bluuebunny/crossref_metadata_2025 huggingface dataset:
Prevention of Graft-Versus-Host Disease in Mouse Model Using Anti-Mouse C5 Antibody.
Authors: Nishimura Jun-ichi , DeOliveira Divino , Chen Benny J. , Kanakura Yuzuru , Rother Russell P. , Chao Nelson J.
Published: October 2019

Graft versus-host disease (GVHD) is a major cause of morbidity and mortality after bone marrow transplantation (BMT). An allogeneic GVH reaction is a response of donor lymphoid cells to host minor or major histocompatibility antigens. Donor T cells can be activated through the innate and the adaptive immune mechanisms. Donor B cells produce antibodies directed to host cells. These mechanisms may activate complement pathways. Thus, complement may have a crucial role in inflammation during a GVH reaction, but direct evidence for this has not been shown. In this study, we investigated the possibility of complement inhibitor, anti-mouse C5 antibody (BB5.1), to ameliorate the symptoms of GVHD using an acute GVHD mouse model: C57BL/6 (H2b) →BALB/c (H2d). One million T cells were injected together with 1 x 107 T-cell-depleted bone marrow (TCD BM) cells via tail vein into lethally irradiated BALB/c (8.5 Gy) recipients. Anti-mouse C5 antibody or its isotype matched control was administered intraperitoneally at a dose of 1 mg/mouse, 3 doses/week, for 4 weeks. Recipients were weighed weekly, and their survival was monitored daily. Average body weight of C5 antibody treated mice was 15.8 g at day 84 (19.2 g at day 0, N=12), whereas average weight of control mice was 13.3 g (19.2 g at day 0, N=12) (P=0.05, Student’s t-test). Kaplan-Meier survival curves were also compared as shown in the Figure. Eight of 12 mice were alive at day 84 in the treated group, as compared to only 2 of 12 in the control group (P=0.03, Logrank test). A second experiment showed similar data. We, thus, observed the effect of anti-mouse C5 antibody to reduce the symptoms of GVHD using an acute GVHD mouse model. These results might open a new window for the prevention of acute GVHD. Further experiments are currently ongoing to clarify the exact mechanism between complement and GVHD.

Figure Figure
Damage to Aging Thymic Stroma and De Novo T Cell Development.
Authors: Prockop Susan , Beesen Ana , Myers Aaron , O’Reilly Richard J.
Published: October 2019

Successful T lymphopoiesis is essential for maintaining resistance to infection, mediating graft versus tumor activity, and as a platform for some immune based therapies. Among the most profound age-related change affecting the hemopoietic system is involution of the thymus and a decline in T cell production. Coordinated interaction of marrow-derived lymphoid progenitors with thymic stromal cells is required for successful de novo T lymphopoiesis. Current models of thymic involution indicate that aging alters the capacity of thymic stromal cells to support this de novo T cell lymphopoiesis. Loss of specific sets of thymic stromal cells, or loss of specific lympho-stromal signaling interactions may partially explain age-based impaired T cell reconstitution. Prior models examining the potential to reconstitute the aged thymus have been conducted either in vitro or in cyto-reduced hosts, and thus largely risk confounding cytotoxic damage with age-related damage. We have established an in vivo functional assessment of the ability of thymic stroma to support de novo T lymphocyte development. In the absence of radiation or chemotherapy this system supports robust thymic reconstitution after transplantation of limited numbers of T cell depleted wild type precursors. We have previously demonstrated that the capacity for reconstitution in this model system depends on the presence of normal, but empty, stromal niches. Using this model we have demonstrated that a variety of chemotherapeutic agents damage the thymic stroma and impair robust reconstitution. We now present data demonstrating that with increasing age reconstitution becomes less robust. Thus in mice older than 16 months unlike in those younger than twelve months, donor derived T lymphocytes can be identified, but the absolute cellularity of the thymus does not increase after transplant. In addition, we demonstrate that there are agent specific defects in the stromal compartments of treated aged mice. We have proceeded to identify a subset of agents (including split dose irradiation) that impair reconstitution in aged recipients but do not do so in the young. Thus while reconstitution of the thymus of young mice subjected to hyperfractionated total body irradiation declines 28 +/− 3% that of old mice treated in the same manner declines 91 +/− 6%. This last finding has enabled us to identify specific “at risk” stromal populations that normally appear to support the DN2 to DN3 transition in T lymphopoiesis. In a series of experiments designed to ask whether these at risk stromal populations can be protected from injury we identify agent specific differences in mechanisms of injury. It is anticipated that results from these investigations will augment our understanding of lympho-stromal interactions crucial to normal intra-thymic T cell development.
Ancestim (r-metHuSCF) Plus Filgrastim for the Mobilization and Collection of Peripheral Blood Progenitor Cells for Autologous Transplant after Failure of Prior Mobilization: The French Experience of Compassionate Use.
Authors: Lapierre Valerie , Chabannon Christian , Makowski Caroline , Dumesnil Cecile , Valteau-Couanet Dominique , Vekhoff Anne , Caillot Denis , Azar Nabih , Divine Marine , Blakolmer Karin , Cailliot Christian , Moreau Philippe , Rossi Jean-Francois
Published: October 2019

Background: Ancestim (r-met human stem cell factor; Amgen Inc, CA, USA) is available in France for compassionate use in patients who have failed prior mobilization or an insufficient number of CD34+ cells for autograft. We retrospectively reviewed data from these patients to evaluate the efficacy of ancestim combined with filgrastim in achieving an adequate stem cell yield.

Patients and methods: Between January 1998 and July 2007, permission for ancestim use was granted by the French health authority and Amgen to 550 patients (pts) at 82 sites (median age 55 yrs (range, 18–74); 50% male). Non-Hodgkin’s lymphoma (NHL) accounted for 50% of pts, multiple myeloma (MM) 28%, chronic lymphocytic leukaemia 6%, Ewing’s sarcoma 4%, neuroblastomas 3%, ovarian carcinoma 1%, osteosarcomas 1%, and other tumours 7%. Of the 508 pts for whom data were available and analysed, 378 had undergone prior mobilization and 117 were considered to have insufficient harvests; mobilization history was unknown for 13 pts. Fifty-eight percent of the pts were treated with ancestim (20 microg/kg/day s.c.) and filgrastim (10 microg/kg/day s.c.) alone for a median of 6 days (range, 2–21), while the other pts received chemotherapy followed by ancestim (20 microg/kg/day s.c.) and filgrastim (5 microg/kg/day s.c.) for a median of 11 days (range, 1–33).

Results: The target threshold of 2x106 CD34+ cells/kg was reached in 198/508 pts (39%) (including 51% of the 144 MM pts and 31% of the 254 NHL pts) after a median of 2 leukaphereses (range, 1–6). 35% of pts with prior mobilization failure and 52% of pts with prior insufficient harvests reached the target of 2 x106 CD34+ cells/kg, with a median number of CD34+ cells harvested of 3.4 x106/kg (range, 0.5–39.5). Of these, 150 pts (76%) proceeded to autologous transplantation. Data on engraftment are available for 137 pts: the median times to reach 0.5 x 109/L neutrophils and 20 x 109/L platelets were 12 days (range, 6–40) and 13 days (range, 0–31), respectively. All pts receiving ancestim were premedicated and treatment was generally well tolerated. Four pts were reported to have experienced severe mast cell-mediated reactions, none of which were life-threatening.

Conclusion: Ancestim plus filgrastim, alone or with chemotherapy, successfully rescued adequate CD34+ cell mobilization and collection in preparation for autologous transplantation in approximately 40% of pts.
Cryopreservation of Peripheral Blood Stem Cells (PBSC) with Hydroxyethylstarch (HES) and Dimethylsulfoxide (DMSO) Results in Faster Granulocyte Recovery Than Using Dimethylsulfoxide Alone.
Authors: Chan Wai Shun , Ma Michael , Bhatty Muhammad U.A. , Preti Robert , DeVito Marie , Pecora Andrew L. , Donato Michele L. , Feldman Tatyana , Goldberg Stuart L. , Goy Andre , Siegel David S. , Rowley Scott D.
Published: October 2019

PBSC components are routinely frozen for storage before autologous transplantation in the treatment of malignant diseases. We have used 5% DMSO, 3.75% human serum albumin (HSA), 0.2% dextrose with 6% HES for cryopreservation of cells (DMSO/HES), but the lack of a commercial source of HES required us to evaluate other cryopreservative solutions. A solution containing 5% DMSO, 2.25% HSA in normal saline (DMSO) was selected from a range of cryoprotectant solutions based on in vitro data demonstrating equivalence in preservation of hematopoietic colony forming ability to the HES solution previously used at this center. Fifteen months after the change, we performed a retrospective review of engraftment kinetics in autologous PBSC transplantation for the treatment of patients classified by cryoprotectant solution used. PBSC collected before 13-June-2006 were frozen in the DMSO/HES solution. PBSC collected after that date were frozen in DMSO. Patients underwent transplantation using dose-intense preparative regimens appropriate to diagnosis. Filgrastim was administered after transplantation to hasten granulocyte recovery. All patients after the change date were evaluated and patients treated in a similar time frame before the change were selected for comparison. Patient demographics and mean CD34+ cell number infused were similar for these two groups.

Demographics N Age CD34+ Cells/Kg (xE6) Gender (M:F) Diagnosis (MM:NHL:Other) Shown are median age and mean CD34+ cells/kg transplanted. Diagnosis is MM=multiple myeloma, NHL=non-Hodgkin’s lymphoma, and Other = other hematological malignancies. DMSO/HES 122 56 7.6 59:63 75:34:13 DMSO 103 58 7.1 55:48 79:14:10 p Value 0.26 0.56 0.17 0.84

Patients who received PBSC frozen in DMSO/HES achieved a significant one-day faster granulocyte recovery but similar platelet recovery compared to patients receiving PBSC frozen in DMSO.

Engraftment Kinetics N ANC>500 ANC>1000 Platelet>20,000 Shown are median days to achieve particular endpoint of engraftment. DMSO/HES 122 11 12 13 DMSO 103 12 13 14 p Value <0.001 0.002 0.83

Despite similar in vitro and patient demographic profiles, these data indicate that the combination cryoprotectant solution results in a clinically significant enhancement in speed of engraftment, resulting in a one (1) day reduction in hospital stay associated with the transplant regimen. The effect appears to be lineage-specific. Other modifications of cryoprotectant solutions may achieve yet faster engraftment, benefiting patients undergoing autologous PBSC transplantation by shortening the period of marrow hypoplasia. These results agree with another study of cryopreservation with and without HES (Rowley, et al. Bone Marrow Transpl2003;31:1043–51).
Molecular Studies of Neutropenia in Barth Syndrome.
Authors: Aprikyan Andrew A. , Makaryan Vahagn , Dale David C.
Published: October 2019

The Barth syndrome is a rare mitochondrial disease that causes dilated cardiomyopathy, skeletal myopathy, and severe neutropenia. It has been reported that at least two patients with Barth syndrome died from septicemia attributed to neutropenia. The disease is an X-linked autosomal recessive disorder caused by mutations in the G4.5 or TAZ (tafazzin) gene, which introduce truncations, substitution, or alternative splice sites. Hematological investigations revealed normal chemotaxis, but the presence cytoplasmic vacuoles in myeloid cells and maturation arrest of neutrophil development at the myelocyte stage. Thus, a defect in neutrophil formation appears to be the primary reason for neutropenia and the susceptibility to infections in the Barth syndrome patients. Recent studies were mostly focused on cardiac, genetic and metabolic abnormalities associated with the disorder. However, the neutropenia aspect of the Barth syndrome remains unclear. The TAZ gene mutations appear to truncate the tafazzin protein likely resulting in the loss of function. Although a drosophila model of the Barth syndrome was recently reported, the cellular or mouse models of this disorder are not available yet. Thus, the link between TAZ mutations and severe neutropenia remains unknown. We hypothesized that TAZ mutations, which lead to the loss of function of tafazzin protein, trigger impaired cell survival and reduced production of neutrophils and their neutrophil precursors, thus resulting in severe neutropenia in Barth syndrome. To test this hypothesis, we attempted to knock-down the expression of the tafazzin gene in human myeloid progenitor cells using TAZ-specific shRNA. Four shRNA sequences specific to exons 4 through 7 were used for transfection of human myeloid progenitor cells that were later examined by flow cytometry and Western blot analyses. At least 2 of the shRNA constructs resulted in a substantial down-regulation in the expression level of the tafazzin protein in transfected myeloid progenitor cells as determined by Western blot. Flow cytometry analyses revealed that the knock-down of TAZ gene expression was associated with approximately 40–50% increase in proportion of apoptotic annexin-positive cells compared with cells transfected with control scrambled shRNA. These data suggest that the loss of function of TAZ gene is cytotoxic to hematopoietic cells and that severe neutropenia is due to the accelerated apoptosis of myeloid progenitor cells in patients with Barth syndrome. Further studies needed to elucidate the specific signaling pathways and to identify potentially therapeutic agents capable of controlling accelerated apoptosis of myeloid cells in Barth syndrome.
Predictors of Transformation to Myelodysplasia/Acute Myelogenous Leukemia (MDS/AML) in Severe Congenital Neutropenia (SCN).
Authors: Boxer Laurence A. , Bolyard Audrey Anna , Newburger Peter E. , Bonilla Mary Ann , Kannourakis George , Dror Yigal , Link Daniel C. , Alter Blanche P. , Rosenberg Philip S. , Dale David C.
Published: October 2019

Severe congenital neutropenia (SCN) is a heterogeneous disorder of myelopoiesis characterized by an absolute neutrophil count (ANC) persistently below 0.50 x 109/L (500/[um]L), with maturation arrest of neutrophil precursors of the bone marrow at the promyelocyte/myelocyte stage. G-CSF treated and untreated SCN patients are at risk of developing MDS/AML. Clinicians caring for these patients must vigilantly observe for evidence of evolution to malignancy and continually consider hematopoietic transplantation as an alternative therapy. We have previously reported that SCN patients requiring higher daily doses of G-CSF treatment (> 8 mcg/kg/day) are at an increased risk of MDS/AML, and that this risk is not predicted by pre-treatment bone marrow examinations or the pre-treatment ANC. We have reviewed clinical data for 46 patients over a 15-year period who were referred to the Severe Chronic Neutropenia International Registry (SCNIR) and developed MDS/AML. Four patients referred with a history of SCN had increased blasts on the bone marrow evaluation prior to G-CSF treatment or enrollment in the SCNIR. Blood counts were not available for two patients. For the other 40 patients, all treated with G-CSF for a median of 79 months (range 0.8 to 182 months), comparison of blood counts for the periods 12–24 months and 0–3 months before the diagnosis of MDS/AML showed:

Changes in blood counts for the patient population may suggest a potential for malignant transformation. In these 40 cases, the principal clinical findings leading to the diagnosis of MDS/AML were: routine bone marrow surveillance 38%, decreased peripheral blood counts 26%, increased infections 10%, increased blasts in the blood 5%, decreased ANC with requirement for increased G-CSF dosage 2%, hepatosplenomegaly 2%, other (1 septal panniculitis and 1 leukemia cutis) 5%, and unknown 12%. The finding of mutations in the ELA2 gene in 10 of 15 cases (67%) did not influence the likelihood of MDS/AML. Previously we found that the duration and dose of G-CSF and alterations of the ANC on G-CSF therapy, suggest a high risk of transformation over time. Based on these data, the SCNIR recommends that patients with SCN have regular surveillance of blood counts every 1 to 2 months and annual bone marrow examinations with cytogenetic studies. Our findings suggest that a decline or change in blood counts of individual patients over a 3-month period may be informative in recognizing transformation to MDS/AML.
Cost-Effectiveness Analysis in Canada of Dasatinib Versus Imatinib for the Treatment of Chronic Myelogenous Leukemia in Patients with Imatinib Resistance.
Authors: Sambrook Jennifer C. , Zou Denise , Taylor Matthew J. , Davis Catherine C. , Joyeux Alexandre , Levy Adrian R.
Published: October 2019

Objective: To estimate the cost-utility of dasatinib versus high-dose imatinib (HDI, imatinib 800mg/day) for chronic myelogenous leukemia (CML) in patients with resistance to imatinib at standard doses.

Methods: We adapted to the Canadian setting a Markov model with a monthly cycle length that followed hypothetical patients until death. Cost effectiveness was evaluated during three phases of CML: chronic (86% of Ontario patients in 2006), accelerated (9%) and blast crisis (5%). Efficacy of dasatinib and HDI (cytogenetic and hematologic response) and adverse events (AEs) were taken from randomized trials. Canadian costs of medications, laboratory tests, professional fees, hospitalizations, and AEs were obtained from published sources. Societal utility scores were estimated from a Canadian time trade-off study. Analyses were performed from the perspective of the Ontario Ministry of Health. Costs (2006 CDN$) and outcomes (QALYs) were discounted at 5% annually after the first year. Univariate and probabilistic sensitivity analyses were carried out to quantify uncertainty for the incremental cost-effectiveness ratios (ICERs).

Results: Dasatinib dominated HDI in chronic phase patients with additional QALYs (3.92 vs. 3.47) and a lower lifetime cost ($379,678 vs. $444,934). The ICERs of dasatinib vs. HDI in accelerated and blast phase were $88,098/QALY and $173,922/QALY respectively. The higher cost/QALY of dasatinib in the advanced phases is explained primarily by the increased predicted survival of dasatinib patients and therefore the longer duration of drug therapy for these patients. Parameters with the greatest influence on results were the time horizon, drug costs and utility values. The interpretation of results remained robust in univariate and probabilistic sensitivity analyses.

Conclusion: From an economic perspective dasatinib is an attractive treatment choice for the majority of imatinib resistant CML patients and provides better value for money than HDI.
Multisite Phosphorylation of NIPA at G2/M.
Authors: Illert Anna L. , Bassermann Florian , von Klitzing Christine , Seipel Petra , Morris Stephan W. , Peschel Christian , Duyster Justus
Published: October 2019

The regulated oscillation of protein expression is an essential mechanism of cell cycle control. The SCF class of E3 ubiquitin ligases is involved in this process by targeting cell cycle regulatory proteins for degradation by the proteasome, with the F-Box subunit of the SCF specifically recruiting a given substrate to the SCF core. We previously reported the cloning of NIPA (Nuclear Interaction Partner of ALK) in complex with constitutively active oncogenic fusions of ALK, which contributes to the development of lymphomas and sarcomas. Subsequently we characterized NIPA as a F-Box protein (FBP) that defines an oscillating ubiquitin E3 ligase. The SCFNIPA complex targets nuclear cyclin B1 for ubiquitination in interphase while phosphorylation of NIPA in late G2 phase and mitosis inactivates the complex to allow for accumulation of cyclin B1. Here, we identify the region of NIPA that mediates binding to its substrate cyclin B1. In addition to the recently described serine residue 354, we specify 2 new residues, Ser-359 and Ser-395, implicated in the phosphorylation process at G2M within this region. Moreover, we found cyclin B1/Cdk1 to phosphorylate NIPA at Ser-395 in mitosis. Mutation of both Ser-359 and Ser-395 impaired effective inactivation of the SCFNIPA complex, resulting in reduced levels of mitotic cyclin B1. Furthermore, we aimed to identify the kinases involved in the initial phosphorylation of Ser-345. Therefore, we tested a panel of different kinases active at the G2M transition such as GSK3?, Casein kinase 2, PLK-1 and Erk1. Effective in vitro phosphorylation of NIPA could only be demonstrated with Erk-1. Moreover, we demonstrate an interaction of Erk-1 and NIPA at G2M but not in interphase cells. Binding of Erk-1 and NIPA led to phosphorylation at Ser-354 in vivo and could be blocked by the MEK-1/MEK-2 inhibitor PD98059. Together these data suggest a process of sequential phosphorylation where NIPA is initially phosphorylated by Erk-1 leading to the dissociation of NIPA from the SCF core complex. Once Ser-354 is phosphorylated, cyclin B1/CDK1 amplifies phosphorylation of NIPA, thus contributing to the regulation of its own abundance in early mitosis. In ALK positive lymphomas enhanced phosphorylation of NIPA at Ser 354 can be observed. We demonstrate that NPM-ALK leads to the activation of Erk-1, thereby phosphorylating and inactivating the SCFNIPA E3 ligase. Inactivation of SCFNIPA may have an important impact on the cell cycle turnover of lymphoma cells and thus for the pathogenesis of NPM-ALK induced lymphomas.
Systems Biology Analysis of Human Primary T Cells Identifies SAP145 as Rate Limiting for the G1→S Phase Transition.
Authors: Orr Stephen J. , Wang Rong , Lea Nicholas C. , Chronis Constantinos , Ramani Arun K. , Mufti Ghulam J. , Marcotte Edward M. , Thomas N. Shaun B.
Published: October 2019

We identified a G0→G1 commitment point in primary human T cells that controls entry into the cell cycle from quiescence. We demonstrated proof of principle that cellular pathways regulating cell cycle progression and effector functions that normally coincide during CD3/CD28 stimulation can be uncoupled experimentally. We have now used systems biology approaches to identify nuclear protein networks in primary human T cells that are regulated during the transition from quiescence into the cell cycle (G0→G1→S-phase). First we sequenced proteins that became bound to chromatin & nuclear matrix in G1 but were not bound in G0 and vice versa by mass spectrometry. Bioinformatic analysis identified 76 proteins specifically bound in G0 not G1 and 254 bound in G1 not G0. 179 of the 254 proteins bound in G1 not G0 (i.e. dynamic protein changes) were mapped to the 55,000 human protein interaction dataset. These are involved in numerous cellular functions, including epigenetics, transcription, RNA splicing & transport, and others. Cell cycle regulated chromatin/matrix binding of a subset was verified by western blotting (2/2 bound in G0 not G1 and 22/23 bound in G1 not G0). One of the proteins induced and bound in G1 was SAP145 (SF3B2). This is a component of the ubiquitous SF3b RNA splicing complex, involved in both major (U2-type) and minor (U12-type) spliceosomes. Since SAP145 is induced during G1 we investigated whether there was a role for SAP145 in regulating cell cycle progression. T cells depleted of SAP145 by siRNA enter G1 from G0 but progress poorly through S phase and die, probably by apoptosis. The same occurs if another component of the SF3B complex, SAP49 (SF3B4) is depleted with siRNA, indicating that the effect is due to depleting the complex rather than the individual SF3B protein. Proteins that are induced during G1 by CD3/CD28 stimulation e.g. cyclin D3, Cdc6 and cdc2 are produced normally when SAP145 is depleted, suggesting that their pre-mRNAs are spliced normally. In contrast, the expression of p107 and cyclin A2 are reduced markedly when SAP145 is depleted. Therefore, a systems biology approach to analysing cell cycle transitions identifies the splicing protein, SAP145 as rate-limiting for the G1 →S phase cell cycle transition but not for the transition from G0→G1.
Cyclin-Dependent Kinase Phosphorylation of RUNX1/AML1 on Three Sites Increases Trans-Activation Potency and Stimulates Cell Proliferation.
Authors: Friedman Alan D. , Zhang Linsheng , Fried Florence Bernardin
Published: October 2019

RUNX1/AML1 regulates lineage-specific genes during hematopoiesis and also stimulates G1 cell cycle progression. CBFβ-SMMHC or AML1-ETO dominantly inhibit RUNX1 and slow G1 progression in hematopoietic cell lines or in murine or human marrow progenitors, cdk4, cyclin D2, or c-Myc overcome inhibition of proliferation by these CBF oncoproteins, exogenous RUNX1 stimulates G1 progression, and stimulation of G1 via deletion of p16INK4a or expression of E7 cooperates with CBFβ-SMMHC or TEL-AML1 to induce acute leukemia in mice. Induction of cdk4 or cyclin D3 transcription may underlie stimulation of G1 progression by RUNX1. Remarkably, the C. elegans ortholog of RUNX1, RNT-1, also stimulates G1 progression and couples stem cell proliferation with differentiation. Not only does RUNX1 regulate cell cycle progression, but in addition RUNX1 levels increase as hematopoietic cells progress from G1 to S and from S to G2/M. Within RUNX1, S48, S303, and S424 fit the cdk phosphorylation consensus, (S/T)PX(R/K). Phosphorylation of RUNX1 by cyclin dependent kinases on serine 303 was shown to mediate destabilization of RUNX1 in G2/M. We now find that S48 and S424 are also phosphorylated by cdk1 or cdk6. S48, S303, or S424 phosphopeptide antiserum that we developed specifically recognized kinased GST-RUNX1 and interacted with RUNX1 expressed in 293T cells or in the Ba/F3 hematopoietic cell line. S48 phosphorylation of RUNX1 paralleled total RUNX1 levels during cell cycle progression, S303 was more effectively phosphorylated in G2/M, and S424 in G1. Single, double, and triple mutation to alanine or to the partially phosphomimetic aspartic acid progressively diminished or increased trans-activation, such that the tripleA mutant activated a RUNX1 reporter 5-fold less potently than the tripleD mutant. Aspartic acid does not perfectly mimic serine phosphorylation, as illustrated by the much greater affinity of our antisera for wild-type RUNX1 versus RUNX1(tripleD), suggesting that the biologic effect of RUNX1 cdk phosphorylation is even more significant. The p300 co-activator retained interaction with the tripleA variant. The tripleD RUNX1 mutant rescued Ba/F3 cells from inhibition of proliferation by CBFβ-SMMHC more effectively than the tripleA mutant. Cdk phosphorylation of RUNX1 on three sites increases its ability to active transcription and to stimulate proliferation, potentially coupling entry of stem/progenitors into cycle with induction of genes required for hematopoietic lineage progression, such as those encoding myeloperoxidase, neutrophil elastase, the M-CSF receptor, and PU.1.
High Expression of MDR1 and BCRP1 in Leukemic Stem Cells Correlates with Reversible Drug Resistance Ex Vivo and Predicts Failure of Remission Induction Chemotherapy in Patients with Acute Myeloid Leukemia.
Authors: Ho Maria M. , Hogge Donna E. , Ling Victor
Published: October 2019

Although overexpression of members of the ATP-Binding-Cassette (ABC) transporter superfamily has been implicated in chemotherapy failure in acute myeloid leukemia (AML) in some studies, results have been conflicting. Previously we found no consistent difference in pre-treatment ABC transporter expression when comparing total blasts of 18 patients achieving complete remission (CR) and 13 patients who were refractory to induction chemotherapy (NR). However, among the CD34+CD38− AML cells (enriched for candidate “leukemic stem cells” which engraft in immunodeficient mice), as assessed by quantitative RT-PCR, elevated expression of MDR1 and/or BCRP1, two ABC transporters associated with drug resistance, was found in 8/10 NR patients as compared to 0/7 CR patients. No difference between CR and NR samples was observed in the more differentiated CD34+CD38+ or CD34− AML cells while none of the subpopulations showed a significant difference in MRP1 expression between CR and NR patient samples. To determine if this difference in gene expression was associated with altered functional activity of MDR1/BCRP1, the relative sensitivity of subpopulations of AML cells to induction of apoptosis following ex vivo treatment with daunorubicin with or without ABC inhibitors (PSC-833 and Verapamil) was assessed. All subpopulations of AML cells isolated from 2 CR patients showed similar drug sensitivity profiles (IC50 <0.04 μg/ml). In contrast, CD34+CD38− cells from 6 NR patients were more resistant to daunorubicin-induced apoptosis (p<0.05 for comparison of IC50s between CR and NR samples). ABC transporter inhibition in CD34+CD38− cells did not change daunorubicin sensitivity in CR samples, but increased drug sensitivity in a dose-dependent manner in NR samples, particularly in the CD34+CD38− fraction. This suggests a role for ABC transporters in sustaining a population of chemotherapy resistant leukemic stem cells which may allow survival of the malignant clone in patients following induction therapy. If the value of measuring MDR1/BCRP1 expression among these cells in predicting chemotherapy outcome is confirmed in a larger patient population, it may be possible to use such analysis to modify therapy for individual AML patients to overcome drug resistance.
Expression of ROR1 in Chronic Lymphocytic Leukemia Cells Enhances Cells Survival by Wnt5a Signaling.
Authors: Chen Liguang , Tang Li , Fukuda Tetsu , Kipps Thomas J.
Published: October 2019

ROR1 is a type I membrane-receptor tyrosine kinase with intracellular tyrosine kinase domains and extracellular Frizzled-like cysteine-rich domains (CRDs) and kringle domains, which are common to receptors of members of the Wnt-family. We found that ROR1 might serve as an orphan receptor for Wnt5a, which can activate NF-κB via a non-canonical Wnt-signaling pathway. In prior studies we found that CLL cells expressed ROR1 and high-levels of Wnt3, Wnt5b, Wnt6, Wnt10a, Wnt14, and Wnt16, but not Wnt5a, which is found expressed on dendritic cells and other stromal cells that can support leukemia-cell survival in vitro, and presumably in vivo. As such, we examined whether cells could support the survival of CLL cells in a Wnt5a-dependent manner. For this we cultured CLL cells alone or together with Chinese Hamster Ovary (CHO) cells or CHO transduced to express Wnt5a (CHO-Wnt5a) and monitored the viability of CLL cells over time. We found that the viability of CLL cells co-cultured with CHO cells or CHO-Wnt5a cells was greater than that of CLL cells cultured alone. However the viability of CLL cells co-cultured for two days with CHO-Wnt5a cells (71% ± 18% S.D) was significantly greater than that of CLL cells co-cultured with CHO cells (45% ± 17% S.D) (N=10, P<0.01). The capacity of CHO-Wnt5a to enhance the viability of CLL cells relative to that of CHO cells could be neutralized by anti-ROR1 antisera generated in patients who received autologous CLL cells transduced to express the CD40-ligand (CD154), but not by normal human serum or pre-treatment human serum. CHO cells transduced to express ROR1, but not CHO cells transduced with a control vector, could absorb the capacity of the anti-ROR1 antisera to bind ROR1 and removed its capacity to neutralize the capacity of CHO-Wnt5a cells to provide for a protecive advantage over CHO cells for CLL cell survival in vitro. These studies provide the first evidence that the survival of CLL cells can be enhanced by cells expressing Wnt5a, let alone Wnt factors in general, and might account in part for the enhanced survival of CLL cells in tissue microenvironments containing cells that express Wnt5a. Conceivably, the anti-ROR1 antibodies induced by autologous CLL cells transduced to express CD154, or the administration of antibodies or antagonists that can inhibit Wnt5a-ROR1 signaling, could have therapeutic activity in patients with CLL.
Hospital Readmission within 30 Days: A New Benchmark for Quality Care among Children with Sickle Cell Disease.
Authors: Frei-Jones Melissa J. , DeBaun Michael R.
Published: October 2019

The National Association of Children’s Hospitals and Related Institutions (NACHRI) has established benchmarks for measuring quality of care in children that are hospitalized. Readmission within 30 days of discharge is the benchmark chosen to assess the quality of care for patients with Sickle Cell Disease (SCD). Limited data exist to determine risk factors for re-admission and whether such risk factors are modifiable. We performed a retrospective case-control study to identify risk factors for hospital readmission in children with SCD. All hospital admissions of patients with SCD for one year were reviewed. Cases were defined as children with SCD who were readmitted within 30 days of their first admission during the 12 month study period. Controls were defined as children with SCD who were not readmitted within 30 days of their first admission. A total of 30 cases and 70 controls were identified. The average time between admissions was 10.7 days with 50% readmitted within 8 days and 77% readmitted within 21 days. No difference in demographic data was found between cases and controls. The most common admission and readmission diagnosis was pain, 78% and 70%, respectively. The greatest risk factor for readmission was no follow-up appointment within 30 days after discharge in the SCD clinic (OR 7.7, 95% CI 2.4–24.4). The second highest risk factor was severity of disease, defined as patients with ≥ 3 hospitalizations in the previous 12 months versus patients with ≤ 2 hospitalizations in the previous 12 months (OR 7.3, 95% CI 2.8–18.9). A diagnosis of asthma was also a risk factor for readmission (OR 2.9, 95% CI 1.2–7.3). Patients who initially required supplemental oxygen to maintain their oxygen saturation in the normal range and were subsequently on room air for ≤ 24 hours at discharge were also more likely to be readmitted (OR 3.3, 95% CI 1.1–9.7). Steroid administration was not a risk factor for readmission (OR 1.2, 95% CT 0.5–3.2). Potential modifiable risk factors exist to decrease the rate of readmission. Specifically, strategies targeted at the modification of disease severity, aggressive management of asthma, and outpatient follow-up after hospitalization may decrease the 30 day readmission rate.
Zanolimumab (HuMax-CD4™), a Fully Human Monoclonal Antibody: Efficacy and Safety in Patients with Relapsed or Treatment-Refractory Non-Cutaneous CD4+ T-Cell Lymphoma.
Authors: d’Amore Francesco , Radford John , Jerkeman Mats , Relander Thomas , Tilly Herve , Osterborg Anders , Morschhauser Franck , Gramatzki Martin , Dreyling Martin , Bang Bo , Baadsgaard Ole , Hagberg Hans
Published: October 2019

Combination chemotherapy has been the basis of treatment for peripheral T-cell lymphoma (PTCL) for many years, but relapses are frequent, often rapid and usually associated with a poor outcome. New treatment strategies are therefore required. Zanolimumab is a fully human monoclonal cytotoxic IgG1κ antibody, targeting the CD4 molecule on T-cells. The present open-labeled exploratory clinical trial investigated the efficacy and safety of Zanolimumab in 21 patients with biopsy-proven, treatment-refractory or relapsed CD4+ PTCL of non-cutaneous type. Zanolimumab was administered by i.v. infusion at a dose of 980 mg once weekly for 12 weeks. Patients (11 male, 10 female, median age 69 years) were enrolled with the following diagnoses: enteropathy-associated T-cell lymphoma (n=1), anaplastic large T-cell lymphoma (ALTL, n=4, 3 ALKneg subtype, 1 unknown), angioimmunoblastic T-cell lymphoma (n=9) and PTCL-unspecified (n=7). Patients had received a median of 1 previous treatment (range, 1–5) and 3 had undergone high dose chemotherapy with autologous stem cell rescue. The primary endpoint was objective tumor response. Responses were based on CT scan and clinical examination and classified according to the Cheson criteria. Objective tumor response was obtained in 5 out of 21 patients (23.8%; 95% CI: [8.2; 47.2]). Three patients (1 Anaplastic Large T-cell lymphoma and 2 angioimmunoblastic T-cell lymphoma) obtained a partial response of 43 and 51 days duration, respectively, one had not relapsed after 182 days, no further data available. Two patients (PTCL-unspecified and angioimmunoblastic T-cell lymphoma) obtained a complete response (unconfirmed) of 46 days duration and in the latter no relapse after 252 days was observed. 27 serious AEs were reported of which 6 were assessed as related to Zanolimumab treatment. Four infusion related AEs, one transient thrombocytopenia, one febrile neutropenia in a patient that was neutropenic at trial entry. All patients with serious AEs had complete recovery. Two non-serious grade 3 infusion related AEs were reported and one joint pain. In conclusion, zanolimumab was found to be active and well tolerated in heavily pretreated patients with aggressive PTCL. Zanolimumab in combination with CHOP is now being investigated in the setting of previously untreated patients with nodal T-cell lymphoma.
Preliminary Pharmacokinetic (PK) Analysis of Eastern Cooperative Oncology Group Protocol E4402: Rituximab Extended Schedule or Re-Treatment Trial (RESORT).
Authors: Kahl Brad S. , Williams Michael E. , Hong Fangxin , Gascoyne Randy , Horning Sandra J.
Published: October 2019

Background: E4402 is a randomized phase III study comparing two different rituximab dosing strategies for patients (pt) with low tumor burden, indolent histology non-Hodgkin’s lymphoma. Eligible pt receive rituximab weekly x 4 and responders are randomized to receive either re-treatment at progression or a scheduled dose every 3 months (mo). Each strategy is continued until the development of rituximab resistance with a primary endpoint of time to rituximab failure. Administration of a single rituximab dose every 3 mo was based on PK data derived from the Pivotal trial (ref) that suggested 25 mcg/mL may be an important threshold to maintain response, and a PK study (ref Gordon) that suggested this level could be maintained with a single dose administered every 3 mo.

Methods: Rituximab levels were measured within 30 minutes of completion of the 4th infusion and at 12 weeks (time of response assessment). Pt randomized to re-treatment have trough levels measured at 6 mo, at 1st relapse, and at the first restaging after re-treatment. Pt randomized to scheduled rituximab have trough levels measured at 6 and 12 mo and upon progression. Presented here is an analysis of serum rituximab levels in 159 and 161 follicular lymphoma pt with available data of week 4 and week 12, respectively.

Results: The median level after the 4th infusion is 316 mcg/mL (range 56.4–754.0 mcg/mL). The median level at 12 weeks is 26.9 mcg/mL (range 0.0 -87.7). Fifty-three percent of the patients had levels ≥ 25 mcg/mL at 12 weeks. The median week 4 levels are somewhat lower than the Pivotal trial and a comparison of mean levels indicates a statistically significant difference, (312.9 mcg/mL vs. 450 mcg/mL, p-value < 0.0001). However the week 12 levels are similar (table). No correlation between week 4 and week 12 levels was observed in the 132 follicular lymphoma pt with paired data.

Time N Median (mcg/mL) STD Min (mcg/mL) Max (mcg/mL) E4402 week 4 159 316.0 108.6 56.4 754.0 Pivotal week 4 141 460.7 187.0 115.4 996.6 E4402 week 12 161 26.9 19.6 0.0 87.7 Pivotal week 12 104 20.2 20.8 0.0 96.8

Conclusions: Despite the fact that the E4402 pt are untreated and have a low tumor burden, the week 4 serum drug levels are somewhat lower and the week 12 levels are very similar to the Pivotal trial. The week 4 level is not predictive of the week 12 level. These data suggest that serum levels in nearly half of patients receiving a single rituximab dose every 12 weeks will be below a 25 mcg/mL threshold. Future analysis will correlate week 4 and week 12 levels with initial response to rituximab. Later PK time points will be correlated to the time to rituximab resistance.
R-ESHAP as Salvage Therapy for Patients with Relapsed or Refractory Diffuse Large B-Cell Lymphoma: A Spanish GEL-TAMO Multicenter Study.
Authors: Martin Alejandro , Conde Eulogio , Arnan Montserrat , Canales Miguel A. , Deben Guillermo , Andreu Rafael , Salar Antonio , Garcia-Sanchez Pedro , Nistal Sara , Requena Maria J. , Donato Eva M. , Gonzalez Jose A. , Leon Angel , Ruiz Concepcion , Grande Carlos , Arranz Reyes , Duran Soledad , Ortegon Sergio , Rodriguez Juan N. , Sandoval Virgilio , Andreu Maria A. , Bernal Teresa , Hernandez Miguel T. , Gonzalez-Barca Eva , Caballero Maria D.
Published: October 2019

The addition of Rituximab to CHOP-like chemotherapy (CT) regimens has improved survival in diffuse large B-cell lymphoma (DLBCL). For this reason, Rituximab is also extensively used in combination with salvage CT regimens, although the information published on this setting is scarce. In the present study, we have analysed a large series of patients (pt) treated with R-ESHAP. 151 pt with relapsed or refractory DLBCL received R-ESHAP (Rituximab, etoposide, cytarabine, cisplatinum and methylprednisolone) as salvage therapy between May 2000 and March 2007. Median age was 54 years (19–70). 22% of pt were refractory to front-line therapy, 19% were partial responders, and 56% had relapsed disease. 56% of pt had received Rituximab in addition to CT as first-line treatment. 15 pt had been treated with autologous stem-cell transplantation (ASCT) prior to R-ESHAP. Patients received a median of 3 cycles of R-ESHAP (1–6). Overall response (OR) rate was 72%, with 69 pt (46%) achieving complete response (CR) and 40 (26%) partial response (PR). Factors with significant influence on CR rates in multivariate analysis were: absence of bulky disease (RR: 3.1), PR to first-line treatment (RR: 3.9) or relapsed disease (RR: 5.8), low-risk IPI (RR: 6.6), and administration of ≥3 cycles (RR: 5). Patients who had received Rituximab prior to R-ESHAP had lower CR (39 vs 54%) and OR (66 vs 80%) rates than patients who had not received it, but the difference did not reach statistical significance. In total, 94 out of 151 pt underwent transplantation after the salvage therapy (91 autologous, and 3 allogeneic). With a median follow-up of 22 months (3–73), the 4-year freedom from treatmet failure (FFTF) and overall survival (OS) were 49% and 48%, respectively. Patients who received Rituximab prior to R-ESHAP had a significantly worse FFTF (26 vs 67% at 4 years) and OS (30 vs 63% at 4 years) as compared with patiens who did not receive it. On multivariate analysis, factors significantly associated with a poor OS were: prior exposure to Rituximab (RR: 2.8), high-risk IPI (RR: 5.2), thrombocytopenia <100x109/l at the time of R-ESHAP (RR: 9.5); and achievement of PR (RR: 6.4) or progressive disease (RR: 17) after R-ESHAP. Our results show the efficacy of R-ESHAP prior to ASCT for refractory or relapsed DLBCL. Although prior exposure to Rituximab was associated with a worse outcome, still a significant number of patients responded to the regimen. Future studies are needed in order to determine which patients will benefit from Rituximab re-treatment.
Outcome of Limited-Stage Peripheral T-Cell Lymphoma: Results from the Nebraska Lymphoma Study Group.
Authors: Bierman Philip , Vose Julie M. , Bociek R. Gregory , Loberiza Fausto , Bast Martin , Weisenburger Dennis D. , Armitage James Olen
Published: October 2019

The outcome of treatment for the majority of patients with peripheral T-cell lymphoma who have advanced-stage disease is worse than similar patients with diffuse large B-cell lymphoma. Patients with limited-stage diffuse large B-cell lymphoma have a good prognosis and are often treated differently than patients with advanced-stage disease. There is little information on results of treatment for patients with limited-stage peripheral T-cell lymphoma. We have performed a retrospective review of patients with limited-stage peripheral T-cell lymphoma who were identified in the database of the Nebraska Lymphoma Study Group. Patients with masses ≥10 cm. and those with B symptoms were excluded. We identified 36 patients with non bulky stage I-A (n=21, 58%) or II-A (n=15, 42%) disease who were diagnosed between and April, 1984 and February, 2006. The median age was 59 (range 18–87) years. There were 20 (56%) males. Histology was classified according to the World Health Organization (WHO) classification. Three patients (8%) had angioimmunoblastic T-cell lymphoma, 25 (69%) had peripheral T-cell lymphoma, unspecified, and 8 (22%) had anaplastic large cell lymphoma. Thirty patients (83%) were treated with anthracycline- or mitoxantrone-based therapy, 5 (14%) were treated with radiation, alone, and 1 (3%) received other chemotherapy. Seventeen patients (47%) were treated with combined chemotherapy and radiation. The median follow-up of surviving patients is 108 (range 12–235) months. The progression-free survival and overall survival are displayed in the figure, below. The actuarial 5-year and 10-year progression-free survival rates are 77% and 63%, respectively. The corresponding values for overall survival are 71% and 56%, respectively. Univariate analysis did not demonstrate significant differences in the risk of treatment failure or death when the influence of histology, stage (I vs. II), and type of treatment (chemotherapy vs. chemotherapy + radiation; <3 cycles of chemotherapy vs. ≥3 cycles) were analyzed, although the numbers in each group were relatively small. Similarly, the outcome was not influenced by age, gender, performance status, LDH level, bulk, or the presence of extranodal disease. These results demonstrate that long-term progression-free survival can be observed for patients with limited-stage peripheral T-cell lymphoma. These results are better than the results of treatment for higher stages of peripheral T-cell lymphoma. Currently available therapy for limited-stage peripheral T-cell lymphoma can achieve results that are close to the results of treatment for limited-stage diffuse large B-cell lymphoma.

Figure Figure