bluuebunny/crossref_metadata_2025 huggingface dataset:
Kinetics of Plasma-Cell Chimerism after Allogeneic Stem Cell Transplantation by Real-Time PCR and Its Value To Quantify Minimal Residual Disease in Patients with Multiple Myeloma.
Authors: Kroeger Nicolaus , Zagrivnaja Maria , Schwartz Sabine , Badbaran Anita , Zabelina Tatjana , Ayuk Francis , Zander Axel , Fehse Boris
Published: April 2020

To investigate lineage-specific chimerism of plasma cells after allogeneic transplantation by real-time PCR based on bi-allelic single-nucleotide polymorphism (SNP) or, in case of female-to-male transplantation, on the detection of the DFFRY-gene, 48 samples from bone marrow samples and peripheral blood from 34 non-myeloma patients were analyzed at different times after transplantation. Plasma cells and T-cells were obtained with CD138- or CD3-microbeads and MiniMACS columns. The median chimerism for T-cells at day +100 was > 99.9 % and remained stable on day +180 and one year after transplantation. In contrast, the median donor-plasma-cell chimerism at day +100 was 95.5 %, at day +180 98,6 %, at day +360 99.8 %, and two or more years after transplantation > 99.9 %. There was a trend for more patients with full donor-plasma-cell chimerism at day 180 who received standard conditioning in comparison to reduced-intensity conditioning (56 % vs. 28 %, p=0.3) and who experienced acute graft-versus-host disease (43 % vs. 18 %, p=0.2).

To evaluate whether the quantitative measurement of donor-plasma-cell chimerism after allogeneic stem cell transplantation for patients with multiple myeloma might be a useful tool to determine minimal residual disease, we investigated 48 samples from 21 myeloma patients at different times after allogeneic stem cell transplantation, and we compared it with immunofixation and, in some cases, with molecular methods by using patient-specific primers. All patients with molecular remission measured by patient-specific primers had a plasma-cell chimerism of more than 99.5 %. Furthermore, in all but one patient plasma-cell chimerism of more than 99.3 % was associated with a negative immunofixation. In six patients with positive immunofixation after allogeneic stem cell transplantation treatment with bortezomib or donor-lymphocyte infusion was started and monitored by plasma-cell chimerism and immunofixation. All patients experienced an increase of donor-plasma-cell chimerism with negative immunofixation if chimerism exceeded > 99.3 %. Dilution experiments with the TIB-196 cell line showed sensitivity of 104 using real-time PCR and SNPs, 105 in case of Y-PCR and 105 for nested PCR with patient-specific primers. We conclude that plasma-cell chimerism after allogeneic stem cell transplantation is delayed in comparison to T-cell chimerism, suggesting a reduced graft versus hematopoiesis effect on plasma cells. Quantitative measurement of plasma cells after allogeneic stem cell transplantation with highly sensitive real-time PCR allows monitoring of residual host-tumor cells in patients with multiple myeloma and allows guiding adoptive immunotherapy strategies to enhance remission status and to prevent relapse.
The Aurora Kinase Inhibitor AZD1152 Causes Perturbation of Cell Cycle Distribution in Cell Lines and Primary AML Samples.
Authors: Walsby Elisabeth , Walsh Val , Pepper Chris , Mills Ken , Burnett Alan K.
Published: April 2020

Aurora kinases (AK) participate in chromosome separation during mitosis and are essential for mitotic processes and completion of cytokinesis. AK-A is found at the centrosome and spindle apparatus during prophase to telophase, while AK-B is seen in the midzone during anaphase of the cell cycle. Inhibition of these kinases may be a viable therapeutic strategy. AZD1152 is a specific AK inhibitor, designed to target cell division in proliferating tumour cells, with potential for activity in a wide range of tumours. In order to evaluate the potential utility of the AK inhibitor AZD1152 in acute myeloid leukaemia (AML), the expression levels of AK-A, -B, and -C were evaluated in a panel of myeloid cell lines (HL-60, NB4, NB4R2, U937, KG1, and K562) and in cells from 10 normal bone marrows and 240 AML patients using the Affymetrix gene expression system (Affymetrix Inc.) validated by real-time quantitative polymerase chain reaction in 105 patients. AK-C was absent in all samples, while AK-A and -B were present in 5% and 34%, respectively, of the AML patients and both were present in normal bone marrow samples. Expression levels did not correlate with age, sex, French-American-British classification or cytogenetic risk group. AML cell lines were incubated with a variable concentration of AK inhibitor (0.01 μM to 10 μM) for up to 72 h and the effects on viability, cell growth, cell cycle, and DNA content measured after 24 and 48 h exposure. This resulted in the accumulation of cells with 4N DNA content, indicating an increased proportion of cells in G2, and development of cells with DNA content greater than G2/M phase cells, on occasions showing 8N ploidy. This effect was time and dose dependent. At 24 h AK inhibitor caused a significant increase (p≤0.05) in the number of cells with 4N DNA content, suggestive of G2, or with greater than 4N DNA content at concentrations as low as 0.01 μM. Growth of HL-60 and NB4 cells was inhibited, following 72 h treatment with AK inhibitor (0.01 μM), by 50% and 90% (p<0.05), respectively. Incubation of AML samples (n=28) with AK inhibitor also showed an effect on the cell cycle, with a significant increase in G2 population (p≤0.05), and occasionally an increase of DNA content (p≤0.05) after 24 and 48 h at a minimum concentration of 1.0 μM. Flow cytometric analysis showed histone H3 phosphorylation was decreased, from around 3–5% in untreated cells, only in a proportion (~35%) of primary samples. The growth inhibition effects seen are combined with increased DNA content, which is consistent with the endoreduplication effects associated with these agents. AK-B is expressed in around one-third of AML patients, and agents targeted against this kinase may have therapeutic potential in AML.
Expression of Vascular Endothelial-Cadherin on Leukemic Cells Mediates Their Interaction with Vascular Endothelium.
Authors: Ellis Leslie R. , Vincent Loic , Shmelkov Sergey , Hooper Andrea , Avecilla Scott , Young Lauren , Rafii Shahin
Published: April 2020

It has already been established that subsets of leukemic cells express receptors for pro-angiogenic factors, such as vascular endothelial growth factor receptor-2 (VEGFR-2). Further studies have shown that these same leukemic cells also produce the ligand for these VEGF receptors, VEGF-A. This autocrine loop supports the invasion and proliferation of these particular leukemic cells. The VEGFR-2 signaling pathway is further dependent upon the co-activation of other pro-angiogenic factors, such as vascular endothelial (VE)-cadherin. VE-cadherin is an endothelial cell-specific transmembrane cellular adhesion protein that when bound results in the dephosphorylation of VEGFR-2 and contributes to neo-vessel formation. Recent studies have suggested that VE-cadherin may be expressed by a unique subset of hematopoietic cells, raising the possibility that leukemic cells may express VE-cadherin as well. We therefore sought to identify the expression of VE-cadherin on leukemic cell lines and primary samples, and further determine its role in the interaction with VE-cadherin-positive endothelial cells. Leukemic cell lines and primary leukemias were screened for the presence of VE-cadherin by both RT-PCR and Western blot analysis. Primary leukemic samples, as well as established cell lines for human erythroblastic leukemia (HEL) and acute myelogenous leukemia (KG-1a) were found to express VE-cadherin. VE-cadherin expression was further confirmed by flow cytometry and immunocytochemistry, which demonstrated that approximately 15% of the total population was VE-cadherin-positive. Proliferation and migration assays utilizing neutralizing monoclonal antibodies to VE-cadherin were performed with no obvious effects. However, immunofluorescent staining of a co-culture performed with the above leukemic cells grown on a layer of human umbilical vein endothelial cells (HUVECs) demonstrated that the leukemic cells and HUVECs interact via VE-cadherin. Furthermore, when these leukemic cells were injected into NOD-SCID mice subcutaneously, the VE-cadherin-positive cells localized around the vessels present within the leukemic chloroma. These data set forth the concept that a subset of leukemic cells expresses the protein VE-cadherin and that VE-cadherin is involved in the cell-to-cell interaction between a subset of leukemic cells and vascular endothelial cells. Heterotypic VE-cadherin interaction between leukemic cells and neo-vessels may increase the survival of leukemic cells and contribute to the generation of minimal residual disease. Therefore, inhibition of VEGFR-2 in conjunction with VE-cadherin may provide a novel strategy to eradicate minimal residual disease.
Advanced Glycation End Products (AGEs)-Mediated Cell Growth of Human Acute Myelogenous Leukemia Via Mitogen-Activated Protein (MAP) Kinase Pathways.
Authors: Kim Ju Young , Park Hyun Ki , Yoon Jin Sun , Kim Eun Shil , Ahn Kwang Sung , Yoon Sung Soo , Kim Byoung Kook , Lee Young
Published: April 2020

Advanced glycation end products (AGEs) are products of non-enzymatic glycation/oxidation of proteins/lipids that accumulate slowly during natural aging and at a much accelerated rate in a variety of disorders such as diabetes, renal failure, and Alzheimer’s disease. AGE modifications do not only change the physicochemical properties of the afflicted molecules, but also induce cellular signaling, activation of transcription factors and subsequent gene expression in vitro and in vivo. Most of the biologic activities associated with AGEs have been transduced by receptor for AGE (RAGE). Recently, AGEs are known to be in association with diverse cancers in terms of cellular proliferation and metastasis. However, little is known about the role of AGEs in acute myelogenous leukemia (AML). Here we examined the effects of the AGEs-RAGE interaction on the cell proliferation and intracellular signaling of AGEs in human leukemia cell lines. Expression of RAGE was observed in 8 AML cell lines examined, and up-regulated by treatment of AGE. AGE induced the proliferation of AML cell lines, HL60 and HEL, in a dose-dependent manner. Treatment with 5 μM of antisense S-ODN for RAGE did effectively inhibit cell growth of HEL cells. Exposure of HL60 and HEL with AGE induced a significant increase in the numbers of cells in S phase of cell cycle in a dose-dependent manner. AGE enhanced the expression of cell cycle regulatory proteins such as cyclin-dependent kinase (CDK) 2/4/6, cyclin D1/E/B in a dose- and a time-dependent manner. In addition, the protein levels of the cyclin-dependent kinase inhibitor (CDKI), p21 and p27, were decreased by 24 hr exposure of AGE from 10 to 200 μg/ml in HEL. Furthermore, treatment of HEL with 200 μg/ml of AGE triggered activation of mitogen-activated protein (MAP) kinases, Erk, Akt, and p38, pathways and in nuclear translocation of transcription factors NF-kB. These results indicated that AGE induced the cell growth of human AML cells, HL60 and HEL, via augmentation of cell cycle and activation of MAPK kinase pathways. Up-regulation of RAGE by exposure of AGE suggested that cellular proliferation of AML cells might be mediated in autocrine fashion.
Regulation of T Cell Homeostasis and Cell Cycling in Patients with Acute Myeloid Leukemia.
Authors: Hunger J. Katrin , Pfoertner Susanne , Ivanyi Philipp , Krauter Juergen , Ganser Arnold , Buer Jan , Franzke Anke
Published: April 2020

Background and Aims: Although leukemic cells express tumor-associated antigens, suppression of an effective T cell immune response is a well known phenomenon in acute myeloid leukemia (AML). In principle, the malignant cells may escape immune surveillance by (1) incomplete T cell activation due to the absence of costimulatory molecules on the leukemic cell types, (2) secretion of soluble immunosuppressive factors, which may lead among others to (3) induction of anergic and/or regulatory T cell phenotypes. A better understanding of the underlying mechanism would have important implications for the development of immunotherapeutic strategies.

Methods: CD3+ T cells were analyzed for activation, proliferation and Th1 cytokine production after coculturing with primary AML cells or leukemic cell supernatant. In addition, self-developed human Treg microarrays were used to reveal a gene expression profile of CD4+ T cells isolated from untreated AML patients (n=7) in comparison to CD4+ T cells from healthy volunteers (n=6) and AML patients after achieving a complete remission (n=4). The specific regulation of selected canditate genes was confirmed by realtime RT-PCR. Based on microarray results cell-cycle analyses were performed in order to study the effect of primary AML cells on the cell-cycle progression of stimulated T-cells.

Results: Our studies could show that primary AML cells suppress T cell activation, proliferation and Th1 cytokines production mainly in a cell-contact dependent manner and to a minor degree via soluble factors. Nevertheless, cell-cycle analyses demonstrated that soluble factors from leukemic cell supernatants may inhibit the cell-cycle entry of T cells after mitogenic stimulation with an arrest in G0/G1 phase. Interestingly, the molecular profile of CD4+ T cells isolated from untreated AML patients could identify a profound dysregulation of molecules involved in the regulation of cell-cycle (i.e., CDK4, Vav2, Egr-1, Id2) and apoptosis (i.e., Bcl-2, Bax). In addition, genes important for the immune response (i.e., IL2R, CTLA-4), especially various chemokines/-receptors (i.e., RANTES, CXCL10, CCR2) were dysregulated, whereas the expression pattern of genes specific for regulatory T cells (i.e., FoxP3, PD-1, GITR) were broadly unaffected.

Conclusion: AML cells (in-)/directly modulate normal T cell function such as activation, proliferation, cytokine/chemokine production and cell-cycle regulation. One might speculate that the lack of an effective anti-tumor response is associated with a cell-cycle arrest resulting in T cell anergy. Cell-cycle regulators may serve as potential targets for novel immunotherapeutic strategies to eradicate minimal residual disease in patients with AML.
4E-Binding Protein 1 (4E-BP1) in CD34+ Cells as Measured by Quantitative Flow Cytometry Is an Independent Prognostic Factor in Patients with Acute Myeloid Leukemia.
Authors: Gorre M. , Jilani I. , Chang R. , Bareng J. , Chan H. , Keating M. , Kantarjian H. , Estey E. , O’Brien S. , Giles F. , Albitar M.
Published: April 2020

The mammalian target of rapamycin (mTOR) is a serine/threonine kinase involved in the regulation of cell growth and proliferation. Once activated, mTOR can phosphorylate its downstream targets. One of these targets is the 4E-binding protein 1 (4E-BP1), which is phosphorylated and inactivated by mTOR in response to a growth signal. Phospho-4E-BP1 dissociates from the eukaryotic initiation factor 4E (eIF-4E), a translation initiation factor that subsequently binds the cap structure of 5′ mRNAs and initiates the translation of transcripts encoding genes involved in cell cycle control. Rapamycin and its analogs are immunosuppressant drugs that exert their activity by specific inhibition of mTOR. mTOR inhibition induces cell cycle arrest not only in normal lymphocytes but also in malignant cells. Using flow cytometry, we quantified the levels of 4E-BP1 and phosopho-4E-BP1 in CD34+ and CD3+ cells from bone marrow samples collected from patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). We then measured the antibody binding capacity of 100 CD34+ or CD3+ cells using QuantiBRITE and PE (phycoerythrin)-labeled antibodies in a 1:1 ratio. We demonstrated that CD34+ cells express significantly higher levels of 4E-BP1 (P=0.005) and phosphor-4E-BP1 (P=0.0001) as compared with CD3+ cells in patients with AML (n=49). In contrast, there was no significant difference in the levels of 4E-BP1 or phospho-4E-BP1 between CD34+ and CD3+ cells in patients with MDS (n=15). More importantly, in patients with AML, high expression of 4E-BP1 in CD34+ cells was associated with shorter survival (P=0.003) as well as shorter complete remission duration (CRD) (P=0.03). This association between survival and levels of 4E-BP1 was independent of cytogenetic abnormalities in this group of patients. This data not only suggests that the 4E-BP1 level, as measured in the CD34+ cells, can be an important prognostic indicator in AML but also suggests that 4E-BP1 plays a role in the biology of AML. Furthermore, targeting 4E-BP1 by mTOR inhibitors, or other means of down modulating 4E-BP1 levels, is a rational therapeutic approach in AML.

Figure Figure
Tipifarnib (Zarnestra, R115777) as Maintenance Therapy for Adults in Complete Remission (CR) Following Induction and Consolidation Therapies for Poor-Risk Acute Myelogenous Leukemia (AML): A Phase II Trial.
Authors: Karp Judith E. , Gojo Ivana , Greer Jacqueline , Smith B. Douglas , Klein Maureen , Lancet Jeffery E. , Gore Steven D. , Morris Lawrence , Thibault Alain , Rybak Mary Ellen , Wright John J.
Published: April 2020

Disease-free survival (DFS) is short in patients ≥ age 60 or with secondary AML, adverse cytogenetics, or leukostasis at presentation. In such patients, median CR duration is ≤ 8 mos and only 20% achieve 1 yr DFS. Historically, maintenance therapy with low doses of cytotoxic chemotherapy has not prolonged DFS. We tested the hypothesis that Tipifarnib (T) might be active as maintenance therapy for adults with poor-risk AML in first CR after induction and consolidation therapies. Oral T 400 mg bid for 2/3 wks was begun median 2.4 mos (range 1.0–4.1) after start of final consolidation cycle and given for up to 48 wks (16 cycles) to 36 adults with median age 63 (range 27–82), secondary AML 31%, adverse cytogenetics 47%, leukostasis 17%, ≥ 2 risk factors 40%. T was well-tolerated, with only 4 of 36 unable to complete 2 cycles because of constitutional symptoms (1 rash, 3 non-compliant). To date, 256 cycles have been administered (median 8 per pt, range 1–16), with hospitalization required during only 4 (1.5%) cycles (infection 3, bowel obstruction 1). Dose reductions for myelosuppression (400 mg bid to 300 mg bid) occurred in 17/32 (53%) by cycle 3, and 2 (6%) needed platelet transfusions. A total of 9 patients completed all planned 16 cycles of T with a median CR duration of 24 mos (range 15–36+). Five of the 9 remain in continuous CR (CCR) 19+-36+ mos, median 26+), compared with 4 who relapsed after CR of 15–24 mos (median 22). There are 8 additional pts in CCR and still receiving T (2–12 cycles) with CCR 4+-12+ mos. A total of 15 patients progressed while on T at median 6.5 mos CR (range 3.5–12). Median CR duration for all patients is 10+ mos (range 3.5–36+), with 88% ≥ 6 mos and 48% ≥ 12 mos. In 13 “comparable” poor-risk pts (age ≥ 60 46%, secondary AML 25%, adverse cytogenetics 46%, leukostasis 23%) who were eligible for but declined T, 4 are in unmaintained CCR at 14+-25+ mos, 9 have relapsed at median 7.5 mos (5–13 mos). Treatment with T did not have a negative impact on reinduction chemotherapy at relapse, as 6 of 9 patients achieved second CR. Administration of T in CR after induction and consolidation therapy has low toxicity and is associated with prolonged DFS in some adults with poor-risk AML. Phase 3 studies are warranted in this patient population.
SU11657, a FLT3-Targeted Tyrosine Kinase, Has Pro-Apoptotic Activity on Leukemia Cells In Vitro.
Authors: Grafone Tiziana , Ferraretti Laura , Ottaviani Emanuela , Mancini Manuela , Palmisano Michela , Malagola Michele , Terragna Carolina , Renzulli Matteo , Poerio Angela , Amabile Marilina , Soverini Simona , Colarossi Sabrina , Iacobucci Ilaria , Baccarani Michele , Martinelli Giovanni
Published: April 2020

Fms-related tyrosine kinase3 (Flt3) is the most commonly mutated gene in human acute myeloid leukemia (AML) and has implicated in its pathogenesis. Constitutive activation of the Flt3 receptor tyrosine kinase, have been linked either by internal tandem duplication (ITD) of the juxtamembrane region or by point mutation in the second tyrosine kinase domain (TKD). To investigate the effect in vitro of SU11657, a new compound FLT3 kinase inhibitors, we analyzed human cell lines from AML patients (MV4-11 and HL60) and blast from patients AML using a wide range of concentrations (1nM-10μM) of this novel agent. In HL-60, FLT3-wt cell line, used as negative control does not show relevant effect after treatment with SU11657. Instead, in MV4-11, FLT3-ITD cell line, we observed a decrease dose-dependent in cell viability after treatment with SU11657. The effects of this compound on cell cycle progression show an accumulation of G1/S phase and an induction of apoptosis at 1-10nM concentration after 24h of treatment. First we observed a dephosphorylation of FLT3 on Tyr591 in whole cell extracts from MV4-11 cells after treatment with SU11657 100nM. We also demonstrated a hypophosphorylation of AKT on Ser473 and a consequently dephosphorylation of BAD on Ser136 at nanomolar concentration. We observed a dephosphorylation of STAT-5 to 100nM of SU11657 at 24h.

We evaluated the effects of this new compound in AML primary progenitors that showed FLT3-ITD, FLt3-TKD and FLT3-wt. In the patients with mutation ITD and TKD was evident a modification of cell cycle progression with a decrease in G2/M phase and an increase of subdiploid peak. The effect of SU11657 in patients FLT3-wt was not relevant.

Our study thus showed a potential therapeutic usefulness of the drug in treatment of AML. Study of signal transductions and gene profile expression will contribute to further understanding of the drug mechanisms.
Imaging of Peripheral Blood Mononuclear Cells (PBMC) Trafficking in Patients with Non-Hodgkin’s Lymphoma (NHL) Prior to and Following Therapy.
Authors: Weiner George , Wiseman Gregory , Witzig Thomas , Link Brian , Wooldridge James , Ponto James , Juweid Malik
Published: April 2020

Antibody-dependent cellular cytotoxicity (ADCC) mediated by PBMC’s is believed to play an important role in the anti-tumor effects of rituximab in patients (pts) with NHL but little is known about the trafficking of PBMC’s into the involved nodes. The primary objective of this pilot study is to demonstrate the feasibility of imaging PBMC trafficking in pts with NHL prior to and following various forms of therapy, including rituximab. To date, autologous PBMC’s were harvested in 9 pts (7M, 2F, age 49–83 y) using 100 ml of peripheral blood and Ficoll-Paque density gradient for cell separation and then radiolabeled with 400–450 mCi of 111In-oxine. Total number of leukocytes in the final product ranged from 1.5–12x107 (mean=5.5x107) with %PBMC’s ranging from 40–98 (mean= 75). The radiolabeling efficiency ranged from 53 to 87% (mean = 71) Viability of PBMC’s by trypan blue exclusion was 99–100%. Eight patients were imaged at baseline and six of these also following 2 cycles of rituximab (n=5) or one cycle of R-CHOP (n=1). Imaging was performed at approximately 4, 24, and 48 h after injection of 165–300 mCi of 111In-PBMCs. No significant adverse events were observed. All 8 pts showed the expected biodistribution of PBMC’s in normal organs, such as lung (primarily at 4h) and liver and spleen (at all timepoints). There was no evidence of PBMC localization in normal lymph nodes prior to or posttherapy. In contrast, PBMC localization was seen prior to therapy in ≥ 1 known tumor lesion per pt in all 8 pts and in the 6 pts imaged posttherapy, similar or greater PBMC tumor accumulation was observed. In conclusion, our initial results demonstrate the feasibility of imaging PBMC trafficking in pts with NHL prior to and following therapy with promising initial imaging findings. Our studies could eventually help better understand the role that PBMC play in the anti-tumor effect of rituximab and prove useful in the development of combination treatments such as the use of moAbs and cytokines.
Clinical Significance of Serum Th1-, Th2- and Regulatory T Cells-Associated Cytokines in Adult T-Cell Leukemia/Lymphoma: High Interleukin-5 and -10 Levels Are Significant Unfavorable Prognostic Factors.
Authors: Inagaki Atsushi , Ishida Takashi , Ishii Toshihiko , Komatsu Hirokazu , Iida Shinsuke , Yonekura Kentaro , Takeuchi Shogo , Takatsuka Yoshifusa , Utsunomiya Atae , Ueda Ryuzo
Published: April 2020

Purpose & Experimental Design: Patients with adult T-cell leukemia/lymphoma (ATLL) are in a severely immunocompromised state, leading to frequent and severe infectious complications. Therefore, it is assumed that ATLL cells either express particular cytokines or induce their expression in host immune cells, disrupting the balanced production of cytokines and causing the host’s immune system to break down. We examined the levels of serum cytokines including T helper type 1- (Th1-)associated cytokines (IFN-γ, TNF-α, and IL-2), Th2-associated cytokines (IL-4, -5, and -6), and regulatory T cell-associated cytokines (IL-10 and TGF-β1) in 94 ATLL patients, 39 asymptomatic human T-cell lymphotropic virus type-1 (HTLV-1) carriers, and 50 healthy adult volunteers, in order to clarify whether elevated levels of particular cytokines are associated with the prognosis of ATLL patients.

Results: On multivariate analysis, high IL-5 and IL-10 levels were independent and significant unfavorable prognostic factors among the ATLL patients. The IL-10 level significantly increased with disease progression at each step from asymptomatic HTLV-1 carrier to ATLL of the indolent variant (chronic and smoldering subtypes) to ATLL of the aggressive variant (acute and lymphoma subtypes). Furthermore, high IL-10 was significantly associated with high LDH, indicating that the IL-10 level reflects the tumor burden. The IL-5 level was not associated with disease progression nor LDH. Among ATLL patients with the aggressive variant, high IL-5, but not high IL-10, was an independent and significant unfavorable prognostic factor on multivariate analysis.

Conclusions: The present study provide novel insights into the immunocompromised state of ATLL patients, and show that measurement of serum IL-5 and IL-10 levels is useful for predicting the prognosis and for determining a suitable treatment strategy for ATLL patients.
Rituximab-Mediated Inhibition of the PI3K-Akt Survival Signaling Pathway in B-NHL Cell Lines Leading to Downregulation of Bclxl Expression and Chemosensitization.
Authors: Suzuki Eriko , Jazirehi Ali R. , Bonavida Benjamin
Published: April 2020

Rituximab (chimeric anti-CD20 monoclonal antibody) has been used in the treatment of B-NHL. We have reported in vitro that rituximab treatment signals B-NHL cell lines Ramos and Daudi and inhibits both the ERK 1/2 MAPK and NF-κB signaling pathways leading to selective inhibition of Bclxl expression and sensitization to drug-induced apoptosis. The inhibition of the NF-κB signaling pathway by rituximab was shown to be due, in part, to the induction of the Raf Kinase Inhibitor Protein (RKIP) (Jazirehi, et al., 2005 Cancer Research 65:264–276). The PI3K-Akt signaling pathway is a key regulator of cell survival and aberrant activation of the PI3K-Akt pathway has been implicated in both drug resistance and resistance to apoptosis-inducing stimuli. Akt can promote cell survival by indirectly activating the proximal transcription factor NF-κB through the phosphorylation of I-kappa B kinase (I-κB) (Ozes et. al. Nature401:82–85, 1999). This study investigated whether NF-κB inhibition by rituximab and downregulation of Bclxl expression was also the result of rituximab-mediated inhibition of the PI3K-Akt pathway. Ramos and Daudi B-NHL cell lines were treated with rituximab (20 ug/ml) and cell lysates were prepared and both Akt and phospho-Akt (p-Akt) expression were examined by western blot. The findings demonstrate that both cell lines show constitutively activated p-Akt and treatment with rituximab significantly inhibited p-Akt but not Akt. Time kinetics analysis demonstrated that inhibition of p-Akt was first detected at 3–6 hours following rituximab treatment and inhibition was maintained up to 24 hours. Concomitantlly, a similar time kinetics revealed inhibition of NF-κB activity as assessed by EMSA. Since the inhibition of NF-kB activity resulted in significant downregulation of Bclxl expression, we also examined the role of the Akt pathway in the regulation of Bclxl expression. Tumor cells were treated with the Akt inhibitor LY294002 and analysis of cell lysates showed significant downregulation of Bclxl expression. Rituximab was previously shown to sensitize B-NHL cells to drug-induced apoptosis via inhibition of NF-κB activity and Bclxl expression. We examined if inhibition of the Akt pathway also chemosensitized the cells. Treatment of Ramos cells with the Akt inhibitor LY294002 significantly sensitized the cells to CDDP-induced apoptosis and synergy was achieved. Altogether, these findings demonstrate, for the first time, that rituximab inhibits the Akt pathway and that this pathway is involved in the regulation of tumor- cell resistance to chemotherapeutic drugs. This study also proposes that the Akt pathway is a potential targeting pathway for therapeutic intervention in the treatment of rituximab and drug-resistant B-NHL.
A Very Short Mutational Hot-Spot in the IGH 5′Sμ Region Suggests That the Acquisition of a Class Switch Recombination Defect Is an Important Step Toward Lymphomagenesis.
Authors: Ruminy Philippe , Jardin Fabrice , Penther Dominique , Parmentier Françoise , Buchonnet Gérard , Picquenot Jean-Michel , Tilly Hervé , Bastard Christian
Published: April 2020

The t(14;18), which occurs during VDJ joining, is usually seen as the primary genetic event in follicular lymphoma (FL). Pre-lymphoma cells would then migrate to the secondary lymphoid organs and acquire secondary genetic abnormalities. The mechanisms leading to these secondary lesions are unclear, but both somatic hypermutation (SHM) and class switch recombination (CSR) processes may be involved. Surprisingly, while many FL still express an IgM, aberrant switch junctions were reported on both IGH alleles, indicating dysfunctions of CSR during the onset of the neoplasm. Recently, AID mediated point mutations were shown to accumulate in the switch μ (Sμ) regions of stimulated B cells undergoing CSR. To get insights into a possible CSR deregulation in FL, we sequenced the 5′ end of the Sμ region for 73 t(14;18) positive tumours. 152 point mutations and 5 deletions were identified, for a mutation frequency of 0.31/100 bp. Notably, mutations were found for a majority of cases (49/73, 67.1%) rather than being restricted to few tumours. Significant targeting of RGYW motifs (p<10−4) and bias for transitions over transvertions (p<10−4) both suggested the involvement of AID. More importantly, our data showed a highly significant clustering effect, with 22 mutations (13,9%) found on a 4 bp cluster on the GTAA splicing donor site of the Iμ exon (p<10−6). Those 22 mutations were found in different tumors so that 30.1% of the lymphomas in our series had a mutated splicing site. Analysis of whole blood genomic DNA from 14 patients and 20 healthy donors indicated that these mutations are not polymorphisms but are instead acquired by tumor cells. Importantly, allele specific PCR experiments showed no clustering effect on the translocated alleles, while 15/79 (19%) mutations on the functional alleles were found on the Iμ splicing site (p<10−6). Such mutations were also found, but on the translocated alleles, for two additional lymphomas with complex t(3;14;18)(q27;q32;q21) and t(8;14;18)(q24;q32;q21) translocations, suggesting that they may be linked to downstream rearrangements on the IGH locus. Finally, RT-PCR experiments showed that a mutation of the Iμ splicing site leads to the expression of aberrant germline transcripts. Together, our data thus indicate that mutations leading to an incorrect splicing of the germline Sμ transcripts are probably positively selected during lymphomagenesis, and therefore that an acquired perturbation of the CSR process may have a major impact during the onset of the disease, maybe by preventing pre-lymphoma cells from switching their surface immunoglobulin and/or by favouring subsequent genetic lesions.
CD56+ Monomorphic Enteropathy-Type T-Cell Lymphoma Is a Genetically Distinct Entity Characterized by 8q Gains and Lack of 1q and 5q Gains.
Authors: deLeeuw Ronald J. , Zettl Andreas , Trottier Magan , Gascoyne Randy D. , Chott Andreas , Mueller-Hermelink Hans K. , Lam Wan L.
Published: April 2020

Introduction: Enteropathy-type T-cell Lymphoma (ETL) is a highly aggressive extranodal T-cell non-Hodgkin’s lymphoma that is often associated with underlying celiac disease. In this study we used whole genome tiling resolution array comparative genomic hybridization (CGH) to delineate genomic changes that may be critical in disease pathogenesis in 30 well-characterized cases of ETL, among them 14 cases of CD56+ monomorphic small to medium-sized ETL, so-called small cell variant (CD56+smETL).

Experimental Approach: Whole genome tiling array CGH was used to generate high resolution segmental copy number profiles of 30 ETL specimens, the array platform consisting of 26,819 bacterial artificial chromosome (BAC) derived amplified fragment pools spotted in duplicate.

Results: Alignment of these profiles with the human genome map has resulted in the identification of both previously reported and novel regions of recurrent genomic alteration. The most frequently identified recurrent chromosomal gains were 9q34.11-qter (approx. 10.2 Mb; 70%), 9q33.2-q33.2 (6.6 Mb; 67%), 9q31.3-q33.2 (13.8 Mb; 60%), 5q34-q35.2 (6.5 Mb), 7q33-q34 (2.4 Mb, 47% each), 1q25.3-q31.2 (8.4 Mb), 1q32.2-q41 (7.9 Mb), 7q22-q31.1 (5.6Mb), 7q36.1-qter (9.6Mb, 43% each), 8q13.3–8q21.11 (3Mb), and 8q22.1-q24.3 (46.2 Mb, 40% each). Frequent recurrent chromosomal losses were observed on 8p22-p23.2 (15.3Mb, 37%), 10q26.2-q26.3 (5.5Mb, 27%), and 9p21.2-p21.3 (4.9Mb, 23%). Interestingly, the most frequent gains at 9q34.11-qter were inversely correlated with losses at 16q12.1 (2.5Mb, 23%, p=0.0139), suggesting these regions may accomplish similar biological effects. CD56-positive monomorphic small cell ETL were characterized by highly recurrent gains of 8q13.3-q21.11 and 8q22.1-q24.3 (71.4% in CD56+smETL vs 18.8% in non-monomorphic ETL, p=0.0086) and the rare occurrence of gains on 1q25.3-q31.2 and 1q32.2-q41 (21.4% in CD56+smETL vs 62.5% in non-monomorphic ETL, p=0.0329) and 5q34-q35.2 (21.4% in CD56+smETL vs 68.8% in non-monomorphic ETL, p=0.0136) which are common within non-monomorphic ETL.

Conclusions: ETL show highly recurrent and characteristic gains and losses of chromosomal material. While gains of chromosome 9q (detected in 70% of ETL) appear to be the hallmark genetic alteration in ETL, ETL, in line with differences in morphology, immunophenotypes and clinical features, seems to be also genetically subdivided into two distinct sub-groups: CD56+monomorphic small to medium sized ETL characterized by gains of 8q and CD56-negative non-monomorphic ETL characterized by gains of 1q and 5q.
The Leukemia Oncogene E2a-Hlf Impairs Checkpoint Activation.
Authors: Porter Christopher C. , DeGregori James V.
Published: April 2020

The E2a-Hlf oncogene results from the translocation of chromosomes 17 and 19. This is a rare translocation found in pro-B cell leukemia cells of older children and adolescents. However, the resulting leukemia has been fatal in all reported cases despite intensive therapy. Evidence suggests that E2a-Hlf contributes to leukemogenesis by impairing apoptosis, but the precise molecular mechanisms of malignant transformation are not well understood. We have observed that E2a-Hlf confers a survival disadvantage relative to un-transduced cells and hypothesize that impaired checkpoints play a major role in genetic instability, malignant transformation, and resistance to therapy. In retrovirally transduced murine pro-B lymphocytes (FL5.12) we have observed that when co-cultured with un-transduced cells, the percentage of E2a-Hlf expressing cells diminishes over time. Furthermore, after retroviral transduction of wild-type primary hematopoietic progenitor cells and transplantation into lethally irradiated recipients, E2a-Hlf expressing cells are undetectable after 2 weeks. In contrast, after transduction and transplantation of replication impaired hematopoietic progenitors (E2F1 and E2F2 mutated), E2a-Hlf expressing cells persist for up to 6 months. We have also observed a survival disadvantage in fibroblasts, indicating that the effect is not tissue specific. When exposed to genotoxic agents such as bleomycin, E2a-Hlf expressing cells have substantially decreased activation of the checkpoint proteins p53, Chk1, and Chk2 as determined by Western blot analysis using phospho-specific antibodies. This effect appears to be downstream of DNA damage focus formation, as levels of ATR and phosphorylated ATM (Western blotting) and gamma-H2AX foci formation (immunofluorescence) are not affected by E2a-Hlf expression. Future studies will be directed at determining how checkpoint activation is inhibited by E2a-Hlf. A better understanding of the molecular mechanisms of E2a-Hlf mediated malignant transformation may lead to targeted and more effective therapy.
Dominant Negative Effect of Kinase-Inactive Bcr-Abl on Cell Survival and DNA Damage Response Pathways in Imatinib-Treated Cells.
Authors: van der Kuip Heiko , Fiesel Fabienne , Moehring Alexandra , Aulitzky Walter E.
Published: April 2020

Imatinib inhibits cell proliferation and induces cell death in BCR-ABL positive leukemic cells in the absence of exogenic growth factors in vitro. In this study, we investigated the effects of physiological growth factors on cell survival and DNA damage response in Imatinib treated Bcr-Abl positive and Bcr-Abl negative cells from murine and human origin. To this end, we used 32D and 32D/bcr-abl, BaF3 and BaF3/bcr-abl, and M07 and M07bcr-abl cell lines. All bcr-abl positive cell lines were highly sensitive to Imatinib in the absence of growth factors. mIL3 protected Bcr-Abl transformed murine cell lines from Imatinib-induced cell death but was not capable to restore the Imatinib-dependent blockade of the p53 response to cisplatinum or gamma irradiation in Bcr-Abl positive cells. Despite prolonged pre-incubation with mIL3, Imatinib led to a transient inhibition of PI3K-, MAPK-, and STAT5-pathways selectively in Bcr-Abl positive murine cells. Importantly, this transient blockade of survival pathways by Imatinib was completely abolished after simultaneous siRNA-mediated suppression of Bcr-Abl synthesis. In contrast to the murine cell lines, Bcr-Abl positive human megakaryocytic M07 cells can not be rescued from Imatinib-induced cell death. Imatinib treated cells died even when pre-incubated simultaneously with a growth factor mix consisting of hGM-CSF, hSCF, and hIL3. Suppression of Bcr-Abl synthesis by RNAi using a breakpoint-specific siRNA selectively inhibited Bcr-Abl-dependent growth of M07/bcr-abl cells to an extent comparable to that observed with Imatinib. Importantly, growth factor stimulation enabled survival of M07/bcr-abl cells after suppression of Bcr-Abl synthesis by siRNA but not after Imatinib-mediated inhibition of Bcr-Abl kinase activity.

In summary, our results indicate a dominant negative effect of kinase-inactive Bcr-Abl both on survival and on DNA damage response pathways.
Transplantation of Haplo-Identical Bone Marrow-Derived Mesenchymal Stem Cells Together with Hematopoietic Stem Cells To Promote Engraftment in Children. A Phase I/II Multicenter Study.
Authors: van Velzen-Ball Lynne Margaret , Le Blanc Katarina , Lankester Arjen C. , Roelofs Helene , Egeler Maarten , Frassoni Francesco , Ringdén Olle , Fibbe Willem E.
Published: April 2020

Graft failure or rejection is an identified problem in haplo-identical or second attempt transplantation. In the bone marrow, mesenchymal stem cells (MSCs) have been identified and shown in animal models to enhance hematopoietic stem cell (HSC) engraftment. Advances in techniques and higher quality culture components have allowed the development of an MSC expansion procedure resulting in sufficient MSCs for clinical application. Adult studies have suggested that co-transplantation of MSCs and HSCs in the HLA-identical setting, is feasible and safe.

Here we present the first combined clinical experience within the EBMT MSC expansion consortium with respect to co-transplantation of haplo-identical MSCs in the pediatric allogeneic transplantation setting.

Six weeks before the SCT, 50 cc of bone marrow are sampled under sterile conditions. Density gradient-separated MNCs are plated into tissue culture flasks in low-glucose DMEM supplemented with 10% fetal calf serum and incubated at 370C with 5% CO2. At 70% confluence, the cells are trypsinized and re-plated at 4x103 cells/ cm2 until the target dose of 1–2 x106/kg recipient body weight is obtained. Enrichment and expansion of MSCs is performed under GMP conditions in nationally accredited laboratories. MSCs (either fresh or cryopreserved) are administered i.v. 4 hours before infusion of donor HSCs. To date 4 children have undergone co-transplantation. Patient characteristics are summarized in the table. All toxicities associated with the procedure were documented, as were the engraftment kinetics and immune recovery. The study was carried out with approval of the respective local ethical committees.

The data indicate that expansion and co-transplantation of MSCs is feasible and well tolerated. While the study is ongoing initial engraftment and immune recovery data (compared to historical data) is encouraging and to date there have been no episodes of graft rejection or severe adverse events related to this treatment.

Patient characteristics Patient demographics HSC characteristics MSC characteristics UPN Gender Age Diagnosis Donor CD34 dose (106/kg) Donor Cell dose (106/kg) 1 M 15 ANLL mother-haplo 16 mother 1.5 2 M 2 XLPD father-haplo 20 father 1.9 3 M 2 XLPD father-haplo 18.7 father 1.56 4 M 8 SAA URD (2 loci mismatched) 2.65 mother 1.0
The Role of GM-CSF in Haplo-Identical Transplant Patients Who Do Not Benefit from NK Alloreactivity.
Authors: Lewalle Philippe , Delforge Alain , Nowak Berengere , Misplon Virginie , Bennani Jalil , Bron Dominique , Martiat Philippe
Published: April 2020

Haplo-identical transplant is now established as a procedure of choice for patients who lack a compatible donor. It might even be the best choice for AML, provided there is a GvH NK allo-reactivity. However, patients are still referred too late, heavily pre-treated, at very advanced stages. We initiated a three-step phase I study trying improve transplant related mortality, relapse rate and immunity:

: G-CSF + DLI, : GM-CSF + DLI, : patient and disease adapted strategy.

Thirty-six consecutive leukemia patients, aged 18–55, were investigated (20 very poor risk, 12 poor risk and 4 better risk). GvH type NK alloreactivity was chosen when possible (21/36) and balanced across the 3 groups. In the first 9 patients, G-CSF was used pot-transplant and prophylactic DLI were given at month 1, 2 and 3. The use of G-CSF and 1 to 3 DLI (10exp4 CD3/kg) was found safe. It resulted in faster CD4 recovery and a low rate of infections. However, it was insufficient to induce a protective GVL effect. In the next 12 patients, GM-CSF was used plus 1 DLI (104 CD3/kg) at day 30 unless aGVHD (3 pts). The comparison between the 2 first groups can be summarized as follows: G-CSF + DLI: TRM at day 100: 0, RR: 6/9, severe aGVHD:0. GM-CSF + 1 DLI group: RR: 1/12, TRM at day 100: 3, aGVHD grade 2 or more: 9/12; price to pay: GVHD resulting in 5 deaths in total. Median time to relapse in the 21 first patients was 6 months range (4 – 9). Step 3 (17 patients) consists of a patient adapted strategy: no more aspecific DLI (selected anti-CMV and aspergillus DLI planned in all patients); in myeloid disorders with NK allo-reactivity: no GF. In the other cases, GM-CSF (at a reduced total dose of 500 mcg) is given from day 5 to day 9. The follow-up of patients alive in CCR (12), although promising (3 relapses), is currently short (median 8 months), compared to the median of relapse in the 2 first groups (6 months). Overall, TRM at day 100 is 2/29, reflecting the good tolerance of the conditioning in a heavily pre-treated population (median age: 43). Overall relapse rate for all patients treated with GM-CSF, without the benefit of NK-alloreactivity, is 6/16 (median FU: 15 months). We conclude that the third strategy might improve the outcome and the relapse rate without exposing patients to unnecessary severe GVHD. These data will be updated with 6 months more follow-up and 3 more patients.
Outcome of Non-T-Cell-Depleted Hematopoietic Stem Cell Transplantation between HLA-Haploidentical Non-Inherited Maternal Antigen (NIMA)-Mismatched Family Members in Childhood.
Authors: Yoshihara Takao , Okada Keiko , Yabe Hiromasa , Kobayashi Michihiro , Kikuta Atsushi , Kato Koji , Adachi Naoto , Kikuchi Akira , Hirota Yasuzou , Kuroda Hiroshi , Nagatoshi Yoshihisa , Inukai Takeshi , Koike Kazutoshi , Kigasawa Hisato , Tanaka Ryuhei , Watanabe Arata
Published: April 2020

Sporadic cases of successful non-T-cell-depleted (TCD) hematopoietic stem cell transplantation (SCT) from HLA-haploidentical family members mismatched for noninherited maternal antigens (NIMAs) have been reported over the last few years. This kind of SCT is based on the hypothesis that long-term feto-maternal microchimerism is associated with acquired immunologic hyporesponsiveness to NIMA or inherited paternal antigens (IPAs). To confirm the effectiveness and safety of NIMA-mismatched SCT in a large cohort, we retrospectively surveyed the outcomes of 76 children (44 boys, 32 girls; median age 7 years, range, 0–18) with either advanced non-malignant disorders (n=10), hematological malignancies (n=62) or solid tumors (n=4) who underwent T-cell-replete HLA-2-loci- or HLA-3-loci incompatible SCT from NIMA-mismatched donors (mother, n=53; NIMA-mismatched sibling, n=12) or other family donors (father/NIPA-mismatched sibling) (n=11) between 01/2000 and 12/2004. Disease status of malignant disease at SCT was as follows: CR1/CR2/CP in 19 and chemorefractory in 47. Types of grafts were bone marrow in 40 and peripheral blood stem cells in 35. Feto-maternal michrochimerism was detected in 32 out of 35 mothers tested and 8 out of 8 NIMA-mismatched sibling donors. GVHD prophylaxis consisted of tacrolimus-based regimen in 73. All but two patients achieved sustained neutrophil engraftment at median of 16.5 days (range, 10–29). Grade II to IV acute GVHD occurred in 36 of 73 evaluable patients (49%) between days 7 and 36 (median, 17). In non-malignant disorders, no severe (grade III/IV) acute GVHD was observed, while in malignant disorders, severe acute GVHD occurred in 21 (32%) of 65 evaluable patients. Twenty-two out of 41 evaluable patients (54%) who survived more than 6 months had extensive chronic GVHD. As of 04/2005, in non-malignant disorders, all 9 patients who obtained engraftment were alive. In malignant disorders, twenty-nine out of 66 patients (44%) were alive and 25 of them were disease-free with median follow-up of 25 (range, 4 to 57) months. Death were due to disease progression (n=22), infection (n=6), GVHD (n=4) and others (n=4). These results suggest that pediatric patients who lack immediate access to a conventional stem cell source can obtain successful results with non-TCD transplants from an HLA-haploidentical NIMA-mismatched donor.