bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of Endothelial Progenitor Cells in Healthy Subjects: Effects of Different Factors on Immunophenotype and Clonogenic Potential.
Authors: Giorgetti Alessandra , Ciulla Michele M. , Cortiana Michela , Silvestri Ilaria , Montelatici Elisa , Giordano Rosaria , Paliotti Roberta , Cortelezzi Agostino , Rebulla Paolo , Lazzari Lorenza
Published: October 2019

There are experimental evidences that Endothelial Progenitor Cells (EPCs) is a pool of cells that could proliferate and differentiate into mature endothelial cells and that may contribute to endothelial repair, but no systematic studies exist regarding physiologic variations in EP circulating pool and in CFU-Endo capacity in healthy subjects. In this regard, since the level of EPCs may be a biologic marker for vascular function, we decided to characterize primitive and circulating EPCs and their relation with gender, aging and the capacity to form CFU-Endo in a selected healthy population. Each subject underwent a screening evaluation to exclude any underlying cardiac diseases. A 20mL sample of peripheral blood was obtained (h 11:00–12:00 AM) from 15 male and 17 female age matched subjects (mean age 41.6±15.9yrs). EPCs were assessed by flow cytometric analysis on 100,000 events per sample (FACScan, Becton Dickinson). Primitive EPCs, defined as CD45−/34+/133+ and CD45−/KDR+/144−, were 8±1.8SE/uL. Circulating EPCs, defined as CD45−/146+/31+/FCSlow and CD45−/146+/144+, were 18.72±2.5SE/uL. No correlation was found between these populations and aging, gender and BMI. Instead a significant decrease in CD45−/34+/133+ (2.9±0.6SE/uL) subpopulation was observed with aging (r = 0.45; p = 0.009). CFU-Endo were performed as described by Hill JM et al. (NEJM 2003) and scored by microscopy in blind by two operators. The confirmation of endothelial-cell lineage was performed by immunostaining for endothelial-specific antibodies (CD31+, CD105+, CD144+, CD146low, CD133−, vWf-). CFU-Endo were expressed as the mean number of colonies per well in 8–25 separate determinations for each subjects and only 15 subjects gave colonies (mean 2.1±0.05SE). CFU-Endo do not correlate with aging and gender in healthy subjects confirming that endothelial injuries and cardiovascular risk factors are a crucial endothelium stimulus. In fact, recently it has been reported that higher colony counts were found in subjects with various degrees of cardiovascular risks. In conclusion CD45-/34+/133+ cell population correlates with aging but since this population represents Hemato-Endo Precursors that include a majority of endothelial progenitors and a small number of hematopoietic stem cells, further studies will be needed to identify a subpopulation strongly predictive of CFU-Endo capacity and to assess its endothelial lineage switch.
Modulation of Cord Blood CD8+ T Cell Effector Differentiation by 4-1BB and CD30: Cross-Talk with TGF-β1.
Authors: Kim Young-June , Stringfield Teresa M. , Chen Yan , Broxmeyer Hal E.
Published: October 2019

Transforming growth factor-β1 (TGF-β1), an immunosuppressive cytokine, potently suppressed cytotoxic T lymphocyte (CTL) differentiation of human cord blood naïve CD8+ T cells as determined by reduced induction of characteristic phenotypes of effector cells and cytotoxic activity. In contrast, 4-1BB (CD137), a costimulatory molecule in the TNF receptor family, promoted effector and memory CTL differentiation. We present evidence that the suppressive TGF-β1 effects can be effectively reversed by anti-4-1BB costimulnation during in vitro CTL differentiation driven by IL-15 from exclusively naïve cord blood CD8+ T cells. 4-1BB may counteract TGF-β1 effects at the level of TGF-β1 signaling by inhibiting Smad2 phosphorylation induced by TGF-β1. In contrast to 4-1BB, recent studies show that CD30, another member of the TNF receptor family, may play an important role in down-modulating effector or memory CTL responses. Thus, we hypothesized that CD30 may interact with 4-1BB in responding to the TGF-β1 suppression. TGF-β1 inhibits 4-1BB expression on CTLs. Conversely, TGF-β1 greatly up-regulates CD30 expression. IL-4 augments the TGF-β1-mediated up-regulation of CD30 expression resulting in further increase of CD30 expression. In contrast, IL-12 abrogates TGF-β1-induced CD30 expression. Such IL-4 and IL-12 effects on CD30 expression appear to be exactly opposite for 4-1BB expression: down- and up-regulating 4-1BB expression, respectively. Co-engagement of CD30 abrogates 4-1BB expression as well as its activity for reversing TGF-β1-mediated suppression of CTL proliferation particularly in the presence of IL-4. Expression of NKG2D, an important stimulatory receptor for NK and CTL cytotoxic activity is inhibited by TGF-β1. 4-1BB stimulation restores the NKG2D expression inhibited by TGF-β1. Again, co-engagement of CD30 significantly inhibits the 4-1BB activity for restoration of NKG2D expression. Taken together, our data suggest that 4-1BB promotes effector CTL differentiation and CD30 acts as a negative feed-back molecule, promoting TGF-β1-mediated CTL suppression and counteracting 4-1BB activity. In conclusion, we have delineated a novel link between CD30 expression and TGF-β1 which is pivotal to the down-regulation of 4-1BB-induced CTL effector differentiation.
Interleukin-7 Over-Expression Regulates T Cell Versus B Cell Lineage Development in the Thymus.
Authors: El Kassar Nahed , Lucas Philip J. , Flomerfelt Frank , Merchant Melinda , Bare Catherine V. , Mahmud Semhar , Mackall Crystal L. , Bhandoola Avinash , Gress Ronald E.
Published: October 2019

We have shown that high IL-7 transgene (Tg) over-expression (39-fold at day 1 in thymic tissue) under the T cell specific, proximal lck promoter had a dose effect on TCRαβ that was accompanied by active B cell development in the thymus. To further characterize these affects in the thymus of IL-7 transgenic mice, we analyzed thymi from day 18 embryos and newborn Tg mice, as well as fetal thymic organ culture (FTOC) derived from using day 16 embryos. We show that arrested T-cell and increased B-cell thymic development is initiated during fetal development. Using mixed bone marrow chimeras and anti-IL-7 monoclonal antibody injection, we further demonstrate that abnormalities in thymic T and B cell development are non-cell autonomous and are due to IL-7 over-expression. Recently, it was shown that only the early thymocyte progenitor (ETP, c-kit+IL-7R−/lo) fraction within the DN1 subpopulation had a T-cell proliferative potential in contrast to the c-kit−IL-7R+ DN1 subset. Here we show that in Tg mice the ETPs were decreased, while the c-kit−IL-7R+ cells are increased in both percentage and absolute count when compared to normal controls. In order to explore the T vs. B ETP potential, we seeded re-aggregate thymic organ cultures with sorted lin−CD44+CD25−c-kit+IL-7R+ cells. While ETPs derived from normal controls were able to proliferate and produce 83% of DP thymocytes, ETPs sorted from Tg mice developed poorly (10-fold less) into DP cells (30%) and produced 14% of B220+ cells vs. 6% in controls. Moreover, sorted Pro/Pre B derived thymic B cells from Tg mice, but not BM-derived Pro/Pre B cells had the TCRβD-J rearrangement, suggesting a T-specific origin. Since the B-cell differentiation pathway in normal mice is selectively inhibited by thymic presentation of Notch ligands, we hypothesized that IL-7 down-regulates Notch signaling. To test this hypothesis, we analyzed thymocyte progenitors (DN1-DN4) in normal and Tg mice for the intra-cytoplasmic part of Notch, that is cleaved upon Notch/Notch-ligand activation. Notch staining was decreased in the lin−CD44+CD25inter representing the only DN2 population present in these Tg mice. These data favour a decrease of Notch signalling in mice with high IL-7 Tg over-expression, inducing a block in TCRαβ development, and skewing of thymic B cell development by T vs. B lineage subversion. These conclusions may have implications for IL-7 in the clinical setting.
The Peripheral Cannabinoid Receptor CB2 and CD40 Are Novel Biological Markers That Predict Outcome in Diffuse Large B-Cell Lymphoma of Elderly Patients.
Authors: Rayman Nazik , Lam King H. , Mulder Dries A.H , van der Holt Ronnie B. , Doorduijn Jeanette , Lowenberg Bob , Sonneveld Pieter , Delwel Ruud H.R
Published: October 2019

Since diffuse large B-cell lymphomas (DLBCL) are biologically and clinically heterogeneous, various attempts have been made to distinguish subtypes of DLBCL. Using gene expression profiling two main subtypes have previously been identified, i.e germinal center B-cell like (GCB) with a favorable prognosis and non-GCB-cell like DLBCL with an unfavorable outcome (Alizadeh et al. Nature403, 503–511, 2000). Applying immunohistochemical analysis, using antibodies directed against CD10, BCL-6 and MUM1, the same investigators were capable of identifying the same subclasses of favorable (GCB) and non-favorable (Non-GCB) DLBCL phenotypes (Hans et al. Blood103, 275–282, 2004). To investigate the applicability of these three markers in de novo DLBCL patients at an age of 65 years or older with advanced stage disease, a cohort of 138 cases was studied. All patients were included in a multicenter clinical trial setting and received uniform treatment. Using the markers CD10, BCL-6 and MUM1, we assigned 40 cases (29%) into the GCB (CD10+ and/or BCL6+, MUM1−) and 98 cases (71%) into the non-GCB-like (CD10−, BCL6+, Mum1+ or CD10−, BCL6−, MUM1+) DLBCL group. In contrast to previous reports, no clinical significance was observed between the two subtypes (overall survival (OS), event-free survival (EFS), progression-free survival (PFS) and disease-free survival (DFS) P-values >0.2), indicating that the prognostic significance of this classification was not evident in this cohort of elderly patients. We previously identified a novel combination of markers that could discriminate between GCB and non-GCB in normal lymphoid tissue, i.e. CB2 and the activation protein CD40. B-cells present in the germinal center (GC) of healthy individuals appeared highly positive for both CD40 and CB2, a previously identified proto-oncogene that encodes for the peripheral cannabinoid receptor. Non-GCB-lymphocytes were mainly CB2 and CD40 negative or weakly positive. We used these two markers for immunohistochemical analyses in the same cohort of novo DLBCL. Single expression of CB2 (PFS (P<0.04) and DFS (P=0.02)) and CD40 (P<0.04) was associated with a favorable clinical outcome. A combined immunophenotypic profile of N-CB2 and CD40 resulted in improved outcome prediction (PFS, P=0.02). Moreover, our data show that CB2 and CD40 are novel markers that recognize DLBCL with a particular GC-like phenotype and have impact on the clinical outcome in a cohort of elderly DLBCL patients. We suggest that CB2 and CD40 may serve as novel markers in DLBCL outcome prediction.
An IELSG International Survey of Primary Effusion Lymphoma (PEL).
Authors: Conconi Annarita , Spina Michele , Ascoli Valeria , Lopez-Guillermo Armando , Cortelazzo Sergio , Alessandro Re , Ichinohasama Ryo , Sata Tetsutaro , Luppi Mario , Vallisa Daniele , Bergonzi Cesare , Provencio Mariano , Rossi Davide , Levine Alexandra , Raphael Martine , Klepfish Abraham , Gloghini Annunziata , Prevot Sophie , Gaidano Gianluca , Carbone Antonino
Published: October 2019

PEL is a rare B-cell neoplasm characterized by a preferential involvement of fluid-filled body spaces, consistent infection of the tumor clone by human herpesvirus type-8 (HHV-8) and a close relationship with underlying immunodeficiency status of the host. The International Extranodal Lymphoma Study Group (IELSG) coordinated a retrospective survey involving 15 international institutions to determine the clinico-pathological features and patterns of outcome of PEL. Fourty-three patients (38 males and 5 females) were registered. Median age at diagnosis was 59 years (range 27–102). In 23 (53%) patients an associated human immunodeficiency virus (HIV) infection was reported, in one case the diagnosis of PEL was made after a solid organ transplantation, in two patients other immunodeficiency conditions were present. The tumor HHV-8 infection was demonstrated in 35 out of the 39 tested cases, Epstein-Barr virus infection in 13 of 30 cases. CD4 count was lower than 200/μl in 18 of the 25 cases in whom the data was available. An ECOG perfomance status score ≥ 2 was observed in 29 patients and the presence of B-symptoms in 20 patients. Serum LDH was elevated in 21 of the 39 tested patients. In 4 patients nodal involvement at diagnosis was reported, in 4 cases at least one extranodal site of localization other than serous cavities was present. A low/low-intermediate risk score according to International Prognostic Index was reported in 10 cases, an intermediate-high/high risk score in 29 cases. Twenty-one patients received systemic chemotherapy, in 17 cases an anthracycline-based regimen. Intrapleural cidofovir was administered in 3 patients. Twelve HIV+ patients received highly active anti-retroviral therapy (HAART), four of them as single therapy. Among the 39 patients for whom adequate follow-up data were available, median overall survival was 6.7 months, median cause-specific survival was 13.6 months and median progression-free survival was 8.3 months. Interestingly, cases of tumor complete regression after implementation of the sole HAART, after intrapleural administration of cidofovir or without any treatment were reported. Our data confirm the poor prognosis of PEL but suggest a possible heterogeneity of this entity with respect to its biological and clinical features. A review of the pathological, phenotypical and virological features is forthcoming to validate these preliminary results.
HLA-DR Protein Status Predicts Survival in Patients with Diffuse Large B Cell Lymphoma (DLBCL) Treated with the MACOP-B Chemotherapy Regimen.
Authors: Rimsza Lisa M. , Farinha Pedro , Fuchs Deborah A. , Masoudi Hamid , Connors Joseph M. , Gascoyne Randy D.
Published: October 2019

Background: HLA-DR is one of the MHC Class II antigen presenting molecules. Loss of HLA-DR expression on DLBCL tumor cells has been associated with poor survival in patients on Southwest Oncology Group (SWOG) studies as well as the recent Leukemia and Lymphoma Molecular Profiling project (Rosenwald et al, NEJM 2002). This study was conceived to examine the effect of HLA-DR status on a completely different cohort of patients from a single institution treated uniformly with a single chemotherapy regimen, MACOP-B, in an attempt to validate HLA-DR as an important biomarker in DLBCL.

Methods: HLA-DR immunostaining was performed using a tissue microarray block containing two 0.6mm cores of paraffin embedded tissues from 97 patients treated on the MACOP-B regimen (1981–86) at the BC Cancer Agency in Vancouver, Canada (Semin Hematol 1988, suppl 2: 41–6). Only cases of B cell lineage were used. We used the HLA-DR antibody Class II DR, (IgG2b) at a 1:50 dilution (Novocastra, UK) with epitope recovery, on an automated immunostainer (Ventana Benchmark System) using a biotin-avidin-diaminobenzidine-based detection system. Two pathologists (LR and DF) scored all slides for positive or negative results.

Results: All patients had advanced-stage disease and were treated uniformly with the MACOP-B chemotherapy regimen. The median follow-up of living patients is 17 years. The IPI was predictive of overall survival (OS) in the study group (p = 0.023). Of the 97 B cell cases, 82 had interpretable staining. The other cases were eliminated for lack of tumor (2), necrotic tissue (1), or lack of internal positive control cells in the tissue core (12). Of the remaining 82 cases, 52 were positive for HLA-DR protein with a median OS of 16.2 years while 30 were negative with a median OS of 4.2 years, (p=0.037, figure below). A Cox multivariate model established both IPI (p = 0.031) and HLA-DR status (p = 0.04) as independent predictors of OS.

Discussion: The importance of loss of antigen presenting molecules on cells of DLBCL was a key finding of the previous LLMPP work, and is now further confirmed as impacting OS in a separate set of patients treated uniformly with a different chemotherapy regimen. These findings lend credence to the hypothesis that loss of immunogenicity is of key importance in patient response to treatment and OS and suggest that specific therapies focused on this pathway may benefit patients with DLBCL regardless of their treatment regimen.

Figure Figure
The Novel De-Immunized Anti-CD20-IL-2 Immunocytokine DI-Leu16-IL-2 Is Effective in SCID Mice Bearing Rituximab-Resistant Human Lymphoma Xenografts.
Authors: Staak Jan O. , Colcher David , Lauder Scott , Wang Jianyi , Gillies Stephen D. , Raubitschek Andrew A.
Published: October 2019

Objectives:

Tumor-targeted MAb-cytokine fusion proteins, such as IL-2 based immunocytokines, have demonstrated potent anti-tumor activity in several pre-clinical models as well as early stage clinical trials. For the engineering of anti-CD20-IL-2, the parental murine anti-CD20 MAb Leu16 was deimmunized by removal of potential helper T cell epitopes and then humanized by combination of recombinant V regions with human L chain and H chain constant regions. Fusion of the H chain to human IL-2 resulted in the de-immunized immunocytokine DI-Leu16-IL-2 which retained full CD20 binding activity and showed enhanced ADCC effector function relative to a control MAb (rituximab). This novel molecule has been demonstrated to be effective in vivo in a Daudi Burkitt lymphoma model. We investigated the therapeutic potential in a rituximab resistant CD20+ human lymphoma xenograft mouse model.

Materials & Methods:

The mature plasmocytic B-cell line ARH77 was stably transfected with the firefly luciferase-gene using electroporation. 2x106 luciferase-transfected ARH77 cells (ARH77:luc) were inoculated s.c. into the right flank of 6–8 week old SCID mice. Groups of 8 ARH77:luc tumor bearing mice were injected i.v. with 3 doses of DI-Leu16-IL2 (25μg), Rituximab (20μg) or PBS on day 0, 2, and 4. Tumor burden was monitored by both physical measurements and bioluminescence imaging. Mice were to be sacrificed at a tumor volume > 2000mm3.

Results:

In this regimen DI-Leu16-IL2 treated mice demonstrated significant reduction in tumor growth whereas rituximab or the PBS control had no effect on ARH77:luc tumor progression. An increase of survival from 28 to 44 days, relative to rituximab treated mice and PBS-control, was seen. Re-growth of tumors in the DI-Leu16-IL2 group was observed after an average delay of 16 days. There were no signs of toxicity. Multiple regression analyses between tumor volume and light emission were performed which revealed a correlation between tumor mass and bioluminescence for the DI-Leu16-IL-2 group (r2=0.64), rituximab group (r2=0.61), PBS-group (r2=0.82), and the combined data sets (r2=0.7).

Conclusions:

DI-Leu16-IL2 demonstrated significant anti-tumor activity against rituxan-resistant human lymphoma xenografts using both physical measures and bioluminescence imaging. Despite a shorter half-life it was more effective than rituximab. Previous studies have shown the anti-tumor effects of DI-Leu16-IL2 were not entirely dependent on FcR binding suggesting that both ADCC and IL-2 targeting contribute to the superior anti-tumor activity of the immunocytokine. Ongoing studies include the use of repeat dosing cycles of this novel immunocytokine for best response using the SCID mouse/ARH77:luc tumor model. DI-Leu16-IL2 may offer promising therapeutic potential with presumed low immunogenicity for patients with CD20+ lymphoma.
The Human Anti-CD30 Antibody 5F11 Activates NF-kB and Sensitizes Lymphoma Cells to Bortezomib Induced Apoptosis.
Authors: Pogge Elke , Boell Boris , Tawadros Samir , Reiners Katrin , Borchmann Peter , Hansen Hinrich , Graziano Robert , Yellin Michael , Engert Andreas
Published: October 2019

Purpose: We recently developed a fully human monoclonal antibody (5F11) directed against the CD30 receptor, which is an excellent target for antibody based immunotherapy of malignant lymphoma cells. The antibody is cytotoxic for lymphoma cells in vitro and in vivo, however a partial or even complete resistance of several Hodgkin cell lines has been observed. In this study we analysed, whether the efficacy of 5F11 can be improved by combination with bortezomib.

Methods and Results: Bortezomib is an inhibitor of the ubiquitin-proteasomes pathway that is toxic to multiple solid and hemotologic tumor cells. Using XTT viability assays, TUNEL assays and Facs analysis we demonstrate a synergistic toxic effect of 5F11 and bortezomib on the Hodgkin cell lines tested (L540, L1236, L428) and the CD30 expressing ALCL Karpas299. Moreover the growth of subcutaneous L540 derived tumors in SCID mice is inhibited by 5F11 in combination with bortezomib. The synergy depends on the regime of the drugs and is only seen when a pre-incubation with the antibody is followed by exposure to bortezomib. Immunofluorescence analysis demonstrates that the sensitization of the tumor cells correlates with a 5F11 dependent localisation of the survival factor NF-kB into the cell nuclei. A transfected NF-kB responsive reporter gene is 2–3 fold activated in 5F11 treated L428 cells and enhanced NF-kB binding is detected performing EMSA.. We furthermore measured an increased expression of the NF-kB target gene c-flip, that is down regulated after additional incubation of the tumor cells with bortezomib.

Conclusion: Our data indicate that initial activation of NF-kB and downstream anti-apoptotic proteins in response to 5F11 via CD30 sensitizes the tumor cells to bortezomib induced cell death. The in vitro and in vivo activity of the combination of 5F11 and bortezomib suggests a therapeutic value for the treatment of HD patients.
Patterns of Growth Factor (GF) Usage and Febrile Neutropenia (FN) among Older Patients (pts) with Diffuse Large B-Cell Lymphoma (DLBCL) Treated with CHOP or R-CHOP: An Intergroup Experience (CALGB 9793, ECOG-SWOG 4494).
Authors: Morrison Vicki A. , Weller Edie A. , Habermann Thomas M. , Li Shuli , Horning Sandra J. , Fisher Richard I. , Peterson Bruce A.
Published: October 2019

The use of anthracycline-based combination chemotherapy to treat DLBCL in older pts is complicated by the more common occurrence of therapy-related myelosuppression, which may lead to dose reductions that might impair efficacy. The preliminary results of initial therapy with cyclophosphamide, doxorubicin, vincristine, prednisone alone (CHOP) or with rituximab (R-CHOP) as induction therapy in patients age 60 years (yrs) or greater with DLBCL have been reported (Blood2003;102:6a, Abstr 8). Pts were randomized at induction to receive 6–8 cycles of CHOP or R-CHOP. Responding patients were then randomized to receive either no treatment or maintenance rituximab. Per protocol, GF was not to be given with the 1st cycle of therapy, and thereafter ASCO guidelines were to be followed regarding use of GF. The objectives of this study were to determine the frequency of and indications for GF usage and the incidence of FN. GF usage was defined as the use of either GCSF or GMCSF for the purpose of preventing dose reduction/dose delay or as prophylaxis in pts with a prior hospitalization for FN. Of the 546 eligible pts, adequate data was available to report on 528 patients. Among these pts, 75% were >=65 yrs, 41% hemoglobin <12 g/dl, 50% male, 59% elevated LDH and 59% HI/High IPI. 264 of 528 pts (50%) used GF in at least 1 cycle of therapy (GF used in 307 cycles to treat neutropenia). 181 of the 528 pts (34%) used GF per our definition with the 93% using GCSF only in at least 1 cycle of therapy. For these 181 pts, the median number of cycles of GF use was 5 (range 1–8) and the median duration of GF use was 9 d (SD=3). GF was used in 552 of 3256 cycles (17%). Significantly more patients used GF at later cycles of therapy (65/513,13% at cycle 2 vs 98/429, 23% at cycle 6, p<0.001). The use of GF was evaluated by study entry characteristics (age, sex, performance status, IPI score). The only significant difference was that pts age 60–64 yrs were less likely to have received GF than pts >=65 yrs (25% vs 37%,p=0.01). 217 pts (41%, 95% CI [37,45%]) had at least one FN event. FN occurred in 267/3247 (8%) cycles of therapy with 103 (39%, 95% CI [33,45%]) FN events occurring in cycle 1. The median time to FN was d 11 (SD=3) of the cycle. In this older pt population treated with CHOP+/− R therapy for DLBCL, GF was used in 17% of the cycles, with usage more common in later cycles of therapy. FN was most common in the first cycle of therapy occurring mostly within the first two weeks. This early occurrence of FN supports consideration of GF use to prevent FN from the 1st cycle of CHOP-based therapy in older pts (>=60 yrs), in addition to any other usage per ASCO guidelines. Results will be presented on the impact of delivered dose intensity on GF usage and FN.
Reduced Relative Dose Intensity (RDI) in Patients with Aggressive Non-Hodgkin’s Lymphoma (NHL).
Authors: Dale David C. , Goldschmidt Jerome H. , Crawford Jeffrey , Lyman Gary H. ,   for the ANC Study Group
Published: October 2019

Introduction: A nationwide survey of oncology practices has previously demonstrated that half of patients with aggressive NHL receive substantial reductions in RDI potentially compromising response rates and long term outcomes. Further study is needed of those factors associated with treatment-related complications and reduced RDI in this population.

Methods: A nationwide survey of 567 oncology practices was undertaken between June 1999 and December 2001 with data extracted from the records of 1,620 adult patients with aggressive NHL treated with CHOP (N=1,367), CHOP-R (N=117) or CNOP (N=136). Outcomes included febrile neutropenia (FN), reduced RDI and disease response to treatment. Multivariate logistic and proportional hazards regression analyses based on common demographic, treatment and disease variables and prophylactic colony-stimulating factor (CSF) provided adjusted odds ratios and hazards ratios, respectively.

Results: FN was reported in 319 (20%) patients of which 80% were hospitalized. The initial episode occurred within the first two cycles of treatment in 60% of patients with FN. Independent predictors of FN risk included age >60 (1.5), female gender (1.5), ECOG >2 (1.9) and liver disease (2.0) with a reduction in risk with prophylactic CSF (0.77). The risk of FN was significantly reduced with prophylactic CSF in patients >60 years of age (HR=.51 [.29,.88]). The overall RDI was 79% with planned and unplanned reductions of 12% and 9%, respectively. RDI <85% of standard dose intensity occurred in 52% of patients including: age >60 (58%), ECOG >2 (56%), stage ≤2 (58%), cardiovascular disease (60%), liver disease (71%), renal disease (58%) and diabetes (59%). Independent predictors of planned reductions in RDI were older age, stage ≤2, heart disease, liver disease, regimen and poor chemotherapy response with less reduction with the addition of prophylactic CSF (R2=0.40; P<.0001). Independent predictors of unplanned reductions in RDI were younger age, diabetes and stage ≤2 with less reduction with the addition of prophylactic CSF (R2=0.13, P<.001).

Conclusions: This practice-based study confirms previous reports of a high level of planned and unplanned reductions in RDI in patients with aggressive NHL. Factors associated with reduced dose intensity include age, comorbid conditions, regimen, stage and no prophylactic CSF.
Role of Allogeneic Stem Cell Transplantation in Lymphoplasmacytic Lymphoma. Analysis of 61 Cases from the European Bone Marrow Registry (EBMT).
Authors: Kyriakou Charalampia A. , Goldstone Anthony H. , Sureda Anna , Yong Kwee , Taghipour Goli , de Elvira Carmen Ruiz , Linch David C. , Mackinnon Stephen , Schmitz Norbert
Published: October 2019

Despite effectiveness of standard chemotherapy regimens, complete response is infrequent in Lymphoplasmacytic Lymphoma (LPL) patients and there is no cure. The role of allogeneic stem cell transplantation (ASCT) has not been explored extensively and the available data are limited. We studied retrospectively LPL patients from 47 European centres who underwent conventional (CT) and non-myeloablative (NMT) allogeneic stem cell transplantation between 1989 and 2003. Sixty-one patients (42 male) of median age at transplant of 41 years (38–57), underwent allogeneic transplantation for LPL. Thirty-two patients were treated with CT and 29 NMT. Thirteen patients received 1, 25 received 2 and 23 more than 2 previous treatment lines. Seven patients were in CR1, 20 in CR2, 19 in PR and 15 with PD. Conditioning used was a combination of TBI and cyclophosphamide or busulfan for the CT and Fludarabine based regimens for the NMT. Twenty-six NMT and 25 CT patients received stem cells from an HLA-identical sibling, while 3 NMT and 7 CT from a matched unrelated donor. For GVHD prophylaxis or treatment cyclosporin A, Methotrexate, ATG, MoAB, T-cell depletion were used. Nine NMT patients received donor lymphocyte infusion for either residual disease or mixed chimera. Median follow up is 7 months (1 to 168). Twenty-nine patients developed a-GVHD (47.5%) [Grade I-II (n=25), Grade III-IV (n=4)] with no statistically significant difference between the 2 groups. Eleven CT patients (34.4%) and 10 NMT (34.5%) patients died from regimen toxicity. Non-relapse mortality was the same for both groups. One CT and 2 NMT patients relapsed and died from disease progression. The progression free survival (PFS) is 51%, at 1 and 41% at 3 years for the entire group and comparison between CT and NMT has not shown a statistical significant difference. Relapse rate at 2 years for CT and NMT is 23% and 34% respectively. Overall survival for CT is 60% at 1 and 55% at 3 years and is similar in the two groups. In conclusion allogeneic stem cell transplantation is safe with moderate non-relapse toxicity and good overall response in this group of advanced and multitreated LPL patients.
The Presence and Number of Activatory KIR (R+D−) Impact the Infection and Survival Status of Patients Transplanted with an HLA Matched Sibling Donor.
Authors: Loiseau Pascale , Chen Chun , Appert Marie-Lorraine , Rocha Vanderson , Busson Marc , Filion Alain , Lepage Virginia , Toubert Antoine , Gluckman Eliane , Charron Dominique
Published: October 2019

Killer immunoglobulin-like receptor (KIR) ligand incompatibilities (=absence on recipient tissues of a ligand for KIRs present in the graft) have been associated with decreased relapse, rejection and acute GVHD, after T cell depleted haplo and unrelated donor Hematopoietic Stem cell transplantation, mainly for myeloid hematopoietic malignancies. The association of donor and recipient KIR genotypes with outcomes, including infections, after HLA genoidentical HSCT has not been studied. We have analysed genotypes for ten KIR genes (2DS1, 2DS2, 2DS3, 2DS4, 2DL1, 2DL2, 2DL3, 3DS1, 3DL1, 3DL2) in 131 donor-recipient pairs from HLA-matched sibling bone marrow transplantations performed for lymphoid (53 cases) and for myeloid malignant diseases (78 cases). We classified the pairs according to KIR-ligand incompatibilities and donor-recipient KIR matching (=concommitant expression of KIRs in donors and recipients). Median age was 29 years. 105 patients were transplanted for a low risk disease, 12 for intermdiate and 14 for high risk disease. Conditioning was myeloablative in all cases and consisted in TBI based (n=63). Cyclosporine and Methotrexate for GVHD prophylaxis was used in 118 cases. Cumulative incidence (competing rsik analysis) of acute GVHD at day 100 and CI of 180 days-bacterial, CMV and fungal infections were 48%, 19%, 34%, 11%, respectively. 5 years overall survival was 70%. We have not found any significant association of “KIR-ligand incompatibilities”, donor KIR genotype, number of activatory KIR genes present in the donor and donor-recipient matching for each KIR with aGVHD, relapse, bacterial, viral or fungal infection and survival, except for KIR2DS2. In fact, donor-recipient mismatching for KIR2DS2 was associated with the incidence of acute GVHD (II-IV p=0.05). Although not significant, we have also observed a trend with decreased survival in patients homozygous for group 2 HLA-C and grafted for myeloid malignant disease with a HLA-matched sibling donor carrying the activatory KIR2DS2 (63 % survival at 20 months (N=6) versus 86% (N=9)). Finally,. the higher is the number of activatory KIRs present in the recipient (R+) and absent from the donor (D−), the higher is the risk of fungal (p=0.03) and CMV infection (p=0.05) and the worse is the survival (at 5 years: 78% survival in case of 0 KIR R+D−, 64% in case of 1 or 2 KIR R+D− and 44% in case of 3 or 4 KIR R+D−, p=0.001). A multivariate analysis for survival confirmed the association of number of R+D− activatory KIR (p = 0.001, HR = 3.13) among other clinical factors such as gender (p=0.03), ABO compatibility (p=0.003), recipient age (p=0.01) and status of the disease (p = 0.0001).
CD45 Defines Signaling Thresholds Critical for Proliferation and Apoptosis in Myeloma Cells.
Authors: Liu Shangqin , Ishikawa Hideaki , Tsuyama Naohiro , Abroun Saeid , Li Fu-Jun , Otsuyama Ken-ichiro , Zheng Xu , Ma Zi , Maki Yasuko , Obata Masanori , Kawano Michio M.
Published: October 2019

Expression of the common leukocyte antigen, CD45 is quite variable in human myeloma cells and cell lines, such as U266, and CD45+ U266 proliferates in response to a growth factor, interleukin-6 (IL-6). IL-6 as a mitogenic stimulus, activated a src family kinase, Lyn and phospholipase C (PLC)-g2 followed by Ca2+ influxes and protein kinase C (PKC) activation in CD45+ U266. Here we show that compared with CD45− U266, CD45+ U266 was more sensitive to various apoptotic stimuli, such as oxidative stress and endoplasmic reticulum (ER)-stress. Both release of apoptosis-inducing factor or cytochrome c and the activation of caspase-9 and caspase-3 were enhanced in CD45+ U266 more than in CD45− U266. Increased susceptibility to the stress-induced apoptosis was also observed in other CD45-expressing myeloma cell lines and the CD45-transfected U266. Reactive oxygen species (ROS) and calcium ion (Ca2+) seem to be involved in the susceptibility to apoptosis of CD45+ U266 because either an anti-oxidant, DTT or a Ca2+ chelating agent, BAPTA blocked the apoptosis. As the oxidative stress-induced Ca2+ influxes and apoptosis of CD45+ U266 were blocked by a src family kinase inhibitor, PP2, CD45 phosphatase regulating the src family kinase activity plays an important role in the augmented apoptosis in CD45+ U266 by oxidative stress. The intracellular accumulation of ROS may activate src associated with CD45, and the activated src kinases accelerate the Ca2+ influxes followed by calicineurin activation, mitochondrial translocation of Bad, and inhibition of Bcl-2 function. These results indicate that the CD45-expression renders myeloma cells competent with not only mitogenic but also apoptotic stimuli, resulting in either proliferation or apoptosis of CD45+ myeloma cells dependently upon the stimuli. Furthermore, voltage-dependent anion channel (VDAC)-1 was identified as a gene highly expressed in CD45+ U266 by cDNA subtraction. The increased expression of VDAC-1 seemed to augment the sensitivity to the ER-stress because the VDAC-1-transfected CD45− U266 was confirmed to be susceptible to the thapsigargin-induced apoptosis. Thus, CD45 may define the signaling thresholds that are critical for the IL-6-induced proliferation and the oxidative stress-induced apoptosis of myeloma cells, and the CD45 expression accompanied by the increased VDAC-1 expression sensitizes myeloma cells to the various extracellular stimuli triggering apoptosis via the mitochondrial pathways.
The Coexpression of CD11a and CD45bright Is the Hallmark of Proliferating Myeloma Cells.
Authors: Pellat-Deceunynck Catherine , Robillard Nelly , Bataille Regis
Published: October 2019

To identify new potential therapeutical targets in multiple myeloma (MM), we have defined the phenotype of the subset of proliferative myeloma cells (n=66) in comparison with that of normal PC (n=25). Proliferation was evaluated by ex vivo incorporation of BrdU (labeling index, LI). Surface PC phenotype was performed in a four-color assay with CD38, CD45, CD138 and the mAb indicated. For intracellular BrdU staining, cells were first labeled with CD38, CD45 and CD138, fixed and permeabilized before BdrU staining. At least 1000 normal PC and 10000 myeloma cells were analyzed. We show that all bone marrow PC, either malignant or normal, always included a subset of proliferative PC (BrdU+) that was always located within the CD45++subpopulation. Indeed, CD45++ myeloma cells (median 12%) had a labeling index 7.5-fold higher of that of CD45+/− myeloma cells (7.1% versus 0.94%). Actually, in all cases of MM, CD45++ myeloma cells were always the most proliferative myeloma cells. As observed for myeloma cells, LI of normal PC was heterogeneous i.e., higher in the CD45++ population of PC: CD45++ PC (median 65%) had a LI 5.7-fold higher of that of CD45+/− PC. Compared to myeloma cells, LI of PC were higher in both subsets, of 20.5% and 3.6% for CD45++ and CD45+/−, respectively. Non-malignant PC from blood or tonsil were homogeneously CD45++ and did proliferate (LI> 10% and up to 45% for reactive PC). In all PC (normal, reactive, malignant), we found an inverse correlation between CD45 and Bcl-2, confirming a known inverse correlation between proliferation and Bcl-2 expression. Our data suggest that a minor cycling Bcl2lowCD45++ population of myeloma cells differentiate into a no more cycling major Bcl2high CD45+/− population of myelom a cells that accumulates.

We further characterized the phenotype of the CD45++ myeloma cells population: we found that CD11a and to a less extend HLA-DR were expressed by CD45++ myeloma cells only in contrast to CD40 and CXCR4 that were expressed by all myeloma cells. Moreover, all CD45++ myeloma cells coexpressed CD11a. Thus, the-to-be-killed population of myeloma cells could be targeted through CD45 or CD11a.
The Tyrosine Kinase Inhibitor Adaphostin (NSC 680410), but Not Imatinib Mesylate, Inhibits Survival and Src Tyrosine Kinase Family- Triggered Signaling Pathways of MM Cells.
Authors: Podar Klaus , Simoncini Melissa , Tai Yu-Tzu , Sattler Martin , Ishitsuka Kenji , Le Gouill Steven , Hamasaki Makoto , Burger Renate , Raje Noopur , Hideshima Teru C. , Chauhan Dharminder , Anderson Kenneth
Published: October 2019

The tyrosine kinase inhibitor adaphostin is a member of the tyrophostin family of small molecules that interfere with peptide binding rather, than targeting the kinase ATP-binding site. Adaphostin has therefore been examined as an alternative to the 2-phenylaminopyrimidine derivate imatinib mesylate, with remarkable efficacy in the treatment of chronic myeloic leukemia (CML). Previous studies show that adaphostin induces apoptosis: (1) in Bcr/Abl+ cells more rapidly than imatinib mesylate; (2) in imatinib mesylate resistant cells; and (3) in Bcr/ Abl - cells.

Imatinib mesylate has minimal, if any activity in MM; the efficacy of adaphostin in multiple myeloma (MM) is unknown. Here we compare the effects of adaphostin and imatinib mesylate against human MM cells. Our results show concentration-dependent apoptosis in MM.1S, U266, OPM-2, INA-6, RPMI8226 and RPMI-Dox40 MM cells after treatment with adaphostin, but not with imatinib mesylate. Imatinib mesylate induced more than 50% apoptosis in K562 cells using concentrations as low as 1mM, which served as a positive control. Moreover, adaphostin, but not imatinib mesylate, induced caspase-9, caspase-8, and PARP cleavage, as well as downregulation of Mcl-1, in MM cells. Further results demonstrated that adaphostin induces peroxide production and DNA strand breaks after long-term treatment. Importantly MM cell proliferation induced by MM cell binding to BMSCs was abrogated by adaphostin- treatment. IL-6 and IGF-1 signaling and sequelae triggered by these cytokines are important growth, survival, and drug resistance factors in MM; conversely, adaphostin but not imatinib mesylate, inhibited phosphorylation of Src tyrosine kinase family, Akt-1, and ERK. Taken together, our studies in MM cells show that (1) adaphostin- inhibits IGF-1- and IL-6- triggered signaling pathways as well as (2) induces reactive oxygen species and apoptosis. These studies therefore provide the preclinical framework for its clinical evaluation to improve patient outcome in MM.
Bmps(Bone Morphogenetic Proteins) Inhibit Growth in Multiple Myeloma Cells by p53 Activation.
Authors: Sundan Anders , Ro Torstein B. , Bonhorst Janne , Waage Anders , Borset Magne
Published: October 2019

Only a few naturally occurring cytokines are able to inhibit myeloma cell growth, and among them BMP-4 is a potent inhibitor of growth as well as an inductor of apoptosis in myeloma cells. To study the mechanism behind BMP-4-induced growth inhibition, we performed gene expression profiling by microarrays in two human myeloma cell lines after BMP-4 stimulation for 4 hours. We found that BMP-4 upregulated several known p53 target genes like p21/Cip1, Bax, cyclin G, Gadd45 and dual specificity phosphatases, suggesting a role for p53 in BMP-mediated growth inhibition of myeloma cells. p53 is activated by several post-translational modifications, including phosphorylation at several serine/threonine residues. We found that BMP-4 treatment lead to rapid phosphorylation of p53 at Serine 15. Furthermore, we were able to co-precipitate phosphorylated Smad-1/5/8 and p53 from nuclear extracts of cells treated with BMP by applying a p53 binding element from the Gadd45 promoter. Because some myeloma cell lines are resistant for BMP-4-induced growth inhibition, we examined whether this BMP-4 resistance was associated with p53 mutations in the BMP-4-resistant cell lines. We sequenced the p53 cDNA from eight myeloma cell lines, and resistant cells all had mutated or deleted p53. Introduction of normal p53 by adenoviral constructs in the p53 mutated cell lines restored BMP-4 responsiveness to some degree. Furthermore, in a myeloma cell line with a temperature-sensitive p53 mutation, the cells were sensitive towards BMP-induced growth inhibition at the permissive temperature but resistant when p53 was inactive. In conclusion, we show that normal p53 activity is involved in BMP-4-induced growth inhibition of multiple myeloma cells.
Depletion of the Intracellular Akt Kinase, and Its Downstream Signaling, Mediates Synergy between the Proteasome Inhibitor MG-132 and the Heat Shock Protein 90 Inhibitor 17-AAG in the Multiple Myeloma Cell Line U266.
Authors: Maisel Christopher , Bahlis Nizar , Miao Yanling , Liu Lili , Gerson Stanton
Published: October 2019

While proteasome inhibitors are effective therapy for multiple myeloma (MM), their efficacy could be improved by synergistic targeting of apoptosis. The intracellular serine/threonine kinase Akt has been demonstrated to have a central role in MM cell growth, survival, and drug resistance. Akt is activated by extracellular cytokines such as IGF-1 and IL-6, and contributes to MM resistance by ameliorating the apoptotic effects of proteasome inhibition. Akt requires chaperone proteins for proper stability and function, including the 90 kD molecule Heat Shock Protein 90 (HSP-90). HSP-90 function is abrogated by geldanamycin and its derivative, 17-allylamino-17-demethoxygeldanamycin (17-AAG).

In the U266 MM cell line, the IC50 of the proteasome inhibitor MG-132 and 17-AAG was 100 nM and 800 nM, respectively. Following exposure of U266 MM cells to either drug alone, or the combination at a fixed-ratio of their IC50s (1:8), apoptosis was determined by Annexin V staining and FACScan analysis. Synergy analysis was performed using Calcusyn (Biosoft, Cambridge, UK). We found that the combination index (CI) was synergistic (CI<1) throughout the dose range, with a CI = 0.449 ± 0.298 at the combination IC50 (highly significant). For example, the apoptotic effect of 50 nM MG-132 and 400 nM 17-AAG was 6 ±2 % and 23 ±3 %, respectively, whereas the 50:400 nM combination produced apoptosis in 68 ± 2 % of the cells.

To analyze effects on Akt and its substrates, we incubated U266 MM cells with MG-132 (50 nM), 17-AAG (400 nM), or the combination. We harvested lysates after zero, two, six, and 24 hours incubation, and Western blot analysis was performed. Co-incubation with MG-132 and 17-AAG, but not either alone, depleted Akt by 24 hours post-therapy. Co-treatment also produced significantly greater upregulation of HSP-90 and HSP-70 than 17-AAG alone, thus demonstrating greater functional inhibition of Akt. The combination also demonstrated the greatest abrogation of Akt-mediated effects on mitochondrial apoptosis: Co-treatment produced the greatest expression of BAD, decreased BCL-XL expression, reduced phosphorylation of GSK-3, and produced the greatest activation of caspase 3.

Monotherapy with 17-AAG upregulated HSP-90 and HSP-70, reduced BCL-XL expression, and activated caspase 9. MG-132 monotherapy produced none of these effects. These findings demonstrate that synergy between proteasome inhibitors and 17-AAG is mediated by Akt depletion and abrogation of Akt signaling, predominantly by MG-132 augmentation of 17-AAG-mediated decay of Akt. Down-regulation of Akt-mediated resistance allows dual-apoptotic signaling and synergistic effect of combination therapy. These findings demonstrate a mechanistic rationale for utilizing heat shock protein inhibitors in combination with proteasome inhibitors as therapy for MM.
Homeodomain Expression in AML and T-ALL Cell Lines.
Authors: Drabkin Harry , Gadgil Sharvari , Zeng Chan , Baron Anna , Bernard Olivier
Published: October 2019

HOX genes are frequent targets of chromosomal translocations and retroviral integrations in human and murine acute leukemia, often involving genes at the 5′-end of the HOX clusters. We previously reported that HOX expression patterns in AML were related to prognostic cytogenetic subsets. We also identified a distinct subset of patients with intermediate cytogenetics based on high levels of HOX and FLT3 expression, frequent FLT3 mutations and a low incidence of C/EBPa mutations. Certain cases of T-ALL also have rearrangements of homeodomain genes and some T-ALLs express limited myeloid markers. To further explore the spectrum of homeodomain gene expression, we developed qRT-PCR assays for nearly all clustered HOXA-D genes, selected homeodomain genes on chromosomes often altered in AML, and selected polycomb (Pc) genes, FLT3 and MLL. Altogether, 52 genes were analyzed in 32 AML and T-ALL cell lines. FLT3 expression was confined to a subset of AMLs. HOX11, HOX11L2 and NKX2.5 were expressed only in cases involving rearrangements of these genes. The Pc and MLL genes were uniformly expressed. Among HOX clusters, the frequency of gene expression was HOXA>B>C>D. Genes more highly expressed in the HOXC and D clusters were those at the 5′-ends (e.g., D13, C10). Marked or selective overexpression of individual genes suggests their possible involvement in the disease process, immortalization or differentiation. Examples include EN1 (SUPT1), D13 (MEGA1, B9 (PEER). A hierarchical cluster analysis based on homeodomain genes successfully identified subsets of related cell lines. Thus, the analysis of quantitative HOX expression may provide an important new tool to better understand the biology of acute leukemia.