bluuebunny/crossref_metadata_2025 huggingface dataset:
Adoptive Transfer of In Vitro Generated T Cell Precursors Enhances Donor T Cell Reconstitution and Graft-Versus-Tumor Activity in Allogeneic Hematopoietic Stem Cell Transplantation Recipients.
Authors: Zakrzewski Johannes L. , Kochman Adam A. , Lu Sidney X. , Terwey Theis H. , Kim Theo D. , Hubbard Vanessa M. , Muriglan Stephanie J. , Suh David , Cabrera-Perez Javier , Radhakrishnan Radhika , Heller Glenn , Zuniga-Pflucker Juan Carlos , Alpdogan Onder , van den Brink Marcel R.M.
Published: April 2020

Allogeneic hematopoietic stem cell transplantation (HSCT) is associated with a varying period of immunoincompetence that particularly affects he T cell lineage resulting in significant morbidity and mortality from opportunistic infections. Recent studies have shown that murine T cells and their precursors can be generated from hematopoietic stem cells (HSC) in vitro using a OP9-DL1 coculture system consisting of OP9 bone marrow stromal cells expressing the Notch 1 ligand Delta-like 1 and growth factors (interleukin 7 and fms-like tyrosine kinase-3 ligand). In this study we determined the effects of adoptively transferred in vitro generated T cell precursors on T cell reconstitution after allogeneic HSCT. We selected HSC (Lin- Sca-1hi c-kithi) from bone marrow (BM) of C57BL/6 mice and cultured these cells on a monolayer of OP9-DL1 cells in the presence of growth factors. These HSC expanded 2,000–5,000-fold within 3–4 weeks and consisted of >95% CD4-CD8-double negative (DN) T cell precursors after 16–28 days of culture. We infused these cells (8x106) with T cell depleted (TCD) BM (5x106) or purified HSC into allogeneic recipients using minor antigen mismatched and MHC class I/II mismatched transplant models. Control mice received TCD BM or purified HSC only. Progeny of OP9-DL1 derived T cell precursors were found in thymus and spleen increasing thymic cellularity and significantly improving thymic and splenic donor T cell chimerism. This effect was even more pronounced when purified HSC instead of whole BM were used as allograft. T cell receptor repertoire and proliferative response to foreign antigen (determined by third party MLR) of in vivo differentiated OP9-DL1 derived mature T cells were intact. Administration of in vitro generated T cell precursors did not induce graft-versus-host disease (GVHD) but mediated significant graft-versus-tumor (GVT) activity (determined by in vivo bioluminescence imaging) resulting in a subsequent significant survival benefit. This advantage was associated with better cytokine responses (IL-2, INF-g, TNF-a) in T cells originating from OP9-DL1 derived T cell precursors compared to BM donor derived T cells. We conclude that the adoptive transfer of OP9-DL1 derived T cell precursors significantly enhances post-transplant T cell reconstitution and GVT activity in the absence GVHD.
AML1-ETO9a, an Alternatively Spliced Form of AML1-ETO, Collaborates with AML1-ETO To Promote Leukemogenesis.
Authors: Yan Ming , Wang Yang , Peterson Luke F. , Kanbe Eiki , Boyapati Anita , Zhang Dong-Er
Published: April 2020

t(8;21) is a common chromosome translocation in acute myeloid leukemia (AML). AML1-ETO is the fusion protein produced by t(8;21). Results from our work and others indicate that full length AML1-ETO is not leukemogenic and needs additional mutations to promote leukemia. Most recently, we have identified an alternatively spliced form of AML1-ETO, AML1-ETO9a, that lacks the C-terminal NHR3 and NHR4 domains of AML1-ETO. Here, we report that unlike the negative effect of full length AML1-ETO in cell cycle progression, AML1-ETO9a enhances cell proliferation by shortening G1 phase of cell cycle in the IL-3 dependent multipotent hematopoietic progenitor cell line FDCPmix. Using retroviral mediated gene expression in murine hematopoietic cells and bone marrow transplantation in mice, we have demonstrated that AML1-ETO9a is highly leukemogenic. Most importantly, we show that AML1-ETO9a expression in transgenic MRP-8 AML1-ETO hematopoietic cells results in earlier onset of AML than AML1-ETO9a expression alone controls. Furthermore, the presence of full length AML1-ETO also alters the phenotype of leukemia progenitor cells from CD34- to CD34+. These results suggest that the alternatively spliced form AML1-ETO9a cooperates with full length AML1-ETO in leukemogenesis when coexpressed in t(8;21) patients.
The Incidence of Leukemia and Mortality from Sepsis in Patients with Severe Congenital Neutropenia Receiving Long-Term G-CSF Therapy.
Authors: Rosenberg Philip S. , Alter Blanche P. , Bolyard Audrey A. , Bonilla Mary A. , Boxer Laurence A. , Cham Bonnie , Fier Carol , Freedman Melvin , Kannourakis George , Kinsey Sally , Schwinzer Beate , Zeidler Connie , Welte Karl , Dale David C.
Published: April 2020

Background: In patients with severe congenital neutropenia (SCN), mortality from sepsis is reduced by treatment with granulocyte colony-stimulating factor (G-CSF), but myelodsyplastic syndrome and acute myeloid leukemia (MDS/AML) have been reported in treated and untreated patients.

Methods: We studied 374 patients with SCN and 29 patients with Shwachman-Diamond Syndrome (SDS) on long-term G-CSF enrolled in the Severe Chronic Neutropenia International Registry.

Results: In SCN, mortality from sepsis was stable at 0.9%/year. The hazard of MDS/AML increased significantly over time, from 2.9%/year after 6 years to 8.0%/year after 12 years on G-CSF. After 10 years, the cumulative incidence was 8% for mortality from sepsis and 21% for MDS/AML. The hazard of MDS/AML increased with the dose of G-CSF. Twenty-nine percent of SCN patients received more than the median dose of G-CSF (≥8 μg/kg/day), but achieved less than the median absolute neutrophil count (ANC) response (ANC <2188 cells/μL at 6–18 months). In these less responsive patients, the cumulative incidence of adverse events was highest: after 10 years, 40% developed MDS/AML and 14% died of sepsis, compared to 11% and 4%, respectively, of more responsive patients whose ANC was above the median on doses of G-CSF below the median. An intermediate group achieved an ANC above the median on doses of G-CSF above the median; among them, the 10 year cumulative incidence was 15% for MDS/AML and 3% for mortality from sepsis. In secondary analyses, we found that pre-treatment blood cell counts could not predict the subsequent clinical outcome. Furthermore, on therapy, patients who were less responsive vis-à-vis their neutrophil counts had similar increases in eosinophils, basophils, monocytes, and lymphocytes, and similar decreases in platelets, as other patients maintained on ≥8 μg/kg/day. Consistent with the SCN results, in SDS patients, the limited available data do not suggest that G-CSF therapy is a risk factor for MDS/AML in SDS.

Conclusions: The risk of MDS/AML was similarly low in all patients who achieved an ANC above the median on any dose of G-CSF. It appears that G-CSF has reduced mortality from sepsis, and revealed the underlying leukemic predisposition of SCN.
Gene Expression Profiling Identifies Distinct Subclasses in Core Binding Factor Acute Myeloid Leukemia.
Authors: Bullinger Lars , Kurz Stephan , Dohner Konstanze , Scholl Claudia , Frohling Stefan , Schlenk Richard F. , Dohner Hartmut , Pollack Jonathan R.
Published: April 2020

Recurrent cytogenetic aberrations have been shown to constitute markers of diagnostic and prognostic value in acute myeloid leukemia (AML). However, even within well-defined cytogenetic AML subgroups with an inv(16) or a t(8;21) we see substantial biological and clinical heterogeneity which is not fully reflected by the current classification system. Therefore, we profiled gene expression in a large series of adult AML patients with core binding factor (CBF) leukemia [inv(16) n=55, t(8;21) n=38] using a whole genome DNA microarray platform in order to better characterize this disease on the molecular level. By unsupervised hierarchical clustering based on 8556 filtered genes we observed that our CBF leukemia samples separated primarily into three different subgroups. While two of the subgroups were characterized by inv(16) and t(8;21) cases, respectively, the third subgroup contained a mixture of both cytogenetic groups. There was no obvious correlation with known secondary aberrations or molecular marker like FLT3-ITD, NRAS and KIT mutations between the cases in the mixed subgroup and the others. However, the newly defined inv(16)/t(8;21)-subgroup (n=35) was characterized by distinct clinical behavior with shorter overall survival times (P=0.029; log rank test) compared to the other two groups. Unsupervised analyses within the inv(16) and t(8;21) cases also revealed corresponding inv(16) and t(8;21) subgroups with a strong trend towards inferior outcome (P=0.11 and P=0.09, respectively; log rank test). Since the primary translocation/inversion events themselves are not sufficient for leukemogenesis, distinct patterns of gene expression found within each of these cytogenetic groups may suggest alternative cooperating mutations and deregulated pathways leading to transformation. Therefore, we performed a supervised analysis to determine the characteristic gene expression patterns underlying the cluster-defined subgroups. We identified 528 genes significantly differentially expressed between the newly defined inv(16)/t(8;21)-subgroup and the other CBF cases (significance analysis of microarrays, false discovery rate < 0.001). Potential candidates for cooperating pathways characterizing the mixed inv(16)/t(8;21)-subgroup included e.g. AVO3, a member of the mTOR pathway, oncogene homologs like LYN and BRAF, as well as FOXO1A and IL6ST which have been previously identified to correlate with outcome in AML (Bullinger et al., N Engl J Med350:1605, 2004). In conclusion, while the observed signatures remain to be validated for their functional relevance, both supervised and unsupervised methods provide numerous insights into the pathogenesis of CBF AML, identifying clinically significant patterns of gene expression, as well as candidate target genes involved in leukemogenesis.
The Impact of Histologic Grade on the Outcome of High-Dose Therapy and Autologous Stem Cell Transplantation for Follicular Lymphoma.
Authors: Pham Rosalyn N. , Gooley Ted A. , Keeney Grant E. , Press Oliver W. , Pagel John M. , Petersdorf Stephen H. , Maloney David G. , Gopal Ajay K.
Published: April 2020

Follicular lymphomas (FL) represent approximately one-fourth of all non-Hodgkins lymphomas (NHL) and are the most common indolent lymphomas. Studies suggest that patients (pts) with relapsed FL treated with high-dose therapy (HDT) and autologous stem cell transplantation (ASCT) have prolonged progression-free survival (PFS) and overall survival (OS) as compared to those treated with standard chemotherapy alone, yet the impact of histologic grade on these outcomes has not been established. In order to address this issue, we evaluated all FL pts that underwent HDT and ASCT at our center from Dec 1985 to June 2005 (n=219). Pts with transformation to diffuse large B-cell lymphoma were excluded. Histologic grades 1, 2, and 3 were present in 106 (48%), 75 (34%), and 38 (17%) pts, respectively. Other baseline characteristics at the time of transplant for this cohort included: Female = 39%, median age = 48 (range 24 – 66), stage III/IV = 93%, elevated LDH = 27%, median number of prior regimens = 3 (range 1 to 11), prior radiation therapy (RT) = 16%, bulk ≥5cm = 19%, chemosensitive = 68%, complete remission = 18%. 53% of pts are alive and 36% are alive without relapse at last contact leading to 5- and 10-year estimates of 60% and 42% for OS and 39% and 28% for PFS, respectively. The median follow-up for surviving patients was 7.8 years. The 5- and 10-year OS estimates for the histologic grades were grade 1: 57% and 42%, 2: 60% and 36%, and 3: 65% and 55% (Figure). Five- and 10-year PFS estimates were grade 1: 35% and 27%, grade 2: 44% and 25%, and grade 3: 45% and 40% (Figure). In order to adjust for any potential imbalances of critical prognostic factors between the three grades, we performed a multivariable analysis adjusting for criteria that were found to be associated with OS and/or PFS (age, # of prior regimens, prior RT, chemosensitivity, and LDH) and obtained the hazard ratios (HR) and p-values noted (Table). These data suggest that 1) prolonged OS and PFS can be attained via HDT and ASCT in FL pts, 2) histologic grade does not statistically significantly impact outcomes, and 3) other factors noted above should be utilized to predict outcome and counsel patients.

Grade HR for Death (95% CI) p HR for Death or Progression (95% CI) p 1 1 (– –) – 1 (– –) – 2 1.00 (0.66–1.54) 0.98 0.92 (0.63–1.34) 0.69 3 0.66 (0.35–1.24) 0.20 0.79 (0.48–1.33) 0.38 Figure Figure
Crystal Structure of the Interaction of Human Urokinase Plasminogen Activitor with Its Receptor.
Authors: Huai Qing , Mazar Andrew P. , Parry Graham , Kuo Alice , Cines Douglas B. , Li Yongdong , Yuan Cai , Bian Chuanbing , Chen Liqing , Furie Bruce , Furie Barbara C. , Huang Mingdong
Published: April 2020

Urokinase-type plasminogen activator (uPA) and its cellular receptor (uPAR) mediate plasminogen activation. The uPA binds to uPAR with high affinity (Kd 0.1–1nM), thus localizing the generation of plasmin from plasminogen on the surface of a variety of cells. uPA-uPAR binding is also involved in other cellular functions and diverse pathophysiological processes such as tissue remodeling during wound healing, atherosclerosis, angiogenesis and tumor metastasis. We have determined the structure of uPAR complexed with the amino terminal fragment (amino acid residues 1–143) of uPA (ATF), which contains the uPAR binding domain, at 1.9 Å resolution by X-ray crystallography. Soluble uPAR (suPAR) and the ATF were expressed separately in stably transfected Drosophila Schneider 2 (S2) cells. The suPAR-ATF complex was crystallized by the sitting-drop vapor diffusion method. However, the diffraction of this crystal was limited to 3.1 Å resolution. Therefore, the Fab fragment of an antibody raised against suPAR, ATN-615, was used to facilitate suPAR-ATF crystallization. Crystals of the suPAR-ATF-ATN615 ternary complex were generated by microdialysis with 4% PEG4K, 5% ethylene glycol, 5% methanol, 0.05% sodium azide, 50 mM cacodylate pH 6.5. A complete data set of the ternary complex to 1.9 Å was collected using synchrotron radiation at the Advanced Photon Source (APS), Argonne National Laboratory. The crystals belong to the monoclinic space group, with unit cell parameters a=51.79 Å, b=86.81 Å, c=124.69 Å and β=94.54°. uPAR is comprised of three consecutive domains (D1, D2 and D3) that form the shape of a thick-walled teacup with a diameter of about 52 Å and a height of 27 Å. At the center of teacup and surrounded by three suPAR domains is a cone shape cavity with a wide opening (25 Å) and large depth (14 Å). ATF consists of a growth factor domain (GFD) and a kringle domain. Both domains pack more tightly in the complex structure compared with their unbound state. The GFD domain of uPA occupies part of the uPAR cavity and is primarily responsible for uPAR binding. The D1 domain of uPAR forms three hydrogen bonds and many hydrophobic interactions with the GFD domain of uPA, thus playing an important role in the binding of uPA. However, D2 and D3 of uPAR also have direct interactions with the GFD domain of uPA. The kringle domain of uPA sits outside the uPAR pocket, but forms some direct contacts with the D1 domain of uPAR. Therefore, the three domains of uPAR and two domains of uPA form a complementary interaction, which describes the structural basis for the high affinity binding of uPA to uPAR. This structure presents the first high resolution view of uPA-uPAR interaction, and may provide a new platform to design de novo uPA-uPAR antagonists.
Tissue Factor Activation Involves Disulfide Switching.
Authors: Chen Vivien M. , Ruf Wolfram , Berndt Michael C. , Hogg Philip J.
Published: April 2020

Tissue factor (TF), the essential cofactor for FVIIa, is required for activation of FX and FIX to generate thrombin. Transmembrane TF resides in a cryptic configuration on the cell surface with low procoagulant activity, however TF can be rapidly switched to an active configuration on exposure to ionomycin. The precise nature of this switch is unknown, however it is known to be an extracellular event (Wolberg et al Biophys Biochem Comm.272, 2000). The extracellular part of TF consists of 2 fibronectin type III domains. The disulphide-bond in the membrane proximal domain (Cys186–Cys209) is atypical for fibronectin domains in that it links adjacent strands in the same β-sheet, a cross-strand bond. The Cys186-Cys209 TF bond has the same unusual configuration as the disulphide-bond in the second domain of CD4, which controls CD4 function by switching between oxidized (disulphide) and reduced (dithiol) states (Matthias et al. Nature Immunol.3, 727, 2002). Ablation of the cross-strand bond severely impairs procoagulant activity (Rehemtulla et al. J. Biol. Chem.266, 10294, 1991). Labeling with a biotinylated maleimide, we demonstrate that cryptic tissue factor is reduced at the domain 2 disulfide and oxidised on activation. In HL60 cells, membrane based tissue factor procoagulant activity is blocked by the mono-thiol alkylator N-ethylmaleimide but increased by formation of the disulfide via the thiol oxidiser, HgCl2 or thiol cross-linkers, bismaleimidohexane and bismalemidoethane. These results indicate that the activation of TF involves a change of conformation of the domain 2 of TF caused by formation of the cross-strand cys186–cys209 disulfide bond. This is likely to be the physiological change that facilitates productive binding of FIX and FX in coagulation.
Rituximab Plus GM-CSF for Patients with Chronic Lymphocytic Leukemia.
Authors: Ferrajoli Alessandra , O’Brien Susan M. , Faderl Stefan H. , Wierda William G. , Ravandi-Kashani Farhad , O’Neal Brandi , Schlette Ellen , Albitar Maher , Kantarjian Hagop M. , Keating Michael J.
Published: April 2020

Treatment with single agent rituximab is associated with overall response (OR) rate of 15–35% in previously treated patients and up to 44% in untreated patients with CLL. The majority of the responses are partial (PR). GM-CSF was found to increase surface CD20 expression in vitro and potentiate ADCC and therefore improve the efficacy of rituximab. The combination of rituximab and GM-CSF has been investigated in follicular lymphomas and found to be associated with significant responses (McLaughlin et al. Ann. Oncol16:v68, 2005). Therefore, we designed a phase II study of rituximab plus GM-CSF in CLL. Patients were eligible for this study if they belonged to one of the following: Group 1: previously untreated patients, Rai stage 0-II and b2M >3 mg/mL, B symptoms or significant fatigue. Group 2: previously untreated patients ≥ 70 years with indication for treatment (NCI-WG criteria). Group 3: previously treated patients with evidence of active disease (NCI-WG criteria). All patients received 4 weekly infusions of rituximab 375 mg/m2 plus GM-CSF 250 mcg sc three times a week for 8 weeks. Prior to study entry patients were evaluated for genomic aberration (FISH), IgVH mutation status, ZAP70 expression, and number of CD20 antigen sites. Changes in self-reported fatigue were measured using the FACT-An fatigue scale. Eighty-two patients have been enrolled at this time and 55 are evaluable for response and toxicity. Responses (NCI-WG):

Pts CR (%) NPR (%)* PR (%) OR (%) *Nodular PR:residual lymphoid nodules present Group 1 9 7 7 (78) Group 2 20 3 2 12 17 (85) Group 3 26 2 3 9 14 (54) OVERALL 55 5 (9) 5 (9) 28 (51) 38 (69)

Toxicities attributable to the administration of GM-CSF consisted in Grade 1 injection site reactions in 25% and Grade 1–2 bone pain in 13%. Grade 3 thromobocytopenia was observed in 2% of the patients and Grade 3–4 neutropenia in 4%. Grade 3 infections were observed in 9% of the patients.

The following characteristics were correlated with responses.

Characteristic Patients CR% OR% FISH 11q-/17p-11 1 (9) 4 (36) 13q-/+12/normal 38 4 (11) 32 (84) p<.001 IgVH Unmutated 23 1 (4) 11 (48) Mutated 28 4 (14) 23 (82) p=.01 ZAP70 Positive 30 1 (3) 17 (57) Negative 23 4 (17) 17 (74) p=NS CD20 sites/cell ≤10,000 20 10 (50) >10,000 16 3 (19) 13 (81) p=.05

Treatment with rituximab plus GM-CSF was associated with a reduction of self-reported fatigue (median score reduction 7 points) in two thirds of the patients.

In conclusion, the combination of rituximab plus GM-CSF is well tolerated and associated with an encouragingly high response rate. Favorable genomic profile, mutated IgVH status and high level of CD20 expression predicted for response to this combination. Ongoing correlative studies aim to clarify the mechanism of action of this combination. Accrual to this study is ongoing.
The Interaction of the Wnt and Notch Pathways Modulates NK vs. T Cell Commitment.
Authors: Aoyama Keisuke , Varnum-Finney Barbara , Moon Randall T. , Bernstein Irwin D.
Published: April 2020

The Wnt and Notch signaling pathways have critical roles in cell fate decisions. However, the interaction of these pathways is poorly understood. Using highly purified Wnt3a and immobilized Notch ligand, Delta1ext-IgG, we investigated the mechanisms involved in Wnt and Notch signaling interactions during hematopoietic stem cell differentiation. When CD34+CD38- cord blood stem cells were cultured for 2 to 3 weeks with five growth factors (SCF 300ng/ml, Flt-3L 300ng/ml, TPO 100ng/ml, IL-6 100ng/ml, and IL-3 10ng/ml), most precursor cells lost CD34 expression and differentiated into mature cells, most of which were monocytes. However, as we previously reported, when cells were cultured with Delta1ext-IgG, we found an increased percentage of lymphoid progenitors (CD34+CD7+) and more mature lymphoid precursors (CD34-CD7+) in the cell population. The addition of purified Wnt3a (100ng/ml) alone without Delta1ext-IgG did not significantly change the percentage of CD7+ cells (1% vs. 3%). However, when both Wnt3a and Delta1ext-IgG were added, we saw an increased percentage of CD7+ cells (11% with Delta1ext-IgG alone to 27% with both) (Fig. 1A). Wnt3a also enhanced gene expression of CD3ε and preTα induced by Delta1ext-IgG. These results suggest that Wnt3a enhances the effect of Notch signaling on T-cell lineage development. To test the role of endogenous Wnt signaling, we added Dickkopf1, an inhibitor of Wnt signaling. When CD34+CD38- cells were cultured with Dickkopf1 (300ng/ml) alone for a 2 week, the percentage and number of CD56+ NK cells was unaffected. However, when cultured with Dickkopf1 and Delta1ext-IgG, the percentage and number of CD56+ NK precursor cells were increased (2% with Delta1ext-IgG alone vs. 17% with Dickkopf1 and Delta1ext-IgG; 0.2 ×105 vs. 1.5×105, p<0.01) (Fig. 1B). This result shows that decreased endogenous Wnt signaling enhances the generation of NK cells in the presence of Notch signaling. To address whether Wnt signaling affects Notch signaling by modulating protein levels, we assessed the amount of activated Notch1 intracellular domain with or without Wnt 3a by Western blot (Fig. 2A). After stimulation for 24 h, Wnt3a increased the amount of activated Notch1 intracellular domain induced by Delta1ext-IgG. Wnt3a also enhanced the expression of a primary target gene of Notch signaling, Hes1, determined by quantitative RT-PCR (Fig. 2B). In contrast, Dickkopf1 reduced Delta1ext-IgG-induced Hes1 expression. These results suggest that Wnt signaling directly modulates Notch signaling. Thus, these studies suggest that Wnt signaling is a key factor in cell fate determination at the point of NK/T cell commitment that is mediated via an interaction with Notch signaling. These studies also suggest that Wnt directly regulates Notch signaling by modulating protein turnover.

Fig. 1, Fig. 2 Fig. 1, Fig. 2.
Decreased Responsiveness of Mature Dendritic Cells from Cord Blood to Chemotaxis Induced by CXCR4 Ligand CXCL12, and CCR7 Ligand CCL19 May Explain Lowered GVHD Associated with Cord Blood Tranplantation.
Authors: Li Geling , Han Myung-Kwan , Broxmeyer Hal E.
Published: April 2020

Cord blood has been successfully used as a transplantable source of hemopoietic stem cells with a relatively low incidence of graft-versus-host disease (GVHD). Mature dendritic cells (DC), as the most efficient antigen presenting cells, play a critical role in the pathogenesis of GVHD and their function is intimately linked to their capacity to migrate. Chemokines and their G-protein linked receptors are of particular importance in DC migration. CCR7 and CXCR4 are two chemokine receptors expressed on mature DC. Binding of CCR7 ligand, MIP-3β/CCL19 and CXCR4 ligand, SDF-1/CXCL12 induce chemotaxis of mature DC and are pivotal for DC to initiate their functions in vivo. We hypothesized that the relatively lowered level of GVHD elicited by cord blood, compared to adult cells, might be due, at least in part, to a reduced capability of cord blood DC to migrate to lymphoid organs to initiate immune reactions and to organs such as liver and skin where GVHD pathogenesis usually occurs. To begin testing this hypothesis, we randomly picked 16 cord blood donors and 14 adult blood donors and did paired comparison study on chemotactic responses of cord blood and adult blood DC to CXCL12 and CCL19. Monocytes were isolated from both cord blood and adult blood and cultured with GM-CSF and IL-4 for 5 days to generate immature DC. LPS was added into cell culture for an additional 1 day to induce the maturation of these DC. Mature DC were harvested on day 6. We found that cord blood DC expressed significantly reduced surface levels of CCR7 compared with that of adult blood DC (p = .00067) and migrated at significantly lower efficiency towards the CCR7 ligand, CCL19 than did adult blood DC (p=0.029). Cord blood DC expressed significantly higher surface expression of CXCR4 than adult blood DC (p = .0018), but migrated at significantly lower efficiency towards CXCR4 ligand, CXCL12 than adult blood DC (p=0.00012). The lowered responsiveness of mature cord blood DC to chemotaxis by CXCL12 may reflect the enhanced truncation of CXCR4 we found on these cells. Also, cord blood DC migrated less well than adult blood DC toward the combination of CXCL12 and CCL19 than adult blood DC (p = .0014). These results suggest that low incidence and pathogenesis of GVHD observed in cord blood might be due, at least in part, to reduced capability of cord blood mature DC to migrate to critical organs where immune reaction and inflammation occur, effects reflecting lowered expression of CCR7 and enhanced truncation of CXCR4 on mature cord blood DC.
Individuals Expressing FcγRIIIA-158 V/V and V/F Show Increased NK Cell Surface Expression of FcgRIIIA (CD16), Rituximab Binding, and Demonstrate Higher Levels of ADCC Activity in Response to Rituximab.
Authors: Hatjiharissi Evdoxia , Santos Daniel Ditzel , Xu Lian , Verselis Sigitas , Modica Michael , Leleu Xavier , Hunter Zachary , Ho Allen W. , Manning Robert , Patterson Christopher , Anderson Kenneth C. , Fox Edward , Treon Steven P.
Published: April 2020

The homozygous expression of phenylalanine (F/F) at codon 158 of FcγRIIIa (CD16) is associated with inferior clinical responses to the anti-CD20 monoclonal antibody rituximab in patients with indolent non-Hodgkin’s lymphoma, in contrast to higher response rates observed for Waldenstrom’s macroglobulinemia patients expressing at least one valine (V/F, V/V) and follicular NHL patients who are homozygous for valine (V/V). We attempted elucidate the potential basic mechanism(s) behind these clinical observations by analyzing all the potential implications of this polymorphism among the 3 polymorphic subgroups at FcγRIIIA-158 (F/F, V/F, and V/V). We therefore used peripheral blood isolated natural killer (NK) cells from a pool of 52 unrelated healthy donors who were genotyped for FcγRIIIA-158. We evaluated allele-specific differences for FcγRIIIa gene expression by quantitative RT-PCR and demonstrated higher transcript levels among V/V (23.2 ng/mL) versus V/F (6.7 ng/mL) and F/F (6.2 ng/mL) (p<0.0001). We then determined protein levels for CD16 in the same subgroup of donors using quantitative flow cytometry. The number of CD16 receptors per NK cell was 105,947, 94,863, and 69,130 for the V/V, V/F and F/F donors, respectively and was significantly higher among donors who expressed at least one valine (V/V and V/F) versus F/F (p=0.033). We next determined rituximab binding affinity to NK cells from V/V, V/F and F/F donors following incubation at concentrations of 10–200 ug/ml, and use of an indirect competitive assay with the anti-CD16 monoclonal antibody 3G8 (Cancer Research 64:4664). Rituximab binding to NK cells was higher among donors expressing at least one valine at all concentrations evaluated, with mean rituximab binding (defined as % of inhibition of mean fluorescence intensity for 3G8) as follow: V/V (72%); V/F (53%); and FF (37%); (p=0.017). Lastly, we assessed rituximab dependent NK cell mediated cytotoxicity (ADCC) using CD20 expressing ARH-77 B-cells and observed higher levels of ADCC killing among V/V (82%) and V/F (80%) versus F/F (23%) donors at an effector: target cell ratio of 20:1. Taken together, these studies suggest that individuals who express at least one valine at FcγRIIIA-158 might have better clinical outcomes to rituximab therapy on the basis of increased FcγRIIIA-158 receptor expression, rituximab binding and ADCC mediated killing of tumor cells.
Bortezomib Therapy Alone and in Combination with Dexamethasone for Patients with Previously Untreated Multiple Myeloma.
Authors: Jagannath Sundar , Durie B. , Wolf J. , Camacho E. , Irwin D. , Lutzky J. , McKinley M. , Gabayan E. , Mazumder A. , Crowley J. , Vescio R. , Schenkein D.
Published: April 2020

Introduction: Bortezomib ± dexamethasone (dex) has demonstrated impressive activity in the treatment of relapsed and refractory myeloma. Bortezomib is currently approved in the U.S. for multiple myeloma patients who have received at least 1 prior therapy. We are conducting a phase 2 trial evaluating bortezomib alone or in combination with dex as first-line therapy in patients with myeloma.

Methods: Eligible patients had measurable disease and a Karnofsky performance score (KPS) ≥ 50%. Bortezomib 1.3 mg/m2 was administered by IV bolus on days 1, 4, 8, and 11 of a 3-week cycle for a maximum of 6 cycles. Oral dex 40 mg was given to patients who achieved < partial response (PR) after 2 cycles or < complete response (CR) after 4 cycles. European Group for Blood and Marrow Transplantation criteria were used to assess response, with the addition of a near CR (nCR) category, defined as nondetectable M-protein by electrophoresis but positive immunofixation and normal bone marrow.

Results: Fifty patients have been accrued to the study, and 40 are evaluable for response. Median age was 58 years, and 52% were men. The major myeloma subtypes were IgG (64%) and IgA (19%). The majority of patients were Durie-Salmon stage II (29%) or IIIA (43%). The major response rate (CR + nCR + PR) was 85%. The best response was observed after cycle 2 in 41%, after cycle 4 in 75%, and after cycle 6 in 85% of patients. Dex was added for 28 patients (70%): 17 patients at cycle 3, 10 patients at cycle 5, and 1 at cycle 6. An improved response after combination therapy was observed in 18 patients: 11 improved from MR to PR, 5 improved from stable disease (SD) to PR, 1 improved from SD to CR, and 1 improved from SD to minor response (MR). Twelve patients underwent stem cell transplantation, and all had complete hematologic recovery. Thirteen patients discontinued from the study: 9 due to adverse events, 2 due to progressive disease, and 2 withdrew. The most common adverse events (grade ≥ 2) were neuropathy (26%), fatigue (22%), constipation (17%), and neutropenia (14%). Two patients developed grade 4 events, 1 with neutropenia and 1 with thrombocytopenia.

Conclusion: Bortezomib alone or in combination with dex was highly active in the first-line treatment of patients with myeloma. The combination of bortezomib + dex demonstrated additional benefit. Toxicities were manageable and reversible. Although experience has been limited to date, stem cell transplantation was successful in all attempts, indicating that bortezomib-based therapy should be further explored as an induction regimen before transplantation.
Treatment Outcome of Children over 10 Years and Young Adults with ALL in Sweden: A Comparison between Paediatric- and Adult Protocols.
Authors: Heyman Mats , Gustafsson Goran , Smedmyr Bengt , Soderhall Stefan , Hallbook Helene
Published: April 2020

On behalf of the Swedish Adult ALL Group and the Swedish Childhood Leukemia Group. Several recent studies have shown superior outcome for older teenagers and young adults with acute lymphoblastic leukemia (ALL) treated in pediatric oncology departments compared with adult hematology departments. However, some of these comparisons have been hampered by either biased risk-group stratification (adult protocols considering young adults as lower risk and pediatric protocols considering the same age-group as high-risk), or a markedly higher treatment-related mortality for the adult treatment. During the 1990s in Sweden, adolescents with ALL were treated in a pediatric oncology unit or in an adult haematological unit, depending on the initial referral. For both groups there were well established national (and for children Nordic) treatment protocols designed by the Swedish Adult ALL Group and the Nordic Society of Pediatric Haematology and Oncology (NOPHO), but with different treatment strategies. In the NOPHO protocol, the teenagers were excluded from the lowest risk-group, but were only treated as high-risk if additional risk-factors were present, whereas age was not a risk-factor in the adult protocol.

The clinical characteristics, treatment and outcome for patients with ALL aged 10–40 years treated either according to the NOPHO protocol NOPHO ALL-92 (1992–2000), or the Swedish Adult ALL Group protocol 1994–2000, were reviewed. The impact of these treatment protocols on remission rate and survival was analysed. In total, 243 patients with B-precursor and T-cell ALL were treated according to the protocols. There was a significant difference in remission rate between the pediatric protocol (99%, n=144) and the adult protocol (90%, n=99) (p<0,01). Furthermore, the event-free survival (EFS) was superior for patients treated according the pediatric protocol compared to patients treated according to the adult protocol (p<0.01). Both protocols included response to induction treatment in the treatment stratification, but the difference in outcome was significant also when only pre-treatment risk-criteria, not including initial treatment response, were considered. Patients aged 15–25 had a superior outcome compared to patients aged 26–40 within the adult protocol group (p=0.01). Both protocols had treatment related mortality rates well below 5%. Treatment protocol was identified as an independent risk-factor in multiple regression analyses and was the only independent predictor of outcome in the 15–20 year age group. The results indicate that adolescents may benefit from treatment according to the pediatric protocol. Future trials are called for to determine whether young adults could benefit from similar treatment.
Adoptive Transfer of Adenovirus Specific T-Cells in Children with Systemic Adenovirus Infection after Allogeneic Stem Cell Transplantation.
Authors: Feuchtinger Tobias F. , Matthes-Martin Susanne , Richard Celine , Lion Thomas , Hamprecht Klaus , Peters Christina , Schumm Michael , Beck Robert , Niethammer Dietrich , Handgretinger Rupert , Lang Peter
Published: April 2020

Allogeneic stem cell transplantation (SCT) has become an increasing treatment option for a variety of malignant and non-malignant disease. During immune reconstitution the host is at significant risk for viral infections. Human adenovirus (HAdV) infection is especially in children an important and serious complication. Virus-specific T-cells are essential for the clearance of HAdV, since antiviral chemotherapy has been insufficient to date. We present a new treatment option using virus-specific donor T-cells for adoptive transfer of immunity to patients with systemic HAdV-infection. We isolated in 6 patients with systemic HAdV-infection after SCT virus-specific T-cells of the donor, according to INF-γ secretion after short in vitro stimulation with viral antigen, resulting in a combination of CD4+ and CD8+ T-cells. Between 5-50x103/kg T-cells were infused for adoptive transfer. For follow-up, the infection and the in-vivo expansion of infused T-cells were evaluated. Isolated cells showed high specificity and markedly reduced but residual alloreactivity in-vitro. In three of four evaluable patients the infused T-cells underwent an in-vivo expansion and in these three patients the viral load decreased in peripheral blood after adoptive T-cell transfer. In-vivo expansion of specific T-cells was dose-independent. T-cell infusion was well tolerated. One patient experienced GvHD°II of the skin after T-cell transfer. In conclusion specific T-cell immunotherapy as a new treatment approach for children was performed in 6 cases of systemic HAdV-infection after allogeneic SCT. Induction of a specific T-cell response through adoptive transfer has been shown feasible and effective to protect from HAdV-related complications.
Simultaneous Loss of Mouse Dematin and beta-Adducin Results in Severe Erythrocyte Fragility and Hemolytic Anemia.
Authors: Khan Anwar A. , Chen Huiqing , Gilligan Diana M. , Peters Luanne L. , Messick Joanne , Haschek-Hock Wanda M. , Liu Fei , Ostafin Agnes E. , Chishti Athar H.
Published: April 2020

The mechanical strength and stability of the erythrocyte membrane are regulated by a network of proteins that participate in both horizontal and vertical interactions. The actin-containing junctional complexes, located at the tail ends of spectrin molecules, serve as the critical regulatory nodes for the maintenance of membrane stability. Dematin and beta-adducin, two actin-binding proteins of the junctional complex, are known to play essential roles in the regulation of erythrocyte shape and membrane stability, as revealed recently by the development of mouse knockout models. Here, we show that simultaneous loss of functional dematin (headpiece domain deletion) and beta-adducin results in severe fragility and abnormal shape of erythrocytes, despite the presence of major skeletal proteins. Adducin/Dematin Double Knockout (ADKO) mice are viable and can be distinguished at birth by their pallor with pronounced spleomegaly and regenerative hematopoiesis. Hematological evaluations show a reduction of erythrocytes, reduced hematocrit and hemoglobin, and a ~52% increase in the number of reticulocytes. The presence of a variety of misshapen and fragmented erythrocytes in the ADKO mice correlates with increased osmotic fragility and reduced erythrocyte life span in vivo. Despite an apparently normal composition of ghosts and skeletal proteins, the retention of spectrin in the ADKO erythrocyte plasma membrane is significantly compromised. Atomic force microscopy (AFM) revealed similar volume parameters in the four genotypes examined, but an increased grain size, and a decreased filament number in the ADKO erythrocyte membrane. In addition, highly aggregated, disassembled, and irregular features were visualized by AFM in the ADKO erythrocyte membrane. Staining of filamentous actin provided further evidence for the existence of large protein aggregates in the ADKO erythrocyte membrane. Together, these results demonstrate a crucial function of dematin and beta-adducin in the maintenance of erythrocyte shape and membrane stability, and more importantly, suggest the existence of an alternate mechanism for the linkage of junctional complexes to the plasma membrane.
Comparison of Three Radiation Dose Levels after EBVP Regimen in Favorable Supradiaphragmatic Clinical Stages (CS) I-II Hodgkin’s Lymphoma (HL): Preliminary Results of the EORTC-GELA H9-F Trial.
Authors: Eghbali Houchingue , Brice Pauline , Creemers Geert-Yan , van Marwijk Kooij Marinus , Carde Patrice , Van’t Veer Mars B. , Lugtenburg Elli , Sonet Anne , Sebban Catherine , Blanc Michel , Raemaekers John M. , Voillat Laurent , Rieux Chantal , Noordijk Ed M. , Henry-Amar Michel
Published: April 2020

Preventing late toxicity has become the cornerstone in the management of HL as global results greatly improved regarding event-free (EFS) and overall survival (OS) rates. With this aim, the EORTC and GELA groups conducted a trial (H9-F) comparing three radiation doses in patients in complete remission (CR/CRu) after chemotherapy. Adult patients with supradiaphragmatic CS I-II HL and favorable features (age < 50, and CS I-II2–3, and A + ESR < 50 or B + ESR < 30, and MT ratio < 0.35) were eligible. After 6 cycles of EBVP (epirubicin, bleomycin, vinblastine, prednisone), CR/CRu patients were randomized between 36 Gy involved-field radiotherapy (IF-RT), 20 Gy IF-RT and no RT. From September 1998 to May 2004, 783 patients were enrolled in 111 institutions from 10 European countries. Inclusion of patients in the no-RT arm was stopped in May 2002 because stopping rules were met (i.e. > 20% of events). After 6 EBVP response rates were 50% CR, 26% CRu, 18% PR, 3% no change or progression, and 3% unspecified. Of the 591 CR/CRu patients, 13 (2%) were not randomized (6 refusals, 3 protocol violations, 4 causes unspecified). After a median follow-up of 51 months (range 14–81), 130 events (36 progressions, 91 relapses, 1 toxic death, 2 deaths of second cancer) were observed. At July 2005, the 4-year EFS and OS rates are as follows:

Treatment No. Pts. Prog. /Rel. /Death 4-yr EFS 4-yr OS 6 EBVP + IF-RT 36 Gy 239 2 /18 /0 88% 98% 6 EBVP + IF-RT 20 Gy 209 5 /16 /0 85% 100% 6 EBVP + no RT 130 1 /35 /1 69% 98% P value < 0.001 0.241 6 EBVP, no random. 205 28 /22 /2 68% 93%

Non-randomized patients experienced a significantly worse OS than did complete responders (P=0.0012). Relapses in nodal sites were more frequent in the no RT arm (35 in unirradiated sites) than in RT arms (6 + 10 in irradiated sites). 15 patients have died, 10 of progressive disease, 2 of treatment-related complication, 1 of intercurrent disease and 2 of second cancer (1 AML, 1 NHL). In favorable early stage HL patients who achieve CR(u) after 6 cycles of EBVP, omission of IF-RT leads to an unacceptable failure rate; In contrast, with the current follow-up, IF-RT 20 Gy provides results equivalent to those of IF-RT 36 Gy.
Risk Adapted BEACOPP Regimen Based on Early Scintigraphy Can Reduce the Cumulative Dose of Chemotherapy for Standard and High Risk Hodgkin Lymphoma (HD) with No Impairment of Outcome.
Authors: Dann Eldad J. , Shalom Rachel Bar , Haim Nissim , Tamir Ada , Ben Shachar Menachem , Avivi Irit , Zuckerman Tsila , Kirschbaum Mark , Goor Odelia , Libster Diana , Rowe Jacob M. , Apelbaum Ron
Published: April 2020

A prospective study evaluating the outcome of HD pts whose chemotherapy was tailored based on results of scans performed after 1or 2 cycles of chemotherapy reducing the cumulative chemotherapy for early responders and maximizing the dose for late responders. Study was initiated in 1999 for patients with classical HD age 18–65y. Eligibility criteria were either stage I, II with unfavorable features (see table) or any stage III or IV HD. Those with I-II B or bulky or any Stage III-IV were defined according to IPS. Patients with standard risk were treated with 2 cycles of standard BEACOPP (SB). Those with high risk were treated with 2 cycles of escalated BEACOPP (EB). GA67 or FDG PET scan was performed at diagnosis and after the 1st or 2nd cycle for 57 and 55 pts respectively. If the early scan remained positive then additional 4 cycles of EB were administered, otherwise, SB was given. 108 pts 44 F64 M age 18–63y (median 33) who had at least 1 year of follow up are reported. The CR rate was 97%. The 4-yr disease free (DFS) and overall survival (OS) is 84% and 90% respectively at a median follow up of 35-month (5-70). The 4-yr DFS and OS are similar for standard and high risk patients. The disease progressed in 3/10 pts with an interim positive PET versus 1/40 negative scans (p<0.03) and in 1/13 (8%) interim positive Ga67scan versus 7/44 (16%) negative scan (NS). Negative predictive value of early normal PET and GA67 scans are 98% and 84% respectively. Only one patient developed secondary leukemia following salvage therapy and high dose chemotherapy. One patient died during therapy from unrelated cause.

Conclusion: PET is a useful tool for early interim decision regarding dose of chemotherapy. Early PET allowed for chemotherapy reduction in 80% of high-risk pts. Only 14% of standard risk patients required dose intensification. Similar DFS and OS can be obtained regardless of IPS. 6 cycles of risk adapted BEACOPP are effective.

Standard Risk High Risk Standard BEACOPP Escalated BEACOPP Stage I, II ≥ Age 50yrs; ≥ 4 sites; “B” symptoms or Bulky, stage III,IV IPS 0-2 IPS 3-7
Mechanisms of γ-Globin Gene Promoter Activation by HBF-Inducing Short Chain Fatty Acid Derivatives.
Authors: Mankidy Rishikesh , Faller Douglas V. , Boosalis Michael S. , Bohacek Regine , Perrine Susan P.
Published: April 2020

High-level γ-globin gene expression for definitive therapy of beta globin disorders likely requires γ-globin gene promoter activation, dissociation of repressor complexes, and/or local chromatin modification. To define the molecular mechanisms employed by high-potency γ-globin inducers, the effects of butyrate on transcriptional co-regulatory protein recruitment to the human γ- and β-globin gene promoters were defined and compared to those of novel SCFAD γ-globin inducers recently identified through computational modeling. One particularly potent SCFAD inducer, RB7, which has no inhibitory effect on class I HDAC enzymes (HDAC1, HDAC2, and HDAC3), demonstrated 5-fold induction, (2.5 to 3-fold greater γ-globin gene-inducing activity than butyrate), in a transcriptional reporter assay which detects only strong γ-globin gene inducers. Chromatin immunoprecipitation (ChIP) assays performed on the integrated human gamma and beta globin gene promoters demonstrated that RB7, butyrate, and other transcriptionally-active SCFADs, induced dissociation of HDAC-3, and its adaptor protein NCoR, from the γ-globin gene promoter, with no such effect on the β-globin promoter, coincident with recruitment of RNA Polymerase II to the γ-globin promoter and initiation of γ-globin transcription. In a reciprocal fashion, as HDAC-3 dissociated, the chromatin-modifying ATPases Brg-1 and Brm were recruited specifically to the γ-globin gene promoter in response to the active SCFADs. The ability of the novel SCFADs to induce dissociation of HDAC-3 (but not HDAC-1 or -2) from the γ-globin promoter was proportional to the degree of γ-globin promoter induction by these agents. Knockdown of HDAC-3 by siRNA induced transcription of γ-globin specifically, demonstrating the functional importance of the activity of SCFADS in dissociating HDAC-3 from the γ-globin gene promoter. These studies demonstrate new dynamic alterations in transcriptional regulatory complexes specifically-associated with high-level activation of γ-globin and also identify essential molecular targets for future therapeutic interventions.