bluuebunny/crossref_metadata_2025 huggingface dataset:
Bortezomib Resistant Constitutive NF-κB Activation in Mantle Cell Lymphoma.
Authors: Yang David T. , Young Ken H. , Kahl Brad S. , Miyamoto Shigeki
Published: October 2019

Bortezomib is a proteasome inhibitor whose antineoplastic effects include inhibition of NF-κB, a transcription factor whose deregulation may play a central role in mantle cell lymphoma (MCL) pathogenesis. Bortezomib has shown clinical efficacy in relapsed and refractory cases of MCL with response rates of 40%. NF-κB can be activated through several pathways, including a proteasome inhibitor resistant pathway. It remains unknown whether MCL harbors bortezomib resistant constitutive NF-κB activity, but characterization of this may have important implications in elucidating bortezomib resistance and also in establishing rational therapeutic combinations. We investigated the effect of bortezomib on constitutive NF-κB activity in 3 EBV-negative MCL cell lines (Jeko, Rec-1 and Z138) and 20 MCL patient samples. Electrophoretic mobility supershift assay demonstrated that each of the cell lines had distinct NF-κB complexes with the Jeko and Rec-1 containing mainly p50/p65 and p50/cRel heterodimers, and Z138 comprised almost entirely of p52/RelB heterodimers. At a physiologically achievable dose of bortezomib (20nM), a live cell-based proteasome inhibition assay demonstrated greater than 80% proteasome inhibition in all three cell lines. Treatment of Jeko cells with such a dose resulted in a 50% decrease of NF-κB DNA binding, in contrast to a 10 to 30% increase of DNA binding in Rec-1 and Z138 cells by electrophoretic mobililty shift assay. Of 10 MCL patient samples from which results could be obtained, only 2 demonstrated a greater than 50% decrease in NF-κB DNA binding after treatment with 20nM and 100nM of bortezomib, whereas the remainder showed either no inhibition or even increased binding. Thus, bortezomib resistant constitutive NF-κB activity appears to be present in Rec-1, Z138, and a majority of MCL cases. Cytotoxicity assessed by flow cytometry following staining with propidium iodide showed Rec-1 and Z138 cells had greater resistance to bortezomib induced apoptosis (82 ± 5% and 69 ± 5% viability) than Jeko cells (47 ± 6% viability) after 20nM bortezomib treatment for 24 hours. Combining bortezomib with perillyl alcohol, a known suppressor of proteasome inhibitor resistant NF-κB activation, resulted in a synergistic cytotoxic effect in all 3 cell lines as assessed by the combination index (CI) method with CIs of 0.31, 0.32, and 0.60 for Jeko, Rec-1, and Z138 cells respectively, where CI of 0.1–0.3 is strong synergism, 0.3–0.7 is synergism, 0.7–0.85 is moderate synergism and 0.85–0.9 is slight synergism. In conclusion, our findings suggest that bortezomib resistant NF-κB activity is present in a significant subset of MCL cases, and the combination of bortezomib with a suppressor of proteasome inhibitor resistant NF-κB activity may elicit a synergistic cytotoxic effect in MCL.
Dysregulation of Frizzled 6 Is a Critical Component of B Cell Leukemogenesis in a Mouse Model of Chronic Lymphocytic Leukemia.
Authors: Wu Qingli , Claudia Zierold , Ranheim Erik A.
Published: October 2019

Wnt/Fzd signaling is known to play a key role in development, tissue specific stem cell maintenance, and tumorigenesis, particularly through the canonical pathway involving stabilization of β-catenin. We have previously shown that Fzd9−/− mice exhibit a decrease in pre-B cells at a stage when self-renewing division is occurring in preference to further differentiation, prior to light chain immunoglobulin recombination. To determine whether pathologic usurpation of this pathway plays a role in B cell leukemogenesis, we examined the expression of Wnt/Fzd pathway genes in the Eμ-TCL1 mouse model of chronic lymphocytic leukemia (CLL). We find that in the course of leukemogenesis, the expression of Wnt16, Wnt10b, Fzd1, and most dramatically, Fzd6, are progressively upregulated in the transformed CD5+ B cells of these mice, as are β-catenin protein levels. Fzd6 expression is increased an average of 35-fold in tumor B cells in comparison to CD5- normal B cells in the same mouse. In 3 animals we were able to compare oligoclonal, preleukemic CD5+ B cells with monoclonally transformed CD5+ B cells and noted between 4 and 340 fold incremental increases in Fzd6 expression. Elimination of Fzd6 gene expression by crossing Eμ-TCL1 into Fzd6−/− mice significantly delays or eliminates development of CLL in this model, while crossing into Fzd9−/− mice has no effect. Greater than 50% of Fzd6+/+ X Eμ-TCL1 mice have evidence of CLL in the peripheral blood at 5 months of age, while in Fzd6−/− X Eμ-TCL1 mice, this occurs at 8 months (p < 0.001). Even at 1 yr. of age, >30% of Fzd6−/− X Eμ-TCL1 mice remain disease free while >95% of Fzd6+/+ X Eμ-TCL1 mice have leukemia at 7 months of age. Tumors that do arise in Fzd6−/− mice are morphologically similar to the usual Eμ-TCL1 CLL cells, but do not show upregulation of β-catenin protein by flow cytometry, suggesting that Fzd6 is responsible for this aberrant expression. Further, rather than upregulating Wnt16, Wnt10b, and Fzd1, these genes are down-modulated in tumor cells lacking Fzd6, suggesting a balance between positive and negative signals in CLL B cells mediated by the Wnt/Fzd pathway with Fzd6 acting as an oncogene and other family members potentially as tumor suppressors. We and others have noted increases in LEF-1, Wnt10b, Wnt16, and Fzd3 in human CLL B cells as well, mirroring the findings in the mouse model. Our findings suggest that the self-renewal signals mediated by Wnt/Fzd that are enlisted during B cell development may be pathologically reactivated in the neoplastic transformation of mature B cells; and that agents targeting specific members of the beta-catenin signaling pathway may have therapeutic impact that could be tested in the Eμ-TCL1 model.
Glucocorticoid Induced Cell Death Is Mediated by Its Effect on Cellular Metabolism and Is Independent of Caspase Activity.
Authors: Gurbuxani Sandeep
Published: October 2019

Glucocorticoids (GCs) such as prednisone and dexamethasone are a crucial component of acute lymphoblastic leukemia (ALL) therapy protocols. Multiple studies in childhood ALL have demonstrated that resistance to GC mediated cell death in vitro and in vivo is the single most important predictor of treatment outcome in childhood ALL. However, the mechanisms of GC mediated cell death as well as the mechanisms of resistance are poorly understood. The present study was undertaken to better understand the mechanism of GC induced cell death and to delineate possible mechanisms of resistance. In the initial experiments performed, multiple ALL cell lines, when treated with dexamethasone, underwent a reduction in the amount of reactive oxygen species (ROS) followed by cell cycle arrest and finally cell death which was unaffected by the presence of a pan-caspase inhibitor z-VAD-fmk. Since the amount of ROS present in a cell is an indicator of the metabolic state of the cell, specifically the amount of oxidative phosphorylation going on in the mitochondria, additional experiments were performed to directly estimate the mitochondrial mass as well as the metabolic state of the cells treated with GCs. While the mitochondrial mass measured by Mitotracker green labeling of mitochondria in the viable cells remained unchanged in cell lines susceptible to low concentrations (nano or micromolar) of dexamethasone, there was a prominent reduction in mitochondrial mass 36 hours after dexamethasone exposure in MOLT-4 cell line that requires several fold higher (millimolar) concentration of dexamethasone to induce cell death. The reduction in ROS was not accompanied by an increase in glycolysis as determined by the measurement of lactate concentration in the culture supernatants either in the susceptible or the resistant cells. Since one possible mechanism of reduction in ROS is increased scavenging by molecules that are dependent on the presence of NADPH generated during glucose metabolism via the pentose phosphate pathway (PPP), additional experiments were performed to determine if chemical inhibition of this pathway could augment dexamethasone induced cell death in ALL cell lines. Indeed, addition of transandosterone, an inhibitor of G6PD, the rate limiting enzyme of the PPP, resulted in significantly increased dexamethasone toxicity. Based on these experiments it can be concluded that GC induced cell death is mediated by its effect on cellular metabolism. Furthermore, this cell death is caspase independent and likely proceeds via a pathway mechanistically distinct from classical apoptosis. Finally, cells resistant to GC induced cell death have evolved mechanisms to adapt to GC induced changes in cellular metabolism and may maintain energy production via alternative pathways such as the PPP shunt that are independent of mitochondria.
Vitamin K2 Induces Autophagy and Apoptosis Simultaneously in Leukemia Cells and Bcl-2 Expression Level Determines the Phenotype of Their Cell Death.
Authors: Miyazawa Keisuke , Yokoyama Tomohisa , Naito Munekazu , Toyotake Juri , Tauchi Testuzo , Itoh Masahiro , Yuo Akira , Kondo Yasuko , Kondo Seiji , Ohyashiki Kazuma
Published: October 2019

Vitamin K2 (menaquinone-2: VK2) is now known to be a potent inducer for apoptosis in leukemia cells in vitro. HL-60bcl-2 cells, which are derived from a stable transfectant clone of human bcl-2 gene into HL-60 leukemia cell line, show 5-fold greater expression of Bcl-2 protein compared with that in HL-60neo cells, a control clone transfected with vector alone. Although HL-60neo cells are induced apoptosis in response to VK2, HL-60bcl-2 cells are resistant against apoptosis induction but still show cell growth inhibition along with an increase of cytoplasmic vacuoles during exposure to VK2. Electron microscopy revealed autophagosomes and autolysosomes formation in HL-60bcl-2 cells after exposure to VK2. An increase of acid vesicular organelles (AVO) detected by acridine orange staining for lysosomes as well as conversion of LC3B-I into LC3B-II by immunonoblotting and an increased punctuated pattern of cytoplasmic LC3B by fluorescent immunostaining all supported enhanced autophagy induction in response to VK2 in HL-60bcl-2 cells. However, during shorter exposure to VK2, autophagosome formation was rather prominent in HL-60neo cells although nuclear chromatin condensations and nuclear fragments were also observed at the same time. These findings indicated the mixed morphologic features of apoptosis and autophagy. Inhibition of autophagy by either addition of 3-methyladenine, siRNA for Atg7, or Tet-off Atg5 system all resulted in attenuation of VK2-incuded cell death, indicating autophagy-mediated cell death in response to VK2. These data demonstrate that autophagy and apoptosis can be simultaneously induced by VK2. However, autophagy becomes prominent when the cells were protected from rapid apoptotic death by higher expression level of Bcl-2.
Different Cooperating Mutation Patterns of Receptor Tyrosine Kinases/Ras/JAK2 between De Novo AML1-ETO and CBFβ-MYH11 Acute Myeloid Leukemia.
Authors: Liang Der-Cherng , Shih Lee-Yung , Huang Chein-Fuang , Lai Chang-Liang , Chang Ya-Tzu , Lin Tung-Huei , Wu Jin-Hou , Kou Ming-Chung , Lin Tung-Liang , Yang Chao-Ping , Liu Hsu-Che
Published: October 2019

Background. Two-hit model of leukemogenesis has been proposed for AML; class I mutations that drive proliferation and survival, and class II mutations that block differentiation. Core-binding factor (CBF) AML consists of AML with AML1-ETO and AML with CBFβ-MYH11, that are class II mutations.

Aim. We sought

to determine the frequencies of cooperating mutations of class I including receptor tyrosine kinases (RTK)/Ras/JAK2 signaling pathways in CBF-AML, and to compare the patterns of cooperating mutations between AML with AML1-ETO and AML with CBFβ-MYH11.

Patients and methods. By RT-PCR analysis, 130 adult and 45 children were identified to have CBF-AML, 129 with AML1-ETO and 46 with CBFβ-MYH11. Bone marrow samples at diagnosis were analyzed for FLT3-LM, FLT3-TKD, c-KIT, c-FMS, N-ras, K-ras and JAK2 mutations.

Results. Sixty-six of 129 patients (51.2%) with AML1-ETO had RTK/Ras/JAK2 mutations compared with 30 of 46 patients (65.2%) with CBFβ-MYH11 (p=0.121). The frequencies of RTK/Ras/JAK2 mutations in 129 AML1-ETO AML were 3.9% (n=5) for FLT3-LM, 6.2% (n=8) for FLT3-TKD, 2.3% (n=3) for N-ras, 3.9% (n=5) for K-ras, 35.7% (n=46) for c-KIT, and1.6% (n=2) for JAK2 mutation. The frequencies of RTK/Ras/JAK2 mutations in 46 CBFβ-MYH11 AML were 2.2% (n=1) for FLT3-LM, 19.6% (n=9) for FLT3-TKD, 21.7% (n=10) for N-ras, 23.9% (n=11) for c-KIT, and none for K-ras or JAK2 mutations. No c-FMS mutations were detected in both subtypes of CBF-AML. All RTK/Ras/JAK2 mutations were mutually exclusive except three, one each with N-ras and K-ras mutations, FLT3-TKD and c-KIT mutations, c-KIT and JAK2 mutations, respectively. Patients with CBFβ-MYH11 had a significantly higher frequency of FLT3-TKD and N-ras mutations than patients with AML1-ETO (p=0.017 for FLT3-TKD, and p<0.001 for N-ras). Taken together, c-KIT mutations accounted for 32.6% in CBF-AML, the frequency of c-KIT mutations in patients with AML1-ETO was significantly higher than that of CBFβ-MYH11 subtype. Of the 46 patients with AML1-ETO and c-KIT mutations, 34 had mutations located at kinase domain (exon 17), 7 in exon 8, 1 in exon 9, and 4 in exon 11. Of the 11 patients with CBFβ-MYH11 and c-KIT mutations, 5 had mutations in exon 8, 2 in exon 11 and 4 in exon 17. Patients with AML1-ETO were more frequently associated with c-KIT mutations at kinase domain compared with patients with CBFβ-MYH11 (p=0.031), whereas those with CBFβ-MYH11 had a higher frequency of c-KIT mutations in exon 8 than patients with AML1-ETO (p=0.042).

Conclusion. Our results showed that occurrence of cooperating mutations of RTK/Ras/JAK2 pathways are common in patients with CBF-AML, but the patterns of mutations were different between AML1-ETO and CBFβ-MYH11 subtypes.
The In Vivo Impact of Human Regulatory T Cells on the Graft Versus Tumor Effect.
Authors: Mutis Tuna , Rozemuller Henk , Emmelot Maarten E. , Aarts-Riemens Tineke , Verweij Vivienne , Ebeling Saskia , Verdonck Leo F. , Lockhorst Henk , Martens Anton
Published: October 2019

The curative Graft-vs-Tumor effect (GvT) of allogeneic Stem cell transplantation (SCT) is frequently complicated with life threatening Graft-vs-Host Disease (GvHD). In mice, prevention of GvHD, without abrogation of GvT is possible by co-transplantation of naturally occurring regulatory T cells (Tregs) with SC grafts. Consistent with these murine studies, we recently demonstrated that also human Tregs possess potent GvHD-downregulatory capacities in a xenogeneic(x) model, where x-GvHD is induced by infusion of autologous human T cells in RAG2−/−γc−/− mice (Mutis et al. Clin. Cancer Res.2006, 12: 5520–5525). Towards clinical application of Tregs, we now explored the impact of human Treg-administration on GvT in a bioluminescence imaging (BLI) based human-GvT model in the RAG2−/−γc−/− mice. In this model, mice inoculated with luciferase (LUC)-transduced human myeloma (MM) cell lines developed BLI-detectable, progressive, MM-like multifocal tumors exclusively in the bone marrow (BM). Full blown tumors were effectively eliminated by infusion of allogeneic human PBMC. This treatment also caused lethal x-GvHD as expected. In this setting, co-infusion of human PBMC with autologous, in vitro cultured Tregs at a 1:1 Treg: T effector cell ratio had no adverse effects on the development of GvT while significantly reducing the lethality of x-GvHD. In vitro analyses of sacrificed mice at day 21 revealed that administered Tregs homed to BM and spleen, significantly downregulated the total numbers of IFN-γ-producing CD4+ and CD8+ T cells responding to CD3 mediated signals, but had no downregulatory effect on the frequencies of IFN-γ-producing T cells responding to tumor cells. There was also no downregulation of cytotoxic activity against tumor cells in Treg-treated mice. Conclusively, these results showed that Tregs, at doses which are inhibitory for x-GvHD-inducing T cells, could maintain the GvT effect by allowing T cell reactivity against tumor cells. Human Tregs thus still hold promise as attractive cellular tools for separating GvT from GvHD.
NF-kB as a Regulator of FA/BRCA Gene Expression in Multiple Myeloma.
Authors: Oliveira Vasco A. , Mathews Linda , Yarde Danielle , Wang Xingyu , Boulware David , Hazlehurst Lori A. , Chen Dung-Tsa , Beg Amer , Dalton William S.
Published: October 2019

Results to date argue compellingly that disruption of FA/BRCA gene expression plays a pivotal role in human somatic carcinogenesis. Melphalan, a DNA cross-linker, is one of the most widely used and effective drugs in the treatment of multiple myeloma (MM). Although most patients respond to standard and high dose melphalan, eventually patients acquire resistance and develop progressive disease. In 1991, our laboratory reported that acquired resistance in a human myeloma cell line was associated with reduced DNA crosslinks, elevated glutathione levels, and increased radiation survival (Cancer Res. 5:993; 1991). Most recently, we reported that the melphalan-resistant myeloma cell lines, 8226/LR5 and U266/LR6, showed a significant increase in several FA/BRCA genes compared to drug-sensitive cells, and that enhanced interstrand crosslink (ICL) repair via this signaling pathway contributes to acquired drug resistance in melphalan resistant cell lines (Blood 10:698; 2005). Here, we report that IKKa is constitutively phosphorylated in unstimulated 8226/LR5 cells, but not in melphalan-sensitive control cells. The specific phosphorylation of IKKa leads to an increase in basal NF-kB DNA binding activity, and 8226/LR5 cells are found to be markedly sensitive to BMS-345541 (a highly selective inhibitor of IkB) relative to control cells. Importantly, a cytotoxic dose of BMS-345541 induces a dramatic decrease in FA/BRCA gene expression, and a concomitant inhibition of NF-kB DNA binding activity in both 8226/S and 8226/LR5 cells. Furthermore, we show that 8226/LR5 cells experience the highest degree of direct binding between FANCD2 promoter and NF-kB/Rel family members, which, in turn, leads to an increase in basal FANCD2-specific NF-kB activity. Small-interfering RNA (siRNA)-mediated depletion of RelB and p50, but not other NF-kB subunits, in 8226 cells results in impaired NF-kB binding activity, and visible decrease in FANCD2 protein expression. Studies designed to dissect the role of NF-kB in acquired melphalan resistance are in progress, and the results will be presented. Our findings suggest that NF-kB functions as a regulator of FA/BRCA expression, and that this pathway represents a new target for preventing acquired drug resistance in myeloma patients.
Serum Concentrations of DKK-1 Correlate with the Extent of Bone Disease in Multiple Myeloma.
Authors: Kaiser Martin , Mieth Maren , Liebisch Peter , Rötzer Susanne , Jakob Christian , Fleissner Claudia , Braendle Edgar , Stover David , Peters Malte , Sezer Orhan , Heider Ulrike
Published: October 2019

Objectives: Lytic bone disease is a hallmark of multiple myeloma (MM) and is caused by osteoclast activation and osteoblast inhibition. Secretion of Dickkopf (DKK)-1 by myeloma cells was reported to cause inhibition of osteoblast precursors. DKK-1 is an inhibitor of the Wnt/β-catenin signaling, which is a critical signaling pathway for the differentiation of mesenchymal stem cells into osteoblasts. So far there is no study showing a significant difference in serum DKK-1 levels in MM patients with or without lytic bone lesions.

Methods: DKK-1 serum levels were quantified in 184 previously untreated MM patients and 33 MGUS patients by ELISA, using a monoclonal anti-DKK-1 antibody. For the evaluation of bone disease, skeletal X-rays were performed.

Results: Serum DKK-1 was elevated in MM as compared to MGUS (mean 11,963 pg/mL versus 1993 pg/mL, P < 0.05). Serum DKK-1 levels significantly correlated with myeloma stage according to Durie and Salmon (mean 2223 pg/mL versus 15,209 pg/mL in stage I and II/III, respectively; P = 0.005). Importantly, myeloma patients without lytic lesions in conventional radiography had significantly lower DKK-1 levels than patients with lytic bone disease (mean 3114 pg/mL versus 17,915 pg/mL; P = 0.003). Of interest, serum DKK-1 correlated with the number of bone lesions (0 vs. 1–3 vs. >3 lesions: mean 3114 pg/mL vs. 3559 pg/mL vs. 24,068 pg/mL; P = 0.002).

Conclusion: This is the largest study of DKK-1 serum levels in multiple myeloma patients and data show for the first time a correlation between DKK-1 serum concentration and the amount of lytic bone disease, suggesting that DKK1 is an important factor for the extent of bone disease and supporting the hypothesis of DKK-1 as a therapeutic target in myeloma bone disease.
Biological Sequelae of TRAF2 Downregulation in Waldenstrom Macroglobulinemia Cells.
Authors: Leleu Xavier , Xu Lian , Hunter Zachary R. , Adamia Sophia , Hatjiharissi Evdoxia , Moreau Anne-Sophie , Roccaro Aldo , Sacco Antonio , Ciccarelli Brian , Manning Robert J. , Patterson Christopher J. , Ghobrial Irene M. , Treon Steven P.
Published: October 2019

Background. Several TNF family members (CD40L and BAFF/BLYS) have been implicated in Waldenstrom’s Macroglobulinemia (WM) cell growth and survival. More recently, abnormalities in the APRIL-TACI pathway have been demonstrated by us in WM cells (Hunter, ASH2006, #228). TRAFs (TNFR-associated factor) are a family of adaptor proteins that mediate signal transduction from multiple members of the TNF receptor superfamily. In particular, TRAFs facilitate pro-apoptotic signaling from the TACI receptor, and TRAF2 is of importance among the TRAF adapter proteins since this protein is required for TNF-alpha-mediated activation of SAPK/JNK MAPK known to be involved in drug-induced death of tumor B cells. We therefore examined the role of TRAF2 in WM growth and survival.

Method. We investigated TRAF2, 3 and 5 gene expression in WM patient bone marrow (BM) CD19+ cells and cell lines (BCWM.1, WSU-WM) and compared their expression to BM CD19+ cells from healthy donors. Expression of human TRAF transcripts were determined using real time quantitative RT-PCR (qPCR) based on TaqMan fluorescence methodology. To evaluate the role of TRAF2, a knockdown model was prepared in BL2126 B-cells and BCWM.1 WM cells using electroporation, with resulted ≥50% knockdown efficiency using RT-PCR and immunoblotting.

Results. We found that TRAF3 and 5 gene expression was higher in WM versus healthy donors, while TRAF2 expression was lower in 8/13 (60%) patients, using qPCR. TRAFs gene expression did not correlate with tumor burden or WM prognostic markers. We next sought to understand the biological sequelae of TRAF2 deficiency in BL2126 and BCWM.1 cells and found that TRAF2 knockdown induced increased survival at 72 hours in both cell lines. We next studied sequence analysis of 20 WM patients CD19+ BM cells to determine whether there was a TRAF2 genomic alteration, and found heterozygous early termination mutation in exon 5 in 1 (5%) patient.

Conclusion. Our data demonstrate that TRAF2 is a commonly dysregulated TNF family adapter protein in patients with WM, with important consequences in WM cell growth and survival.
Amyloidogenic lambda Light Chain Renal Toxicity: Oxidative Stress Implicated.
Authors: Zeldenrust Steven R. , Blanco Ellen W. , Nath Karl A.
Published: October 2019

The kidney is the most common organ involved in systemic light-chain amyloidosis (AL), seen in up to 40% of patients. Renal involvement can result in proteinuria and/or renal insufficiency. The mechanism of renal injury in amyloidosis remains unclear. We present data supporting a central role for oxidative stress in amyloid-induced renal damage. Soluble free light chains(LCs) were isolated from plasma of AL and myeloma patients obtained at the time of stem cell collection. LCs were purified by liquid-phase iso-electric fractionation of pooled plasma samples from each patient. Purified LC were incubated with cultured rat renal epithelial cells, both wild-type and heme oxygenase-1 (HO-1) over-expressing, for 16 hours. Macrophage chemoattractant protein-1 (MCP-1) induction was measured in the media by ELISA. LC from myeloma patients were used as negative controls. Significant induction of MCP-1, a sensitive indicator of oxidative stress, was seen in 4/5 patients with lambda light-chain amyloidosis and 0/4 kappa light-chain amyloidosis patients. Induction of MCP-1 was seen in only 1/8 myeloma patients tested (4 lambda and 4 kappa). Induction of MCP-1 was reduced in the presence of HO-1 overexpression, implicating oxidative stress as a critical pathway of toxicity of amyloidogenic lambda light chains in this system. These results suggest that lambda and kappa light chains may cause renal injury through distinct pathways in the kidney.
Immunophenotypic Analysis of Waldenstrom’s Macroglobulinemia: A Study of 63 Cases.
Authors: Toma Andrea , Le Garff-Tavernier Magali , Brissard Martine , Bonnemye Patrick , Musset Lucile , Azar Nabih , Leblond Veronique , Merle-Beral Helene
Published: October 2019

The immunophenotypic characterization of Waldenstrom’s macroglobulinemia (WM) is still unclear, despite being an essential tool in the diagnosis of hematological malignancies. We retrospectively reviewed the immunophenotypic profile of 63 cases of WM showing monoclonal IgM in the serum and morphological lymphoplasmacytic bone marrow infiltration, and of 26 cases of other chronic B-cell lymphoproliferative disorders having monoclonal IgM in the serum (LPD-M), including marginal zone (n=16), mantle cell (n=8) and follicular (n=2) lymphomas. The median age at diagnosis was 64[46–92] years. Immunophenotypic analysis was performed by flow cytometry between January 1998 and July 2007, using four or six monoclonal antibody combinations. In WM and LPD-M groups, all patients showed a monoclonal tumoral B-cell population, detected and studied in blood (21 and 23 patients, respectively), blood and bone marrow (19 and 2 patients, respectively) or bone marrow samples (23 and 1 patient, respectively). Patients with a Matutes score > 3 were excluded. Neoplastic cells, in all cases, expressed a monoclonal immunoglobulin light chain (kappa for 70% WM and 73% LPD-M, lambda for 30% WM and 27% LPD-M). The intensity of expression of the monoclonal light chain was particularly heterogeneous in both groups: high, normal or low expression in 43%, 27% or 30% of WM, and in 52%, 33% or 15% of LPD-M, respectively. Pan-B antigens (CD20, CD19, CD79b) were positive for at least 97% of patients. The results obtained with other antigens in WM compared to LPD-M were: CD10 = 10 versus 7%, CD23 = 33 versus 56%, CD5 = 14 versus 26%, FMC7 = 76 versus 89%, CD38 = 56 versus 41%, CD25 = 86 versus 84%, CD43 = 12 versus 16%, and CD11c = 10 versus 36%. The intensity of expression of these antigens was heterogeneous in both groups. Among the antigens only tested in the WM group, CD1c and CD27 were positive for 70% of patients, IgM and IgD for 95% of patients, and CD103 as well as CD117 were negative in all cases. Considering all (blood and/or bone marrow) WM samples, plasma cells (CD38/CD138 positive cells) were found at low levels (less than 2.5% of B-cells) for 46% of WM. Comparing blood versus bone marrow WM samples, no differences were found for all previous antigens except the higher frequency of low counts of plasma cells in bone marrow (observed in 71% of WM) versus blood samples (28% of WM). Among the 10 WM patients tested for ZAP-70 expression, nine were negative and one showed a low intensity expression. In conclusion, our results show that the immunophenotypic analysis usually performed with standard antigens in WM overlaps with other B-cell lymphoproliferative disorders. Studies involving the expression of new antigens and/or other biological approaches are required to identify the WM among the B-cell malignancies and are ongoing in our group.
Phase I Trial of Multiple Infusions of Autologous Activated T Cells with Anti-CD3 x Anti-CD20 Bispecific Antibody (CD20Bi) after Autologous Peripheral Blood Stem Cell Transplant for NonHodgkins Lymphoma To Improve Graft-vs-Lymphoma Effects.
Authors: Lum Lawrence G. , Uberti Joseph P. , Al-Kadhimi Zaid , Skuba Cassara , Steele Pat , Sandborg Rebecca , Ratanatharathorn Voravit
Published: October 2019

Relapse rates after high dose chemotherapy (HDC) autologous peripheral blood stem cell transplant (PBSCT) for high risk refractory or relapsed non-Hodgkin’s lymphoma (NHL) patients remain unacceptably high. In order to augment the anti-lymphoma effect and decrease relapsed rates, our strategy combines 1) the cytotoxicity mediated by anti-CD3 activated autologous T cells (ATC) armed with a bispecific antibody (BiAb) that redirects ATC to kill CD20 + lymphoma cells and circumvents rituximab resistance (Exp Hemat33:452, 2005) and 2) autologous PBSCT after high dose chemotherapy. Arming ATC with anti-CD3 x anti-CD20 (CD20Bi) converts each ATC into a CD20-specific cytotoxic T cell. Our phase I trial tests whether multiple infusions of armed ATC armed given after HDC followed by PBSCT are safe and whether such infusions will provide an anti-lymphoma effect to improve overall survival and disease free survival after PBSCT. On day +4, the patients receive immune consolidation consisting of 3 infusions of CD20Bi-armed ATC per week for 3 weeks and then 1 infusion per week for 6 additional weeks. The dose levels are 5, 10, 15, and 20 x 109 cells/infusion with total armed ATC doses equaling 75, 150, 225, and 300 x 109 CD20Bi-armed ATC. Subcutaneous IL-2 (300,000 IU/m2/day) will be given daily beginning d+4 and ending after the last dose of armed ATC. The dose of CD34+/kg ranged from 1.04 to 3.4 x 106. T cells in the leukopheresis product were activated with anti-CD3 and expanded in low dose IL-2, harvested, armed with CD20Bi, and cryopreserved for infusions after PBSCT. The ATC harvest ranged from 119–140 billion with >94% viability with 96–99% CD3+, 33–67% CD4+, 32–53% CD8+, and <6.5% CD4+CD25+ and CD8+CD25+ cells. All three patients received all of their infusions. Three patients completed the first dose level (total of 70 x 109 CD20Bi armed ATC) without any dose limiting toxicities. All of the patients are alive 780, 650, and 521 days after PBSCT. Two are free of disease and one was transplanted with persistent disease went into remission after PBSCT and relapsed 8 months after PBSCT. He is now in remission following chemotherapy and a MUD PBSCT. The cytotoxicity mediated by the patients’ ex vivo expanded, armed ATC against the B9C cell line was significantly higher than unarmed ATC. CTL activity directed at B cell targets was detected by 3 weeks after PBSCT. These results suggest that large numbers of armed autologous ATC can be infused after PBSCT without major side effects related to the armed targeted ATC. This strategy may provide a unique opportunity to increase the GVL effect without increasing toxicities after autologous PBSCT for CD20+ lymphomas.
In Vitro Characterization of OP-R003-1, a New Fully Human Anti-IL6 Antibody.
Authors: Vermot-Desroches Claudine , Wang Wei , Subiger Olivier , Abribat Thierry , Alberici Gilles , Zauderer Maurice , Ernest Smith
Published: October 2019

Potential applications for drugs targeting IL-6 include a range of inflammatory and proliferative disorders, including rheumatoid arthritis, multiple myeloma, post transplant lymphoma, Crohn’s disease and Systemic Lupus. Using the ActivMab® technology, a high-affinity, fully human antagonistic monoclonal antibody (MAb) OP-R003-1 was structurally derived from elsilimomab (B-E8, a murine anti-IL-6 MAb) which had previously been shown to be effective in several proof-of-concept clinical trials. In vitro studies demonstrated that OP-R003-1 shares similar biological properties, including affinity and epitope specificity with its “parent” murine antibody. OP-R003-1 detected human and monkey IL-6 but not murine or rat IL-6. No binding was observed with other IL-6 superfamily members. This novel fully human MAb inhibited the binding of IL-6 to its receptor as evidenced by flow cytometry using U266 cells and also inhibited IL-6 induced cell proliferation in murine B9, human TF1 or U266 cells. The conversion of the murine B-E8 MAb into a fully human MAb will result in an improved therapeutic MAb clinical profile, including reduced immunogenicity and improved half-life, while preserving the specificity and clinical effectiveness of the murine Mab.
Cytotoxicity of Genetically Engineered Fusion Protein with CD154 Peptide Mimetic (OmpC-CD154p) on B-NHL Cell Lines.
Authors: Martinez-Miguel Bernardo , Martinez-Paniagua Melisa A. , Huerta-Yepez Sara , Hernandez-Pando Rogelio , Gonzalez-Bonilla Cesar R. , Bonavida Benjamin , Vega Mario I.
Published: October 2019

The interaction between CD40, a member of the tumor necrosis factor super family, and its ligand CD154 is essential for the development of humoral and cellular immune responses. Selective inhibition or activation of this pathway forms the basis for the development of new therapeutics against immunologically-based diseases and malignancies. CD40 is expressed primarily on dendritic cells, macrophages and B cells. Engagement of CD40-CD154 induces activation and proliferation of B lymphocytes and triggers apoptosis of carcinoma and B lymphoma cells. Agonist CD40 antibodies mimic the signal of CD154-CD40 ligation on the surface of many tumors and mediate a direct cytotoxic effect in the absence of immune accessory molecules. CD40 expression is found on nearly all B cell malignancies. Engagement of CD40 in vivo inhibits B cell lymphoma xenografts in immune compromised mice. Several clinical trials have been reported targeting CD40 in cancer patients using recombinant CD154, mAbs and gene therapy, which were well tolerated and resulted in objective tumor responses. In addition to these therapies, CD54 mimetics have been considered with the objective to augment and potentiate the direct cytotoxic anti-tumor activity and for better accessibility to tumor sites. This approach was developed by us and we hypothesized that the genetic engineering of a fusion protein containing a CD154 peptide mimetic may be advantageous in that it may have a better affinity to CD40 on B cell malignancies and trigger cell death and the partner may be a carrier targeting other surface molecules expressed on the malignant cells. This hypothesis was tested by the development of a gene fusion of Salmonella typhi OmpC protein expressing the CD154 Trp140-Ser149 amino acid strand (Vega et al., Immunology2003; 110: 206–216). This OmpC-CD154p fusion protein binds CD40 and triggers the CD40 expressing B cells. In this study, we demonstrate that OmpC-CD154p treatment inhibits cell growth and proliferation of the B-NHL cell lines Raji and Ramos. In addition, significant apoptosis was achieved and the extent of apoptosis was a function of the concentration used and time of incubation. The anti-tumor effect was specific as treatment with OmpC alone had no effect. These findings establish the basis of the development of new fusion proteins with dual specificity (targeting the tumor cells directly or targeting the tumor cells and immune cells). The advantages of this approach over conventional CD40-targeted therapies as well as the mechanism of OmpC-CD154p-induced cell signaling and cell death will be presented.
MTHFR 677CC/1298CC Genotypes Are Highly Associated with Chronic Myelogenous Leukemia: A Case-Control Study in Korea.
Authors: Moon Hee Won , Kim Tae Young , Oh Bo Ra , Min Hyun Chung , Cho Han Ik , Lee Jae Hoon , Yoon Sung Soo , Lee Dong Soon
Published: October 2019

Methylenetetrahydrofolate reductase (MTHFR) is an enzyme involved in folate metabolism and DNA methylation. Studies on MTHFR polymorphism in leukemia have largely focused on the protective role of MTHFR polymorphism in ALL. We evaluated the C677T and A1298C polymorphisms using the TaqMan® allelic discrimination assay in various malignancies. The study population included 115 subjects with CML, 200 with AML, 196 with MM and 434 healthy control subjects. The frequency of 1298 CC was statistically significantly higher in subjects with CML than that of the controls (OR = 5.12, 95% CI: 1.75–14.9, P value =.003). Of note, the frequencies of 677CC/1298CC genotype was statistically significantly higher in subjects with CML, AML and MM than that of the controls (OR = 8.8, 3.5, 3.83, P value =.002, 0.036, 0.023, respectively). Our results demonstrate that the MTHFR 1298CC homozygote variant is strongly associated with an increased risk of CML, while MTHFR C677T does not significantly affect the risk of CML. Moreover, we demonstrated that MTHFR 677CC and 1298CC genotype might have combined effect on risk of CML, AML and MM and it is inferred that the A1298C may play a different role in carcinogenesis, depending on the types of organs involved, the types of disease entities and the genotype of C677T.
Efficacy of Lenalidomide for Treatment of Anemia in Secondary Myelodysplastic Syndrome (MDS) with Chromosome 5q Deletion.
Authors: Mintzer David Michael , Billet Shira N.
Published: October 2019

Lenalidomide can be effective in treatment of MDS, particularly in patients with isolated 5q deletion, as well as in patients with 5q− with other karyotypic abnormalities (List et. al. NEJM355, 1456; 2006). However, there is little data for secondary MDS. We report two patients with chemotherapy-induced MDS with 5q−, both treated with lenalidomide, one of whom responded. Case 1: An 81-year-old female with multiple myeloma who had been treated with melphalan and prednisone developed worsening anemia despite continued biochemical remission of her myeloma. Three years after melphalan treatment, a bone marrow revealed MDS with only 2% plasma cells. Cytogenetics showed: 46, XX, del(5)(q13q33), del(14)(q1?3), add(22)(q13)[1]/46, sl, del(20)(q13.1) [9]/47, sdl1, +8[10]. The patient was transfusion dependent despite treatment with EPO. She was treated with lenalidomide 10 mg daily and began responding after eight weeks of therapy. Her anemia improved, and she became transfusion independent until her death 6 months later from infection.

Figure 1. HCT before and after lenalidomide treatment (patient 1) Figure 1. HCT before and after lenalidomide treatment (patient 1)

Case 2: A 54-year-old male developed MDS after multiple therapies for low grade non-Hodgkins lymphoma, including rituximab, ibritumomab tiuxetan, chlorambucil, and CHOP. While in remission for lymphoma, he developed pancytopenia. A bone marrow revealed no lymphoma, but demonstrated evidence of MDS. Cytogenetics showed: 46, XY, del (5)(q13q33) [3]/44, idem, −7, add(19)(q13), −21[3]/46, XY [14]. The patient was transfusion dependent. He was treated with lenalidomide 10mg daily, demonstrating no significant benefit after 10 weeks. We report that one of our two patients with secondary MDS and 5q− responded to lenalidomide with a resolution of her transfusion requirement. Two prior cases of improvement in secondary MDS with 5q− after lenalidomide treatment have been reported (Melchert et. al. Leukemia21: 1576–78, 2007). Lenalidomide is active in some cases of secondary MDS with 5q−. Further studies are needed to better define the activity of lenalidomide in secondary MDS with and without 5q−.
JAK2 (V617F) Mutation Levels and Marrow Fibrosis in Patients Affected by Budd-Chiari Syndrome and Non-Cirrhotic Extra-Hepatic Portal Vein Obstruction.
Authors: Rago Angela , Marzano Chiara , Mammì Corrado , Laganà Carmelo , Fama Angelo , Bizzoni Luisa , Tomei Gabriella , Trasarti Stefania , Gioisa Francesca , Breccia Massimo , Carmosino Ida , Latagliata Roberto , Alimena Giuliana , Foa Robert , Riggio Oliviero , Tafuri Agostino
Published: October 2019

Budd-Chiari syndrome (BCS) and Non-Cirrhotic Extra-Hepatic Portal Vein Obstruction (EHPVO) are two rare vascular diseases of the liver. Inherited and acquired disorders including Myeloproliferative Disorders (MPD) have been frequently reported associated with these diseases, although the large majority of cases is still defined as “idiopathic disease”. The somatic mutation in the tyrosine kinase JAK2 has been described in MPD, specifically in the majority of patients with polycythemia vera (PV), in about half of cases with essential thrombocythemia (ET) and in one third of idiopathic myelofibrosis (IMF), suggesting its pathogenetic role in these diseases. Since the diagnosis of MPD is often difficult in patients with BCS or EHPVO because of spleen enlargement and secondary pancitopenia that can mask erythrocytosis and thrombocytosis, recent observations have included in their diagnostic work-up the analysis of JAK2 mutations. In fact, recently, a high prevalence of JAK2 mutations has been described in splanchnic vein thrombosis. The aim of this study was to evaluate the prevalence and the levels of JAK2 mutation in the patient population affected by BCS and EHPVO followed at our Hepatology Division. The JAK2 mutation was evaluated by allele-specific real-time TaqMan polymerase chain reaction. We enrolled in this study 18 patients (median age: 38 ± 9.8 years) affected by BCS (7 patients), EHPVO (10 patients), or both (1 patient). In 9 of them the JAK2 mutation was absent, while it was demonstrated in the remaining cases. Among these, an heterozygous deletion (JAK2 mutation ranging between 2.7% and 48%) was detected in 5 and an homozygous deletion (55.7%–88%) in 4. A diagnosis of PV was made in 2 patients with JAK2 heterozygous deletion affected by BCS and EHPVO. The 3 IMF were all characterized by homozygous deletions: 1 had BCS and 2 EHPVO. In the remaining 3 patients, in the absence of other diagnostic criteria, only the bone marrow biopsy revealed the presence of initial marrow fibrosis: grade 0–1 (2 patients both affected by EHPVO with heterozygous and homozygous deletion) and grade 1 (in a heterozygous patient affected by EHPVO). In summary, the JAK2 mutation was demonstrated in half of the cases with vascular liver diseases, regardless of the JAK mutation levels. The marrow fibrosis was, in these cases, frequently associated with both BCS and EHPVO, suggesting a common potential pathogenetic role in these vascular diseases of the liver.
ZAP-70 Expression in Chronic Lymphocytic Leukemia Is Associated with Altered Signal Transduction.
Authors: Wolniak Kristy L. , Marvin James , Goolsby Charles
Published: October 2019

B-cell chronic lymphocytic leukemia (CLL) is a malignancy of mature B-cells with a variable clinical course. Given the heterogeneity of disease course, identification of prognostic factors is imperative. One recently identified negative prognostic indicator in CLL is the T-cell tyrosine kinase ZAP70. ZAP70 is analogous to, and can functionally substitute for, the B-cell tyrosine kinase Syk and is activated following B cell receptor (BCR) crosslinking in CLL cells. However, the role of ZAP70 expression in CLL remains elusive. Utilizing flow cytometric analyses of signal transduction molecules, this study examined if ZAP70 expression affects signal transduction in CLL. Peripheral blood samples from ZAP70+ and ZAP70− CLL patients were analyzed. The levels of phosphorylated ERK, STAT5, and S6 (p-ERK, p-STAT5, p-S6) were measured by flow cytometry with and without BCR stimulation. Kinase inhibitors were used to verify the measured phosphorlyated protein levels. Increased constitutive levels of p-ERK, p-STAT5, and p-S6 were seen in ZAP70+ CLL cells as compared to ZAP70− CLL cells. BCR crosslinking gave variable responses in both ZAP70+ and ZAP70− CLL. The majority of ZAP70− cases showed no significant activation or a predominant activation of ERK with rare p-S6 activation. ZAP70+ cells showed activation of either, or both, ERK and S6 pathways and fewer showing no activation. In summary, though the role of ZAP70 in B cell signaling is poorly understood, expression of ZAP70 in CLL correlates with a constitutively active phenotype. Additionally, differential response in ZAP70+ versus ZAP70− cases following BCR crosslinking suggests a possible modulatory role of the BCR pathway. Current studies are underway to determine if these measures carry prognostic information and if the varying signal responses to BCR stimulation correlate with reported differences in apoptotic versus proliferative response.
Epidemiology and Clinical Outcomes of the M-Component Immunoglobulin Types of Multiple Myeloma in an Inner City Hispanic Population.
Authors: Batty George Nicolas , Brower Jonathan , Aziz Adel , Maddukuri Neelima , Tadros James , Uhm Kyudong , Maroules Michael
Published: October 2019

Introduction: Different types of myeloma are classified by the type of immunoglobulin. From prior analysis, we know that IgG is the commonest class of myeloma and that this type seems to be the most benign. While IgA myeloma appear to be of intermediate outcome, IgD myeloma and lambda light-chain disease are the most aggressive types. Breakdown by immunoglobulin type reveals higher prevalence of IgG myeloma among Whites, compared to Blacks. This study examines the type of immunoglobulin and clinical implication in Hispanics compared to other ethnic groups.

Methods: In a single inner city institution, we retrospectively examined patients diagnosed with multiple myeloma over a 5-year period. This investigation defined Hispanic patients as those of Latin American descent. We identified the M-component immunoglobulin type from immunoelectrophoresis as reported in medical records. New onset renal failure and disease related mortality are considered as adverse clinical outcomes. Various ethnic groups were compared between the type of immunoglobulin and adverse clinical outcomes using chi-square test.

Results: A total of 203 patients were examined, of which 63 (30.9%) were Hispanics, 87 (42.9%) Caucasians and 53 (26.2%) African American, (104 males, 99 females, age range 34 – 93 yrs, mean age 67.45 ± sd 10.28 yrs). IgG and IgA M-components predominated among all ethnic groups respectively Hispanics 36 (57.9%), 6 (10.5%), Caucasians 51 (59%), 18 (22.7%) and African Americans 25 (50%), 20 (37.5%). A higher proportion of light-chain cases were seen among Hispanics since 21 (31.6%) had no peak on immunoelectrophoresis compared to Caucasians 31 (18.3%) and African Americans 8 (12.5%) (chi-square test, two-tailed p=0.426). Adverse clinical outcomes of new onset renal failure developed correspondingly in 20 (31.6%) Hispanics, 31 (36.3%) Caucasians and 15 (28.6%) African Americans while disease related mortality occurred in 20 (31.6%) Hispanics, 18 (21.4%) Caucasians and 9 (18.2%) African Americans (chi-square test, two-tailed p= 0.132). However, differences were not statistically significant.

Conclusion: Although Hispanics exhibited more myeloma of light chain type and worse clinical outcomes compared to other ethnic groups, our study was unable to detect a statistical disparity. Further prospective large population based studies via the new light chain immunodiagnostic assay are warranted to define the epidemiology of this disease.