Gene expression data obtained using Affymetrix HG Focus microarrays were normalized by variance stabilization on probe level, and genes consistently changed in GC-treated vs -untreated cells were identified using a SAM-like analysis of log ratios in an unbalanced permutation test. By these procedures, a group of 195 genes (false discovery rate = 0.005) was identified. Furthermore, the T-ALL samples were investigated for GC-specific apoptosis (10 μM dexamethasone, 20 hr). Apoptotic response to dexamethasone was highly heterogeneous with an apoptosis rate varying from 0% to 56% and a mean value of 23% (assessed by annexin V/propidium iodide staining). To identify genes with the highest impact on GC-specific apoptosis, we investigated the correlation between GC-specific gene expression changes and apoptosis rate in T-ALL samples. Expression changes of 25 genes were found to correlate significantly with the rate of apoptosis (Spearman regression analysis; correlation coefficients rs=0.54–0.81, p<0.05). First, we found that the GC-specific apoptosis was associated with a downregulation of genes encoding for cell cycle-promoting proteins (PTTG1, CDC7) and an upregulation of antiproliferative factors (ID2, BTG1), thus indicating that antiproliferative and cytotoxic activities of GCs are coupled in T-ALL. Furthermore, the rate of apoptosis correlated with a suppression of the commonly known survival signaling pathways, as indicated by the upregulation of the NFkappaB inhibitor IkappaB-alpha and downregulation of c-myc. In addition, we observed a downregulation of Bcl11A, a survival factor in the T-cell development. Finally, GC-specific apoptosis correlated with an upregulation of genes encoding for an actin depolymerizing factor DSTN and catabolic enzymes ISG20 (exonuclease) and CLPP (protease). Taken together, investigation of gene expression changes in the context of apoptotic cellular response identified common and T-cell specific survival pathways as GC-targets in T-ALL. In addition, our data suggest a novel GC-specific mechanism of cytotoxicity via direct transcriptional regulation of genes encoding for cellular catabolic proteins.
Obesity and Body Weight Independently Predict Relapse and Survival in Preadolescents and Teenagers with Acute Lymphoblastic Leukemia (ALL). A Retrospective Analysis of Five Children Cancer Group (CCG) Studies.
Authors: Butturini Anna , Dorey Fred , Gaynon Paul , Fu Cecilia , Franklin Janet , Siegel Stuart , Sacks Nancy , Siebel Nita , Henry David , Rogers Paul , Lange Beverly , Sather Harland , Trigg Michael , Bleyer Archie
Published: October 2019

We analyzed data from 4356 patients with ALL diagnosed from 1988 to 1995 at age 2–20 years and enrolled in CCG studies 1881, 1891, 1922, 1882 or 1901. Patients with Down Syndrome and those with CNS disease at diagnosis were excluded. Obesity was defined as a body mass index (BMI)>95th percentile by the Center for Disease Control growth charts (www.cdc.gov/growthcharts). Obesity at diagnosis did not affect outcome in patients younger than 10 years but independently predicted 5y risk of leukemia relapse and event free survival (EFS) in the 1026 patients aged 10 years or older. Multivariate analysis in patients aged 10 years or older showed that obese patients had increased risk of leukemia relapse (hazard ratio-HR- 1.5, p=0.013) and of all the events (HR 1.5, p=0.009). Other independent predictors of poor outcome were high WBC at diagnosis and failure to achieve bone marrow M1 status by day 7 of therapy. Age had borderline significance; sex, race and type of therapy were not significant. To assess whether the effect of obesity on outcome was due to absolute body weight, we studied the effect of weight in Kg at diagnosis in patients aged 10 years or older. Patients >60kg had increased risk of leukemia relapse (HR 1.4, p=0.002) and of all the events (HR 1.4, p=0.006). High WBC at diagnosis and bone marrow status at day 7 maintained their significance; age, sex, race and therapy were not significant. The effects of obesity and weight on outcome only partially overlapped: obesity still predicted outcome when only patients <60 kg were analyzed as well as weight >60 kg predicted outcome when obese patients were excluded from the analysis. Obesity and weight >60 kg at diagnosis were not associated with differences in early toxic deaths, lenght of initial phases of therapy or days of hospitalization. In conclusion, preadolescents and teenagers with ALL who were either obese or >60 kg of weight at diagnosis had higher risk of leukemia relapse and poorer EFS.
von Willebrand Factor Protects Ca2+-Dependent Structure of the Factor VIII Light Chain.
Authors: Takeyama Masahiro , Shima Midori , Nogami Keiji , Okuda Masahiro , Sakurai Yoshihiko , Tatsumi Kohei , Takeda Tomohiro , Tanaka Ichiro , Yoshioka Akira
Published: October 2019

Factor VIII (FVIII) cofactor function is dependent on the presence of divalent cation, since its active form is metal-linked heterotrimer as well as factor V (FV). The activity of FVIII (FVIII:C) and FV in normal plasma were completely and selectively inactivated by the treatment with divalent cation exchange resin, imidinoacetate. However, FVIII antigen (FVIII:Ag) was preserved, suggesting that deprivation of FVIII:C by the resin was not due to absorption of FVIII but due to inactivation. Both FVIII:Ag and FVIII:C of recombinant FVIII were decreased by treatment with the resin. However, FVIII:Ag but not FVIII:C, was preserved in FVIII and von Willebrand factor complex (FVIII/VWF). Sandwich ELISA using anti-A3 and anti-A2 monoclonal antibodies revealed that association with heavy and the light chains were impaired in the resin-treated FVIII. These results suggested that FVIII was inactivated by deprivation of the metal ion by the resin and that VWF protected cation-dependent FVIII structure. Therefore, we tested if the resin deprives the metal ion such as Ca2+ from FVIII or FVIII/VWF. [Ca2+] in FVIII preparation was decreased from 1.30 to 0.07 mM by the resin. Furthermore, [Ca2+] was decreased from 0.39 to 0.01 mM in FVIII/VWF preparation. [Ca2+] was recovered completely by elution with 1 N HCl from the resins used in both preparations. FVIII:C of the resin-treated FVIII/VWF was partially recovered by addition of Ca2+, whilst FVIII:C of the resin-treated FVIII was not recovered. When FVIII was treated with the resin after addition of [Ca2+], the inactivation of FVIII was dose-dependently inhibited by ~20 % (at [Ca2+]: ~75 mM). On the other hand, the inactivation of FVIII was inhibited by ~60 % (at [Ca2+]: ~25 mM) in FVIII/VWF preparation. Present results demonstrated that FVIII was selectively inactivated by the cation exchange resin due to deprivation of Ca2+. Kinetic experiments by surface plasmon resonance using BIAcore demonstrated that the resin-treated FVIII as well as treated C2 didn’t interact with phospholipid and VWF. Furthermore, immunoblot analysis using the resin-treated FVIII revealed that anti-A2 monoclonal antibody reacted with the heavy chain, whilst anti-A3 and anti-C2 antibodies failed to react with the light chain. On the other hand, these antibodies reacted with the light chain in the experiment using FVIII/VWF, indicating that VWF protects antigenic conformation of the FVIII light chain. Present findings suggest another protection mechanism of VWF on FVIII through stabilization of Ca2+-dependent structure of the FVIII light chain.
C6PS Regulates the Inactivation of Factor Va by Activated Protein C.
Authors: Majumder Rinku , Wang JinMing , Lentz Barry R.
Published: October 2019

Protein C is a vitamin K-dependent, anti-thrombotic protein that is proteolytically cleaved by thrombin to produce the active serine protease, activated protein C (APC). APC inactivates co-factors Va and VIIIa, leading to down-regulation of thrombin generation. Factor Va requires phosphatidylserine (PS) for full cofactor activity. APC inactivates bovine factor Va by catalyzing cleavage in its heavy-chain at Arg505, Arg 662 and Arg306. The cleavage at Arg 306 is stimulated by PS-containing membranes. In this paper, we use water-soluble 2-dicaproyl-sn-glycero-3-phospho-L-serine (C6PS) to ask whether a membrane or molecular PS regulates inactivation of factor Va by APC. Synthetic substrate titration suggested that APC has two Ca2+-requiring binding sites for C6PS: one site increased the APC amidolytic activity (Kdeff ~ 1.3 μM) while the other site decreased it (Kdeff ~ 2 μM) in the presence of 2 mM Ca2+. The effect of C6PS on APC amidolytic activity was PS-specific, with C6PE having no effect. However, titration of both intrinsic fluorescence and DEGR ([5-(dimethylamino)-l-naphthalenesulfonyl] glutamylycylarginyl chloromethyl )-labeled APC fluorescence showed only one Ca2+-requiring C6PS binding site (Kdeff ~ 0.8 μM). The fluorescence anisotropy of DEGR-APC in the presence of 200 μM C6PS revealed C6PS-dependent 1:1 binding to both factor Va isoforms (Va1or Va2) with Kdeff of 1.13x10-9 M (Va1) and 0.3x10-9 M (Va2). The inactivation of factors Va1/Va2 by APC was also promoted by C6PS. In the presence of 200 μM C6PS to saturate sites on both APC and factor Va, both factors Va1/Va2 were fully inactivated by APC (indicating cleavage at Arg 306). However, in the absence of C6PS or presence of only 4 μM C6PS (sufficient to saturate sites only on APC), only partial inactivation (48–52%) of factor Va1/Va2 was observed. These results suggest that PS binding to APC may have some effect on cleavage at Arg505 or Arg 662 but that PS binding to factor Va was needed to promote cleavage at Arg 306. Supported by USPHS grant HL072827 to BRL.
CD34+ Selection before Autologous Peripheral Blood Stem Cell Transplantation: Comparison of the Nexell Isolex 300i® and Miltenyi Biotec CliniMACS® Systems.
Authors: Mantha Simon , Savage David G. , Nichols Gwen L. , Skerrett Donna , Hesdorffer Charles S.
Published: October 2019

Autologous stem cell transplant has been used with variable success rates in the treatment of different malignancies. One of the potential causes of relapse is contamination of the stem cell collection by neoplastic cells. Purging the stem cells by CD34+ selection has been used to reduce such contamination. In this study two different devices were compared in their efficiency to achieve CD34+ selection of peripheral blood stem cells collected in individuals undergoing autologous transplant for breast cancer, neuroblastoma, plasma cell dyscrasia, lymphoma (both Hodgkin and non Hodgkin), rhabdomyosarcoma and amyloidosis. A total of 28 patients were randomized to CD34 selection on the Nexell Isolex 300i® system (NIS) or the Miltenyi Biotec CliniMACS® (MCMS) system. The prevalence of plasma cell dyscrasia was higher in the MCMS group (57%) than in the NIS group (22%). On the other hand, there were 2 cases of Hodgkin lymphoma and 2 cases of breast cancer in the NIS group, in comparison to none in the MCMS group. Average values for parameters of selection efficacy for the 19 patients transplanted are shown in table 1 (recovery = number of cells post / number of cells pre-selection X 100, viability measured with the trypan blue exclusion test). Transplant was done as per local protocols according to the type of malignancy. CD34+ cell dose was determined by institutional guidelines. Average values for engraftment are shown in table 2 (two patients who died before engraftment in the MCMS group were excluded from calculation of the mean values). One patient died of graft failure in the MCMS group in contrast to none in the NIS group. Although the numbers of patients are relatively low, this is the first randomized study in which the selection efficiency of the NIS and MCMS devices have been compared in patients receiving CD34-selected autografts. CFU-GM recovery and cell viability were lower, while neutrophil engraftment was slower in the MCMS group, but these differences were not statistically significant. Our data do not show any clinically significant advantage for either the NIS or MCMS selection device.

Table 1 Device CD34+ recovery (%,range) CFU-GEMM recovery (%,range) CFU-GM recovery (%,range) Viability pre (%,range) Viability post (%,range) P-value calculated with Student’s t-Test. NIS 52 (16–76) 5 (1–14) 9 (0–42) 95 (91–99) 95 (90–99) MCMS 66 (48–75) 4 (1–5) 5 (0–9) 94 (80–99) 88 (60–100) P 0.17 0.71 0.57 0.69 0.16 Table 2 Device CD34+ infused (X 106/kg, range) Time to ANC ≥500 (days, range) Time to plt ≥20 (days, range) # Units plt P-value calculated with Student’s t-Test. NIS 3.6 (2.0–5.2) 12.2 (9–20) 15.9 (11–38) 19.4 (0–30) MCMS 3.6 (1.5–5.1) 15.1 (11- ∞) 16.8 (9-∞) 40.2 (6–150) P - 0.22 0.79 0.23
The Critical Role of Survivin in the Survival and Proliferation of Human Myeloma Cells.
Authors: Romagnoli Mathilde , Bataille Regis , Barillé-Nion Sophie
Published: October 2019

Multiple myeloma (MM) is characterized by the accumulation of malignant plasma cells with an enhanced proliferation and survival capacity within the bone marrow. Myeloma cells often develop drug resistance leading to treatment failure in the patients. Survivin is a member of the inhibitors of apoptosis (IAP) gene family that has been implicated in both cell viability and cell cycle regulation and described as overexpressed in most cancers. We have observed that survivin is expressed in human myeloma cell lines (HMCL) from moderate level in both HMCL XG-6 and MM1S to strong level in U266. Survivin was also detectable in primary myeloma cells. Moreover, survivin expression peaked at G2/M phase and was induced by IL-6 and IGF-1 through JAK/STAT and PI3K/AKT signalling pathways. In order to elucidate the role of survivin in myeloma cells, MM1S and XG-6 HMCL were transfected with human survivin cDNA. First, The survivin transient transfectants MM1S were significantly less sensitive to different drugs (dexamethasone, melphalan, paclitaxel, vincristine, doxorubicin) than the control vector transfectants MM1S. Secondly survivin stable transfected clones established from the HMCL XG-6 that is dependent on IL-6 for its growth, could be maintained in culture without IL-6 longer than control stable clones. Moreover these survivin clones proliferated without exogenous IL-6 in contrast to control clones that kept their IL-6 dependent growth. Furthermore, in vivo preliminary data suggest that survivin overexpressing clones could develop tumor in the SCID-human MM model in contrast to control clones. These data tend to prove that survivin participates in drug sensitivity, escape from IL-6 dependence and tumor formation capacity of HMCL. In summary, our findings suggest that survivin plays an important role in the pathogenesis of MM. A more defined understanding of survivin biology should enhance the rational development of drugs to inhibit its function in myeloma cells.
Risk Factors for Second Malignancies after Transplantation Differ between Allogeneic and Autologous Transplantation.
Authors: Friedman Debra L. , Leisenring Wendy , Flowers Mary E. , Holmberg Leona , Schwartz Jeffrey , Deeg H. Joachim
Published: October 2019

Survivors of hematopoietic stem cell transplantation (HSCT) are at risk for second malignant neoplasms (SMN). We undertook this analysis to ascertain whether risk of SMN was comparable in survivors of allogeneic and autologous transplantation. Among 8662 transplant recipients treated at the Fred Hutchinson Cancer Research Center between November 1969 and April 2004, who survived at least 100 days post transplant, there were 1743 autologous and 6919 allogeneic HSCT recipients. Within this cohort, there were 56 SMNs among the autologous and 224 among the allogeneic recipients. Cumulative incidence of SMN at 10 years post-HSCT was 2.6% in the allogeneic and 4.2% in the autologous HSCT survivors. A multivariate Cox regression model adjusted for current age, TBI, gender and length of follow-up was fit and the hazard ratio (HR) for SMN for the allogeneic transplant survivors was 0.7 (95% confidence interval {CI} 0.5, 1.0) compared to the autologous transplant survivors (reference group), suggesting that the adjusted hazard of SMN is higher for autologous than for allogeneic HSCT recipients. Risk factors differed between the two groups. For survivors of autologous transplantation, in multivariate Cox regression models, only age > 18 years at transplant was associated with decreased risk of SMN (<18 years HR = 1.0; 18–39 years HR = 0.04; 40+ years HR = 0.004). Use of total body irradiation (TBI) was not significantly associated with risk among the autologous HSCT recipients. For allogeneic transplant survivors, increased risk of SMN was associated with TBI and the effect of TBI was stronger for younger (<18 years at HSCT: HR = 4.6; 95% CI 1.6, 13.5) than for older (≥18 years; HR = 1.5; 95% CI 1.0, 2.3) HSCT recipients (interaction p = 0.04) in multivariate Cox regression models. Risk was also increased after acute graft versus host disease (HR = 1.4; 95% CI 1.0, 1.9) and with ongoing follow-up time, with HRs of 1.7 (95% CI 1.1, 2.5) at 10 – 14 years, 2.2 (95% CI 1.3, 3.7) at 15–19 years and 2.6 (95% CI 1.2, 5.4) at 20+ years of follow-up. Unrelated HSCT also increased risk of SMN (HR = 1.4; 95% CI 1.0, 2.0). In adjusted analyses, risk of SMN thus appears slightly lower in allogeneic versus autologous transplant survivors, and risk factors appear different between the two groups. Attention should focus on modifiable risk factors in both groups. Particularly in autologous transplant survivors, the relative contribution of pre-transplant exposures must be established.
HLA Identical Hematopoietic Stem Cell Transplantation for Acute Leukemia in Children - A Single Center Experience.
Authors: Willemze Annemieke J. , Geskus Ronald B. , Noordijk Evert M. , Vossen Jaak M.
Published: October 2019

Between 1980 and 2002, 132 children and adolescents (median age 8.5 years, range 1.1 to 17.3) got an allogeneic HLA-identical hematopoietic stem cell transplantation (SCT) from a sibling donor for acute leukemia. The patients suffered from ALL in 1st remission (n=24), ALL in 2nd remission (n=53) and AML in 1st remission (n=55). The mean follow-up time after SCT was 11.5 years (range 1.3 to 23.3 years). The source of the stem cells was bone marrow in all but 3 cases of cord blood-derived stem cells. The number of nucleated marrow cells in the graft varied between 0.5 and 6.8x108/kg (median 2.75x108/kg). Most patients, 118 of 132, were pre-treated with a total body irradiation (TBI) and cyclophosphamide-based regimen. TBI doses were 1x 7 Gy for children of 3 and 4 years, 1x 7.5 Gy for >4 and <10 years, and 1x 8 Gy (before 1990) and 2x 6 Gy on two consecutive days (after 1990) for children older than 10 years. Initially, graft-versus-host disease (GvHD) prophylaxis consisted of long-course methotrexate (MTX) only (n=24), later a short-course MTX-Cyclosporine A prophylaxis was given (n=102). All patients were nursed in strict protective isolation, using positive-pressure laminar-air flow cubicles or rooms, and got total gut decontamination with non-absorbable antimicrobials. Engraftment, transplant-related complications, relapse of the disease and survival were evaluated retrospectively in relation to disease- and transplant-related characteristics. None of the patients failed to engraft. The mean 5-year probability of overall survival was 63%, 53% and 74% for ALL1, ALL2 and AML1 patients, respectively. Transplant-related mortality was 6%. The incidence of acute GvHD in evaluable cases was 17% (21/124), of whom 7 (6%) got grade II to IV. Chronic GvHD occurred in 10 patients out of 125 evaluable patients (8%). The mean 5-year probability of relapse was 35%, 49% and 26% for ALL1, ALL2, and AML1 patients, respectively. Most relapses (70%) occurred within the first year post-SCT. The patient age and dose of TBI were significantly related with post-transplant relapse (p=0.007). Overall survival was significantly higher in children that received a higher dose of TBI (see figure). Overall, acute GvHD was not significantly associated with relapse, but none of the AML patients (n=9) with acute GvHD got a relapse (likelihood ratio test, p=0.02), which was not the case for ALL first or second remission patients. This retrospective single-center study on HLA-identical SCT for acute leukemia in 132 consecutively enrolled children confirms the beneficial role of slight to moderate acute GvHD in the suppression of relapse of AML, and draws attention to the potential antileukemia effect of a high (fractionated) dose of TBI in the conditioning.

Figure Figure
Transcriptional Regulation Via GATA Switches: Proteasome-Mediated Exchange of Chromatin-Bound GATA Factors.
Authors: Lurie Louis J. , Boyer Meghan E. , Bresnick Emery H.
Published: October 2019

The transcription factors GATA-1 and GATA-2 confer both shared and unique activities to regulate hematopoiesis. GATA-2 is essential for the survival and proliferation of hematopoietic stem cells and multipotent hematopoietic precursors, whereas GATA-1 is critical for erythropoiesis. GATA-1 and GATA-2 are expressed reciprocally during erythropoiesis, with GATA-2 expression preceding that of GATA-1. Erythroid maturation appears to require GATA-2 repression. We have shown that chromatin-bound GATA-2 is displaced by GATA-1, which instigates a distinct transcriptional output at the respective locus. We hypothesize that such “GATA switches” are an important transcriptional mechanism that drives erythropoiesis. Their distinct transcriptional outputs suggest that GATA-1 and GATA-2 have unique biochemical properties. However, biochemical analyses have primarily identified similarities between the factors. For instance, the two zinc fingers of GATA-1 are highly homologous to those of GATA-2, and therefore both factors interact with the cofactor FOG-1 and bind an identical WGATAR sequence with high affinity. We found that GATA-2 is considerably less stable than GATA-1. Studies using cycloheximide to block protein synthesis in erythroid precursor cell lines derived from embryonic stem cells indicate that the half-life of endogenous GATA-2 is ~45–90 min, whereas that of endogenous GATA-1 is >4 h. Biosynthetic labeling studies to measure the stabilities of endogenous GATA-1 and GATA-2 expressed in the same cell confirmed that GATA-2 is considerably less stable than GATA-1. GATA-2 levels increase in cells treated with the proteasome inhibitors MG132 or clasto-lactacystin β-lactone. Importantly, we have found that rapid GATA-2 proteolysis is essential for the proper temporal control of erythroid-specific gene transcription. Quantitative chromatin immunoprecipitation analysis revealed that proteasomal inhibition attenuated the ability of GATA-1 to displace GATA-2 from target genes, which are regulated by GATA switches. Proteasomal inhibition resulted in impaired GATA-2 displacement from GATA-2, and consequently a significantly lower degree of GATA-2 repression. Proteasomal inhibition also diminished GATA-1-mediated displacement of GATA-2 from GATA-1, α-globin, and ALAS-2, genes at which GATA switches precede transcriptional activation. We have therefore identified the 26S proteasome as a key regulator of GATA factor exchange on chromatin. Our studies indicate that rapid proteasomal degradation of GATA-2 is required for GATA-1 and GATA-2 to establish unique patterns of gene expression during erythropoiesis. Studies are underway to determine whether GATA-2 is similarly unstable in multipotent hematopoietic precursors, or if its instability is established upon commitment to the erythroid lineage.
Normalisation of ADAMTS 13 Activity and Disappearance of ADAMTS 13 Antibody Following Rituximab in Acute Refractory Thrombotic Thrombocytopenic Purpura.
Authors: Scully Marie , Cavenagh Jamie , Hagger Diane , Benjamin Sylvia , Mackie Ian , Mchin Samuel J. , Cohen Hannah
Published: October 2019

Thrombotic thrombocytopenic purpura (TTP) is a life threatening disorder characterised by thrombocytopenia and microangiopathic haemolytic anaemia associated with a deficiency in ADAMTS 13. Plasma exchange (PEX) remains the primary treatment modality in acute TTP, with various immunosuppressive agents (e.g. methylprednisolone, vincristine, ciclosporin (CSA)) and other disease modifying agents(e.g. defibrotide), added in refractory cases. However, mortality remains at 15–20%. Rituximab is a monoclonal CD 20 antibody, which acts specifically to clear peripheral B lymphocytes involved with antibody production. We have treated 15 patients with acute refractory TTP; 12 female and 3 male, with 2–8 weekly Rituximab infusions, 375mg/m2. Fourteen had neurological features and 3 had cardiac TTP at presentation. Patients received Rituximab because of progressive clinical disease or deterioration of laboratory parameters despite intensive therapy. On admission, median haemoglobin was 8.2 (range 5.1–12) g/dl, platelet count 14 (range 3–70) X 109/L, lactate dehydrogenase (LDH) 1620 (range 411–>2832) IU/L. All patients attained normal laboratory parameters and clinical remission within 22 days of starting Rituximab therapy. Three patients proceeded to further Rituximab based on ADAMTS 13 activity following 4 treatments. Twelve patients had initial ADAMTS 13 activities <5% (normal range (NR) 66–126% by collagen binding assay) and thirteen patients had normal ADAMTS 13 activity (median 78%) following Rituximab. IgG antibodies to ADAMTS 13 pre Rituximab were detected in thirteen patients but were undetectable post Rituximab. The median number of PEX pre Rituximab was 14 (1–35) and following the first Rituximab treatment, 6 (1–22), including weaning with an alternate day regime following clinical and laboratory remission. This difference was significant (p=0.042, Wilcoxon signed ranks test). To date (follow-up 1–27 months) there have been no TTP relapses. No significant decrease in CD 19 levels, immunoglobulin levels or infectious complications have been documented. These data suggest that patients with acute refractory TTP respond promptly to Rituximab, associated with a significant reduction in the requirement for PEX. Clinical and haematological remission was demonstrated and maintained within 3 weeks of Rituximab treatment in all patients. Only one patient failed to increment ADAMTS 13 activity. In all patients with IgG antibodies to ADAMTS 13, these disappeared within 28 days of Rituximab treatment. Rituximab therefore appears to be a safe, effective, targeted therapy for acute refractory TTP.