Haematologica 2003 - Supplements

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368 PHARMACOGENOMIC STUDIES OF BORTEZOMIB (VELCADETM) TREATMENT IN LATE STAGE MULTIPLE MYELOMA G.Mulligan, B. Bryant, J.Stec, S. Kim, M.Morrissey, A.Damokosh, A.Singh, A.Bolt, A.Schmitt, J.Metivier, J.Larsen-Gallup, D.Esseltine, J.Adams, D.Schenkein, A. Boral, J. Brown, G. Linette, J. Ross, and the SUMMIT Myeloma Study Group* Millennium Pharmaceuticals, Inc. Cambridge, MA The clinical development of the proteasome inhibitor, bortezomib (VELCADETM) for the treatment of late stage multiple myeloma has included a transcriptional profiling-based pharmacogenomics strategy to identify genomic predictors of response and further elucidate the mechanism of action of the drug. Through a collaborative effort with the Phase II SUMMIT clinical trial sites, patient bone marrow aspirates were subjected to a rapid negative selection enrichment protocol to purify myeloma cells before being frozen and shipped for centralized pharmacogenomics assessment. Sixty-two percent of 202 patients agreed to the optional expression profiling analysis. The intrinsic gene expression patterns of these samples were determined on Affymetrix U-133 oligonucleotide microarrays prior to treatment with bortezomib. Multiple bioinformatic marker selection algorithms with supervised learning were used to identify genes expression patterns that characterized the differences between responders and non-responders to bortezomib using the Blade response criteria. Predictive accuracy as determined by 5 fold cross validation was significant (71-78%, range) in multiple models. These pharmacogenomic studies of late stage multiple myeloma patients is continuing with the evaluation of new samples obtained from patients in the Phase III clinical trial designed to further discover new markers and validate the current predictive gene sets. 369 Effects of PS-341 and PS-1145 on cytokine-mediated proliferation and anti-apoptosis in myeloma cells. Unn-Merete Fagerli, Vadim Baykov, Torstein Baade Ro, Anders Waage, Anders Sundan and Magne Borset. Norwegian University of Science and Technology, Dept. of Cancer Research and Molecular Medicine; Dept. of Oncology and Section of Haematology, Trondheim University Hospital, Trondheim, Norway. Several cytokines have been implicated in the pathogenesis of multiple myeloma (MM). Interleukin (IL)-6 is an important growth and anti-apoptotic factor for MM cells. However, other cytokines may substitute for IL-6, for example tumor necrosis factor (TNF), IL-10, IL-15, insulin-like growth factor (IGF)-1 or IL-21. For instance, in the human myeloma cell line OH-2, combinations of TNF with IL-10, IL-15 or IL-21 give synergistic effects on proliferation and a stronger response than IL-6 alone. We examined in vitro effects of the proteasome inhibitor PS-341 and the IkB kinase inhibitor PS-1145 on the cytokine-dependent myeloma cell lines OH-2 and IH-1 as well as on the cytokineindependent cell line RPMI 8226. We wanted to see if the two substances discriminated between MM cells stimulated to grow by different cytokines and combinations of different cytokines. We examined proliferation by 3H-thymidine incorporation and induction of apoptosis by Annexin V-FITC/propidium iodide flow cytometry. PS-341 inhibited proliferation and induced apoptosis in all cell lines, irrespective of which cytokine stimulated the growth. In the proliferation assay ED50 was 1.3 nM. PS-1145 inhibited TNF-induced proliferation of OH-2 cells by more than 50% in a dose-dependent manner, whereas growth stimulated by other cytokines was inhibited less than 25%. In the apoptosis assay, PS-1145 totally inhibited the anti-apoptotic effect of TNF in OH-2 cells, whereas little or no inhibition was seen against anti-apoptosis mediated by IL-6 or IL-21. When growth of OH-2 cells was supported by a combination of TNF and other cytokines, proliferation or anti-apoptosis was inhibited to the level seen in controls without TNF. Because of the striking synergy between TNF and other cytokines in OH-2 cells, the effect of PS-1145 was most pronounced under these conditions, both in absolute and in relative sense (e.g. more than 60% reduction of proliferation stimulated by TNF and IL-21). Conversely, in the IH-1 cell line, which is not growth-stimulated by TNF, PS-1145 gave a less than 10% reduction in proliferation of cells stimulated with a combination of TNF and IL-6. In the cytokine-independent cell line RPMI-8226, PS-1145 was pro-apoptotic only in the presence of TNF, showing that TNF became cytotoxic when added together with PS-1145. In conclusion, PS-341 induces apoptosis irrespective of which cytokine is promoting growth of the cells, suggesting that its proapoptotic effect is not linked to any specific signaling pathway. In contrast, PS-1145 seems to be specific for TNF-mediated growth, and in fact, TNF itself becomes cytotoxic in the presence of PS- 1145. The anti-proliferative effect of PS-1145 is most conspicuous when TNF is combined with other growthpromoting cytokines. We have earlier suggested that TNF might be more important to myeloma growth as a synergistic stimulant than its effect as a single agent suggests. Inhibition of TNFinduced and NF-kB-mediated proliferation and anti-apoptosis is promising, suggesting a place for PS-1145 or other agents targeting NF-kB in treatment of MM. 370 Proteasome inhibitors induce growth inhibition and apoptosis in human myeloma cells irrespective of chromosome 13 deletion Ivana Zavrski, Christian Jakob, Ulrike Heider, Claudia Fleissner, Jan Eucker, Kurt Possinger, Orhan Sezer Department of Hematology and Oncology, Universitätsklinikum Charité, Berlin, Germany. The proteasome is a ATP ubiquitin-dependent protease which plays a crucial role in degrading cytosolic and nuclear proteins, which are strongly involved in basic cellular functions, like the cell cycle regulation, transcription of growth factors and apoptosis. The inhibition of the ubiquitin-proteasome pathway leads to an uncoordinated expression and degradation of several short living proteins required for appropriate cell cycle progression and survival in malignant cells and activates apoptosis. In this study, we investigated the effects of cellpermeable proteasome inhibitors MG-132, MG-262, PSI and lactacystin on multiple myeloma cell lines OPM-2, U266, RPMI8226 and freshly isolated plasma cells with or without deletion of chromosome 13 from patients with multiple myeloma or plasma cell leukemia, and CD34+ human hematopoietic stem cells. The effects of proteasome inhibitors on cell cycle progression, cell growth and apoptosis were determined. Using the MTT-assay, we found PSI with the half maximal cytotoxicity (IC50) of 5.7 nM to be the most potent proteasome inhibitor among those tested, followed by MG-262 (31 nM), MG- 132 (177 nM) and lactacystin (>1 µm). The growth inhibition occurred despite of deletion of chromosome 13, there was no significant difference for IC50 between myeloma cells with and S253
without chromosome 13. Mean IC50 for PSI was 5.4 and 6.1 for myeloma cells with and without chromosome 13 deletion, respectively. Mean IC50 for MG-262 was 24 and 38 for myeloma cells with and without chromosome 13 deletion, respectively. Cell cycle arrest occurred either in G0, G1 or in G2-M in a dose and time dependent manner, as shown by 7-AAD, Ki-67 staining and flow cytometry. Sub-apoptotic dosages led to a partial loss of Ki-67 antigen and arrested cells in the G0, whereas higher dosages led to a reduction of Ki-67 levels, growth inhibition and induction of apoptosis. As shown by Annexin-V staining, apoptosis was partially dependent on activation of caspase-3, since Ac-DEVD-cho, a caspase-3 inhibitor, could reduce the apoptosis significantly. The cytotoxicity of the four proteasome inhibitors tested was significantly lower on human progenitor stem cells than in myeloma cells. In conclusion, our results show that proteasome inhibitors induce cell cycle alterations, growth inhibition and apoptosis in human myeloma cells and importantly, chromosome 13 status did not affect the response to proteasome inhibitor treatment. 371 Phase I study of the proteasome inhibitor bortezomib and pegylated liposomal doxorubicin in patients with advanced hematologic malignancies Robert Z. Orlowski, Peter M. Voorhees, Reynaldo A. Garcia, Melissa D. Hall, Fred J. Kudrik, Catherine M. Hogan, Elizabeth Humes, Anandhi Johri, Hendrik W. Van Deventer, Don A. Gabriel, Thomas C. Shea, Beverly S. Mitchell, Julian Adams, Dixie-Lee Esseltine, Natalie D. Depcik-Smith, Henry Bell, Mary Jo Lehman, Celeste M. Lindley, and E. Claire Dees University of North Carolina at Chapel Hill, Chapel Hill, NC, and Millennium Pharmaceuticals, Inc., Cambridge, MA. Introduction: The proteasome is involved in intracellular ubiquitin-dependent and –independent protein degradation, and is a novel target for cancer therapy. Bortezomib (VELCADE, formerly PS-341), a novel specific and selective inhibitor of the proteasome, has shown significant single-agent activity against multiple myeloma in both preclinical and clinical studies. Proteasome inhibitors also block several survival pathways activated by anthracyclines, including nuclear factor-B and p44/42 mitogen activated protein kinase, that may limit their own effectiveness, suggesting such combinations might have enhanced anti-tumor efficacy. Study Aims: We therefore sought to evaluate the maximum tolerated dose (MTD), dose limiting toxicity (DLT), pharmacokinetics, and pharmacodynamics of bortezomib and pegylated, liposomal doxorubicin (Doxil®) in patients with hematologic malignancies. Methods: Bortezomib was given as an intravenous bolus at doses that were to range from 0.90 to 1.50 mg/m2 on days 1, 4, 8, and 11 of a 3 week cycle. Doxil® was given as an intravenous infusion following bortezomib on day 4 at 30 mg/m2. The MTD was defined based on toxicities occurring during cycle 1, while responses were evaluated every 2 cycles. Patients: 25 patients have been enrolled and treated to date, with 18 of these having had advanced multiple myeloma. Results: A mean of 3.9 cycles (range 1-10) has been administered, with 22 patients evaluable for toxicity. At the 0.90 mg/m2 dose level a patient with Crohn’s disease had grade 3 diarrhea, hypotension, confusion and syncope, but no other DLTs were seen at this level, nor at 1.05, 1.20, and 1.30 mg/m2. At the 1.40 mg/m2 level one patient with multiple myeloma and plasma cell leukemia had prolonged grade 4 neutropenia, but additional patients in this cohort did not experience a DLT. All other nonhematologic drug-related toxicities during cycle 1 have been grade 1 or 2 in intensity. Grade 3 or 4 toxicities in later cycles included thrombocytopenia, neutropenia, febrile neutropenia, fatigue, neuropathy, and palmar plantar erythrodysesthesia. Of 13 evaluable multiple myeloma patients complete responses (CR) have been observed in 4, near-CR with a residual paraprotein seen only by immunofixation in 1, partial responses in 3, a minor response has been seen in 1 patient, and 3 patients have had stable disease, while one patient with non-secretory myeloma progressed by marrow criteria. Six of these patients, including two of the CRs, had disease that previously progressed or didn’t respond to anthracycline-based therapy. One patient with multiple cytogenetic abnormalities, including a deletion of chromosome 13, achieved a CR after four cycles with clearance of cytogenetic abnormalities after two cycles. Five of these patients are continuing treatment, the MTD has yet to be defined, and accrual is continuing at the 1.50 mg/m2 dose level. Conclusions: Early results from this study suggest that bortezomib/Doxil® may be well tolerated and active in patients with advanced multiple myeloma, and phase II testing of this regimen may be warranted. 372 Identification of Molecular Markers that Determine Sensitivity (or Resistance) to PS-341 / Velcade in Multiple Myeloma Reshma Shringarpure, Dharminder Chauhan, Yu-Tzu Tai, Renate Burger, Laurence Catley, Klaus Podar, Teru Hideshima, Nicholas Mitsiades, Constantine Mitsiades, Nikhil Munshi and Kenneth Anderson Jerome Lipper Multiple Myeloma Center, Dana-Farber Cancer Institute, Harvard Mdical School. PS-341 (VelcadeTM/Bortezomib) is a highly potent therapeutic agent for multiple myeloma. Although some of its downstream molecular targets have been defined, the mechanisms of sensitivity and resistance to PS-341 are not known. We compared sensitivity of cell lines derived from various hematologic malignancies to PS-341. Our preliminary experiments showed that two B-cell lines, both derived from patients with diffuse histiocytic lymphoma (DHL), had remarkably different responses to PS-341 treatment. While the SUDHL-6 cell line was very sensitive (IC50 = 2.5 nM), the SUDHL-4 cell line had a 20-fold higher IC50 (>50nM). Interestingly, PS-341-resistant SUDHL-4 cells survived (for at least 48 hours) in the presence of 20nM PS- 341, despite the fact that greater than 80% of the proteasome chymotrypsin-like activity was inhibited by 6 hours. Proteasome inhibition in resistant SUDHL-4 cells was also confirmed by intracellular accumulation of p27Kip1, a proteasome substrate, within 6 hours after treatment with 20nM PS-341. Since the two cell lines are phenotypically quite similar (CD20-positive, CD138-negative) and derived from a similar developmental stage, we are using these cell lines as models to determine markers of PS-341 sensitivity and resistance. We have profiled these two cell lines as well as the myeloma cell line MM.1S, to define changes in gene expression before and after PS-341 treatment. We are currently analyzing these data to identify genes, which are uniquely altered in SUDHL-4 or SUDHL-6 cells upon PS-341 treatment, to define molecular mechanisms conferring the “resistant” versus “sensitive” phenotype. S254
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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
Page 211 and 212: sustained response, lasting from 52
Page 213 and 214: proportion of bone abnormality. IgD
Page 215 and 216: 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
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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: 11.5 CC 4047, PS 341 and arsenic tr
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 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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