bluuebunny/crossref_metadata_2025 huggingface dataset:
Gadd45a and Gadd45b Protect Haematopoetic Cells from Ultraviolet Radiation Induced Apoptosis Via Distinct Signaling Pathways.
Authors: Gupta Mamta , Gupta Shiv K. , Hoffman Barbara , Liebermann Dan A.
Published: April 2020

Gadd45 expression, which is stress inducible, has been associated with growth arrest, but the exact role of gadd45 family genes in apoptosis still remains unclear. We have found that myeloid progenitor cells from gadd45a and gadd45b-deficient mice are more sensitive to ultra-violet radiation, VP-16 or daunorubicin induced apoptosis. indicating that gadd45a or gadd45b protect haematopoetic cells from DNA damaging agents. To determine, how gadd45a or gadd45b proteins exert their anti-apoptotic function, bone marrow cells from wild-type and gadd45a or gadd45b deficient mice were exposed to ultraviolet radiation (UV) and analyzed for expression of stress responsive kinases, including JNK and p38. It was observed that P38 and JNK were activated in wt bone marrow cells in response to UV but not in bone marrow cells defecient in gadd45a. Also, the transcription factor NF-kB was activated in wt bone marrow cells, but not in gadd45a−/− cells. The pharmacological inhibitor SB203580 specific for p38, increased apoptosis in reponse to UV, indicating that p38 is implicated in signaling myeloid cell survival. SB203580 was observed also to inhibit the expression of certain NF-kB target genes, including cIAP-1, c-IAP-2, bcl-2 and bcl-xl, in gadd45a+/+ cells but not in gadd45a deficient bone marrow cells. Taken together this data provides first evidence for the role gadd45a plays in the control of hematopoietic cell survival in response to UV, via modulation of P38 MAPK and NF-kB signaling pathways. Unlike in gadd45a−/− bone marrow cells, p38 activation appeared not to be impaired in gadd45b−/− cells, indicating that gadd45b is not involved in p38 activation in myeloid cells. However, UV induced JNK activation was sustained in gadd45b−/− myeloid cells compared to wt cells, indicating that gadd45b is a negative modulator of UV induced JNK signaling in myeloid cells. UV induced activation of MKK4 an upstream regulator of JNK also was impaired in gadd45b−/−. NF-kB was also found activated in wt cells, but not in gadd45b−/− cells. This data indicates that in bone marrow cells exposed to UV, NF-kB induced expression of Gadd45b plays a protective role against UV induced apoptosis via inhibition of MKK4 kinase which in turn results in suppression of JNK activity. Taken together this data provides evidence that Gadd45a and Gadd45b protect haematopoetic cells from genotoxic-stress induced apoptosis via distinct signaling pathways.
A Novel Sphingomyelin Synthase Gene, SMS1-Regulated Sphingomyelin/Ceramide-Rich Microdomain Plays a Crucial Role in Fas-Mediated Apoptosis.
Authors: Miyaji Michihiko , Yamaoka Shohei , Xiong Jin Zhe , Amakawa Ryuichi , Mimori Tsuneyo , Fukuhara Shirou , Umehara Hisanori , Okazaki Toshiro
Published: April 2020

Lipid microdomain consisting of sphingolipids, glycosphingolipids and cholesterol plays an important role in mediating cell death signals. Although functional analysis of lipid microdomain is conventionally performed by exclusion of cholesterol tightly binding with sphingomyein (SM), the exact role of SM in microdomain in signal transduction remains to be cleared. Since we for the first time obtained cDNA (SMS1) responsible for SM synthesis, it became possible to investigate the role of SM in the membrane without affecting cholesterol content. We here examine the role of SM/ceramide-rich microdomain in Fas-induced apoptosis using the SM-deficient cells and its revertant cells by transfection with SMS1.

SM synthase-defective WR19L cells transfected with human-Fas gene (WR/Fas-SM(-)), and its functional revertant cells by transfection with SMS1 (WR/Fas-SMS1) were established. Both SMS1 cells and SM(-) cells expressed similar levels of CD44, CD90, CD95 (Human) and LFA-1 by FACS analysis. Although the levels of cholesterol and GM1-ganglioside on the surface of the membrane were similar in both cells, SM was detected only on the surface of SMS1 cells by FACS and confocal microscopy analysis. Fas crosslinking induced not only higher rate of apoptotic cell death, but also translocation of Fas into TritonX-100-insoluble fractions with a significant increase of ceramide in SMS1 cells as compared with SM(-) cells. After increase of SM and ceramide in microdomain, more efficient aggregation of Fas was detected with increase of DISC formation, resulting in activation of caspase-8 and 3 and decrease of mitochondrial membrane potential in SMS-1 cells than SM(-) cells. Our results clearly demonstrate that SM and ceramide in microdomain of plasma membrane plays a crucial role in Fas clustering and aggregation requires for activation of apoptosis-inducing signals.
Casein Kinase Iε Downregulates PI3K/Akt Signaling, Followed by Genotoxic Stress-Induced Apoptosis of Hematopoietic Cells.
Authors: Okamura Atsuo , Yamamoto Katsuya , Yakushijin Kimikazu , Urahama Norinaga , Minagawa Kentaro , Sada Akiko , Hato Akio , Nishikawa Shinichiro , Yokoyama Kazuaki , Ito Mitsuhiro , Matsui Toshimitsu
Published: April 2020

Casein kinase I (CKI) ε is involved in cytokine-induced hematopoietic cell differentiation by directly interacting with the suppressor of cytokine signaling 3 (SOCS3) and/or β-catenin. Here, we show that CKIε plays an important role in hematopoietic cell survival through modifying Phosphatidylinositol 3-kinase (PI3K)/Akt signaling. Introduction of wild-type (WT)-CKIε into murine interleukin-3 (IL-3)-dependent myeloid progenitor 32D cells increased the sensitivity to genotoxic stress, such as γ-irradiation, anti-cancer drug and cytokine-deprivation, whereas kinase-negative (KN)-CKIε suppressed it. While IL-3-induced Akt phosphorylation at Ser473 was sustained by KN-CKIε, it was attenuated by WT-CKIε. Similarly, in human promyelocytic leukemia HL-60 cells, the increase of phosphorylated Akt induced by all-trans retinoic acid was accompanied by the decrease of CKIε expression. A specific inhibitor for CKIε, CKI-7 also increased the phospho-Akt as well as phospho-PTEN (Ser380/Thr382/383; an inactive form of PTEN). Functional expression of CKIε activated PTEN by their physical association, following the decrease of phospho-Akt. The present studies suggest that the expression of CKIε downregulates PI3K/Akt signaling through PTEN, increasing the sensitivity to apoptotic signals. Taken together with the fact that CKIε is ubiquitously expressed in normal hematopoietic cells, CKIε might be critical for prevention against genotoxic stress-induced leukomogenesis. In other words, CKIε could be a candidate of the tumor suppressor in hematological malignancies as well as cancer.
Expression of Complex Junction Proteins in Hematopoietic Progenitor Cells.
Authors: Straub Beate K. , Eckstein Volker , Grund Christine , Miesala Katrin , Horsch Kerstin , Wuchter Patrick , Gottschling Sandra , Franke Werner W. , Ho Anthony D.
Published: April 2020

The interaction of hematopoietic stem cells with the microenvironment plays a pivotal role in regulating self-renewal and differentiation. N-cadherin-based adherens junctions have been reported to be involved in this process (Puch et al., 2001; Zhang et al., 2003). However, the molecular composition of such junctions in hematopoietic progenitor cells (HPC) remains to be defined as well as their function. CD34+ cells and CD34+/CD38− cells were isolated from human umbilical chord blood (CB). In immunoblotting, CD34+ cells were positive for the transmembrane proteins N- and E-cadherin, cadherin 11 (low amounts) and the cytoplasmic plaque proteins α- and β-catenin, protein p120ctn, vinculin, spectrin/fodrin and actin as markers for adherens junctions as well as for ezrin, moesin and drebrin, actin-binding proteins known to be enriched in cellular protrusions. Other classical cadherins such as VE-, P-, R-cadherin or cadherin-6 were negative. Moreover HPCs were positive for the intermediate filament cytoskeletal protein vimentin, the integral heparan sulphate proteoglycans syndecan-1 (CD138), syndecan-2 (fibroglycan), syndecan-3 (N-syndecan) and syndecan-4 (ryudocan, amphiglycan) and the gap junction protein connexin 43. CD34+/CD38− cells showed positive reactions with antibodies against vimentin, ezrin, vinculin, α- and β-catenin and with a pan cadherin antibody, with no obvious differences to CD34+/CD38+ and Kg1a cells. In parallel, we established single- and double-immunohistochemistry with antigen-retrieval-treated, formalin-fixed, paraffin-embedded bone marrow using peroxidase, alkaline phosphatase and glucose oxidase coupled secondary antibodies. The architecture of human, rat and bovine bone marrow tissue was analyzed with a panel of antibodies against components of adhering, gap and tight junctions. Immunohistochemical sections of bone marrow incubated with the various antibodies remarkably showed the presence of all these antigens in their natural microenvironment. Intercellular junctions of rat and bovine bone marrow were also observed with transmission electron microscopy. Our results show that HPC are equipped with components of cadherin-catenin-based adherens junctions. Immunoprecipitation and immunolocalisation experiments are underway to further elucidate the homo- and heterotypic intercellular junctional complexes between HPC and stromal cells in vitro and in vivo.
Promotion of Chimerism and Tolerance by Flt3 Ligand-Mobilized Facilitating Cells Is Associated with Upregulation of CXCR4 and SDF-1.
Authors: Huang Yiming , Kucia Magda , Rezzoug Francine , Ratajczak Mariusz Z. , Tanner Michael , Schanie Carrie , Xu Hong , Fugier Isabelle , Ildstad Suzanne T.
Published: April 2020

Facilitating cells (FC) are a CD8+/TCR− subpopulation of bone marrow (BM) cells that enhance engraftment of hematopoietic stem cells (HSC) in allogeneic recipients without causing graft-versus-host disease. They also significantly enhance engraftment of limiting numbers of HSC in syngeneic recipients. Treatment of mice with Flt3 ligand (Flt3-L) induces significant expansion of FC and HSC in the BM and their mobilization into peripheral blood (PB). In the present study, we evaluated the function of Flt3-L expanded FC and potential mechanism of action. Flt3-L treatment resulted in an 8.5-fold increase of FC in BM and a 100-fold increase in PB compared to untreated mice. When FC from the PB and BM of Flt3-L-treated donors were transplanted with HSC into allogeneic recipient mice, BM FC exhibited significantly impaired function while PB FC were potently functional (Fig. a). Strikingly, this correlated with an increase in mRNA for the chemokine SDF-1 and its receptor, CXCR4, which were significantly increased in PB FC (≥10 fold) and decreased in Flt3-L-expanded BM FC as compared to untreated control BM FC (Fig. b). The impaired function for BM FC was restored within 5 days after cessation of Flt3-L-treatment. Taken together, these data suggest that FC can be mobilized efficiently with Flt3-L and their engraftment facilitating function is associated with upregulation of transcripts for SDF-1 and CXCR4, suggesting that FC may chemoattract HSC by secreting SDF-1 and enhance their homing to the new microenvironment after transplantation. The fact that FC express CXCR4 suggests that they may co-migrate with HSC to the hematopoietic microenvironment after transplantation, being chemoattracted by BM-derived SDF-1. In summary, we provide evidence that FC are mobilized with HSC; the SDF-1-CXCR4 axis is involved in FC:HSC interaction; and FC co-migrate with HSC in response to an SDF-1 gradient.

Figure Figure
Early Kinetics of Bone Marrow Cell Engraftment in Bone after Transplantation: Insights into the Mechanism of Engraftment.
Authors: Garlits John E. , Dominici Massimo , Hofmann Ted J. , Horwitz Edwin M.
Published: April 2020

Despite their anatomic proximity and recognized physiologic relationship, the interactions between bone and bone marrow (BM) are incompletely understood. Consistent with our data from clinical BMT in children with osteogenesis imperfecta, murine models show significant engraftment of transplanted donor BM cells in the osteogenic compartment early after BMT, which declines to a nearly undetectable level by 9 months post-BMT. To gain insight into the mechanism of donor BM cell engraftment in bone, we studied the kinetics of hematopoietic and osteopoietic engraftment. Lethally irradiated (1100 cGy) FVB/N mice were transplanted with 2x106 BM cells transduced with a retroviral vector encoding the green fluorescent protein (GFP). Transduction efficiency, assessed by flow cytometry, was 53%. Engraftment in the hematopoietic and osteopoietic compartments was analyzed at 2, 4, or 6 weeks post-transplant (wpt). Flow cytometric analyses of blood leukocytes, as a marker of functional hematopoietic marrow, showed 66% ± 2% GFP+ (mean ± SEM) at 2 wpt (n = 8 for each time point), increased at 4 wpt to 73% ± 2% and plateaued at 77% ± 2% by 6 wpt (p < 0.01). Erythrocytes and platelets exhibited a similar profile. BM cellularity, estimated by microscopic evaluation, increased from approximately 50% to 80% while GFP expression was observed in about 50% of marrow cells and remained at that level from 2 to 6 wpt. Analysis of 5 immunostained bone sections per animal revealed 40% ± 4% GFP+ osteoblasts at 2 wpt with a decline to 20% ± 2% by 6 wpt (p < 0.01). Mature osteocytes were 20% ± 3% GFP+ at 2 wpt, then declined to 10% ± 3% by 6 wpt (p < 0.05). Thus, both hematopoietic and osteopoietic engraftment are early events; however, donor-derived hematopoietic BM proliferates over the first 6 weeks post-BMT while the donor-derived osteopoietic cell population declines. Our data suggest that either the transplantable marrow osteopoietic cell is a progenitor capable of a single burst of differentiation or that bone specific mechanisms related to the BMT procedure underlie the capacity of BM cells to engraft in the osteopoietic environment. Elucidating this cellular pathway will be a key element to the development of BM cell therapy for mesenchymal tissues.
G-CSF-Stimulation of Human Marrow Stromal Cells Induces In Vitro Migration of Hematopoietic Progenitor Cells Involving MMP-2 and MMP-9 but Not MMP-1.
Authors: López Adriana , Chabot Valérie , Vaudin Pascal , Clément Nathalie , Hérault Olivier , Charbord Pierre , Domenech Jorge
Published: April 2020

The mechanism of G-CSF-induced hematopoietic progenitor/stem cell (HPC/HSC) mobilization from bone marrow microenvironment to peripheral blood has been related to local production by neutrophils of proteases, including elastase, cathepsin G and metalloproteinases (MMPs). We previously showed that in vitro migration of hematopoietic cells across a layer of human marrow stromal cells (MSC) is induced when stimulated by G-CSF. In the present study, we investigated the role of MMPs produced by MSC used for the trans-stromal migration of MO7e line and marrow CD34+ cells. Normal human MSC were previously grown to confluence on Transwell® filters (pore diameter 5-μm) and then stimulated or not by IL-1 (15 U/mL/Day) or G-CSF (15 ng/mL/Day) for three consecutive days. In a second step, MO7e or marrow CD34+ cells, put in the upper chamber, were allowed to migrate through the layers (4 hrs, SDF-1 100 ng/mL in the bottom chamber). The contribution of MMPs was evaluated in this in vitro trans-stromal migration assay using specific blocking monoclonal antibodies (mAb), anti-MMP-1, anti-MMP-2 and anti-MMP-9 (each at 5 μg/mL). The amounts of antigenic MMP-2 and MMP-9 produced by MSC were evaluated in cell supernatants by enzyme-linked immunosorbent assay (ELISA) and gelatinolytic activity of MMPs by zymography. mRNA expression of MMPs in MSC was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR). None of the MSC layers used contained detectable hematopoietic cells since they were all CD45-. Migration of MO7e cells across unstimulated or G-CSF-stimulated stromal cells was significantly inhibited by specific mAb. Percentages of inhibition were 25+/−5 % and 54+/−9 % for anti-MMP-2 mAb, and 18+/−6 % and 53+/−9 % for anti-MMP-9 mAb, respectively. Using the combination of anti MMP-2 and anti-MMP-9, inhibition reached 71+/−24 % across stimulated layers. In contrast, anti-MMP-1 mAb did not show any significant inhibition. Migration of CD34+ across G-CSF-stimulated stromal cells was similarly inhibited with percentages of 38+/−18 % for anti-MMP- 2 mAb, and 34+/−11 % for anti-MMP-9 mAb, while migration was not inhibited again, using anti-MMP-1 mAb. In parallel, we demonstrated that MSC did express G-CSF receptor by RT-PCR. Production of MMPs by unstimulated and G-CSF-stimulated MSC was detected by ELISA, zymography and RT-PCR for MMP-2, and by zymography and RT-PCR for MMP-9. In conclusion, G-CSF-induction of HPC trans-stromal migration involves MMP-2 and MMP-9 but not MMP-1. These findings suggest that microenvironment, in the absence of neutrophils, could have a significant role in G-CSF mobilization of HPC/HSC. A local production of MMP-2 and MMP-9 by marrow stromal cells could be critical in the egress of HSC out of the hematopoietic niche.
The Role of CXCR4 Inhibitors as Novel Antiangiogenesis Agents in Cancer Therapy.
Authors: Ghobrial Irene M. , Timm Michael M. , Hedin Karen E. , Witzig Thomas E.
Published: April 2020

Angiogenesis plays an important role in tumor growth. Endothelial cells express the chemokine receptor CXCR4, and interact with its ligand SDF-1/CXCL12. Previous studies have demonstrated that the ERK MAP kinase and the PI3Kinase pathways are activated in response to SDF-1 stimulation. In this study, we investigate the effect of the role of inhibitors of CXCR4, ERK MAP kinase and PI3Kinase on angiogenesis. The AngioKit (TCS Cellworks, U.K) is a 24 well plate in which human endothelial cells are co-cultured with other human myoblasts and fibroblasts in a specially designed medium. Control wells in the kit include media alone, VEGF (+control) and suramin (-control). Test samples were added on the day the kits arrive, then changed on days 4, 7, and 9, and stained on day 11 with CD31 (PECAM). The wells are then photographed and subjected to image analysis. The software measures angiogenesis as total tubule length per well in microns. Test samples can then be compared to the control wells to determine the drugs affect on angiogenesis in vitro. The following drugs were tested in this angiogenesis model system, a human CXCR4 neutralizing antibody (MAB 171, R&D systems, MN), SDF-1, the MAP kinase inhibitor PD098059, and the PI3Kinase inhibitor LY294002. Treatment with the CXCR4 inhibitory antibody, PD098059, and LY294002 caused marked decrease in angiogenesis (below the level of the negative control suramin). Inhibition of angiogenesis below the level of suramin was first detected at 1mg/ml CXCR4 antibody, and10mg/ml CXCR4 antibody resulted in complete inhibition of angiogenesis. The effect of PD098059 on angiogenesis was dependent on its concentration; 20mM PD098059 inhibited angiogenesis while lower concentrations did not. These results are consistent with the drug’s known concentration-dependent inhibition of MEK-1 and indicate that the MEK-1 inhibitor leads to angiostasis secondary to its specific inhibitory effect on MEK-1. The lowest level tested of 1μM LY294002 led to inhibition of angiogenesis, and 50μM of LY294002 led to complete abrogation of angiogenesis. SDF-1 has been reported to be angiogenic. In this model system, the effect of SDF-1 alone on angiogenesis was subtle. However, the endothelial cells used in this model system may be secreting endogenous SDF-1 leading to the saturation of the CXCR4 receptor and minimal effects of exogenous SDF-1 stimulation. This was demonstrated by the significant effect of the CXCR4 inhibitor on angiogenesis without the addition of exogenous SDF-1. These results indicate that CXCR4 inhibition and its downstream pathways PI3K and ERK MAP kinase lead to significant inhibition of angiogenesis, and suggest that selective inhibitors of CXCR4 may be useful agents to inhibit angiogenesis.
Reduction of Combined Modality Treatment Intensity in Early Stage Hodgkin’s Lymphoma: Interim Analysis of the HD 10 Trial of the GHSG.
Authors: Diehl Volker , Brillant C. , Engert A. , Wolf J. , Nogova L. , Mueller R.-P. , Eich H. , Mueller-Hermelink H.K. , Herrmann R. , Doerken B. , Kanz L. , Greil R. , Pfistner B. ,   for the German Hodgkin′s lymphoma study group (GHSG),   the Competence Network Malignant Lymphoma.
Published: April 2020

Background: Combined modality treatment of chemotherapy (usually ABVD) followed by involved field irradiation (IF) is today a standard treatment regimen for early stage Hodgkin′s lymphoma (HL)in stages I-II without risk factors in most study groups. The complete remission rates achieved with this treatment strategy are more than 90%. However, the question arises, whether treatment intensity of chemotherapy, radiotherapy or both may be further reduced in these patients.

Aim: In the HD10 trial of the GHSG, reduction of ABVD cycles as well as reduction of IF- RT dose were investigated.

Methods: Patients were randomized between 4 cycles of ABVD followed by 30 Gy IF radiotherapy as standard treatment (arm A), 4 cycles ABVD + 20 Gy IF (arm B), 2 cycles ABVD + 30 Gy IF (arm C) and 2 cycles ABVD + 20 Gy IF (arm D).

Results: Between May 1998 and May 2002, 1131 HL patients with early stage disease (CS I,II without risk factors) were randomized. 847 patients (75%) were evaluable for this second interim analysis performed in August 2003. Patient characteristics were well balanced between the treatment arms. In total, 98.4% of the patients reached complete remission. Progressive disease or no change were observed in 0.9% of patients, relapse rate was 2.5%. In total, thirteen patients died during the study (1,5%). During chemotherapy there were 19% leukopenia as the most common WHO grade III/IV toxicity (4xABVD: 22 %, 2xABVD: 15%). Radiotherapy was well tolerated with dysphagia in 2.3%. Altogether ten secondary neoplasias were seen: 1AML, 4 NHL and 5 solid tumors.

After a median observation time of two years, overall survival (OS) and freedom from treatment failure (FFTF) rates were 98.5% and 96.6%, respectively. No statistical differences in FFTF and OS were detected between 4xABVD and 2xABVD. In addition, there was no statistical difference between the different doses of radiotherapy (30Gy vs. 20Gy).

Conclusions: Since only few events were observed, the results of this interim analysis can be interpreted as a trend that therapy intensity in combined modality treatment of early stage might be further reduced in terms of the number of chemotherapy cycles as well as the radiotherapy dose, even in IF-RT setting.
Intensification of Chemotherapy and Concomitant Reduction of Radiotherapy Dose in Intermediate Stage Hodgkin′s Lymphoma: Results of the Fourth Interim Analysis of the HD 11 Trial of the GHSG.
Authors: Diehl Volker , Brillant C. , Engert A. , Wolf J. , Nogova L. , Mueller R.-P. , Eich H. , Hermelink H.K. Mueller , Herrman R. , Markova J. , Ho A. , Hiddemann W. , Doerken B. , Greil R. , Pfistner B. ,   for the German Hodgkin′s Lymphoma Study Group (GHSG),   the Competence Network Malignant Lymphoma.
Published: April 2020

Background: Combined modality treatment consisting of chemotherapy (CT) followed by involved field radiotherapy (IF-RT) is the standard treatment for intermediate stage (early unfavourable) Hodgkin′s lymphoma (HL). Despite high complete remission (CR) rates comparable to early favorable stage HL, failure rates are remarkably higher in this group of patients.

Aim: In the HD 11 multicenter trial of the GHSG, introduction of the BEACOPP regimen for intensification of chemotherapy in combination with dose reduction of IF-RT on 20 Gy was tested in an attempt to improve therapy outcome.

Methods: Between May 1998 and July 2002, 1363 patients aged 16–75 suffering from intermediate stage HL (CS I, IIA with risk factors or IIB with elevated ESR and/or 3 3 nodal areas) were randomised according to a factorial design between: 4 cycles of ABVD followed by 30 Gy IF-RT (arm A - standard treatment), 4 ABVD + 20 Gy IF-RT (arm B), 4 BEACOPP basis + 30 Gy IF-RT (arm C) and 4 BEACOPP basis + 20 Gy IF-RT (arm D).

Results: In the fourth interim analysis in August 2003, 1047 (77%) patients were evaluable for chemotherapy and 982 (72%) for radiotherapy evaluation. Patient characteristics were well balanced between the treatment arms in regard to age, gender, clinical stage, histopathology and performance status. 95.1% patients reached CR, 2.0% suffered progression, the relapse rate was 5.9%, the mortality rate 3.2%. The most frequent haematological toxicity was leucopenia observed in 33% of patients (ABVD: 27%, BEACOPP: 40%). Infection rate was 5% (ABVD: 4%, BEACOPP:7%). The most frequent toxicity during radiotherapy was dysphagia in 5.6%. Nine secondary neoplasias were observed: 2 AML, 3NHL, 4 solid tumors.

After a median observation time of two years overall survival (OS) was 97.4% (95%-CI: 96-98) and freedom from treatment failure (FFTF) was 89.9% (95%-CI: 88-92). Both for FFTF and OS, there was no sequential significant difference either between ABVD and BEACOPP arms nor between 30 Gy and 20 Gy IF-RT arms.

Conclusions: At two years of median observation time, no differences in treatment outcome were detected either between chemotherapy regimes nor between the different doses of radiotherapy. The low FFTF (89,9 % at two years) led us to further intensification of treatment by the introduction of 2 cycles of escalated BEACOPP + 2 cycles ABVD +30 Gy IF-RT in the next study generation (HD14 study).
Phase I/II Study of Fractionated Radioimmunotherapy (RIT) in Relapsed Low Grade Non-Hodgkin’s Lymphoma (NHL).
Authors: Illidge Tim M. , Bayne Mike C. , Zivanovic Maureen , Du Yong , Lewington Valerie , Peter Johnson W.
Published: April 2020

RIT produces high rates of durable complete responses in “low grade” NHL. Clinical studies have thus far been limited to a single infusion of a radiolabeled anti-CD20 mAb (131I tositumomab or 90Y ibritumomab tiuxetan) in patients with < 25% bone marrow involvement. In this study we have tested the safety and efficacy of 4 weekly infusions of Rituximab followed by 2 fractions of 131I labelled rituximab given 8 weeks apart in relapsed "low grade" NHL and have included patients with higher levels than 25% bone marrow infiltration with lymphoma. Whole body dose (WBD) calculations have been used to allow the total dose given in 2 fractions to be increased in cohorts of 30cGy, 60cGy, 90cGy and 120cGy. A unique anti-rituximab idiotype mAb has been generated enabling serial analysis of serum rituximab concentrations (Cragg et al Blood 2004 Jun 22 Epub). This tool has allowed us to accurately quantify the serum levels of rituximab during the entire treatment schedule. We have also analysed the impact made by induction rituximab, the pre-dose of cold antibody and effects of fractionated RIT on the clearance and biodistribution of the 131I labeled rituximab. Sequential pharmacokinetic analyses have identified wide variation in the effective half-life of 131I-rituximab not only between patients but also within the same patient over the course of the treatment protocol. We found that the mean effective half-life of 131I rituximab increased from 43 hours prior to induction rituximab to approximately 106 hours prior to the second fraction of 131I-rituximab. A strong inverse correlation was found between the patients’ disease burden and the clearance of rituximab, both in the same patient as the tumor burden decreased and between different patients. We have demonstrated that higher cumulative WBD doses can be safely delivered by RIT dose fractionation than can be given with a single dose of anti-CD20, with no grade 3–4 myelotoxicity at the 90 cGy dose level. All 12 patients in the first 3 dose cohorts experienced responses and response durations equal to, or in the majority of cases, superior to that seen with their previous chemotherapy regimen. Data on all 16 patients including 120 cGy dose level will be available at the time of presentation. In conclusion, this fractionated RIT protocol is feasible, efficacious and enables larger WBD doses to be safely delivered than has previously been achieved with non-myeloablative RIT.
The Superior Prognosis of Female Patients with Hodgkin’s Lymphoma Is Related to More Treatment-Induced Leucopenia and Warrants a More Individualized Therapy.
Authors: Klimm Beate , Reineke Thorsten , Haverkamp Heinz , Eich Hans T. , Josting Andreas , Pfistner Beate , Diehl Volker , Engert Andreas
Published: April 2020

Purpose: Several scores have described gender as an important prognostic factor in patients with Hodgkin’s lymphoma (HL), particularly in advanced-stages. However, so far there is no larger analysis to systematically evaluate possible gender-specific factors. The purpose of this analysis was to investigate sex differences with regard to pre-treatment and therapy-related variables and to evaluate their influence on the outcome of HL patients.

Patients and Methods: From 1988 to 1998 the GSHG conducted two generations of clinical trials for early, intermediate and advanced HL (HD4 - HD9). The present analysis comprises 4626 patients of all stages, aged 15 to 75 years, who were enrolled into these multicenter studies and treated according to the GHSG-study protocols.

Results: At a median observation time of 5.5 years, 2050 female and 2576 male patients were suitable for this retrospective analysis. Patients in each group had very similar profiles in terms of age, performance status, stage, histological subtype, clinical risk factors and prognostic factors. Slightly more nodular sclerosis subtypes and mediastinal masses were observed in women. In contrast, more men had a mixed cellularity subtype, older age, advanced stage of disease, and B-symptoms at diagnosis. Acute chemotherapy-related toxicity was distributed almost equally. However, there was more hematotoxicity in females. This difference was most obvious for leucopenia [WHO grade III/IV: 69.9 % versus 55.2 %]. Importantly, there was no higher risk of infections in female patients. Response rates being similar in both groups, however, a lower rate of relapse and death was observed in females.

Univariate analyses revealed significant better outcome in terms of FFTF and OS for females. In multivariate analyses, sex was not identified as an independent prognostic factor, however, lower stages of disease (p<.0001), less B-symptoms (p<.0001), younger age (p<.0001), and leucopenia grade III/IV (p<.0001) were significant variables to which better outcome in females can be related. Particularly, the high prevalence of leucopenia grade III/IV during chemotherapy has a clear impact on better FFTF in females. The smaller proportion of males expressing severe leucopenia also had better outcomes. In addition, when only those patients were included who developed leucopenia grade III/IV within the first two cycles of chemotherapy, the factor maintains its protective role.

Conclusion: In this large retrospective analysis of the GHSG database, a better outcome is observed for female patients compared to male patients with HL. The protective role of severe leucopenia supports the rationale for a more individualized therapy, that may be tailored in a response-adapted manner depending on the individual toxicity profile within the first cycles.
R-DHAOX as Salvage Regimen in Patients (pts) with Relapsed/Resistant Non-Hodgkin[spos]s Lymphoma (NHL).
Authors: Rupolo Maurizio , Spina Michele , Michieli Mariagrazia , Berretta Massimiliano , Abbruzzese Luciano , Mazzucato Mario , Gattei Valter , Tirelli Umberto
Published: April 2020

Rituximab is active as single agent in relapsed CD20-positive NHL. Second line chemotherapeutic regimens in NHL are mainly based on cisplatin (DDP) and are most frequently employed as cytoreductive regimens for high dose chemotherapy and for peripheral blood CD34-positive (PBSC) harvesting. On the basis of preclinical and clinical studies, the substitution of DDP with oxaliplatin (OXDDP) in regimens like DHAP can result in a lower toxicity and higher activity. We studied the tolerability and efficacy of R-DHAOX regimen in the treatment of relapsed and refractory NHL. From November 2002 until June 2004, 33 pts affected by relapsed/refractory NHL were treated with R-DHAOX regimen (Rituximab 375 mg/m2, e.v. day 1, Desametazone 40 mg e.v. days 1–4, Oxaliplatin 130 mg/m2 e.v. day 2 and Cytarabine 2000 mg/m2 e.v. twice a day, day 3). The median age was 51 yrs (range 19–74). Twenty-one pts were in first relapse, 7 pts >1 relapse and 5 pts refractory to the first line. The results are summarized in table 1. These data suggested that the R-DHAOx regimen is a novel combination in salvage therapy for relapsed/refractory NHL. It has clinically significant activity with an acceptable toxicity profile, even in HIV-positive pts. R-DHAOX permits adequate stem cell harvest with G-CSF mobilisation and in our experience no delay in engraftment occurred. Supported by ISS and AIRC grants. Table 1

°30 pts evaluable; ^5 HIV-positive pts, 1 HCV-positive pts, 22 elegible for ASCT M/F 21/12 PS 1 (0–2) Median age 51 (19–74) HIV-positive 10 HCV-positive 1 Histology Lymphocytic 2 Follicular 8 (24%) Mantle 2 Diffuse large cell 18 (55%) Burkitt 1 Large cell CD30+ 2 Stage II 1 III 6 (18%) IV 26 (79%) s-IPI 0–1 10 (30%) >1 23 (70%) # previous lines 1 (1–2) Dose reduction 65 (55%) Toxicity Neutropenia G3/G4 22 (66%) Anemia G3/G4 8 (24%) Thrombocytopenia G3/G4 21 (64%) Mucositis G3/G4 2 Neurotoxicity G3/G4 1 Response° CR 8 (27%) PR 13 (43%) SD 1 (3%) PRO 8 (27%) Day of PBSC peak +14 (12–18) CFU-GM x10 4 /kg 93 (10.9–230) CD34+ 10 6 /kg 7 (0.65–20) # apheresis 1 (1–2) High dose chemotherapy^ 15 (68%)
GD2 Peptide Mimotope DNA Vaccines for Anti-Neuroblastoma Immunotherapy.
Authors: Fest Stefan , Hilt Kerstin , Huebener Nicole , Zeng Yan , Strandsby Anne , Weixler Silke , Gaedicke Gerhard , Riemer Angelika , Foerster-Waldl Elisabeth , Jensen-Jarolim Erika , Lode Holger N.
Published: April 2020

The tumor-associated antigen disialoganglioside-GD2 is expressed on neuroblastoma and melanoma and is an established target for passive immunotherapy. The aim of this study was to develop an active immunization strategy leading to the induction of a humoral anti-GD2 immune response. However, carbohydrates and glycolipids are T cell-independent antigens (TI) and usually evoke a poor immune response in tumor-bearing hosts.

Here, we describe the identification, characterization and in vivo efficacy of cyclic peptides mimicking the structure of glycolipid GD2, i.e. GD2 mimotopes, in order to overcome T-cell independency. First, GD2 peptide mimotopes were identified by biopanning experiments of a phage-display library displaying circular decapeptides against the human/mouse chimeric anti-GD2 antibody (Ab) ch14.18. Thirteen independent phage clones were isolated which bind to ch14.18 with high specificity. Competitive binding of phages expressing GD2 peptide mimotopes to ch14.18 antibody revealed two superior peptide candidates, mimotope A (MA) and and mimotope D (MD), which were subjected to further evaluation. Second, two plasmid DNA minigene vaccines were generated by overlapping PCR encoding for MA and MD, respectively. The plasmids were based on pSecTag2-A also including a kappa leader sequence, a T-cell helper epitope from HIV-1 gp 120 (T1) and a myc-tag. Minigene expression was demonstrated following transfection of COS-7 cells in western-blots and GD2 mimikry was determined in solid phase ELISA experiments. Third, the efficacy of these mimotope DNA vaccines to induce a tumor protective anti-GD2 immune response was tested in the syngeneic NXS2 model of neuroblastoma expressing ganglioside GD2. The DNA vaccination was accomplished with attenuated Salmonella typhimurium (SL 7207) used as an oral vaccine carrier. Only mice receiving the mimotope DNA vaccines revealed a decrease in primary tumor growth by 50% and a dramatic reduction of spontaneous liver metastases with a mean liver weight of 1g in both groups (MA and MD) in contrast to negative controls (3g). Interestingly, mice immunized with KLH conjugated peptide mimotopes A and D revealed an increased rate of s.c. tumor growth and spontaneous liver metastasis with average liver weights of 5 (MA) and 7 (MD), respecively, suggesting the induction of tolerance using this peptide vaccine approach. Finally the highest anti-GD2 humoral immune response was observed in sera of mice from both GD2 mimotope DNA vaccine groups, consistent with the anti-tumor reponse observed in vivo. Based on these data, we belive that GD2 mimotope DNA vaccines may provide a useful strategy for active immunization against neuroblastoma.
Transfection of Dendritic Cells with In Vitro Transcribed RNA Induces Polyclonal CMV-Specific CD8+ and CD4+ Mediated T Cell Responses.
Authors: Heine Annkristin , Holderried Tobias , Grünebach Frank , Appel Silke , Weck Markus M. , Reichardt Volker L. , Brossart Peter
Published: April 2020

Transfection of dendritic cells (DC) with in vitro transcribed RNA was shown to be a highly efficient method to generate antigen specific T cells, probably due to the induction of a polyclonal T cell response directed against multiple antigens presented on different HLA allels. However, the experimental evidence of this assumption remains to be demonstrated. To accomplish this, we used monocyte derived DC that were electroporated with RNA coding for the CMV pp65 antigen. The induction and expansion of antigen specific CD8+ and CD4+ T cells was assessed using a pannel of peptides derived from this antigen and presented on HLA-A2, -A1, -A11, -A24, -B35 and -B7 in IFN-g ELISPOT, 51Cr-release and proliferation assays. Autologous DC generated from CMV positive healthy donors were pulsed with peptides or transfected with pp65 RNA and utilized as stimulators. Autologous purified CD8+ and CD4+ lymphocytes were used as effector cells. By applying this approach we found that transfection of DC with pp65 RNA induces an expansion of polyclonal CD8+ mediated T cell responses that recognized peptide antigens presented on different HLA molecules. These in vitro generated cytotoxic T cells were able to efficiently lyse DC pulsed with pp65 derived peptides or transfected with the cognate RNA in an antigen specific manner after several in vitro restimulations. Furthermore, this experimental approach allowed the identification of the immunodominace of T cell epitopes presented upon RNA transfection. The HLA-2 directed responses were more pronounced as compared to the HLA-A1, -A11, -A24 or -B35 allels. In contrast, in 7 out of 7 HLA-A2 and HLA-B7 positive donors B7-peptides elicited a stronger T cell response than the A2-peptide, indicating the immunodominance of HLA-B7 epitopes. Interestingly, transfection of DC with pp65 RNA resulted in the induction of CD4+ antigen specific T cells that produced IFN-g and proliferated upon stimulation with transfected DC. In the next set of experiments we analyzed the possible induction of CMV specific T cells that recognize epitopes deduced from different antigens. Co-transfection of DC with a mixture of RNAs coding for the CMV pp65 and IE1 antigens elicited polyclonal T lymphocytes specific for peptides derived from both antigens. More importantly, polyclonal cytotoxic T cells could be elicited in peripheral blood of 2 out of 3 CMV negative donors demonstrating the efficiency of this approach. Our results demonstrate that DC transfected with RNA can elicit polyclonal T cell responses and have implications for the development of immunotherapeutic strategies to target viral or tumor associated antigens.
Molecular Characterization of AML1 (RUNX1) Amplification: A Poor Risk Chromosomal Marker in Acute Lymphoblastic Leukaemia (ALL).
Authors: Strefford Jon C. , van Delft Frederic , Raghavan Manoj , Robinson Hazel M. , Moorman Anthony V. , Young Bryan D. , Saha Vaskar , Harrison Christine J.
Published: April 2020

We have recently defined a new recurrent chromosomal abnormality in patients with acute lymphoblastic leukemia (ALL) involving the duplication of chromosomal band 21q22 and amplification of the AML1 (RUNX1) gene. Currently, fluorescence in situ hybridization (FISH) with probes directed to the AML1 gene is the only reliable detection method. AML1 signals are seen as clusters in interphase cells and in tandem duplication on the long arm of chromosome 21 in metaphase. To date, we have identified 46 patients with this abnormality: 26 males and 20 females. The median age of the 44 children was 9 years (range 4–16 years): two adults were 20 and 30 years old. Their median white cell count was 3.7x109/L (range 1−80x109/L) and all had a common/pre-B immunophenotype. The 3 year event free survival for the 24 children treated on MRC ALL97 was 51% (95% confidence interval 27%–71%). Hence, this abnormality has been associated with older children with a low white cell count and, most significantly, a poor prognosis. The NCRI UK Childhood Leukaemia Working Party has categorized these children into a high-risk group and recommended that they be treated on a more intensive protocol, making their accurate detection vital. To achieve this goal, we have attempted to define the amplified region on chromosome 21 and to identify the genes involved, employing a range of molecular techniques. Microarray analysis (genomic and expression) in conjunction with FISH and quantitative PCR were used to investigate DNA and/or RNA samples from 11 patients. Comparative genomic hybridization (Spectral Genomics 1Mb) (aCGH) showed variable regions of amplification (common minimal region ~10Mb DNA).

Complementary FISH, using a range of specific probes corresponding to the aCGH clones, confirmed the extent of the amplified region on chromosome 21, while indicating the precise copy number changes for each clone. FISH also revealed unexpected chromosomal rearrangements of the amplified chromosome, including loss of the 21q telomeric region. These observations confirm the chromosomal instability associated with this abnormality, as previously indicated from the variable chromosome morphology seen by cytogenetic analysis. Expression profiling (Affymetrix HG-U133) detected 60 over-expressed genes in these patients, of which six were located within the chromosome 21 amplicon. This study has defined the extent of the chromosome 21 amplified region surrounding AML1, and highlighted the over-expression of specific genes within the amplicon. This combined molecular approach will help to identify the most appropriate test for the detection of this high-risk abnormality.
Mechanisms of IL-21 Enhancement of Rituximab Efficacy in a Lymphoma Xenograft Model.
Authors: Hughes Steve D. , Bannink Ken , Krejsa Cecile , Heipel Mark , Johnson Becky , Holly Rick , Sivakumar P.V. , Henderson Katherine , Clegg Chris , Sievers Eric , Kindsvogel Wayne , Foster Don
Published: April 2020

Interleukin 21 (IL-21) is an IL-2 family cytokine produced by activated CD4+ T cells. Potent effects of IL-21 have been observed on the growth, survival, and functional activation of T cells, B cells, and natural killer (NK) cells. A Phase I clinical trial of IL-21 in metastatic melanoma and renal cell carcinoma is currently in progress. We recently reported that IL-21 significantly enhanced rituximab mediated clearance of CD20+ lymphoma cell lines both in vitro and in vivo, and that these effects were potentially mediated through IL-21 enhancement of NK cell capacity to effect antibody dependent cellular cytotoxicity (ADCC). Specifically, NK cells treated with IL-21 showed increased cytotoxicity, granzyme B and IFNg production. Current studies aim to further evaluate the mechanisms by which IL-21 enhances ADCC. A number of observations suggest a multi-factorial basis for IL-21 synergy with rituximab. In a xenograft tumor model, SCID mice were injected IV with HS Sultan cells on day 0. Treatment with recombinant murine IL-21 (mIL-21; starting day 1) combined with rituximab (starting day 3) resulted in significantly increased survival (70% vs. 20% on day 100), compared to rituximab alone. In separate studies, the spleens of mice treated with mIL-21 showed increased numbers of activated macrophages and granulocytes. As macrophages and granulocytes can participate in ADCC, IL-21 synergy with rituximab in vivo may be partly dependent on its activation of these cell types. We have also evaluated whether direct effects of IL-21 on lymphoma cells contribute to enhancement of rituximab efficacy. The xenogeneic B lymphoma models in which IL-21 plus rituximab exhibited enhanced survival are highly aggressive and these models were not shown to respond to treatment with mIL-21 alone. In vitro studies were performed to determine if IL-21 could potentiate the growth inhibitory and pro-apoptotic effects of rituximab. In the absence of effector cells synergistic interaction was not observed. In addition, we tested the ability of IL-21 to enhance cytotoxicity when combined with antibodies targeting non-hematopoietic tumor cells (e.g. trastuzumab). Human NK cells treated with IL-21 displayed significantly increased cytotoxicity in ADCC assays using trastuzumab to target breast cancer cells expressing varying levels of HER-2 antigen. In summary, the current evidence suggests that IL-21 can enhance antibody-mediated tumor cell lysis through activation of multiple effectors of ADCC. Thus IL-21 may prove to be broadly applicable to monoclonal antibody therapy of cancer.
First-Line Treatment with Rituximab Improves Survival of Patients with Post-Transplant Lymphoproliferative Disease (PTLD).
Authors: Gonzalez-Barca E. , Domingo-Domenech E. , Gomez-Codina J. , Capote F. , Flores E. , Briones J. , Salar A. , Panizo C. , Montalban C. , Ribera J. M. , Caballero D. , Muñoz A. , Gallur L. , Canales M.A. , Fernandez P. , Encuentra M. , de Sevilla A. Fernandez
Published: April 2020

Purpose: to compare the response rate and survival between patients diagnosed of PTLD and treated with front-line rituximab and those not treated with rituximab.

Patients and Methods: 108 patients with PTLD have been studied from January 1996 to January 2004. Survival curves were expressed as Kaplan-Meier plots and were compared by the Log-rank test. A multivariate Cox regression analysis was performed to asses the effect of prognostic factors on survival.

Results: median age was 55 years (limits: 18–73). 70% were males. The transplanted organ was: kidney 46%, liver 28%, heart 16%. Median time between transplant and PTLD was 59 months, 25% were diagnosed during the first year after the transplant. The most frequent histological subtypes were: large B-cell lymphoma 53% and polymorphic SLPT 13%. 70% were EBV +. Clinical characteristics at diagnosis were: disseminated disease: 52%, extra-nodal disease: 81%, ECOG 3 2: 43%, LDH >N: 60%, IPI 3 3: 40%. Treatments used were: reduction of immunosuppression 91%, chemotherapy 59%, rituximab 33%, antiviral 13%. Response to treatment was: CR 46%, PR 13% failure 11%, not evaluable (early deceased): 29%. With a median follow-up of 15,2 months, survival was: OS 21% and EFS 15%. Forty-six (43%) patients died. The causes of death were: lymphoma progression 15 (33%), infection 15 (33%), toxicity 16 (34%). Survival of patients treated with rituximab was significantly better than the general group: OS 76% (p=0.007) and EFS 70% (p=0.02). Among patients treated with rituximab, 8 (23%) patients died. The significant bad prognostic factors for EFS in the multivariate analysis were: disseminated disease (RR: 2, 95% IC:1,02–3,8; p=0,04), ECOG 3 2 (RR: 5, 95% IC:2,6–9,8; p=0,0001), not been treated with rituximab (RR: 3,8, 95% IC: 1,7–10; p=0,001). IPI did not have prognostic impact.

Conclusions: survival of patients with PTLD is low with conventional therapy, and the main causes of death are toxicity and infections. Treatment with Rituximab significantly improves their survival. Patients with disseminated disease and bad performance status have worse prognosis. IPI is not a useful index of prognosis in patients with PTLD.