Haematologica 2003 - Supplements

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even higher in monoclonal gammopathy of unknown significance (MGUS). This lack of detectable chromosomal abnormalities is mainly due to a low level of bone marrow infiltration and the low mitotic index of plasma cells in vitro, but also to the presence of cytogenetically cryptic translocations affecting the immunoglobulin heavy chain (IGH) locus at 14q32. Such limitations can be bypassed by interphase cytogenetic techniques, like fluorescence in situ hybridization (FISH). Common chromosomal abnormalities in PCD are translocations affecting the IGH locus, and less frequently, the immunoglobulin light chain loci lambda (IGL) at 22q11 and kappa (IGK) at 2p12. In the present study, we aimed to systematically evaluate the incidence of chromosomal translocations with breakpoints in the IG loci by FISH in 50 MM and 50 MGUS with normal karyotype. From all these patients, cytogenetic suspensions from bone marrow samples were studied by interphase cytogenetics. A triple-color assay for the IGH locus and double-color assays for the IGL and IGK loci (Martín-Subero et al., Int J Cancer 2002) were applied. In sixteen out of 50 MM patients (32%), FISH with these probes detected at least one structural or numerical chromosomal alteration. Ten cases (20%) carried translocations affecting the IGH locus whereas 5 (10%) displayed IGL breakpoints. In contrast, no IGK translocation was detected. Four (8%) cases and 1 (2%) case displayed three copies of the intact IGK and IGH locus, respectively. No gain of IGL was detected. The 50 MGUS cases were less informative. Only 4 cases (8%) presented signal patterns indicative for a translocation affecting the IGH locus. Neither evidence for illegitimate IGL or IGK rearrangements nor for numerical aberrations was found. Our results support the hypothesis that interphase FISH in bone marrow specimens should be an important adjunct to conventional cytogenetics in MM with normal karyotype, but probably not in MGUS. Because of the frequent low bone marrow infiltration with malignant plasma cells particulary in MGUS, combined immunophenotyping and FISH (FICTION technique) or FISH after MACS (Magnetic Cell Sorting) might be more suited than conventional interphase FISH for the diagnosis of PCD with normal karyotype. 024 Multicolor interphase FISH for the detection of IGH translocations in multiple myeloma Borja Saez, José I. Martín-Subero, María D. Odero, Juan C. Cigudosa, María J. Calasanz, Reiner Siebert 1. Department of Genetics, University of Navarra, Pamplona, Spain 2. Institute of Human Genetics, University Hospital Schleswig- Holstein, Campus Kiel, Germany 3. Deparment of Human Genetics, Spanish National Cancer Center, Madrid, Spain Translocations involving the immunoglobulin heavy chain (IGH) locus at 14q32, or its variants, are among the most common chromosomal abnormalities in multiple myeloma (MM). These translocations juxtapose IG regulatory sequences next to various oncogenes, whose expression is subsequently altered. IGH partners in MM targeted by such translocations are CCND1 (11q13), FGFR3/MMSET (4p16), MAF (16q23), CCND3 (6p21), IRF4 (6p25), MAFB (20q11) and IRTA1/2 (1q21). Some of these translocations affecting IGH in MM are difficult to detect by conventional chromosome analyses because they involve subtelomeric regions on both affected chromosomes, e.g. t(4;14)(p16;q32), t(14;16)(q23;q23) or t(6;14)(p25;q32). This limitation can be overcome by fluorescence in situ hybridization (FISH) using suitable locus-specific probes. Here, we present new multicolor interphase FISH (MI-FISH) assays for the rapid and simultaneous detection of IGH translocations in MM. Probes flanking the chromosomal breakpoints of the most common translocation partners were designed and labeled in a dual-color fashion. These probes were validated in healthy donors and the cut-off for false-positive results was calculated. Their suitability to detect their respective chromosomal breakpoints was proven by using cytogeneticallypositive controls. After this thorough validation process, the probes for each gene were pooled and differentially labeled with DEAC, Spectrum Orange, Texas Red or Cyanine 5. The first multicolor assay was made of a probe for the IGH locus labeled in Spectrum Green together with probes for the most frequent gene partners: CCND1, FGFR3/MMSET, MAF and CCND3. By means of this assay, the t(11;14)(q13;q32), t(4;14)(p16;q32), t(14;16)(q32;q23) and t(6;14)(p21;q32) translocations can be detected in a single experiment. The second multicolor assay included a probe for the IGH locus together with the new probes for the less frequent IG gene partners: IRF4, MAFB and IRTA1/2 to detect the t(6;14)(p25;q32), t(14;20)(q32;q11) and t(1;14)(q21;q32) translocations. These MI-FISH assays were tested in negative and positive controls and hybridized in a small series of MM cases with normal karyotypes where IGH breakpoints were previously detected. Thus, we could validate the capacity of the new probe sets to simultaneously detect the most common translocations in MM. Furthermore, the modular probe design allows easy combination with probes for IGL or IGK instead of IGH to detect variant translocations. In the near future, in order to increase the sensitivity of the MI- FISH approach, multicolor combined immunophenotying and FISH assays will be established. This technique will pave the way for the accurate study of low infiltration with chromosomally altered plasma cells, as it occurs in monoclonal gammopathy of unknown significance (MGUS). 025 Genomic characterisation of the chromosomal breakpoints of t(4;14) and t(11;14) Multiple Myeloma suggest distinct mechanisms of recombination. JAL Fenton, G Pratt, AC Rawstron, AM Dring, FE Davies, JA Child and GJ Morgan University of Leeds Translocations involving the IgH locus are the commonest genetic lesion in multiple myeloma (MM), with recent reports suggesting they are a virtually universal event. Studying the nature of the breakpoints will help us to understand the molecular mechanism leading to MM. We have characterised the genomic breakpoints of nine MM patients, seven t(4;14) translocations, and two t(11;14), using inverse and long range PCR techniques. By convention chromosome 14q32 breakpoints in MM are believed to be located in the IgH S switch region on der(4) or der(11) and a further downstream switch (S) region on der(14), with deletion of intervening 14q32 DNA occurring as a result of aberrant class switch recombination (CSR). Our analysis showed such breakpoints did occur, but there was also evidence of more complex recombination events in four of the eight patients examined. In two t(4;14) patients it was possible to demonstrate that rearranged hybrid switch region sequence was joined to DNA from chromosome 4p16. For the first patient it was found, on der(4), that Ssequence had recombined with S3 sequence before a further recombination site with 4p16 was observed. Similarly for the second case S had apparently recombined with S before being joined to 4p16 sequence. We also provide S98
evidence from two t(11;14) cases where DNA from 11q13 was joined to recombined hybrid (S/S) switch region sequences. In the first case S sequence and S1 sequence, on der(11), had apparently recombined before a 3’ join to 11q13. However in the second case 11q13 sequence was joined to sequence 5’ of a S/S1 hybrid switch region on der(14). There was also evidence of another non-CSR-mediated rearrangement having occurred at the t(11;14) recombination site on der(14), with 5’S sequence inverted from germline orientation for a run of 40 bases before being aligned in the conventional orientation running for approximately 1kb, until the recombination point with S1 sequence. For this case the reciprocal der(11) breakpoint was also isolated and sequenced, comparison between the two breakpoints showed that 21 bases of 11q13 sequence was common to both, i.e had been apparently duplicated in the recombination process associated with the translocation event. Both this data and the fact the breakpoint on 14q32 involved the same region 5’ of S and not another switch region suggests that the translocation did not occur by an aberrant CSR process. The four cases described above suggest that primary IgH translocations in MM can occur via different mechanisms. We speculate that legitimate CSR rearrangements can occur before a later translocation event between 4p16 or 11q13 and 14q32, which implies that primary IgH translocations can occur at different time points. Therefore the crucial neoplastic event which juxtaposes the IgH enhancers to the critical oncogene(s) associated with immortalisation of plasma cells is independent of translocation partner and may not occur via a CSR mechanism, but rather a later undefined event. 026 t(4;14)(p16;q32) as a Model for Unbalanced Immunoglobulin Heavy Chain Translocations in Multiple Myeloma Jonathan J. Keats, Tony Reiman, Christopher A. Maxwell, Michael J. Mant, Andrew R. Belch, Linda M. Pilarski Department of Oncology and Medicine, University of Alberta and Cross Cancer Institute, Edmonton, AB Canada Translocations involving the immunoglobulin heavy chain (IgH) locus are common and recurrent events in multiple myeloma (MM). The most common IgH translocations identified to date are t(11;14)(q13;q32), t(4;14)(p16;q32), and t(14;16)(q32;q23). The frequency of each event in patient populations has become apparent in the last few years with t(11;14) occurring in 15-20%, t(4;14) 10-15%, and t(14;16) in approximately 5% of patients. Moreover, multiple independent research groups have now reported similar results regarding the clinical impact of IgH translocations in MM. We and others have found that t(4;14) predicts for a poor outcome while other groups have shown a similar prognosis for t(14;16) patients, however, t(11;14) seems to predict for an improved outcome. Interestingly, as shown by our group, the clinical outcome associated with t(4;14) appears to be independent of FGFR3 expression, the proposed target gene of t(4;14), as a subset of patients lack FGFR3 expression. These observations lead us to hypothesize that in some patients t(4;14) may be unbalanced resulting in the selective loss of der(14), which has recently been validated by Santra et al. Moreover, this phenomenon does not seem to be localized to t(4;14) as Fonseca and colleges have recently shown that unbalanced translocations also occur in both t(11;14) and t(14;16) MM. Therefore, we are beginning to investigate the importance of genes that flank either side of the t(4;14) breakpoints to determine their expression levels and potential contribution to MM. To access the importance of flanking genes we have updated our cohort, which now includes 272 MM and 77 MGUS patients. Using an RT-PCR screening assay we have found 38 (14.0%) t(4;14) positive MM patients and 1 (1.3%) positive MGUS patient. This strategy can identify three major breakpoint clusters, termed MB4-1, 2, and 3. The 38 MM patients segregate into 26 MB4-1, and 6 each of MB4-2 and MB4-3, while the MGUS patient is an MB4-1. FGFR3 is expressed in 27/37 (73.0%) positive MM patients and in the one MGUS patient. All FGFR3 non-expressers lacked a detectable der(14) RT-PCR product. Furthermore, for 54 negative and 11 t(4;14) positive patients we have tested multiple BM samples, primarily diagnosis and relapse, and have yet to find any differences in t(4;14) status or FGFR3 expression. To define the level of gene expression we have chosen to use quantitative RT-PCR. We are particularly interested in genes that are not interrupted by any of the breakpoints or genes that may promote the oncogenic process by either overexpression or haplo-insufficiency. One gene that falls into the latter category is the Response Element-II Binding Protein (RE-IIBP). Transcription of RE-IIBP initiates downstream of all known t(4;14) breakpoints and thus, unlike MMSET type I & II, RE-IIBP could be overexpressed in its native form by the mu enhancer within all t(4;14) patients. To date we have observed an 8-15 fold increase in the expression of RE-IIBP in t(4;14) positive cell lines compared to other human myeloma cell lines. Work is ongoing to identify other genes located on chromosome 4 that are deregulated by t(4;14). 027 The recurrent translocation t(14;20)(q32;q12) in multiple myeloma results in aberrant expression of MafB and PPP1R16B (TIMAP); a molecular and genetical analysis of the chromosomal breakpoint Gienke R Boersma-Vreugdenhil, Jeroen Kuipers, Ton Peeters, Esther van Straalen, Anne Hagemeijer, Peter L Pearson, Hans C. Clevers, Bert J.E.G. Bast Depts Immunology & Medical Genetics, UMC UtrechtCenter for Human Genetics, KU Leuven Chromosomal translocations of the Immunoglobulin Heavy gene region at 14q32 are regularly involved in B lymphoid malignancies. They are thought to be important in initiating transformation either by deregulation of existing (proto) oncogenes or creation of new hybrid genes with transforming properties. Previously, we reported a novel translocation, t(14;20)(q32;q12), found in the myeloma cell line UM3 by means of double color FISH analysis. In this cell line, the t(14;20) is the only translocation involving the IgH locus. Using a recently developed sensitive double color Immuno FISH, we found the t(14;20) in the diagnostic bone marrow sample as well, excluding a possible in vitro artifact. We cloned the regions containing the breakpoints in the der(14) and der(20) chromosomes from the UM3 cell line, and analyzed ectopic mRNA expression of genes and expressed sequence tags (EST’s) in the breakpoint regions of both derivative chromosomes. Ectopic gene expression was observed for the transcription factor MafB in der(14), and aberrant expression levels of PPPIR16B, also known as TIMAP (putatively involved in the regulation of apoptosis), was detected in der(20). In addition, we characterized the breakpoints in a similar if not identical t(14;20) in 3 other cell lines; the breakpoint scatter is comprised within a region of 0,8 Mb, explaining the aberrant expression in all situations. We also have found such a t(14;20) in additional patient material. S99
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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
Page 55 and 56: presumably through the circulation,
Page 57 and 58: 9. Chemotherapy, maintenance treatm
Page 59 and 60: stratified according to their antim
Page 61 and 62: explore higher doses of zoledronic
Page 63 and 64: initial therapy. However, the role
Page 65 and 66: P10.2.2 SINGLE VERSUS TANDEM HIGH D
Page 67 and 68: With a median follow-up of 38 month
Page 69 and 70: cells could be harvested following
Page 71 and 72: 11. What is the role of allogeneic
Page 73 and 74: found that 75% of patients who were
Page 75 and 76: patients with responsive disease 3
Page 77 and 78: 9. Giralt S, Estey E, Albitar M, et
Page 79 and 80: Badros A, Barlogie B, Siegel E, et
Page 81 and 82: Figure 3b. Event-free Survival 4-Ye
Page 83 and 84: improve patient outcome in MM. Impo
Page 85 and 86: myeloma and in 40% of previously un
Page 87 and 88: degrade OPG in the same way as myel
Page 89 and 90: P12.2.5 MONOCLONAL ANTIBODY THERAPY
Page 91 and 92: myeloma. Blood 2001; 98:210-216. 6.
Page 93 and 94: MHC molecules, in effectively prese
Page 95 and 96: mice demonstrate that class II-spec
Page 97 and 98: detected in 293 and NIH3T3 cells. S
Page 99 and 100: hypothesis that (1) CCND1 and FGFR3
Page 101 and 102: patient samples. In addition, the p
Page 103 and 104: 016 NEUTRAL ENDOPEPTIDASE (CD10) KN
Page 105: 2. Genetic heterogeneity in MM: imp
Page 109 and 110: immunoglobulin heavy chain (IgH) lo
Page 111 and 112: 034 Instability of Pericentromeric
Page 113 and 114: clusters were found, the first was
Page 115 and 116: MGUS but most likely not crucial fo
Page 117 and 118: Objective: To compare the impact on
Page 119 and 120: 4 patients had MMSET/IgH but did no
Page 121 and 122: and clonal PC from MGUS, MM and PCL
Page 123 and 124: 059 VEGF receptor expresion in Mult
Page 125 and 126: two HLA-A2+ myeloma patients with C
Page 127 and 128: molecules expressed on the surface
Page 129 and 130: Recognition of these antigens appea
Page 131 and 132: Diagnosis requires a single area of
Page 133 and 134: 081 Incidence of solid tumors in co
Page 135 and 136: Growth Factor (VEGF), Hepatocyte Gr
Page 137 and 138: PC-AI levels of MGUS and SMM patien
Page 139 and 140: 093 The predominant pathway of tran
Page 141 and 142: was associated with I.V. line, whil
Page 143 and 144: 103 Vascular amyloidosis induces se
Page 145 and 146: 5. Signal transduction pathways and
Page 147 and 148: integrin in IGF-1-triggered MM cell
Page 149 and 150: 118 IL-6- Induced Phosphorylation o
Page 151 and 152: 123 THE IGF/IGF-1R SYSTEM IS A MAJO
Page 153 and 154: human myeloma cell line (HMCL) to a
Page 155 and 156: 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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non-CR after the first transplant a
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296 PERIPHERAL BLOOD STEM CELL TRAN
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References Effect of dose-intensive
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with cyclosporine A and methotrexat
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11.Role of novel therapies targetin
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312 Thalidomide as Rescue of Relaps
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patients, light chain in 1 patients
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platelets of 207 (76-402), B2M of 3
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325 Low Dose Thalidomide plus Dexam
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in 5 pts (11%), M protein decreased
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time from diagnosis was 3.7 years a
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339 Thalidomide, Clarithromycin and
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344 COMBINATION OF THALIDOMIDE, DEX
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experienced early death during the
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untreated patients with high tumor
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357 Thalidomide protects endothelia
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362 EOSINOPHILIA IS A VERY COMMON F
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11.5 CC 4047, PS 341 and arsenic tr
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without chromosome 13. Mean IC50 fo
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myeloma patients. Phase I data indi
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11.6 Other drugs 380 EFFECT OF STI5
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significant inhibition of cell prol
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which acts to prevent the synthesis
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of B and its metabolites, however,
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and 10 months after start of therap
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terminal kinase (JNK) pathway which
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lymphocytes was tested by Ellispot.
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411 Dendritic Cell-Based Idiotype V
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Author Index Abe M 74 160 Abelman W
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De La Serna J 291 De Laurenzi A P11
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Hull DR 338 Hullen C P10.2.1 Humes
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Morra E 249 280 359 Morris CM 226 M
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Siegel D 70 115 250 Sierra J 304 30
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