bluuebunny/crossref_metadata_2025 huggingface dataset:
Poly-Glutamine Induces Bax-Mediated Cell Death by Dissociating Ku70 from Bax through Ku70-Acetylation.
Authors: Gama Vivian , Yoshida Tomoyuki , Cohen Haim , Sinclair David , Yokota Takanori , Distelhorst Clark , Matsuyama Shigemi
Published: April 2020

Poly-glutamine (PolyQ) diseases (e.g. Huntington’s disease and Machado-Joseph disease) are caused by the amplification of CAG repeats in the genes of proteins normally expressed in the cell. PolyQ induces caspase-independent cell death associated with vacuolization in the cytoplasm that is distinct from the previously known cell death types such as apoptosis and necrosis. PolyQ-induced cell death is considered a new type of cell death and the elucidation of its mechanism is being awaited. At present, there is no effective treatment to rescue cells from polyQ’s toxicity. Previously, we developed cell permeable cytoprotective peptides designed form Bax-inhibiting domain of Ku70, and these peptides were named Bax Inhibiting Peptides (BIPs). We found that BIP (50 – 200 uM range) blocked polyQ-induced cell death in human epithelial kidney (HEK) 293T cells and human neuroblastoma cell line (Neuro2). PolyQ was expressed by plasmid transfection and cell death was detected by trypan blue exclusion and the release of Lactose Dehydrogenease (LDH) from dead cells into the medium. BIP almost completely suppressed polyQ-induced cell death and vacuolization while caspase-inhibitor (z-VAD-fmk) did not. In Bax-deficient human prostate cancer cell (Du145), polyQ did not induce cell death, but the expression of sub-lethal level of Bax restored the toxicity of polyQ in Du145. Bax changes its conformation when it is activated to mediate cell death and the “active” Bax can be detected by a specific monoclonal antibody (6A7 antibody). By immunohistochemistry Bax activation was confirmed in the neurons of human patients suffering from PolyQ disease as well as in the neurons of a transgenic mouse model of the disease. Previously we found that Ku70 binds and inhibits Bax activation, and that the acetylation of Ku70 releases Bax from Ku70. We found that polyQ binds Ku70 and a Histone Acetyl-Transferase (Creb Binding Protein-CBP), and thus polyQ mediates Ku70 acetylation in the cells. We confirmed that Ku70 overexpression protects cells from polyQ expression whereas Ku70 mutants mimicking acetylated Ku70 did not. Based on these in vivo and in vitro studies, we propose that Bax plays a key role in polyQ-induced cell death and that BIP and Ku70 may be utilized to rescue cells from this kind of death in patients suffering from polyQ diseases.
Outcomes after HLA-Identical Sibling Allogeneic Peripheral Blood Stem Cell Transplantation Using Reduced Intensity Conditioning Compared to Autologous Peripheral Blood Stem Cell Transplantation for Elderly Patients with De Novo Acute Myeloid Leukemia.
Authors: Herr Andree-Laure , Labopin Myriam , Blaise Didier , Potter Mike , Ferrara Felicetto , Carreras Enric , Arcese William , Nagler Arnon , Gorin Norbert C. , Frassoni Francesco , Rocha Vanderson
Published: April 2020

Results of allogeneic hematopoietic stem cell transplantation (HSCT) in elderly patients are associated to high TRM mainly due to toxic effects of myeloablative preparative regimen allogeneic and GVHD. Therefore, chemotherapy or autologous (auto) HSCT are often treatment alternatives. Recently, allogeneic peripheral blood SCT (PBSCT) with reduced-intensity conditioning (RIC) has emerged as an alternative to decrease TRM and to add a GVL effect. We have conducted a registry-based retrospective risk-adjusted comparative analysis of outcomes of 204 RIC-PBSCT to those of 954 auto-PBSCT in adult patients aged 50 years or older with de novo AML. Transplants were performed from 1997 until 2003 and reported to the EBMT. Median age was similar in both groups (58 y in RIC and 57y auto PBST), however RIC patients were transplanted more recently, had more advanced disease at time of transplant and poorer cytogenetics compared to the auto-PSCT group (p<0.05). The majority of conditioning regimens used in the RIC group was fludarabine-based associated to low dose TBI or Busulfan (<8mg/kg). Median follow-up was 13 months in both groups. Median time to engraftment was 16 days in the RIC group and 12 days in the auto group (p <0.0001). Unadjusted one-year probability of TRM was 15 ±3% in RIC and 8±1% in auto (p=0.002) and one-year probability of relapse were 34 ±3% in the RIC group versus 40 ±2% in the auto group (p = 0.31). However after adjustment, multivariate analysis showed a lower risk for relapse (RR 0.75, p = 0.04) but a trend for a higher TRM (RR 1.57, p = 0.07) for RIC patients. Major causes of non leukemic death in the RIC group were infections and graft-versus-host disease; and in the auto group, infections. Unadjusted one-year probabilities of leukemia-free survival (LFS) and overall survival (OS) were not statistically different in both groups: LFS of 50±4% in the RIC group versus 52±2% in the auto group (p = 0.31), and OS of 62±4% versus 66±2% (p = 0.23), respectively. In a multivariate analysis, OS and LFS were not statistically different for both groups. In the subgroup analysis, unadjusted one-year probability of LFS was 62±8% for patients in 2nd complete remission receiving RIC transplants compared to 43±6% (p=0.02). Conclusion: Allogeneic HLA identical RIC PBSCT is an effective treatment option for older patients with AML, with acceptable overall survival and decreased relapse incidence compared to autologous PBSCT. These results suggest that RIC PBSCT are feasible in a high risk population and should be evaluated in prospective trials in elderly AML.
Properties of MSC Derived Stromal Cell Lines Developed from Bone Marrow of TNF Knockout Mice.
Authors: Nifontova Irina N. , Rosov Fedor N. , Beliavsky Alexander V. , Drize Nina J.
Published: April 2020

Mesenchymal stem cells (MSC) forming adherent stromal cell lines have been revealed in suspension fraction (SF) of 70+ weeks old long-term bone marrow culture (LTBMC) of TNF knockout mice.

The proportion of CD45+ cells in such old cultures decreased significantly while cellularity of SF increased, accompanied by the first successful generation of cell lines. More than 100 cell lines were established.

Almost all cell lines were Sca1 positive, expressed SDF1, had fibroblast-like morphology and expressed collagens type II and IV. Most of cell lines could maintain hematopoiesis (CFU-S, CFU-GM and CAFC production) for 10 weeks. Some of the cell lines were able to maintain growth without differentiation of murine ES cells and some expressed LIF or LIF-receptor, VEGF and FGF1. All cell lines produced hematopoietic growth factors - their supernatants stimulated growth of CFU-GM in semisolid media. The expression of SCF was revealed in all cell lines tested for growth factors production.

Quantitative RT-PCR analysis revealed that the expression of hematopoietic growth factors - M-CSF, G-CSF and TPO increased sometimes up to 6 times in comparison with the expression of these factors in adherent cell layer (ACL) of LTBMC. The expression of N-cadherin - one of the main regulators of hematopoietic stem cells (HSC) in niche - increased 7–32 fold in 7 tested cell lines regarding the expression of this RNA in ACL of LTBMC. Whereas the expression level of N-cadherin was lower in wild type bone marrow, NIH3T3 fibroblasts and MS5 cells, maintaining the hematopoiesis and CAFC growth, showed the expression on the higher level than in ACL. The expression of ICAM 1, the molecule also regulating HSC, was reduced in all cell lines. The expression level of genes participating in activating of hematopoietic stem cells in niche such as Jagged, Dll 1 and Ang1 more than halved in 5 out of 7 tested lines. The production of Wnt 5a RNA fluctuated from 0,5 to 12 fold and did not correlate with any other changes in gene expression and the ability of cell lines to maintain hematopoiesis. The level of Ihh, Shh was downregulated in cell lines.

The cell line 26dD3 developed in the media containing dexamethasone demonstrated 70 fold increased expression of Kirre - gene supporting HSC but there was no difference in maintaining hematopoiesis and ES cells. High expression of human homolog of this gene (NEPH2) was known to be detected in brain only. At the same time this cell line expressed tubulin b3. These data support a model of lineage specification in which unilineage commitment is prefaced by a “promiscuous” phase of multilineage locus activation. Thus the developed stromal cell lines are not terminally differentiated and use different pathways to maintain hematopoiesis. The TNF deficiency leads to dramatic alterations in regulation of MSC. Mechanisms of this regulation should be scrutinized.
Regeneration of Hematopoietic Microenvironment (Bone Marrow) Using Adipose-Derived Stem Cells - The 2nd Report.
Authors: Ogawa Rei , Fujimura Juri , Mizuno Hiroshi , Hyakusoku Hiko , Shimada Takashi
Published: April 2020

Backgrounds: We have reported bone marrow regeneration using adipose-derived stem cells (ASCs) since 2003. In our study using mice, we showed that not only bone matrix but also bone marrow could be regenerated subcutaneously in vivo. Transplanted ASCs proliferated and differentiated into osteoblasts in micropores on the surface of scaffolds made of hydroxyapatites (HA). We have not yet obtained incontrovertible proof that the ASCs differentiated into hematopoietic cells. However, we could regenerate a subcutaneous microenvironment in which hematopoietic cells survived, which may lead to new treatments for fibrotic bone marrow diseases, such as idiopathic myelofibrosis and osteopetrosis.

Purposes: In this report, we examined the optimal shapes of scaffolds for bone and bone marrow regeneration using ASCs.

Methods: Taking advantage of homogeneously marked cells from green fluorescent protein (GFP) transgenic mice, ASCs were isolated from the inguinal fat pads of GFP transgenic mice. ASCs were cultured in vitro and after three passages were seeded into the small pores of scaffolds of various shapes made of HA. The ASC-containing scaffolds were then subsequently implanted into immunocompetent mice subcutaneously. Two months later, the scaffolds were extirpated for histological, immunohistochemical and flow cytometry analyses.

Results: Histological examination showed that the pores were filled with bone matrix and that the bone marrow was composed of adipocytes, hematopoietic cells, and vasculatures. Bone marrow regeneration on small, thin scaffolds of high porosity was better than on other scaffolds. Immunohistochemical analysis confirmed that the GFP+ ASCs that had differentiated into osteoblasts were composed of bone matrix. Flow cytometry analyses showed that the bone marrow was composed of blood cells, including populations of monocytes, lymphocytes and granulocytes.

Conclusions: Bone marrow engineering has great potential for hematopoietic disease therapy. The regeneration of the hematopoietic system using ASCs might be useful in the future for treating hematopoietic stem cell diseases such as leukemia as well as fibrotic bone marrow diseases. We plan to continue looking for suitable scaffolds and methods for bone and bone marrow regeneration using ASCs.
A Novel Method of Isolation of Mesenchymal Stem Cells from Human Umbilical Cord Tisssues.
Authors: Lu Lulu , Liu Yong-jun , Xu Zhen-shu , Fan Cun-gang , Wang Han , Gong Wei , Han Zhi-bo , Lu Yong-xin , Liu Delong , Chen Zhi-zhe , Han Zhong-chao
Published: April 2020

Mesenchymal stem cells (MSCs) have been isolated from venous endothelia and subendothelia of the human umbilical cord using a tedious procedure. We established a simple method to isolate abundant MSCs from human umbilical cord tissues (UC). 36 full-term umbilical cords were obtained. MSCs were isolated after enzyme digestion of minced cord fragments. The mean nucleated cells isolated from UC was 1.13±0.37×106/cm UC. A total of 1×10e10 MSCs was obtained after several passages over 4 weeks. CFU-F frequency is 1:1609. The population doubling time was approximately 28.02±10.53 h in passage 2 cells. The MSC cells were positive for CD13, CD29, CD44, SH-2, SH-3, CD166 and HLA class I (A, B, C), but were negative for CD34, CD38, CD45, CD31 and HLA-DR. More than 80% cells were in G0-G1 phase, whereas a small population of cells was engaged in proliferation (S+G2+M=9.16%). Under specified culture conditions, the MSC cells differentiated into adipocytes, osteoblasts and neural cells. The MSCs were also found to express cytokines of SCF, LIF, M-CSF, Flt3-ligand, IL-6, GM-CSF, G-CSF, VEGF, and SDF-1. When co-cultured with CD34+ cells from UC blood, the UC-derived MSCs were able to support the hematopoiesis of long-term culture-initiating cells. These findings suggested that abundant MSCs can be isolated simply and effectively from the whole cord tissue. Umbilical cords may be an attractive source of MSCs for tissue engineering, cord blood expansion and cord blood transplantation.
Mesenchymal Stem Cells from Bone Marrow of Children with Malignant and Non Malignant Hematological Disorders Are Not Quantitatively and Qualitatively Affected.
Authors: Dimitriou Helen , Linardakis Emmanouela , Martimianaki Georgia , Stiakaki Eftichia , Fillipidi Anthi , Kaparou Maria , Kalmanti Maria
Published: April 2020

Mesenchymal Stem Cells (MSCs) are multipotent progenitor cells within the bone marrow (BM) capable of differentiating into various tissue specific cells including adipocytes (A), osteoblasts (O), chondrocytes (C), myoblasts, hepatocytes and possibly neuronal tissue. MSCs form an integral part of the BM stroma, have immunomodulatory functions and play an important role in the support of hematopoeisis. Their multipotentiality and ease of ex vivo expansion has raised great interest in the clinical use of MSCs for tissue repair and gene therapy. In order to evaluate if malignant and non malignant hematological diseases quantitatively and qualitatively affect BM derived MSCs, bone marrow from children with Acute Lymphoblastic Leukemia (ALL diagnosis n=9, different stages of treatment n=14, end of therapy n=9), Idiopathic Thrombocytopenic Purpura (ITP, n=12), autoimmune neutropenia (n= 9) and control patients (solid tumors without bone marrow involvement, n=19) was harvested and the mononuclear cell (MNC) fraction isolated. MSCs were expanded in aMEM supplemented with 10% selected FCS, characterized and compared in terms of their phenotypic characteristics, clonogenicity and ability to differentiate into A, O and C lineages. MNCs at day 0 of culture expressed high levels of CD34, CD45, CD29 and CD44, and very low levels of CD14, CD105 and CD90. Expression of hematopoietic markers (CD34, CD45 and CD14) on cells at passage 1 (P1) and thereafter progressively diminished while expression of CD29, CD44, CD90 and CD105 increased approaching 100%. High clonogenicity was observed in all samples at all passages as shown by the presence of CFU-F colonies (>50 cells) with the exception of ALL samples at diagnosis which showed impaired proliferation and clonogenicity that returned to normal at the following stages of treatment till the end of therapy. At P2 or P3, MSCs were differentiated towards the A, O, and C lineages by using specific induction media. Differentiation was assessed by histochemistry (Oil red O for A, von Kossa and Alkaline phosphatase (ALP) for O and Alcian Blue for C) and RT-PCR (LPL and aP2 for A, osteoprotegerin, osteocalcin and ALP for O, aggrecan and Col II for C). P2 or P3 MSCs from all groups exhibited bi- or tri-lineage differentiation. These results indicate that blood diseases of childhood do not affect the characteristics of MSCs and therefore could have clinical uses particularly in hematopoietic reconstitution following allotransplantation.
A Comparison of Mesenchymal Cells from Different Tissues To Suppress T-Cell Activation.
Authors: Keyser Kirsten A. , Beagles Karen E. , Kiem Hans-Peter
Published: April 2020

Graft failure and graft-versus-host-disease (GvHD) remain significant complications of allogeneic stem cell transplantations. To reduce the risk of GvHD, patients often receive a graft that has been depleted of T-cells, which in turn increase the risk of graft failure. Studies have suggested that co-transplantation of ex vivo expanded marrow derived mesenchymal stem cells (MSCs) with allogeneic hematopoietic stem cells (HSC) reduces the risk of GvHD and graft rejection. Additionally, it has been shown that infusion of expanded marrow derived MSCs improves ongoing acute GvHD and facilitates engraftment across MHC-barriers. The effect of MSCs is thought to be due to their ability to suppress T-cell activation. Here we wished to examine whether inhibition of T-cells could be enhanced by MSCs from different tissues. We were particularly interested in MSCs derived from adipose tissue, because of its abundance and accessibility, which is especially important for repeat applications. We investigated and compared the immunosuppressive potential of MSCs derived from multiple mesenchymal tissues of different origins, such as adipose tissue, muscle tissue, omentum, and bone. Cells from the different tissues were enriched for MSCs and cultured and expanded for two to three weeks to deplete hematopoietic cells. MSCs from all tissues differentiated into multiple lineages and stained positive using antibodies against common MSC markers (CD29, CD44, CD105, CD106) and negative for hematopoietic markers (CD3, CD14, CD34, CD45). Mixed lymphocyte reactions (MLRs) using either a mitogen (Concanavalin A) or allogeneic T-cells as inducers of T-cell activation were performed in the presence or absence of MSCs from the different tissues. Mitogen-induced T-cell activation, as well as allogeneic T-cell responses were significantly reduced in MLRs with MSCs compared to MLRs without MSCs. The most pronounced effects were observed with MSCs from muscle tissue (90% reduction) in the mitogen-induced MLR and with MSCs from adipose tissue (85% reduction) in the allogeneic MLRs. These results suggest that MSCs from adipose tissue, which is easily optained and abundantly available, could be used to modulate GvHD and facilitate engraftment across MHC barriers. In conclusion our data demonstrate that MSCs from multiple tissues efficiently reduce T-cell activation and suggest that MSCs from adipose tissue can serve as an alternative source for MSCs to modulate GvHD or facilitate engraftment across MHC barriers. In vivo studies to test the effects on GvHD are currently under way.
Mesenchymal Cancer Cells Can Arise from Ex Vivo Modified Mesenchymal Stem Cells.
Authors: Tolar Jakub , Osborn Mark J. , Panoskaltsis-Mortari Angela , McElmurry Ron T. , Bell Scott , Xia Lily , Riddle Megan , Schroeder Tania M. , Westendorf Jennifer J. , McIvor R. Scott , Oseth LeAnn , Hirsch Betsy , Yant Stephen R. , Kay Mark A. , Peister Alexandra , Prockop Darwin J. , Blazar Bruce R.
Published: April 2020

Mesenchymal stem cells (MSCs) can differentiate into non-hematopoietic cell types, including adipocytes, chondrocytes and osteocytes. MSCs have been isolated from multiple species, including humans, and multiple organs, including bone marrow, adipose tissue and umbilical cord blood. The beneficial effects of MSCs are being tested clinically in attempts to: improve hematopoietic engraftment, to treat osteogenesis imperfecta, graft-versus-host disease and autoimmune diseases, and as antitumor agents to deliver therapy for malignancies. Phase I clinical studies have not been associated with toxicities. We aimed to investigate the capacity of MSCs to aid in tissue healing after radiation induced injury in irradiated bone marrow transplant (BMT) recipients. To study the biodistribution of MSCs, we labeled adult murine C57BL/6 MSCs with firefly luciferase and DsRed2 fluorescent protein using non-viral Sleeping Beauty transposons, and co-infused them with allogeneic bone marrow into irradiated reipients. Using in vivo whole body bioluminenscent imaging luciferase signals were shown to be increased between weeks 3 and 12 indicating expansion of MSCs. Unexpectedly, some mice (N=8/17) with the highest luciferase signals died and all surviving mice (N=9/17) developed foci of ectopic ossification in lungs. Two of mice also developed osteosarcomas in their extremities. This prompted us to characterize the transformed MSCs that originated from the donor MSCs. The transformed cells were aneuploid, lost their capacity to differentiate into mesenchyme-derived adipocytes and chondrocytes, and histologically identified as osteosarcomas. In addition, infusion of tumor cells resulted in malignant lesions in secondary recipients. Mapping of transposition sites in the genome and karyotype analysis indicated that the critical transformation event(s) occurred before infusion of the MSCs. Even though we have not encountered a transformation event in >100 mice infused with MSC manipulated with transposons, we speculated that mutation by transposition was the inciting event. None of the identifiable transposition events occurred in a known proto-oncogene or tumor suppressor gene. This does not discount the possibility of insertional mutagenesis as the genomic lesion may have occurred on the chromosome which was subsequently disrupted or lost. Alternatively, genomic instability could have been a result of spontaneous unrepaired chromosomal lesion(s) that preceded the transposon insertion and resulted in osteosarcoma. These findings provide evidence of evolution of MSCs with osteogenic capacity into osteosarcoma in vivo and are clinically relevant as they document the potential of ex vivo manipulated MSCs for transformation into malignant disease.
Charcterization of a Newly Established Megakaryo/Erythroid Leukemia Cell Line, JAS-R.
Authors: Yamada Hisashi , Horiguchi-Yamada Junko , Sekikawa Tetsuaki
Published: April 2020

A few leukemic cell lines which express megakaryo/erythroid markers are available. We recently established a new cell line, designated JAS-R, from a 64-year-old patient with acute megakaryocytic leukemia (AML M7). Immunophenotyping showed that JAS-R cells were positive for CD4, CD7, CD13, CD33, CD41, CD61 and glycophorin A. Chromosome analysis was composite karyotype, but no major translocation abnormalities were observed. Electron-microscope examination disclosed that JAS-R had bleb like surface margin and a-granules in cytoplasm. Major four proteins which exist in a-granule were expressed high levels in JAS-R by RT-PCR. To further characterize JAS-R from four other megakaryo/erythroid cell lines (MEG-01, CMK, K562 and KU812), the comparison of gene expression profiling was studied by using oligo-DNA array. JAS-R was categorized as most different cell line among them. Of note, JAS-R secreted erythropoietin and expressed erythropoietin-receptor. But erythropoietin-neutralizing antibody failed to inhibit the growth of JAS-R cells. JAS-R may be useful for the further understanding of megakaryo and erythroid regulation and for the study of megakaryo/erythroid leukemogenesis.
Analysis of Both Clonal Functional and Non-Functional Immunoglobulin Heavy Chain (IgH) Rearrangements in Waldenström’s Macroglobulinemia.
Authors: Martin-Jimenez Patricia , Balanzategui Ana , Ocio Miguel E. , Alcoceba Miguel , Sarasquete Maria E. , Aguilera Carmen , Portero Jose A. , Calvo Javier Fernandez , Frade Javier Garcia , Chillon Maria C. , Diaz Guadalupe , Armellini Adriana , Garcia-Sanz Ramon , Gonzalez Marcos , Miguel J.F. San
Published: April 2020

Introduction: Waldenström’s Macroglobulinemia (WM) is an uncommon lymphoproliferative disorder characterized by bone marrow infiltration of the lymphoplasmocytic cells and IgM monoclonal gammopathy. The normal counterpart of the WM malignant cell is believed to be a post germinal-center B cell. However, the low frequency of WM has hampered investigation into Immunoglobulin Heavy Chain (IgH) rearrangements in large series of patients and whether preferential use of specific genetic segments or non-functional rearrangements exits.

Aim: Molecular characterization in a large cohort of WM patients analyzing their functional, complete VDJH, and non-functional, incomplete DJH, IgH rearrangements, the pattern of somatic hypermutation (SHM) and gene segment usage.

Patients and methods: 47 patients with unequivocal diagnosis of WM were included in the study. Bone Marrow samples (always with more than 10% of tumor cell) were used for amplification of clonal rearrangements employing the Biomed-2 strategy (Leukemia2003; 17; 2257), followed by direct automated sequencing in ABI 377 DNA sequencer using Big-Dye terminators (Applied Biosystems, Foster City, CA).

Results: All patients showed a monoclonal amplification of at least one VDJH or DJH rearrangement. VDJH monoclonal rearrangements were detected in 42/47 (89.3%) patients while DJH were observed in 20 (42.5%) patients.

VH, DH and JH gene segment usage: VH3 gene segment was the most commonly represented family (76,2%) while the other gene segments were scarcely detectable, and VH5 was totally absent. In the complete VDJH rearrangements, DH6 was the family most commonly represented while DH2 (45%) and DH4 (30%) were the most common in the incomplete DJH rearrangement. Regarding JH elements, JH4 (38%) was the most represented in the complete rearrangements and JH5 (46%) in the incomplete rearrangements.

Somatic hypermutation: SHM were observed in all except two of the cases (94%, 34 of 36 cases), in which the study could be performed. (median 9,38; range2.9–16.6). In the remaining clonal cases (6 cases, 14.3%) no amplification of FR1 region was obtained, also suggesting the presence of SHM.

Conclusions: In this series of patients, we observed the presence of incomplete rearrangements with an incidence similar (44%) to that described in multiple myeloma. The preferential usage for determined families of genetic segments, in VDJH and in DJH, suggests the presence of positive and negative selection processes. Finally, the majority, (94 %) of the cases, presented SHM, although the presence of two patients lacking in SHM suggests a pre-follicular origin for some WM cases.
NOTCH1 Is Targeted by Ig Translocations in NHL.
Authors: Wlodarska Iwona , De Wolf-Peeters Chris , Cools Jan , Minnei Francesca , Bosly Andre , Delos Monique , Michaux Lucienne , Vandenberghe Peter , Hagemeijer Anne , Marynen Peter
Published: April 2020

NOTCH1 signaling is required for normal T cell development. Its aberrant activation by either the rare t(7;9) translocation or frequent mutation(s) in T-ALL points to an important role of this gene in T cell lymphomagenesis. So far, a pathogenetic role for NOTCH1 in B-cell malignancies is unknown. We report here evidence for NOTCH1 rearrangements in B-cell lymphoma. Two novel 9q34 translocations involving either the 14q32/IGH or the 22q11/IGL locus were identified in 2 patients with respectively, Richter syndrome (RS) and follicular lymphoma (FL). The first case showed a clone with a sole trisomy 12 detected by FISH in 25% of interphase cells and a subclone with an additional dic(9;14)(q34;q32) in 60% of cells from a lymph node sample. The karyotype of the FL case was characterized by the typical t(14;18)(q32;q21)/IGH-BCL2 accompanied by additional t(8;14)(q24;q32)/IGH-CMYC and t(9;22)(q34;q11). FISH analysis of dic(9;14) and t(9;22) using a set of BAC clones mapped both 9q34 breakpoints to the 5′end of NOTCH1. These breakpoints were different from the breakpoint of t(7;9)(q35;q34) (intron 24), as demonstrated by FISH analysis of the T-ALL SUPT1 cell line. NOTCH1 rearrangements showed to be rare in B-NHL. Any of the additional 30 lymphoma cases with structural aberrations of 9q34 analyzed by FISH showed rearrangement of this gene. So far, aberrant activation of NOTCH1 in the reported cases with dic(9;14) and t(9;22) could not be documented: both lymphomas but also 9 control CLL and FL cases without 9q34 aberrations revealed expression of NOTCH1 mRNA by RT-PCR, its ligand JAGGED2 and its transcriptional target HES1. This expression pattern indicates a ligand-driven NOTCH1 signaling in all analyzed cases, possibly reflecting its physiological activation in progenitor B cells and thus, masking the presumed aberrant activation of NOTCH1 in present lymphomas. Whether IGH/L-NOTCH1 translocations in B-NHL lead to the generation of truncated/activated NOTCH1 proteins, similar to these found in T-ALL, remains to be determined. Particularly interesting is the association of the dic(9;14) with Richter transformation in the first case suggesting that rearrangement of NOTCH1 could be responsible for a rapid progression of the underlied CLL. In the second case of FL, t(9;22) seemed to be associated with the evolution of t(14;18)-positive karyotype. These findings contrast with t(7;9), considered as a primary oncogenic event in development of T-ALL. Further molecular and immunohistochemical investigations of the reported cases are in progress.
Transcriptome Analysis of Pediatric cALL Versus Normal Fetal B Cells Reveals a Novel Signature of the Malignant Phenotype.
Authors: Richter Guenther , Hattenhorst Uwe E. , Roessler Sabine , Staege Martin S. , Hansen Gesine , Burdach Stefan
Published: April 2020

Differential gene expression profiling of pediatric common acute lymphoblastic leukemia (cALL) versus non-malignant tissues enables identification of aberrantly expressed genes in malignant cells, facilitating discrimination of leukemic from normal cells and possibly revealing specific disease mechanisms. Expression patterns of 29 pediatric cALL samples were analyzed by use of high-density DNA microarrays HG-U133A. Leukemic patients’ bone marrow samples were compared to sorted B cells from cord blood of healthy donors expressing CD19 and CD10 surface antigens. Principal component analysis clearly distinguished leukemia samples from normal controls. Significance analysis of microarrays revealed 723 genes significantly up-regulated, and 617 down-regulated genes in leukemic cells. Independent validation of deregulated genes by RT-PCR was chosen to address enrichment limitations of controls. A comparison to previous publications investigating genetically defined subsets of cALL revealed only 5 – 22% match with our differentially expressed genes. Furthermore, class prediction with only 14 differentially expressed genes correctly classified tumors and controls not included in the training set and hitherto not investigated samples including 3 leukemic tumor lines (NALM-6, CALL-2, 697) as cALL. Interestingly, terminal deoxynucleotidyl-transferase (DNTT) as well as in the context of cALL unknown genes, were found to be the strongest predictive genes for the malignant phenotype signifying the diagnostic value of our approach.
Transcriptional Profiling of Epstein-Barr Virus (EBV) Genes and Host Cellular Genes in Nasal NK/T-Cell Lymphoma and Chronic Active EBV Infection.
Authors: Zhang Yu , Ohyashiki Junko H. , Takaku Tomoiku , Shimizu Norio , Ohyashiki Kazuma
Published: April 2020

Nasal NK/T-cell lymphoma is a uniquely aggressive and incurable subtype of non-Hodgkin lymphoma (NHL) that is closely associated with Epstein-Barr virus (EBV). Since necrosis is the common feature of nasal NK/T-cell lymphoma, the biological features of lymphoma cell are poorly understood because of the difficulty in obtaining sufficient viable specimens. The clonal expansion of EBV-infected NK or T cells is also seen in patients with chronic active EBV (CAEBV) infection, suggesting that two diseases might share a partially similar mechanism by which EBV affects host cellular gene expression. Although the molecular biology of EBV and B cell transformation has been extensively studied for years, little is known about the host-virus interaction in EBV-related NK- and T-cell malignancies. This study aimed to investigate the virus-host interaction in two types of EBV-associated NK/T-cell LPD which may clarify the possible association between infection and cancer. We attempted transcriptional profiling of EBV genes using a novel viral DNA microarray, HHV-4 Viruchip®, in addition to host cellular gene expression profiling using human oligonucleotide DNA microarrays, seeking to evaluate the possible role of EBV-host interaction in nasal NK/T-cell lymphoma and CAEBV infection. We used six cell lines with EBV-associated T/NK-cell LPD: SNK-1, SNK-6 and SNT-8 were isolated from nasal NK/T-cell lymphoma, and SNK-10, SNT-13 and SNT-16 were isolated from the peripheral blood of patients with CAEBV infection (Zhang Yu, et al., Br. J. Haematol., 121: 805–814, 2003). We found that expression of BZLF1, which encodes the immediate-early gene product Zta, was preferentially high in cell lines from CAEBV infection. We also analyzed the gene expression patterns of host cellular genes using a pathway-focused human oligonucleotide DNA microarray (NCBI GEO accession number: GPL1919 and GPL 1920). We identified a subset of pathogenically and clinically relevant host cellular genes, including TNFRSF10D, CDK2, HSPCA, IL12A as a common molecular biological properties of EBV-associated NK/T-cell LPD, and a subset of genes, such as PDCD4 as a putative contributor for disease progression. This study describes a novel approach from the aspects of viral and host gene expression which could identify novel therapeutic targets in EBV-associated NK/T-cell LPD.
Efficacy and Safety of a Single-Course of Yttrium-90 (90Y) Ibritumomab Tiuxetan (Zevalin®) in Patients with Relapsed or Refractory Mantle Cell Lymphoma (MCL) After/Not Appropriate for Autologous Stem Cell Transplantation (ASCT) - A Phase II Trial of the European MCL Network.
Authors: Weigert Oliver , von Schilling Christoph , Rummel Mathias J. , Pezzutto Antonio , Unterhalt Michael , Hiddemann Wolfgang , Dreyling Martin
Published: April 2020

Radioimmunotherapy (RIT) has demonstrated high clinical efficacy in the treatment of B-cell non-Hodgkin’s lymphoma (B-NHL) and has been approved for relapsed indolent and transformed B-NHL. However little is known about RIT with Zevalin® in patients with mantle cell lymphoma (MCL), a lymphoma subtype characterized by indolent morphology but aggressive clinical course with a median survival of only 3– 4 years and virtually no long-time survivors. Patients with relapsed or refractory CD20 positive MCL after/not appropriate for ASCT, WHO performance status <2, age < 75 years and adequate function of the bone marrow (ANC >1,500/mm3, platelets >100,000/mm3), liver and kidneys were eligible for this trial. Patients were excluded if they had > 25% bone marrow involvement, prior allogeneic stem cell transplantation or RIT, known CNS lymphoma, HIV infection or other concurrent severe medical disease. Patients with normal platelet counts received a dose of Zevalin® at 14.8 MBq 90Y/kg (to a maximum dose of 1,184 MBq), whereas those with platelet counts <150,000/mm3 received 11.1 MBq 90Y/kg. Currently, 14 patients have been enrolled and 6 are eligible for evaluation of treatment response. The median age was 68 years (range 56 – 71), 9 patients were male. All patients had been previously treated with Rituximab containing regimens. The median number of prior regimens was 4 (range 2–6). Zevalin® treatment was generally well tolerated, with the most common toxicities being hematologic. Thrombocytopenia grade 3 and 4 was observed in 2 and 4 patients, respectively, without significant bleeding complications. Granulocytopenia grade 3 and 4 occurred in 3 and 2 patients, respectively and 1 patient developed a grade 4 infectious complication. This patient also developed non-hematologic toxicities grade 3 and 4 consisting of sepsis-associated hepatotoxicity, diarrhoea and pleural empyema. Partial responses were observed in 2 of 6 patients (33.3 %), another patient experienced a stable disease. Median progression free survival (PFS) was 3.9 months in this high risk group. Preliminary analysis suggests that patients with bulky disease did not respond to radioimmunotherapy. The clinical course of the total group of this ongoing trial will be presented at the meeting. The observed responses to Zevalin® in heavily pretreated patients with MCL are promising, however median PFS was short-lived. Thus, a prior debulking strategy with combined immuno-chemotherapy is recommended to potentially achieve a longer duration of remission. Accordingly, in future trials RIT should be applied earlier in the treatment algorithm of MCL.
A Pilot Study of Yttrium 90 (90Y) Ibritumomab Tiuxetan (Zevalin®) in Patients with Relapsed and Refractory Classical Hodgkin Lymphoma (HL): Delivering Cross Fire Radiation by Targeting Reactive B Cells in the Microenvironment.
Authors: Younes Anas , Romaguera Jorge E. , McLaughlin Peter , Wong Franklin
Published: April 2020

Both non-Hodgkin lymphoma (NHL) and HL are radiosensitive tumors. Although the efficacy of radioimmunotherapy (RIT) targeting CD20 antigen has been established in several types of B-cell NHL, no data exisit on the potential use of this approach in classical HL. The malignant Hodgkin and Reed-Sternberg (HRS) cells of HL infrequently express CD20 suggesting that classical HL may not be the optimal tumor to be targeted by this approach. However, HRS cells are frequently surrounded by reactive B lymphocytes that express CD20. Therefore, we hypothesized that targeting reactive B cells in HL microenvironment by RIT could deliver effective radiation dose to HL lymph nodes killing HRS cells by cross-fire effect. To test this hypothesis, we treated 4 patients with relapsed classical HL with Zevalin RIT. During the imaging phase, patients received a slow intravenous infusion of rituximab (250 mg/m2) followed by an intravenous injection of 4–5 mCi of In-111 labeled anti-CD 20 (In-111 Zevalin). Whole-body scans were performed at 2–4 hours and 40–48 hours after injection using a Siemens dual-head gamma camera. Simultaneous anterior and posterior projections were obtained at these time points. After ruling out any unfavorable biodistribution of Zevalin was confirmed on images at both time points, patients underwent the treatment phase which consisted of rituximab (250 mg/m2) followed by Y-90 anti-CD 20 (Y-90 Zevalin). To-date 4 patients are enrolled on study (ages 25, 31, 65, and 74 years). Two patients received prior ASCT, and 2 were treated after failing ABVD chemotherapy alone. All patients received 0.4 mCi/Kg up to a maximum dose of 32 mCi. Normal biodistribution of Zevalin was confirmed in all 4 patients during the imaging phase. No Zevalin-avid lesions was noted in one patient. There was faint visualization of some of the known lesions (by Ga-67 SPECT, CT or F-18 FDG PET) in the axilla and abdomen in 2 of the 4 patients. Intense Zevalin-uptake sites were identified in the supraclavicular, infraclavicular, mediastinal, hepatic and abdominal lymphatic chains in another patient. Imaging results and treatment responses are shown in the following table:

Patient Response Prior ASCT In-111 Zevalin-avidity Clinical 1 Yes none PD 2 No faint, right axilla PD 3 Yes very faint, abdomen NC 4 No intense NC

Treatment was well tolerated with hematologic toxicity similar to those observed in NHL patients. Our preliminary results suggest that RIT targeting reactive B cells in classical HL is feasible and safe. Future studies should incorporate pretreatment evaluation of a selected lymph node by fine needle aspiration to determine the fraction of reactive B cells in the microenvironment prior to treatment with RIT targeting CD20.
Dichotomy of All-Trans Retinoic Acid Inducing Signals for Adult T-Cell Leukemia.
Authors: Maeda Yasuhiro , Yamaguchi Terufumi , Hijikata Yasuki , Morita Yasuyoshi , Hirase Chikara , Takai Syunsuke , Miyatake Jun-ichi , Matsuda Mitsuhiro , Kanamaru Akihisa
Published: April 2020

We previously reported that all-trans retinoic acid (ATRA) inhibits growth in HTLV-1-positive T-cell lines and fresh cells from patients with adult T-cell leukemia. However, the mechanism of this inhibition is not clear. In the present study, we observed that NF-κB transcriptional activity as well as cell growth decreased significantly in HTLV-1-positive T-cell lines in the presence of ATRA. Furthermore, we observed that ATRA reduced HTLV-1 proviral DNA, HTLV-1 genes (gag, tax or pol mRNA) using the real time quantitative polymerase chain reaction. SIL-2R was reduced by ATRA in both protein level (culture supernantant) and mRNA level in HTLV-1-positive T-cell lines. Interestingly, ATRA significantly inhibited RT activity similar to azidothimidine (AZT) in HTLV-1-positive T-cell lines. Moreover, AZT was inhibitory of proviral DNA but not NF-kB transcriptional activity and sIL-2R on HTLV-1, however ATRA was inhibitory of NF-kB, proviral DNA and sIL-2R on HTLV-1. These results suggested that the decrease of sIL-2R induced by ATRA may be caused by the actions of a NF-kB inhibitor acting on the NF-kB/sIL-2R signal pathway. These results suggested that ATRA could have two roles, as a NF-kB inhibitor and as a RT inhibitor.
Chemosensitization of Drug-Resistant Ramos B-NHL to Drug-Induced Apoptosis: YY1 Expression Is Decreased in Response to Cytoskeletal-Interacting Drugs.
Authors: Neshat Mehran , Jazirehi Ali R. , Bonavida Benjamin
Published: April 2020

The transcription factor Yin Yang 1 (YY1) regulates cellular differentiation, hematopoiesis, response to apoptotic stimuli, pathogenesis of cancer and its increased expression is associated with inhibition of differentiation of progenitor cells. We and others have previously shown that expression levels of YY1 also correlate with drug sensitivity in cancer cells. A comparison between the wild type (wt) Ramos, with the recently generated rituximab-resistant Ramos (Ramos RR) clones, revealed that, unlike wt Ramos, Ramos RR1 cannot be chemosensitized by rituximab and exhibited higher drug resistance. Further, there was enhanced YY1 expression in Ramos RR1. We hypothesized that overexpression of YY1 may be, in part, responsible for drug-resistance in Ramos RR1 and its inhibition can reverse resistance. This study investigated whether the heightened expression of YY1 in Ramos RR1 cells can be reversed by a panel of drugs used in combination. Ramos and Ramos RR1 cells were treated with vincristine, VP-16, CDDP, and ADR, and the NF-κB inhibitors Bortezomib and DHMEQ. Treatment of Ramos RR1 with the NF-κB inhibitor, in combination with any of the above chemotherapeutic drugs, reversed the acquired drug-resistance and synergy was achieved. Noteworthy, in Ramos RR1 cells, only vincristine (or in combination with NF-κB inhibitors) significantly abrogated or diminished YY1 expression. Similarly, in the prostatic cell line PC-3, 2-methoxyestradiol, another cytoskeletal interacting drug, resulted in marked reduction of YY1 expression level and activity (assessed by a luciferase reporter assay). These results suggest that YY1 overexpression may regulate the resistance of B-NHL to a selected group of drugs but not all drugs. The studies also suggest that agents that can modulate YY1 expression/activity may be potential therapeutics when used in combination with chemotherapeutic drugs in the treatment of drug and rituximab-resistant B-NHL. Currently, we are examining potential mechanisms that underlie the downregulation of YY1 expression by vincristine and 2-methoxyestradiol.
Suppression of Cell Growth and Induction of Apoptosis of HTLV-I-Infected T-Cell Lines and Primary ATL Cells by Galectin-9.
Authors: Okudaira Taeko , Tomita Mariko , Hirashima Mitsuomi , Mori Naoki
Published: April 2020

Adult T-cell leukemia (ATL) is a fatal malignancy of T lymphocytes caused by infection with human T-cell leukemia virus type I (HTLV-I), and remains incurable. Therefore, novel treatments are urgently needed. Galectins are a family of animal lectins with diverse biological activities. They function both extracellularly, by interacting with cell-surface and extracellular matrix glycoproteins and glycolipids, and intracellularly, by interacting with cytoplasmic and nuclear proteins to modulate signaling pathways. The distribution of galectins is quite diverse, and their expression in various leukocytes has been observed. To determine whether expression of galectins in T cells correlates with HTLV-I infection, we surveyed a number of uninfected and HTLV-I-infected T-cell lines for galectin-3, -8, and -9 expression by RT-PCR. Expression of galectin-8 did not correlate with HTLV-I infection. Galectin-3 was abundantly expressed in HTLV-I-infected T-cell lines and primary ATL cells, but not in uninfected T-cell lines. In contrast, galectin-9 was abundantly expressed in uninfected T-cell lines and normal PBMCs, but not in HTLV-I-infected T-cell lines and primary ATL cells. HTLV-I transformed protein, Tax, did not affect the expression of galectin-3 and -9. It was previously shown that galectin-8 and -9 are proapoptotic proteins. We found that galectin-9 prevented cell growth of HTLV-I-infected T-cell lines and primary ATL cells compared with galectin-8. Galectin-9 induced cell cycle arrest by reducing the expression of cyclin D1, cyclin D2, cyclin B1, Cdk1, Cdk4, Cdk6, and Cdc25C, and apoptosis by reducing the expression of XIAP and c-IAP2. Most of these genes are known to be regulated by NF-κB, which plays a critical role in oncogenesis by HTLV-I. Galectin-9 suppressed phosphorylation of IκBα. Most importantly, treatment with galectin-9 reduced tumor formation from an HTLV-I-infected T-cell line, HUT-102, when these cells were inoculated s.c. into severe combined immunodeficient mice. Our results suggest that galectin-9 may be a new approach for management of ATL.
Arsenic Trioxide in Mantle Cell Lymphoma.
Authors: Kwong Yok Lam , Chow Chit , Kumana Cyrus R. , Srivastava Gopesh , Au Wing Yan
Published: April 2020

Background. Mantle cell lymphoma (MCL) is incurable for many patients. Arsenic trioxide (As2O3) has activity in vitro against lymphoid malignancies. The effects of As2O3 on MCL in vitro and in patients with refractory disease were investigated.

Materials and methods. Mantle cell lymphoma (MCL) lines (Jeko-1, Granta-519) were treated with As2O3, in combination with mitoxantrone (MTZ) and ascorbic acid (AA). Consenting patients with refractory MCL were treated with oral-As2O3 (10 mg/day), AA (1 g/day) and chlorambucil (4 mg/day) as outpatients until maximum response or the disease judged refractory. Responses were defined by standard NCI criteria. In patients showing an initial response, vincristine (2 mg intravenously) and prednisolone (30 mg/day) might also be added. After achievement of maximum response, patients were maintained with As2O3 (10 mg/day) and AA (1 g/day) for two weeks every month, for a planned two years.

Results. As2O3 and MTZ but not AA induced a dose dependent apoptosis in the MCL lines, as shown by flow cytometry and MTT assays. As2O3, MTZ and AA were tested in various combinations in MTT assays. Synergistic interactions were observed only in the combinations As2O3 (1 uM) + MTZ (0.2 mg/L), and As2O3 (1 uM) + MTZ (0.2 mg/L) + AA (100 uM). Western blotting showed that As2O3-induced apoptosis was associated with a dose and time dependent down-regulation of cyclin D1. However, quantitative polymerase chain reaction showed no change in cyclin D1 gene transcription during As2O3-induced apoptosis. Eleven patients (10 men, 1 women) at a median age of 69 (51–70) years with refractory MCL were studied, at a median of 33 (8–85) months from diagnosis. At the time of As2O3 treatment, they had already had a median of 2 (1 – 4) relapses managed with a median of 2 (1 – 6) previous chemotherapeutic regimens. Eight of eleven patients were evaluable (for the other three, one died after 4 days of treatment, and the two were still receiving therapy). At a median follow up of 9 (4 – 20) months, 4 patients had reached complete remission (CR) or probable CR (CRu), two were in good partial remission, and two had died with progressive disease.

Conclusion. As2O3 induced apoptosis of MCL cells by post-transcription down-regulation of cyclin D1. Synergistic interactions were observed with AA and cytotoxics. Oral-As2O3, AA and chlorambucil were an active regimen for relapsed and therapy-refractory MCL, and treatment results compared favorably with other salvage regimens. This entirely oral regimen has several attractions, including outpatient treatment, low toxicity and cost.