Haematologica 2003 - Supplements

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1/MAGE-A3 immunotherapy can be used as secondary prevention to prevent relapse/disease progression in NY-ESO- 1/MAGE-A3 negative patients receiving chemotherapy or in smoldering/indolent myeloma. 403 Dendritic cell numbers and their subsets during treatment of multiple myeloma 1KOVAROVA, Lucie, MSc; 1,2HAJEK, Roman, MD; 3SVOBODNIK, Adam, MSc; 1KLABUSAY, Martin, MD, PhD; 1VORLICEK, Jiri, Professor, MD 1Department of Internal Medicine-Hematooncology, Masaryk University Hospital Brno-Bohunice, Jihlavska 20, 625 00 Brno, Czech Republic2Department of Clinical Hematology, Masaryk University Hospital Brno-Bohunice, Jihlavska 20, 625 00 Brno, Czech Republic3Centre of Biostatistics and Analysis, Masaryk University Brno-Bohunice, Kamenice 126/3,625 00 Brno Background and objective: In this study, the proportion of dendritic cell (DC) subsets (DC1, DC2) in peripheral blood of patients with multiple myeloma (MM) was evaluated before and during treatment. Design and Methods: Flow cytometric determination of DC subsets in peripheral blood was based on positive expression of the surface antigen CD83 in combination with HLA-DR and either CD11c or CD123. Results: No significant differencies were found in initial values between the group of healthy volunteers (n = 15; mean count of CD83+ cells was 0.62±0.06%; ratio DC1/DC2 = 2.5) and the group of patients before treatment (n = 15; 0.59±0.13% CD83+; DC1/DC2 = 2.17). In a group of patients (n = 10) treated with VAD regimen (vincristine, adriamycin, dexamethasone), the mean percentage of DC after induction treatment (0.84±0.4% CD83+ cells; DC1/DC2 = 1.59) was higher than initial values. Administration of G-CSF reduced the total DC numbers (0.66±0.6%; DC1/DC2 = 3.71). The lowest total DC counts were in the apheresis products (0.36±0.2%; DC1/DC2 = 4.75). Administration of GM-CSF increased DC numbers (0.56±0.3%; DC1/DC2 = 1.59). Pretreatment DC values were achieved within six months after transplantation (0.83±0.6%; DC1/DC2 = 3.92). Interpretation and Conclusions: The highest number of total DC was found after induction treatment and within six months of transplantation. The ratio DC1/DC2 showed a the highest value in the apheresis products and in peripheral blood within the first six months after transplantation. 404 IL-12 corrects the dendritic cell defect of patients with myeloma Ross Brown1, Allan Murray1, Belinda Pope1, Daniel M Sze1, John Gibson1, P Joy Ho1, Derek Hart2, Doug Joshua1 1Institute of Haematology, Royal Prince Alfred Hospital, Sydney; 2Mater Medical Research Institute, Brisbane, Australia. The poor response to immunotherapy in patients with multiple myeloma indicates that a better understanding of the immune defects in this disease is required before more effective therapeutic strategies can be developed. Recently we reported that high potency (CMRF44+) dendritic cells (DC) in the peripheral blood of patients with multiple myeloma failed to upregulate the expression of the B7 costimulatory molecules, CD80 and CD86, in response to an appropriate signal from huCD40LT. During antigen presentation to T cells the level of expression of the B7 molecules provides an important “second signal” that determines the fate of each cell – apoptosis, anergy or productive immunity. We have demonstrated that this defect is caused by TGF1 and IL-10 produced by malignant plasma cells and that it is possible to neutralise the defect in vitro with anti- TGF1. If this defect has an impact on immunotherapy strategies, it would be important to identify a more suitable agent than anti-TGF1 for in vivo use. The number of high potency DC (CMRF44+, CD14-, CD19-, PI-) in the blood of patients with myeloma (0.03-0.8% of mononuclear cells; n= 26) was not significantly different from normal controls (0.05-0.8% of MNCs; n=13). The expression of the costimulatory molecules CD80 and CD86 on blood DC of these patients (2917% and 8510% of MNCs respectively) was also normal (2917% and 8616% of MNCs). Incubation with huCD40LT stimulated upregulation of CD80 expression on the DC and B cells of normal controls but there was either reduced or no upregulation of CD80 on the DC of the patients with myeloma. Less than 10% of malignant plasma cells expressed CD80 and huCD40LT failed to significantly upregulate CD80 expression on mature plasma cells (n=6). Upregulation of CD80 on DC of normal controls was inhibited by rTGF-in a dose dependent manner. CD86 expression on DC was high both before (86%) and after (89%) stimulation. IL-12 but not interferon-could replace anti-TGF1 as an agent capable of neutralising the failure to stimulate CD80 upregulation by huCD40LT. DC subsets stimulated by IL-12 were predominantly myeloid (CD11c+ and CDw123- ), suggesting that they could initiate a Th1 type response. Thus patients with myeloma have a normal number of DC but may fail to upregulate CD80 expression in the presence of huCD40LT due to tumour-derived TGF-and/or IL-10. This defect can be corrected with IL-12. 405 IN VITRO STIMULATION OF LYMPHOCYTES FROM MULTIPLE MYELOMA PATIENTS WITH AUTOLOGOUS DENDRITIC CELLS PULSED WITH PLASMOCYTE LYSATES. L. Garderet, C. Mazurier, C. Funck-Brentano, A. Karim, S. Bouchet, J. Frick, NC. Gorin, M. Lopez. Department of Hematology, CIC, EFS, LTCRA, Inserm U 76-CHU Saint Antoine, 27 rue de Chaligny- 75012, Paris; 92-Fontenay Aux Roses, France. Tumor vaccination using dendritic cells (DCs) pulsed with tumor antigens has proven useful mainly in melanomas, prostate cancers, and lymphomas.Experimental data in mouse models or in in vitro human models have established that multiple myeloma (MM) is immunogenic and can induce an immune T response when the tumor antigen is adequately presented to T lymphocytes by DCs. In a pre-clinical study, we are evaluating the possibility to generate in MM cytotoxic T lymphocytes directed against the autologous patient’s tumor cells, i.e. the plasmocytes in a protocol that fits GMP procedures. Until now, 8 patients (pts) have been collected. Plasmocytes were isolated from bone marrow at diagnosis (except for 2 pts, for whom the monoclonal immunoglobulin was used as tumor antigen), selected with anti- CD138 magnetic beads and cryopreserved. Monocytes and lymphocytes were purified by elutriation from apheresis products of pts at remission . Monocytes were differentiated into DCs by culture during 5 days in teflon bags in the presence of GM- CSF+IL-13+fetal calf serum. DCs were pulsed with autologous plasmocyte lysates or monoclonal Ig, matured using poly-IC + anti-CD40, and then cocultured with autologous T lymphocytes. The cytotoxicity of T lymphocytes against autologous plasmocytes (or Ig-pulsed DCs) was tested using a 51chromiumrelease assay. The production of interferon-γ by stimulated S268
lymphocytes was tested by Ellispot. The entire experiment could be achieved for only 5 pts. Immature DCs had the same phenotype than DCs obtained from healthy donors, i.e. CD14-, CD1a+, HLA-DR++, CD86++, CD40++, CD83-. Until now, only one patient’s lymphocytes showed a specific antitumor activity. In conclusion, although feasible, our procedure is not easy to achieve for all patients. However, we now have experience, and think it would help us to get relevant data to confirm the encouraging results recently reported by Dhodapkar et al (PNAS, 2002) and Wen et al (Blood, 2002) 406 INDUCTION OF MYELOMA SPECIFIC CYTOTOXIC T CELLS USING DENDRITIC CELLS TRANSFECTED WITH TUMOR-DERIVED RNA Reichardt V., Milazzo C., Müller M., Grünebach F. and Brossart P. Medizinische Klinik II, Eberhard-Karls-Universität Tübingen, Germany We investigated myeloma RNA transfection of dendritic cells (DC) to induce myeloma specific cytotoxic T cell (CTL) responses in vitro. By this methodology we hope to bypass the need for the identification of shared MM associated antigens or specific antigens such as idiotype (Id). Monocyte derived DC from buffy coats, which were matched in the HLA class I haplotype to the myeloma cell lines LP-1(HLA- A3/A24) and U266 (HLA-A2/A3), were used for RNA transfection. DC were electroporated with total myeloma cell line RNA and were used as antigen presenting cells for the induction of myeloma specific CTL. After a single restimulation with RNA transfected DC, cytotoxic activity of induced T cells was analyzed in 51-Cr release assays. We found that myeloma RNA transfected DC induce CTL that lyse the LP-1 myeloma cells. Cell line specificity was demonstrated by cold target inhibition assay and MHC class I restriction was revealed by antibody blocking studies. Total LP-1 RNA transfected DC also served as suitable targets in a 51-Cr release assay, being equivalent to the respective original tumor cell line. To analyze whether Id specific cytotoxicity contributed to the robust lysis observed, we purified Id protein from LP-1 supernatants and used autologous Id pulsed DC as targets in 51- Cr release assays. Interestingly, LP-1 specific CTL showed no specificity for the idiotype. U266 specific CTL were also successfully induced by the same methodology. We furthermore analyzed whether MUC1 specificity added to the lytic activity of U266 (MUC1+) specific CTL. As corresponding epitopes we tested the recently described HLA-A2 restricted peptides M1.1 and M1.2 and found a striking fine specificity for M1.2, assuming a possible immunodominance of this peptide. We report here on the successful induction of myeloma specific CTL by RNA transfection of DC, which could serve as a potential vaccine. 407 IL-15 generated Dendritic Cell Vaccines to Elicit Specific Immunity to Multiple Myeloma S. N. Bykovskaia, S. Marks, W.J. Storkus, J. Shogan Departments of Surgery and Heamotologic Oncology, University of Pittsburgh Cancer Institute, Hillman Cancer Center, Pittsburgh, PA 15213 We have evaluated the anti-tumor efficacy of IL-15-induced Dendritic Cell (DC) vaccines against multiple myeloma (MM) in preclinical studies. MM is a malignancy involving the uncontrolled expansion of clonal B plasma cells secreting a monoclonal idiotypic immunoglobulin (IdIg). This unique antigen contains epitopes that can be specifically recognized by MHC-restricted T cells. When fed to Dendritic Cells (DC), peptides derived from Ig protein can be presented in MHC complexes on the cell surface of the antigen-presenting cell, thus serving as a highly-specific immunogen. We have recently shown that DC can be generated from CD34+ stem cell precursors in cultures containing IL-15. IL-15-induced DCs appear to represent most powerful stimulators of T cell proliferation in mixed lymphocyte reactions (MLR) than DC generated in the presence of GM-CSF and TNF (S. Bykovskaia e.a., 1999). In current work we evaluated the effects of rIL-15 to enhance the ability of DCs to stimulate MM-specific T cells in vitro as a prelude to subsequent clinical development. CD14+ monocytes were isolated from both MM patient’s and normal donor PBMCs using antibody-coated magnetic beads (MiltinyiBiotec) and cultured with the two sets of cytokines: rhGM-CSF + rhIL4 or rhGM-CSF + rhIL-15. Increased frequencies of DCs bearing the CD1a and CD83 maturation markers were noted for cultures containing IL-15. MM patient’s DCs generated in the presence of IL-15 induce enhanced primary responses in CD8+ T cells. MM patient’s T cells were isolated by specific antibody coated-MACS, co-cultured with autologous DCs generated with both IL-4- and IL-15-supplemented medium, and pulsed with the patient’s IdIg purified by Protein A affinity chromatography from the patient’s serum. Our data suggest specific proliferation of CD8+ T cells in both stimulation groups, however, IL-15-generated DC induced higher rates of proliferation vs. DC cultured in presence of IL-4. We next evaluated specific T cells for secretion of interferon gamma (IFNγ;i.eh1-type) in response to autologous DCs pulsed with the autologous IdIg protein. CD14+-derived DCs generated in medium supplemented with both sets of cytokines were pulsed by autologous IdIg and cultured with the patient’s own T cells. T cells were then analyzed for IFN secretion in ELISPOT assays. IL-15-generated DCs were superior in stimulating the development of specific Th1-type responses, particularly among CD8+ T cell responders. The results of this study should provide the basis for a perspective clinical vaccine formulation designed to treat MM. 408 DNA fusion vaccine against a cancer testis antigen provides protection in a murine tumor model: relevance for human multiple myeloma Jane Watkins, Surinder S. Sahota, Jason Rice and Freda K. Stevenson Molecular Immunology Group, Tenovus Laboratory, Southampton University Hospitals UK. Modern chemotherapeutic regimens and transplantation strategies are able to bring multiple myeloma (MM) into remission, and open the possibility of active vaccination strategies to raise immunity against tumor cells. In this strategy, vaccination with naked DNA provides an attractive option, able to activate both innate and specific immune responses against encoded tumor associated antigen (TAA). For myeloma, the tumor specific idiotype is one such target. We have shown previously that fusing V gene sequences in a single chain variable fragment (scFv) format to a pathogen-derived sequence from tetanus toxin (fragment C, FrC) promotes idiotype specific CD4+ T cell responses, which provide protection from tumor challenge in the S269
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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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yielding a median of 3x106/kg CD34
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patients(pts) aged ≥60 yrs. We co
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PBSC mobilizing regimen utilizing C
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their leukocytes and platelets. No
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25 Gy to marrow. The tracer dose wa
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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 and 268: 11.6 Other drugs 380 EFFECT OF STI5
Page 269 and 270: significant inhibition of cell prol
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: terminal kinase (JNK) pathway which
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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