bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluation of the Specificity and Cytotoxicity of Three Proteasome Inhibitors.
Authors: Crawford Lisa J.A. , Ovaa Huib , Walker Brian , Chauhan Dharminder , Anderson Kenneth C. , Morris Treen C.M. , Irvine Alexandra E.
Published: April 2020

The proteasome is a mutlicatalytic protease with three main catalytic activities - chymotrypsin-like (CT-L), trypsin-like (T-L) and peptidylglutamyl peptide hydrolising (PGPH). Proteasome inhibition is an emerging therapy for many cancers and is a novel treatment for multiple myeloma (MM). The CT-L activity, considered to be the rate-limiting step in protein degradation, is the primary target of many proteasome inhibitors. We have compared the specificity and potency of the novel proteasome inhibitor BzLLLCOCHO to the previously characterised inhibitors PS-341 (Velcade, bortezomib) and MG-132. Specific fluorogenic substrates were used to measure proteasome proteolytic activity in the presence and absence of the inhibitory compounds. An active site directed probe with a dansyl-sulfonamidohexanoyl hapten tag was used in conjuction with immunoblotting to determine the subunit specificity of the proteasome inhibitors (Nature Methods2005;2:357–362). MM cell lines (U266, OPM-2, KMS-11, KMS-18) were incubated with 10 μM BzLLLCOCHO, 5 nM PS-341 or 1 μM MG-132 for 24 hrs and proteasome activity was measured. Addition of BzLLLCOCHO reduced CT-L activity by 83 ± 13 % in the fluorogenic assay, and T-L and PGPH activities were reduced by 93 ± 6 % and 92 ± 2 % respectively. Immunoblot results revealed a similar pattern, the T-L and PGPH subunits were completely inhibited by BzLLLCOCHO and there was only weak labeling of the CT-L subunit with the active site probe. In contrast, treatment with PS-341 completely inhibited the CT-L and PGPH activities and incubation with MG-132 resulted in weak inhibition of the CT-L and PGPH activities, neither inihibitor significantly affected T-L activity. The ability of the different inihibitors to induce apoptosis in MM cell lines was then evaluated. All three inhibitors were demonstrated to act through both the caspase-8 and caspase-9 signalling pathways. Using Mitosensor™ and Hoescht/Propidium Iodide staining we found that MM cells were more sensitive to the induction of apoptosis by PS-341 and MG-132 than BzLLLCOCHO (U266 cells treated for 72 hrs with BzLLLCOCHO 51 % apoptosis, PS-341 79 % apoptosis and MG-132 84 % apoptosis). BzLLLCOCHO is a cell permeable and potent inhibitor of all three proteolytic activities of the proteasome. PS-341 and MG-132 inhibited only two of the three proteasome activities but were more efficient than BzLLLCOCHO at inducing apoptosis in MM cell lines. MG-132 is known to inhibit non proteasomal proteases such as Cathepsin B and Calpain 1 which may contribute to its potency. Further investigation on the effects of these inhibitors on gene and protein expression in the cell may lead to the development of more specific and targeted inhibitors.
IRF-4/MUM-1 Expression Is a Critical Switch in the Generation of Plasma Cells Versus Memory B-Cells.
Authors: Klein Ulf , Casola Stefano , Cattoretti Giorgio , Shen Qiong , Lia Marie , Mo Tongwei , Ludwig Thomas , Rajewsky Klaus , Dalla-Favera Riccardo
Published: April 2020

Most types of human B-cell lymphomas harbor somatically mutated Ig variable region genes, reflecting their origin from cells that have undergone the germinal center (GC) reaction of T-dependent immune responses. The lymphomas exhibit diverse phenotypes and clinical behaviors, likely as a consequence of differences both in the mechanisms of transformation and in the specific target cell. We have recently identified two distinct subsets of GC B-cells that may represent late stages of the GC-reaction and may be the precursors of plasma cells and memory B-cells. The corresponding subsets are characterized by downregulation of the GC-marker BCL6 and the alternative expression of IRF-4/MUM-1 or nuclear c-Rel. These two subsets seem to reflect distinct cellular programs which are altered during B-lymphomagenesis in various tumor subtypes which co-express BCL6, IRF-4 and nuclear c-Rel, an event never observed in normal B-cells. In order to gain insights into the physiologic role of IRF-4/MUM-1 and nuclear c-Rel in GC-development, we are ablating their expression specifically in mouse GC B-cells. Transgenic mice were generated that carry an IRF-4/MUM-1 null allele and a conditional IRF-4/MUM-1 allele which, following Cre-mediated deletion of the loxP-flanked promotor region and exons 1 and 2, expresses eGFP, thus allowing to track the development of the IRF-4/MUM-1 deficient cells at the single cell level. IRF-4/MUM-1fl/- mice were crossed with transgenic mice that express the Cre-recombinase specifically in B-cells undergoing class-switch to IgG1, an event occurring in a large fraction of GC B-cells. Upon immunization with sheep red blood cells or nitrophenyl-(NP)-KLH, mice unable to express IRF-4/MUM-1 in late GC B-cells (IRF-4/MUM-1fl/-/Cγ 1Cre/+), in contrast to control mice (IRF-4/MUM-1fl/+/Cγ 1Cre/+), did not develop plasma cells (IgG1+CD138+) in the peripheral lymphoid organs, blood, and bone marrow. On the other hand, the generation of memory B-cells appears normal since antigen-specific B-cells were present in the spleen (eGFP+B220+PNA-IgG1+) and blood (eGFP+B220+CD38+IgG1+). These results suggest that the IRF-4/MUM-1 gene product is required for the development of antigen-selected GC B-cells into plasma cells. We suggest that the expression of IRF-4/MUM-1 in a GC centrocyte is the critical event in the commitment of B-cells to differentiate into a plasma cell versus a memory B-cell, and are currently testing the role of nuclear c-Rel in the same process.
Zoledronate Exerts Direct Antiproliferative and Apoptotic Effects on Human Myeloma Cells In Vitro and In Vivo.
Authors: Guenther Andreas , Gordon Sharon , Bakker Frank , Burger Renate , Tiemann Markus , Green Jonathan R. , Rogers Michael , Gramatzki Martin
Published: April 2020

Zoledronate (ZOL) is the most potent nitrogen-containing bisphosphonate and is effective at preventing osteolytic bone disease in patients with multiple myeloma (MM) and solid tumors. ZOL inhibits the enzyme farnesylpyrophosphate synthase and thus blocks the prenylation of small GTPases. In vitro studies have demonstrated that ZOL can also directly affect the growth and viability of myeloma cells, however, the molecular mechanisms underlying this activity have not been fully elucidated. The goal of our study was to investigate direct antimyeloma effects of ZOL in vitro and in vivo. In five myeloma cell lines (RPMI8226, L363, U266, JK-6L, and the IL-6 dependent INA-6), growth was inhibited and apoptosis induced by ZOL in a dose-dependent manner (IC50’s between 30 μM and 285 μM). Similar results were obtained in the presence of bone marrow stromal cells, IL-6 (20 ng/mL), IGF-1 (200 ng/mL), or a combination of both cytokines. The potential antitumor effect of ZOL on myeloma cells in vivo was studied in the INA-6 SCID model, in which mice are injected intraperitoneally with INA-6 cells and subsequently develop plasmacytomas. Mice treated with ZOL had reduced tumor burden and a significant survival benefit compared to the control group (p=0.002). Histological examination of plasmacytomas explanted 72 hours after a single injection of 8 μg ZOL revealed extensive apoptotic/necrotic areas while no such areas were found in tumors of untreated animals. Induction of apoptosis was confirmed by Western blot analysis of tumor lysates, which revealed increased levels of cleaved poly (ADP-ribose) polymerase (PARP) in tumors of ZOL treated vs. untreated animals. This correlated with an accumulation of the unprenylated form of the small GTPase Rap1A, which was virtually absent in tumors of untreated mice. Our findings demonstrate a direct and specific effect of ZOL in plasmacytomas in vitro and in vivo and point to a therapeutic potential in MM beyond the prevention of osteolytic lesions.
Macrofocal Multiple Myeloma in Young Patients: A Distinct Entity with Favorable Prognosis.
Authors: Dimopoulos Meletios A. , Pouli Anastasia , Angnostopoulos Athanasios , Repoussis Panagiotis , Symeonidis Argyris , Delimbasi Souzana , Tsolakis Fotis , Zervas Constantinos
Published: April 2020

Introduction: Most patients with multiple myeloma (MM) present with multiple lytic bone lesions, extensive marrow plasmacytosis and anemia. Furthermore in several of them hypercalcemia and renal impairment are evident at diagnosis. Over the years, we have seen occasional young patients with MM who presented with multiple lytic bone lesions but without intervening infiltration of bone marrow i.e. a pattern consisting of macrofocal MM.

Patients and methods: In order to assess the clinical and laboratory features and the outcome of patients with macrofocal MM we performed a retrospective analysis of symptomatic patients with MM ≤40 years of age at diagnosis who received primary treatment over a 20 year period (from January 1, 1985 to December 31, 2004). The diagnosis of macrofocal MM required the presence of lytic bone lesions and the absence of obvious bone marrow plasmacytosis (BMPC<10%) on bone marrow aspirate and/or biopsy.

Results: Among 51 patients ≤40 years at the time of initial treatment, 10 patients fulfilled the criteria of macrofocal MM. Patients’ median age, gender, myeloma heavy and light chain were similar among patients of the 2 groups. When compared with patients with typical MM, patients with macrofocal pattern were less anemic (p=0.018), none had hypercalcemia (p=0.1), renal impairment (p=0.17), elevated serum LDH (p=0.14), or stage 3 according to the International Staging System (ISS) (p=0.13). Four of the 10 patients with macrofocal MM versus 0 of 41 patients with typical MM (p=<0.01) had a prior diagnosis of solitary bone plasmacytoma. Approximately 80% of patients in both groups received primary treatment based on high-dose dexamethasone regimens (VAD or VAD-like regimens). An objective response was noted in 55% of patients with macrofocal MM and in 50% of patients without this pattern. The median survival of patients with typical MM was 57 months and has not been reached in patients with macrofocal MM (p=0.087). In the latter group of patients, median survival is projected to exceed 8 years. The 10 year survival rate is 20% and 5% in patients with or without macrofocal myeloma respecrively.

Conclusion: Our analysis indicates that macrofocal MM is a distinct entity in young patients with MM. Despite multiple lytic bone lesions, such patients have features of low tumor burden and an improved survival when compared with young patients with typical MM. Future studies including gene profiling may reveal potential biological differences among the two groups of patients.
Serum Pleiotrophin Is a New Multiple Myeloma Tumor Marker That Also Predicts Clinical Status.
Authors: Yeh Howard S. , Chen Haiming , Manyak Steven J. , Swift Regina A. , Campbell Richard A. , Wang Cathy , Li Mingjie , Jin Hee , Gordon Melinda S. , Bonavida Benjamin , Berenson James R.
Published: April 2020

Pleiotrophin (PTN), an 18 kD heparin-binding protein, has been found to be expressed in some solid tumors whereas it has not been previously evaluated in hematologic malignancies. Constitutive expression of PTN promotes tumor expansion and may increase metastatic potential. This protein binds to syndecan-1 (CD138) and stimulates angiogenesis. Its role in multiple myeloma (MM), thus far, has not been evaluated. Recently, we showed that PTN was strongly expressed in MM cell lines and malignant plasma cells from fresh bone marrow samples but not in normal control bone marrow specimens. Since PTN has been shown to be elevated in the serum of some solid tumor patients, we determined whether PTN may be elevated in the serum of MM patients, and correlated these levels with the patients’ disease status. By using a highly sensitive and specific enzyme-liked immunosorbent assay (ELISA) for PTN, we tested 270 different serum samples [MM (n=194), MGUS (n=18) and age-matched healthy control subjects (n=58)]. Serum samples from MM patients were obtained at the time of diagnosis as well as during the course of their disease. Serum PTN levels were higher in MM patients than in the control subjects (median=1.44 ng/ml vs 0.42 ng/ml, p <0.0001). In addition, among patients with MGUS and smoldering myeloma, serum PTN was also higher than in the controls (median=1.14 ng/ml vs 0.42 ng/ml, p <0.0001) but lower than among patients with active MM (p <0.0001). Patients with untreated MM showed serum PTN levels similar to those patients who received prior treatment. Among previously treated patients, the group with progressive disease at the time of evaluating their PTN level had higher serum concentrations of this protein than patients with responsive or stable disease (median=1.73 ng/ml vs 1.16 ng/ml, p <0.0001). Evaluation of patients who had a change in disease status showed that the serum PTN level increased at the time of progression when compared with their baseline level (median=1.76 ng/ml vs 1.01 ng/ml, p=0.009). Conversely, patients who responded to anti-MM therapy exhibited significant decreases of PTN as compared with their pre-treatment PTN values (median=0.95 ng/ml vs 1.90 ng/ml, p=0.007). These results suggest that serum PTN may be a new tumor marker for MM, and monitoring of this protein may be useful to follow the responsiveness to therapy and disease status of MM patients. We are now undertaking studies to evaluate PTN as a prognostic marker in a large cohort of newly diagnosed MM patients.
Mutations of Myeloid Transcription Factors AML1, CEBPα and PU.1 in Patients with De Novo Myelodysplastic Syndrome: An Analysis of Paired Bone Marrow Samples at Diagnosis and at AML Transformation.
Authors: Shih Lee-Yung , Liang Der-Cherng , Huang Chein-Fuang , Wu Jin-Hou , Wang Po-Nan , Kuo Ming-Chung , Dunn Po , Tang Tzung-Chih , Lin Tung-Liang , Chang Hung
Published: April 2020

Transcription factors AML1, PU.1 and CEBPα are essential for normal hematopoiesis. Recently, somatic mutations of genes encoding these transcription factors have been described in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). The role of mutations of these transcription factors in the progression of MDS into AML remained to be determined, as no matched paired samples of both phases have been analyzed in the previous studies. We analyzed mutations of AML1, CEBPα and PU.1 on paired bone marrow samples at initial diagnosis of de novo MDS (8 RCMD, 12 RAEB-1, and 17 RAEB-2) and at AML transformation. Mutation analysis was performed by direct sequencing of all RT-PCR or DNA PCR products using different primer pairs which cover the entire coding sequences of AML1, PU.1 and CEBPα genes. Eight of 37 patients (21.6%) had AML1 mutations and one of them had 2 mutations; 3 mutations were located in Runt homology domain (RHD) (exons 3–5), whereas 6 mutations were located in the C-terminal region (exons 6–8) at initial presentation of MDS. Another 2 acquired C-terminal mutations at AML transformation. The 11 AML1 mutations detected in 10 patients during their disease course, included 4 missense mutations, 3 nonsense mutations, 1 silent mutation, and 3 frameshift mutations. Taken together, 30% (3/10) of AML1 mutations detected in MDS patients were located in RHD and were exclusively missense compared with 70% at C-terminal region with frequent nonsense or frameshift mutations (86%) (P=0.033). Patients with AML1 mutations were older than those without mutations (P=0.019). No differences were observed between patients with and without AML1 mutations with respect to sex, blood counts, percentage of blasts in bone marrow or peripheral blood, morphologic subtype, and cytogenetic risk group. There was no difference in the time to AML transformation and overall survival with respect to the status of AML1 mutation. Only one out of 34 MDS patients had CEBPα mutation at initial diagnosis, she lost the mutation after chemotherapy and no mutation was detected at AML transformation. Another one did not have CEBPα mutation at diagnosis but acquired CEBPα mutation at AML transformation, the patient also had AML1 mutation at both phases. PU.1 mutation was not detected in 32 patients examined at both MDS and AML phases. The present study showed that AML1 mutations were common in patients with MDS, especially at C-terminal region and frequently with frameshift or nonsense mutations, and might be acquired during AML transformation; whereas CEBPα mutations were infrequent and PU.1 mutation was absent.
Lenalidomide Effects on NK Function in Patients with Myelodysplastic Syndrome (MDS).
Authors: Epling-Burnette P.K. , Bai Fanqi , Painter Jeffrey S. , Djeu Julie Y. , List Alan F.
Published: April 2020

Lenalidomide, which is a 4-amino-glutarimide analogue of thalidomide, has significant erythropoietic activity in patients with lower-risk MDS (List et al, NEJM, 351:26,2004). Although its precise target of action in MDS is not known, lenalidomide modulates cellular response to varied stimuli including inhibition of angiogenic response and endotoxin induction of inflammatory cytokines and enhancement of antigen-induced immunologic response and erythropoietin receptor signaling. Clinical investigations in multiple myeloma indicate that the immunomodulatory effects of thalidomide and lenalidomide extends to the expansion of natural killer (NK) cells. We recently found that MDS patients have defective NK function, araising in part to reduced expression of activating NK receptors (NKRs) NKp30, CD244 (2B4), and NKG2D. To determine if lenalidomide may restore NK function in MDS, we investigated the effects of in vitro treatment with lenalidomide on NK function and phenotype. Lytic function was studied using peripheral blood mononuclear cells (PBMCs) as effector cells and the leukemia cell line, K562, as a target in standard 4-hr 51Cr-release assays at 12:1 and 25:1 effector:target (E:T) ratios. Among eight MDS patient’s specimens evaluated, five patients had significant increase in tumor lysis after treatment with 1 μM lenalidomide for 72 hours (p ≤ 0.01, T-test). In similar experiments using PBMCs from normal donors, we found that NK lysis of K562 was 42% ± 15 (25:1 E:T ratio) pre-treatment which increased to 71% ± 17 (25:1 E:T ratio) after treatment, which was statistically significant (p ≤ 0.01, T-test). We also examined the in vitro effects of lenalidomide on lytic activity by the NK cell lines, NK92 and NKL, which was significantly increased after drug treatment. To discern the mechanisms of lenalidomide action in NK cell lines and normal NK cells, we evaluated NKR display by flow cytometry, and NKR function by antibody redirected cytotoxicity using the FcγR+ murine mastocytoma (P815) target cell line. Using NK92 and NKL cells, treatment with lenalidomide 1 μM for 72 hrs increased lysis by anti-NKG2D and anti-CD244 activating antibodies. We also found that NKG2D surface expression was increased on normal NK cells after lenalidomide treatment in vitro. These results suggest that some MDS patients may have improved NK function through the immunomodulatory effects of lenalidomide. The relationship between in vitro NK responsiveness to lenalidomide and in vivo hematological response warrants investigation in patients with MDS.
Comprehensive Promoter Methylation Analysis of Genes on 5q31.1 in MDS and AML.
Authors: Oki Yasuhiro , Jelinek Jaroslav , Shen Lanlan , Issa Jean-Pierre
Published: April 2020

Interstitial deletion or loss of chromosome 5 is a frequent chromosomal abnormality in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Previous studies uncovered a 1.5–2Mb region on 5q31.1 to be a common deleted region (CDR) in those diseases. It is believed that there exists one or more of critical tumor suppressor gene(s) in this CDR. Mutation analysis, however, failed to identify target gene(s). With a hypothesis that epigenetic silencing may play a significant role as a “second hit” in MDS/AML, we performed a comprehensive methylation analysis of genes in this CDR on 5q31.1. Among 25 genes that are located in this region (SPOCK-AF116688, covering CDR determined by Horrigan et al. Blood 2000 and Fang et al. Genomics 2000), 14 genes had promoter regions in CpG islands that were therefore susceptible to methylation dependent silencing. Methylation status of multiple CpG sites in promoter regions of these those 14 genes (SPOCK, HNRPA1, PKD2L2, C5orf5, KIF20A, GFRA3, CDC25C, C5orf6, JMJD1B, C5orf19, EGR1, ETF1, HSPA9B, CTNNA1, SIL1) was analyzed using bisulfite-PCR followed by pyrosequencing. Analyzed samples were 13 leukemic cell lines as well as peripheral blood mononuclear cells from 5 patients with AML with 5q deletion (5qAML), 9 AML without 5q deletion (non-5qAML), 17 MDS with 5q deletion (5qMDS), 9 MDS without 5q deletion (non-5qMDS) and 10 individuals without leukemia. Significant hypermethylation (>15% by pyrosequencing) was observed in 5 genes in patient samples (SPOCK, PKD2L2, GFRA3, HSPA9B, and ETF1, see Table 1) and 3 other genes in cell lines only (CDC25C, C5orf19, CTNNA1). In 5qMDS, the most frequently methylated genes were ETF1 (5/17), GFRA3 (4/17), and HSPA9B (11/17), but HSPA9B also showed methylation in normal blood. We then evaluated the expression levels of 5 genes that showed significant methylation in patients’ samples using quantitative RT-PCR. SPOCK, PKD2L2, GFRA3 were not expressed in normal PBMC. HSPA9B expression levels did not correlate with the degree of methylation. On the other hand, ETF1 expression was inversely correlated with the degree of methylation. ETF1 is a eukaryotic translation termination factor, which plays a role in recognizing stop codon in mRNA and terminates translation. Based on specific hypermethylation and silencing in 5qMDS, we propose ETF1 as a candidate tumor suppressor gene in the 5q31.1 region. Functional analysis of ETF1 in leukemic cells is underway.

Frequency of promoter CpG sites methylation Samples N SPOCK PKD2L2 GFRA3 HSPA9B ETF1 Cell lines 13 62% 46% 77% 38% 0% 5qAML 5 0% 0% 0% 20% 0% Non-5qAML 9 33% 0% 0% 0% 0% 5qMDS 17 6% 6% 24% 65% 29% Non-5qMDS 9 11% 11% 33% 33% 11% Normal 10 0% 0% 0% 90% 0%
Systemic Treatment with the Antiangiogenic Agent ABT-510 Inhibited Myeloma-Induced Microvessels and Tumor Growth in Myelomatous SCID-hu Mice.
Authors: Yaccoby Shmuel , Ling Wen , Saha Rinku , Yata Kenichiro , Tricot Guido
Published: April 2020

Myeloma cells induce bone marrow angiogenesis and increased microvessel density (MVD) is associated with poor prognosis in this disease. The aim of this study was to investigate whether neovascularization is required for myeloma growth by testing the anti-angiogenesis and anti-tumor efficacy of thrombospondin-1 (TSP-1)-derived mimetic peptide, ABT-510 (Abbott Laboratories), in the SCID-hu model for primary myeloma. TSP-1 is a matricellular glycoprotein with multiple biological functions, including antiangiogenic activity. ABT-510 is a capped nonapeptide based on the linear TSP-1 heptapeptide sequence. Like TSP-1, ABT-510 can be targeted to a specific receptor on vascular endothelial cells and efficiently inhibits angiogenesis in vitro and after systemic injection in experimental models of human malignancies. SCID-hu mice were constructed as previously described (Yaccoby et al., Blood 1998; 1999; 2002). Myelomatous SCID-hu mice were prepared by injection of myeloma cells from 6 patients into the implanted human bone in the hosts. Changes in levels of tumor burden were monitored by weekly measurements of human monotypic immunoglobulins (hIg) using ELISA and histologically confirmed by immunohistochemical staining of human bone sections for cIg. MVD, visualized by CD34 staining, were counted in 4 non-overlapping areas. Upon establishment of myeloma growth (401±146 μg/ml pre-treatment levels) mice were intraperitoneally injected with ABT-510 (100 mg/kg, twice a day) or with the vehicle (5% dextrose) for 4–10 weeks. Treatment was well tolerated and no sign of toxicity or reductions in body weight were observed. Whereas all dextrose -treated mice had increased hIg levels during the experimental period, ABT-510 treatment resulted in marked reduction of tumor burden in 2 experiments by 48% and 20%, respectively, retardation of myeloma growth in 3 additional experiments and no response in one experiment. Overall, tumor burden in control- and ABT-510-treated mice was increased by 3696%±3264 and 448%±169 from pre-treatment levels, respectively (p<0.03). MVD in control- and ABT-510-treated mice were 21±6/mm2 and 11±4/mm2, respectively (p<0.02). Intriguingly, MVD in the non-responding mouse was reduced by >35% (11±2 vs. 17±1 microvessels/MM2 in control mouse, p<0.04), suggesting that certain myeloma cells are not dependent on neovascularization. We conclude that myeloma cells from the majority of patients require angiogenesis for growth and that ABT-510, which is undergoing a phase II clinical trial for treating solid tumors, is a potential agent for treating myeloma.
Treatment of Bortezomib Increases Osteoblast Function in Patients with Multiple Myeloma.
Authors: Heider Ulrike , Kaiser Martin , Müller Christian , Schulz Carsten-Oliver , Jakob Christian , Zavrski Ivana , Eucker Jan , Sterz Jan , Kleeberg Lorenz , Possinger Kurt , Sezer Orhan
Published: April 2020

Myeloma bone disease is caused by an enhanced osteoclast activation and impaired osteoblast function. Until now, there is no specific treatment to restore osteoblast activity, and anti-myeloma therapies that lead to a disease remission are usually not associated with an increase of osteoblast markers. Recently, preclinical data suggested that proteasome inhibitors may enhance osteoblast function. Bortezomib (Velcade) represents the first substance from this group which is clinically used in relapsed multiple myeloma. To evaluate whether there is clinical evidence for an osteoblast stimulation under bortezomib treatment, we analyzed serum levels of two specific osteoblast markers, i.e. bone-specific alkaline phosphatase (BAP) and osteocalcin, in 25 multiple myeloma patients treated with bortezomib alone or in combination with dexamethasone. 56 percent of patients achieved a complete or partial remission. In the whole group of patients, mean serum levels of osteocalcin significantly increased from 6.3 μg/l before treatment to 10.8 μg/l after three months of therapy (P=0.024). In parallel, mean levels of BAP increased from 19.7 U/l to 30.2 U/l (P<0.0005). The increase in BAP was irrespective of the combination with dexamethasone and was noted both in responders and in non-responders. This is of special interest, since it implicates that the increase in osteoblast function may be a direct effect of bortezomib on osteoblasts and not an indirect consequence of the reduced myeloma burden. Proteasome inhibition may modulate the Wnt/b-catenin pathway, a major signalling pathway in osteoblasts. Myeloma patients with osteolytic lesions have been shown to overexpress DKK-1, an inhibitor of the Wnt/b-catenin pathway. Recent experiments on mesenchymal cells showed that proteasome inhibitors decreased the DKK-1 production. Moreover, proteasome inhibition elevates cytoplasmatic b-catenin levels by inhibition of its degradation. In addition, animal models gave evidence that proteasome inhibitors stimulate the bone morphogenetic protein (BMP)-2 mediated osteoblast differentiation. Taken together, these preclinical observations suggest that proteasome inhibition may enhance osteoblast activity. Our study gives clinical evidence for a significant improvement of osteoblast function under bortezomib. This is of special interest, since it demonstrates additional effects of proteasome inhibitors and may provide a novel treatment approach in myeloma bone disease.
Open-Label Study of Ibandronate in Elderly Myeloma Patients with Pre-Existing Renal Deterioration.
Authors: Henrich Dirk , Bergner Raoul , Hoffmann Martin , Honecker Andrea , Nagel Dietmar , Uppenkamp Michael
Published: April 2020

Bisphosphonates effectively reduce the number of skeletal-related events and relieve metastatic bone pain, improving quality of life in patients with malignant bone disease. However, clinical studies have highlighted that some, but not all intravenous (i.v.) bisphosphonates are associated with an increased risk of renal toxicity. The renal safety of bisphosphonates is particularly important in patients with multiple myeloma or in the elderly, who often have some degree of underlying renal damage and are at high risk of renal failure. In phase III trials, the renal safety profile of i.v. ibandronate was comparable to placebo. This agent may therefore be suitable for use in patients with impaired renal health. The current open-label study assessed the renal safety of ibandronate in elderly patients with multiple myeloma and varying stages of pre-existing renal impairment. Patients with multiple myeloma (creatinine clearance 8–139mL/min) received i.v. ibandronate (6mg, infused over 30 minutes). For each patient, deterioration in renal function was graded at baseline using creatinine clearance (grade 0: ≥80, 1: 50–79, 2: 30–49, 3: <30mL/min). Urinary markers of tubular damage (α-glutathione-S-transferase [α GST] and β-N-acetyl-glucosaminidase [βNAG]) were measured at baseline and at 24 and 72 hours following ibandronate infusion. Follow-up toxicity data was collected over 6 months for a subset of patients (n=6). A total of 29 patients (15 females and 14 males; mean age 71.1 ± 5 years) were included in this study. At baseline, four patients had a normal renal function (grade 0). The remaining 25 patients had varying degrees of renal insufficiency (grade 1: n=6, grade 2, n=8, grade 3: n=11). Mean proteinuria was 1799 ± 2140mg/24 hours. Serum creatinine and levels of urinary markers (α GST and βNAG) remained stable throughout the study. There was a statistically non-significant decrease in βNAG, and a positive correlation between ibandronate elimination and creatinine clearance. In the subset of six patients receiving six additional monthly infusions, ibandronate had no negative impact on renal function. To conclude, in this study of elderly patients with multiple myeloma, ibandronate was well tolerated and did not compromise renal health, despite 86% (25/29) of patients having pre-existing renal insufficiency. This was evidenced by markers of tubular damage and renal function, which remained unchanged throughout the study period. Furthermore, a subset of patients who received repeated ibandronate infusions did not experience renal toxicity. The management of drug-related nephrotoxicity needs special attention in patients with multiple myeloma, and in the elderly. These data suggest that ibandronate may be suitable for use in high risk multiple myeloma patients with pre-existing renal impairment. Further studies in larger groups of patients are warranted.
Bisphosphonates and Osteonecrosis of the Jaws: Incidence in a Homogeneous Series of Patients with Newly Diagnosed Multiple Myeloma Treated with Zoledronic Acid.
Authors: Tosi Patrizia , Zamagni Elena , Cangini Delia , Tacchetti Paola , Offidani Massimo , Ronconi Sonia , Perrone Giulia , Ceccolini Michela , Baccarani Michele , Tura Sante , Cavo Michele
Published: April 2020

Osteonecrosis of the jaws (ONJ) is a rare complication that has been described as osteoradionecrosis in patients who had previously undergone radiotherapy for head and neck cancer or similar disorders. It has been recently reported that the incidence of ONJ has significantly increased since the wide application of bisphosphonates as specific therapy of cancer related bone disease. No consensus on diagnostic criteria does presently exist, the pathogenesis of this complication is unknown and the relative contribution of repeated trauma from dental procedures, poor oral hygiene, concomitant chemotherapy, and cancer bone involvement has not been clarified yet. All the same, no data have so far been presented concerning the incidence of ONJ in a homogeneous series of patients with respect to disease status and treatment received. For this purpose, we have retrospectively evaluated the occurrence of ONJ in patients with symptomatic newly diagnosed multiple myeloma enrolled in the “Bologna 2002” clinical trial. According to treatment protocol, all patients received four months of combined thalidomide (100mg/d for two weeks and 200mg/d thereafter) and dexamethasone (40mg/d on d 1–4, 9–12, 17–20/28d on odd cycles and on d 1–4 on even cycles) followed by cyclophosphamide 7g/m2 + G-CSF and PBSC collection, and two courses of Melphalan 200mg/mq and PBSC transplantation. Zoledronic acid was administered at 4mg/28 d throughout the whole study period and afterwards until disease progression. Out of 225 patients analyzed so far (median follow-up = 26 months), ONJ was diagnosed in 6 cases (2.7%), median time from start of treatment and occurrence of dental symptoms was 23 months (range 13–32 months). All the patients underwent dental extraction and/or oral/maxillofacial surgery that resulted in areas of non-healing bone with persistent purulent discharge. Bone biopsies confirmed bone necrosis and inflammation; in 1 case, however, a concomitant infiltration of monoclonal plasma cells was detected in the site of necrosis. In conclusion, results of this retrospective analysis indicate that the occurrence of ONJ among newly diagnosed MM patients receiving long-term (median: 2 years) zoledronic acid treatment was approximately 3%. Surgery can significantly impact patients outcome, so that, according to currently available recommendations on ONJ, dental procedures should be avoided. In addition, the role played by disease itself in the pathogenesis of this complication should not be overlooked.
Dysfunctional T Regulatory Cells in Myeloma: Molecular Mechanisms of Dysregulation.
Authors: Prabhala Rao H. , Neri Paola , Tassone Pierfrancesco , Bae Jooeun E. , Shammas Masood A. , Daley John F. , Song Weihua , Bertheau Robert , Chauhan Dharminder , Bachu Ramesh B. , Thatte Hemant , Anderson Kenneth , Munshi Nikhil C.
Published: April 2020

Multiple myeloma (MM) is characterized by production of monoclonal immunoglobulin, associated with suppressed uninvolved immunoglobulins and dysfunctional T cell responses. The biological basis of this dysfunction remains ill defined. Since T regulatory (Treg) cells play an important role in suppressing normal immune responses, we have here evaluated the potential role of Treg cells in immune dysfunction in MM. We observed a significant increase in CD4+CD25+ T cells in individuals with monoclonal gammopathy of undetermined significance (MGUS) and patients with MM compared to normal donors (25% and 26% versus 14%, respectively); however, Treg cells as measured by Foxp3 expression are significantly decreased in both MGUS (1.6±0.5%, p<0.01) and MM (1.6±0.5%, p<0.01) compared to normal donors (6.0±0.8%). Additionally, these Treg cells also do not function normally. Treg cells from patients with MM and MGUS even when added in higher proportions are unable to suppress anti-CD3-mediated T cell proliferation. This decreased number and function of Treg cells in MGUS and in MM may account, at least in part, for the non-specific increase in CD4+CD25+ T cells, thereby contributing to dysfunctional T cell responses. We have further analyzed the molecular basis for the Treg cell dysfunction in myeloma. Based on the preliminary results suggesting a role of IL-6 in Treg cell function and since both serum IL-6 and soluble IL-6 receptor levels are significantly elevated in MGUS and MM, we evaluated the role of IL-6 and its soluble receptor on Treg cell function. We observed that the addition of IL-6 and/or sIL-6 receptor to the culture leads to loss of Treg cell activity in normal donor cells similar to one observed in myeloma patients; and conversely, when Treg cells from MM patients are treated with the anti-IL-6 antibody or IL-6 receptor super antagonist, sant 7, the suppressive activity of Treg cells is restored. Additionally, we have preliminary evidence of expansion of Foxp3+ cell numbers in PBMC from MM patients following in vitro treatment with anti-IL-6 antibody. This data suggests a role of IL-6 and bone marrow microenvironment in dysfunctional Treg cells in MM and that inhibition of IL-6 signaling results in beneficial effects on T cell activity by increasing Treg cell activity. A blockade of IL-6 signaling thus emerges as a potential approach to establish immune homeostasis to improve immune function in MM.
Dendritic Cell Vaccines Are Superior to Idiotype-KLH Protein Vaccines at Priming a Therapeutic, Tumor-Specific Immunity Against Multiple Myeloma.
Authors: Yi Qing , Wang Siqing , Wezeman Michele , Qian Jianfei , Yang Jing
Published: April 2020

Idiotype (Id) protein secreted by myeloma cells is the best-characterized tumor-specific antigen and has been used in clinical vaccination trials administered either as Id-keyhole limpet hemocyanin (KLH) protein vaccines or as Id-pulsed dendritic cell (DC) vaccines. In this study, we used the 5TGM1 myeloma murine model to compare the efficacy of Id-KLH (protein vaccines) versus DCs pulsed with Id-KLH (DC vaccines), two formulas commonly used in clinical trials, to induce tumor-specific immunity, to protect mice from developing myeloma, and to treat myeloma-bearing mice. Vaccinations consisted of three weekly, subcutaneous injections of the protein conjugates (100 μg/injection) or Id-KLH-pulsed, bone marrow-derived mature DCs (106 cells/injection). Following each vaccination, GM-CSF (200 ng/day/mouse) was injected subcutaneously (adjacent to the vaccination sites) for three consecutive days. We found that, although the protein vaccines induced significantly higher titers of specific antibodies, DC vaccines were superior at inducing tumor-specific, cellular immune responses, evident by increased IFN-γ production, T cell proliferation, and cytotoxic T cell activities against the tumor cells. As prophylactic treatments, protein and DC vaccines were equally efficient at protecting mice from subsequent tumor challenge. However, only DC vaccines induced therapeutic immunity in tumor-bearing mice: DC vaccinations not only retarded tumor growth but also eradicated established tumors in more than 50% of mice, which was associated with an induction of potent, tumor-specific type-1 (IFN-γ) and type-2 (IL-4) T-cell responses. DC-vaccinated mice also showed a more pronounced increase in the percentages of CD8+ T cells in their spleens after in vitro stimulation with irradiated myeloma cells and increased tumor-specific cytotoxicity in enriched CD8+ T cells over the whole splenic cells, suggesting that CD8+ T cells play a major role in killing tumor cells in vivo. Furthermore, surviving mice were also protected from rechallenge with the myeloma cells, indicating that memory T cells existed and were functional. Thus, our results show that Id-based DC vaccines may be a favorable vaccine for immunotherapy in MM. Further studies are required to optimize DC vaccination protocols to achieve therapeutic efficacy in clinical trials.
Expression of Cancer/Testis Antigens CT10 and SSX in Multiple Myeloma: Promising Targets for Antigen-Specific Immunotherapy.
Authors: Atanackovic Djordje , Arfsten Julia , Cao Yanran , Wolschke Christine , Bartels Katrin , Faltz Christiane , Hossfeld Dieter K. , Zander Axel R. , Bokemeyer Carsten , Kroeger Nicolaus
Published: April 2020

Background: The introduction of autologous and allogeneic stem cell transplantation has resulted in improved responses to treatment in patients with multiple myeloma (MM). However, despite high remission rates, recurrences are frequent due to remaining minimal residual disease. Myeloma-specific immunotherapies would represent a potentially useful treatment option in these patients. Cancer/testis (CT) antigens are highly attractive targets for T cell-mediated immunotherapy of cancer, due to their immunogenicity and restricted tissue expression. We investigated the expression of a larger number of CT antigens, the expression of which is characteristically restricted to cancer and normal testis, in patients with MM.

Methods: We analyzed the expression of 9 different CT antigens (SSX-1 to SSX-5, CT10/MAGE-E1, BAGE, MAGE-3, NY-ESO-1) in 10 myeloma cell lines, 54 bone marrow samples from patients with stage II-III myeloma (plasma cell infiltration 10–100 %), and 18 bone marrow samples from healthy donors using rtPCR.

Results: Myeloma cell lines showed a remarkably high expression of the majority of CT antigens. All antigens, with the exception of SSX-3, were expressed in 80–100% of cell lines. SSX-3 was not expressed in any cell line. Analyzing bone marrow samples from MM patients, we observed that CT10 showed a very high expression level with 55% of all malignant samples expressing this antigen. The SSX gene family evidenced high expression levels with 40% (SSX-1), 17% (SSX-2), 13% (SSX-4), and 15% (SSX-5) of patient samples expressing the given SSX family member. SSX-3 was not expressed in any samples. In agreement with previous findings we observed a high expression of MAGE-3 (61%), an intermediate expression of BAGE (18%), and a comparably low expression of NY-ESO-1 (7%). With the exception of SSX-4, which was expressed in 18% of healthy bone marrow samples, none of the CT antigens were expressed in non-malignant samples.

Conclusions: We show here for the first time that CT10 and members of the SSX gene family are strongly and specifically expressed in multiple myeloma. Importantly, both CT10 and SSX have been shown to elicit spontaneous immune responses in patients with solid tumors. Therefore, CT10 and SSX-1, SSX-2, and SSX-5 might represent valuable targets for antigen-specific immunotherapy of multiple myeloma.
The Telomerase Template Antagonist GRN163L in Combination with Chemotherapeutics Reduces Tumor Volume in Multiple Myeloma Xenograft Models.
Authors: Ertem Server A. , Wu Kaida , Ponomarev Vladimir , Chin Allison C. , Moore Malcolm A.S.
Published: April 2020

Primary hematopoietic malignancies are characterized by short telomeres, suggesting that they would be acutely susceptible to telomerase inhibition. We have previously shown that treatment with GRN163L, a lipid-conjugated 13-mer thio-phosphoramidate oligonucleotide (Geron Corporation), inhibits the growth of human multiple myeloma (CAG) and ovarian carcinoma (OVCAR3) cell lines in vitro and in vivo (AACR 2005 Annual Meeting). The CAG multiple myeloma cell line (telomere length 2.7 Kb) was transfected with a retroviral vector encoding a triple fusion gene for HSV-TK, firefly luciferase and green fluorescence protein, and whole animal bioluminescence imaging was undertaken in either a subcutaneous or disseminated NOD/SCID model with extensive spinal cord and bone marrow tumor infiltration. In a subcutaneous model with CAG, GRN163L was administered three times per week (tiw) at 36 mg/kg for 4 weeks alone or in combination with either a single dose of melphalan (5 mg/kg) or 9 doses of Velcade (0.25 mg/kg tiw). Tumor mass was reduced 56% with GRN163L alone (p<0.001), 60% with melphalan alone (p<0.001), 89% with the combination of GRN163L and melphalan (p<0.001). At the dose used, Velcade alone showed no efficacy, but the combination of GRN163L with Velcade reduced the tumor mass by 68% (p<0.001) compared to controls. Data on the treatment with a mismatch control oligonucleotide in addition to dose optimization of GRN163L in combination with Melphalan and Velcade in subcutaneous and disseminated myeloma models (CAG, MM.1S) will be presented. Geron initiated a Phase I/II trial with GRN163L in chronic lymphocytic leukemia in July 2005. These data support the development of GRN163L as an effective and safe agent for therapy of hematopoietic malignancies.
Acquired Resistance to Activated Protein C (aAPCR) Is Associated with Increased Risk of Deep Vein Thrombosis in Multiple Myeloma.
Authors: Elice Francesca , Fink Louis , Tricot Guido J. , Milner Teresa J. , Barlogie Bart , Zangari Maurizio
Published: April 2020

Non factor V Leiden APC resistance (aAPCR) has been described in cancer patients and found to be associated with an increased risk of deep venous thrombosis (DVT). We analyzed the incidence and clinical impact of APC resistance in a large group of multiple myeloma patients. A total of 1178 myeloma patients were tested for APC resistance using an aPTT-based assay in the presence of excess of factor V-deficient plasma and the ratio with or without APC was calculated (≤ 2.00 was considered abnormal). PCR amplification of genomic DNA was used to detect Factor V Leiden.

Abnormal APC resistance was found in 109 patients (9.3%), 83 of those were tested for factor V Leiden, 31 had the mutation and 52 (63%) did not have it. Analyzing a subgroup of 254 chemotherapy naïve patients, APC ratio was abnormal in 11% of patients and two third of them were not carriers of factor V Leiden mutation. The presence of aAPC resistance was associated with an increased risk for DVT: 27.9% in patients with aAPCR vs.12.3% in the others (P = 0.008); 22.6% in patients with factor V Leiden mutation.

In 32 patients with abnormal aAPCR, the test was repeated: 31/32 patients normalized their APC ratio in sequential testing. Correlation between myeloma baseline markers (serum and urine M-component, beta2-microglobulin, CRP, IL-6), response to treatment and APC activity were studied. In this analysis active disease emerged as the most important factor associated with aAPCR, as 19 patients with normalization of the APC ratio had a concomitant clinical response to therapy. We concluded that aAPCR is a transient finding in myeloma patients that showed a significant correlation with development of DVT.
The JAK2 V617F Tyrosine Kinase Mutation in Myelofibrosis with Myeloid Metaplasia: Clinical Correlates and Prognostic Relevance in 157 Patients.
Authors: Tefferi Ayalew , Lasho Terra L. , Schwager Susan M. , Steensma David P. , Mesa Ruben A. , Li Chin Yang , Wadleigh Martha , Gilliland D. Gary
Published: April 2020

Background: The occurrence of an activating JAK2 mutation (JAK2 V617F) has been reported in a variable proportion of patients with bcr/abl-negative myeloproliferative disorders including myelofibrosis with myeloid metaplasia (MMM). In the current study, we report mutational frequencies in each of the 3 MMM variants as well as examine the prognostic relevance of the mutation in a large group of patients from a single institution.

Methods: Mutation analysis for JAK2 V617F was performed in DNA derived from peripheral blood mononuclear cells (PBMC) from all study patients (n=157). Concomitant analysis was also performed in DNA derived from purified granulocytes (n=57), CD34-positive cells (n=25), and T cells (n=19). Genomic DNA was amplified by PCR and fluorescent dye chemistry sequencing was performed using the same primers used for amplification.

Results: The study cohort included 157 consecutive patients with MMM including 117 with agnogenic (AMM), 22 with post polycythemic (PPMM), and 18 with post thrombocythemic (PTMM) myeloid metaplasia. The overall detection rate for JAK2 V617F in PBMC was 51% (homozygous in 5.7%) and was significantly (p=0.0002) higher in PPMM (91%; homozygous in 18%) compared to either AMM (45.3%; homozygous in 2.6%) or PTMM (38.9%; homozygous in 11.1%). In AMM (n=117), the presence of JAK2 V617F was significantly associated with an older age at diagnosis (p=0.03), and a history of both thrombosis (p=0.01) and pruritus (p=0.005). No other statistical associations were evident and the presence of the mutation did not predict leukemic transformation. Furthermore, multivariate analysis for overall survival of patients with AMM identified only age and the Dupriez prognostic score as independent prognostic factors; JAK2 V617F had no prognostic significance. Finally, the mutation was not found in T cells and its detection rate in either granulocytes or CD34-positive cells was similar to that in PBMC.

Conclusion: JAK2 V617F is a myeloid lineage-specific event, its incidence in MMM is significantly higher with an antecedent history of polycythemia vera, and its presence in AMM does not affect prognosis but is associated with PV-characteristic clinical features.