Abstracts 126–224 (includes Monday presentations)

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123 STEM CELLS EXPRESSING FAS-LIGAND RESIST IMMUNE REJECTION Civin C. Baltimore, MD, USA Abstract not received at time of printing.

124 FINDING NEW AVENUES TO PROMOTE EXPANSION OF PRIMITIVE HEMATOPOIETIC CELLS Humphries K.R. Vancouver, BC, Columbia Abstract not received at time of printing.

125 ENGINEERING STEM CELLS AS THERAPEUTICS Cavazzana-Calvo M. Paris, France Abstract not received at time of printing.

126 CORD BLOOD VERSUS UNRELATED HEMOPOIETIC STEM CELL TRANSPLANTATION IN ACUTE LEUKEMIA Gluckman E. Paris, France Abstract not received at time of printing.

127 CLINICAL TRANSPLANT — STATE OF THE ART Martelli M.*,1, Aversa F.1, Tabilio A.1, Velardi A.1 1 University of Perugia, Perugia, Italy Eighty-one AML and 69 ALL patients at high-risk because of bad-risk CR I (30), CR II or later (48) or in relapse (72) received megadoses of T cell depleted hematopoietic stem cells. Before 1995 36 patients conditioned with TBI, thiotepa, ATG and CY received lectin-separated bone marrow and PBPCs. Afterward, fludarabine replaced CY. PBPCs were positively selected using CellPro (44 patients) or CliniMacs (70 patients without post-transplant G-CSF). In the 114 transplants after 1995 primary engraftment improved from 80% to 91%. Without post-transplant prophylaxis, the incidence of acute GvHD dropped from 18% to 8%. The probability of relapse in ALL was 0.89, 0.60 and 0.17 for the 36 patients transplanted in relapse, the 19 in CR II-III and the 14 transplanted in CR I, respectively. In AML, relapse was 0.40, 0.50 and 0.19 for the 36 patients transplanted in relapse, the 29 in CR II-III and the 16 in CR I. A non-NK-cell alloreactive donor was the major factor for relapse in AML (0.08 for the 36 recipients of NKalloreactive donors vs 0.68 for the other 42). Before 1995 18/36

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patients died of non-leukemic causes, mainly infections and 44/ 114 died after 1995. 23 (13 infections) of the 70 transplants with Clinimacs and no G-CSF post-transplant died of non-leukemic causes. Improved immunologic reconstitution since 1999 has reduced mortality in 30 AML transplanted in any CR (0.24 vs 0.70 in 12 patients at the same stage of disease transplanted pre-1999). Ten-year EFS was 0.05, 0.24 and 0.19 for ALL transplanted in relapse, in CR II-III and in CR I, respectively; 0.22, 0.28 and 0.63 for AML transplanted in relapse, in CR II-III and CR I, respectively. A non- NK-cell alloreactive donor was the major risk factor for EFS (0.65 for the 36 recipients of grafts from NK-alloreactive donors and 0.06 for the other 42).

128 PRIMED MARROW FOR AUTOLOGOUS AND ALLOGENEIC TRANSPLANTATION Elfenbein G.*,1 1 Roger Williams Medical Center, Providence, RI, USA There are at least 13 publications in the autotransplant and 23 publications in the allotransplant literature comparing blood stem cells (BSC) and marrow stem cells (MSC) with respect to pace of engraftment. We have performed summary analyses of these publications to answer the question, “Which stem cell source produces more rapid engraftment?” For autologous G-CSF mobilized BSC (n ⫽ 407), the weighted median day to recovery of AGC ⬎ 500/µL and PLT ⬎ 20,000/µL were 12.2 and 16.1, respectively. For steady state MSC (n ⫽ 347), the weighted medians were 16.7 and 27.4. These data support the hypothesis that BSC produce more rapid engraftment than MSC. However, for GCSF primed MSC (n ⫽ 159), the medians were 14.7 and 20.8. These data support the hypothesis that G-CSF accelerates engraftment for MSC just like it does for BSC. Furthermore, in 2 randomized trials comparing G-CSF mobilized BSC and G-CSF primed MSC head-to-head, MSC and BSC produced equivalent engraftment times. Despite these results, a controversy exists, resolution of which would be better by observing allotransplants, because hematopoiesis of healthy donors is presumably normal. In allotransplants, for recovery of AGC and PLT, the weighted medians of G-CSF mobilized BSC (n ⫽ 1950) were 14.4 and 16.2 days; for steady state MSC (n ⫽ 3782) they were 19.2 and 26.4 days; and for G-CSF primed MSC (n ⫽ 108) they were 14.4 and 16.4 days. In one randomized trial comparing G-CSF mobilized BSC to GCSF primed MSC, engraftment times were equivalent for AGC and PLT. AGC recovery is delayed by methotrexate in GVHD prophylaxis. G-CSF pretreatment is the reason why mobilized BSC appear to engraft quicker than steady state MSC. For autotransplants, relapse rates and survival are similar. For allotransplants, overall survival and disease-free survival are similar. There is much less chronic GVHD for recipients of any allo-MSC and potentially less acute GVHD for recipients of G-CSF primed allo-MSC.

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129 HIGH-DOSE CYCLOPHOSPHAMIDE (CY) FOR ACQUIRED SEVERE APLASTIC ANEMIA (SAA): LONG-TERM FOLLOW-UP IN 56 PATIENTS Brodsky R.A.*,1 1 Sidney Kimmel Comp. Cancer Center at Johns Hopkins, Baltimore, MD, USA SAA is a life-threatening autoimmune bone marrow failure disorder. Bone marrow transplantation (BMT) is the preferred therapy for young patients with an HLA-matched sibling donor. Patients who are not suitable BMT candidates are commonly treated with antilymphocyte globulin or antithymocyte globulin in combination with cyclosporine (ATG/CSA). Despite a high response rate to ATG/CSA (60% to 80%), most responses are incomplete, with many patients suffering long-term complications: relapse, secondary clonal disorders, and avascular necrosis. CY (200 mg/kg) has been shown to induce a high rate of complete remissions (CR) in SAA with little risk of relapse; however, concern has been raised regarding the safety of this approach. We have treated 56 SAA patients with CY: Group A consists of 10 previously untreated patients who received CY between 1977 and 1984; Group B consists of 30 previously untreated patients (median age, 42 years) who received CY between 1996 and 2003; and Group C patients (n ⫽ 16, median age 32 years), treated between 1996 and 2003, had all failed 1 or more courses of ATG/CSA. In group A, 7 of 10 patients achieved a CR. One patient in CR died of AIDS 4 years after treatment; the remaining 6 maintain a CR without relapse or secondary clonal disease with follow-up ranging from 16–26 years. In group B, overall survival is 83.3% with a median followup of 32 (range, 1 to 83) months. Early mortality (⬍100 days) for Group A and B combined was 12.5%. Overall survival for group C is 56% with a median follow-up of 26 (range, 1 to 70) months; early mortality was 25%. CY is highly effective therapy for SAA with most patients (72.5%) achieving durable drug-free remissions; early mortality low, comparable to ATG/CSA. None of the 40 patients who received CY as primary therapy have developed secondary clonal diseases.

130 MOLECULAR ANATOMY OF THE GRANULOCYTE: HALF OF THE EXPRESSED GENES ARE STILL UNKNOWN Bertrand G.*,1, Segarra C.1, Schved J.F.1, Commes T.2, Marti J.2, Coste J.1 1 Etablissement Franc¸ais du Sang P.M., Montpellier, France, 2 Institut de Ge´ne´tique Humaine, Montpellier, France Bacteria, yeast and fungal infections are an expected consequence of both neutropenia and disorders of abnormal Polymorphonuclear Neutrophil (PMN) function. Granulocyte transfusions are useful as treatment for infections, but they are associated with frequent, often severe complications. Our goal is to improve the safety of granulocyte transfusions, by studying the molecular anatomy of the granulocytes (essentially represented by PMNs) in order to identify new membrane markers potentially associated with adverse immunological reactions. Granulocytes from 12 different donors were purified by Magnetic Cell Sorting using magnetic

beads coated with anti-CD15 antibodies, in order to obtain a high purity degree (99.55 ⫾ 0.19%) an a low activation of the cells. The gene expression profile of the purified cells was performed with the Serial Analysis of Gene Expression (SAGE) method, based on random sequencing of short cDNA tags (14 bp). Twenty thousand seven hundred eighty-seven tags were sequenced, corresponding to 8547 distinct mRNA. 34.6% of these mRNA were identified thanks to databases, and corresponded to a large extent to genes involved in immunity and inflammatory responses (cell mobility/ diapedesis, phagocytosis of pathogens). 13.8% were mRNAs completely sequenced but without known function (Expressed Sequence Tags). Finally, half of the tags (47.8%) were never sequenced and totally unknown. This transcriptome of reference will enable to identify new antigens and molecular markers of the granulocyte, in order to improve the security of granulocyte transfusions, and more generally to better understand diverse pathologies associated to this cell.

131 EOSINOPHIL-COMMITTED PROGENITORS ARE GENERATED FROM GRANULOCYTE/MONOCYTE PROGENITORS BY INSTRUCTIVE ACTION OF GATA-2 Iwasaki H.*,1, Mizuno S.1, Shigematsu H.1, Takatsu K.2, Akashi K.1 1 Dana–Farber Cancer Institute, Boston, MA, USA, 2Institute of Medical Science, Tokyo, Japan Eosinophils play an important role in allergic disorders, but its origin remains unclear. We analyzed the developmental pathway of eosinophils within the hierarchical scheme of hematopoietic development. We first found that eosinophil potential is observed in progenitors along the granulocyte/monocyte development, such as common myeloid progenitors and granulocyte/monocyte progenitors (GMPs), but not in megakaryocyte/erythrocyte progenitors or common lymphoid progenitors. Only a fraction of GMPs (∼1%) gave rise to colonies containing eosinophils in the presence of IL5. We then identified eosinophil-committed progenitors within the GMP cultures by using IL-5Ra expression as a positive marker. IL5Ra⫹Lineage⫺CD34⫹c-Kitlo cells isolated within the day 3 GMP cultures possessed immature blastic morphology, and displayed eosinophil-restricted differentiation potential, indicating that eosinophil progenitors are located downstream of GMPs. We also isolated the Lineage⫺CD34⫹IL-5Rα⫹c-Kitlo cells in steady-state bone marrow, which exclusively generated pure eosinophil colonies at ⬎80% of plating efficiency. We then evaluated the role of IL-5 signals in eosinophil commitment. GMPs with retrovirallytranduced IL-5R did not display the increase in the frequency of eosinophil development, suggesting that IL-5R in normal eosinophil progenitors might transduce “permissive” but not “instructive” signals to eosinophil differentiation. In contrast, GATA-2 can instruct GMPs to commit to the eosinophil lineage: GMPs with retrovirally-transduced GATA-2 expressed IL-5Ra and gave rise only to pure eosinophil colonies at ⬎70% of plating efficiency. These data indicate that the vast majority of GMPs possess eosinophil potential that can be induced by instructive action of GATA2. The newly identified murine eosinophil progenitors might be useful to analyze the developmental mechanisms of allergic disorders and to search for a human counterpart subset, which is a

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potential therapeutic target to control allergic disorders and hypereosinophilic syndrome.

132 PHOSPHORYLATION BY AURORA-B CONVERTS MgcRacGAP TO A RhoGAP DURING CYTOKINESIS Yukinori Minoshima1,2, Toshiyuki Kawashima2,*, Koichi Hirose2,*, Tetsuya Nosaka2,*, and Toshio Kitamura1,2 1 Division of Cellular Therapy, 2Division of Hematopoietic Factors, The Institute of Medical Science, University of Tokyo, Tokyo, Japan An antisence cDNA that encodes full-length mouse MgcRacGAP blocked differentiation of murine M1 leukemia cells into macrophages. In human HL-60 leukemic cells, overexpression of a full-length form of human MgcRacGAP and a GAP-inactive mutant also induced growth suppression and macrophage differentiation. These data indicated that MgcRacGAP plays some role in differentiation of hemopoietic cells. However, we recently found that the GAP activity of MgcRacGAP is essential for cytokinesis, indicating that MgcRacGAP plays a role in both differentiation and cell division through distinct function. We here report the molecular mechanism by which MgcRacGAP induces completion of cytokinesis. Cell division is finely controlled by various molecules including small G proteins and kinases/phosphatases. Among these, Aurora-B, RhoA, ECT2 and MgcRacGAP have been implicated in cytokinesis, but the underlying mechanisms have remained unclear. Here, we show that MgcRacGAP colocalizes with AuroraB and RhoA, but not Rac1/Cdc42, at the midbody. Aurora-B phosphorylates MgcRacGAP on serine residues and that this modification induces latent GAP activity toward RhoA in vitro. Expression of a kinase defective mutant of Aurora-B disrupts cytokinesis and inhibits phosphorylation of MgcRacGAP at Ser387, but not its localization to the midbody. Overexpression of a phosphorylationdeficient MgcRacGAP-S387A mutant, but not phosphorylationmimic MgcRacGAP-S387D mutant, arrests cytokinesis at a late stage and induces cell polyploidy. Together, these findings indicate that during cytokinesis MgcRacGAP, previously known as a GAP for Rac/Cdc42, is functionally converted to a RhoGAP through phosphorylation by Aurora-B. It would be interesting to investigate how cell division and differentiation is controlled through regulation of MgcRacGAP.

133 EXPRESSION OF ANGIOTENSIN-CONVERTING ENZYME (ACE) MARKS THE EMERGENCE OF THE HUMAN HEMATOPOIETIC SYSTEM AND IDENTIFIES CANDIDATE HEMATOPOIETIC STEM CELLS IN NEONATAL AND ADULT TISSUES Jokubaitis V.*,1, Tavian M.2, Haylock D.1, Bertoncello I.1, Williams B.1, Driessen R.1, Peault B.2, Simmons P.1 1 Peter MacCallum Cancer Institute, Melbourne, Australia, 2 INSERM U506, Villejuif, France Previous studies from this laboratory have demonstrated that monoclonal antibody BB9 identifies a subpopulation of CD34⫹

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cells in adult human bone marrow (BM). We now demonstrate that the cell-surface molecule identified by BB9 is expressed throughout ontogeny of the human hematopoietic system. The origin of definitive hematopoietic stem cells (HSCs) in man is the aorta-gonad-mesonephros region of the developing embryo. BB9 also identifies rare cells scattered in the mesoderm of the para-aortic splanchonpleura (PaS), a pattern of expression that is maintained until the 26th day of development when BB9⫹ cells are localized around the dorsal aorta. Notably, at these early stages none of the BB9⫹ cells express CD34 or CD45. At later stages hematopoietic cells emerging on the ventral surface of aorta and surrounding endothelial cells express BB9. These data therefore suggest that expression of BB9 is correlated with the emergence of blood forming potential in the PaS. BB9 binds at high level to ⱖ90% of CD34⫹ cells in human fetal liver, to ∼50% of CD34⫹ cells in human umbilical cord blood (UCB) and to ∼20% of the CD34⫹ population in adult BM. To investigate expression of BB9 on transplantable HSCs, CD34⫹ cells from UCB were fractionated by FACS into CD34⫹BB9⫹ and CD34⫹BB9⫺ populations. Following transplantation into NOD/SCID mice, only CD34⫹BB9⫹ cells demonstrated multi-lineage engraftment. To identify the cellsurface molecule identified by BB9, immunoprecipitated protein was subjected to mass spectrometric analysis. BB9 demonstrated identity with the somatic form of Angiotensin Converting Enzyme (ACE/ CD143), a mammalian zinc-dependent metalloprotease expressed as a ∼160kD cell-surface glycoprotein. ACE, although well documented for its role in the regulation of blood pressure and maintaining fluid/electrolyte homeostasis, has not been documented on primitive hematopoietic cells. Collectively, these data are therefore consistent with an unrecognised role for ACE in the ontogeny and regulation of the hematopoietic system.

134 HUMAN MYELOMA CELLS PRODUCE ANGIOPOIETIN-1 BUT NOT ANGIOPOIETIN-2: POTENTIAL RELATIONSHIP WITH MYELOMA-INDUCED ANGIOGENESIS Colla S.1, Giuliani N.*,1, Roti G.1, Lazzaretti M.2, Mancini C.2, Bonomini S.1, Hojden M.1, Morandi F.1, Sala R.3, Lunghi P.4, Bataille R.5, Rizzoli V.1 1 Cattedra di Ematologia, Parma, Italy, 2Anatomia Patologica, Parma, Italy, 3Patologia Generale, Parma, Italy, 4Patologia Medica, Parma, Italy, 5INSERM U463, Nantes, France Multiple myeloma (MM) patients have an increased bone marrow (BM) angiogenesis, however the pro-angiogenetic properties of myeloma cells and the mechanisms of MM-induced angiogenesis are not completely clarified. The angiopoietin system has been identified as critical in the regulation of vessel formation. In this study we have demonstrated that myeloma cells express angiopoietin-1 (Ang-1), but not its antagonist Ang-2, was expressed by several human myeloma cell lines (HMCLs) at both mRNA and protein level by RT-PCR and Western blot analysis, respectively. Immuhistochemistry and immunoprecipitation confirmed that HMCL produced and secrete Ang-1 but not Ang-2. In a transwell co-culture system, we observed that myeloma cells up-regulated the ang-1 receptor Tie2 in human BM endothelial cells. Moreover, in an experimental model of angiogenesis the conditioned medium

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of HMCLs significantly stimulated vessel formation as compared to control or to VEGF treatment. The presence of anti-Tie2 blocking Ab completely blunted the pro-angiogenetic effect of XG-6. Our in vitro results were supported by the in vivo finding of Ang1 but not Ang-2 mRNA and protein expression in purified MM cells obtained from about 47% of 23 patients analyzed. BM angiogenesis was evaluated in bone biopsies obtained from 15 out of 23 patients. The number of microvessels per field was higher in Ang-1 positive patients in comparison with Ang-1 negative ones (mean ⫾ SE: 6.23 ⫾ 0.2 vs. 2.94 ⫾ 0.1, median: 6.21 vs. 2.79; p ⫽ 0.001,) and the micrivascular density was significantly increased (32.98 ⫾ 1.7 vs. 14.55 ⫾ 1.3, median: 34.69 vs. 13.04; p ⬍ 0.01; capillaries: 26.73 ⫾ 1.3 vs. 10.42 ⫾ 0.8, median: 24.06 vs. 9.04; p ⬍ 0.01, small venules: 9.56 ⫾ 0.5 vs. 4.14 ⫾ 0.5, median: 10.60 vs. 3.65; p ⬍ 0.01). Furthermore a significant positive correlation between Ang-1 expression and MVD was found in our cohort of patients (Pearson’s chi-square: p ⫽ 0.036, Cochran’s Linear Trend: p ⫽ 0.01). In conclusion our data indicate that myeloma cells produced Ang-1 that it is involved in MM-induced angiogenesis.

135 MUTATIONS OF VON HIPPEL-LINDAU TUMOR SUPPRESSOR AND CONGENITAL POLYCYTHEMIA Pastore Y.D.*,1, Liu E.2, Jedlickova K.2, Guan Y.2, Hasle H.3, Prchal J.2 1 Texas Children Cancer Center, Baylor College of Medicine, Houston, TX, USA, 2Med Hem/Onc, Baylor College of Medicine, Houston, TX, USA, 3Vibrog Hospital, Viborg, Denmark The Von Hippel-Lindau protein (pVHL) plays an important role in hypoxia sensing. It serves as recognition component of an E3-ubiquitin ligase complex, binding to the hydroxylated form of the hypoxia-inducible factor 1 αsubunit (HIF-iα). Hypoxia or mutation in VHL results in accumulation of HIF-1 and activation of hypoxia-inducible genes, including erythropoietin (EPO). The autosomal dominant cancer-predisposition Von Hippel-Lindau syndrome is due to heterozygosity for VHL germline mutation. In contrast, we found that homozygous germline R200W VHL mutation leads to Chuvash polycythemia (CP), an autosomal recessive polycythemic disorder (Ang et al, Nat Genet 2002). We subsequently found VHL mutations in three unrelated non-Chuvash individuals, one of them was compound heterozygous for the CP and another VHL mutations (Pastore et al, Blood 2003). We have evaluated 13 additional polycythemic patients of non-Chuvash origin and found VHL mutations in both alleles in 7 of them. Among the seven, a Croatian boy was homozygous for a H191D VHL mutation; this is the first discovered homozygous VHL germline polycythemia-causing mutation (VPC) besides CP mutation. Of all six others, three were homozygous for the CP, and three compound heterozygous for the CP and another VHL mutation. By analyzing a large series of polymorphic markers within the VHL gene, same haplotypes were found in patients with CP mutations of Chuvash and non-Chuvash origin, suggesting a founder effect. Interestingly, we have not observed VHL syndrome associated tumors in our patients. While this needs to be evaluated by longitudinal studies, it is likely that VPC differ from tumors predisposing mutations. Interestingly, VPC are mostly located in the C terminal

portion of VHL and does not appear to affect directly HIF-iα or Elongin B or C binding, and may result in a tissue, erythroid specific phenotype. Overall, we found that up to half of patients with apparent congenital polycythemia and increased serum EPO have VHL mutations. In conclusion, inherited VHL mutations are the most frequent cause for congenital polycythemia. The R200W is the most frequent VPC, for which a founder effect is being demonstrated. Potential evolutional advantage of this mutation is being evaluated.

136 Bmi-1 IS REQUIRED FOR MAINTENANCE OF ADULT SELF-RENEWING HEMATOPOIETIC STEM CELLS Park I.1, Qian D.1, Kiel M.1, Becker M.1, Prohaska S.2, Weissman I.2, Morrison S.1, Clarke M.*,1 1 University of Michigan, Ann Arbor, MI, USA, 2Stanford University, Palo Alto, CA, USA A pool of hematopoietic stem cells (HSCs) constantly replenishes the mature cells of the lympho-hematopoietic system. A central issue in stem cell biology is to understand the mechanisms that regulate self-renewal of HSCs, which is required for hematopoiesis to persist for the lifetime of the animal. We found that adult and E14.5 fetal mouse and adult human hematopoietic stem cells express the proto-oncogene bmi-1. The number of fetal liver HSCs, as measured by flow cytometry, was normal in loss of function bmi-1 mice, and the bmi-1⫺/⫺HSCs were able to migrate normally towards a chemokine gradient. In post-natal bmi-1⫺/⫺ mice, the number of HSCs was markedly reduced. Both fetal liver and bone marrow cells obtained from bmi-1⫺/⫺ mice were able to contribute only transiently to hematopoiesis when transplanted into lethally irradiated recipients. There was no detectable selfrenewal of adult hematopoietic stem cells, indicating a cell autonomous defect in bmi-1⫺/⫺ mice. A gene expression analysis revealed that the expression of stem cell associated genes, cell survival genes, transcription factors, and genes modulating proliferation including p16INK4A and p19ARF was altered in bone marrow cells of the bmi-1 null mice. Expression of p16INK4A and p19ARF in normal HSCs resulted in proliferative arrest and p53-dependent cell death, respectively. This study indicates that expression of bmi-1 is essential for the generation of self-renewing adult hematopoietic stem cells.

137 P-SELECTIN DELAYS THE IN VITRO DIFFERENTIATION OF MURINE LINⴚ Sca-1ⴙ c-KITⴙ CELLS WHEREAS E-SELECTIN ACCELERATES THEIR DIFFERENTIATION Eto T.1, Winkler I.1, Purton L.1, Simmons P.1, Levesque J.P.*,1 1 Peter MacCallum Cancer Institute, Melbourne, Australia We have previously shown that adhesion of human haemopoietic progenitor cells (HPC) to P-selectin mediated by PSGL-1/ CD162, a transmembrane receptor for both P- and E-selectins, results in HPC growth inhibition in vitro. We now report the effects of P-/E-selectin-mediated adhesion on the growth and differentiation of murine HPC. Bone marrow HPC were isolated according to their expression of c-KIT and PSGL-1 and transplanted

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into lethally irradiated congenic recipients. All long-term competitive repopulating cells (LTCRC) were comprised in the lineage⫺ c-KITbright PSGL-1medium fraction, indicating a potential role for Pand E-selectin on these primitive stem cells. Cultures of lin⫺ Sca1⫹ c-KIT⫹ cells (LSK) were then established on immobilised P-/ E-selectin for 4 weeks in the presence of IL-11, SCF, Flt-3L and TPO. During the first two weeks of culture, cell proliferation was significantly inhibited compared to control cultures on immobilised BSA. Phenotypic analyses revealed that after 3 weeks of culture on E-selectin, 80% of the cells expressed the granulocyte maturation marker, Gr-1, and produced minimal numbers of CFC and CFU-S. In marked contrast, 60% of cells cultured on P-selectin were Gr-1⫺ Sca-1⫹, and had increased numbers of CFC and CFUS above both input numbers (uncultured LSK) and LSK cultured on BSA. Despite expansion of CFC and CFU-S on P-selectin, neither P-, E-selectin nor BSA increased or maintained LTCRC. These results suggest that while E-selectin accelerates the differentiation of HPC, P-selectin delays their differentiation, allowing exvivo expansion of CFC and CFU-S.

138 THE CD34 FAMILY: KEY REGULATORS OF ADHESION AND CELL–CELL CONTACT Mcnagny K.*,1, Nielsen J.1, Drew E.1, Winters M.1, Tan P.1, Lam M.1, Chelliah S.1, Merkens H.1, Doyonnas R.1 1 University of British Columbia, Vancouver, BC, Canada CD34 is the founding member of a family of three cell surface sialomucins: CD34, Podocalyxin and Endoglycan. Although these molecules act as glycosylation-dependent selectin ligands when expressed by high endothelial venules (HEV), their function on all other vasculature and hematopoietic lineage cells is unknown. Here we have used gain- and loss-of-function experiments to evaluate the role of these molecules in three different hematopoietic settings: mast cell maturation, anemia-induced erythropoiesis, and hematopoietic progenitor cell migration. In all three scenarios, we have found that these molecules play a dynamic role in blocking inappropriate cell adhesion. Thus, CD34-deficient mast cells show increased adhesiveness and a propensity to aggregate in vitro and this effect is fully reversible by the re-expression of CD34. Likewise, we have found that Podocalyxin is upregulated during anemia-induced erythropoiesis and that loss of this molecule leads to impaired erythroid progenitor homing due to excessive adhesion. Finally, analysis of hematopoietic progenitors lacking CD34, Podocalyxin or both molecules suggests that these molecules are required for short-term homing to bone marrow in vivo. In vitro analyses of these progenitors suggest that this impaired homing reflects a gene–dose-dependent defect in the ability of these cells to cross endothelial barriers. Our results suggest that, in contrast to their well-documented role in selectin-dependent adhesion to HEV, under virtually all other circumstances CD34-type proteins act as molecular “Teflon” to inhibit inappropriate cell adhesion and increase invasiveness. In addition, our data suggest that this anti-adhesive effect is tightly regulated by cell activation and the spatial organization of CD34-type proteins in the plasma membrane.

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139 IMMOBILIZATION OF NOTCH LIGAND, JAGGED1, IS REQUIRED FOR RENEWAL OF PRIMITIVE HAEMATOPOIETIC PROGENITOR CELLS IN VITRO Vas V.*,1, Paloczi K.1, Szilagyi L.2, Hajdu M.1, Uher F.1 1 National Health Center, Budapest, Hungary, 2Eotvos Lorand University of Science, Budapest, Hungary The expression of Jagged1 in BM suggests that they may influence haematopoietic stem cells (HSC), inhibiting differentiation programs, permitting self-renewal. To study the effect of Jagged1 on HSC we used recombinant soluble-Jagged1 extracellulardomain (JG1ECD) and JG1ECD bound to Sepharose-4B-particles as a model for Jagged1-presenting cells. We applied soft-gel cultures to evaluate progenitor cell numbers and stroma-dependent cobblestonearea-forming cell (CAFC) assays to estimate the HSC hierarchy from BDF1 mice. Using BM suspension, immobilized and nonimmobilized JG1ECD — along with growth factors IL-3, CSFs — support the proliferation of progenitors and increase the number of CFU-GM, BFU-E and CFU-GEMM in methylcellulose culture. Similarly, in the first two weeks soluble-JG1ECD (5–10µg/ml) increases the colony number in CAFC culture. But after the third week soluble-JG1ECD inhibits colony-formation in this concentration. Only low concentrations of soluble-JG1ECD (50ng/ml) stimulate the proliferation of primitive HSC and enhance the number of CAFC at 28—35 days. In another experimental setting we preincubated lineage-negative cells with GFs (SCF, Flt3-ligand, TPO) and sepharose-bound or soluble-JG1ECD before placing them onto irradiated stroma. We found that the multivalent JG1ECD increased the number almost ten times of primitive HSC — calculated from the 35-day CAFC — as much as soluble-JG1ECD. Notch signaling is one of the potential cell-fate decision regulation pathways of adult HSC. In presence of GFs multivalent insoluble-Jagged1 stimulates the proliferation of HSC and is able to enlarge HSC compartment without worsening their quality based on CAFC-assay. The soluble form inhibits these self-maintenance effects in the same situation. Jagged1 is a weak GF of heamatopoietic progenitors.

140 DRAMATIC CELL CYCLE PROGENITOR EXPRESSION SHIFTS THROUGH CELL CYCLE Colvin G.*,1, Dooner M.1, Lambert J.2, Demers D.1, Abedi M.1, Quesenberry P.1 1 Roger Williams Medical Center, Providence, RI, USA, 2 Division of Hematology, University Hospital, Geneva, Switzerland Work on cell cycle related hematopoietic stem cell (HSC) function has indicated that HSC are in a continuum, with constant reversible phenotypic variation through cycle. We have previously shown that cytokine-cultured synchronized HSC show a fluctuating engraftment phenotype tightly and inversely linked to marked and reversible fluctuations in 7-factor-responsive, high-proliferativepotential colony-forming-cells (HPP-CFC) during first cycle transit, termed, progenitor/stem cell inversions. We have evaluated progenitor numbers through G1 and the differentiation capacity of purified progenitors through cycle. B6.SJL marrow cultured in FLT3-ligand, thrombopoietin and steel-factor (FTS) showed

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marked fluctuations in total HPP-CFC during the G1 phase of cycle. HPP-CFC was at 35% of input at 2 hours and returned to 96% of input values after 8-hours of cytokine-culture. There was a second nadir (17% of input) at 10-hours. Differentiation studies of purified progenitors [lineagenegativerhodaminelowHoeschtlow(LRH) cells] were performed. LRH cells were primarily cultured in FTS for cycle initiation from resting G0 state and then serially sub-cultured (1,000 cells/vessel) in inductive differentiation cocktails prior to cell division. GM-CSF and G-CSF were used together at three different log-dilutions; steel-factor was present in all cultures at 50ng/ml. Marked variations in differentiated cell production were seen with the first cell cycle transit. Megakaryocyte differentiation was amplified from 3.5 × 103 cells at G0 to 9.1 × 104 cells in mid-S phase. Proliferative granulocyte differentiation was amplified from 6.0 × 104 cells at G0 to 2.4 × 105 cells in mid-S phase. Non-proliferating granulocyte differentiation was amplified from 8.2 × 104 cells at G0 to 4.6 × 105 cells in late-S phase. These data further support a flexible system for hematopoietic regulation in which multiple different outcomes could occur dependent on cell cycle phase and specific microenvironmental influences. Early primitive marrow stem/progenitor cells represent a continuum of reversible phenotypic shifts as opposed to a hierarchy, with continuous change in a reversible fashion.

141 TWO DISTINCT POPULATIONS OF FETAL LIVER CELLS ARE REQUIRED FOR EXPANSION OF IMMATURE HEMATOPOIETIC PROGENITORS IN VITRO Takeuchi M.*,1, Kasahara D.1, Sekiguchi T.1, Minehata K.1, Miyajima A.1 1 IMCB, The University of Tokyo, Tokyo, Japan During mammalian development, definitive hematopoietic stem cells (HSC) arise in the aorta-gonad-mesonephros (AGM) region and then colonize the fetal liver (FL). In late gestation, FL is the major hematopoietic site where HSCs are amplified and mature hematopoietic cells are massively produced. To investigate the mechanism of FL hematopoiesis, we previously established a co-culture system between AGM-originated HSCs and the FL non-hematopoietic cells. In this system, FL stromal cells support expansion of HSCs and production of mature as well as immature hematopoietic cells. Therefore this co-culture system can be used as a model system to dissect FL hematopoiesis. FL is composed of various types of cells that include immature hepatocytes, endothelial cells and hematopoietic cells. However, due to lack of appropriate surface antigens to fractionate FL cells, little is known about the cells responsible for hematopoietic supporting activity in FL. Recently, we found that delta-like (Dlk) is expressed exclusively on immature fetal hepatocytes. We also found that CD31 is expressed in the non-hematopoietic Dlk- cells in FL. To examine hematopoietic supporting activity of Dlk⫹ immature hepatocytes and CD31⫹ cells, we isolated Dlk⫹ cells from FL and co-cultured them with AGM-HSCs. The numbers of hematopoietic cells as well as CFU-C generated from AGMHSCs in co-culture with Dlk⫹ cells were comparable to those with unfractionated FL cells. However, the number of immature multipotent colonies (CFU-Mix) was significantly reduced in co-culture with Dlk⫹ cells. Interestingly, the combination of CD31⫹ cells

and Dlk⫹ cells supported the generation of CFU-Mix to the level comparable to whole FL cells. These findings indicate that both fetal hepatocytes and endothelial cells are required for expansion of multi-potent progenitors from AGM-HSCs.

142 HISTONE DEACETYLASE INHIBITORS ARE STRONG INDUCERS OF GAMMA GLOBIN GENE EXPRESSION Cao H.*,1, Stamatoyannopoulos G.1, Jung M.2 1 University of Washington, Seattle, WA, USA, 2 Wilhelm-Universita¨t, Mu¨nster, Germany We investigated the induction of human γ globin gene activity by three classes of histone deacetylase inhibitors: amide analogues of Trichostatin A; hydroxamic acid analogues of Trapoxin; and Scriptaid and its analogues. The screening consisted of measuring the effects of these compounds on human globin gene promoter expression in GM979 cells stably transfected with a construct containing a γ promoter linked to firefly luciferase and a β promoter linked to renilla luciferase. Compounds that increased the γ/γ⫹β luciferase ratio were further investigated by measuring their effects on the level of γ mRNA and on the frequency of fetal hemoglobin containing erythroblasts in human BFUe cultures. Compounds belonging to all three classes induced γ gene expression in low micromolar concentrations. These results indicate that further investigation of HDAC inhibitors may lead to the discovery of new clinically useful inducers of fetal hemoglobin. The induction of γ gene expression by HDAC inhibitors was not uniform and compounds which displayed very similar degrees of inhibition of the HDAC activity differed strikingly on their effects on γ gene promoter activity, or failed to induce the γ gene promoter. The lack of correlation between HDAC inhibition and γ gene induction raise the possibility that compounds which fail to induce γ gene expression inhibit HDACs which fail to interact with the γ gene promoter.

143 LOW-FREQUENCY OF CELLS WITH THE CD34ⴙ/ CD45ⴚ PHENOTYPE IN APHERESIS SAMPLES OBTAINED FROM PATIENTS WITH A VARIETY OF CANCERS Chabannon C.*,1, Alario T.1, Bechlian D.1, Imbert A.M.1, Calmels B.1 1 Institut Paoli-Calmettes, Marseille, France The presence of activated circulating endothelial progenitors was recently reported in cancer patients; in addition to other markers, these progenitors express the “stem cell antigen” CD34, but lack the pan leukocyte membrane phosphatase CD45. Because the presence of these cells bear important implications for the physiopathology of cancers, we screened apheresis samples obtained from cancer patients who were candidates for high-dose chemotherapy with autologous blood transplantation, for the presence of cells with the CD34⫹/CD45⫺ phenotype. Ninety-six samples from 46 patients with a variety of hematological and non-hematological malignant diseases were studied with a FITC-conjugated anti CD45 murine MoAb and a PE-conjugated anti CD34 MoAb. Forty-two additional samples from 22 NHL patients and 7 patients with breast

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cancer were studied with an APC-conjugated anti CD45 MoAb and a PerCP-conjugated anti CD34 MoAb. In the first series, the percentage of CD34⫹/CD45⫺ cells was below 0.05% (0–0.05%), whereas the average percentage of CD34⫹ cells was 1.49 ⫾ 1.52% (0.09–8.5%). In the second series, the average percentage of CD34⫹/CD45⫺ cells was 0.03 ⫾ 0.1% (0–0.66%), while the average percentage of CD34⫹ cells was 1.25 ⫾ 1.44% (0.1–5.8%); there was no relation between the percentages of total CD34⫹ cells and of CD34⫹/CD45⫺ cells. CD34⫹ cells were immuno-selected from 18 apheresis samples; as a control, CD34⫹ cells were immunoselected from 9 cord blood samples. The average percentage of CD45⫺ cells within the CD34⫹ cell subset was 0.08 ⫾ 0.03% for apheresis samples, as compared with 1.55 ⫾ 0.37% for cord blood samples. The frequency of CD34⫹/CD45⫺ cells in apheresis samples obtained from cancer patients undergoing mobilization with chemotherapy and/or rhG-CSF is low, and phenotypic detection of this rare population is difficult. The mobilization regimen may contribute to lowering the frequency of this population.

144 ENHANCED HEMOPOIETIC RECOVERY FOLLOWING TRANSPLANTATION WITH EX VIVO EXPANDED MOBILIZED BLOOD CD34ⴙ CELLS Haylock D.*,1, Prince H.1, Whitty G.1, Wall D.1, Barber L.1, Mints-Kotowska L.1, Simmons P.1 1 Peter MacCallum Cancer Cente, Melbourne, Australia This study evaluated the efficacy of ex vivo expanded, CD34⫹-selected peripheral blood progenitor cells (PBPC) to support repetitive cycles of high-dose cyclophosphamide (4gm/m2), thiotepa (300mg/m2), and paclitaxel (175mg/m2) (CTP) for treatment of metastatic breast cancer. The protocol involved 3 cohorts, each consisting of 3 patients with each receiving 3 cycles of CTP supported by PBPC with or without ex vivo expanded CD34⫹ cells. Apheresis collections were either cryopreserved as an unmanipulated product or as an Isolex-300i selected CD34⫹ fraction. Each CTP cycle was supported by transplantation with unmanipulated PBPC and/or ex vivo expanded (EXE) CD34⫹ cells. CD34⫹ cells were cultured for 12 days in stericell flasks containing X VIVO10 media supplemented with 0.5% human serum albumin, G-CSF, SCF and MGDF (each at 100ng/ml) in GMP conditions. Infusion of EXE cells (5.82 × 109 ⫾ 3.23, mean ⫾ SD, n ⫽ 21) resulted in fewer days with ANC ⬍0.1 × 109/L (2.0 ⫾ 1.3 vs 4.5 ⫾ 1.6, mean ⫾ SD, p ⫽ 0.0056; expanded vs unmanipulated PBPC), faster neutrophil recovery and fewer episodes of febrile neutropenia. Regarding the impact on platelets (plt), the ex vivo expanded product contained 15.5 ⫾ 8.75% (mean ⫾ SD) megakaryocytic cells (CD61⫹CD42a⫹) and CTP cycles supported by EXE cells (n ⫽ 21) required fewer plt transfusions with 29% of cycles requiring no plt support (cf to 0% of the 6 unmanipulated cycles and 0% in a historical control group, n ⫽ 124). Furthermore, time to last plt transfusion (7d v 9d; p ⫽ 0.036) and time to spontaneous platelet recovery (8d v 12d; p ⫽ 0.043) was improved. Number of days to platelets to ⬎20 × 109/L was no different (med ⫽ 12d, p ⫽ 0.6).

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145 RANDOMIZED PHASE II STUDY OF MAINTENANCE CHEMOTHERAPY VERSUS HIGH-DOSE CHEMOTHERAPY WITH AUTOLOGOUS PERIPHERAL BLOOD STEM CELL TRANSPLANTATION AS POSTREMISSION THERAPY OF AML Harada M.*,1, Imajo K.1, Shinagawa K.1, Gondo H.1, Kawano F.1, Ogawa M.1, Takaku F.1 1 Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan To evaluate the efficacy of high-dose chemotherapy (HDC) supported by autologous peripheral blood stem cell transplantation (PBSCT) as a postremission therapy of acute myelogenous leukemia (AML), we conducted a randomized phase II study of conventional maintenance chemotherapy versus HDC with autologous PBSCT for treatment of patients with AML in first complete remission (CR). During 1996 and 2002, 123 patients with AML in first CR were enrolled to receive either 6 courses of maintenance chemotherapy or HDC consisting of busulfan, etoposide, and AraC with G-CSF-priming plus autologous PBSCT. Both groups of patients were compatible in regard with age and FAB classification. Of 42 patients of the chemotherapy group, 7 patients were excluded because of the complications and disease conditions. Fourteen of 44 patients in the PBSCT group were also excluded from the analysis for some reasons. At a median follow up of 30 (4–56) months, continuous CR was obtained in 18 patients (51%), while relapse and non-relapse mortality were observed in 15 (43%) and 2 (6%) patients, respectively in a group of chemotherapy. In a group of PBSCT, continuous CR, relapse and non-relapse mortality were observed in 16 (53%), 10 (33%), and 4 (13%) patients, respectively. Three-year relapse-free survival was 38% in the chemotherapy group and 60% in the PBSCT group (N.S.). Although a relapse rate is lower in a PBSCT group than in a chemotherapy group, a role of HDC with autologous PBSCT as a postremission therapy has not evaluated at this time. The final analysis will be performed in April at a median follow up of 42 (15–71) months.

146 MARROW VERSUS PERIPHERAL BLOOD FOR GENO-IDENTICAL ALLOGENEIC STEM CELL TRANSPLANTATION IN ACUTE MYELOCYTIC LEUKAEMIA: INFLUENCE OF THE DOSE AND THE SOURCE OF STEM CELLS; BETTER OUTCOME WITH RICH MARROW N.C. Gorin, M. Labopin, F. Frassoni, on behalf of the Acute Leukaemia Working Party of the European Group for Blood and Marrow Transplantation (EBMT) Several studies have compared bone marrow (BM) and peripheral blood (PB) as sources of stem cells in allografted patients, but the dose of stem cells infused has not been taken into account especially for marrow. We compared retrospectively on the ALWP/ EBMT registry the outcome of patients with Acute Myelocytic Leukaemia (AML) allografted in first remission (CR1) using marrow or PB as a source of stem cells, focusing on the dose of

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stem cells infused.We studied 881 patients with AML, older than 16 years of age, who received a non T depleted allogeneic bone marrow (n ⫽ 515) or mobilised peripheral blood (n ⫽ 366) standard transplant, in CR1, from an HLA identical sibling, over a five year period from January 1994 (date of the first allogeneic PB transplant) to January 2001. Multivariate analyses were adjusted on centre (since a centre effect had been observed in a previous EBMT study). The bone marrow cell dose ranged from 0.17 to 29 × 108/kg recipient weight with a median of 2.7 x108/Kg. The peripheral blood cell dose ranged from 0.02 to 77 × 108/kg with a median of 9.3 × 108/kg. The median dose for patients receiving BM (2.7 × 108/kg) gave the greatest discrimination for all endpoints studied. In multivariate analyses, high dose BM compared to PB, was associated with lower TRM (RR ⫽ 0.61 ; 95% CI 0.39– 0.98; p ⫽ 0.04), better Leukemia Free Survival (RR ⫽ 0.65; 95% CI 0.46–0.91; p ⫽ 0.013), and better overall survival (RR ⫽ 0.64 ; 95% CI 0.44–0.92; p ⫽ 0.016). No significant association was found for RI. The present study, which concerns patients with AML allografted in first remission, indicates a better outcome with marrow as compared to PB, when the dose of marrow infused is rich.

147 PERIPHERAL T CELL LEVELS CORRELATE WITH OUTCOME OF PATIENTS UNDERGOING AUTOLOGOUS PERIPHERAL BLOOD PROGENITOR CELL (PBPC) TRANSPLANT Rosinski S.1, Nieto Y.1, Shpall E.2, Clough N.1, Russell P.1, Blunk B.1, Mcniece I.*,3 1 University of Colorado HSC, Denver, CO, USA, 2MD Anderson, Houston, TX, USA, 3Johns Hopkins Oncology Center, Baltimore, MD, USA The immune status of cancer patients, both pre- and posttransplant, may affect the proliferation of tumor cells. The existence of anti-tumor T cells has been demonstrated in the allogeneic transplant setting. However, its importance in the autologous setting remains unclear. We prospectively evaluated peripheral T cells in sixty autologous PBPC transplant recipients. Fifty percent of patients demonstrated a deficiency in CD4 T cells prior to transplant and CD4 T cells remained below pre-transplant levels in the majority of patients for six months or longer post-transplant. Pretransplant levels of CD4 T cells and CD4⫹CD45RA-CD62Lmemory T cells correlated with their post-transplant levels (p ⫽ 0.007 and p ⫽ 0.04, respectively). Pre-transplant CD4 T cell levels correlated with relapse free survival (RFS) and overall survival (p ⫽ 0.003 and p ⫽ 0.008, respectively). Pre-transplant CD4⫹CD45RA⫺CD62L⫺ memory T cells correlated with RFS in the overall patient group (p ⫽ 0.01) and patients with breast cancer (BC) (p ⫽ 0.01). Additionally, post-transplant CD4 counts on day ⫹30 correlated with RFS (p ⫽ 0.03). Multivariate analysis including, T cell levels, tumor type, stage of disease, responsiveness to chemotherapy, and HER2/neu status for breast cancer patients, demonstrated that pre-transplant memory T cells subsets (CD4⫹CD45RA⫺CD62L⫺ and CD4⫹CD45RO⫹) predicted an independent value for RFS (p ⫽ 0.04 and 0.04, respectively). These data suggest that the immune status of patients prior to transplant impacts the long-term outcome, and that memory CD4 T cell subsets play a critical role in immune control of minimal residual disease.

148 AUTOLOGOUS TRANSPLANTIONS VERSUS CONVENTIONAL CHEMOTHERAPY IN CHRONIC LYMPHOCYTIC LEUKEMIA (CLL): A EUROPEAN BLOOD AND MARROW TRANSPLANT (EBMT) AND FRENCH CLL COOPERATIVE GROUP STUDY M. Michallet1, S. Chevret2, R. Brand1, A. van Biezen1, V. Le´vy2, P. Dreger1, D. Milligan1, A.S. Michallet, E. Kimby1, J. Esteve1, G. Bandini1, M. Leporrier2, B. Cazin2, N. Boudjerra2, P. Feugier2, B. Desablens2, M.J. Rapp2, J. Jaubert2, B. Autrand2, M. Divine2, B. Dreyfus2, K. Maloum2, P. Travade2, J.L. Binet2, G. Dighiero2, D. Niederwieser1 1 French CLL Cooperative Group, Paris, France, 2EBMT Registry, Leiden, The Netherlands We performed a retrospective cohort study using a Proportional Hazard (PH) Cox regression model to assess the potential benefit of AutoT in terms of overall survival from first line response, adjusting for age, sex, time from CLL diagnosis and response to first-line therapy. Secondly, a case-control analysis has been performed on the sub-samples of paired data on age (⬍, ⱖ 50 years), sex, and first line response, using a PH Cox model with frailty. Among 743 patients reported to the EBMT registry, 621 patients fulfilled the inclusion criteria. Of the 937 patients enrolled in the CLL90 protocol, 630 fulfilled the inclusion criteria. Concerning the whole cohort (n ⫽ 1251) we did not observe any significant difference of survival between the 2 groups (HR ⫽ 1.15, 95%CI:0.88–1.51) with no significant impact of age and previous chemotherapy (CAP, CHOP or FAMP). Considering timedependent effects, we demonstrated a significant over-mortality of AutoT over the first 24 months (HR ⫽ 1.37, 95%CI:0.99–1.89) without difference in survival between AutoT and Chemo after 24 months. A total of 493 cases and controls could be paired and we demonstrated also a significant over-mortality of AutoT over the first 24 months (HR ⫽ 1.49, 95%CI: 1.06–2.09), without difference after 24 months, but when we restricted the analysis to one-line therapy paired comparison (n ⫽ 81 AutoT paired to 81 Chemo patients) we demonstrated a significant over-mortality of AutoT over the first 24 months (HR ⫽ 1.72, 95%CI: 1.34–2.20) but also a significant benefit of AutoT after 24 months (HR ⫽ 0.54, 95%CI: 0.36–0.81). Because of the retrospective aspect of this study, it remains until now difficult to estimate the real impact of AutoT on CLL survival. In consequence, prospective randomized clinical trials are mandatory, in first line CLL patients, either in response or not with controlling for base line differences in patient populations and without introducing any selection bias.

149 OUTCOMES OF HLA IDENTICAL SIBLINGS ARE COMPARABLE TO UNRELATED BONE MARROW TANSPLANTS IN ADULTS WITH ACUTE LYMPHOBLASTIC LEUKEMIA Rocha V.*,1, Labopin M.1, Ruutu T.1, Basara N.1, Jouet J.P.1, Gluckman E.1, Ringden O.1, Arcese W.1, Polge E.1, Gorin N.C.1, Hoelzer D.1, Frassoni F.1 1 EBMT, Institut des Cordeliers, Paris, France In order to compare HLA identical siblings bone marrow transplants (genoid-BMT) with HLA matched unrelated BMT (MUD)

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we analysed patients with ALL older than 16 years receiving a non-manipulated BMT from 1994 to 2001. We studied 623 patients, 428 received a genoid-BMT and 195 a MUD. Univariable and multivariable analyses, using relapse and death as competing event, were performed to study risk factors for transplant-related mortality (TRM) and relapse incidence (RI). Event-free survival (EFS) and other times to event were estimated using Cox models after adjustment for patient-, disease- and transplant-related factors that could potentially affect outcomes. The main statistical differences between the two groups were: MUD were transplanted more frequently in ⱖCR3 or relapse compared to genoid-BMT (38% vs 18%, p ⬍ 0.01) and with Philadelphia positive (Phi⫹) chromosome (35% vs 24%, p ⫽ 0.004), respectively. Unrelated donors were older than HLA identical-sibs (p ⬍ 0.0001). Other characteristics such as WBC, patients age and gender, number of nucleated cell dose, GvHD prophylaxis were not statistically different between the two groups. Acute GvHD (ⱖII) was observed in 39% of genoid-BMT and 41% of MUD (p ⫽ 0.83). After statistical adjustment for patient, disease and transplant related characteristics, there was a trend of higher TRM (p ⫽ 0.08) and a lower incidence of relapse in MUD (p ⫽ 0.05) compared to genoid-BMT. Therefore LFS was similar between the two groups (p ⫽ 0.82). Estimated 2y-LFS non adjusted for patients with ALL Phi+ transplanted in CR2 or more was 8 ⫾ 5% for genoid-BMT (n ⫽ 31) compared to 30 ⫾ 8% for MUD (n ⫽ 34). In conclusion, relapse incidence is decreased after MUD compared to genoid-BMT probably due to a stronger GvL effect after unrelated transplants. LFS is similar between the two types of transplants, however it is apparently better for patients with ALL ph⫹ transplanted in CR2 or more. Thus the indications for a MUD in adults with ALL should be the same as the indications for a genoid-BMT.

150 AUTOLOGOUS BONE MARROW TRANSPLANT IN A PATIENT WITH SICKLE CELL DISEASE AND DIFFUSE LARGE B-CELL LYMPHOMA Onitilo A.*,1, Lazarchick J.2, Brunson C.2, Frei-Lahr D.2, Stuart R.2 1,2 Hem-Onc Division/Medical University of South Carolina, Charleston, SC, USA As the life expectancy of patients with homozygous sickle cell anemia (SCA) improves, SCA care providers are confronted with diseases of the adult SCA population rarely seen before. We report here a 40-year-old woman with SCA who developed diffuse large B-cell non-Hodgkin’s lymphoma (NHL) that was treated with 8 cycles of chemotherapy consisting of cyclophosphamide, doxorubicin, vincristine, prednisone, and etoposide (CHOPE), without complete remission. She subsequently underwent high-dose chemotherapy with autologous bone marrow transplantation (BMT), and remains in remission 12 years post-transplant. To our knowledge, this case study is the first reported case of autologous BMT in a patient with SCA. This poster presentation includes the rationale for our treatment decision, particularly the use of granulocytecolony stimulating factor (G-CSF) in patients with SCA pre- and post- transplantation. Our patient illustrates that SCA in itself does not preclude autologous stem cell transplantation for lymphoma in selected patients; of course more patients need to be studied. While a cure

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for sickle cell disease is still elusive, studies on ways of treating the other chronic and malignant diseases of the adult patient with sickle cell disease need to be conducted. This presentation should encourage others to consider autologous BMT in adults with SCA where it represents a life-saving therapy for malignant diseases.

151 HIGH-PERFORMANCE MULTI-COLOUR FLUORESCENT ANALYSIS FOR HAEMATOPOIETIC CHIMAERISM AFTER STEM CELL TRANSPLANTATION Hattori H.*,1, Oda S.1, Yoshida M.1, Miyashita K.1, Yamada Y.1, Uike N.1, Nagatoshi Y.1, Matsuzaki A.2, Hara T.2, Okamura J.1 1 National Kyushu Cancer Center, Fukuoka, Japan, 2Department of Pediatrics, Kyushu University, Fukuoka, Japan We have developed a new system for monitering haematopoietic chimaerism after stem cell transplantation where multi-colour fluorescent PCR for STRs is used. The advantages of this system are (1) originally designed primer sequences, (2) quantitativity highly comparable with conventional assay systems and (3) high throughput and high resolution derived from the tri-colour fluorescence technique, all of which have inevitably increased the efficacy and the cost-effectiveness of the assay for this purpose. Four STR markers (D5S818, D7S820, D13S317, FGA) were selected, from the mass genetic point of view. A set of three different fluorescence (NED, 6-FAM, HEX)-labelled primers was originally designed on each STR marker. These primer sets clearly discriminated all of 73 recipient/donor pairs tested. The linearity of detection was assessed using serial dilutions of mixed DNA and cell references. The relationship between signal and input was highly linear (R2 ⫽ 0.99) in both experiments. These results are also highly comparable with those obtained using a FISH assay done accurately (R2 ⫽ 0.99). Finally, in this system, donor, recipient and post-transplantation samples are subjected to electrophoresis at the same time, which increases the efficacy of the assay and is timesaving. Furthermore, combination of three different colours provides a platform where the chimaeric state in a given sample is highly intelligible. We show several typical instances that have been clearly demonstrated as a clinically different status using this system. Application of such systems may facilitate an accurate and immediate assessment of the haematopoietic chimaerism in patients who underwent stem cell transplantation.

152 GENOIDENTICAL PBSCT WITH REDUCED INTENSITY CONDITIONING COMPARED TO AUTOLOGOUS PBSCT FOR OLD PATIENTS WITH ACUTE LEUKEMIA: AN EBMT MATCHED PAIR ANALYSIS Rocha V.*,1, Labopin M.1, Boiron J.M.1, Ferrant A.1, Garban F.1, Polge E.1, Gluckman E.1, Gorin N.C.1, Frassoni F.1 In order to compare outcomes of autologous-PBSCT (autoPBSCT) and allogeneic genoidentical PSCT using reduced intensity conditioning regimen (reduced-PBSCT) in old patients with AL

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(⬎50 y), we conducted a matched pair analysis. Patients receiving TBI in the group of reduced-PBSCT were not included. Reduced conditioning regimen consisted more frequently in the presence of Fludarabine and low dose Busulfan. Thus, 78 patients with AL receiving reduced-PBSCT from 01/96 to 01/2002 were matched for age, diagnosis, and status of disease at transplant with 156 auto-PBSCT. The median follow-up time was 10 months. For the whole group of patients (n ⫽ 234) the median age was 57 y (5069), 85% were transplanted for AML and 15% for ALL, in first CR (38%), CR2 (26%) and advanced disease (36%). There were not statistically differences between the two groups regarding gender, interval from diagnosis to transplant, AML FAB classification. Hematopoietic recovery was 90% in reduced-PBSCT and 92% in auto-PBSCT (p ⫽ 0.63). Incidence of acute GVHD (ⱖII) was 24% in the reduced-PBSCT. One year -TRM, -RI and -LFS were respectively in the reduced-PBSCT compared to the auto-PBSCT: 23 ⫾ 6% versus 17 ⫾ 5% (p ⫽ 0.19), 36 ⫾ 7% vs 58 ⫾ 5% (p ⫽ 0.04); and 50 ⫾ 7% vs 35 ⫾ 5% (p ⫽ 0.28). In patients transplanted in first CR, 1y-TRM was 23 ⫾ 4% in the reduced-PBSCT and 5 ⫾ 3% in the auto-PBCST (p ⫽ 0.007) with 1y-RI of 25 ⫾ 10% and 44 ⫾ 8% (p ⫽ 0.23) respectively. However for patients in second CR, 1y-TRM was 12 ⫾ 8% in the reduced-PBSCT and 23 ⫾ 9% in the auto-PBCST (p ⫽ 0.32) with 1y-RI of 14 ⫾ 9% and 60 ⫾ 10% (p ⫽ 0.02) and 1y-LFS of 76 ⫾ 10% and 31 ⫾ 9% (p ⫽ 0.01), respectively. For patients transplanted in advanced phase no statistically difference of outcomes were observed between the 2 modalities of transplant. In conclusion reduced-PBSCT is a good option to treat old patients with AL when a HLA identical sibling is available with a comparable TRM of auto-PBSCT and a higher GvL effect.

153 INTERFERON-GAMMA (IFN-GAMMA) NON-MYELOABLATIVE CONDITIONING REDUCES STEM CELL REPOPULATING ABILITY AND ENHANCES ENGRAFTMENT IN FANCONI ANEMIA TYPE C FANCC ⴚ/ⴚ MICE Li X.*,1, Yang Y.1, Yuan J.1, Clapp D.W.1 1 Indiana University School of Medicine, Indianapolis, IN, USA Fanconi anemia (FA) is a heterogeneous disorder characterized by bone marrow (BM) failure, cancer susceptibility, and hypersensitivity to bifunctional alkylating agents. Nearly 90% of patients will ultimately die of hematologic complications. Treatment with genetically corrected stem cells could provide a cure for these individuals, however genotoxic conditioning regimens may predispose patients to the development of secondary malignancies. We and Rathbun et al have previously shown that Fancc ⫺/⫺ myeloid progenitors have increased apoptosis following treatment with IFNgamma in vitro. We hypothesized that IFN-gamma conditioning may be useful as a nongenotoxic conditioning regimen. To test this hypothesis, Fancc ⫺/⫺ mice, originally developed by Buchwald and colleagues, were utilized as a model system. A dosedependent decrease in peripheral blood cell counts and bone marrow cellularity in both Fancc ⫺/⫺ and WT mice. However, the decline in both primitive myeloid progenitors (HPP-CFC) as well as mature hematopoietic progenitors (LPP-CFC) was greatly reduced in Fancc ⫺/⫺ mice. In addition, in competitive repopulating assays, stem cell repopulating ability was profoundly reduced in

Fancc ⫺/⫺ mice following IFN-gamma treatment. To test whether IFN-gamma conditioning is sufficient as a myelopreparative regimen, Syngeneic WT cells that express the CD45.1 antigen were transplanted into Fancc ⫺/⫺ and WT recipients. Donor chimerism following transplantation consistently achieved 25–50% in Fancc ⫺/⫺ recipient mice. Collectively, these data demonstrate that Fancc ⫺/⫺ progenitors and stem cells have increased apoptosis to IFN-gamma in vitro and in vivo and that IFN-gamma conditioning may be a useful nongenotoxic strategy for myelopreparative conditioning.

154 PHASE I STUDY OF BBR-2778 IN BSHAP FOR RELAPSED AGGRESSIVE NON-HODGKIN’S LYMPHOMA (NHL) Levine A.*,1, Fayad L.2, Modiano M.3, Camboni G.4, Cabinallas F.2 1 USC Norris Cancer Center, Los Angeles, CA, USA, 2 MD Anderson Cancer Center, Houston, TX, USA, 3Arizona Clinical Research Center, Tucson, AZ, USA, 4Novuspharma SpA, Milan, Italy ESHAP [etoposide (E), methylprednisolone (S), high-dose cytarabine (HA), cisplatin (P)] is a standard reinduction regimen for transplant-suitable patients with relapsed aggressive NHL. BBR2778 (B) is a novel aza-anthracenedione that is synergistic with P, has no delayed cardiotoxicity in animal models, and has singleagent activity in NHL patients. We substituted B for E to create the BSHAP regimen. B was to be administered on a dose-escalation schedule to determine the dose-limiting toxicity (DLT) and maximum tolerated dose (MTD) of B when combined with SHAP. A secondary objective was to determine efficacy of the combination. B was infused over 1 hour on day 1 every 21 days in combination with fixed doses of S (500 mg/day over 15 minutes IV days 1–5), HA (2000 mg/m2 over 2 hours on day 5), and P (25 mg/m2 over 30 minutes days 1–4 with hydration). Reported are preliminary results with BSHAP in 15 patients with relapsed or refractory aggressive NHL. Six patients received the starting dose of 80 mg/m2; 2 DLTs (grade 3 infection and grade 3 febrile neutropenia) were observed. Another 9 patients were added at the same dose level, with bone marrow stimulatory factors used prophylactically; one additional DLT (grade 1 gingival bleeding with grade 3 thrombocytopenia) was observed. The MTD of B in BSHAP was 80 mg/m2. No clinically significant cardiac events were noted. Encouraging preliminary evidence of efficacy (best response) included 4 CR (2 proceeded to BMT before confirmation), 2 CRu, 3 PR, 5 SD and 1 PD among 15 patients.

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155 AUTOLOGOUS PERIPHERAL BLOOD STEM CELL TRANSPLANTATION WITH GRANULOCYTE-COLONY STIMULATING FACTOR COMBINED PREPARATIVE REGIMEN FOR ACUTE PROMYELOCYTIC LEUKEMIA IN FIRST REMISSION Hayashi S.*,1, Kamimura T.1, Miyamoto T.2, Nagafuji K.2, Taniguchi S.3, Gondo H.2, Harada M.2 1 Harasanshin General Hospital, Fukuoka, Japan, 2Kyushu University, Fukuoka, Japan, 3Hamanomachi Hospital, Fukuoka, Japan We evaluated the safety and therapeutic efficacy of myeloablative therapy following autologous peripheral blood stem cell transplantation (auto-PBSCT) for adult patients with acute promyelocytic leukemia (APL) in first complete remission (CR). Between April 1992 to February 2003, we evaluated 20 consecutive patients with APL in first CR, who received myeloablative therapy following auto-PBSCT. Peripheral blood stem cells (PBSC) were collected during hematopoietic recovery after consolidation chemotherapy of intermediate-dose cytosine arabinoside (AraC). The preparative regimen for all patients consisted of continuous infusion of AraC 100mg/sqm/day on day ⫺12 to ⫺6, busulfan 4mg/kg/day on days ⫺8 to ⫺5, etoposide 20mg/kg/days ⫺4 and ⫺3, AraC 3g/m2/ 12hours on days ⫺3 and ⫺2. Concurrently, granulocyte-colony stimulating factor (G-CSF) was administered on days ⫺14 to ⫺4 to increase chemosensitivity of leukemic cells to the S-phase specific anti-leukemic AraC. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis of PML/RARα were performed to detect minimal residual disease (MRD) of PBSC in eleven patients, and bone marrow cells in four patients who remain CR after autoPBSCT. Twenty patients with median age of 45 (range 16–68) years were enrolled to the study. No engraftment failure was seen. There was no transplant-related mortality. The median follow-up time was 57 (range 9–115) months and all 20 patients are alive without relapse. We demonstrated negative results of MRD in PBSC and BM samples in all patients analyzed. Our result is striking that all of 20 APL patients are surviving without relapse. Auto-PBSCT for APL in first CR is a promising strategy to minimize leukemic relapse. It is a safe procedure as evidenced by no TRM.

156 TRANSFORMATIONS AND CLINICAL IMPORTANCES OF HEMATOPOIETIC CHIMERISM IN NONMYELOABLATIVE ALLOGENEIC STEM CELL TRANSPLANTATION Hui-Sheng Ai, Cun-Hua Zao*, Guo-Mei, Chang-Lin Yu, Dan-Hong Wang, Jian-Hui, Qiao, Qi-Yun Sun, Wan-Jun Sun, Zhang-Shi. Department of Hematology, North Tai Ping Road Hospital, Beijing, China *Tian Jing Research Institute of Hematology, Tian Jing, China Tranditional myeloablative allogeneic stem cell transplantation is always associated with full donor chimerism (FDC) and others. So we studied the Transformations and clinical importances of the

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donor-recipient hematopoietic mixed chimerism in nonmyeloablative allogeneic stem cell transplantation. Nonmyeloablative pretreatment such as CD3 monoclonal antibody, cyclosporine A, cyclophosphamide and cytarabine was used for nonmyeloablative allogeneic stem cell transplantation (NST) in 52 patients with hematological diseases. All the 52 patients passed smoothly the hematopoietic suppression stage and achieved engraftment of the donor cells. There were 15 cases of full donor chimerism, 37 cases of mixed chimerism (MC) (26 cases converted to FDC, 7 cases remained at stable chimeric state and 4 cases developed graft rejection). Of the 52 cases, 8 (15.4%) developed acute GVHD (aGVHD). Of them, 6 belonged to group FDC (6/15, 40%) and 2 belonged to group MC (2/37, 5.4%). The incidence of aGVHD in group FDC was significantly higher than that in group MC (P ⬍ 0.05). Eight of the 52 cases (15.4%) developed chronic GVHD. Of them, 4 belonged to group FDC (4/15.26.6%) and 4 belonged to group MC (4/37.10.8%), with no significant statistical difference between the two groups. All the 52 patients had a smooth transplantation process. Both the minimum values and recovery time of neutrophils and platelet of group FDC, and group MC had no significant difference (P⬎ 0.05). Of the 52 cases, 42(80.3%) still live and 10 (19.7%) died. Of them, 13 surviving patients belonged to group FDC (13/15, 86.7%), 29 surviving patients belonged to group MC (29/37, 78.4%). With nonmyeloablative pretreatment, most patients with hematological diseases can form FDC or MC engraftment. Both FDC and MC can rapidly restore the hematopoiesis of 3 blood series. However, MC has obviously decreased GVHD incidence and mild complications. Most MC patients can convert to FDC and have relatively high survival rate. After NST, the patients with hematological diseases form MC first and then convert gradually to FDC, which not only does not influence the recovery of hematopoiesis, but also obviously alleviates GVHD and does not reduce GVL effect. This may be the ideal mode of engraftment.

157 OUTCOME AFTER REDUCED-INTENSITY STEM CELL TRANSPLANTATION (RIST) VERSUS CONVENTIONAL STEM CELL TRANSPLANTATION (CST) FOR PATIENTS WITH REFRACTORY HEMATOLOGICAL DISORDERS Onishi Y.*,1, Kami M.1, Murashige N.1, Usubuchi N.1, Kishi Y.1, Kimu S.1, Makimoto A.1, Tanosaki R.1, Mineishi S.1, Masuo S.2, Miyakoshi S.3, Takaue Y.1 1 National Cancer Center Hospital, Tokyo, Japan, 2JR Tokyo General Hospital, Tokyo, Japan 3Toranomon Hospital, Tokyo, Japan Most physicians believe that RIST is insufficient in controlling refractory hematological malignancies, and that intensification of preparative regimens is required to improve their prognosis. However, we have little information on efficacy and toxicities of RIST for refractory hematological diseases. We retrospectively analyzed 34 consecutive patients who had received RIST (n ⫽ 12) or CST (n ⫽ 22) for refractory hematological malignancies. Underlying diseases were acute myelogeneous leukemia (n ⫽ 20), acute lymphoblastic leukemia (n ⫽ 6), chronic myelogeneous leukemia (n ⫽ 3), and myelodysplastic syndrome (n ⫽ 5). Although RIST recipients were significantly older than CST recipients (54 vs. 41

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years), the other backgrounds were not different between the two groups. Preparative regimens of RIST were fludarabine-based (n ⫽ 11) or cladribine-based (n ⫽ 1). Those of CST were CY/ TBI (n ⫽ 21) or BU/CY (n ⫽ 1). GVHD prophylaxes of RIST and CST were cyclosporine alone, and combination of cyclosporine and methotrexate, respectively. All of the RIST recipients engrafted, while 3 of the 22 CST recipients died before engraftment. Twenty-one CST and four RIST recipients developed grade II to IV RRT according to the Bearman’s criteria (p ⫽ 0.0002). Grade II to IV acute GVHD occurred in 12 CST and eight RIST recipients. Six CST and one RIST recipients died without relapse within 100 days. Estimated one-year overall survivals in RIST and CST recipients were 65% and 25%, respectively (p ⫽ 0.054). Causes of deaths in 16 CST recipients were relapse (n ⫽ 8), interstitial pneumonitis (IP) (n ⫽ 3), GVHD (n ⫽ 2), organ failure (n ⫽ 2), and bacterial infection (n ⫽ 1). Those in four RIST recipients were relapse (n ⫽ 1), GVHD (n ⫽ 2) and IP (n ⫽ 1). The Cox multivariate model revealed that CST regimens was a independent poor prognostic factor (relative risk, 4.78: 95% confidence interval, 1.38–16.5). The reduced RRT is likely to contribute to the better survival in RIST. Furthermore, the unexpectedly low relapse rates following RIST suggests that it has a strong anti-tumor activity through allogeneic immunity.

or myeloablative regimens (p ⬍ 0.05). The analysis for NK cells disclosed same trend, while the analysis of intracellular cytokines after SEB stimulation did not show any characteristic difference between the regimens. Immune recovery after RIST without ATG was comparable to those after conventional myeloablative transplantation, while this was retarded with the use of ATG.

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Although a proportion of patients with MDS can be cured with allogeneic HSCT, the clinical benefits are not clearly discussed comparing PBSCT and BMT for MDS. From forgoing database, we performed subgroup analysis on outcomes of Japanese patients with MDS (25 PBSCs and 46 BM), who received myeloablative HSCT from an HLA-identical (n ⫽ 65) or one-locus mismatched related donor (n ⫽ 6) between 1999 and 2001. There were 24 cases of refractory anemia (RA), 19 RA with excess blasts (RAEB), 19 RAEB in transformation, and 4 others. Patient status was not significantly different between two groups: RA (37% with PBSCs and 28% with BM, respectively), TBI-based regimen (44% and 59%), median age (45 years and 39.5 years), and GVHD prophylaxis (cyclosporin and short-term methotorexate: 88% and 91%). With a median follow-up of 13 months, the incidence of acute and chronic GVHD was similar in both groups (acute grade II–IV: 36% and 39%, chronic: 73% and 53%, respectively, in PBSC and BM). Clinical outcomes were not significantly different in PBSCs and BM: The 2-year OS 72% and 66%, 2-year EFS 69% and 64%, and TRM 20% and 25%, respectively. Such parameters were similar between PBSCs and BM even in the patients with RA. In a Japanese population with MDS, the clinical outcome is not associated with the use of PBSCs or BM. However, this is a limited retrospective analysis with a relatively short follow-up. These findings should be confirmed in the prospective randomized study that is underway in Japan.

IMMUNE RECONSTITUTION FOLLOWING ALLOGENEIC ENGRAFTMENT: COMPARISON BETWEEN REDUCED-INTENSITY TRANSPLANTATION (RIST) WITH OR WITHOUT ANTI-THYMOCYTE GLOBLIN (ATG), AND CONVENTIONAL MYELOABLATIVE TRANSPLANTATION (CST) Hamaki T.*,1, Heike Y.1, Nakai K.1, Shimada M.1, Harada Y.1, Kishi Y.1, Sakiyama M.1, Chizuka A.1, Onishi M.1, Kami M.1, Tanosaki R.1, Takaue Y.1 1 National Cancer Center Hospital, Tokyo, Japan There has been an assumption that a rapid hematopoietic engraftment associated with RIST may have a concomitant association of earlier recovery of immune function. We assessed the expression of cell surface antigen and intracellular cytokine excretion form peripheral blood mononuclear cells (PBMC) from patients treated with RIST or conventional myeloablative transplantation to evaluate the association between immune reconstitution and clinical outcome following RIST. Thirty-three patients received RIST for hematological or non-hematological malignancies, while 32 patients underwent conventional transplantation. Conditioning for RIST consisted of fludarabine plus low-dose busulfan (n ⫽ 23), or with additional anti-thymocyte globulin (ATG) (n ⫽ 20). The drug used for prophylaxis of graft-versus-host disease (GvHD) was cyclosporin. The regimen for CST was cyclophosphamide combined with total body irradiation or busulfan, and GVHD prophylaxis was cyclosporin and methotrexate. Immunological parameters of the patients who received RIST were compared to those who received CST. The results disclosed a decrease in the absolute number of CD4⫹ cells with a relative increase in CD8⫹ cells, which resulted in an inverted CD4/CD8 counts. Notably, RIST with ATG resulted in lowered CD4/CD8 ratio during the early period after transplant, compared to RIST without ATG

159 RETROSPECTIVE COMPARISON OF ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANTATION (HSCT) WITH BONE MARROW (BM) AND GRANULOCYTE COLONY-STIMULATING FACTORMOBILIZED PERIPHERAL BLOOD PROGENITOR CELLS (PBSCS) IN JAPANESE PATIENTS WITH MYELODYSPLASTIC SYNDROMES (MDS) Hamaki T.*,1, Tanimoto T.2, Ohno Y.3, Nishiwaki K.4, Tanaka J.5, Kobayashi T.6, Takaue Y.1, Harada M.1 1 Japanese Group for Blood and Marrow Transplant, Tokyo, Japan, 2Department of Medicine and Biosystemic Science, Gr, Fukuoka, Japan, 3Kita-kyushu Medical Center, Kita-Kyushu, Japan, 4Kashiwa Hospital, The Jikei University, Kashiwa, Japan, 5Department of Hematology, Hokkaido University, Sapporo, Japan, 6Tsuchiura Kyodo General Hospital, Tsuchiura, Japan

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160 ALLOREACTIVE DONOR LYMPHOCYTE INFUSIONS FOLLOWING ALLOGENEIC HEMATOIPETIC STEM CELL TRANSPLANTATIONS (HSCT) AFTER REDUCED INTENSITY CONDITIONING (RIC) REGIMEN: EFFICACY AND TOXICITY A.S. Michallet1, F. Nicolini1, A. Thiebaut1, C. Audigier-Valette1, J.P. Tremisi, J.P. Bourgeot, X. Thomas1, J. Troncy, C. Vigouroux1, V. Dubois, L. Ge´buhrer2, M. Michallet1 1 Service d’He´matologie, Hoˆpital Edouard Herriot, Lyon, France, 2Laboratoire d’Histocompatibilite´, Etablissement Franc¸ais du Sang, Lyon, France We analyzed the results of DLI given either for persistent or progressive disease (group 1) or for persistent mixed chimerism (MC) (group 2) after RIC HSCT. Before DLI, group 1 concerned 23 patients who presented an evolutive disease [10 patients had a full donor chimerism (FDC) and 13 a MC] and group 2 concerned 8 patients who were in MC and all in disease response. DLI were given either as a bulk dose (BD) [median dose: 5 × 107 CD3+/kg (1 × 106⫺2.64 × 108)] or escalating doses (ED) (from 1 × 106 to 2.65 × 108 CD3⫹/kg). In group 1, 5/13 (36%) MC converted to FDC and we observed 6 disease response (26%): 1 CR in a NHL, 5 PR (3 AML, 2 MM) after 4 BD and 2 ED (2 DLI) with a median delay of 1.5 months. Two patients developed an acute GVHD grade III (2ED) and 1 grade IV (1 BD) and 3 a limited chronic GVHD. At the last follow-up, 3 patients are alive [1 MM in PR (30 m), 1 LAM in CR (24 m) and 1 NHL in CR (39 m)] and 20 patients died (2 from TRM and 18 from disease progression). In group 2, we observed 5 chimerism conversion (62.5 %), 3 after BD and 2 after ED (3 DLI) after a median delay of 2.3 months (1-29.5). Two patients developed an acute GVHD grade III and 1 a chronic limited GVHD. At the last follow-up, 4 patients are alive [3 MM in CR (21, 24 and 33 months) and 1 LAM in relapse (14 months)] and 4 patients died (2 from TRM in CR and 2 from disease progression). This study showed that DLI (1) permit chimerism conversion (2) induce a long-term GVM effect but results are disappointing when DLI were given for a burden malignant disease.

161 COMPARIED WITH THE SIGNIFICANCE BY FLUDARABINE CONDITION PROTOCOLE AND NO FLUDARABINE CONDITION PROTOCOLE IN NONMYLOABALATIVE ALLOGENEIC PERIPHERAL BLOOD STEM CELLS TRANSPLANTATION Mei G.*,1 1 Department of Hematology, Beitaipinglu Hospital, Beijing, China We studied the significance of fludarabine condition protocole and no fludarabine condition protocole in nonmyloabalative allogeneic peripheral blood stem cells transplantation in the treatment hematological diseases. Thirty-six patients with acute leukaemia, severe aplastic anaemia, MDS and myelofibrosis received G-CSF mobilized peripheral blood stem cells from HLA matched donors after Nonmyloabalative regimens. The conditioning therapy consisted of CTX, Ara-C, CSA, CD3 antibodies or anti-thymocyte globuline and fludardabine or no fludarabine. GVHD prophylaxis

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was performed with cyclosporine combined methyotreate (no MMF group n ⫽ 5) or mycophenolate mofetil (MMF group n ⫽ 31). Results: All of the treatment was generally well tolerated and achieved engrafted of the donor cells. In fludarabine group, Engraftment was observed in 87.5% (14/16) patients with complete donor chimerism; Graft failure was 12.5% (2/16). In no fludarabine group, Engraftment was observed in 80% (16/20) patients with complete donor chimerism; Graft failure was 20% (4/20). The incidence of acute GVHD (grade I–IV) was 27.8% patients (10/36) and chronic GVHD was found in 22.2% patients (8/36). In fludarabine group, I–II⬚aGVHD was 37.5%; In no fludarabine group, I–II⬚aGVHD was 20%. In fludarabine group, cGVHD was 12.5%; In no fludarabine group, cGVHD was 30%. The interstitial pneumonia (IP) was observed in 16.7% (6/36) patients; In fludarabine group, IP was 18.7% (3/16); In no fludarabine group, IP was 15% (3/20), Overall actual survival rate 80.5% (29/36) with a median follow-up of 13 months. The fludarabine-based condition protocole (n ⫽ 16) and non fludarabine-based condition protocole (n ⫽ 20) in nonmyeloablative allogeneic hematopoietic stem cell transplantation in the treatment hematological diseases was no significant difference in incidence of GVHD, IP, engraftment and survival.

162 NONMYELOABLATIVE ALLOGENEIC PERIPHERAL BLOOD STEM CELL TRANSPLANTATION FOR THE TREATMENT OF CHRONIC LEUKEMIA: A REPORT OF SEVEN CASES Changlin Y.*,1, Huisheng A.1, Danhong W.1 1 Department of Hematology, Beitaipinglu Hospital, Beijing, China The use of traditional bone marrow transplantation (BMT) or peripheral blood sem cell transplantation (PBSCT) with myeloablative conditioning regimen from a fully matched donor for chronic myeloid leukemia (CML) and chronic lymphoid leukemia is considered a effective curative method. For the purpose of elimination of malignant hematopoietic cells, Immune-mediated graftversus-leukemia (GVL) effects play more important role than myeloablative conditioning in the course of BMT or PBSCT. Reduced-intensity or nonmyeloablative stem cell transplantation (NST) is designed to induce host-versus-graft tolerance by engraftment of donor stem cells and to avoid or minimize procedure-related toxicity and mortality. The rationale behind NST is to induce optimal graft-versus-leukemia (GVL) effects for elimination of all malignant cells by donor alloreactive immunocompetent cells as an alternative to standard high-dose myeloablative chemoradiotherapy. It seems reasonable to exploit a new reduced-intensity nonmyeloablative conditioning regimen for establishment of hostto-graft transplantation tolerance with optimal GVL. This paper investigate the use of nonmyeloablative allogeneic peripheral blood stem cell transplantation for the treatment of chronic leukemia and establish a new nonmyeloablative conditioning regimen which summarizes our cumulative experience in a cohort of 7 consecutive patients with CML in first chronic phase and 1 patient with CLL in third phase who were transplanted beween July 1999 and September 2000. The enrolled 6 consecutive patients with Philadelphia-positive CML in first chronic phase and 1 patients with CLL in third

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phase were diagnosed according to previously published criteria. All the patients were male aged from 26 to 42 (medium 28) years old and received transplants from sibling donors fully matched for human leukocyte antigen (HLA) class I and II. Conditioning regimen of NST consisted of intensive immunosuppression with intravenously administered fludarabine (30mg/m2/d on days ⫺5 to ⫺1), cyclosporine (CSP) (6mg/kg/d on days ⫺5 to ⫺1), monoclonal antibody CD3 (McCD3) (10mg/d on days ⫺5 to ⫺1, made in Cuba), cyclophosphamide (CTX) (45mg/kg/d on days ⫺3 and ⫺2) and cytarabine (Ara-C) (200mg/ m2/d on days ⫺4 to ⫺1). Prior to NST, all patients received trimethoprim/sulfamethoxazole (10mg/kg/d trimethoprin) on days ⫺10 to ⫺2 against Pneumocystis carinii infection and ganciclovir (10mg/kg/d) × 7d) agaist Cytomegalovirus (CMV) infections. From day ⫺1, Patients were treated in reverse isolation rooms equipped with high-efficiency particulate air (HEPA) filters and received a regular diet. Blood component transfusions were administered as necessary. Donors were injected subcutaneously with granulocyte-colony stimulating factor (Kirin, 5µg/kg twice daily for 5 days) and mobilized peripheral blood stem cells were collected on days 5 and 6 with Baxter. Graftversus-host disease prophylaxis consisted of low-dose, short-term cyclosporine (CSP) (3mg/kg daily, administered orally in 2 divided doses) and additional mycophenolate mofetil (MMF) (35mg/kg/d) which started on day 0 and tapered after 12 weeks after transplantation, according to chimeric status and evidence of GVHD. Acute and chronic GVHD were graded according to the Glucksberg et al criteria. Immediately upon the appearance of signs and symptoms of GVHD, methylprednisolone (2 to 4mg/kg) and CSP were administered intravenously as first line drug when ATG and FK506 as second line drug. In order to assess engraftment, degree of chimerism, minimal residual disease, and early relapse, patients were monitored at regular intervals by cytogenetic analysis of donorand host-specific DNA markers by short tandem repeated PCR (STR-PCR) assay. Cytogenetic analysis for the Philadelphia chromosome and the bcr/abl reverse transcriptase (RT-PCR) test were applied at the time of diagnosis and for detection of relapse during follow-up. The protocol used for conditioning was well tolerated by all 7 patients without oral mucositis, fever, hepatic veno-occlusive disease (VOD), and thus maintained normal oral intake. After conditioning , the nadir of WBC count (0.2 × 109/L) occurred during ⫹3d–⫹5d while the nadir of platlet count (8.0 × 109/L) occurred during ⫹5d–⫹7d. Donor mononuclear cells (MNC 4.9 to 8.1 7times; 108/kg) with CD34(⫹) cells (3.7 to 12.2 × 106/kg) were transfused on 0d. Mixed chimerism was detected in all patients and lasted from 2 to 24 (media 13) months. All patients displayed evidence of engraftment shortly after NST, four with full of donor cells grafted, three with mixed chimerism. Two mixed chimerism converted to full donor chimerism, the other one (CML) rejected after ⫹63d and came back to chronic phase. All recovered hematopoiesis (WBC recoveried to more than 0.5 × 109/L during ⫹9d–⫹ 21d and platelet recoveried to more than 30 × 109/L during ⫹ 11d–⫹28d.). One of them died due to aGVHD of degree IV after the first and second line drug therapy agaist GVHD; another developed curable aGVHD of degree I after the first line drug therapy. Six patients keep disease-free survival for 5 to 24 months. NST has much improved in recent years, but the above mentioned conditioning regimen without TBI has never been reported in the past years. These findings indicate the procedure is much safe, effective and of less complications than the myeloablative condioning regimens and may represent another new approach in the management of patients with chronic leukemia.

163 EFFICACY OF EXTENDED LAMIVUDINE THERAPY AGAINST HEPATITIS B VIRUS (HBV) INFECTION IN HEMATOPOIETIC STEM CELL TRANSPLANT (HSCT) RECIPIENTS Hsiao L.T.*,1, Chiou, T.1, Liu J.1, Yen C.1, Wang W.1, Lin Y.1, Chu C.1, Chao T.1, Yang M.1, Chen P.1 1 Taipei Veteran’s General Hospital, Taipei, Taiwan HBV reactivation causes non-relapse deaths in HSCT recipients, and Lamivudine is effective to treat and even prevent these flares. Extended Lamivudine therapy (LAM) through HSCT period is not uncommon; however, its safety is not understood. HSCT recipients who received LAM in our institute were enrolled and clinical outcomes were recorded. Stored serums were examined for HBV markers, quantitative serum DNA (sDNA), variants and YMDD mutants. Between 2000/03 and 2002/11, total 18 HBsAgpositive recipients (M/F ⫽ 14/4; 5AL/5CML/6NHL/1MM/1HD; median age 43 [19–56]; auto/allo ⫽ 9/9) received LAM since pre- (n ⫽ 13) and post-HSCT period (n ⫽ 5) as prophylaxis or salvage therapy of HBV reactivation. Virological parameters at baseline indicated 5 HBeAg-positive, 8 basic core promoter (T1762/A1764) and 11 precore (1896A) variants, and median sDNA of 19.9 pg/ml (0.025–9988). Median duration of LAM was 9.6 months (4–30). During therapy, sDNA became undetectable (⬍0.001 pg/ml, 100 copies) in 6 cases, and the other 12 cases had a median decline of 2.57 log sDNA (0–5.59), with serum ALT normalization. Only 1 case lost eAg (1/5). YVDD mutant was detected in 4 cases after 8, 8.7, 12, and 23 months of therapy, in which 2 had only virological breakthrough. At the end of FU, 8 cases stop LAM and 3 complicated with HBV flare, including virological only, nonicteric and icteric hepatitis in each one. No cases died from HBV-associated hepatic failure. Extended LAM is safe and effective as the treatment and prophylaxis of HBV reactivation in HSCT recipients. Regular follow-up is important to early detect the flares due to YMDD mutants (during the period of therapy) and those due to withdrawal.

164 ENGRAFTMENT POTENTIAL OF MARROW HARVESTED AFTER CHEMO-GROWTH FACTOR MOBILIZED BLOOD COLLECTION Elfenbein G.*,1, Lum L.1, Rathore R.1, Colvin G.1, Abedi M.1, Quesenberry P.1, Morgan D.1, Falvey M.1 1 Roger Williams Medical Center, Providence, RI, USA Failure to mobilize sufficient CD34⫹ cells to assure rapid engraftment (i.e., ⱖ2 × 106 cells/kg) into the bloodstream occurs frequently. Among 34 autotransplant patients in 2001–2002, only 17 had ⱖ2 × 106 CD34⫹ cells/kg collected. Previously (Blood 1999;94(suppl 1):558a), we expanded marrow ex vivo (utilizing the AastromReplicell쑓) to accelerate engraftment of CD34⫹ low yield, blood stem cells (BSC). However, the AastromReplicell쑓 is not yet available in the USA. We performed a pilot study in 3 patients to determine if marrow stem cells (MSC), harvested after 3 leukaphereses contained cumulatively a low yield of CD34⫹ BSC, could accelerate engraftment of low yield BSC. Two patients had 1.5 and 1.9 × 106/kg CD34⫹ BSC collected upon recovery

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of the leukocyte count above 3 × 109/L after cyclophosphamide (3500mg/m2 IV over 2 days) and prednisone (2mg/kg/day for 4 days PO) followed by G-CSF (10mcg/kg/day SC starting on day ⫹ 3). Three to six days after leukaphereses (days ⫹22 and ⫹21 of chemotherapy), while still on G-CSF, these patients had 0.9x106/ kg CD34⫹ MSC, each, harvested. After receiving BSC and MSC, these 2 patients both recovered granulocytes ⬎0.5 × 109/L on day ⫹ 12 and platelets ⬎20 × 109/L on days ⫹42 and ⫹21, respectively, after autotransplant. A third patient, unfortunately, could not have BSC collected due to bacterial infection. After antibiotic therapy, this patient had 2.4 × 106/kg CD34⫹ MSC harvested 15 days after initiation of chemotherapy while still on G-CSF. This patient recovered granulocytes on day ⫹12 and platelets on day ⫹25 after autotransplant. The 3 marrow collections were somewhat hypocellular with median (range) of mononuclear cells being 147(50– 224) × 106 cells/kg. To our knowledge, this is the first demonstration that MSC, collected after chemotherapy and G-CSF were used to mobilize BSC, are capable of assisting CD34⫹ low yield BSC to repopulate hematopoiesis rapidly. More striking is the observation that MSC collected after chemo-growth factor therapy can repopulate hematopoiesis by itself.

165 POST-GRAFTING G-CSF ACCELERATES GRANULOCYTE ENGRAFTMENT AFTER G-CSF PRETREATED ALLOGENEIC STEM CELL TRANSPLANTATION Elfenbein G.*,1, Oblon D.1, Falvey M.1, Morgan D.1, Sackstein R.2 1 Roger Williams Medical Center, Providence, RI, USA, 2 Massachusetts General Hospital, Boston, MA, USA Now that we have demonstrated that G-CSF primed allogeneic marrow stem cells (MSC) engraft as rapidly as do G-CSF mobilized blood stem cells (BSC) (Blood 2001;98:413a), we have turned our attention to the impact of other factors upon allo-engraftment. Because donors are healthy family members, we may safely presume that they have normal hematopoiesis. Engraftment for granulocytes is defined as the first of 3 days when the absolute granulocyte count (AGC) exceeds 0.5 × 109/L and for platelets the first of 3 days when the platelet count (PLT) exceeds 20 × 109/L without transfusion support. In 28 consecutive patients, 21 received genotypically HLA-identical and 11 received syngeneic sibling grafts. 11 patients received transplant regimens containing busulfan, 11 received regimens including TBI, and 6 received regimens containing neither busulfan nor TBI. 17 patients received BSC and 11 received MSC. All patients received cyclosporine for acute GVHD prophylaxis but no methotrexate. 11 (39%) received G-CSF postgrafting. The median time (range) to AGC ⬎0.5 × 109/L was 11 days (6–23) for HLA-identical grafts and 11 days (8–15) for syngeneic grafts. Similarly, the median time to recovery of PLT ⬎ 20 × 109/L was 18 days (12–49+) and 19 days (8–39), respectively. In autotransplantation for comparison, after preparation with a busulfan regimen, the median day for AGC ⬎0.5 × 109/L is day 11 and day 20 for PLT ⬎20 × 109/L (using our best mobilization regimen of cyclophosphamide, prednisone, and G-CSF). Transplant regimen had no influence upon the pace of allo-engraftment. The most striking finding, in the setting of G-CSF pretreatment

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before allogeneic stem cell collection, is post-grafting G-CSF further accelerated granulocyte recovery, on average, by 2 days (twotailed p ⫽ 0.02) and platelet recovery, on average, by 6 days (p ⬍ 0.15) as compared to patients receiving no post-grafting GCSF. This finding suggests the need for a prospective, randomized, controlled, clinical trial to determine the true value of post-grafting G-CSF.

166 HAPLOTYPE MISMATCHED STEM CELL TRANSPLANTATION FROM RELATED DONORS USING STRATIFIED CONDITIONINGS IN PATIENTS WITH HIGH-RISK ACUTE LEUKEMIA Kim H.*,1, Min W.S.1, Park Y.1, Kim Y.1, Lee S.1, Kim D.1, Lee J.1, Kim C.1 1 Catholic HSCT Center, Seoul, Korea We tried to reveal the possible role of a full haplotype mismatch transplantation in addition to 1 or 2 major antigen(s) mismatch settings. We performed 11 transplants (9AML, 2ALL), 3 haploidentical, five 5/6, three 4/6 HLA-matched. Median age was 34 (range 16–44). Conditioning regimen for patients who received stem cells from related 2 or 3 major HLA antigen-mismatch donors included fractionated TBI of 12Gy in addition to the administration of busulfex (intravenous busulfan; Bu), ATG, and fludarabine in 5 patients and 1 patient who relapsed after allogeneic stem cell transplantation received Bu, melphalan, fludarabine, and ATG. They all received G-CSF primed PB CD34⫹ cells using CliniMACS consecutive 4 days without either any GvHD prophylaxis or post-transplant G-CSF. Four patients transplanted from 1 major antigen-mismatched donors received megadose CD34⫹ cells containing G-CSF primed PB CD34⫹ cells and G-CSF primed BM with TBI⫹cyclophosphamide(Cy) or TBI⫹Bu and the other patient received Bu⫹Cy. They all received tacrolimus⫹short-term methotrexate GvHD prophylaxis with post-transplant G-CSF. The infused CD34⫹ cell dose was median 16.7 × 106/kg (range 6.8–32.4). The CD3⫹ cells infused in patients who received greater than 2 major antigen-mismatched transplant ranged from 0.4 × 104/kg to 1.6 × 104/kg. The median day required to reach ANC ⬎500/µl, platelet ⬎20,000/µl were 11 (range 10–13), 13 (range 11–15), respectively. All patients showed complete donor chimerism using multiplex fluorescent short tandem repeat analysis at post-transplant D⫹21. The most frequent RRT was severe mucositis, but it was controllable in all cases. One treatment-related death resulted from ARDS that was related to old pre-transplant pneumonic scar. Nobody showed acute GvHD except 1 patient who received 1 major antigen-mismatched transplantation developed treatable chronic hepatic GvHD at post-transplant 6 months. Three AML of 11 patients who were in far-advanced disease status relapsed early after transplantation and one of them died. The other 8 patients have been in good clinical condition with follow-up duration of 3∼10 months. We could see that at least in patients in CR who were categorized as poor-risk acute leukemia, this treatment modality should be considered actively as early as possible.

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167 AUTOLOGOUS PERIPHERAL BLOOD STEM CELL TRANSPLANTATION FOR TREATMENT OF PATIENTS WITH NON-HODGKIN’S LYMPHOMA — REPORT OF 182 CASES IN CHINA. Wanming D.*,1, Ky L.1, Dp L.1 1 Peking University Institute of Hematology, Beijng, China To evaluate the efficacy of autologous peripheral blood stem cell transplantation (APBSCT) for treatment of patients with nonHodgkin’s lymphoma. One hundred eighty-two patients with non-Hodgkin’s lymphoma were enrolled in a clinical study carried in 34 hospitals of China. At the time of APBSCT, 112 patients were in first completely remission (CR1), and 70 patients in partial remission (PR) or relapse. Autologous peripheral stem cell (APSC) mobilized by high-dose cyclophosphamide (HD-CY) (n ⫽ 55), high dose Ara-C (HD-Are-C) (n ⫽ 7), increased dose CY based regimens with rh-G-CSF (n ⫽ 102) and G-CSF only (n ⫽ 18). One hundred twelve patients in CR1 received high dose chemotherapy with (n ⫽ 39) or without total body irradiation (TBI) (n ⫽ 73). 70 patients in PR or relapse received high dose chemotherapy with (n ⫽ 29) or without TBI (n ⫽ 41). After APBSCT, median time for hematopoietic recovery of WBC ⱖ1.0 × 109/L, and platetetsⱖ 20.0 × 109/L was 12 (range 10–30) days and 12 (range 0–181) days, respectively. With a median follow-up of 24 months, the probability of 3-year disease free survival (DFS) in CR1 and in PR or relapse was 69.7% and 44.9%, respectively. The probability of 3-year overall (OS) and DFS for patients in CR1 group was 76.2% and 70.1% following high-dose chemotherapy with TBI. However, the probability of 3-year OS and DFS for patients PR or relapse group was 76.5% and 57.5% following high dose chemotherapy with TBI and 58.4% and 40.1% following high dose chemotherapy without TBI. After transplantation, 18 patients were died of original disease (16.1%) in CR1 group and 19 patients (27.1%) in PR or relapse group. Transplantation related mortality was 2.6%. These results suggest that APBSCT is a safe and efficacious therapeutic measure in patients with NHL.

168 NON-MYELOABLATIVE STEM CELL TRANSPLANTATION IN 18 CASES OF HEMATOLOGICAL DISEASES Xiao M.1, De-Pei W.*,1, Ai-Ning S.1, Zheng-Zheng F.1, Xiao-Wen T.1, Hui-Ying Q.1, Miao M.1, Yuejun L.1 1 JiangSu Institute of Hematology, SuZhou, China We explored the efficiency and toxicity of non-myeloablative stem cell transplantation (NST) for hematological disease. Eighteen patients with chronic myelogenous leukemia (7/18), acute myeloid leukemia (3/18), severe aplastic anemia (4/18), non-Hodgkin’s lymphoma (2/18), multiple myeloma (1/18), myelodysplastic syndrome (1/18) received non-myeloblative stem cell transplantation from HLA-identical sibling donors. Risk factors include: chronic myelogenous leukemia-accelerate phase (3/18), chronic myelogenous leukemia ⫺2nd chronic phase (1/18), old age (8 patients over 40 years), hepatitis B virus or other pathogen carrier (6/18), female donor and male recipient (10/18). Peripheral blood stem cells were mobilized by G-CSF 300µg per 12 hours × 5 d.

2.15 to 10.01 × 106 CD34⫹ cell/kg were transplanted. A nonmyeloablative conditioning regimen included Fludarabine 30mg/ m2.d⫺1 × 6; Busulfan 4mg/kg.d⫺1 × 2 or Cyclophosphamide 50 mg/ kg.d⫺1 × 2 and Antilymphocytic Glubin 12∼15mg/kg.d⫺1 × 4. Cyclosporin A was used to prevent graft versus host disease (GVHD) alone and no G-CSF was administered after NST. Hematopoietic reconstitution obtained on days 8 to 19 (day 13 on average). Severe mucositis was absent. Venoocclusive live disease did not occur. Infection complications were rare. Acute GVHD was present in 5 patients. Chronic GVHD occurred in 5 patients. Idiopathic pneumonia was developed in 5 patients. In the followup duration of 90 to 425 days, no early hematological relapse developed; the blood and bone marrow smear were normal and BCR-ABL (7/18) transcripts disappeared. Most of these patients (16/18) had a stable mixed chimerical state. Fifteen of 18 patients are alive. The NST has promise for reducing the morbidity and mortality associated with conventional high-dose chemo-radiation therapy, and they allow allogeneic transplants in elderly or medically infirm patients who are at present not candidates for transplantation. NST is an effective therapy in the treatment of hematological disease.

169 FLUDARABINE-INCLUDE CONDITIONAL REGIMEN FOR EIGHTEEN PATIENTS WITH HEMATOLOGY DISEASE Xiao M.1, De-Pei W.*,1, Ai-Ning S.1, Zheng-Zheng F.1, Xiao-Wen T.1, Hui-Ying Q.1, Miao M.1, Yue-Jun L.1 1 First Affiliated Hospital of SooChow University, SuZhou, China We explored the efficiency and toxicity of non-myeloablative stem cell transplantation (NST) for hematological disease. Eighteen patients with chronic myelogenous leukemia (7/18), acute myeloid leukemia (3/18), severe aplastic anemia (4/18), non-Hodgkin’s lymphoma (2/18), multiple myeloma (1/18), myelodysplastic syndrome (1/18) received non-myeloblative stem cell transplantation from HLA-identical sibling donors. Risk factors include: chronic myelogenous leukemia-accelerate phase (3/18), chronic myelogenous leukemia ⫺2nd chronic phase (1/18), old age (8 patients over 40 years), hepatitis B virus or other pathogen carrier (6/18), female donor and male recipient (10/18). Peripheral blood stem cells were mobilized by G-CSF 300µg per 12 hours × 5 d. 2.15 to 10.01 × 106 CD34⫹ cell/kg were transplanted. A nonmyeloablative conditioning regimen included Fludarabine 30mg/ m2.d⫺1 × 6; Busulfan 4mg/kg.d⫺1 × 2 or Cyclophosphamide 50mg/ kg.d⫺1 × 2 and Antilymphocytic Glubin 12∼15mg/kg.d⫺1 × 4. Cyclosporin A was used to prevent graft versus host disease (GVHD) alone and no G-CSF was administered after NST. Hematopoietic reconstitution obtained on days 8 to 19 (day 13 on average). Severe mucositis was absent. Venoocclusive live disease did not occur. Infection complications were rare. Acute GVHD was present in 5 patients. Chronic GVHD occurred in 5 patients. Idiopathic pneumonia was developed in 5 patients. In the follow-up duration of 90 to 425 days, no early hematological relapse developed; the blood and bone marrow smear were normal and BCRABL (7/18) transcripts disappeared. Most of these patients (16/18) had a stable mixed chimerical state. Fifteen of 18 patients are

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alive. The NST has promise for reducing the morbidity and mortality associated with conventional high-dose chemo-radiation therapy, and they allow allogeneic transplants in elderly or medically infirm patients who are at present not candidates for transplantation. NST is an effective therapy in the treatment of hematological disease.

170 COMPARISON OF TWO APHERESIS PROTOCOLS FOR COLLECTION OF AUTOLOGOUS PERIPHERAL BLOOD PROGENITOR CELLS Ch.Giraud1,3, H. Sardet2, M. Renaud1, J.C. Chomel3, A. Sadoun1, E. Randriamalala1, L. Lacotte Thierry1, V. Delwail1, J. Guilhot1, F. Guilhot1 1 Service d’Oncologie He´matologique et The´rapie Cellulaire CHU, Poitiers, France, 2Service Pharmacie CHU, Poitiers France, 3Service Pre´le`vement de CSP et Cryobiologie, EFS-CA, Poitiers France Version 4.7 and AutoPBSC using a cell separator (Spectra, Cobe) were compared in terms of stem cells quality, transplantation and cost. AutoPBSC is less operator intensive than V4.7 but depends on device settings determinated before apheresis. First apheresis and transplantation (CD34 infused ⬎2.106/kg) for 111 myeloma patients (pts) (65 male, 46 female) and 127 lymphoma pts (84 male, 43 female) were analysed. Stemcells volume, CD34 values and CFUGM assays were compared before, after cryopreservation (10% DMSO in autologous plasma) and thawing. Mann and Withney’s test was used to compare the two groups. There was no difference concerning sex ratio and age in V4.7 (n ⫽ 65) or AutoPBSC (n ⫽ 45) for myeloma and lymphoma pts (n ⫽ 80 and n ⫽ 47). However, body mass index was significantly higher for myeloma pts in AutoPBSC. Product volume was higher in V4.7 for myeloma pts (median 163ml, range 120–240 and median 122ml, range 107–197 ) compared to AutoPBSC (p ⬍ 0.0001) and in lymphoma pts (median 168ml, range 137-240 and 121ml, range 61–180 ) (p ⬍ 0.0001). No difference was recorded for CD34 106/ kg and CFUGM 104/kg for myeloma pts before cryopreservation and after thawing, whereas for lymphoma pts CFUGM 104/kg collection was increased after thawing with AutoPBSC ( p ⫽ 0.03). Duration of hospitalisation and number of red cells and platelets transfusion were similar for myeloma pts (after Melphalan 140). However, significant higher number of platelets transfusion was noted for lymphoma pts with version 4.7 ( p ⫽ 0.02) ( BEAM 400 for all pts). AutoPBSC and V4.7 are both efficient. AutoPBSC’s advantages are safety, reduced component volume, thawing standardisation, automatisation, and reproductibility. Reduction of transfusion with AutoPBSC in lymphoma has to be confirmed.

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171 AUTOLOGOUS BONE MARROW TRANSPLANT IN A PATIENT WITH SICKLE CELL DISEASE AND DIFFUSE LARGE B-CELL LYMPHOMA Onitilo A.*,1, Lazarchick J.1, Brunson C.1, Frei-Lahr D.1, Stuart R.1 1 Hem-Onc Division/Medical Uniersity of South Carolina, Charleston, SC, USA As the life expectancy of patients with homozygous sickle cell anemia (SCA) improves, SCA care providers are confronted with diseases of the adult SCA population rarely seen before. We report here a 40-year-old woman with SCA who developed diffuse large B-cell non-Hodgkin’s lymphoma (NHL) that was treated with 8 cycles of chemotherapy consisting of cyclophosphamide, doxorubicin, vincristine, prednisone, and etoposide (CHOPE), without complete remission. She subsequently underwent high-dose cyclophosphamide and total body irradiation followed by autologous bone marrow transplantation (BMT). To reduce the risk of sickle cell crisis precipitated by G-CSF, she underwent hypertransfusion to maintain a low % hemoglobin S throughout her treatment course. Although she has required iron chelation therapy and shows no sign of modification of her underlying SCA, she remains in remission from NHL 12 years post-transplant. To our knowledge, this is the first reported case of autologous BMT in a patient with SCA. Our patient illustrates that SCA in itself does not preclude autologous stem cell transplantation or G-CSF use in selected patients with SCA, and this report should encourage others to consider autologous BMT in adults with SCA where it represents a lifesaving therapy for malignant diseases.

172 TANDEM AUTO-ALLO-TRANSPLANTATION IN PATIENTS WITH PRIMARY REFRACTORY PERIPHERAL T-CELL-LYMPHOMA (PTCL): CASE REPORT Melkova K.*,1, Vorobyeva S.1, Frolov G.1, Ogorodnikova E.2, Baranov A.1 1 State Research Centre — Institute of Biophysics, Moscow, Russia, 2Oncolog.RC — RAMN n.N.N.Blochina, Moscow, Russia A-39-yr-old male with PTCL (without BM involvement) diagnosed in 5/01, no achieved CR after 7 course of chemotherapy. Time to transplant was 9 months. The interval between autologous and allogeneic transplantation was 46 days. The patient first received autografting after TBI (12 Gy) and cyclophosphamide (Cy, 120 mg/kg) followed by a dose-reduced regimen consisting of cisplatin 25 mg/m2/d (×4d), fludarabine 30 mg/m2/d (×2d), Cy 500 mg/m2/d (×2d) and allografting from related sister, 46 years, to induce a graft versus lymphoma effect. The source of the stem cell was autoPBSC (CD34⫹ 5 × 106/kg) and G-CSF (Filgrastim) stimulated alloBM. Neutrophil recovery (⬎1.5 × 109/L) occurred on day 15 and platelet recovery (⬎50 × 109/L) on day 14 after autoPBSCT. Catheter-related bloodstream infection was complication of autoPBSCT, duration of hospitalization was 21 day after transplantation. GVHD prophylaxis post-alloBMT was cyclosporin A (to day 24) plus “short” methotrexate and folinic acid. Acute GVHD was not. We observed a CMV reactivation in the first

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month after alloBMT. The fever, focal neurologic symptoms and nodule of a cerebellum on MRI scan of a head were detected on day 56. The therapy of liposomal amphotericin 5 mg/kg was effective and the clinical signs were not determined within one month. The MRI scan shoved absence of pathological changes after 4 months. Results of hematological recovery, chimerism and outcome after alloBMT are: to ANC ⬎1 × 109/L — day 29, to platelet ⬎ 50 × 109/L — day 23, chimerism: ⬍ day 56 — 60–70% of donor cells, ⬎ days 120 — full donor chimerism, status after 12 months: CR, limited chronic GVHD. The tandem auto-allo-transplantation provides engraftment with complete donor chimerism and allows to achieve CR at primary refractory PTCL.

173 CLINICAL EFFECT OF FETAL CELL TRANSPLANTATION IN PATIENTS WITH SEVERE LIVER DYSFUNCTION Manukian G.*,1, Azatian C.1, Yeramishantsev A.1, Zhdanov A.1, Shtil A.1, Aivazyan A.1, Jarigin K.1, Aprikyan A.2, Sukhikh G.1 1 Research Center for Obstetrics and Perinatology, Moscow, Russia, 2University of Washington, Seattle, WA, USA Fetal cell transplantation (FCT) aimed at restoration of impaired organ function emerges as a perspective strategy for treatment of liver dysfunction. We performed FCT in 22 patients with liver cirrhosis and portal hypertension. FCT was used as a component of multimodality treatment (prior to or after surgery; 13 cases) and solely as the method of choice (9 patients). Cells for FCT were obtained from human fetal liver at the second trimester of gestation. The protocol included three consecutive transplantations (300– 500 × 106 fetal cells per injection) one week apart. Cells were delivered into portal vessels during surgery or via the transfemoral way using endovascular technique. The patients were monitored for up to 24 months after transplantation. Overall, FCT improved patients’ status in 19 cases (86.3%). FCT had little or no effect in 3 patients. The major features of improvement were gain of weight (an increase up to 15 kg), loss of weakness and fatigue, disappearance of encephalopathy, jaundice and ascites. Biochemical criteria of improved status were balanced content of total protein and partially restored levels of serum albumin, bilirubin and aminotransferases. Patterns of liver scintigraphy were normalized in 13 (59.9%) patients. In 10 cases the morphologic examination of liver biopsies confirmed minimal-to-null activity of the pathologic process after FCT. No complications of FCT were observed. Importantly, the status of all patients with positive results of FCT remains stable. We conclude that FCT alone and in combination with conventional treatment may be an efficient approach in patients with severely compromised liver functions.

174 ABSENCE OF HOXA9 CORRELATES WITH B-CELL DEVELOPMENT IN THE PRESENCE OF MLL/AF4 EXPRESSION Bertrand F.*,1, Spengeman J.1, Lebien T.2 1 Brody Medical School at East Carolina University, Greenville, NC, USA, 2University of Minnesota Cancer Center, Minneapolis, MN, USA The t(4;11) variant of infant acute leukemia is characterized by expression of the MLL/AF4 fusion oncoprotein and expansion of cells with a pro-B/monocyte phenotype. We have established a novel t(4;11) cell line, BLIN-3, that expresses the MLL/AF4 fusion protein and has retained features of normal B-lineage development, namely, bone marrow (BM) stromal cell/growth factor dependency, apoptotic sensitivity, and B-lineage developmental capacity. We used gene chip microarray analysis to identify unique patterns of gene expression in BLIN-3 that may contribute to its retention of developmental features consonant with normal B-lineage development. These data were compared with published array data examining eight t(4;11) patient samples. BLIN-3 exhibited increased or unique expression of several genes known to be involved in B-lineage development (e.g., 14.1/lambda 5, Btk, CD24, and OBF1) consistent with its ability to differentiate from the pro-B to preB cell stage. Expression of myeloid lineage associated genes CD44, MIR-10 and MNDA was only found in t(4;11) patient samples. Remarkably, BLIN-3 displayed a pattern of Hox gene expression distinct from other t(4;11). HOXA5, HOXA7 and HOXA9 were only expressed by t(4;11) patient samples and not by BLIN-3. Absence of HOXA7 and HOXA9 expression in BLIN-3 was verified via RT-PCR and Western blot analysis. We conclude that B-cell development can occur in the presence of the MLL/AF4 oncoprotein in the absence of HOXA5, HOXA7 or HOXA9 expression.

175 ABERRANT EXPRESSION OF HELIOS CAUSES PARTIAL ARREST OF B CELL DEVELOPMENT, AUGMENTED IMMUNE RESPONSE AND DEVELOPMENT OF LYMPHOMA Dovat S.*,1, Montecino-Rodriguez E.2, Schuman V.3, Dorshkind K.2, Smale S.3 Helios is a T cell-restricted member of the Ikaros family of zinc finger proteins. Previous experiments suggested that Helios might play a role in lineage switching during lymphopoiesis. To test this hypothesis, we created transgenic mice with Helios overexpressed in B cells and T cells, under the control of the immunoglobulin enhancer. Analysis of B cell subfractions in bone marrow showed decreased numbers of pre-B cells in transgenic mice compared to wild type controls. Pro-B cells in transgenic mice had decreased colony-forming ability upon stimulation by IL-7. These data suggest that expression of Helios in B cells partially arrests B cell development at the transition from the pro-B to pre-B cell stage. Bone marrow and peripheral B cells had prolonged survival and highly elevated expression of anti-apoptotic gene Bcl-XL. Peripheral B cells had augmented proliferative response, decreased activation

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threshold to antigen stimulation, and altered isotype switching following T cell-independent immune response to polysaccharide antigens. Aging transgenic mice frequently developed B cell lymphoma. Malignant cells were CD45R⫹ and CD5⫹ suggesting mature B cell origin. Preliminary analysis of tumor samples of patients with lymphoid malignancies showed that overexpression of Helios is associated with malignant transformation in humans. Our data suggest that negative regulation of Helios expression is necessary for normal B cell development. Ectopic expression of Helios alters differentiation and immune response of B cells and may have an oncogenic role in development of lymphoma.

176 A NOVEL SERUM-FREE CULTURE SYSTEM FOR ACUTE LYMPHOBLASTIC LEUKEMIA REVEALS THE EXISTENCE OF AN AUTOCRINE PROLIFERATION INDUCING FACTOR Goselink H.*,1, Nijmeijer B.A.1, Hoogeboom M.1, Willemze R.1, Falkenburg J.H.F.1 1 Leiden University Medical Center, Leiden, The Netherlands Study of the biology of ALL is hampered by the fact that ALL cells can not be cultured in vitro. We established a serum-free culture system in which a high proportion of primary ALL can be cultured longterm. In 10 out of 25 primary precursor B-ALL samples longterm proliferation (currently more than 2 years) could be induced. Karyotypic analysis demonstrated that all proliferating cultures originated from the primary leukemic clone. All proliferating ALL samples were immune phenotypically identical to the primary cells. The cultured leukemic cells proliferated with a constant doubling time of 2.0 ⫾ 0.3 days, but only in cultures of moderate to high cell densities, suggesting the production of an autocrine proliferation inducing soluble factor or the dependence of proliferation on cellcell contact. To study the possible autocrine production of a proliferation inducing soluble factor, low cell concentrations (1-250 cells/well) were cultured in the presence or absence of autologous conditioned medium (CM). In the absence of CM, no proliferation was observed as determined by 3H-thymidine uptake as well as in a limiting dilution assay in 9 out of 10 ALL samples. In the presence of CM, proliferation was induced in all ALL samples demonstrating the presence of an autocrine factor. Cross-testing of CM revealed that non-autologous CM also induced identical proliferation in all ALL samples demonstrating that the samples proliferated and responded to the same autocrine factor. The auticrine factor demonstrated to be specifically produced by lymphatic cells since CM, derived from the bladder carcinoma cell line 5637 cells did not exert any effect on the proliferation of ALL cells. We conclude that our serum free culture system enables the longterm culture of primary ALL. Furthermore, this system revealed the existence of a lymphatic growth factor. The factor will be further characterized.

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177 COMPLEX REGULATION OF KILLER INHIBITORY RECEPTORS (KIRs) LEAD TO SKEWED NK RECEPTOR EXPRESSION IN PATIENTS WITH LYMPHOPROLIFERATIVE DISEASE OF GRANULAR LYMPHOCYTES Epling-Burnette P.K.*,1, Painter J.1, Chaurasia P.1, Yu S.2, Bai F.1, Wei S.2, Djeu J.Y.2, Loughran T.P.1 1 Hematologic Malignancy, Moffitt Cancer Center, Tampa, FL, USA, 2Immunology, Moffitt Cancer Center, Tampa, FL, USA The NK type of Lymphoproliferative Disease of Granular Lymphocytes (LDGL) is associated with the expansion of CD3⫺, CD16⫹ and/or CD56⫹ lymphocytes. Expansion of NK cells in patients with LDGL has been attributed to an undefined antigen that enhances cell survival. We have examined the repertoire of NK receptors expressed on these cells and delineated the pathway that mediates target cell lysis. In 11/12 patients with NK LDGL, we found that the NK receptor expression was skewed toward a loss of Killer Inhibitory Receptors (KIR) by flow cytometry. Additionally, reactivity with a single anti-KIR antibody was found in 85–95% of the NK cells from 6 of 12 patients. Interestingly, the regulation of KIR expression has not been previously observed in NK cells of normal individuals. In patients with NK-LDGL, the loss in KIR antibody reactivity did not result from a loss in mRNA or DNA expression. Interestingly, there was evidence of limited transcriptional and predominantly post-transcriptional regulation, which contributed to the restricted KIR phenotype. In contrast to KIR, CD94 and its heterodimerization partner NKG2A were both invariably expressed at high levels on these NK cells. Despite expressing NKG2A, PBMC from these patients had enhanced capacity to lyse tumor targets. Unlike normal NK cells, target lysis by patient NK cells was inhibited by incubation with the anti-CD94 antibody. In conclusion, the skewed pattern of KIR on NK cells from patients with LDGL is under the control of undefined in vivo signals that restrict KIR receptor surface protein expression suggesting antigenic education. Additionally, it has always been difficult to assess clonality in this disorder. The expression of a full range of KIR mRNAs with very skewed surface protein expression suggests that this disease represents polyclonal amplification of NK cells rather than a monoclonal process.

178 SELENIUM IN OXIDATION STATE ZERO IS A POTENT AND SELECTIVE ANTI-LEUKEMIA/LYMPHOMA AGENT Sieber F.1 1 Medical College of Wisconsin, Milwaukee, WI, USA A long-held dogma has been that selenium in oxidation state zero (Se(0)) is biologically inert. We report here that conjugates consisting of photochemically generated Se(0) and serum albumin or lipoproteins are highly cytotoxic to leukemia/lymphoma and selected solid tumor (e.g. neuroblastoma, medulloblastoma, breast cancer) cells but are well tolerated by normal CD34-positive hematopoietic stem cells and granulocyte/macrophage progenitors. A 1-hour exposure to micromolar concentrations of Se(0)-protein conjugates is sufficient to deplete most leukemia cells at least

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100,000-fold while preserving all CD34-positive normal hematopoietic stem cells. The (lipo)protein component of the Se(0)-protein conjugates serves as a delivery vehicle whereas Se(0) is the cytotoxic entity. Differential conjugate uptake is a major but probably not the only determinant of selectivity. Small (subnano) particle size appears to be crucial for the cytotoxic activity and selectivity of colloidal Se. The endocytosis of Se(0)-protein conjugates causes a rapid and extensive (up to 80% in 1 hour) depletion of intracellular glutathione, a loss of transmembrane asymmetry, a breakdown of mitochondrial potential, and the activation of several caspases. Se(0)-protein conjugates potentiate (often synergistically) the cytotoxic effects of ionizing radiation and several standard chemotherapeutic agents. Drugs modeled after our Se(0)-protein conjugates may prove useful for the systemic therapy of leukemia and selected solid tumors.

179 IL-7 IS PRODUCED BY MYELOMA CELLS IN PRESENCE OF IL-6 Giuliani N.*,1, Colla S.1, Bonomini S.1, Hojden M.1, Roti G.1, Morandi F.1, Sammarelli G.1, Bataille R.2, Rizzoli V.1 1 Cattedra di Ematologia, Parma, Italy, 2INSERM U463, Parma, Italy It has been recently shown that IL-7 stimulates IL-6 secretion by bone marrow (BM) stromal cells that express IL-7 receptor (IL7R) as well as long lived plasma cell progenitors. On the basis of this evidence the production of IL-7 and its potential role in multiple myeloma (MM) has been investigated in this study. We found that human myeloma cell lines (HMCL) and fresh MM cells obtained from 12 patients were positive for IL-7 mRNA. On the other hand IL-7R mRNA was not expressed in any HMCL tested while the EBV positive cell line ARH-77 was positive for IL-7R. Using an ELISA assay IL-7 was detected in the supernatant of HMCL, in contrast IL-7 was undetectable in conditioned medium of mononuclear cells or normal CD19⫹ B cells or B leukemic cell line REH. IL-7 levels were significantly up-regulated when the HMCL RPMI-8226, U266, XG-6 were cultured in presence of IL-6 but not normal or leukemia B cells as well as in EBV positive cells. A stimulatory effect of IL-7 on ARH-77 proliferation was found (⫹12% ⫾ 1; p ⬍ 0.05). In addition IL-7 induced the production of the critical osteoclastogenetic factor RANKL by T lymphocytes and blocking anti IL-7 Ab inhibited the stimulatory effect of HMCL on RANKL production by T lymphocytes. Following we tested IL-7 levels in MM patients. IL-7 serum levels were significantly higher in MM patients in comparison with healthy subjects (median: 12.15 pg/ml; range: 2.41–29.5 pg/ml vs. 1.91 pg/ml; range: 0– 3.43 pg/ml, p ⬍ 0.05). Similarly, IL-7 levels in BM plasma were significantly increased in MM patients in comparison with normal subjects (median: 8.67 pg/ml; range: 2.68–36,8 pg/ml vs. 0.40 pg/ ml; range 0–0.46 pg/ml; p ⬍ 0.05). In conclusion, our data indicate that human myeloma cells produce IL-7 in presence of IL-6 and that IL-7 could be involved in the physiopatology of MM.

180 T CELL SIGNALLING MOLECULES IN MULTIPLE MYELOMA-INCREASED DYSFUNCTION BY ADVANCING STAGE Mozaffari F.*,1, Hansson L.1, Kiaii S.1, Ju X.1, Rossmann E.1, Rabbani H.1, Mellstedt H.1, Osterborg A.1 1 Karolinska Hospital, Stockholm, Sweden T cell immune dysfunction in patients with malignant tumors has been attributed to abnormal signal transduction, partly through altered expression of components of the TCR/CD3 complex and their associated intracellular protein tyrosine kinases. In this study, four-colour flow cytometry was applied to study the surface bound molecules TCRab, CD28, CD152 and CD154 involved in T cell signalling and the intracellular components of the TCR/CD3 complex CD3-z chain, p56LCK, p59fyn, ZAP-70 and PI3-k as well as production of the cytokines IFN-g, IL-4 and IL-2 of blood T cells in multiple myeloma patients at different stages of the disease. Multiple abnormalities were demonstrated in vivo of CD4 and CD8 populations as well as after stimulation with the TCR binding molecule Staphylococcus enterotoxin B (SEB) a superantigen activating T cells. There was a marked reduction, particularly in advanced-stage numbers of T cells expressing CD28, CD152, CD3z, p56LCK, ZAP-70 and PI3-k (p ⬍ 0.01–0.007). The cytokine production of IFN-g and IL-4 in resting T cells was significantly higher (p ⬍ 0.05) in patients than in controls and the T cells did not react normally on SEB stimulation. The results demonstrated profound and multiple T cell dysfunctions especially in advanced stage which should be taken into consideration when developing T cell therapeutic approaches e.g. vaccination.

181 TWO INHIBITORS OF HMG-COA REDUCTASE, FLUVASTATIN AND SIMVASTATIN, INDUCE APOPTOSIS OF CHRONIC LYMPHOCYTIC LEUKEMIA CELLS: CRITICAL ROLE OF PROTEIN PRENYLATION AND CASPASE-3 ACTIVATION Lagneaux L.1, Delforge A.*,1, Dejeneffe M.1, Massy M.1, Bernier M.1, Bron D.1 1 Experimental Hematology, Bruxelles, Belgique Statins block de novo synthesis of cholesterol by inhibiting the enzyme, HMG-CoA reductase. The product of this reaction, mevalonic acid, is also a precursor of isoprenoids, molecules required for the activation of signaling G-proteins involved in cell survival. Recently, it has been demonstrated that statins induce a significant apoptotic response in acute myeloid leukemia cells. We have investigated the apoptotic effect of Fluvastatin (FV)(openring structure) (Novartis Pharma) and Simvastatin (SV) (closed-ring structure) (Merck & Co) on CLL cells. Exposure of CLL cells (n ⫽ 15) to these two drugs resulted in a time- (0–48h) and dose- (0– 100 mM) dependent inhibition of cell survival. The mean LC50 (concentration of drug required to produce 50% cytotoxicity) was respectively 46 ⫾ 6 mM and 16 ⫾ 5 mM for FV and SV, determined by trypan blue exclusion and MTT assay. No effect was observed with the ethanol/water vehicle tested at similar dilutions. The cytotoxic effect of statins resulted from apoptosis as evidenced by the increase in annexin V binding, caspase-3 activation, PARP cleavage, DNA fragmentation and decrease of the

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mitochondrial transmembrane potential (DYm). FV and SV can thus induce a specific apoptotic response in CLL cells at concentrations corresponding to the therapeutic range. In addition, bcl-2 expression was significantly inhibited by these statins. The FV and SV-induced apoptosis can be specifically prevented by mevalonate (100 mM), the immediate endproduct of the reaction catalyzed by HMG-CoA reductase. The observation that the treatment of CLL cells with the mevalonate metabolites, farnesylpyrophosphate (FPP) (5mM) and geranylgeranylpyrophosphate (GG-PP) (5mM), prevents also apoptosis induced by the lipophilic statins suggest that the reduction of isoprenoid concentrations and the subsequent protein prenylation are implicated in the cytotoxic activity of these statins. FV and SV bring new promises in the therapeutic approach of CLL patients.

182 COMBINED APPLICATION OF SIMVASTATIN WITH EITHER CHEMOTHERAPY OR CYTOKINE STIMULATION TO MYELOMA CELL LINES Drucker L.*,1, Osadchi A.2, Afensiev F.2, Radnay J.3, Shapira H.3, Lishner M.4 1 Oncogenetic Lab, Sapir Medical Center, Kfar-Saba, Israel, 2 Hematology Lab, Sapir Medical Center, Kfar-Saba, Israel, 3 Oncogenetic Lab & Med Department, Sapir Med Center, KfarSaba, Israel, 4Oncogenetic Lab & Med Department, Sapir Med Center, Kfar-Saba and Tel Aviv University, Tel Aviv, Israel The myeloma (MM) clone is characterized by intensive interactions with its surroundings modulated by membranal embedded components that promote cell growth and survival. We have previously demonstrated that simvastatin (Sim) widely prescribed for hypercholesterolinemia, has significant anti-myeloma activity in an in vitro setting. In the present work we examined Sim’s activity when administered in combination with cytokines that promote growth and survival in vivo and evaluated the potential application of Sim combined with conventional cytotoxic drugs used for treatment of MM. RPMI8226, U266 and ARH77 seeded in culture plates pre-coated with fibronectin (FN)/agarose/none were treated with Sim, IGF-I, IL-6 or combinations for 5 days. The cell lines were also exposed to Sim combined with melphalan (Mel) or Dexamethasone (Dex). Next, we assessed- cell morphology; viability (WST1); cell cycle (PI staining and flow cytometric analysis); total cell count and cell death (trypan blue exclusion); and DNA fragmentation. Reduced viability was demonstrated with Sim in all treated cell lines with and without co administration of IGF-I or IL-6 (p ⬍ 0.05). The extent of inhibition did not vary between Sim only and combinations (NS). FN did not influence cell response to Sim alone or combined with IL-6/IGF-I (NS). We conclude that IL-6, IGF-I and FN do not afford myeloma cell lines protection from Sim modulation. Moreover, redundancy of proliferation and survival pathways supports the possibility that Sim attenuates a joint upstream cellular component, such as PI3K/AKT. Pretreatment of myeloma cell lines with Sim resulted in sensitization to Dex and an additive effect with Mel (p ⬍ 0.05).

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183 DOWN-MODULATION OF ERK PROTEIN KINASE ACTIVITY SUPPRESSES HUMAN MYELOMA CELL PROLIFERATION AND MYELOMA-INDUCED ANGIOGENESIS BY VEGF INHIBITION Giuliani N.*,1, Lunghi P.2, Bonomini S.1, Hojden M.1, Colla S.1, Morandi F.1, Rizzoli V.1, Bonati A.2 1 Cattedra di Ematologia, Parma, Italy, 2Istituto di Patologia Medica, Parma, Italy The mitogen-activated protein (MAP) cascade leading to the activation of ERK (Extracellular signal-regulated kinase) is critical for regulating myeloma cell growth, however the relationships of ERK1/2 protein kinases activity with vascular endothelial growth factor (VEGF) production and the effects of their down-modulation in myeloma cells are not completely investigated. By immunoenzymatic MAP kinase assay using Elk1 as substrate (Elk1-p) or dualphosphorylation-ERK1/2 specific antisera (p-ERK1/2) we found that steady-state levels of phosphorylated ERK 1/2 (p-ERK) were stronger in U266 than RPMI-8226, and XG6. The total amounts of ERK1 and ERK2 proteins were comparable in U266, XG6 and OPM-2 whereas in RPMI-8226 the expression of ERK1 was significantly higher. IL-6 stimulation significantly increased ERK1/ 2 activity and VEGF secretion in human myeloma cell line (HMCL) XG-6, U266, RPMI-8226. The treatment with an inhibitor of MEK PD98059 (40µM) produced a reduction of p-ERK1/2 levels of more than 80% and prevented the increase of p-ERK1/2 induced by IL-6 treatment. PD98059 induced a significant inhibition of HMCL proliferation (⫺33 ⫾ 5%) and blunted the stimulatory effect induced by IL-6. A more potent inhibition on HMCL proliferation was observed with the ERK inhibitor PD184352 at 2µM (⫺70% ⫾ 4%). PD184352 but not PD98059 induced HMCL apoptosis in combination with Arsenic Trioxide 2µM. A significant inhibition on basal VEGF secretion by HMCL was induced by PD98059 treatment, more evident in RPMI-8226 (771 ⫾ 12 vs 1350 ⫾ 18 pg/ml, ⫺43%; p ⬍ 0.01, after 24 hours). In addition, PD98059 treatment suppressed the stimulatory effect of IL-6 on VEGF secretion by HMCL (RPMI-8226: 887 ⫾ 18 vs 1694 ⫾ 17 pg/ml, ⫺47%; p ⬍ 0.01). Consistently, in an “in vitro” model of angiogenesis, we found that PD98059 inhibits vessel formation induced by HMCL. In conclusion, our data indicate that a constitutive activation of ERK is present in HMCL and that the down modulation of ERK1/2 activity induced an inhibition of cell proliferation and myeloma-induced angiogenesis.

184 COMPARISON OF TWO FLOW CYTOMETRIC METHODS FOR DETECTION OF ANEUPLOIDY IN MULTIPLE MYELOMA: IMPLICATIONS FOR INVESTIGATION OF RESIDUAL DISEASE Oelschlaegel U.*,1, Nowak R.1, Heider T.1, Naumann R.1, Bornha¨user M.1, Ehninger G.1 1 University Hospital, Medical Clinic I, Dresden, Germany Myeloma cells have been flow cytometrically characterized with simultaneous immunophenotyping and aneuploidy detection. Two different preparation approaches have been compared in 266 parallel investigations in 165 patients with Myeloma/MGUS. With

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Cycloscope (Medac) immunophenotyping with CD138/CD38 in one tube was performed after erythrocyte lysing followed by permeabilization with a detergent. Our inhouse Ethanol method included immunophenotyping of mononuclear cells with CD138 or CD38 and a subsequent permeabilization with ethanol. In both approaches propidium iodide was the DNA stain. Both methods revealed aneuploid plasma cells in 60% and diploid cells in 22% of all investigations. First, the CV of G0/G1 peak, influencing the sensitivity of DNA quantification, was significantly higher with Cycloscope (3.16 vs. 2.60; p ⬍ 0.001). Second, DNA index was significantly higher with Cycloscope (1.27 vs. 1.19; p ⬎ 0.001). Despite the different range of indices they correlated well (r ⫽ 0.90). Third, in 18% (n ⫽ 49) of investigations discrepant results were present. Additional aneuploid clones with Cycloscope were often rather false aneuploid (n ⫽ 29). On the one hand the aneuploid clone was not detectable in further measurements of the same patient with active disease. On the other hand comparative multiparameter immunophenotyping showed only CD38⫹⫹ CD19⫹CD56- non-malignant plasma cells. In the remaining 11 investigations no explanation could be given for the additional aneuploid clone with Cycloscope. In further 7/8 measurements with aneuploid cells, present only in the Ethanol method, this aneuploidy was confirmed with both methods in follow up investigations. In the latter patient group a low overall plasma cell content was present or the aneuploid clone represented only a plasma cell subpopulation. In summary, both methods could be used for detection of aneuploidies in Multiple Myeloma at diagosis. Considering residual disease detection the Ethanol method seemed to provide a higher specificity with lower false positive measurements on the one hand and higher sensitivity with fewer false-negative results on the other hand.

185 MICROSATELLITE INSTABILITY AND DNA MISMATCH REPAIR DEFICIENCY IN NON-HODGKIN LYMPHOMA Miyashita K.*,1, Oda S.1, Hattori H.1, Yamada Y.2, Yoshida M.1, Uike N.2, Okamura J.1 1 Institute for Clinical Research, National Kyushu Center, Fukuoka, Japan, 2Department of Hematology, National Kyushu Cancer Center, Fukuoka, Japan Defective DNA mismatch repair (MMR) is associated with various human malignancies, and regarded as an important cause for the ‘mutator phenotype’ that plays a crucial role in carcinogenesis. Microsatellite instability (MSI) is observed in cells with MMR deficiency, because MMR counteracts replication errors in repetitive sequences. Although the role of the ‘mutator phenotype’ in haematopoietic tumourigenesis is controversial, MSI has been reported in various haematological malignancies. In non-Hodgkin lymphoma (NHL), the data in the literature differ widely. Using a newly developed fluorescent system, we have observed two distinct subtypes in MSI in various malignancies. We define Type A alterations as length changes of ⱕ6-base pairs. Type B changes are more drastic and involve modifications of ⱖ8-base pairs. From observations obtained using MMR gene knock-out mice, we concluded that Type A changes are a direct consequence

of defective MMR. MSI that has been observed in various malignancies, including HNPCC, using the conventional assay is predominantly Type B. In tumours obtained from 50 NHL patients, Type A MSI was detected in 16% (8/50). No Type B changes were observed. This finding indicates that in NHL some tumours exhibit an MMR-defective phenotype, suggesting that the ‘mutator phenotype’ may underlie tumourigenesis in NHL.

186 EXPRESSION PATTERNS OF TRAFS AND BIOLOGICAL EFFECTS IN MUTIPLE MYELOMA CELLS AFTER CD40 STIMULATION Qi C.*,1, Zheng L.1, Zhou X.1, Tao Y.1, Gu T.1, Zhang X.1 1 Biotechnology Institute, Suzhou, China Multiple myeloma (MM) is a progressive and fatal neoplasm of B-lineage cells, characterized by the accumulation of malignant plasma cells in the bone marrow. The engagement of CD40 on MM cells produced extensive and complicated biological effects. Delivery of the CD40 signals largely relyed on the mediation of TNF receptor-associated factors (TRAFs) family. Here, we investigated the expression of TRAFs and biological effects in several MM cell lines (XG2, XG7, U266, 8226) stimulated by CD40mAb or rhsCD40L. Our results showed that: different MM cell lines displayed significant differences in distribution and the expression levels of TRAFs; rhsCD40L and CD40mAb stimulation could regulate the expression of TRAFs in different MM cells. In U8226, rhsCD40L and CD40mAb rapidly upregulated TRAF1 (MFI from 2.1 to 5.1). However, in U266, the same stimulation downregulated TRAF1 (MFI from 2.8 to 0.1). RhsCD40L downregulated TRAF2 (MFI from 4.6 to 1.6), TRAF5 (MFI from 3.2 to 0.9) and TRAF6 (MFI from 4.2 to 0.5) in XG2, while rhsCD40L upregulated TRAF2 (MFI from 11.6 to 13.3), TRAF5 (MFI from 2.5 to 11.1) and TRAF6 (MFI from 2.9 to 6.5) in XG7; down-regulation of TRAF2, TRAF5 and TRAF6 in MM cells triggered by rhsCD40L or CD40mAb increased the sensitivity of cells to the apoptosis (20%–25%); whereas the up-regulation of TRAF2 and TRAF6 resisted the apoptosis (3%–5%). Therefore, we suggested that the expression pattern of TRAFs in MM cells after stimulation with rhsCD40L and CD40mAb could result in the different responses of MM cells to CD40 signaling.

187 ISOLATION AND CULTURE OF HUMAN MULTIPLE MYELOMA MEDULLARY PLASMOCYTES Garderet L.*,1, Mazurier C.1, Lagrange M.1, Bouchet S.1, Frick J.1, Gorin N.1, Lopez M.1 1 Hopital Saint Antoine, Paris, France Studies of freshly isolated myeloma patient bone marrow samples are often limited because of massive and rapid cell death. We are developing a plasma cell culture, which keeps some of them alive after a week. Materials and methods: Bone marrow from five myeloma patients (stage III) at diagnosis were collected. Mononuclear cells were isolated by Ficoll Hypaque sedimentation, then plasma cells were purified using the anti-CD138 plasma cell isolation system (Miltenyi-Biotec). Purified plasma cells were used

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immediately or frozen in fetal calf serum. Fresh or thawed plasma cells were maintained in liquid culture in ISCOVE medium with IL-6, GM-CSF, IL-3 and SCF for 8 days. On day 0, 4 and 8, the plasmocyte culture was examined for viability (using trypan blue dye exclusion), morphology (air-dried cytospin stained with Wright-Giemsa) and cell surface phenotype (CD38 and CD138 monoclonal antibodies). After immunomagnetic bead separation, purity was 76% (55%–98%) and yield was 54% (11%–100%). On day 0, the culture was started with a cell range from 0.6 to 4.6 × 106 plasmocytes. After a 4-day culture, 41% of myeloma cells (15%–69%) were viable as assayed by trypan blue and checked by morphological examination. On day 8, the mean plasmocyte number decreased to 15% (4%–24%). In terms of viability, we didn’t observe any difference starting the culture with fresh plasma cells or after thawing. FACS analysis was very heterogeneous, CD138 decreasing steadily during the cell culture as cells were dying and failed to express Syndecan-1. CD38 had a higher expression compared to CD138 because of a more non-specific staining. In this preliminary study, we showed that the combined use of IL-6, GMCSF, IL-3 and SCF enabled some viability of medullary plasma cells during at least an 8-day culture and as such avoided the common early death of freshly uprooted myeloma cells.

188 STUDY OF THE t (12; 21) TRANSLOCATION IN CHINESE PEDIATRIC ACUTE LYMPHOBLASTIC LEUKEMIA PATIENTS Lin D.*,1 1 State Key Laboratory of Experimental Hematology, I, Tianjin, China The t (12; 21) is observed in approximately 20–25% of childhood B-lineage acute lymphoblastic leukemia (ALL) cases. This translocation results in the fusion of TEL gene on 12p13, and AML1 gene on 21q22. However, it is very difficult to identify t (12; 21) by conventional cytogenetic techniques (CCA), but can be easily found by fluorescence in situ hybridization (FISH) and RT-PCR analysis. Recently, we examined the t (12; 21)/TELAML1 fusion gene in bone marrow cells from 51 newly diagnosed Chinese pediatric ALL patients (41 B-lineage ALL, 10 T-ALL) by CCA, dual color I-FISH and RT-PCR. Eleven of them were TEL-AML1 positive by FISH, and four of them confirmed by RTPCR. All of the positive cases expressed B-lineage immunophenotype. None of the T-ALL cases were found with TEL-AML1 positive. The incidence of the t (12; 21) was 21.6% (11/51) in newly diagnosed pediatric ALL and 26.9% (11/41) in B-lineage, except two positive cases with no results. CCA showed 6 cases with normal karyotype, one with deletion of 12 chromosome and 11q23, one with somatic rob (14; 22), and only one showed dubious t (12; 21). In CR duration, four of the positive patients were reexamined by I-FISH, only one showed TEL-AML1 positive. But all of them showed TEL-AML1 positive by RT-PCR. Patients with t (12; 21) are characterized by an age between 1 and 10 years, B lineage immunophenotype, nonhyperdiploid DNA content and an excellent prognosis, making it a distinct subgroup of Chinese pediatric ALL. FISH and RT-PCR are sensitive, specific and reliable methods to be used in the diagnosis and prognosis.

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189 MODULATION OF ERYTHROPOIETIN RECEPTOR EXPRESSION BY HYPOXIA IN MULTIPLE MYELOMA CELL LINES Stewart J.*,1, Maxwell P.1, Lappin T.1 1 Queen’s University–Belfast, Belfast, UK Multiple myeloma (MM) is a malignancy of post-germinal center B lineage cells and is characterized by marked heterogeneity in karyotype and patient outcome. Anemia affects up to 70% of patients with MM and is multifactorial, involving physical replacement of normal hematopoiesis by tumor cells, renal failure and cytokines that contribute to the blunted erythropoietin (EPO) response. Recombinant human erythropoietin (rHuEPO) is now widely used to treat anemia in cancer patients. The EPO receptor (EPO-R) is expressed on the cell line, MM-S1 (Okuno et al., Biochem Biophys Res Comm 1990;170:1128) but few data have been reported concerning the expression of EPO-R in other MM cell lines and whether EPO affects their rate of proliferation. We have investigated the expression of EPO-R in both normoxia and hypoxia on four MM cell lines: U266-B1, KMS-18, KMS-11 and OPM-2. Using immunohistochemistry (IHC) we have shown that in normoxia OPM-2 and KMS-18 had a low level of expression, U266-B1 a moderate level of expression and KMS-11 had a high level of expression of EPO-R. The four MM cell lines were then incubated for 24 hr in hypoxia (1% O2, 5% CO2, balance N2). U266B1, KMS-11 and KMS-18 all showed a moderate upregulation of EPO-R in response to hypoxia whereas, interestingly, KMS-11 showed a marked downregulation in EPO-R expression. RT-PCR analysis showed that EPO-R mRNA levels were similar in cells incubated in either hypoxic or normoxic conditions, suggesting that in these MM cell lines EPO-R is regulated at the protein level in hypoxia. The focus of our current work is to establish whether EPO-R is functional, its effect on cell proliferation and the signal transduction pathways activated in MM cells. It may be important to establish the EPO-R status of myeloma cells of individual patients before treatment with recombinant EPO.

190 THE CORRELATION OF CD23 ANTIGEN EXPRESSION WITH BONE MARROW AND PERIPHERAL BLOOD PROLYMPHOCYTES DEPENDS ON THE ABSOLUTE LYMPHOCYTE COUNT INCREASING IN B-CLL Jurisic V.*,1, Colovic M.2, Colovic N.2, Milenkovic P.3, Kraguljac N.2 1 University of Kragujevac, School of Medicine, Kragujevac, Serbia, 2Institute of Hematology, Clinical Center of Serbia, Belgrade, Serbia, 3Institute for Medical Research, Belgrade, Serbia B-CLL is a malignancy characterized by accumulation of terminally differentiated B cells in different tissues. The different cell membrane molecules were appeared and diversely expressed at cell surface membrane on our cells during malignant process, identified B-CLL. Based on these findings we estimated and correlated percentage expression of CD23 between other membrane molecule in ration of bone marrow and peripheral blood prolymphocytes number, depending on the increase of absolute PBL number. Our

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study was performed in 67 B-CLL patients, including 49 male and 18 female with disease confirmation using routine hematological and biochemistry analyses, bone marrow biopsy, histological evaluation, antigen expression for the CD19, CD20, CD21, CD23, CD38, CD5, CD10 and HLA-DR molecules using panel of monoclonal antibodies (Becton Dickinson, Germany) in direct or indirect immunophenotyping method on gated cells by Flow Cytometry (Becton Dickinson, San Jose, USA), and classified by FAB and Binet clinical stages. The significant correlation between decrease of CD23 antigen expression depending on increase of bone marrow and peripheral blood pro-lymphocytes number (Pearson’s correlation, p ⬍ 0.01) as well as with increase of absolute PBL number were found. B-CLL patients showing PBL up to 40 × 109/L, high percentage of CD23 expressions is significantly higher (p ⬍ 0.01) in comparison to the patients with PBL showing more then 100 × 109/L, and advance clinical stage, where significantly decrease was found. The correlation of the percentage expression of CD23 molecules in terms of CD5 expression (over 70% versus below 70% expression) shows that advance disease and high expression of CD23 were associated with high percentage CD5 expression (Mann-Whitney U-test, p ⬍ 0.05). We concluded that high expression of CD23 molecules in B-CLL exist, but persistence of high tumor mass with high number of malignant cells in peripheral blood was associated with decrease of percentage CD23 expression, probably loss from cell membrane.

191 A CYTOGENIC STUDY OF BURKITT’S LYMPHOMA CELL LINES: DAUDI, NAMALWA AND RAJI M. Salehi1, R. Salehi1, M.H. Goyns2 1 Department of Genetics and Molecular Biology, Medical School, Isfahan University of Medical Sciences, Isfahan, Iran, 2 School of Sciences, Sunderland University, Sunderland, UK Since the Philadelphia chromosome in CLL, the association of specific structural and numerical chromosome abnormalities with certain types of malignancies has been appreciated. Relatively little progress has been achieved in the study of human lymphoma cell lines, especially the cytogenic changes and the degree of heterogeneity observed in different karyotypic variants of the subpopution of these cell lines. The cell lines were cultured in RPMI 1640 supplemented with 10% FBS. After preparing slides from the cells and trypsin treatment, the slides were stained in freshly prepared leishman’s stain. In some cases FISH were applied to confirm results of G-bandings using band specific probes and/or chromosome arm painting (CAP) probes. In Daudi and Raji the chromosome number of majarity of the cells were 46, this number for Namalwa was 45. The most common chromosome abnormality detected was translocation 8:14 (q24;q32), but many other abnormalities were also detected, eg. additional material on the short arm of chromosome 11 (Daudi), additional material on the q35 of chromosome 4 (Raji) and an HSR attached to the long arm of one of the chromosome 1q (Namalwa) among others. Discussion: Specific and non-random chromosome rearrangements in Burkitt’s lymphoma cell lines have been reported previously. Our results demonstrated some well-characterized chromosome abnormalities and also some variations in both the numerical and structural chromosomal abnormalities from those reported in other studies.

Some of these chromosome abnormalities also have reported from Burkitt’s lymphoma patents. Therefore characterizing these abnormalities might be of great importance in understanding the progression of the disease.

192 USE OF T CELL ASSOCIATED ANTIGEN MARKERS FOR PROGNOSIS IN B CELL CLL Berrebi A.*,1 1 Kaplan Medical Center, Rehovot, Israel Prognostic factors of CLL include clinical staging and various parameters such as lymphocyte doubling time, elevated serum beta2-microglobulin (β2-m), soluble CD23, and chromosomal aberrations 11q⫺, 12⫹, 17p⫺ as tested by FISH analysis. Immunophenotype is usually heterogeneous and except for the expression CD38 few markers have an important prognostic significance. During progression of disease the expression of CD5 is more pronounced and the total number of T cells expressed by CD3 decrease. We studied 625 cases of B CLL who presented at 3 Israeli Centers between 1988 and 2002. Following the analysis of the expression of CD5, CD3, CD38 and serum levels of IgG and β2-m we explored the relationships between these parameters and other disease and individual factors, such as gender, age at diagnosis, disease severity and survival time. Statistically significant correlations were found between β2-m and disease severity (p ⬍ 0.0001) (using both the Rai and Binet severity scores), absolute lymphocyte count at diagnosis (p ⬍ 0.0001), hemoglobin levels (p ⬍ 0.0001) and CD38 (p ⬍ 0.05). Several of the disease markers were significantly different for males and females, such as hemoglobin (p ⬍ 0.0001), CD38 (p ⬍ 0.05), and disease severity at diagnosis was significantly different between the sexes, with 62% of males at Binet level A compared with 77% of females (p ⬍ 0.0001). We found statistically significant correlations (univariate) between disease severity (using the Rai & Binet scores) and CD3. We also found a significant inverse correlation between CD3 and CD5. We therefore investigated the expression of T-associated antigen markers, as expressed by the ratio CD5-CD3/CD3. This ratio was significantly associated with disease severity both in a univariate model (p ⬍ 0.001) and in a multivariate regression model (p ⬍ 0.001), after adjusting for gender and disease type (typical and atypical). CD20 expression (known to be low in CLL) has also been tested. Interestingly, 70% of the patients in all stages had ⬎50% CD20⫹ cells; but no correlation was found with disease severity. The implications of the correlation between the high to low CD5-CD3/CD3 ratio, which is a mathematical expression of the inverse correlation between CD3 and CD5, and disease severity, may be used as a sensitive prognostic factor for clinical practice in the management of B-CLL.

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193 FLAVOPIRIDOL INDUCES CASPASE-DEPENDENT CLEAVAGE OF p27kip1 IN LEUKEMIC CELLS FROM B-CLL PATIENTS Billard C.*,1, Quiney C.1, Kern C.1, Tang R.2, Ajchenbaum-Cymbalista F.2, Kolb J.1 1 INSERM U365, Institut Curie, Paris, France, 2INSERM E355, Hopital Hotel-Dieu, Paris, France Flavopiridol, an inhibitor of cdk and other protein kinases, is known to induce in vitro apoptosis of malignant cells from B-cell chronic lymphocytic leukemia. We previously reported that BCLL cells express high levels of p27kip1, a negative regulator of the cell cycle, and that these levels are inverslely related to the ability of B-CLL cells to undergo apoptosis in vitro. As p27 is known to be down-regulated early during apoptosis through caspase-dependent cleavage in various cell lines including of Bcell phenotype, we investigated the effect of flavopiridol on p27 expression in leukemic cells from patients with B-CLL. Cells were induced to apoptosis by treatment with flavopiridol for 18 h, and Western blot experiments were performed on cell lysates with an anti-27 antibody recognizing both p27 and its cleaved form p23. Flavopiridol exposure resulted in inhibition of p27 expression as well as in the concomitant appearance of p23. The extent of these effects, which were observed in all the cases tested (n ⫽ 9), depended on each patient. No such effect of flavopiridol was found with p21cip1, another member of the Kip/Cip family. The p27 cleavage was reverted by simultaneous treatment of cells with zVDA-fmk, a general inhibitor of caspases, indicating that it was caspase-dependent. Flavopiridol-promoted p27 cleavage was found to be associated with several membrane, mitochondrial and nuclear events of apoptosis, and in particular with caspase 3 activation. Furthermore, the production of the anti-apoptotic nitric oxide (NO), due to NO synthase down-regulation, was also associated with p27 cleavage upon flavopiridol exposure. Because p27 can be considered as involved in the cell cycle arrest in B-CLL and thus in the resistance of patients to chemotherapy, our data that p27 is down-regulated by flavopiridol strenghtens the interest in designing new clinical trials of flavopiridol in combination with chemotherapy.

194 RESVERATROL INHIBITS GROWTH OF HUMAN MULTIPLE MYELOMA CELLS: INDUCTION OF DNA DAMAGE AND CELL CYCLE ARREST The Chinese University of Hong Kong, The Prince of Wales Hospital, Hong Kong Multiple myeloma (MM) is a clonal plasma cell malignancy in bone marrow, accounting 15% of all blood cancers. Yet, no effective treatment is available for curing this fatal disease. Most therapies for multiple myeloma patients are palliative. Resveratrol (Rv, 3,5,4′-trihydroxystilbene) exerts a wide variety of biological actions, including antioxidative action, inhibition of platelet aggregation, and anticancer activities. It naturally occurs in many plants, including grapes, peanuts and pines. In this study, we explored the possibility of usage of this natural agent as an adjuvant therapy

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for MM patients. We investigated the effects of Rv on growth of MM derived cells (U266, RPMI8226, OPM2, LP-1, ARH77, and IM9). Treatment of Rv for 4 days inhibited the cell growth and the inhibition apparently showed dose-dependent manner. Ten µM Rv treatment induced statistically significant cell growth inhibition (∼80%, p ⬍ 0.01) in all six cell lines while the treatments at 1 7mu;M showed ∼20% inhibition. To study the mechanism of this Rv-induced cell growth inhibition in MM cells, we examined the effects of Rv on DNA damage in the cells using a COMET assay. Results indicated that Rv significantly increased the tail length of COMET, suggesting the induction of DNA damage by Rv in these MM cell lines. With flow cytometry analysis, treatment of Rv can induce cell cycle arrest in G0/G1 phases in U266 and ARH77 cells while IM9 exhibits cell cycle arrest following with induction of apoptosis. Taken together, Rv induces DNA damage in the MM, leading to cell cycle arrest for DNA repairing or/and programmed cell death. Thus, it results in cell growth inhibition. In conclusion, our findings demonstrated the effective inhibition in MM cell growth by Rv and revealed the potential mechanism of this cell growth inhibition. It sheds light into development of new therapy for curing this disease.

195 THE RARa-PLZF ONCOGENE INHIBITS C/EBPa FUNCTION IN MYELOID CELLS Girard N.1, Haman A.1, Bouchard N.1, Labrecque J.1, Chen B.2, Chen Z.2, Chen S.2, Hoang T.*,1 1 Molecular Biology, University of Montre´al, Montre´al, PQ, Canada, 2State Key laboratory of Medical Genomics, Shanghai, China C/EBPα is essential for the formation of granulocytes and the maturation of myeloblasts. In acute promyelocytic leukemia (APL), the variant t(11;17) translocation produces two fusion proteins, PLZF-RARα and RARα-PLZF, and both proteins are required for leukemogenesis. We show that RARα-PLZF inhibits promyelocyte differentiation through direct interaction with C/EBPα, and recruitment of HDAC1 to silence C/EBPα target genes. Furthermore, interaction with RARα-PLZF relocalizes C/EBPα from punctate nuclear domains into a diffuse nuclear distribution. Finally, our data indicate that C/EBPα activity is severely impaired in leukemic cells from patients with t(11;17) APL, as compared to the t(15;17)APL, which is more amenable to treatment. Since C/EBP7alpha; function is decreased in several other myeloid leukemias, our observations suggest that C/EBPα has a tumor suppressor function in the myeloid lineage. In addition, the present study provides a molecular basis for the collaboration between PLZFRARα and RARα-PLZF in the pathogenesis of APL. Finally, most oncogenes affect pathways that control cell proliferation or apoptosis. Despite the recognition that tumors of diverse histological origins exhibit impaired differentiation associated with distinctive clinical features, differentiation arrest is less well characterized at the molecular level. APL harboring the t(15;17) translocation is responsive to differentiation therapy with retinoic acid or arsenic trioxyde whereas the t(11;17) APL is a more aggressive disease with poor prognosis. While both translocations disrupt the RARα pathway, in t(11;17) APL, C/EBPα function is severely impaired by the RARα-PLZF oncogene, and this inhibition is reversed by

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inhibiting histone deacetylase activity. Together with recent studies, our observations reveal the mechanism through which a new type of modifier oncogenes subverts differentiation, providing support for transcription therapy as an additional approach to APL and possibly other acute leukemias.

196 MYELOID EXPRESSION OF THE BPI (BACTERICIDAL/ PERMEABILITY INCREASING PROTEIN) GENE IS REGULATED BY AML1, Sp3, PU.1 AND C/EBP Lennartsson A.*,1, Pieters K.1, Vidovic K.1, Gullberg U.1 1 Lund University, Lund, Sweden BPI (bactericidal/permeability increasing protein) is a 55kDa cationic protein polypeptide with cytotoxic and opsonic activities against gram-negative bacteria, initially identified in azurophil (primary) granules of neutrophils. BPI is also present in the secondary granules of human eosinophils and on the surface of monocytes and certain epithelia. BPI neutralises the pro-inflammatory effects of LPS and is of potential clinical use in the treatment of fulminant gram-negative infections. A low content of BPI in neutrophils from newborns may be related to increased susceptibility for infections and lack of BPI-expression is included in some cases of specific granule deficiency. The aim of the present work was to functionally characterise the proximal promoter of BPI in myeloid cells. Two alternative transcription start sites at ⫺22 and ⫺52, respectively, from the translation start, were identified by RACE-technique. Progressive deletion of a 2.2 kbp promoter identified 80 bp to be critical for high transcriptional activity. These 80 bp contain potential binding sites for the transcription factors AML-1, C/EBP, PU.1 and Sp1/Sp3. Binding of transacting factors, as judged by EMSA, verified direct binding of AML1, PU.1, Sp3 and C/EBP. Moreover, different C/EBP isoforms, when transiently transfected to HeLa cells, induced transcription from the proximal promoter. The identity of the specifiv isoform of C/EBP that is involved in the activation of the proximal BPI-promoter is unclear, but antibodies to C/EBPbeta and C/EBPdelta dramatically inhibited the protein-oligo interaction, while C/EBPepsilon had a more moderate affect. The previous report that SGD-patients with homozygous inactivation of C/EBPepsilon show absence of BPI-expression, is consistent with a role of C/EBPepsilon in regulation of the BPIgene. In summary, we have characterised the proximal promoter of the BPI-gene and identified AML1, PU.1, Sp3 and C/EBP as important transactivating factors for myeloid expression of BPI.

197 SUPPRESSOR OF CYTOKINE SIGNALLING-3 (SOCS3) IS A KEY NEGATIVE REGULATOR OF IL-6 Nicola N.*,1, Croker B.1, Krebs D.1, Zhang J.1, Wormald S.1, Metcalf D.1, Hilton D.1, Alexander W.1, Roberts A.1 1 The Walter & Eliza Hall Institute, Melbourne, Australia The SOCS family of proteins are attractive candidates as physiological regulators of IL-6 signalling. SOCS1 and SOCS3 are highly homologous and the expression of both is induced by IL-6 in haemopoietic cells. In overexpression systems, both can inhibit

STAT activation after gp130 ligation by IL-6. However, IL-6 signalling is not perturbed in primary cells from SOCS1 knockout mice. We have therefore concentrated on investigating the role of SOCS3 in IL-6 signalling in primary cells. A conditional gene targeting strategy was pursued because SOCS3 knockout mice die early in utero of placental failure. We have generated mice in which either haemopoietic or hepatic cells have no functional SOCS3 alleles, by using mice bearing a Lox-P flanked allele of SOCS3 (fl) and a null allele (o), and intercrossing them with mice expressing Cre recombinase selectively in haemopoietic cells (LysMCre or VavCre) or hepatic cells (AlbCre). Nintey-five percent of bone marrow (BM)-derived macrophages from LysMCre⫹ SOCS3fl/o mice and haemopoietic cells of all lineages from VavCre⫹ SOCS3fl/o mice are totally deficient in SOCS3. Similarly, livers from AlbCre⫹SOCS3fl/o are SOCS3 deficient. In all SOCS3 deficient cells from these mice, marked abnormalities in IL-6 signalling and cellular responses were observed. Following stimulation with IL-6, STAT3 phosphorylation was both increased and prolonged. Microarray analysis of mRNA from livers of IL-6-injected mice confirmed this excess signalling resulted in aberrant target gene transcription. Accordingly, cellular responses to IL-6 were amplified in the absence of SOCS3: (i) IL-6 inhibition of M-CSF-induced macrophage proliferation was increased from 30% of baseline in wildtype cells to 65% in SOCS deficient cells (p ⬍ 0.01); and (ii) in colony formation dose-response experiments, SOCS3 deficient myeloid progenitor cells were hypersensitive to IL-6. These data clearly demonstrate that SOCS3 is a physiological negative regulator of IL-6 signalling.

198 SHIP IS REQUIRED FOR ENDOTOXIN TOLERANCE Sly L.1, Rauh M.1, Kalesnikoff J.1, Krystal G.*,1 1 Terry Fox Laboratory,Vancouver, BC, Canada Lipopolysaccharide (LPS, aka endotoxin), a major glycolipid in the outer membrane of Gram-negative bacteria, stimulates monocytes, macrophages, mast cells and neutrophils to produce proinflammatory cytokines, such as TNFα, IL-1 and IL-6, and nitric oxide (NO). Overproduction of these pro-inflammatory molecules can lead to septic shock and death. Interestingly, however, an initial LPS exposure induces a transient state of cell refractoriness to subsequent LPS exposure, a phenomenon referred to as “endotoxin tolerance”. As a result, cells previously exposed to LPS produce far less pro-inflammatory cytokines and NO upon subsequent challenge with LPS. Since endotoxin tolerance is characterized by a reduced NFκB activation and we recently found that the SH2containing inositol phosphatase, SHIP, is a potent inhibitor of NFκB in bone marrow derived mast cells (BMMCs), we asked if SHIP could perhaps be involved in this phenomenon. Using SHIP⫹/⫹ and SHIP⫺/⫺ bone marrow derived macrophages (BMmφs) and mast cells (BMMCs), we found that SHIP⫺/⫺ cells do not display endotoxin tolerance. Moreover, pretreatment of wild-type BMmφs or BMMCs with LPS increases the level of SHIP, but not SHIP2 or PTEN, and this increase is responsible for the refractoriness to subsequent LPS treatment. Interestingly, this LPS-induced increase in SHIP protein is mediated by the production of autocrine-acting TGFβ. In vivo studies with SHIP⫹/⫹ and ⫺/⫺ mice confirm our ex vivo findings and strongly implicate SHIP as an essential mediator of endotoxin tolerance.

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199 DEFICIENCY OF p21cip1 /WAF1 RENDERS MURINE LTC-IC PRIMITIVE CELLS MORE SENSITIVE TO IL-11 TO PROLIFERATE AND SELF-RENEWED Dumesnil De Maricourt C.1, Sekkaı¨ D.1, Schiavon V.1, Catelain C.1, Vainchenker V.1, Bennaceur-Griscelli A.*,1 1 Institut Gustave Roussy, Villejuif, France The negative regulator p21cip1/Waf1 of the G1 cdk seems to be critical in protecting the stem cell compartment. Increased cell cycle leads to a massive depletion of stem cells in bone marrow from mice p21⫺/⫺ under conditions of stress after serial transplantation. In this work, we asked if p21 could modulate stem cell fate by an intrinsic mechanism or by a differentially sensitivity to microenvironmental determinants. Under homeostatic conditions, the absence of p21 leads to a 2-fold decreased number of bone marrow Lin⫺Sca-1⫹ c-kit⫹ stem cells (6.4% in p21⫹/⫹ versus 13.5% in p21⫺/⫺ mice, p ⫽ 0.003 n ⫽ 10). A same decrease was observed in the CD34⫹ and CD34⫺ cell sub-population derived from Lin⫺ Sca-1⫹ c-kit⫹ stem cells. Although the frequency of LTC-IC was similar in the Lin⫺ Sca-1⫹ c-kit⫹ cells (1/55), the proliferative capacity of LTC-IC was 2 fold decreased in p21⫺/⫺ mice as compared to the p21⫹/⫹ mice (4.4 CFC per LTC-IC versus 8.8, respectively). The viability of Lin⫺ Sca-1⫹ c-kit⫹ cells was not altered in the absence of p21. These data indicate that loss of p21 might induce the differentiation of LTC-IC. In presence of mSCF and FLT3-L, we observed a synergistic proliferative response of the Lin⫺Sca-1⫹ c-kit⫹ cells with low dose of IL-11 (0.1 ng/ml or 1 ng/ml) only in p21⫺/⫺ mice. The increased cell cycling leads to a 5-fold expansion of progenitors and a higher LTC-IC potential after 5 days in culture (13.5 CFC derived LTC-IC from 50 input p21⫺/⫺ cells versus 2 ). In contrast, high dose of IL-11 (50–100 ng/ml), leading to an optimal expansion of wild type hematopoietic stem cells, induced an exhaustion of p21⫺/⫺ LTC-IC. We found similar results when Lin⫺Sca-1⫹ c-kit⫹ cells were cultured with low or high dose of IL-6. These results suggest that p21 migth modulate the gp130/Stat3 signalling pathway by a negative feetback control, and thus renders stem cells more sensititive to this cytokine-familly.

200 GSK3B-MEDIATED POST-TRANSLATIONAL REGULATION OF CyclinD IN NORMAL VS. BCR/ABL HEMATOPOIETIC CELLS Dao M.*,1, Salesse S.1, Verfaillie C.1 1 Stem Cell & Cancer Center, University of Minnesota, Minneapolis, MN, USA GSK3b is a serine/threonine kinase which, upon phosphorylation by AKT, becomes inactive. During S phase, GSK3b phosphorylates nuclear cyclinD prior to cyclinD nuclear export and degradation. We and others have reported enhanced cyclinD transcription in BCR/ABL hematopoietic cells; however, little is known about the role of BCR/ABL in post-translational regulation of cyclinD. Because BCR/ABL activates PI3K/AKT, we hypothesize that BCR/ABL indirectly inactivates GSK3b, interfering with degradation of cyclinD protein. To test our hypothesis, we transduced a human megakaryoblastic cell line with MSCV-BCR/ABLIRES-GFP vs. MSCV-GFP and monitored the regulation of cyclinD

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protein by time-course subcellular fractionation to track the temporal and spatial distribution of cyclinD, metabolic labeling to measure its stability, co-immunoprecipitation to determine its binding partners, and GST-pRB kinase assay to assess its activity. In control cells, total cyclinD decreased as cells progressed through cell cycle; nuclear cyclinD showed patterns of phosphorylation while GSK3b did not. Treatment with LiCl (GSK3b-inhibitor) or with MG132 (proteasome inhibitor) increased detection of cyclinD protein in MO7e cells. In contrast, MO7e/BCR-ABL cells showed maintenance of cyclinD protein level with enhanced phosphorylation of pRb; nuclear cyclinD showed less phosphorylation while GSK3b showed more phosphorylation. Of note, in BCR/ABL cells, cytoplasmic cyclinD sequestered p21Cip1, a cell cycle inhibitor. STI571 treatment reduces detection of phosphorylated GSK3b and reduces cyclinD protein in BCR/ABL-cells; MG132 treatment did not affect cyclinD amount in BCR/ABL cells. We thus conclude that BCR/ABL interferes with cyclinD protein degradation by indirectly inactivating GSK3b, a kinase that phosphorylates cyclinD prior to its nuclear export.

201 REQUIREMENT FOR THE PI3K/Akt PATHWAY IN MEK1-MEDIATED GROWTH AND PREVENTION OF APOPTOSIS: IDENTIFICATION OF AN ACHILLES HEEL IN LEUKEMIA Steelman L.*,1, Shelton J.1, Blalock W.1, Navolanic P.1, Mccubrey J.1 1 Brody School of Medicine, East Carolina University, Greenville, NC, USA The Raf/MEK/ERK kinase cascade plays a critical role in transducing growth signals from activated cell surface receptors. Using ∆MEK1:ER, a conditionally-active form of MEK1 which responds to β-estradiol or the estrogen receptor antagonist 4 hydroxy-tamoxifen (4HT), we previously documented the ability of this protein kinase to abrogate IL-3 dependency of human (TF-1) and murine (FDC-P1 and FL5.12) hematopoietic cell lines. Here we demonstrate the ability of ∆MEK1:ER to activate the phosphatidylinositol 3-kinase (PI3K)/Akt/p70S6K pathway and prevent apoptosis. MEK1-responsive cells could be maintained long-term in the presence of β-estradiol, 4HT or IL-3. Removal of hormone led to rapid cessation of cell proliferation and induction of apoptosis in a manner similar to cytokine-deprivation of the parental cells. Stimulation of ∆MEK1:ER by 4HT or β-estradiol resulted in activation of ERK, PI3K, Akt, p70S6K and cellular proliferation. Treatment with PI3K, Akt and p70S6K inhibitors prevented MEK1responsive growth and the apoptotic effects of these inhibitors were synergistically enhanced 10- to 50-fold upon co-addition of MEK inhibitors. PI3K activity was necessary for MEK1-responsive proliferation as constitutive Akt activity was unable to support growth in the presence of the PI3K inhibitor. Cells transduced by MEK1 or MEK1 ⫹ Akt displayed different sensitivities to signal transduction inhibitors targeting these pathways. These results indicate a requirement for the activation of the PI3K pathway during MEK1-mediated transformation and provide important clues as to why treatment targeting multiple growth/signal transduction pathways may be more efficacious than therapy aimed at inhibiting a single pathway.

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202 DIFFERENTIAL EFFECTS OF KINASE CASCADE INHIBITORS ON NEOPLASTIC AND CYTOKINEMEDIATED CELL PROLIFERATION Shelton J.*,1, Moye P.1, Steelman L.1, Franklin R.1, Mccubrey J.1 1 Brody School of Medicine, East Carolina University, Greenville, NC, USA The effects of Raf, MEK and PI3K inhibitors on conditionally transformed hematopoietic cells were examined to determine if these compounds would display cytotoxic differences between cytokine and oncogene-mediated proliferation and whether inhibition of both pathways was a more effective means to induce apoptosis. Proliferation in the FD/∆Raf:ER cells was conditional and occurred when either IL-3 or the estrogen receptor antagonist 4-hydroxytamoxifen (4HT), which activates the conditional oncogene (∆Raf:ER), were provided. Thus, upon addition of the signal transduction inhibitors and either IL-3 or 4HT, the effects of these drugs were examined in the same cell under “cytokine” and “oncogene” mediated growth conditions. Drug concentrations around the reported IC50 for the Raf inhibitor L-779,450 suppressed DNA synthesis and induced apoptosis in hematopoietic FDC-P1 cells transformed to grow in response to either Raf-1 or A-Raf (FD/ ∆Raf-1:ER and FD/∆A-Raf:ER), but displayed less effects on DNA synthesis and apoptosis when the cells were cultured in IL-3. This Raf inhibitor was less effective in suppressing the proliferation or inducing apoptosis in B-Raf or MEK1 responsive cells demonstrating the specificity of this drug. The PI3K inhibitor LY294002 suppressed Raf-mediated growth indicating that part of the long term proliferative effects mediated by Raf are PI3K-dependent. Simultaneous inhibition of both Raf/MEK/ERK and PI3K/Akt pathways proved a more efficient means to suppress proliferation and induce apoptosis at 10- to 100-fold lower drug concentrations, suggesting that it may be possible to arrest leukemic cell growth at drug concentrations that have less toxic side effects.

203 VERY LOW OXYGEN CONCENTRATION (0.1% O2) FAVOURS THE G0 RE-ENTRY OF CYCLING CD34ⴙ CORD BLOOD CELLS Hermitte F.*,1, Brunet De La Grange P.1, Belloc F.2, Praloran V.1, Ivanovic Z.3 1 Universite´ Victor Segalen, Bordeaux, France, 2Laboratoire d’he´matologie Hoˆpital de Haut-Le´veˆque, Pessac, France, 3 Etablissement Franc¸ais du Sang — Aquitaine — Limou, Bordeaux, France Little is known about oxygen concentration in bone marrow and its role in the regulation of haematopoiesis. Indeed, O2 concentration is around 2–4% in dog bone marrow vessels and an analysis using a Kroghian model proposed the hypothesis of anoxic areas in bone marrow. We previously showed that haematopoietic stem cells (HSC) are better maintained in cultures at 3% to 1% O2 than at 20% O2, but 1% O2 limits CFC expansion. In this work, we investigated the effect of 0.1% O2 concentration on survival and proliferation of HSC, by culturing cord blood (CB) CD34⫹ cells (50000 cells/mL) up to 72 h in a serum free medium with Interleukin-3 (20 ng/mL). Cell viability was evaluated by trypan blue

exclusion, and the rate of apoptosis (Annexin V/Propidium Iodide (PI)) and cell cycle status (Ki-67/PI) assessed by flow cytometry. Culture at 0.1% O2 did not modify cell survival but affected cell cycle after 72 h of culture since 48.7 ⫾ 13.7% of cells remained in G0 (48.2% ⫾ 12.1 at T0) compared to 13.2 ⫾ 6% at 20% O2. Inversely, the percentage of cells in G1 decreased at 0.1% O2 (36.2 ⫾ 16.2%) while it increased to 66.7 ⫾ 5% at 20% O2 (50.4 ⫾ 12.1% at T0). Finally, 0.1% O2 concentration did not modify G1 to SG2M progression. To explore this phenomenon, we separated undivided and divided cells by PKH26 labelling using a cell sorter and analysed their cell cycle. We evidenced that 44.7 ⫾ 21.5% of cells cultured at 0.1% O2 re-entered in G0 after division, against only 8 ⫾ 0.3% at 20% O2. In conclusion, very low O2 concentration, probably related to those found in stem cells areas of bone marrow, induces the G0 re-entry of divided CD34⫹ cells. The mechanisms responsible of these phenomena are under investigation.

204 IDENTIFICATION AND CHARACTERISATION OF THE ASBs AND INTERACTING PROTEINS Debrincat M.1, Zhang J.G.2, Willson T.2, Hilton D.2, Nicola N.2, Martin H.*,1 1 CRC for Cellular Growth Factors, Melbourne, Australia, 2The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia The suppressor of cytokine signalling (SOCS) proteins contain a conserved carboxy-terminal motif termed the SOCS box. This motif couples the SOCS proteins and their binding partners to the elongin B/C complex, thereby targeting them for proteasomal degradation. A sub-family of SOCS proteins, the Ankyrin repeatcontaining SOCS Box proteins (Asbs), contain an ankyrin repeat domain upstream of the SOCS box. The function of these proteins is as yet undefined. The ankyrin repeat is a loosely conserved sequence of approximately 33 amino acids found in proteins that display a wide variety of functions including receptors, cell cycle regulators, secreted proteins, tumour suppressors and transcription factors. Crystallography studies and NMR analysis have shown that the ankyrin repeat motif is involved in protein-protein interactions. Each repeat comprises a V-shaped helix-turn-helix motif, linked together by loops with repeats stacked in bundles. Since the Asb family has been defined, 18 murine Asbs have been identified. Throught a number of biochemical and genetic techniques, Asbs 1–4 have previously been examined. We continue to explore this novel protein family with an emphasis on investigating Asbs and their interacting protein partners. This is being carried out through the generation of GST fusion proteins in bacterial systems and the production of FLAG-tagged protein in mammalian cells. This investigation may reveal whether the Asbs act as negative regulators of signaling like the SOCS proteins, or have other biological roles.

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205 ABILITY OF ACTIVATED PI3K/Akt ONCOPROTEINS TO SYNERGIZE WITH MEK1 AND ABROGATE CYTOKINE-DEPENDENCE OF HEMATOPOIETIC CELLS Shelton J.*,1, Blalock W.1, Steelman L.1, Mccubrey J.1 1 Brody School of Medicine, East Carolina University, Greenville, NC, USA The Raf/MEK/ERK and PI3K/Akt kinase cascades play critical roles in transducing growth signals. Using conditional and constitutively active forms of MEK1 and either PI3K or Akt, we demonstrate synergy between these kinases in relieving cytokine-dependence of FDC-P1 and FL5.12 hematopoietic cell lines. MEK1-responsive cells that grew in the absence of exogenous cytokines were obtained from ∆MEK1:ER-infected cells at a frequency of 5 × 10⫺5, indicating a low frequency of cells became MEK1-responsive. Activated (Act) PI3K or Akt by themselves did not abrogate cytokine-dependence. In contrast, MEK1-responsive cells were recovered approximately 40- to 200-fold more frequently after infection with ∆MEK1:ER and either activated PI3K or Akt. ∆MEK1:ER-responsive cells were grown in the presence of βestradiol or the estrogen-receptor antagonist 4-Hydroxy-Tamoxifen (4HT). Removal of β-estradiol or 4-HT led to the cessation of cell growth, but apoptosis was delayed in cells expressing ∆MEK1:ER and either ∆Akt:ER or PI3K(Act). ∆MEK1:ER⫹ ∆Akt:ER and ∆MEK1:ER⫹PI3K(Act) cells proved more difficult to arrest at G0/G1 after IL-3 and 4HT starvation than cells expressing only ∆MEK1:ER or ∆Akt:ER. These cells were 5- to 20-fold less sensitive to MEK, PI3K, Akt, and p70S6K inhibitors in terms of induction of apoptosis and inhibition of DNA synthesis. Although cytokine-dependent ∆MEK1:ER or ∆Akt:ER cells did not produce any detectable autocrine component, GM-CSF mRNA transcripts were detected in the MEK1-responsive cells indicating that activated ∆MEK1:ER and PI3K/Akt induce a pathway leading to autocrine proliferation. These cell lines will be useful for elaborating the mechanisms by which the Raf/MEK/ERK and PI3K/Akt pathways interact to regulate cell proliferation and apoptosis.

206 NEUTROPHIL PRO-PROTEINASE 3 INDUCES S-PHASE ARREST IN GRANULOPOIETIC PROGENITORS BY INHIBITION OF RIBONUCLEOTIDE REDUCTASE MEDIATED BY PI3-KINASE, Akt/PKB, AND NITRIC OXIDE Sko¨ld S.*,1, Gullberg U.1, Olofsson T.1 1 Department of Hematology, Lund, Sweden Enzymatically inactive proforms of proteinase 3 (proPR3) secreted by promyelocytes can downregulate the proliferation of myeloid progenitors, measured as a reduced fraction of CFU-GM in S-phase. The S-phase downmodulatory effect has a fast onset within 90 min, is reversible, non-cytotoxic and counteracted by G-CSF or GM-CSF. The fast onset suggests a direct interruption of DNA-synthesis, i.e. an S-phase arrest. We now present evidence that the S-phase arrest induced by proPR3 is mediated by a signal transduction pathway engaging phosphatidylinositol 3-kinase (PI3K), Akt/PKB, inducible nitric oxide synthase (iNOS), NO and

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ribonucleotide reductase (RR). Inhibition of PI3K by wortmannin or Akt/PKB by 1L-6-hydroxymethyl-chiro-inositol 2-[(R)-2-Omethyl-3-O-octadecylcarbonate] abrogate the S-phase arrest induced by proPR3. The non-specific NOS-inhibitor NG-monomethyl-L-arginine, the iNOS-specific inhibitor 1400W or the nitric oxide scavenger carboxy-PTIO all abrogate the S-phase downmodulatory effect of proPR3. Furthermore, the effect of proPR3 is abolished by providing 2′-deoxyadenosine and 2′-deoxyguanosine to the cells, suggesting inhibition of ribonucleotide reductase, probably through direct inhibition by NO. The S-phase arrest induced by proPR3 is reproduced by SNAP, a cell-permeable NO-donor, an effect that is readily reversed by carboxy-PTIO or 2′-deoxyadenosine/2′-deoxyguanosine supplementation. In conclusion, we suggest that the S-phase downmodulatory activity of proPR3 towards granulopoietic progenitors is an S-phase arrest caused by inhibition of ribonucleotide reductase by nitric oxide, mediated through PI3K, Akt/PKB, and iNOS.

207 THE ANTIPROLIFERATIVE EFFECT OF A PLANT CYTOKININ ANALOGUE WITH INHIBITORY ACTIVITY ON CYCLIN-DEPENDENT KINASES (CDK) IS ASSOCIATED WITH CELL CYCLE DELAY RATHER THAN WITH CELL CYCLE ARREST Vermeulen K.1, Venken T.1, Strnad M.2, Havlicek L.2, Van Onckelen H.3, Lenjou M.1, Nijs G.1, Van Bockstaele D.1, Berneman Z.*,1 1 Antwerp University Hospital, Edegem, Belgium, 2Palacky University, Olomouc, Czech Republic, 3University of Antwerp, Wilrijk, Belgium The activity of cyclin-dependent kinases (CDK), the key regulators of the cell cycle, is strictly controlled by different mechanisms but is often altered in human cancer. This has stimulated the search for molecules targeting CDK activity as anti-cancer agents. Different types of CDK inhibitors have been described, among them, C2-, N6-, N9-substituted purines such as plant cytokinin analogues. We have previously reported the inhibition of CDK1 and CDK2 activity by a new group of plant cytokinin analogues (Vermeulen et al., Leukemia 2001;16:299). These compounds inhibit proliferation of leukemic cell lines and induce apoptosis, which is initiated by the mitochondrial pathway (Vermeulen et al., Experimen Hemat 2002;30:1107). In this study, the mechanism of CDK inhibitorinduced apoptosis was further characterized. The expression of two Bcl-2 family members (Bcl-2 and Bax) was examined in malignant hematopoietic cell lines after treatment with CDK inhibitors. Bcl2 prolongs cell survival by inhibiting apoptosis, while Bax counteracts the ability of Bcl-2 to inhibit apoptosis. No clear relationship was found between baseline Bcl-2 and Bax protein expression and the inhibition of cell growth after incubation with the CDK inhibitor 2-(1-ethyl-2-hydroxyethylamino)-6-(p-hydroxybenzylamino)-9isopropylpurine (P19). No changes were observed in the expression of the Bcl-2 and Bax protein after treatment with the CDK inhibitor. We also investigated the influence of CDK inhibitors on the progression of cells through the cell cycle. When Jurkat cells were synchronized either at the G1/S or the G2/M boundaries of the cell cycle, addition of the CDK inhibitor markedly delayed but did not block progression through the cell cycle. This probably reflects the

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interaction with cell cycle regulation at both cell cycle transitions following inhibition of CDK1 and CDK2 activity. It also illustrates that, contrary to previously held views, chemical agents active on the cell cycle need not bring about a complete cell cycle block.

208 INDUCTION OF ERYTHROID DIFFERENTIATION THROUGH THE INHIBITION OF THE EPIDERMAL GROWTH FACTOR RECEPTOR KINASE ACTIVITY IN FRIEND ERYTHROLEUKEMIA CELLS Brozik A.*,1, Apati A.1, Magocsi M.1 1 National Medical Centre, Budapest, Hungary Specific-receptor tyrosine kinase inhibitors are promising candidates in cancer therapy by preventing unregulated growth signals typical of malignant cells. On the basis of the recent finding that activation of the ERK mitogen activated kinase pathway counteracts erythroid differentiation, we have tested an EGF receptor tyrosine kinase inhibitor as a possible erythroid differentiation-inducing agent. Tyrphostin 51 turned out to be a potent chemical for that purpose at concentrations of 20–80 µM, as it effectively induced hemoglobin production of Friend erythroleukemia cells (line F46) in 4 days. ERK1 and ERK2 MAP kinases function in a protein kinase cascade that plays a critical role in the regulation of cell growth. ERK proteins require dual phosphorylation at specific tyrosine and serine residues of a TEY motif for maximal enzyme activation. Administration of tyrphostin 51 to F 4-6 cells resulted in a distinct decrease of ERK activity in 6 hours, similar to the MEK (MAPK kinase) inhibitor UO126. The downregulation of the phospho-ERK level in both cases were completely suspended by either tyrosine or serine/threonine phosphatase inhibitors (sodium ortovanadate and ocadaic acid, respectively). In connection with that, phosphatase inhibitors prevented tyrphostin, UO126 and DMSO induced hemoglobin production as well. Investigating the potential involvement of the p38 MAP kinase in tyrphostin-induced erythroid differentiation, we found an elevated phospho-p38 level after tyrphostin 51 treatment. Our results indicate that tyrphostin 51, by inhibiting growth signals that affect the cell through the EGF receptor, causes a significant drop in ERK activity, which is sufficient for induction of hemoglobin production in F4-6 murine erythroleukemia cells.

209 A LETHAL VARIANT OF PYRUVATE KINASE DEFICIENCY ASSOCIATED WITH A MISSENSE MUTATION (G409A) AND A LARGE DELETION IN THE LR-PK GENE Fermo E.1, Bianchi P.*,1, Rodwell R.2, Cowley D.2, Zanella A.1 1 Ospedale Maggiore IRCCS, Milan, Italy, 2Mater Misericordiae Health Service, Queensland, Australia PK deficiency is the most common hereditary enzyme defect of the glycolytic pathway resulting in chronic non spherocytic haemolytic anaemia. More than 130 different mutations have been identified. Among them only one large deletion has been described

in Gipsy resulting in the loss of exon 11. We described a case of severe haemolytic anaemia due to PK deficiency caused by a missense mutation and a deletion of 5006 nucleotides. An Australian baby was studied. At birth he presented hepatomegaly, haemoglobin 8.9 g/dL, bilirubin 275 µmol/L, and ferritin ⬎ 4000 ng/mL. The father was beta-Thalassemia carrier, the mother apparently normal. The baby died during exchange transfusion; the parents refused post-mortem, however the liver histopathology had no evidence of neonatal haemocromathosis. The mother underwent a second pregnancy, interrupted at 8th week of gestation. Both parents showed intermediate values of PK activity compatible with a heterozygous state. The molecular study of LR-PK gene performed on the baby’s DNA led to the detection of a new homozygous mutation G409A (Ala137Thr). It was detected at the heterozygous level in the mother but not in the father. The study of father reticulocytes RNA revealed a large cDNA deletion encompassing exon 4 and exon 11 included. DNA analysis of father and baby showed the presence of a large deletion extending from intron 3 to the last 3 nucleotides of exon 10. The loss of donor consensus sequence between exon 10 and intron 10 probably doesn’t permit the correct splicing of exon 11, resulting in the deletion of exons 4-11. In conclusion this case could be considered one of the most severe reported in PK deficiency. Large deletions may be missed since they are not detected with normal PCR methods.

210 THE INFLUENCE OF FARNESYL PROTEIN TRANSFERASE INHIBITOR, R1157777 ALONE AND IN COMBINATION WITH NEW PURINE NUCLEOSIDE ANALOGUES ON ACUTE MYELOID LEUKEMIA PROGENITORS IN VITRO Robak T.*,1, Korycka A.1 1 Department of Hematology, Medical University, Lodz, Poland The aim of our study was to evaluate the antiproliferative effect of the combination of R115777 (FTI, Janssen Research Foundation, New Jersey, USA) with new purine nucleoside analogues (PNAs): cladribine (2-chlorodeoxyadenosine;2-CdA) and fludarabine (F-ara-A) on myeloid leukemia progenitors (CFU-L) from acute myeloid leukemia (AML) patients in culture in vitro. Our studies were based on the method of semisolid CFU-L and CFU-GM cultures described by Iscove et al. in our modification. Specimens of bone marrow were collected from 10 newly diagnosed patients with AML and from 10 hematologically normal patients. In case of AML CFU-L mononuclear cells were plated in serum free culture and in case of normal CFU-GM 20% of fetal calf serum were used. R115777 at the concentrations of 25; 50 and 100nM were used alone or together with 5, 10 and 20nM of 2-CdA or 0.2; 0.4 and 0.8µM of F-ara-A. We showed that R115777 used alone significantly inhibited the colony growth of AML CFU-L, as compared to normal CFUGM (p ⫽ 0.006; p ⫽ 0.004; p ⫽ 0.0005, respectively). We also observed that the drug used with 2-CdA or F-ara-A at all combinations significantly inhibited the colony growth of AML CFU-L, as compared to the control (p ⬍ 0.0005). Furthermore, the differences between AML CFU-L and normal CFU-GM colony growth inhibition after the use of R115777 either with 2-CdA or F-ara-A were statistically significant (p ⬍ 0.01). In case of AML CFU-L the interaction index was 0.89 for the combination of R115777 with 2-CdA and it was 1.16 for R115777 and F-ara-A.

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In conclusion, the combination of R115777 with both PNAs had as additive effect on AML CFU-L progenitors and it could be more effective than the drugs used alone. However, further experimental and clinical studies seem warranted.

211 EXPRESSION OF TETRASPANINS IN PERIPHERAL BLOOD LEUKOCYTES: A COMPARISON BETWEEN NORMAL AND INFECTIOUS CONDITIONS Tohami T.*,1, Drucker L.2, Radnay J.3, Shapira H.3, Lishner M.4 1 Oncogenetic Lab, Sapir Medical Center, Kfar-Saba, and Tel Aviv University, Tel-Aviv, Israel, 2Oncogenetic Lab, Sapir Medical Center, Kfar-Saba, Israel, 3Hematology Lab, Sapir Medical Center, Kfar-Saba, Israel, 4Oncogenetic Lab & Med Department, Sapir Medical Center, Kfar-Saba and Tel Aviv University, Tel-Aviv, Israel The tetraspanin proteins are involved in diverse processes of cell function and expressed in a variety of cell types but their exact role is not well understood. Aiming to expand the knowledge of tetraspanin cellular function we addressed in our study two primary issues. The first is characterization of the expression levels of a panel of tetraspanins in normal peripheral blood leukocytes (PBL) and the second is the assessment of their expression in a pathological condition. We studied the membranal and cytoplasmatic expression level of six tetraspanins (CD9, CD53, CD63, CD81, CD82 and CD151) in major PBL sub-populations (polymorphonuclear (PMN), monocytes, B and T lymphocytes) employing flow cytometry. Our results demonstrate that PMN are distinguished with dominant expression of cytoplasmatic CD63; Monocytes dominantly express CD53 both on membrane and in cytoplasm; CD82 is primarily expressed on T cell membranes; and B cells are typified with dominant membranal CD53 expression. This work represents the first comprehensive base line of tetraspanins’ expression in normal PBL. Next, we set out to evaluate the expression of tetraspanins on PBL in infections. We assayed their membranal levels and compared them to the normal base-line set in our earlier work. A major trend of down-regulation was demonstrated for the examined tetraspanin proteins, except CD63, in all PBL subtypes (p ⬍ 0.05). This finding is compatible with the necessary motility for leukocyte immune modulation and previous existing knowledge on tetraspanin function as metastasis suppressors.

212 EFFECT OF TGFa ˆ 1 ON DIFFERENTIATION OF CD105ⴙ MESENCHYMAL STEM CELLS FROM VARIOUS FETAL TISSUES Guo H.1, Liao L.*,1, Yang S.1, Cao D.1, Liu J.1, Wang Y.1, Zhao R.C.1 1 SKLEH, TEC, Chinese Academy of Medical Sciences, Tianjin, China CD105, as TGFβ type III receptor, is one of the important phenotypes of bone marrow mesenchymal stem cells (MSCs). CD105 forms a complex with TGFβ receptors I and II and modulates the response of cells to TGFβ1. In order to investigate whether CD105⫹ MSCs reside in diverse tissues and if TGFβ 1 play an

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important role in modulating the differentiation of MSCs, we first analyzed the distribution of CD105 in fetal tissues by immunochemistry, then isolated CD105⫹ MSCs cells from heart, liver, muscle, lung, derma, aortic arch and bone marrow, and identified their immunphenotypes by FACS. CD105, a TGFβ type III receptor, is one of the important phenotypes of mesenchymal stem cells (MSCs). Here antibody against CD105 was used to investigate whether MSCs reside in most tissues. We further isolated CD105⫹ MSCs from heart, liver, muscle, lung, derma, aortic arch and bone marrow(BM), studied their biological characters and investigated whether TGFβ1 affected the differentiation of these cells into adipocytes and osteoblasts. Results showed CD105⫹ MSCs, which could differentiate into adipocyts and osteocytes in vitro, existed in diverse tissues including aorta. It was observed that lipid droplets with membrane were secreted by cells during the processes of adipogenesis, and some needle-shaped crystal calcium deposition similar to bone spicules was also found inside the cytoplasm of induced ostocytes. During differentiation of MSCs into adipocytes, CD105 expression decreased and even terminated, strongly supporting that CD105 was a marker of MSCs. We also showed that triglyceride level in the culture medium increased when the medium was supplemented with dexamethasone. Furthermore, TGFβ1 could inhibit the differentiation of MSCs into adipocytes and osteocytes. Our investigation indicates that MSCs may play a role in atherosclerosis and hyperlipemia.

213 RHO GTPASES REGULATE HEMATOPOIESIS OF DENDRITIC CELLS IN CANCER Payal Watchmaker, Galina V. Shurin, Gudula Schmidt*, Michael R. Shurin Department of Pathology, Division of Immunopathology, University of Pittsburgh Medical Center, Pittsburgh, PA, USA *Institut fur Experimentelle und Klinische Pharmakologie und Toxikologie, Albert-Ludwigs-Universitat, Freiburg, Germany Dendritic cells (DC) perform an essential role in the generation and regulation of specific immune responses including antitumor responses. It is therefore not surprising that many tumors have evolved mechanisms that alter the function of DC. Recent works from this laboratory indicate that the generation, differentiation and maturation of DC (DC hematopoiesis) are markedly inhibited in cancer. However, molecular mechanisms and intracellular pathways involved in inhibition of DC hematopoiesis in cancer have not yet been explored. We have demonstrated that DC differentiation is markedly inhibited by tumors including oral squamous carcinoma. We have shown that expression and activity of Cdc42, a member of the Rho GTPase family, is regulated during DC differentiation. Tumors alter not only the endocytotic, migratory and antigen presenting activity of DC but also expression and activity of Cdc42 in DC’s. This suggests that Rho GTPases are involved during DC differentiation in cancer. Transduction of DC with viral vectors encoding constitutively active or dominant negative Cdc42 or other members of the Rho family, such as Rac1 and RhoA, caused opposite effects on endocytotic, migratory and adhesion of DC or mimicked effect of tumors. Rho GTPases act as a molecular switch, cycling between an active and inactive form, which is controlled by the opposite effects of the regulatory proteins GEFs, GDIs,

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and GAPs. We have shown that DC treated with tumors express altered levels of Rho-GDI-1 and ezrin (Rho GTPase activator). Since active Cdc42 is known to transduce signals to regulate cytoskeleton, endocytosis, transcription, cell cycle, and apoptosis, it will be important to determine which of the Cdc42-related pathways in DC are affected by tumor cells and thus may be responsible for dysregulation of DC hematopoiesis. This will help to direct development of the next generation of tumor vaccines and to correct abnormal hematopoiesis in cancer patients.

214 SELF-RENEWAL PROLIFERATION AND LYMPHOHEMATOPOIETIC DIFFERENTIATION OF HUMAN EMBRYONIC STEM CELLS IN CULTURE Cheng L.*,1, Zhan X.1, Dravid G.1, Ye Z.1, Hammond H.1 1 Johns Hopkins University School of Medicine, Baltimore, MD, USA We have used human embryonic stem (hES) cells to study mechanisms of human stem cell self-renewal and hematopoiesis. 1). We found that culture-expanded human marrow stromal cells (hMSCs) from adults can replace mouse embryonic fibroblasts to support hES cell expansion in an appropriate culture medium. The expanded hES cells (⬎1,000 fold in 7 weeks) retained undifferentiated markers (APase⫹, SSEA-4 & Oct-4), a normal karyotype and differentiation potentials in culture. Thus, adult human cells can also support prolonged expansion of hES cells in serum-free media. 2). We developed a culture condition to differentiate hES cells into both lymphoid and myeloid cells in culture, lasting for ⱖ6 weeks. Erythroid and myeloid colony-forming progenitors were also detected. In addition, We detected lymphoid-like (CD2⫹/CD16⫹ NK and at a lower level CD19⫹ B) cells. With adequate cytokines, hematopoietic cells expressing the MHC II (“professional APCs”) were also generated, which elicited allogeneic human T cell responses in MLR assays. Thus, hES cells can generate large numbers of lympho-hematopoietic cells over an extended time period. 3). We developed a method to stably transduce hES cells at high efficiency (~50%) by lentiviral vectors. Sustained GFP transgene expression was seen after 4-month continuous cultures and hematopoietic differentiation. Among generated CD45⫹ hematopoietic cells, 20% were strongly GFP⫹. Similar percentages of CD2⫹, CD14⫹ and MHC II⫹ cells were also GFP⫹. Taken together, we have developed a system using hES cells to study early events of human lympho-hematopoiesis and to explore new ways to expand postnatal human stem cells in culture.

215 UMBILICAL CORD BLOOD CD34ⴙCD45RAHICD7HI HEMATOPOIETIC PROGENITOR CELLS AS CANDIDATE FETAL THYMUS COLONIZING CELLS Haddad R.*,1, Izac B.2, Dumas B.1, Yagello M.1, Delezoide A.3, Gluckman J.1, Pflumio F.2, Canque B.1 1 INSERM EMI — 13, IUH, Paris, France, 2INSERM U474, Maternite Port-Royal, Paris, France, 3Laboratoire Foetopathologie, Hopital Robert Debre, Paris, France We examined the lymphoid differentiation potential of previously characterized umbilical cord blood (UCB) CD34⫹CD45RAhiCD-

7hi hematopoietic progenitor cells (HPCs) [Canque, Blood 2000;96:3748–3756]. Combining limiting dilution analysis and clonal assays, we show that although this population display a triple natural killer (NK) cell, B lymphocyte (L) and TL potential, it does not correspond to common lymphoid progenitors (CLPs) since their lymphoid differentiation capacity is markedly biased toward NK cells and TL. In contrast, CD45RAhiLin⫺CD10⫹ HPCs, which have been proposed as candidate CLPs, predominantly exhibit BL lineage potential. We show, in addition, that the CD45RAhiCD7hi and CD45RAhiLin⫺CD10⫹ populations are independent since they do not interconvert upon in vitro culture and exhibit different growth factor requirements to differentiate from common lympho-myeloid CD45RAintCD7⫺ HPCs. Analysis of fetal bone marrow, liver and thymus revealed in addition that both HPC populations are present as early as gestation weeks 10–12 in fetal bone marrow where they display similar lymphoid differentiation capacity than in UCB. Of interest, both populations are hardly detectable in weeks 10–21 fetal liver, indicating that, at variance with mice where lymphoid progenitors first differentiate in the liver, fetal bone marrow represents their primary site of emergence in humans. CD45RAhiCD7hi HPCs accumulate in the fetal thymus where they represent the most immature TCR⫺CD4⫺CD8⫺ triplenegative CD34⫹CD1a⫺ cells, and should thus also participate in fetal thymopoiesis. As expected of fetal NK cell and TL progenitors, CD45RAhiCD7hi HPCs wane after birth and are no longer detected in adult bone marrow. Conversely, CD45RAhiLin⫺CD10⫹ BL progenitors persist throughout post-natal life. Altogether, these results indicate that CD45RAhiCD7hi HPCs represent a major population of human fetal NK cell/TL progenitors and that they might correspond to thymus-colonizing cells.

216 INDIVIDUAL ADULT CD34ⴙAC133ⴙ HEMATOPOIETIC PROGENITORS ARE CAPABLE TO GENERATE ENDOTHELIAL PRECURSOR CELLS (EPC) Petzer A.*,1, Wo¨ginger A.1, Wolf D.1, Hoflehner E.1, Ho¨lzler K.1, Gastl G.1, Gunsilius E.1 1 Department of Hematology and Oncology, Innsbruck, Austria It has been proposed that hematopoietic and endothelial cells develop from a common mesodermal precursor, the hemangioblast. In this study, we report that single CD34⫹ hematopoietic progenitors (Colony Forming Units, CFU) from G-CSF mobilized peripheral blood (PB) that are first grown in methylcellulose supplemented with Epo, SCF, IL-3, IL-6 G-CSF and GM-CSF have the capacity to generate endothelial progenitor cells (EPC) when individual colonies are transferred into a specific endothelial growth medium containing vascular endothelial growth factor (VEGF), endothelial-cell growth supplement (ECGS) and stem-cell growth factor (SCGF). We analyzed thousands (2463) of single clones, each derived from individual CD34⫹ PB progenitor cells for their ability to generate EPCs. EPCs mainly generated out of CFU-GM and CFUM, also from more primitive CFU-Mix, but not from erythroid BFU-E and granulocytic CFU-G. The existence of EPCs was demonstrated with immunfluorescence-staining for CD31, CD45, CD146, KDR2 and von Willebrand Factor (vWF). We subsequently found that CD34⫹ cells expressing the surface antigen AC133 define a cell population with the highest frequency of hematopoietic

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progenitors capable to generate EPC. CD34⫹AC133⫹ cells mainly generated granulocytic and/or macrophage progenitors whereas the majority of erythroid BFU-E (up to 98%) derived from CD34⫹AC133⫺ cells. Additional analyses including the expression of KDR2, CD117 or CD45RA on CD34⫹AC133⫹ cells did not further discriminate between hematopoietic colonies capable to generate EPCs out of hemtopoietic colonies or not. These data suggest that adult human mobilized CD34⫹AC133⫹ progenitor cells from peripheral blood have the capability to generate both, hematopoietic cells as well as cells from the endothelial lineage and therefore may have functional hemangioblastic activity, providing a source of blood and endothelial cells in postnatal human life.

217 ONTOGENY AND PLASTICITY OF ADULT HIPPOCAMPAL NEURAL STEM CELLS Sieber-Blum M.*,1 1 Medical College of Wisconsin, Milwaukee, WI, USA We have investigated the ontogenetic origin and the degree of plasticity of adult hippocampal stem cells. Wnt1-expressing cells are located at the dorsal aspect of the embryonic neural tube and some of them are predestined to give rise to neural crest stem cells. Whereas the majority of adult hippocampal neural stem cells does not originate from cells that express Wnt1, a subset does express Wnt1 transiently during embryogenesis, as determined in the double transgenic mouse, Wnt1⫺cre/R26R. Hippocampal stem cells from adult ROSA 26 mice differentiate into chondrocytes, melanocytes (pigment cells) and smooth muscle cells when cocultured with neural crest cells from quail embryos. Neural crest cellgenerated stimuli have a short-range of action and are recognized across species. These observations provide evidence for the heterogeneity in the hippocampal neural stem cell pool with regard to Wnt1 expression. Furthermore, they show plasticity and a remarkably wide range of developmental options of adult hippocampal stem cells.

218 MUSCLE STEM CELLS PROMOTE NERVE REGENERATION AND CONTRIBUTE TO THE DEVELOPMENT OF NEURONAL TISSUES Zhuqing Qu-Petersen1, Jim Cummins1, Aiping Lu1, Arvydas Usas1, Ron Jankowski1, Makato Ikezawa2, Ryosuke Kuroda1, William C. de Groat1, Johnny Huard1 1 Growth and Development Laboratory, Children’s Hospital of Pittsburgh, Department of Orthopaedic Surgery, Departments of Molecular Genetics and Biochemistry and Bioengineering, University of Pittsburgh, Pittsburgh, PA, USA2 University of Kumamoto, Kumamoto, Japan Muscle-derived stem cells (MDSC) isolated from normal neonatal mouse skeletal muscle via the preplate technique display an improved transplantation capacity when implanted in the skeletal muscle of dystrophin-deficient mdx mice. The benefits associated with MDSC transplantation are at least partially attributable to

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their impressive self-renewal ability and their capacity to undergo multipotent differentiation to form myofibers, blood vessels, and peripheral nerves in the injected muscle. We investigated whether the injection of normal MDSC clones can promote the regeneration and repair of 3-mm sciatic nerve defects created in mdx mice. We found that MDSC in the nerve defect area can differentiate into Schwann cells that significantly promote axonal regeneration and subsequently alleviate muscle atrophy in denervated gastrocnemius muscles. The regenerating capacity of the MDSCs in the nerve defect appears to be influenced by the time at which MDSC injection is performed post-injury. Normal MDSC also contribute to the formation of astrocytes and neurons in the hippocampus and cerebral cortex following either intracranial transplantation or intravenous dissemination of the cells. These findings demonstrate that MDSC possess remarkable plasticity in response to environmental cues, and suggest the potential promise of MDSC-based cell therapies for treatment of various neuromuscular diseases.

219 TISSUE-SPECIFIC MUSCLE, NEURAL AND LIVER STEM/PROGENITOR CELLS RESIDE IN THE BONE MARROW, RESPOND TO AN SDF-1 GRADIENT AND ARE MOBILIZED INTO PERIPHERAL BLOOD DURING STRESS AND TISSUE INJURY: A NEW PERSPECTIVE ON STEM CELL PLASTICITY Kucia M.1, Ratajczak J.1, Janowska-Wieczorek A.2, Ratajczak M.*,1 1 University of Louisville, Louisville, KY, USA, 2 University of Alberta, Edmonton, AB, Canada Several reports have suggested that bone marrow hematopoietic stem cells transdifferentiate into tissue-specific stem cells, but the possibility of committed tissue-specific stem cells pre-existing there has not been dealt with adequately. We hypothesized that i) tissue-committed stem cells have to circulate under steady-state conditions in the blood to maintain a pool of stem cells in tissues and their number increases during stress/tissue injury, ii) they are chemoattracted to the bone marrow, and iii) the SDF-1-CXCR4 axis could play an important role in the homing of these cells into stem cell niches in bone marrow as well as in muscle, liver and neural tissues. Supporting this concept we found that mRNA for several early markers for muscle (Myf-5, Myo-D), neural (GFAP, nestin) and liver (CK19, fetoprotein) is detectable in circulating (adherent cell-depleted) peripheral blood mononuclear cells. Using real-time RT-PCR we found that the level of expression of these markers increases in peripheral blood in humans and mice after mobilization by G-CSF. SDF-1 chemotaxis studies combined with real-time RT-PCR analysis of mRNA revealed that i) these early tissue-specific cells reside in the normal human and murine bone marrow, and ii) express CXCR4 on their surface and can be enriched (up to 400x) in humans and mice after chemotaxis to an SDF1 gradient. Our studies were performed on freshly isolated cells to exclude the potential contribution of “transdifferentation” of hematopoietic stem or mesenchymal cells. Our data provide a new perspective on the bone marrow as the “home” not only of hematopoietic stem cells but also of already differentiated tissue-committed stem/progenitors. Moreover, we postulate that tissue-specific stem/progenitors

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circulating in the peripheral blood play an important role in organ and tissue regeneration. They could be mobilized into the blood for therapeutic purposes using similar protocols to those used for mobilization of hematopoietic stem cells.

220 BONE MARROW CELLS PARTICIPATE IN REGENERATION OF SKELETAL MUSCLE BY TRANSDIFFERENTIATION TO MUSCLE CELLS Abedi M.*,1, Greer D.1, Demers D.1, Colvin G.1, Dooner M.1, Cerny J.1, Moore B.1, Quesenberry P.1 1 Roger Williams Medical Center, Providence, RI, USA Bone marrow cells have been shown to be directly involved in regeneration of muscle by conversion to muscle cells. These approaches are clinically relevant only if significant and stable differentiation can be established. We have found increasing levels of marrow conversion or transdifferentiation to skeletal muscle cells by altering the specifics of the transplant regimen including cell number, timing of transplant and mode of cell delivery (i.e. local injection vs. systemic infusion or mobilization of transplanted cells). We have found that the number of conversion events changes significantly with different mobilization regimens and that subsets of marrow cells, i.e. Dexter culture adherent cells and especially lineage negative cells, give higher rates of conversion than unseparated marrow cells. The nature of the skeletal muscle injury, radiation or direct cardiotoxin injection, is also critical in determining the level of transdifferentiation of marrow to muscle cells seen in vivo. Mode of the cell delivery to muscle (i.e. direct injection vs. mobilization of engrafted bone marrow cells vs. intravenous delivery) also changes the percentage of donor derived muscle cells. The relevance of these studies to possible clinical application depends upon the “robustness” of the conversions. In settings without injury or in some other experimental models the levels of conversion have been quite low, at the 0.01% level. However, in preliminary studies using a cardiotoxin muscle injury in previously transplanted mice, combined with radiation and direct injection of different populations of marrow cells we have obtained conversion rates up to 12%, i.e. 12% of the muscle fibers were GFP⫹ skeletal muscle cells. This, to our knowledge, is the highest rate of marrow conversion to muscle cells in the published literature. We have also observed, for the first time, colonies of GFP⫹ muscle cells in the anterior tibialis muscle.

221 ADVANCES IN GVHD PATHOPHYSIOLOGY AND TREATMENT Ferrara J.*,1 1 Cancer Center, University of Michigan, Ann Arbor, MI, USA Graft-versus-host disease (GVHD) is the major complication of allogeneic Bone Marrow Transplant (BMT). Host antigen presenting cells are known to be important stimuli for acute GVHD. We investigated the role of donor cytolytic T lymphocytes in a series of mouse models. We evaluated whether alloantigen expression on host target epithelium is essential for tissue damage induced by GVHD. In bone marrow chimeras, recipients in which either

MHC II or MHC I alloantigen was expressed only on APCs, we found that acute GVHD does not require alloantigen expression on host target epithelium and that neutralization of Tumor Necrosis Factor-alpha and Interleukin-1 prevents acute GVHD. These results were found in models of CD4-mediated GVHD and to a lesser extent in CD8-mediated GVHD. We have evaluated the clinical relevance of these BMT patients, and found levels of TNF receptor proteins elevated in patients with acute GVHD. We also have initiated a trial of soluble TNF receptor (etanercept) in patients with newly diagnosed acute GVHD. These clinical data highlighted the importance of inflammatory cytokines such as TNF alpha to the pathophysiology of acute GVHD. These data suggest that strategies to neutralize these proteins may offer new approaches to this toxic complication of allogeneic BMT.

222 USE OF SUICIDE GENE-EXPRESSING DONOR T CELLS TO CONTROL ALLOREACTIVITY AFTER HAEMATOPOIETIC STEM CELL TRANSPLANTATION Pierre Tiberghien EFS B/FC, INSERM E0119, Besanc¸on, France Conditional ablation of alloreactive donor T cells to prevent or treat graft-versus-host disease (GvHD) after allogeneic hematopoietic stem cell transplantation could significantly contribute to expand the use of alloreactivity as a treatment modality. The prevention and treatment of GvHD induced by Herpes simplex virus 1-thymidine kinase (HS-tk)-expressing donor T cells by ganciclovir (GCV) has been demonstrated in animal models. Available clinical findings suggest that the use of such cells early or late after transplantation is associated with the absence of acute toxicity, GCV-sensitive GvHD and persistent circulation of gene-modified cells (GMC). However, a number of limitations, such as reduced immune function of ex-vivo gene-modified T cells, immunogenicity of GMC as well as the presence of GCV-resistant truncated HS-tk-expressing T cells within circulating GMC, have emerged from these studies and are presently being addressed.

223 MODULATING ANTI-TUMOR IMMUNITY DURING IMMUNE RECONSTITUTION Levitsky H. Baltimore, MD, USA Abstract not received at time of printing.

224 MACROPHAGE AND DENDRITIC CELL-BASED THERAPIES IN CANCER PATIENTS Abastado J.P.*,1 1 IDM, Paris, France Macrophages kill and phagocyte tumor cells in vitro. Dendritic cells (DC) prime naive T cells to become effector cells. Cell therapy based on these two types of cells appears therefore as a promising tool to treat cancer patients. Macrophages were used in patients with

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superficial bladder cancer. Intravesical injections of autologous macrophages were shown to significantly (p ⱕ 0.0005) reduce the risk of recurrence following transurethral tumor resection (Thiounn et al., J Urol 2002). The anti-tumor potential of macrophages is further enhanced in vitro with anti-tumor specific antibody. A phase II clinical trial using macrophages armed with a bi-specific antibody recognizing CD64 and HER2/neu was conducted in ovarian cancer patients. Safety and feasibility of the treatment, as well as signs of clinical activity were demonstrated (de Gramont et al, Gynecol Oncol 2002). Two studies in which macrophages are combined with an anti-CD20 antibody have been initiated in patients with CLL (L. Sutton, Paris) or NHL (M. Favrot, Grenoble). Therapeutic vaccination against tumor has been shown to provide long-term protection in animal models. We are currently developing DCbased vaccines in melanoma, prostate and colorectal cancers. Initial trials prostate cancer and melanoma showed that such vaccines are remarkably well tolerated. Furthermore, DC loaded with tumor extract or recombinant proteins appear to efficiently induce antigenspecific immune responses.

225 STEM CELL PLASTICITY: WILL IT BE USEFUL TO REPAIR INJURY? Sharkis S.*,1 1 Johns Hopkins University, Baltimore, MD, USA In the past few years many reports have been may which either supports the possibility of either adult or embryonic stem cells to engraft multiple germ layers of adult animals and man. We have shown that a single hematopoietic stem cell (HSC) can give rise to cells within several epithelial tissues as well as reconstitute the hematopoietic system of the mouse eleven months following transplantation (Cell 2001;105:369–377). More recent studies have shown that cell fusion may be an alternative explanation for the plasticity of embryonic stem cells in vitro. Our laboratory has investigated the potential for cell fusion in vitro with adult stem cells and has found that this is a most unlikely explanation for transdifferentiation. Our group has gone on to examine the early events (3–48 hours) post-transplant and can demonstrate in the donor cells the expression of transcription factors associated with epithelial development of such tissues as liver, kidney and lung. We have also shown that the homing of adult HSC varies from tissue to tissue but generally if the tissue is damaged prior to transplant the homing is increased to the site of injury. The challenge for the future is first to determine the functional capacity of the cells transplanted and then to derive the best method(s) for increasing the level of transdifferentiation which will allow successful repair of the injury. Finally it is likely to be a formidable task to scale up those studies done in animal models to treatment of Human disease however once the biological principle has been established the experimental therapies must be considered.

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226 TREATMENT OF CARDIAC INFARCTIONS WITH DIRECT STEM CELL INFUSION AND MOBILIZATION D.Orlic Genetics and Molecular Biology Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland, USA We have investigated the potential of bone marrow stem cells (BMSC) to differentiate into cardiac myocytes and vascular smooth muscle cells and endothelium in acute ischemic myocardium. Infarcts produced in the left ventricle of adult female mice were treated with Lin⫺ c-kit⫹ cells isolated from bone marrow from adult male transgenic mice that expressed enhanced green fluorescent protein (eGFP). At 9 days after BMSC transplantation the hearts showed a band of cardiac myosin positive myocytes. These myocytes were also eGFP-positive indicating their origin from the transplanted cells. Several myocyte-specific transcription factors and connexin 43 were observed. Endothelial cells and smooth muscle cells in the developing capillaries and small arterioles were positive for eGFP. We did not find evidence of myocardial repair when as many as 5 × 105 Lin⫺ c-kit⫺ cells were transplanted. Left ventricular end diastolic pressure and left ventricular developed pressure were 30% to 40% improved in mice that had been transplanted with Lin⫺ c-kit⫹ bone marrow cells. In a second series of experiments we investigated the ability of stem cells mobilized by stem cell factor (SCF) and granulocyte colonystimulating factor (G-CSF) to traffic to the infarcted myocardium and promote repair. At 27 days after cytokine treatment and infarction, a new band of myocardium formed. The cardiomyocytes were positive for transcription factors and connexin 43. Regenerating arterioles consisted of endothelial cells and smooth muscle cells that were positive for BrdU and flk1. Left ventricular ejection fraction and survival in the cytokine-treated mice were enhanced. We also tested whether cytokine treatment might be an effective therapy stimulating myocardial repair in a rhesus monkey model. Treatment consisted of 8 daily subcutaneous injections of G-CSF alone or together with SCF. On day 5 the left anterior descending branch of the left coronary artery was ligated to induce a MI. The ligature was removed and the heart reperfused after 2 hours, and cytokines were injected an additional 3 days post-MI. Control animals were subjected to the same protocol but were not given cytokine therapy. After 5 daily injections of G-CSF, the number of mobilized CD34⫹ cells peaked at 31⫹/-13/µL (n ⫽ 6) compared with 71 ⫾ 27/uL (n ⫽ 2) following G-CSF/SCF. In non-treated controls there were 2 ⫾ 2/µL (n ⫽ 5). Two of 7 controls and 1 of 7 animals treated with G-CSF died within two days of the infarction in contrast to 5 of 7 animals treated with G-CSF/SCF. Microscopic examination of the hearts at 12 weeks post-MI indicated collagen deposition with extensive scar formation in the free wall of the left ventricle (LV) with no clear evidence for infarct zone regeneration in any group. The infarction size averaged 24% of the LV in the G-CSF group, 30% in the G-CSF/SCF group, and 21% in the control group. MRI indices of LV volume and ejection fraction showed unfavorable but non-significant trends toward worse outcome in both treatment groups compared to controls. The lack of favorable treatment trends in this pilot study and the unexpected high mortality in the G-CSF/SCF group raise concerns regarding cytokine therapy in the peri-infarction period in this non-human primate trial.