Haematologica 2003 - Supplements

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locked the translocation of Ras to the plasma membrane, Ras activation and ERK1,2 phosphorylation in both cell lines and also in the INA-6/BMSC co-culture model. Treatment with FTI induced apoptosis of MM cells (cell lines and primary cells) and was equally effective in the presence or absence of BMSCs. Conversely, the human Burkitt lymphoma cell line Namalwa, which lacks constitutive ERK1,2 activity, was resistant against FTI-induced apoptosis. Conclusions: Treatment of MM cells with FPT III blocks the Ras/MAPK pathway and induces apoptosis in the absence and in the presence of BMSCs. Our experiments support the hypothesis that the sensitivity of MM cells to FTIs depends on the activation status of the Ras/MAPK pathway rather than on the mutation status of Ras. These findings underscore the potential of FTIs for the development of novel therapeutic strategies for the treatment of MM. 383 Effect of farnesyl transferase inhibitor R115777 alone or in combination with incadronate on the growth of fresh and cloned myeloma cells in vitro Naoya Ochiai, Ryo Uchida, Shin-ichi Fuchida, Akira Okano, Masashi Okamoto, Hideyo Hirai, Eishi Ashihara, Tohru Inaba, Naohisa Fujita, Masao Nakagawa and Chihiro Shimazaki Second Department of Medicine, Kyoto Prefectural University of Medicine, Kyoto, Japan Multiple myeloma (MM) is a malignancy of B cells characterized by the monoclonal proliferation of malignant plasma cells. Several chemotherapeutic regimens and high-dose chemotherapy supported by autologous stem cell transplantation (auto-SCT) have been employed to improve the survival of patients with MM but many patients relapsed for a long time follow up period. Therefore there is an obvious need for new therapeutic strategies. Ras gene mutations occur in 30% to 40% of multiple myeloma (MM), and farnesylation is the first step in the posttranslational modification of Ras proteins. Zarnestra TM (R115777) is one of the potent farnesyl transferase inhibitors (FTIs), but is less effective to K-Ras mutation, which is one of the most common mutations in MM, than N- and H-Ras mutations. It has recently been reported that bisphosphonates have direct antitumor effect in vitro and in vivo, and nitrogen-containing bisphosphonates, such as incadronate, inhibit the mevalonate pathway and prevent posttranslational prenylation of GTP-binding proteins. Mevalonate pathway is essential for the biosynthesis of sterols and long chain isoprenoid lipids, including farnesylpyrophosphate (FPP) and geranylgeranylpylophosphate (GGPP) which are substrates for the FTase and geranylgelanyl transferase (GGTase) 30. Based on these observations, we examined the effect of R115777 alone or in combination with incadronate on the growth of fresh and cloned myeloma cells in vitro. R115777 inhibited the growth of fresh and cloned myeloma cells dose-dependently. Flow cytometric analysis using annexin V and 7AAD showed that R115777 induced apoptosis of two of three myeloma cell lines at the concentration of 1.0×10-8 mol/L. The inhibition of cell growth was intensified when R115777 was combined with incadronate at 1.0×10-6 mol/L and 1.0×10-5 mol/L. R115777 appears to be a potent inducer of apoptosis in fresh and cloned myeloma cells, and the effect was intensified in combination with incadronate. R115777 alone or in combination with incadronate might have some benefit in the treatment of myeloma patients. 384 MOLECULAR CHARACTERISTICS OF APOPTOSIS INDUCED BY THE FARNESYLTRANSFERASE INHIBITOR (FTI) BMS-214662 IN HUMAN MYELOMA CELLS María Gómez-Benito, Pilar Giraldo, Daniel Rubio-Félix, Alberto Anel, Javier Naval & Isabel Marzo Departamento de Bioquimica y Biologia Molecular y Celular, Universidad de Zaragoza, and Servicio de Hematologia, Hospital Miguel Servet Zaragoza (Spain). We have studied the molecular mechanism of apoptosis induced in vitro, by the FTI BMS-214662, in several human multiple myeloma (MM) cell lines (RPMI-8226, NCI-H929, U266 and IM-9). In MM cells treated with BMS-214662 farnesylation of cytosolic chaperone HDJ-2 and of lamin A was clearly observed. BMS-214662 treatment induced the typical morphology of apoptosis, including condensation and fragmentation of chromatin, in all MM cell lines at low doses (0.3-1 µM) after 24 h incubation. Cell death was not prevented by co-treatment with the protein synthesis inhibitor cycloheximide. Apoptosis was accompanied by cell shrinking, phosphatidylserine exposure in plasma membrane, loss of mitochondrial membrane potential (m), activation of caspases 9 and 3 and traslocation of Apoptosis-Inducing Factor (AIF) from mitochondria to nucleus. Cotreatment of cells with the pan-caspase inhibitor Z-VAD-fmk prevented development of apoptotic morphology and death in RPMI8226 and U266 cells. Z-VAD-fmk attenuated some morphological and biochemical characteristics of apoptosis in NCI-H929, but did not prevent cell death. Selective inhibitors of caspase-3 or caspase-9 did not block cell death in any cell line tested. Levels of antiapoptotic proteins Bcl-2 and Mcl-1 decreased in IM-9, RPMI-8226 and U266 cells upon treatment with BMS-214662. This FTI caused a concomitant increase in levels of pro-apoptotic BH3-only proteins Bik and Bim during treatment. Multidomain, proapoptotic Bax protein levels also increased in U266 treated with BMS-214662. These results support the potential usefulness of FTIs as new therapeutic agents for the therapy of MM. 385 Rituximab-mediated signaling and sensitization of 8226/S and 8226/Dox40 MM cell lines to paclitaxelmediated apoptosis. Ali Jazirehi, Gary Schiller, Alan Lichtenstein and Benjamin Bonavida. Departments of Microbiology, Immunology and Molecular Genetics, and Medicine, University of California at Los Angeles, CA 90095, USA. Rituximab is a chimeric mouse anti-human CD20 monoclonal Antibody that has been approved by the FDA for the treatment of patients with low grade and follicular NHL. The response rate is approximately 50%. Following initial treatment, patients with MM develop chemo-resistance. Thus, there is a need for alternative therapeutic approaches. Previous findings from our laboratory have demonstrated that Rituximab sensitizes NHL cell lines to various drug-mediated apoptosis (Clinical Cancer Research, 7: 709, 2001, Cancer Research, 61: 5137, 2001). The objective of this study was to investigate whether Rituximab has an effect on MM. We have used the drug sensitive 8226/S parental and the doxorubicin-resistant variant 8226/Dox40 cell lines as a model system. A fraction of the cells (>20%) expressed CD20 as determined by flow cytometry. Treatment of the 8226/S and 8226/Dox40 cells with Rituximab (20g/ml-24h) resulted in S259
significant inhibition of cell proliferation. Further, pretreatment of the 8226/S and 8226/Dox40 cells with Rituximab followed by treatment with paclitaxel (10nM) for an additional 24h resulted in significant potentiation of cytotoxicity and synergy was achieved. The synergy in cytotoxicity was determined to be due to the induction of apoptosis. The mechanism by which Rituximab sensitized the 8226/Dox40 cells to paclitaxel-mediated apoptosis was investigated. Using western blot analysis it was found that treatment with Rituximab (20g/ml-24h) resulted in selective down-regulation of anti-apoptotic Bcl-xL expression with little effect on other gene products involved in apoptosis (c-IAP1, c- IAP2, XIAP, survivin, Mcl-1, Bcl-2). In addition, treatment with paclitaxel (10nM) arrested the cells at the G2/M phase of the cell cycle and resulted in the down-regulation of Mcl-1, Bcl-2, BclxL, c-IAP1 and survivin. These various gene modifications by Rituximab and paclitaxel resulted in functional complementation and the induction of the apoptotic signaling pathway. These findings suggest that signal I mediated by Rituximab and resulting in down regulation of anti-apoptotic Bcl-xL was important to circumvent the resistance of 8226/Dox40 cells to paclitaxel (signal II)-mediated apoptosis. Further, even though the expression of CD20 was low, nevertheless, the combination treatment resulted in significant percentage of cells undergoing apoptosis. The present findings also demonstrate that Rituximabmediated sensitization of the 8226/Dox40 cells to paclitaxelmediated apoptosis is independent of the MDR phenotype of the cells, as the functionality of the MDR pump was unaffected by single or combination treatments. These findings are of potential clinical significance. (Supported in part by the Jonsson Comprehensive Cancer Center At UCLA (A. J). 386 “An Antibody-avidin Fusion Protein Targeting the Human Transferrin Receptor Inhibits the Growth and Induces Apoptosis in Eight Human Malignant Plasma Cell Lines” Patrick P. Ng1, Xiao-Hu Gan1, Benjamin Bonavida1, Fuyuhiko Tamanoi1, Gary J. Schiller2, Alan K. Lichtenstein2, Dharminder Chauhan3, Kenneth C. Anderson3, Sherie L. Morrison1, Manuel L. Penichet1. 1UCLA Department of Microbiology, Immunology and Molecular Genetics, 2UCLA Division of Hematology and Oncology, Department of Medicine, Los Angeles CA. 3Dana-Farber Cancer Institute, Harvard Medical School, Boston MA We have previously reported that an anti-rat transferrin receptor (TfR) IgG3-avidin fusion protein exhibits anti-proliferative/proapoptotic activity against the rat myeloma cell line Y3-Ag1.2.3 and the rat T-cell lymphoma cell line C58(NT)D.1.G.OVAR.1. This activity was not observed in two rat cell lines of nonhematopoietic lineage (bladder carcinoma BC47 and gliosarcoma 9L) suggesting the potential use of anti-TfR IgG3- avidin for the treatment of hematopoietic malignancies. In addition, an anti-human TfR-IgG3-avidin fusion protein (antihTfR-Av) was shown to inhibit proliferation and induce apoptosis in human erythroleukemia cell line K562 (Ng et al., PNAS, 2002, 99:10706). Recent studies demonstrated that anti-hTfR-Av also inhibits the growth and induces apoptosis in the human malignant plasma cell lines 8226/S, 8226/Dox40 [doxorubicin-resistant and also multidrug resistant], U266, MM.1S, OCI-My5, S6B45, ARH-77, and IM-9, although different levels of sensitivity were observed. Interesting, such anti-proliferative/pro-apoptotic activity seems not to be correlated with the level of TfR expressed on the surface of the cells. We are currently investigating the mechanism by which anti-hTfR-Av mediates its anti-proliferative/pro-apoptotic activity. We will also determine if it can be used in combination with various agents currently used in the treatment of hematopoietic malignancies. Importantly, antihTfR-Av can be further loaded with biotinylated anti-cancer drugs, which may increase its intrinsic anti-cancer activity. These approaches may lead to more effective therapeutics for in vivo eradication of plasma cell malignancies such as multiple myeloma and ex vivo purging of plasma cancer cells in autologous transplantation. 387 A chimeric humanized anti-CD40 antibody renders human multiple myeloma (MM) cells refractory to the mitogenic and protective effects of IL-6 Yu-Tzu Tai,1 Constantine S. Mitsiades,1 Leurence P. Catley,1 Renate Burger,1 Masaharu Akiyama,1 Klaus Podar,1 Reshma Shringarpure,1 Teru Hideshima,1 Dharminder Chauhan,1 Nicholas Mitsiades,1 Nikhil C. Munshi,1 Paul Richardson,1 Steven P Treon, and Kenneth C. Anderson1 1The Jerome Lipper Multiple Myeloma Center, Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Mass., USA CD40 is expressed on B-cell malignancies including human multiple myeloma (MM) and a variety of carcinomas, and there is considerable interest in using CD40 activating agents as novel cancer therapies. Recently, a humanized anti-CD40 Ab (SGN-14) demonstrates a significant anti-tumor effect in SCID mice xenografted with human MM (Cancer Res. 60, 3225-3231, 2000). In this study, we examined the therapeutic impact of SGN-14 in human MM using MM.1S cells (CD138+++CD40+) and plasma cell leukemia patient cells. SGN-14 (0.01-100 µg/ml) did not significantly stimulate proliferation of CD40-expressing MM.1S and MM.1R or two patient plasma cell leukemia cells (p >0.1 for all samples tested). SGN-14 (5 µg/ml) neither significantly induce AKT nor NF-B activation in MM.1S cells, in contrast to 5 µg/ml sCD40L-treated counterparts. ERK activation was induced by SGN-14, although to a lesser extent than sCD40L. SGN-14 did not block nor enhance AKT/NF-B activation induced by sCD40L at the same concentration (5 µg/ml). Notably, 24-hr pretreatment of cells with SGN-14 blocked sCD40L-mediated PI3K/AKT and ERK activation. In contrast, cells treated with sCD40L for 24 h still retained the ability to further activate downstream signaling pathways in response to sCD40L. Importantly, pretreatment of MM.1S cells with SGN-14 rendered them refractory to further proliferation induced by IL-6 (p < 0.05): the two-fold increase of DNA synthesis induced by IL-6 (50 ng/ml) was completely blocked by pretreatment with 10 µg/ml SGN-14 for 48 hrs. Furthermore, IL-6 did not overcome the inhibition of DNA synthesis triggered by Dex and IMiDs (0.01-10 µM) in cells pretreated with SGN-14 for 24-48 hrs. Using oligonucleotide microarray analysis, we first found differential gene expression profile between SGN-14-treated vs sCD40L-treated MM.1S cells and identified an approximately three-fold reduction of interleukin 6 receptor (IL6R) expression in SGN-14-treated MM.1S cells over 6-24 hr. Currently, the mechanism by which SGN-14 down regulates IL6R and possibly CD40 receptor is under further investigation. In addition, VEGF secretion was inhibited in the presence of SGN-14 (0.01- 10 µg/ml) in MM.1S and MM.1R cells, as well as two plasma cell leukemia patient cells, correlating with reduced baseline migration of plasma leukemia cells in the presence of SGN-14. SGN-14 also induces antibody-dependent cell-mediated cytotoxicity and enhanced tumor cell lysis by effectors treated S260
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Focussed Symposia Arsenic trioxide
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assess whether such a program will
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The overall response rate was noted
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Figure 5A Table 1: VEL + THAL for R
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naturally occurring OPG. Present tr
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0.505). Finally, the multiple event
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CLINICOPATHOLOGICAL DEFINITION OF W
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Plenary Sessions 1. Development of
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clonotypic IgH VDJ signature confir
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can be considered as two entities,
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immunofluorescence staining for DKK
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P2.3 DEVELOPMENT OF A MULTIPLE MYEL
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P2.5 MOLECULAR CYTOGENETICS OF MYEL
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clear that the biggest challenge fo
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esponse and have demonstrated that
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variable region of the idiotypic im
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4. From MGUS to symptomatic MM Fig.
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(MRI); some have claimed that level
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P4.5 CYTOKINES IN MGUS: THERAPEUTIC
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preventing phosphorylation of p70S6
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transducing component of the interl
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survive initial drug exposure and a
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quiescent counterpart, the human um
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transcriptional activity of NF-êB;
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manifestations and anomalous protei
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P7.4 ROLE OF SERUM FREE LIGHT CHAIN
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P7.6 IMMUNOPHENOTYPIC INVESTIGATION
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presumably through the circulation,
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9. Chemotherapy, maintenance treatm
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stratified according to their antim
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explore higher doses of zoledronic
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initial therapy. However, the role
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P10.2.2 SINGLE VERSUS TANDEM HIGH D
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With a median follow-up of 38 month
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cells could be harvested following
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11. What is the role of allogeneic
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found that 75% of patients who were
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patients with responsive disease 3
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9. Giralt S, Estey E, Albitar M, et
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Badros A, Barlogie B, Siegel E, et
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Figure 3b. Event-free Survival 4-Ye
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improve patient outcome in MM. Impo
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myeloma and in 40% of previously un
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degrade OPG in the same way as myel
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P12.2.5 MONOCLONAL ANTIBODY THERAPY
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myeloma. Blood 2001; 98:210-216. 6.
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MHC molecules, in effectively prese
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mice demonstrate that class II-spec
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detected in 293 and NIH3T3 cells. S
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hypothesis that (1) CCND1 and FGFR3
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patient samples. In addition, the p
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016 NEUTRAL ENDOPEPTIDASE (CD10) KN
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2. Genetic heterogeneity in MM: imp
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evidence from two t(11;14) cases wh
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immunoglobulin heavy chain (IgH) lo
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034 Instability of Pericentromeric
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clusters were found, the first was
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MGUS but most likely not crucial fo
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Objective: To compare the impact on
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4 patients had MMSET/IgH but did no
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and clonal PC from MGUS, MM and PCL
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059 VEGF receptor expresion in Mult
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two HLA-A2+ myeloma patients with C
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molecules expressed on the surface
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Recognition of these antigens appea
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Diagnosis requires a single area of
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081 Incidence of solid tumors in co
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Growth Factor (VEGF), Hepatocyte Gr
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PC-AI levels of MGUS and SMM patien
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093 The predominant pathway of tran
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was associated with I.V. line, whil
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103 Vascular amyloidosis induces se
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5. Signal transduction pathways and
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integrin in IGF-1-triggered MM cell
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118 IL-6- Induced Phosphorylation o
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123 THE IGF/IGF-1R SYSTEM IS A MAJO
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human myeloma cell line (HMCL) to a
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ligation or dexamethasone (Georgii-
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6. Role of microenvironment 6.1 Cel
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141 Human myeloma cells adhere to f
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145 STUDY OF BONE MARROW ANGIOGENES
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patients, the growth of primary mye
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tibia (con=49.1±0.7mg/cm2 vs 5T2=4
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157 The Nitrogen-Containing Bisphos
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7. New prognostic criteria for clas
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atio of >3. This system has subdivi
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Patient 1 (IgG/κ);IgG - 16.5 days,
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176 Pro-inflammatory and angiogenic
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180 Clinical study on the bone lesi
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7.2 Imaging studies 185 99mTc-MIBI
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189 Factors Predicting Occult Spina
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vivo detected resonances was invest
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Support Unit (CTSU) with flexibilit
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(β2m) & C reactive protein (CRP).
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plasmids carrying the clonotypic in
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newly diagnosed multiple myeloma as
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216 Transgenic expression of Myc an
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221 Prolonged survival in the 5T2MM
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9. Chemotherapy, maintenance, treat
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229 EFFECTIVENESS OF STANDARD CHEMO
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234 Melphalan and Dexamethasone- Is
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Methods. We investigated the VECD p
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9.2 Renal complications. 243 MERIT
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cost of SRE-related care was $10,24
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those with Hb >13 g/dL. Analysis of
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sustained response, lasting from 52
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proportion of bone abnormality. IgD
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disease. The lack of response to in
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Page 219 and 220: patients(pts) aged ≥60 yrs. We co
Page 221 and 222: PBSC mobilizing regimen utilizing C
Page 223 and 224: their leukocytes and platelets. No
Page 225 and 226: 25 Gy to marrow. The tracer dose wa
Page 227 and 228: non-CR after the first transplant a
Page 229 and 230: 296 PERIPHERAL BLOOD STEM CELL TRAN
Page 231 and 232: References Effect of dose-intensive
Page 233 and 234: with cyclosporine A and methotrexat
Page 235 and 236: 11.Role of novel therapies targetin
Page 237 and 238: 312 Thalidomide as Rescue of Relaps
Page 239 and 240: patients, light chain in 1 patients
Page 241 and 242: platelets of 207 (76-402), B2M of 3
Page 243 and 244: 325 Low Dose Thalidomide plus Dexam
Page 245 and 246: in 5 pts (11%), M protein decreased
Page 247 and 248: time from diagnosis was 3.7 years a
Page 249 and 250: 339 Thalidomide, Clarithromycin and
Page 251 and 252: 344 COMBINATION OF THALIDOMIDE, DEX
Page 253 and 254: experienced early death during the
Page 255 and 256: untreated patients with high tumor
Page 257 and 258: 357 Thalidomide protects endothelia
Page 259 and 260: 362 EOSINOPHILIA IS A VERY COMMON F
Page 261 and 262: 11.5 CC 4047, PS 341 and arsenic tr
Page 263 and 264: without chromosome 13. Mean IC50 fo
Page 265 and 266: myeloma patients. Phase I data indi
Page 267: 11.6 Other drugs 380 EFFECT OF STI5
Page 271 and 272: which acts to prevent the synthesis
Page 273 and 274: of B and its metabolites, however,
Page 275 and 276: and 10 months after start of therap
Page 277 and 278: terminal kinase (JNK) pathway which
Page 279 and 280: lymphocytes was tested by Ellispot.
Page 281 and 282: 411 Dendritic Cell-Based Idiotype V
Page 283 and 284: Author Index Abe M 74 160 Abelman W
Page 285 and 286: De La Serna J 291 De Laurenzi A P11
Page 287 and 288: Hull DR 338 Hullen C P10.2.1 Humes
Page 289 and 290: Morra E 249 280 359 Morris CM 226 M
Page 291 and 292: Siegel D 70 115 250 Sierra J 304 30
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