key: cord-0005597-3ljyutre
authors: nan
title: Van Bekkum Award
date: 2005-03-15
journal: Bone Marrow Transplant
DOI: 10.1038/sj.bmt.1704901
sha: 8d2c5e177931ed7511d59d74990542c2ba9da5d9
doc_id: 5597
cord_uid: 3ljyutre

nan

In two ongoing phase I/II trials, patients with hormone resistant prostate cancer and stage IV melanoma are vaccinated with autologous dendritic cells (DC) that have been transfected either with mRNA derived from 3 standard prostate cancer cell lines or from each patient's own tumour (melanoma). Transfection by electroporation results in high expression of proteins encoded by the mRNA. DC are produced under GMP conditions in a closed system with IL4 and GM-CSF for 5 days. After transfection, immature DC are matured for 2 days in standard maturation cocktail with PGE2 and the resulting vaccine is frozen in aliquots. Each patient group was divided into 3 arms. In one arm, DC were given as intradermaly (i.d.) injections, in a second arm the cells were given intranodaly (i.n.). In addition a third group of patients were given i.d. injections in a site pre-treated with imiquimod (prostate cancer) or given DC i.n. in combination with low dose Il-2 by continuous infusion (melanoma). The primary endpoint of the trials were safety, secondary endpoints were immunogenicity and clinical responses. Immune responses were measured in vivo as DTH responses and in vitro as proliferative responses and by the ELISPOT technique. In selected patients, T cells were cloned for further characterization of the immune responses. In the standard regimen, each patient receives 4 weekly vaccines, followed by booster vaccines every 6 months or as long as vaccine is available. Until now 24 patients have received the vaccine intradermaly and 21 i.n. under ultrasound guidance. No serious adverse effects have been seen to date. In 12/15 prostate and 11/19 melanoma patients, an immune response was obtained based on in vitro immunoassays and/or a DTH response. T cell clones specific for mRNA transfected DC could be obtained from several patients and some of these were shown to kill HLA a matched prostate cancer cell line used for mRNA production. Mixed tumor responses, with disappearance of metastatic lymph node and subcutaneous tumors and development of vitiligo were observed in melanoma patients. In the prostate cancer group, immune responses were associated with stable or reduced levels of PSA. In some patients a fall in PSA was only observed after boosting We conclude that the strategy of vaccinating with mRNA transfected DC functions to elicit cellular immune responses, and that such responses may be associated with a clinical benefit.

Monitoring of homing of unfractionated and CD34selected bone marrow cells in the infarcted human myocardium by PET B. Hertenstein, K.C. Wollert, M. Hofmann, G.P. Meyer, A. Menke, L. Arseniev, A. Ganser, W. H. Knapp, H. Drexler Hannover Medical School (Hannover, D) ; Cytonet Hannover GmbH (Hannover, D) Intracoronary transfer of autologous bone marrow cells (BMCs) has been shown to promote recovery of left ventricular (LV) systolic function in patients with acute myocardial infarction. Homing of BMCs to the infarcted LV is probably a crucial early event. Different homing characteristics of selected BMC populations may provide further insights in the cell types involved in myocardial repair. We determined the tissue distribution of labeled BMC in pts. with ST-elevation myocardial infarction, who had undergone stenting of the infarct-related artery (all male; 43 ys; 36 -66). Six pts. received an intracoronary (i.c.) infusion of unselected radiolabeled BMC and 3 pts. received CD34 selected BMC. In addition, 3 pts. received half of the radiolabeled unmanipulated BMC i.v. prior to i.c. infusion. Cells were harvested from the posterior iliac crest during short anesthesia with etomidate and midazolam. For preparation of unselected BMC cells 120-145 ml of BM were harvested and subjected to 4% gelatine polysuccinate sedimentation. 5% of these cells were radiolabeled with 100 MBq 2'-[18F]-fluoro-deoxyglucose (FDG) . For preparation of CD34pos BMC 277-320 ml of BM were harvested. 60% of the BMC were immunomagnetically enriched (Clinimacs, Miltenyi, Germany) and 2-3 x 10 7 CD34pos BMC were labeled with FDG. Cell transfer was performed 5-10 days after stenting. Following application of FDG labeled cells all pts. received 20 + 6 x 10 8 non-labeled BMCs i.c. (i.e. the cell dosage that improved cell function in the BOOST trial). Sixty minutes after cell transfer, all pts. underwent 3D-PET imaging. After i.c. transfer, 1.3 -5.3% of FDG-labeled unfractionated BMCs were detected in the infarcted LV.; the remaining activity was found primarily in the liver and spleen (figure 1: A and B). After i.v. transfer, only background activity was detectable in the infarcted LV. After i.c. transfer of FDG-labeled CD34-enriched cells, 14-39% of the total activity was detectable in the infarcted LV. Unfractionated BMCs engrafted in the infarct center and border zone, while CD34pos cell homing was more pronounced in the border zone (figure 1: C and D). FDG-labeling can be used to monitor myocardial homing and tissue distribution of BMCs after therapeutic application. I.c. transfer is superior to i.v. application in respect to homing of BMC to the infarcted LV. CD34-enriched cells display a higher retention in the infarcted LV as compared to unfractionated BMCs. T-cells cultured with adenovirus peptides recognise adenovirus infected cells: an option for immunotherapy? M. van Tol, B. Heemskerk, C. Sombroek, T. van Vreeswijk, M. Havenga, M. Schilham, L. Veltrop-Duits Leiden University Medical Center (Leiden, NL) The incidence of life-threatening human adenovirus (HAdV) infections is increasing in pediatric recipients of an allogeneic stem cell graft. Infections are mainly caused by HAdV serotypes belonging to three (A, B and C) out of six species known. Since medication is frequently ineffective, the option of adoptive transfer of HAdV-specific donor-derived T cells is under investigation. Culturing of PBMC of healthy donors in the presence of HAdV primarily leads to stimulation of CD4+ T cells, which suggests that a large proportion of the response is directed against structural viral proteins. Instead of stimulating donor T cells with complete virus, it would be more attractive to stimulate with synthetic peptides, which can be used under GMP conditions. However, target proteins and their epitopes are still largely unknown. In order to select dominant peptides, we synthesized overlapping peptides of the main capsid protein, HAdV5 hexon, and determined the response to these peptides by measuring IFN-gamma production (ELISA and ELIspot) in cultures of PBMC from 21 healthy donors with different HLAtypes. This screening resulted in the selection of a panel of 5 peptides, which yielded a response in 13 of 14 donors, apparently independent of HLA types. Long-term cultures of PBMC of healthy donors with different HLA-types stimulated with this panel of 5 peptides resulted in CD4+ T cells, which recognized not only inactivated HAdV5, but also other HAdV serotypes from different species (A, B, C and D) as measured by IFN-gamma production and proliferation. Furthermore, these CD4+ T cells not only responded to inactivated virus but also to active HAdV5. These results suggest that it might be feasible to generate HAdV-specific donor-derived T cells for adoptive transfer by applying a limited set of adenoviral peptides.

This study was financially supported by the Dutch Cancer Society.

Immunosuppressive mesenchymal stem cells as treatment for experimental autoimmune encephalomyelitis E. Zappia, S. Casazza, E. Pedemonte, F. Benvenuto, E. Gerdoni, D. Giunti, F. Frassoni, A. Ceravolo, F. Cazzanti, D. Phinney, G.L. Mancardi, A. Uccelli University of Genoa (Genoa, I) ; San Martino Hospital (Genoa, I); Tulane University (New Orleans, USA) Mesenchimal stem cells (MSCs) have been recently demonstrated to have immunosuppressive properties on activated T cells. Thus, we studied the immunoregulatory properties of MSCs, extracted from C57B/6J mice bone marrow, both in vitro ed in vivo. Starting from bone marrow aspirates, we decreased the haematopoietical component and selected for the stromal staminal one, with serial plating passages. Pure CD45 -Sca1 + CD9 + MSC were obtained after 5 cycles of culture. These cells inhibited the proliferation of murine splenocytes unspecifically stimulated with anti-CD3/anti-CD28 antibodies or specifically stimulated with the encephalitogenic antigen MOG35-55. mMSCs also inhibited allo and xeno MLR reactions. Proliferation inhibition was paired by a decreased IFN-gamma and TNF-alpha production by MSC treated T-cells and was reversed by addition of IL-2 suggesting the induction of an anergic state. Based on these findings, we utilized mMSCs as treatment for MOG35-55 induced EAE in C57B/6J mice. We injected intravenously 1 x 10 6 mMSCs before disease onset on day 3 and 8 p.i (preventive protocol) and at different time points after disease occurrence (therapeutic protocol). mMSCs therapy before disease onset strikingly ameliorated EAE clinical course over a long period of follow-up. The therapeutic scheme was effective when mMSC were administered at disease onset and at the peak of disease while did not ameliorate EAE when given at day 25. Brain and spinal cord pathology showed decreased inflammatory infiltrates, demyelinization and axonal loss in mMSCs transplanted mice. T cells proliferative response to MOG and mitogens and proinflammatory cytokine production by T cells from draining lymph nodes of mMSC treated mice were inhibited and restored by IL-2. Few mMSC, transfected with Green Fluorescent Protein (GFP), were detected in the spine of MSC treated mice. Of relevance, large numbers of GFP+ mMSC were observed in the lymphoid organs of treated mice close to T, B and dendritic cells. These data suggest that the immunosuppressive potential of mMSCs may effectively interfere with the T cell mediated autoimmune attack in the course of EAE inducing an in vivo state of tolerance occurring within secondary lymphoid organs. current study, we analyzed histopathological specimens of 188 out of those 302 patients to analyze prognostic and predictive values of immunohistochemical factors such as Ki-67, p16, Maspin, Bcl-2, Her2/neu and p53. In a univariate analysis of the entire study population after adjustment for therapy, tumor size and estrogen-receptor, Her2/neu-positivity (p=0.001) was a negative and Bcl-2-positivity (p=0.003) was a positive predictive factors for event-free survival. In a multivariate analysis for the whole study population, Her2/neu-positivity (HR 3.68; 95 % CI: 2.01 -6.73; p=0.001) and tumor size > 30 mm (HR 3.04; p=0 .0001) were factors for a lower event-free survival, while p53-positivity (HR 0.57; 95 % CI: 0.34 -0.95; p=0.03) and Bcl-2-positivity (HR 0.35; 95 % CI: 0.19 -0.64; p=0.0006) were associated with a better event-free survival. To evaluate the interaction between immunohistochemical factors and treatment (high-dose vs. standard chemotherapy), for event-free survival only p53 has a positive interaction between the treatment arms (p=0.005). Therefore, a relative risk for high-dose chemotherapy vs. standard chemotherapy is estimated as 2.3 (95 % CI: 0.67 -7.92) in p-53-negative patients and as 0.46 (95 % CI: 0.2 -1.07) in p53-positive patients, which indicates a superiority of high-dose chemotherapy in p53-positive patients and an inferiority in p53-negative patients. No interactive effect could be shown for Her2/neu and Bcl-2. We conclude that Her2/neu and Bcl-2 are prognostic but not predictive factors in patients with high-risk primary breast cancer; p53-positive patients might benefit more from high-dose chemotherapy than from standard chemotherapy, and p-53-negative patients might benefit more from standard chemotherapy than from highdose chemotherapy. This study demonstrates the need to include biological factors within clinical treatment studies.

Severe adverse events of allogeneic peripheral blood stem cell donors -results of nation-wide 2,784 prospectively registered case survey and of its comparison to bone marrow donors in Japan Y. Kodera, M. Harada, S. Kato, S. Shiobara, N. Hamajima, Y. Morishima, M. Tanimoto, S. Asano, Y. Ikeda, H. Dohi, T. Nakahata, M. Imamura, K. Kawa, Y. Takaue, Y. Kanda, K. Yamamoto, A. Gratwohl 

In April 2000, we created a system, which was cooperatively steered by The Japan Society of Hematopoietic Cell Transplantation (JSHCT) and G-CSF producing and/or selling companies, to catch up the types and the frequencies of acute and late severe adverse events (SAE) of peripheral blood stem cell (PBSC) donors in Japan. Every PBSC donor was registered to JSHCT and was given unique donor number (UDN) before the PBSC donation. Every harvest center was mandatory required to submit the day 30 report as well as the immediate report of any severe SAE, and also to ask donors Ereceiving annual health check for 5 years. This time, we report the acute SAE observed among 2, 784 consecutive donors in 223 institutes and the late SAE reported by the forth year of post PBSC harvest among 1,746 donors who agreed with this work of the society. As of March 2004, 47 acute SAE out of 2,784 cases (1.7%) were reported. Those were 12 thrombocytopenia, 9 liver damage, 8 febrile episode, 1 interstitial pneumonitis, subarachnoid hemorrhage (SAH), cholecystitis with stone and others. Late SAE were reported at 27 out of 1,746 cases (1.5%). Those were 2 hematological malignancies (acute myelogenous leukemia and myeloproloiferative disorder), 8 other malignancies (4 breast cancer, 1 gastric cancer, uterus cancer, brain tumor, pharygeal cancer), 5 thyroid disorder, 3 myoma uteri, 2 rheumatoid arthritis, 1 brain infarction and SAH and others. To compare these acute and late SAE of PBSC donors to those of bone marrow (BM) donors, the questionnaires shared with The European Group of Blood and Marrow Transplantation were sent to 378 institutes of JSHCT and 203 institutes (53.7%) answered about 10,701 cases and the comparative results were as followings: BM donors: PBSC donors; Death within 30 days = 0:0, Hematological malignancy = 2:2. These results showed that acute SAE, some of which were close to life-threatening, occurred at PBSC donors with certain frequency and that variable late SAE also occured although the frequency of the occurrence of hematological malignancy was not necessarily high at PBSC donors.

Final results of the IFM9904 protocol: double transplant +/-anti-IL6 monoclonal antibody in high-risk de novo multiple myeloma less than 65 years P. Moreau, C. Hullin, F. Garban, T. Facon, M. Attal, L. Benboubker, G. Marit, J. Fuzibet, C. Doyen, L. Voilat, C. Berthou, P. Casassus, M. Monconduit, M. Michallet, A. Najman, J. Sotto, R. Bataille, J.L. Harousseau Service d'Hematologie (Nantes, F) The median survival of pts with high-risk de novo MM (b2mic > 3 and ch13 deletion ), is approximately 2 years when treated with high-dose therapy. For this subgroup of pts, in 1999, the IFM group initiated 2 trials, IFM9903 & 9904 studies. In both trials, induction regimen consisted of VAD followed by melphalan 200 mg/m² (HDM200) plus APBSCT. When a HLAsibling donor was available, APBSCT was followed by minialloPBSCT: IFM9903 trial. When no donor was available, pts were randomised to receive a second APBSCT prepared by a higher dose of melphalan, HDM220 +/-anti-IL6 moAb (BE-8, 250 mg, Diaclone, France): IFM9904 trial. The aim of this study was to evaluate prospectively the impact of the addition of anti-IL6 moAb to a higher dose of melphalan. From 12/1999 to 08/2004, 225 pts less than 65 years of age with de novo MM with both b2mic > 3 and ch13 deletion at diagnosis without an HLA-id sibling donor were included the IFM9904 trial. 28 pts included after 03/2004 are too early to evaluate. Thus 197 pts are included in this survival analysis. 30 / 197 (15%) were not randomised to receive or not anti-IL6 moAb + HDM220 because of disease progression (9), early death (3) or severe infection (10) during VAD induction therapy, refusal (2), protocol violation (4) or toxic death during the first APBSCT (2). 167 (85%) were randomised, 84 in arm A = HDM220 without anti-IL6 moAb, 83 in arm B = HDM220 + antiIL6 moAb. At the reference date of September 1st 2004, the median EFS and OS of the whole group of 197 pts from the time of diagnosis are 30 and 39 months, respectively. Pts who proceeded to randomisation (arm A + B, n = 167) had a significantly better OS and EFS as compared with pts (n = 30) who were not randomised, median OS 45 months vs 11 (p < .001) and median EFS 31 months vs 10 (p < .001). The addition of anti-IL6 in the conditioning regimen before the 2nd APBSCT did not improve neither EFS nor OS : median EFS 34 months in arm A (without anti-iL6 moAb) vs 30 in arm B (p = .84), and median OS 38 months in arm A vs 46 in arm B (p = .25). These survival rates (> to 50% at 3 years) are superior to what has been previously described in this high-risk subgroup of pts, indicating that a double transplant strategy (HDM200 / HDM 220) may prolong survival. Nevertheless, the addition of anti-IL6 moAb to HDM220 during the 2nd conditioning regimen did not influence the outcome. Final results for the whole cohort of 228 pts will be presented during this EBMT meeting.

Working Party: Acute leukaemia 70 Allogeneic stem cell transplantation for patients with de novo acute myeloid leukaemia in advanced phase: results of a survey from the EBMT J. Esteve, M. Labopin, J. Finke, A. Fassas, C. Cordonnier, A. Fauser, M. Michallet, F. Frassoni, N. Harhalakis, J. Reiffers, E. Alessandrino, G. Ehninger, N. Gorin, V 

Allogeneic stem-cell transplantation (alloSCT) is the postremission strategy with highest antileukaemic potential in patients with acute myeloid leukaemia (AML), resulting in an increased leukaemia-free survival (LFS) and longer survival (OS) in patients with intermediate or high risk AML in earlier phases of the disease. However, the possible role of alloSCT in AML patients in advanced phases of the disease is mostly unknown, and deserves specific investigation. For this purpose, a survey within the EBMT registry including patients who underwent an undepleted alloSCT from an HLA-identical sibling for advanced AML (not in CR) was performed. We analysed 977 adults patients with AML submitted to alloSCT for a primary refractory disease (REF, n=346) , first relapse (REL1, n=506) or second relapse (REL2, n=125) from 1990 to 2002 in EBMT centres. Median age was 40 (16 -73) and 55% of patients were male. FAB classification was: M1/M2, 44%; M4/M5, 33%; M6, 5%; M0, 4%; M3, 3%; M7, 3%. Median time from diagnosis to alloSCT was 136, 219 and 403 days for REF, REL1, and REL2 subgroups, respectively. Bone marrow was the source of stem cells in a higher proportion of patients with relapsed disease as compared to refractory AML (59% for REL1 + REL2 vs 41% for REF, p<0001). Conditioning regimen included TBI in 56% of cases. At 2 years following alloSCT, relapse incidence (RI) was 57*6, 51*4, and 54*8, and transplant-related mortality (TRM) 25*5, 26*4, 31*6 for REF, REL1, and REL2 patients, respectively, without significant differences between subgroups. Similarly, OS and LFS were 25*3, 26*2, 24*2, and 18*2, 23*2, 16*3, respectively (figures 1 and 2). In conclusion, although the overall outcome of HLA-identical sibling allotransplant for patients with advanced AML is poor, a small fraction of patients, of approximately 20%, seems to benefit from the procedure in this otherwise incurable disease. A further analysis is addressed to the recognition of favourable prognostic factors in order to identify subgroups of patients with advanced AML who are candidates to allogeneic transplantation. Allogeneic stem cell transplantation from an haploidentical family donor has been reported from single-center studies as a curative treatment option for patients with high risk acute leukemias. We collected 273 transplants from 75 centers in adults patients with de novo acute myeloid leukaemia (AML) and acute lymphoblastic leukaemia (ALL) reported to the EBMT from 1995 to 2002 and analysed the outcome of this procedure according to the known risk factors. Overall, 170 AML patients underwent transplantation in CR1 (39), CR2 (34) or in advanced disease (97); median age was 38y (16-70). Overall, 103 ALL patients underwent transplantation in CR1 (31), CR2 (22) or in advanced disease (50); median age was 26y (16-56). Graft composition was based on CD34+ cell selection of PBSC, with median CD34+ cells x10 6 /kg of 7.3 (1.1-45.5) in AML and 8.3 (1.14-30) in ALL. Conditioning regimen was TBI-based in 69% and 87% in AML and ALL respectively. Primary engraftment was documented in 87% AML and in 83% of ALL patients, with ANC 0.5 x109/L in a median of 13 days (8-35) and a 2y probability of PLT > 50 x10 9 /L of 80+/-5%. The cumulative incidence of acute GvHD >=II was 16% in AML and 15% in ALL. With a median followup of 19 months (1-85), the estimated leukemia-free survival (LFS) at 2 years in AML was 39 +/-9%, 33 +/-10% and 4 +/-3% and in ALL was 28+/-9%, 16 +/-8, 0% for CR1, CR2 and advanced patients respectively. In multivariate analysis for competing risks, relapse incidence (RI) at 2 y in AML was 8 +/-8%, 9 +/-9%, 25 +/-8% for CR1, CR2 and advanced; RI in ALL was 27 +/-16% and 41 +/-20% in CR1 and CR2. The nonrelapse mortality (NRM) at 2 y in AML was 52 +/-18%, 56 +/-19%, 69 +/-8% for CR1, CR2 and advanced patients; NRM in ALL was 44 +/-18% and 41 +/-21% in CR1 and CR2. On multivariate analysis, factors for LFS were advanced vs CR at transplant (RR 0.4); ALL vs AML (RR 0.5), age >33 (RR 0.4), TBI conditioning (RR 1.6). Significant factors for NMR were advanced vs CR (RR 2.01), year of transplant>=2000 (RR 0.7). Predictive factors for RI were ALL vs AML (RR 2.39) and advanced disease vs CR (RR 1.96). EBMT registry analysis of transplantation from haploidentical donors confirmed relevant LFS at 2 years for adults with high risk acute leukemia. These results suggest that this procedure should be considered for high risk adults patients in less advanced disease status. In adult patients with acute myelocytic leukaemia receiving a conventional myeloablative allogeneic stem cell transplantation, several studies have shown that the higher the doses infused, the better the outcome , with a lower transplant related mortality (TRM) and relapse incidence (RI) and a better leukaemia free survival (LFS). We wondered whether this observation was transposable for non myeloablative transplants with peripheral blood (PB) stem cells where graft versus leukaemia (GVL) is the only anti tumor mechanism. In this particular situation, one might postulate that increasing the doses of peripheral blood T cells infused in an effort to increase GVL might increase GVHD and TRM. Further, one might speculate on the existence of an optimal range of stem cell and T cell doses to infuse. From January 1998 to December 2003, 266 patients with de novo AML, who received a non myelo ablative transplant with PB from a genoidentical donor, were reported to the ALWP registry with the dose of cells infused. The sex ratio was 146 male / 120 female. Patient age was 55 years (18-72). 150 patients were transplanted in first remission (CR1), 49 in second remission (CR2) and 67 patients had refractory disease or were transplanted in relapse. The pretransplant reduced intensity regimens were variable but 92% included fludarabin and Total body irradiation (TBI) was used in 23% of the patients at a dose<5 Gy. The median dose infused was 9.25 x 108 nucleated cells/kg (1.23-24). The follow up was 16 months (2-67).The LFS at 2 years was 42± 4%. For patients transplanted in CR1, CR2 and more advanced disease, the LFS were 49 ± 5%, 48± 8% and 19± 6% and the RI 40±6%, 42±9% and 77±7% respectively. Patients receiving doses of PB nucleated cells above the median had a lower RI (44±6% versus 54±6%; p=0.03) and a higher LFS (48±6% versus 36±5%; p=0.01). By multivariate analysis, the status at transplant (CR1 better) influenced the RI and LFS. A female donor to male recipient combination increased TRM and decreased LFS. Interestingly, the use of TBI was associated to a lower RI (p<0.005, RR=2 (1.22-3.2)) and a higher dose of nucleated cells infused was associated with a higher LFS (p=0.03; RR=1.5 (1.04-2.2)). The relationship of the dose infused to the incidence and severity of GVH is under investigation. We conclude that in the context of non myelo-ablative transplants for adult AML, infusing higher doses of PB nucleated cells is beneficial to the patients. Background: High dose immunosuppressive therapy (HDIT) and hematopoietic stem cell transplantation (HSCT) is a treatment option for patients with severe systemic sclerosis (SSc) based on pilotstudies and a recent registry analysis, which showed durable responses in two thirds of patients up to 3 yrs after HSCT (1). This treatment modality is now further investigated through the ASTIS-trial (autologous stem cell transplantation international scleroderma trial), a prospective, controlled, randomized trial to compare safety and efficacy of HDIT + HSCT versus monthly i.v. cyclophosphamide in SSc patients at risk of major organ failure or early mortality (2). Objectives: to evaluate whether HDIT + HSCT is superior over conventional treatment in terms of safety and efficacy in SSc patients, and to assess potential predictive factors of response. Methods: SSc patients with early active disease with or without major organ involvement are eligible. SSc patients randomized to the transplant arm undergo mobilization with cytoxan 2x2 g/m², conditioning with cytoxan 200 mg/kg, rbATG 7.5 mg/kg, followed by reinfusion of CD34+ selected autologous HSCT. Those randomized to the control arm are treated with 12x monthly i.v. bolus cytoxan 750 mg/kg. The primary endpoint is event-free survival, defined as survival until death or development of major organ failure during 2 years follow-up. Results: Forty-one SSc patients have been randomized in 16 European centers per November 2004: 18 to the transplant arm, 23 to the control arm. No treatment-related mortality (TRM) has yet been observed in either arm. An interimanalysis of safety was done after enrolment of 20 patients. WHO grade III, IV adverse events were observed in 2/11 control patients and 5/9 transplanted patients, all reversible except for renal failure in a transplanted patient. Based on the encouraging results from this interim-analysis, the protocol has been amended to include patients with early diffuse scleroderma. Conclusion: the absence of TRM and unexpected toxicities with 41 SSc patients enrolled underscores the feasibility of the ongoing ASTIS-trial. The next interim-analysis is scheduled at the end of 2004. B. Glass, M. Kloess, A. Engert, W. Berdel, B. Metzner, L. Trümper, M. Loeffler, M. Pfreundschuh, N. Schmitz on behalf of the DSHNHL As a novel approach to primary treatment of aggressive lymphoma, we tested the feasibility and efficacy of a four cycle high-dose chemotherapy protocol including autologous stem cell transplantation after cycles 2, 3 and 4. In a multicenter Phase II study patients with newly diagnosed aggressive NHL, age between 18-60 years, and LDH > N at diagnosis were included. At dose level 1, cycle 1 consisted of Cyclophosphamide (CY) 1500 mg/m², Adriamycin (ADR) 70 mg/m², Vincristine 2mg, Etoposide (ETO) 450 mg/m², and Prednisone 500 mg/m². In subsequent cycles doses of CY and ETO were increased: Cycle II and III : CY 4500 mg/m² and ETO 600mg/m², cycle IV: CY 6000 mg/m² and ETO 1000 mg/m². At dose level 2 ETO was further increased throughout cycles to 600, 960, 960, and 1480 mg/m², respectively. From February 97 to August 99, 124 patients were enrolled; 14 patients had to be excluded mostly due to correction of initial diagnosis. 110 patients were evaluable with a median observation time of 55 months. 81.8% of patients completed therapy as per protocol. There were 5 cases of treatment related mortality (4.5%), and one of these deaths was due to secondary leukemia (0.9%). Overall survival at 5 years was 67.2 % and freedom from treatment failure was 62.1 %. The following factors were tested with respect to their impact on time to treatment failure (TTTF): age, sex, extranodal disease, bulky disease, performance status, B-symptoms, stage, ageadjusted IPI and LDH. In univariate analysis, only the risk factors of the age-adjusted IPI and IPI itself showed a significant impact on TTTF (2 years): LDH (<= 2xUNV/ > 2xUNV): 73.1 vs 48.2% p=0.004, Stage (I, II/ III, IV): 76.5 vs 60.9% p=0.044, Performance status (ECOG 0, 1/ >1), 73.2 vs 51.2% p=0.047 Age-adjusted IPI (1, 2/ 3) 72.4 vs 45.4%, p=0.007. In a Cox regression multivariate analysis, LDH remained the only independent risk factor with a relative risk of 2.0 (p=0.046). Mega-CHOEP is feasible and effective treatment in younger pts. with aggressive lymphoma. Very high LDH compared to moderately elevated LDH has major prognostic significance in patients receiving repetitive highdose therapy and autologous stem cell transplantation. A phase III study comparing Mega-CHOEP + Rituximab to conventional chemotherapy in younger pts. with aggressive NHL is ongoing. Introduction: Recent trials have shown that anti-CD20 monoclonal antibody Rituximab may be effectively associated with high-dose (hd) chemotherapy and peripheral blood progenitor cell (PBPC) autograft. We here report the interim analysis of a prospective, multicenter trial evaluating Rituximab-supplemented hd-sequential chemotherapy (R-HDS) as frontline treatment in patients with high-risk Diffuse Large B-Cell Lymphoma (DLBCL) Patients and Methods: Seven Italian Centers associated to GITIL have participated to the study. Eligibility criteria included: i. biopsy-proven, previously untreated CD20+ DLBCL; ii. age between 16-60 yrs.; iii. advanced stage disease with 2-3 aaIPI score. The R-HDS regimen includes an initial debulkying (3 APO courses) and then the sequential administration, at 15-20 day intervals, of: i. cyclophosphamide 7 gr/sqm + Rituximab (day +2 and +10), followed by PBPC harvest; ii. Ara-C 2 gr/sqm b.i.d. for 6 days (with 1-3x10 6 CD34+ve cells/kg autologous support) + Rituximab (day +8 and +18); iii. etoposide 2.4 gr/sqm + Cisplatin 100 mg/sqm; iv. final myeloablative regimen (Mitoxantrone 60 mg/sqm + L-Pam 180 mg/sqm), with PBPC autograft (5x10 6 CD34+ve cells/kg) + Rituximab (day +30 and +37); v. involved-field radiotherapy on bulky sites Results: So far, 95 patients (median age: 48 yrs.) are evaluable; their aaIPI score was 2 (62) or 3 (33); 29 (30%) had BM involvement. There were 4 early toxic deaths (three sepsis and one JC-virus leucoencephalopathy); one more fatal pneumonia occurred at 10 mos. after R-HDS, for an overall 5.2% TRM. In addition, 21 patients had CMV or VZV reactivation that required antiviral therapy. Overall 74 patients (78 %) reached CR. At a median follow-up of 24 mos., 76 patients are alive and 69 are in continuous CR (CCR), with 5yr OS and EFS projections of 80% and 72%, respectively. A significantly better outcome was seen in aaIPI 2 vs. 3 (5-yr EFS projections: 86% and 63%, respectively). Among BM+ patients, 18 (62%) are in CCR at a median follow-up of 25 mos. Conclusion: R-HDS is feasible at the multicenter level although the occurrence of severe infectious complications should be carefully considered; both CR rate and survival projections compare favorably with the poor outcome usually observed in aaIPI 2-3 patients managed with conventional chemotherapy. The results urge a comparative analysis between conventional vs. intensified Rituximab-supplemented chemo-immunotherapy in younger patients with high-risk DLBCL. 

High-dose therapy (HDT) and autologous stem cell transplantation (ASCT) has been adopted as the standard second-line approach for patients with relapsed or refractory diffuse large B-cell lymphomas (DLBCL); in this retrospective study we investigated, in this setting, the efficacy and toxicity of high-dose sequential chemotherapy including HD-Ara-C and Rituximab followed by autologous stem cell transplantation (R-HDS). Conventional adriamycin or cisplatin containing chemotherapy regimens (APO/DHAP) were performed as debulking. The R-HDS schedule consisted of HD-CTX (7 g/m²) and HD-Ara-C (2 g/m² every 12 hours for 6 days) with 4 infusions of Rituximab (375 mg /m²), twice after HD-CTX and twice after HD-Ara-C.Then Then the patients received etoposide and cisplatin (VP16 2.4 g/m 2 ; CDDP 100 mg/m²) before conditioning regimen and ASCT. Conditioning regimens were HD-Mito/Mel (Mel 180 mg/m² and Mito 60 mg/m²) or BEAM (carmustine BCNU, 300 mg/m²; VP 16, 200 mg/m²; Ara-C, 4000 mg/m²; L-PAM 140 mg/m²); following haematological recovery further two infusions of rituximab were administered at the same dose and, within 3 months post-ASCT, radiationtherapy was performed on initial bulky-disease or posttransplant residual disease. From May 1999 to May 2003, 52 patients with refractory (14) or relapsed (38) DLBCL were treated in six Italian centers associated to GITIL; forthy-nine (94,2%) out of 52 patients were transplanted; at transplant 39 patients (82,9 %) were in CR, 7 (14,3 %) were in PR and 3 (6,1 %) were NR. TRM was 3,8%, with 2 patients died in CR, 1 for ARDS at seven months after ASCT and 1 for cardiac toxicity 6 months after transplant. Forthy (77%) out of 52 patients are alive, 36 (69,2%) in CR and 4 (7,7%) in relapse; 10 patients died of lymphoma. With a median follow-up of 26 months, the 3-year estimate overall survival (OS) and event-free survival (EFS) is 74% and 64% respectively. According to the disease status at enrolment, the 3-year estimate EFS was 71% for the 38 patients in relapse and partial remission and 47% for patients with refractory disease. These results suggested that R-HDS is feasible in these patients, with accettable toxicity and improvement of EFS; further prospective studies are needed to define the role of intensification with HD-Ara-C and Rituximab in relapsed or refractory DLBCL.

Objectives: Rituximab is a monoclonal antibody which binds to the CD20 antigen inducing apoptosis and complement activation as well as effector cell mediated lysis. Treatment of B-cell non-Hodgkin lymphomas (NHL) with Rituximab (Rit) in combination with cytotoxic chemotherapy or alone has been effective without major side effects. If tolerated, Rit maintenance therapy may be able to eradicate minimal residual disease of CD20+ NHL after stem cell transplantation (PBSCT). Methods: We describe a group of 29 patients with stage I-IV B-cell NHL (20 low grade, 9 high grade) who received Rit as maintenance therapy following high dose chemotherapy and autologous PBSCT. Rit was given at a dose of 375 mg/m 2 for a median number of 10 infusions (range 2-37) in time intervals ranging between 4 and 12 weeks. Molecular monitoring of t(14;18) was performed from samples of peripheral blood and bone marrow using nested as well as quantitative real time PCR (qPCR) based on the LightCycler technology. The median time of follow up after transplantation was 30 months (range 8-67). Fifteen of 20 (75%) patients with low grade NHL and 5 of 9 (56%) patients with high-grade NHL were in complete remission at the time of transplantation. Adverse events were documented according to CTC criteria. Results: Except for two patients with cutaneous varicella zoster infection no serious infectious complications (CTC grade III/IV) occurred. One patient had a short episode of subfebrile temperatures after Rit application. Side effects according to CTC-criteria > II did only affect the hematopoietic system. Grade II anemia developed in 4 (14%), leukopenia in 14 (48%, 9 grade II, 5 grade III) and thrombocytopenia in 9 (31%, 3 grade II, 5 grade III, 1 grade IV) patients. No patient died because of treatment related causes. Four patients ( 3 low grade 1 high grade) had progressive disease after a median time of 20 months (range 4-49 months) and two of them died. Three patients with follicular NHL had PCR positive results in peripheral blood and/or bone marrow prior to Rit therapy and converted to negativity in qPCR as well as in nested PCR after in median 12 (range 9-14 months) months of Rit therapy. Conclusions: Long term maintenance therapy with Rit after high dose therapy and PBSCT for patients with B-cell NHL is well tolerated. Major hematotoxicity was seen in 34% of patients. Rit maintenance therapy can induce molecular remission in patients who remain PCR positive after PBSCT. We present data on 156 patients with follicular lymphoma who received high-dose chemotherapy and autologous stem cell transplantation between 1990 and 2002. They were treated if they had relapsed after conventional therapy or if they had high-risk factors. The conditioning regimen consisted either of high dose cyclophosphamide (200 mg/kg) and 14.4 Gy TBI (74% of pts) or BEAM (26%). After a median follow up time of 62 months (1-149 months), 123 pts. were alive (78.8%). 11 pts. died from infection (7.1%), 17 (10.9%) from relapse and 5 pts. (3.1%) from other causes. Secondary malignancies occured in 8 cases (5.1%). Relapse rate was 26.9%. Kaplan-Meier analysis showed a stable plateau after 70 months at 65% relapse free survival. Minimal residual disease was of prognostic relevance in this patient population: the detection of (t(14;18)) by nested PCR in one or more leukapheresis products reduced RFS significantly. Therefore we have evaluated two means of graft purging by matched pair analysis: CD34 selection and in vivo rituximab treatment. 34 patients were grafted with a CD34 selected transplant. For each of them we identified a control patient without CD34 selection to match the variables age, sex, time of transplantation, stage, disease status at time of transplantation as close as possible. Purging of the transplant by means of positive CD34 selection did not result in better OS and RFS. Independent of survival, CD34 selection correlated with prolonged severe thrombocytopenia after HDT (17 vs. 14 days), thus adding to the high cost of purging through an increased need for platelet substitution. A second matched pair analysis was performed for 29 patients treated with 55 applications of rituximab between 0 -8 (mean: 1,9) months before stem cell harvest. Again, we could not identify a significant difference of OS or RFS between patients treated with rituximab or not. This may have been caused by insufficient antibody concentrations at the time of stem cell harvesting due to the longer intervals between rituximab administration and stem cell harvest in some patients.

In conclusion, purging of the peripheral blood stem cell graft by either CD34 selection or in vivo rituximab treatment did not improve OS or RFS in follicular lymphoma. The presence of MRD in the leukapheresis product may be a surrogate marker indicative of aggressive disease or high tumor burden and may not be related to relapses caused by transfer of lymphoma cells during transplantation.

The Follicular Lymphoma International Prognostic Index (FLIPI) as potential tool to select high-risk patients for autologous stem cell transplantation S. Suessmilch, B. Seyfarth, R. Schoch, M. Kneba, N. Schmitz, P. Dreger AK St. Georg (Hamburg, D) ; University of Kiel (Kiel, D) Recently, the FLIPI has been shown to be a useful predictor for the prognosis of patients with follicular lymphoma (FL) (Blood 104:1258). It relies on a score consisting of age, stage, hemoglobin, LDH, and number of nodal sites involved at diagnosis, which defines 3 risk groups with different 5-year survival (5-y OS): Low risk (0-1 factor; 5-y OS >90%); intermediate risk (2 factors; 5-y OS 78%); and high risk (>2 factors; 5-y OS 53%). The purpose of this study was to investigate if SCT could improve the course of FL with high/intermediate risk FLIPI. 91 consecutive patients who received SCT for FL between 1992 and 2002 were analyzed retrospectively. Sufficient baseline information to assess the FLIPI was available for 57 of them. 49 of these 57 patients underwent SCT as part of first-line therapy and were studied for the impact of the FLIPI. Patients were treated with a sequential high-dose therapy comprising 2-8 cycles of a CHOP-like regimen for remission induction, Dexa-BEAM for stem cell mobilization, and TBI/CY (n=32) or BEAM (n=17) for myeloablation. Results: FLIPI scores were age >59 (n=1); stage >2 (n=49); hemoglobin <12g/dl (n=5); LDH elevated (n=5); >4 nodal sites (n=36), assigning 40 patients (82%) to high/intermediate risk and 9 patients (18%) to low risk. Time from diagnosis to transplant was 9 (5-21) months. With a median follow-up of 64 (12-134) months, 5-y OS from diagnosis was 94% (95%CI 85-100%) for the high/intermediate risk group and 89% for the low-risk group (p 0.56). 5-y progression-free survival (PFS) was 65% (95%CI 49-81%) and 89% (95%CI 68-100%), respectively (p 0.13). Conclusions: OS and PFS of patients with high/intermediate FLIPI receiving SCT as part of upfront treatment compare favorably with survival reported for conventional therapy in these risk groups. Therefore the FLIPI might be a useful tool to select those patients for prospective trials on SCT in FL who can be expected to particularly benefit from this procedure. Low grade lymphoma patients (pts) have an indolent evolution with median survival ranging between 8-10 years. During disease's course, high dose therapy (HDT) and autologous stem-cell transplantation (ASCT) can be considered as an alternative to sequential chemotherapies. However, efficacy of this strategy remains controversial. The purpose of our study is to evaluate ASCT efficacy by comparing retrospectively for each pts disease free survival (DFS) after ASCT with DFS observed with pts' last chemotherapy regimen (LCR) just before intensification. Between apr 1988 and feb 2002, 109 low grade lymphoma pts were treated with HDT and ASCT in our department, 61 were male, the median age was 49 yrs [range 28-65]. Histological subtypes were mostly follicular small cell (86 %). At time of diagnosis, LDH were normal for 85 pts; 60 pts had high tumor burden. IPI was 0 for 16 %, 1 for 70 % and 2 for 14 %. Prior to ASCT, pts had experienced a median of 2 progressions (range 1 to 5). At time of graft, 102 pts present complete or partial response and 7 pts present stable disease. Two principal intensification chemo regimens were used before ASCT: VP16/cyclophosphamide in 84 pts and BEAM in 12. TBI was associated for 86 pts. At June 2002, the median follow up was 6.4 yrs from diagnosis and 4.5 yrs from ASCT. 3 years after ASCT, survival rate was 72 % and DFS rate was 50 %. Median DFS decreased with nb of progression (p=0.02) : table 1. Considering pt with more than 1 progression (n=92) as his own control, DFS was longer after ASCT than after LCR for 61 % of patients. Median DFS was 2.5 yrs after ASCT and 2.0 yrs after LCR. At 3 yrs, DFS rate was 48 % after ASCT and 37 % after LCR (p<0,001) : figure 1. This study demonstrates that HDT and ASCT significantly increase DFS in comparison with the LCR for low grade lymphoma patients. Such methodology could be useful to evaluate new strategy incorporating monoclonal antibody. Objectives: The prognosis for patients with relapsed indolent B-cell lymphomas with transformation to aggressive B-cell lymphomas is poor with conventional chemotherapy (CT). The remission rate with conventional chemotherapy (CT) and the role for high dose therapy with stem cell support (HDT) is not well defined, and was addressed. Methods: In a national five-centre prospective phase II study, inclusion criteriae included: -Indolent B-cell lymphomas (excluding lymphocytic lymphoma/chronic lymphocytic leukemia and mantle cell lymphoma) with transformation to diffuse large B-cell lymphoma (DLBCL) or follicular lymphoma grade III (FL3). -One to three previous chemotherapy regimens. Patients were given three to a maximum of six courses of CT without rituximab. Patients in at least partial remission (PR) after three courses, in ECOG 0-1 and with adequate organ function were offered HDT with the BEAM regimen. Results: Altogether 69 patients with a median age of 55 years (range 32-65) were included from July 1999 -July 2004. Forty-nine patients (70%) achieved a remission on induction CT. Seven patients did not achieve an adequate number of CD34+ cells from the blood (PB) and had a bone marrow (BM) harvest. Forty-five patients (65%) were given HDT. There were no treatment related deaths. Remission rates three months after HDT were: CR 29, PR 12, progressive disease 6. Five PR-patients achieved a CR after additional radiotherapy. Altogether 28 of the 45 HDT patients are alive. The median overall survival was 45 months. No survival plateau seems to evolve. Five of seven patients harvested from BM are dead, all of them with a BM relapse/progression. Age of the patient and time from initial diagnosis to inclusion in the study had no impact on survival. Patients with IPI score 0-1 at inclusion had a better survival (both for the whole cohort and for those given HDT) than those with IPI score 2-3. Conclusion: Remission rates on conventional CT and after HDT are satisfactory, but the therapy seems not to be curative for the large majority of patients. Failure to achieve an adequate stem cell number from the PB seems to correlate with an early aggressive bone marrow relapse after HDT. Different RIC regimens aiming to induce an allogeneic graftvs-tumor (GVT) effect have been investigated so far. However, because of the variability of myeloablation or inclusion or not of T cell depleting agents, transplant-related events might vary from one protocol to another. We report here a comparison between two prospective RIC strategies for allo-SCT from an HLA-identical sibling in patients with myeloid malignancies (60 AML, 27 MDS and 21 CML). The first RIC regimen (group I, n=56) was based on fludarabine (Flu), busulfan (Bu) and a combined GVHD prophylaxis with CSA and short course methotrexate (MTX). The second RIC regimen (group II, n=52) included ATG in addition to Flu and Bu, but used a single GVHD prophylaxis with CSA alone. The kinetic of engraftment was strictly comparable between the two groups: median of 16 days for ANC>500/µL; 12 (group I) vs. 13 (group II) days for platelet >20000/µL. 13 patients (23%) experienced grade 2-4 acute GVHD in group I, as compared to 20 patients (38%) in group II (P=NS). The incidence of extensive chronic GVHD was also comparable between both groups (40% in group I vs. 48% in group II; P=NS). Moreover, 10 patients (18%) in group I died from transplant-related mortality as compared to 11 (21%) in group II (P=NS) with disease progression being the major cause of death in both groups. Both protocols could exert a potent GVT effect, and the incidence of relapse or disease progression was comparable (21%, group I vs. 35% group II; P=NS). In multivariate analysis including protocol type (I vs. II), demographic and disease characteristics and transplant-related events, chronic GVHD was the most powerful protective factor against relapse or disease progression (RR=0.11; P<10e-5). Overall, these data establish that regardless of the type of the RIC regimen, RIC-allo-SCT is a valid and beneficial approach for patients with myeloid malignancies. The Flu-Bu-ATG-CSA-RIC approach seems to be comparable to the Flu-Bu-CSA+MTX approach as for major outcomes (engraftment, GVHD, TRM). However, it is unlikely that different RIC strategies are simply interchangeable. A detailed analysis of other outcomes such as chimerism kinetic, immune recovery profile and toxicity (infections, mucositis, liver toxicity…) is still warranted. A patient-tailored approach considering the potential GVT effect benefit and procedurerelated toxicities, adjusted for quality of life may be crucial determinants for the ultimate outcome.

Tandem auto-miniALLO approach for newly diagnosed multiple myeloma: an update of the Italian experience B. Bruno, F. Patriarca, M. Rotta, D. Maloney, N. Mordini, M. Casini, A. Rambaldi, F. Carnevale-Schianca, B. Allione, D. Soligo, P. Bavaro, L. Giaccone, R. Sorasio, V. Montefusco, A. Busca, R. Fanin, A. Gallamini, P. Coser, P. Corradini, A. Levis, M. Aglietta, E. Pogliani, M. Falda, M. Massaia, A. Palumbo, B. Sandmaier, R. Storb, M 

We are conducting a multicentre trial employing a tandem transplant approach for newly diagnosed stage IIA-IIIB multiple myeloma patients (pts) up to the age of 65. Briefly, after induction chemotherapy, pts undergo G-CSF mobilized autografting with high dose melphalan (200 mg/m²) followed 2-4 months later by low dose (2.0 Gy) TBI, PBSC infusion from HLA-identical siblings, and immunosuppression with mycophenolate mofetil (15 mg/kg BID) for 28 days and cyclosporin (6.25 mg/kg BID) for a minimum of 80 days. To date, 92 pts, 53/39 M/F (median age 55, range34-65) from 14 Italian Transplant Centres have entered the study. Seventyseven pts have completed both transplant procedures. Allografts were carried out at a median of 80 (range 44-396) days after autografts. All pts achieved sustained donor engraftment. After a median follow up of 20 months(1-54)post allografting, overall survival is 86%(66/77). The overall response rate evaluated on 75 pts (with a minimal follow up of 84 days)is 79%, with 56%(42/75) complete remissions (CR) and 23% (17/75) partial remissions (PR). Remarkably, in 63/77(82%) pts who were not in CR at allografting, 31/63 (49%)pts attained CR at a median of 94 days (range28-180)showing a gradual graft vs myeloma effect. Ten/59 patients(17%) relapsed after initial response at a median of 13 months (5-23) post-allografting. Ten pts were treated with a median of 2(range 1-4) donor lymphocyte infusions (DLI) + thalidomide and/or bortezomid at a median of 14 months (8-21)post-allografting. Only 3/10 pts responded. Three patients underwent a second allotransplant, 2 from the same donor and 1 from another HLA-matched sibling. Grade II acute graft vs host disease (GVHD) and grade III-IV GVHD developed in 28% and 12%, respectively. In 74 pts with a follow up longer than 100 days, chronic GVHD requiring therapy developed in 47% (35/74) with de novo chronic GVHD in 15/35 (43%). Overall, transplant related mortality (TRM) was 12% (9/77) at a median follow up of 20 months, day 100 TRM was 1%(1/77). We conclude that: the tandem approach allows to increase by a decade the median age of transplant pts compared to conventional allografting (Gahrton et al. 2001 Br J Haematol) ; TRM has dramatically been reduced with early TRM similar to that reported in the autologous setting; though high the response rates, longer follow up is needed to assess the impact of relapse on overall survival and event free survival; DLI were not efficacious to re-induce remission.

Long-lasting remissions in high-risk AML and MDS following sequential therapy with chemotherapy, reducedintensity conditioning for allogeneic transplantation, and prophylactic DLT C. Schmid, M. Schleuning, B. Hertenstein, E. Mischak-Weissinger, D. Bunjes, C. Scheid, U. Holtick, F. Keil, M. Sandherr, H. Kolb Ludwig-Maximilians-University (Munich, D) A novel preparative regimen for allogeneic transplantation in high risk acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) combined a cytoreductive chemotherapy with reduced intensity conditioning. Chemotherapy consisted of fludarabin (4x30 mg/m²), AraC (4x2g/m²) und amsacrine (4x100 mg/m²) (FLAMSA-regimen). After 3 days of rest, the patients received 4 Gy TBI, followed by cyclophosphamide (2x40 resp. 2x60 mg/kg) and ATG (3x10 resp. 3x20 mg/kg, higher doses used with unrelated donors). Inclusion criteria were those of non-myeloablative conditioning regimen. In the absence of GvHD, the Graft-versus-Leukemia effect was augmented by prophylactic transfusions of donor lymphocytes (pDLT), starting at day 120 after transplant.

Here we report on the results of 174 uniformly treated patients (CR1 + unfavorable karyotype, delayed response to induction therapy or secondary AML: n=17, CR2: n=16, 1st or 2nd relapse: n=67, refractory: n=52, untreated progressive MDS or secondary AML: n=22) from 7 transplant centers. 41 patients had a complex, 38 an otherwise unfavorable karyotype. Median age was 50.0 (18.5-65.8) years, 23 patients were older than 60. 73 patients had a family, 101 an unrelated donor. Median follow up was 19.3 months. 13 patients died in aplasia, and 6 patients reconstituted with leukemic blasts. In contrast, 155 patients were free of detectable leukemia at day +35. Non-relapse mortality was 31% at 1 year. 36 patients relapsed at a median of 115 days, 16 achieved a further remission. Overall survival was 53%, 42%, and 39%, current leukemia free survival was 48%, 42% and 39% at 1, 2 and 4 years from transplant. So far, 28 patients received pDLT after fulfilling the criteria of being off immunosuppression and free of GvHD for 30 days. These patients showed an overall survival of 80% at 4 years from transplantation. In a subgroup analysis, most encouraging results were obtained in patients with refractory leukemia and complex karyotype disease: Refractory patients (n=52) showed an OAS of 55% and 44% at 2 and 4 years, patients with complex cytogenetic aberrations (n=41) had a 2-and 4-year OAS of 52% and 40%. We conclude that the approach of of cytoreductive chemotherapy, reduced intensity conditioning and prophylactic DLT is feasible also at the multicenter level. Long term remission can be induced in patients with otherwise most unfavorable prognosis. pDLT in selected patients may be able to further improve the results. In 21 patients with myelofibrosis we evaluated within a prospective pilot-study the effect of reduced intensity conditioning with busulfan (10 mg/kg), fludarabine (150 mg/qm) and anti-thymocyte globulin followed by allogeneic stem cell transplantation from related (n=8) and unrelated donors (n=13). The median age of the patients was 53 (range, 32 to 63) years. No primary graft failure occurred. The median time until leukocyte ( > 1.0 x 10 9 /l) and platelet ( > 20 x 10 9 /l) engraftment was 16 (range, 11 to 26) and 23 (range, 9 to 139) days, respectively. Complete donor chimerism on day 100 was seen in 20 patients (95%). Acute graft-versus host disease (GvHD) grade II-IV and III/IV occurred in 48% and 19 % and of chronic GvHD in 55 % of the patients. Treatment related mortality was 0% at day 100 and 16% (95% CI: 0 to 32%) at one year. Hematological response was seen in 100% and complete histopathological remission was observed in 75% of the patients. 25% of the patients showed partial histopathological remission with still declining grade of fibrosis. After a median follow-up of 22 (range, 4 to 59) months, the 3 years estimated overall and disease-free survival is 84% (95 % CI: 67 to 100 %). Forty patients with relapsed or refractory Hodgkin's disease (HD) underwent allogeneic stem cell transplantation (allo-SCT) following a fludarabine-based conditioning regimen from an HLA-identical sibling (n=20) or a matched unrelated donor (n=20). The median age was 31 years (range 18-58). The median number of chemotherapy regimens received prior to allo-SCT was five (range 2-9). Thirty (75%) and thirty (75%) patients had received prior radiotherapy or a prior autologous SCT, respectively. The median time to progression after autologous SCT was nine months (3-52). Disease status at SCT was refractory relapse (n=14) or sensitive relapse (n=26). The conditioning regimens employed were fludarabine (25 mg/m sq IV x 5 days)-cyclophosphamide (1 g/m sq IV x 3 days) ± antithymocyte globulin (30 mg/kg IV x 3 days) (FC±ATG) (n=14), a less intensive regimen, and fludarabine (25 mg/m sq IV x 5 days) -melphalan (70 mg/m sq IV x 2 days) (FM) (n=26), a more intensive one. The two groups had similar demographics and prognostic factors. Chimerism studies indicated 100% donor-derived engraftment in 26/26 (100%) FM patients and in 9/13 (69%) evaluable FC±ATG patients. Day 100 and cumulative (18-month) transplantrelated mortality (TRM) were 5 % and 22%, respectively for the whole group. The cumulative incidence of acute (grade II-IV) GVHD was 38%. The cumulative incidence of chronic GVHD at 18 months was 69%. There was a non significant trend for a lower relapse rate after the occurrence of GVHD (hazard ratio 0.8; p= 0.6). Progression rates were similar in the FM and FC patients (53% vs. 57% respectively at 18 months, p=0.4). However, disease progression occurred later in FM patients (range 2-34 months) than in FC patients (range 0.7-13 months). In addition the FM group experienced a lower death rate after progression. Twenty-four patients (60%) are alive (fourteen in complete remission) with a median follow-up of 13 months (4-78). Sixteen patients expired (TRM n=8, disease progression n=8). FM patients had significantly better overall survival (73% vs. 39% at 18 months; p=0.03), and a trend towards better progression-free survival (37% vs. 21% at 18 months; p=0.2). We conclude that allo-SCT with fludarabine-based, less intensive conditioning from matched related and unrelated donors is feasible in high-risk HD patients with a low TRM. The intensity of the preparative regimen affects survival.

The upper age limit for allogeneic haematopoietic cell transplantation (HCT) used to be in the range of 50 to 55 years, well below the age with the highest incidence of myeloid malignancies. Here we present data on 72 consecutive patients (pts) older than 60 years of age (median 64; range 60-74) transplanted from a matched sibling (n=23) or unrelated (n=49) donor after conditioning with a uniform, dose-adapted, myeloablative conditioning regimen consisting of fludarabine 150mg/m², carmustine 300 mg/m² and melphalane 110mg/m² (FBM). GvHD prophylaxis consisted of cyclosporine A and mycophenolate mofetil with additional rabbit anti-T lymphocyte globulin (ATG Fresenius) in the unrelated setting. Apart from 5 pts. receiving marrow all other pts received filgrastim mobilized peripheral blood grafts (PBHCT) Pts presented with CLL (3), follicular NHL (2), mantle cell NHL (2), multiple myeloma (2), MPS (3), MDS-RA (3) and AML or MDS-RAEB (n=57). Apart from 3 pts in CR1 and 3 in CR2, all other had active disease, either refractory to prior chemotherapy or were untreated. Acute GvHD >II° occurred in 10 related (50%) and 16 unrelated (45%) transplanted pts. In the related transplantations, 6 pts died due to TRM (30%) and 4 due to relapse (20%). Causes of death were infections (4 pts), acute GvHD (1 pt) and infection in the context of chronic GvHD (1 pt). In unrelated transplantations, there were 8 deaths due to TRM (16%) and 6 due to relapse (12%). Causes of death after unrelated aHCT were graft failure (1 pt), ARDS (1pt), infections (4 pts), acute GvHD (1 pt) and late myocardial infarction (1 pt). After a median follow up of approx. 1 year (range 3-1778 days), the estimated probability of overall survival was 46% and 61% for related and unrelated transplantations, respectively. This difference was not statistically significant. We conclude, that HCT using the doseadapted FBM protocol is a reasonable treatment option for pts >60 years of age with high risk lympho-hematologic malignancies. In contrast to previous reports from transplant registries, we found that HCT from unrelated donors incorporating in vivo ATG results in outcomes comparable to HCT from related donors. Allogeneic HCT in patients 60 to 70 years is indicated in a similar way as in younger patients. Introduction: The main limitations of the myelobalative regimens prior to allogeneic transplant is toxicity. This has led to the development of non-myeloablative procedures which allow for initial engraftment of stem cells and subsequent transfer of immune cells for the induction of graft-versus-tumor reaction. In this setting the impact of the low-dose-TBI parameters on outcome have never been studied.

Material and methods: Data of 106 patients (pts) were recorded from 10 institutions. Median age was 50y (3-72), male to female ratio was 65/41. Diagnoses included AML (n=26), other leukemia (n=30), MPS or MDS (n=12), lymphoma (n=23) and myeloma (n=15). Allogeneic transplant using PB, BM or PB+BM were performed respectively in 82, 20 and 4 pts from HLA identical sibling donor (n=79), MUD (n=23) or unmatched related donors (n=4). TBI delivered 2Gy in 1 fraction in 77 cases whereas 29 pts received a total dose > 2Gy (4 or 6Gy). Median duration of irradiation was 24 (6-40) min. The median dose rate (DR) was 0.105 (0.06-0.57) Gy/min. Results: The median follow-up was of 17 (1-62) m. Engraftment was obtained in 99 patients. Full chimerism was observed in 61% of pts. GVHD grade I to IV was developed in 13, 19, 13, 11, respectively, without any influence of TBI parameters (dose p=0.31; DR p=0.27). In Univariate analyses, the 2-year LFS tends to be higher after TBI delivering 2Gy (50% vs. 20%,) and using higher DR > 0.1 Gy/min (52% vs. 28%) but the level of statistical significance was not reached (p=0.08 and p=0.06 respectively). LFS was significantly better for pts in CR (52% vs. 34%; p=0.006), for female donor to male recipient 52% vs. 36%, p=0.04), HLA id sibling donor (52% vs. 12%; p<0.0001). Multivariate analysis showed 2 independent factors for relapse and LFS : HLA id sibling donor (p=0.0002; RR:12 and p<0.0001; RR: 0.18) and CR (p=0.0009; RR: 0.06 and p=0.0006; RR: 3.46); For overall survival the 3 independent factors were : CR (p=0.001, RR:3.3), HLA id sibling donor (p=0.0003; RR=0.19) and DR (p=0.02; RR:2.07). DR and HLA did not influence TRM. Conclusion: In this multicenter retrospective study we found that RC and HLA sibling donor influence independently the outcome. DR seems to have an impact on OS without any influence on TRM. The results in terms of GVHD incidence, engraftment and chimerism are comparable to the literature. Further analyses are ongoing on higher number of patients recorded recently from other institutions.

Re-188 or Y -90 labeled anti -CD66 antibody as part of a dose-reduced conditioning regimen for patients with acute leukaemia or myelodysplastic syndrome over the age of 55: results of a phase I -II study D. Bunjes, M. Ringhoffer, N. Blumstein, B. Neumaier, G. Glatting, S. Von Harsdorf, I. Buchmann, M. Wiesneth, R. Schlenk, J. Kotzerke, T. Zenz, A. Buck, P. Schauwecker, S. Stilgenbauer, H. Döhner, S. Reske Ulm University Hospital (Ulm, D) Objective: To evaluate the feasibility and safety of using Re -188 or Y -90 labeled anti -CD66 antibodies as part of a reduced -intensity conditioning regimen in patients with acute leukemia in CR or PR and with MDS aged 55 -65 years. Patients and methods: 20 patients (pts) with a median age of 63 years (56 -67y)were treated. The diagnoses at transplant were AML>CR1 (n=7), AML CR1 (n=8), ALL CR1 (n=2), MDS (n=3). All pts had a favorable dosimetry. Radioimmunotherapy was performed with either Re-188 (1999 Re-188 ( -2002 or Y-90 labeled anti -CD antibody (2002 -2004) , n=12) on day -14. Additional conditioning consisted of either 180mg / m² fludarabine plus 40mg / kg ATG for matched related donors (MRD, n= 11) or 150mg / m² fludarabine plus 140mg/ m² for matched unrelated donors (MUD, n= 9). Graft -versus -host disease (GvHD) -prophylaxis was performed with Campath 1H (CP1H) in the bag (MRD) or CP1H i.v. (100mg)plus cyclosporine (MUD). Results: The radiolabeled antibody provided a mean dose of 21,9 Gy to the bone marrow, 5,7 Gy to the kidney, 9,7 Gy to the liver and 2,1 to the whole body. The mean marrow dose and the mean liver dose were significantly higher in pts treated with Y -90 than in those teated with 1 Gy vs. 15, 9 Gy and 12, 9 Gy vs. 5, 1 Gy) . Regimen -related toxicity was low with 2 pts developing 3 episodes of Bearman grade III toxicity, there were no differences in terms of toxicity between the Y -90 and Re -188 groups. No cases of radiation nephropathy have been observed. All pts engrafted and 19/20 became complete donor chimeras. Grade II -IV GvHD was observed in 1 pt (5%) and chronic GvHD in 2 pts (11%). After a median follow -up of 16 mo the cumulative incidence of non -relapse mortality is 27,8%, infections being the most comon cause of death. All pts achieved a CR, and the median duration of remission was 12 mo. The cumulative incidence of relapse was 55%. On univariate analysis the risk of relapse was sigificantly lower in the Y -90 treated group (17% vs. 75%, p= 0,02). The probability of survival was estimated to be 67% at 1 year and 44% at 2 years post transplant. Conclusios: RIT can be safely performed in the context of reduced -intensity conditioning. RIT without additional cytoreductive therapy and without GvL -effect is insufficient to control acute leukemias. RIT should be considere as an adjunct to RIC regimens, preferably with Y -90 as therapeutic nuclide.

Acute leukaemia 1 O108 Haplo-identical haematopoietic stem cell transplantation for secondary leukaemia F. Aversa, T. Aloisi, E. Mastrodicasa, A. Terenzi, A. Carotti, R. Felicini, S. Ballanti, F. Falzetti, M.P. Martelli, R. La Starza, A. Santucci, T. Zei, M.F. Martelli Ospedale Monteluce (Perugia, I) Allogeneic hematopoietic stem cell transplantation, the best treatment for secondary leukemia, has long been restricted to patients under 50 in hematological remission with matched donors. Graft-vs-host disease (GvHD) and rejection, which limited mismatched transplantation, were overcome by using large doses of T-cell-depleted hematopoietic stem cells after a highly immunosuppressive, myeloablative conditioning regimen (Aversa et al. NEJM 1998 ,339,1186 . Here we report the results of full-haplotype mismatched transplantation in 41 patients with secondary leukemia (median age 37 years; range 4-65). Leukemia was secondary to myelodysplastic syndrome (36), Burkitt lymphoma (1), gastric cancer (1), Ewing sarcoma (1), chronic myelomonocytic leukemia (1), uterine leiomyosarcoma (1). Median time from leukemia diagnosis to transplant was 10 months (range 2-60). At transplant, 17 patients were in remission and 24 in relapse. Cytogenetics were normal in 11, unavailable in 11, abnormal in 19: monosomy 7 (5), complex karyotypes (3), trisomy 8 (2), +19, tetraploidy, t8;16, t8;12, t8;10, t6;9, t3;5, t11;12 and -y with t8;21. Conditioning included single fraction TBI, thiotepa, rabbit ATG, cyclophosphamide in the first 5 patients or fludarabine in 36. Median graft content: 13 x 10 6 CD34+ cells/kg (range 5.1-25.9), 1.5 x 10 4 /kg CD3+ cells. No posttransplant immunosuppressive treatment was given as GvHD prophylaxis. Transplant procedure was well tolerated (5 patients had grade III oral-intestinal mucositis; no severe veno-occlusive disease of the liver). 40/41 patients engrafted achieving respectively 1,000 neutrophils and 50,000 platelets 12 and 16 days after transplant. Severe acute GvHD occurred in 1 case and chronic GvHD in 3 (extensive 2, limited 1). Infections were the most common causes of 17 non leukemic deaths (CMV 4, candida 2, EBV 2, bacteria 2, fusarium 1, pn. carinii 1). Other causes of death were GvHD (2), idiopathic pneumonia (2) and rejection (1). 8 patients relapsed (3 were in CR and 5 in relapse at transplant). At a median follow-up of 55 months, range 5-140, 16 patients survive disease-free. The probability of event-free survival (EFS) is 50%,40% and 23% in CRI, CRII and relapse, respectively. The high engraftment rate, low incidence of GvHD and good EFS make mismatched transplantation feasible for patients with secondary leukemia. Haploidentical donors are available for almost all patients without undue delay between decision-making and transplant.

Early allogeneic stem cell transplantation for young adults with acute myeloblastic leukaemia in first complete remission (CR1): an intent-to-treat analysis of the BGMT inter-group experience over 17 years E. Jourdan, J.M. Boiron, N. Dastugue, N. Vey, G. Marit, F. Rigal Huguet, L. Molina, N. Fegueux, A. Pigneux, C. Recher, J.F. Rossi, M. Attal, J.J. Sotto, D. Maraninchi, J. Reiffers, V.J. Bardou, B. Esterni, D 

Since 1984, BGMT inter-group performed allo-SCT as an early consolidation for pts aged <= 45 with CR1 AML. Over 17 years, we carried out 4 successive protocols with the goal of improving the outcome for pts with newly diagnosed AML with or without an allo donor and to prospectively compare the 2 strategies. Of the 472 pts aged under 45 who achieved CR1, 182 (38%) had an HLA identical sibling (donor group) and allo-SCT was performed in 171 (94%) at a median of 60 (11-284) days after CR1. Of the 290 pts without donor (no-donor group), 88% were intended to receive autologous SCT (Auto-SCT). In an intend-to-treat analysis based on donor availability and with a median follow-up of 114 months, the 10-year relapse, non-relapse deaths (NRD), overall survival (OS) and leukemia free survival (LFS) probabilities were 27% vs 55% (p < .001), 24% vs 6% (p < .001), 51% vs 43% (p = .11), and 48% vs 40% (p < .03), respectively for donor and no-donor groups. A Cox analysis performed in the no-donor population indicated that four factors (initial white blood-cell-count, FAB subtypes, cytogenetic risk and number of induction courses) were independent prognostic predictors for survival and allowed to calculate the risk index of each of their combinations. This index reclassified 21% of the pts as compared to usual cytogenetic classification and identified 3 sub-populations with different outcome: low risk (16%), intermediate risk (56%) and poor risk (42%) pts with a respective 10 year survival probability of 74%, 47% and 17%. We then compared the outcome of each subpopulation (low, intermediate and high risk) with the pts presenting the same risk factor characteristics but having a donor. For low risk pts, the 10-year NRD, relapse, LFS and OS probability comparison (donor vs. no-donor) are respectively: 37% vs. 11% (p=.02), 11% vs. 23% (p=NS), 51% vs. 68% (p=NS) and 51% vs. 74% (P=NS). These pts do not benefit from early allo-SCT in CR1. For high risk pts, the 10-year NRD, relapse, LFS and OS probability comparison are respectively: 29% vs. 1% (p<.001), 53% vs. 85% (p=.003), 17% vs. 13% (p=NS)and 28% vs. 17% (P=NS). Early allo-SCT is not the adequate treatment for these pts. For intermediate risk pts, the 10-year NRD, relapse, LFS and OS probability comparison are respectively: 17% vs. 7% (p=.02), 18% vs. 47% (p<.0001), 64% vs. 45% (p=.001)and 63% vs. 47% (P=.02). Conversely allo-SCT leads to a survival advantage in intermediate group and could be refined for better results. Background and objective: The optimal post remission therapy for HR-ALL patients (pts) is not well established. This randomized trial compared chemotherapy (CHT), ALLO or AUTO SCT as post remission therapy in adults with HR-ALL. Patients and methods. Inclusion criteria: one or more of the following: age 30-50 yr., WBC>25x10 9 /L, t(9;22) or BCR/ABL, 11q23 or MLL and t(1;19). After CR and intensification therapy patients with an HLA-identical sibling were assigned to ALLO SCT and the remaining were randomized to AUTO SCT or to delayed intensification CHT followed by standard maintenance CHT up to 2-yr. in CR. Results: Study period: 1993-2004, 254 pts included, 222 evaluable, 35 hospitals, 131 males, mean (SD) age 29(10) yr, WBC count 60(98) x10 9 /L, early pre-B: 43 (19%), common+pre-B: 113 (51%), T: 66 (30%). Cytogenetics: 161 (73%) valid cases after central review, normal: 67 t(9;22): 37, 11q23: 6 cases, t(1;19): 2, other rearrangements: 49 cases. Response to therapy: CR 183 (82%). Slow response to therapy in 40% of cases. 84 pts were assigned to ALLO SCT, 50 randomized to AUTO SCT and 48 to CHT. With a median follow-up of 70 mo.(range 24-133), medians for DFS and OS for the whole series were 17 and 23 mo., and 5-yr DFS and OS probabilities were 35±5% and 34±6% (37±6% and 35±6% after removing Ph+ ALL pts from analysis). Groups of ALLO, AUTO and CHT were comparable for the main clinicobiologic characteristics and the rate of response to therapy. Intentionto-treat analysis showed no differences in DFS and in OS for donor vs. no donor comparison as well as for comparison of AUTO vs. CHT. This lack of differences persisted after removing Ph+ ALL pts from the analysis. The same results were observed when the analysis was performed by actually treated patients. Conclusions: This study failed to prove that, when a family donor is available, allogeneic SCT produces a better outcome than autologous SCT or chemotherapy in adults with HR-ALL.

Results of autologous haemopoietic stem cell transplantation in very good risk patients with acute myelocytic leukaemia, supposedly not candidates for a transplant -an EBMT survey N. Gorin, M. Labopin, M. Vignetti, D. Blaise, F. Locatelli, G Dini, V. Rocha, F 

Common wisdom in the past decade has excluded patients with Acute myelocytic leukaemia (AML) and favourable cytogenetics from hemopoietic stem cell transplantation. However there is no randomized study indicating in these patients the superiority of conventional chemotherapy. The total EBMT data base for the period of January 1990 to January 2002 contained information on 6736 adult patients and 896 children. In adults, the leukaemia free survivals (LFS) at 5 years were 43+/-8% for 5437 patients transplanted in CR1, 32+/-2% for 1117 patients in CR2 and 9 +/-2% in 495 patients transplanted in more advanced stages. In children the figures were 59+/-2% (n= 713) in CR1, 33+/-4% (n=175) in CR2 and 10+/-5% for more advanced disease (n=43). We retrospectively studied the outcome of good risk patients autotransplanted for AML .In a first study, using already identified factors, we defined patient risk groups according to two powerful clinical risk factors , age (< and >35 years) and response to the first induction course (CR achieved in less or more than 40 days). The good risk group (age<35 years; time to CR1<40 days) consisted of 458 patients with a LFS at 5 years of 56+/-2%. The poor risk group(age>35 years; time to CR1>40 days) consisted of 288 patients with a LFS of 38+/-1%. The intermediate group consisted of 2835 patients with a LFS of 41+/-1%. In good risk patients so defined autografted in CR1, the relapse incidence with marrow as a source of stem cells was 33+/-3% versus 47+/-4% with peripheral blood (p=0.01). At time of writing this abstract, further analyses are however underway to confirm or infirm this last finding. Updated results will be presented. In a second step, we focussed on good cytogenetics. Regarding M3, 163 patients were autografted in CR1 with a LFS of 70% at 5 years , and 185 in CR2 leading to a LFS of 45%. 83 patients with inv 16 and 81 patients with t(8;21) were autografted in CR1: the LFS at 5 years were 55+/-7 and 53+/-7 %. 23 patients and 15 respectively were transplanted in CR2 and the LFS at 2 years were 38 +/-12% for both groups. In view of the increasing relapse rates reported in patients with inv 16 treated with conventional chemotherapy and also in view of the results of several prospective randomized studies which indicated indeed better results for autografting in patients with good risk AML, we propose that future prospective studies investigate the role of autografting good risk patients.

Cooperative analysis of patient with acute myeloid leukaemia exhibiting a normal karyotype treated within the German multicentre treatment trials AML-2/95, AML-1/99, AMLHD93 and AMLHD98A R. Schlenk, J. Krauter, K. Döhner, G. Heil, S. Fröhling, H.

Purpose: Karyotype at diagnosis provides the most important prognostic information in adult acute myeloid leukemia (AML). However, approximately 50% of patients lack clonal chromosomal aberrations. The value of different postremission strategies such as intensive chemotherapy (CHEMO), autologous (AUTO) or allogeneic transplantation (ALLO) in these patients remains open and, in particular, if molecular markers such as MLL-partial tandem duplications, activating FLT3-mutations and CEBPA-mutations are included in the analyses. Therefore, we initiated a cooperative analysis of patients exhibiting a normal karyotype at diagnosis treated within four prospective treatment trials. Methods and Patients: All patients (age 16-60 years) received two cycles of induction therapy with standard dose cytarabine (ARAC) combined with etoposide and idarubicin. After a first consolidation therapy, patients were assigned to ALLO, if an HLA-identical sibling donor was available in all four trials. In the AML-2/95 and AMLHD93 trials all other patients were assigned to a high-dose cytarabine based regimen whereas in the AML-1/99 and AMLHD98A trials patients were randomised between AUTO and CHEMO. Results: Between 1993 and 2003 a total of n=795 patients exhibiting a normal karyotype had been registered within one of the four trials. Data from n=524 patients with a good response after first induction cycle and complete remission (CR) after double induction therapy are available. On an intention-to-treat basis, there was no difference between AUTO and CHEMO. Donor versus no-donor analysis revealed a significantly better relapse-free (RFS) (p=0.02) and in trend also a better overall survival (p=0.09). After relapse, second CR rate was 48%, 24% and 39% in the ALLO, AUTO and CHEMO groups, respectively, and survival after relapse was dismal in all three groups. The evaluation of FLT3 mutations revealed a strong negative prognostic impact of FLT3-ITD (p=0.001) but no impact of FLT3-D835/D836 mutations. FLT3-ITD was associated with an unfavourable RFS within the CHEMO (p=0.006) and the AUTO groups (p=0.002), but not in the ALLO group (p=0.82). Conclusions: In AML-patients exhibiting a normal karyotype, allogeneic transplantation from an HLA-identical sibling donor in first CR seems to be beneficial in respect to RFS and the negative prognostic impact of FLT3-ITD seems to be overcome by allogeneic transplantation. Allogeneic haematopoietic cell transplantation (alloHCT) is widely used for the treatment of high risk acute myeloid leukaemia (AML). The results depend on many factors including the center effect. This analysis is based on the EBMT registry data and compares the results in CR1 AML adult patients treated in Western Europe (WE) and in 10 countries in Central and Eastern Europe (CEE). The comparative analysis of matched related donor alloHCT was performed for 2448 patients treated in WE and in 213 patients treated in CEE. The characteristics of WE and CEE groups were comparable except of age (40 vs. 36 y), source of cells (PB; 52% vs. 32%), proportion of patients obtaining TBI conditioning and T depletion (57% vs. 14% and 17% vs. 0.5%), intervals from dgn and from CR1 to HCT (147 vs. 179d and 86 vs. 139d. respectively) -p<0.001. In the whole group the LFS rate in WE and CEE equaled 58+/-1% and 53+/-4% respectively (ns), the relapse rate 26 +/-1% vs. 30 +/-4% (ns) and the non-relapse mortality 22+/-1% vs. 25+/-3% (ns). Subgroup analysis excluding T depleted HCT; WE; n=1456, CEE;n=159: LFS 60+/-2% vs. 53+/-4% (p=0.014), relapse incidence 36+/-8% vs 22+/-2% (p=0.39), non relapse mortality 18+/-2 % vs 23 +/-7% respectively. In multivariate analysis only the difference in non relapse mortality remained significant. A single center analysis for BMT Unit in Katowice. Out of 1160 patients transplanted in this center, 101 were adult AML patients allografted from siblings or unrelated donors (n = 77 and 24). The probability of OS at 11 y and LFS equaled for CR1 patients 81% and 76%, for CR>2 -53% and 53% and for patients in PR 47% and 47%respectively. This comparative analysis demonstrates that the results of alloHCT in CEE countries are comparable to those in WE in spite of longer time from diagnosis and from CR1 to transplant, less frequent use of TBI, PBSC and T depletion in CEE. A comparison of non T depleted subgroups suggests that the LFS is in CEE lower and NRM higher than in WE. This may indicate the need of a better post-transplant care in CEE. The results of single center performing over 150 transplants per year are superior to the registry data confirming the center effect.

The role of reduced-intensity conditioning allogeneic stem cell transplantation in patients with acute myeloid leukaemia: a donor versus no donor comparison H. de Lavallade, M. Mohty, P. Ladaique, C. Faucher, N. Vey, D. Coso, A.M. Stoppa, J.A. Gastaut, D. Blaise Institut Paoli-Calmettes (Marseille, F) Standard myeloablative allo-SCT is a well established therapy for patients with AML. However, because of the high incidence of procedure-related toxicity, this procedure is often limited to younger patients in good medical condition. Using a genetic randomization through a "donor" versus "no donor" comparison, the aim of this analysis was to assess the real benefit of RIC-allo-SCT among 95 adult high risk AML patients. In an intention-to-treat analysis, the Kaplan-Meier estimate of LFS was significantly higher in the "donor" group as compared to the "no donor" group (P=0.01; 54% versus 30% at 4 years). When restricting the analysis to patients who could effectively receive the RIC-allo-SCT, the difference in LFS was also significant between this group of 25 patients ("transplant" group) and the remaining 70 patients ("no transplant" group) who did not receive allo-SCT (P=0.001; 62% versus 31% at 4 years). In the "transplant" group, RICallo-SCT was performed at a median of 209 (range, 119-413) days after diagnosis. No grade 3 or 4 toxicities were encountered during RIC administration, and only 3 patients died from transplant-toxicity, for an overall cumulative incidence of TRM of 12% (95%CI, 3-32%). This relatively low TRM translated towards a significantly higher overall survival (OS) in the "transplant" group as compared to the "no transplant" group (P=0.01) Overall, 41 patients (43%; 95%CI, 33-53%) had relapsed at a median of 295 (range, 116-823) days after diagnosis, with the 4-year cumulative incidence of relapse being significantly higher in the "no transplant" group as compared to the "transplant" group (P=0.0002; 54% versus 12%). After controlling for all relevant factors [demographic characteristics, leukemia features (FAB subtype, leukemia origin (secondary vs. de novo), cytogenetics risk group, history of prior high dose cytarabine or autologous transplantation, and number of chemotherapy induction courses to achieve first CR), identification of an HLA-identical sibling donor, and effective performance of RIC-allo-SCT], in the multivariate analysis, only an intermediate cytogenetic risk group (P=0.01; RR=1.2; 95%CI, 1.2-4.7) and effective performance of RICallo-SCT (P=0.001; RR=4.0; 95%CI, 1.7-9.6), were significantly predictive of an improved LFS. We conclude that if a matched related donor is identified, RIC-allo-SCT should be proposed for AML patients not eligible for standard myeloablative allo-SCT.

A. Urbano-Ispizua, S. Brunet, M. Monzó, G. Perea, A. Navarro, J. Esteve, J. Berlanga, J.M. Ribera, J. Bueno, A. Llorente, J. Besalduch, R. Guàrdia, M. Tormo, C. Pedro, J.M. Sánchez, P. Torres, M.P. Queipo de Llano, L.L. Font, J.M. Moraleda, J.M. Martí, P. Vivancos, M.J. Peñarrubia, J.F. Nomdedéu, E. Montserrat, J. Sierra Institute of Hematology & Oncology (Barcelona, E); CETLAM Group (Barcelona, E) Clinical and laboratory data has allowed the subdivision of AML into three broad prognostic groups. While therapeutical strategy is well defined in both the favorable and unfavorable groups, intermediate group remains a poorly defined category, with patients having different response to chemotherapy, and in whom the decision for either autologous, HLA identical sibling or unrelated allo-SCT is not always clear. One hundred and ten adult patients <60 year-old with intermediate AML (MRC criteria) , enrolled in the AML-99 protocol of the CETLAM group were included in this study. Induction therapy consisted of idarubicin, intermediate dose ara-C (IDAC) and VP16. Intensification included mitoxantrone and IDAC. Patients were following treated with autologous or allogeneic transplantation. The characteristics considered were: age (<50 years vs >50), WBC (<20x10 9 /l vs >20x10 9 /l), FAB classification, MLL rearrangement, internal tandem duplication of FLT3 (ITD-FLT3), induction courses to achieve complete remission (CR) (1 vs 2), and germline polymorphisms of 15 candidates genes. Of the 110 patients, 86 (78%) achieved a CR, 11 (10%) were chemoresistant and 13 (12%) died during induction. After a median follow-up of alive patients of 24 months (range 5-64), overall survival was of 31% at 5 years. In the multivariate analysis, adverse prognostic variables for survival were polymorphism of XPA (RR=3.4; p=0.02) and of MDR1-1 (RR=2.1; p=0.02), and WBC >20x10 9 /l (RR=2.1; p=0.02). Increased risk of relapse was associated with polymorphism of SULT1C2 (RR 4.1; p=0.004), ITD-FLT3 (RR 3.3; p=0.003), VEGF2 (RR 2.8; p=0.04) and MDR1-1 (RR 2.4; p=0.02). Finally, in the multivariate analysis for refractoriness to chemotherapy, XPA polymorphism was the only independent factor increasing the risk (RR=14; p=0.02). In conclusion, germline polymorphisms, which can easily be analyzed from DNA of peripheral blood, have independent prognostic value in intermediate risk AML and might assist in making transplant-decisions.

Where do we stand 6 years after gene therapy? M. Cavazzana-Calvo, S. Hacein-Bey-Abina, C. von Kalle, M. Schmidt, G. de Saint-Basile, F. Le Deist, N. Wulfraat, I. Alexander, P. Landais, S. Blanche, A. Fischer Hôpital Necker-Enfants Malades (Paris, F) For the last 35 years or so, allogeneic hematopoietic stem cell transplantation (HSCT) has been the only curative approach for patients affected by severe combined immunodeficiencies (SCID), a medical emergency. According to the most recent European survey, haploidentical transplants allow a survival rate at 3 years of 75% (taking only into account patients transplanted since 1995). The mortality rate is heavily influenced by age at the time of transplantation (it is very low in young infants), in correlation with the infection burden and also the incidence of acute graft-versus-host reaction (GVHD). Between 1998 and 2003, 6 patients with NK(-) B(+) SCIDs (either gc or JAK3 deficiency) received an haploidentical HSCT at the Necker hospital, Paris. 3 of them died of an infection, one in the context of a severe GVHD. In addition, and despite the improvement in the survival rate, a number of long-term concerns have been detected over time including a frequent persistence of a B cell deficiency and a decline in Tcell functions related to the absence of donor stem cell engraftment and maybe also to a premature decline in the thymus functions. Therefore, several patients have received a second transplant with a low efficiency in an haploidentical setting. These significant limitations set the rationale for the development of an alternative strategy such as gene therapy. From March 1999 up to May 2002, 10 children with gc deficiency under the age of one year were enrolled.

The gc gene transfer into the patients' CD34(+) cells led in 9 out of the 10 treated patients to the emergence of T and NK lymphocytes. It took 10 to 12 weeks to detect mature T cells in the periphery, a time span which is significantly shorter than the one observed after haploidentical HSCT. In all but 2 patients, T cell counts normalized up to 5.3 years after gene therapy. The occurrence of 2 monoclonal lymphoproliferations led to put the trial on hold temporarily in order to understand the pathophysiology of these events and assess the overall risks. One of the 2 patients recently died of a relapse while the other is in a sustained complete remission, easily achieved after the first months of treatment. All the other patients are in a good clinical state without any treatment 6 years after their gene therapy. Following a thorough analysis of the retrospective data and a prospective analysis, including exhaustive site integration analysis in all patients, it was concluded that the benefit/risk balance is in favour of the gene therapy approach at least for patients older than 3 months, hence the reopening of our gene therapy protocol. An update of the clinical and biological data will be provided at the meeting. Patients lacking expression of either RAG-1 or RAG-2 suffer from a Severe Combined Immuno-Deficiency (SCID) disease characterized by an early block in T and B lymphocytes differentiation leading to the absence of both mature lymphocyte subsets. This disease accounts for about 20% of SCID and the only curative treatment is hematopoietic stem cell transplantation, usually successful when an HLAgenoidentical donor is available. In the absence of such a donor, the success rate decreases along with the degree of HLA disparity between donor and recipient. Ex-vivo gene therapy of hematopoietic stem cells can be considered as an alternative treatment as a selective advantage of transgeneexpressing cells is expected. Moreover, constitutive expression of only one of the two RAG proteins should not be harmful as concomitant expression of both genes is required for the recombination activity. We used a lentiviral vecteur containing the RAG-1 cDNA transgene as a therapeutic vector to transduce bone marrow CD34+ cells obtained from RAG-1 deficient patients. The transduced cells were injected into N0D-SCID mice previously irradiated (3Gy) and treated with an anti-TMß1 antibody. Ten weeks after transplantation, in all treated mice, 35±15% of the bone-marrow cells express the human CD45 marker. In this population, 24±2% co-express CD19 and IgM demonstrating that B cell differentiation capacity has been restored. We also detected some CD33+ cells attesting the presence of human myeloid progenitors cells. Altogether, these results suggest that both lymphoid and myeloid precursors have been transduced and demonstrate that gene transfer into hematopoietic cells can reconstitute B cell development in vivo. Our data support the hypothesis that gene therapy could represent a possible alternative to bone marrow transplantation in RAG-1 deficient SCID disease.

Recurrent retroviral vector integration at the MDS1-EVI1 locus in rhesus long-term repopulating haematopoietic stem cells B. Calmels, C. Ferguson, R. Adler, S. Sellers, C. Dunbar NHLBI, NIH (Bethesda, USA) Until recently, the risk of insertional mutagenesis using retroviral vectors for gene therapy has been estimated to be low. Owing to reports of proto-oncogenes activation in mice and humans, this estimation is being re-evaluated. We here report a high frequency of proviral insertions at the MDS1-EVI1 locus in the engrafted gene-modified hematopoiesis of rhesus monkeys. We have recovered vector-genome junction sequences from granulocytes and mononuclear cells of 22 animals that were transplanted with autologous CD34+ cells transduced with an MLV-derived vector containing a neomycin marker gene. Using the LAM-PCR method, we have retrieved and analyzed 702 integration sites. While several genes harbor 2 or 3 proviral insertions, we have identified, in 9 animals, an unexpected 14 integration events within the 2 first introns of the MDS1 gene. MDS1 is adjacent to EVI1, a retrovirally-activated zinc finger transcription factor. We used insertion-specific primers to confirm that the fusion sequences between the MDS1 locus and the 5'-LTR of the vector were detectable in 4 animals for which we had the longest follow-up (4 to 6 years). In order to determine if the cells carrying proviral insertions at this locus have a selective growth advantage, we performed q-PCR experiments with neomycin and MDS1/5'-LTR-specific probes. The animals analyzed to date do not have any evidence of clonal expansion of the MDS1-targeted population, and the number of MLVtransduced circulating cells remains stable 4 to 6 years after transplantation. We also investigated MDS1-EVI1 expression by RT-PCR in neo+ CFU that harbor a proviral insertion at the MDS1 locus. The CFU screened so far do not express any of the MDS1-EVI1 transcripts, suggesting that the transcriptional regulation of the locus has not been altered. Our study suggest that the MDS1-EVI1 locus is particularly susceptible to retroviral integration but the competing hypothesis that proviral insertion within this region favors engraftment and long-term contributions to hematopoiesis will be difficult to eliminate. The long-term follow-up of these primates has revealed completely normal hematopoiesis and lack of any progression towards neoplasia. Systematic analysis of proviral integration sites in this pre-clinical model is essential as it provides decisive information for risk assessment in the development of integrating vectors. Gene transfer into hematopoietic stem cells (HSC) may represent a definitive treatment for severe combined immunodeficiency (SCID) due to adenosine deaminase (ADA) deficiency. We have treated five patients (age: 7-30 months) who lacked a matched sibling donor and for whom PEG-ADA was either not available or had resulted in side effects or insufficient immune reconstitution. In the latter case, PEG-ADA was discontinued to favor the growth advantage for gene corrected cells. Autologous CD34+ cells were harvested from the BM, transduced with a retroviral vector encoding the ADA cDNA, and reinfused after 4 days (mean CD34+ cells/Kg : 5.6 ± 3.5; mean % of gene transfer in CFU-C: 28±12%). On days -3 and -2 the patients were treated with low dose i.v. busulfan (2 mg/Kg/day). Pt2 received the lowest cell dose and oral busulfan. Three patients experienced ANC <0.5x10 9 /L, while only one required platelets transfusions. Long-term engraftment of transduced HSC was demonstrated by stable multilineage marking, persisting up to 4 years from gene therapy, with evidence of common progenitors (lymphoid, erythroid, myeloid) by molecular analyses of vector integration. Marking levels in myeloid cells ranged from 0.1% in Pt2, to 1% in Pt4, and 5-10% in Pt1, Pt3, and Pt5. Within 6 months from treatment, the large majority of T, B and NK lymphocytes were replaced by gene corrected cells. This led to a progressive increase of PB lymphocyte counts, restoration of polyclonal thymopoiesis, and normalization of proliferative responses to mitogens and antigens. Serum Ig levels improved in all patients and production of specific antibodies after antigen vaccination was observed in Pt1 and Pt3 after IVIg discontinuation. Sustained ADA activity in RBC and PB lymphocytes was associated with a dramatic decrease in dAXP toxic metabolites, and restoration of normal growth and development. All the patients are presently at home, thriving and healthy, free from severe infections or adverse effects, with the follow-up ranging from 7 to 50 months. Overall, the level of myeloid engraftment and the speed and degree of immune reconstitution correlated both with the dose of infused transduced CD34+ cells and the actual degree of myelosuppression. In conclusion, these data indicate that HSC gene therapy combined with low intensity busulfan is safe and efficacious and should be considered for the treatment of patients lacking a matched sibling donor. *MGR and CB equally contributed to the work O120 CD133 positive haematopoietic stem cells ''stemness'' genes contain many genes mutated or abnormally expressed in leukaemia A. Toren, B. Bielorai, J. Jacob-Hirsh, T. Fisher, D. Kreiser, S. Zelikovsky, D. Givol, J. Itzkovitz-Eldor, I. Kventsel, E. Rosenthal, N. Amariglio, G. Rechavi Sheba Med Center (Tel-Hashomer, IL) ; Weizmann Institute of Science (Rehovot, IL); Rambam Medical Center (Haifa, IL) Several groups have recently used microarray technology to study the common characteristics of stem cells from different tissues (stemness) and the typical features of stem cells from various sources (tissue specificity). Most groups focused their study on mice, used relatively small cDNA microarrays, and used CD34 as the cell surface marker for hematopoietic stem cells isolation. We studied HSC cells from cord blood (CB) and peripheral blood (PB)characterized by expression of the primitive CD133 antigen, and used the Affymetrix Human Hu133A oligonucleotide arrays to study the gene expression profile of these cells. An unsupervised hierarchical clustering of 14,025 valid probe sets showed a clear distinction between the CD133 + cells representing the stem cell population, and CD133 cells that represent various stages of cell differentiation. Comparison of CD133+ cells isolated from CB and PB to CD133-cells identified 304 genes that were up regulated by at least two folds in CB and 218 genes in PB. These genes were considered as source specific and maybe relevant to the unique properties of CB and PB derived HSC. 244 genes were found to be up regulated by at least two folds in the CD133 positive cells of both CB and PB as compared to the CD133 negative cells. Comparison of these stemness genes, to the lists of stemness genes that were identified by two recent studies that analyzed mainly murine HSC identified 33 (Ramalho et al.) and 65 (Ivanova et al.) common genes.

Twenty-four genes were common to another study that analyzedhuman HSC (Georgantas et al.). Among these common stemness genes we identified several groups of genes that have an important role in hematopoiesis: Growth factor receptors, a group of transcription factors which includes several homeobox genes and TGF-target genes; genes that have an important role in development and genes involved in cell growth.Among these four groups we identified 16 stemness genes (MPL, FLT3, HOXA9, MEIS 1, MLLT3, KIT, TIE, GATA-2, HOXA5, HOXA10, HLF, MYCN, EVI1, MYB, FHL1, and HMGA2) that are known to be mutated or abnormally regulated in acute leukemias. It can be suggested that key hematopoietic stemness machinery genes may lead to abnormal proliferation and leukemia upon mutation or change of their expression.

Haematopoietic cell transplantation in humans results in generation of donor-derived epithelial cells: a mature update A. Spyridonidis, R. Zeiser, Y. Metaxas, P. Faber, V. Degim, M. Follo, H. Bertz, J. Finke Freiburg University Medical Center (Freiburg, D) Several study groups have reported on non-hematopoietic chimerism following allogeneic hematopoietic cell transplantation (aHCT). We recently investigated this phenomenon in humans, described the pitfalls of identification of epithelial chimerism (Spyridonidis et al, Am J Pathol.164,2004) . Recently we performed analyses on epithelial chimerism in various non-hematopoietic tissues derived from female patients after sex-mismatched aHCT. The tissues analysed included colon (12 pts), oral mucosa (14 pts) and skin (2 pts). The biopsies or oral scrappings were obtained 15 to 1964 days after aHCT. Epithelial chimerism was assessed by using a 4-color staining which combines FISH for the Y-or the XY-chromosome, immunofluorescent stain for the epithelial specific marker cytokeratin (CK), for CD45, and cell nuclei stain (CK/ (X)Y / CD45 /DAPI) folllowed by laser confocal microscopy to assess colocalization of the signals and exclude overlapping. Epithelial chimerism was detected in colon biospsies from 10/12 pts analysed (83%). The mean incidence of the engrafted crypts was 7.7% (1.53%-24%) while the mean incidence of the Y+/CK+/CD45-events was 0.12% (0.03%-0.38%). There was a strong correlation between the degree of epithelial chimerism in the colonic crypts and the presence of tissue damage (p=0.0069). In buccal swabs, we detected Y+/CK+/CD45-cells in 12/13 pts (92%), with a mean of 1,8% Y+/CK+/CD45-cells (range 0,5-7,3%). Since cytospins from buccal swabs contain isolated cells, we were able to demonstrate unequivocally that the Y+/CK+/CD45-chimeric events found were due to single cells. The identified Y+/CK+/CD45-cells were counterstained with hematoxylin/eosin and revealed a typical epithelial morphology.

Retrospective review of the patients transplantation documents demonstrated a significant correlation (p=0.0028) between the severity of mucositis in the early post-transplant period (up to day+30) and the numbers of donor-derived epithelial cells found at later time points (d+75 to d+1964) with absent mucositis signs. Our results indicate that aHCT results not only in replacement of the lymphohematopoietic system but also in generation of nonhematopoietic cell populations, aphenomenon which is probably promoted by tissue damage. We never detected XXX+/Y+ epithelial cells, both in colon and buccal samples, making fusion unlikely as the underlying mechanism. The clinical and maybe the therapeutical significance of this phenomenon has to be determined. A. Avigdor, P. Goichberg, I. Petit, O. Perl, E. Rosenthal, I. Hardan, I. Resnick, S. Slavin, A. Nagler, T. Lapidot Chaim Sheba Medical Center (Tel-Hashomer, IL) ; The Weizmann Institute of Science (Rehovot, IL); Hadassah University Hospital (Jerusalem, IL) G-CSF induced hematopoietic stem cell mobilization is regulated by interplay between extracellular matrix, cytokines/chemokines, adhesion molecules, and proteases. Several studies suggest that membrane type 1-matrix metalloproteinase (MT1-MMP) is a key enzyme for normal cell motility and tumor cell invasion. We found that human CD34+ cells express various surface MT1-MMP levels, depending on the cell source and G-CSF treatment. CD34+ cells obtained from BM of healthy donors treated with G-CSF were found to have the highest mean fluorescence intensity (>900 arbitrary units), while the level of expression was lower in CD34+ cells derived from G-CSF mobilized peripheral blood (MPB) from healthy donors (159±40), human steady-state (SS) BM (80±19), and human CB (41±4). Following 48 hr incubation of human SS-BM CD34+ cells with G-CSF, the expression of MT1-MMP increased 2-fold, whereas treatment with other cytokines, such as SCF, SDF-1 and IL-6, had only a minimal impact. Immunocytochemical analysis of human CB CD34+ cells plated on fibronectin or hyaluronate-coated cover slips revealed that in response to SDF-1, MT1-MMP changes its localization in the polarized cells, suggesting a role in the process of human progenitor cell (HPC) directional migration. Indeed, neutralizing antihuman MT1-MMP Ab significantly reduced HPC migration towards a gradient of SDF-1 in transwells. Interestingly, low concentrations of TIMP-2 enhanced SDF-1 induced transwell migration, while higher concentrations hampered this process. In addition, five daily injections of G-CSF to NOD/SCID mice previously engrafted with human cells, up-regulated MT1-MMP expression on CD45+ and CD34+ human cells in the BM and PB of the mobilized chimeric mice compared to untreated, control chimeras. Treatment of chimeric mice with antihuman MT1-MMP Ab on days 3-5 of G-CSF induced mobilization significantly reduced the number of CD45+ and CD34+ human cells in peripheral blood. Finally, the level of MT1-MMP expression on MPB CD34+ cells correlated with the yield of CD34+ cells harvested on the first day of apheresis (r=0.7) in patients receiving G-CSF for PBSC mobilization. In summary, based on our data we suggest that following G-CSF treatment, increased levels of MT1-MMP on the surface of HPCs in the BM facilitate their mobilization most probably due to pericellular ECM degradation and/or activation of other regulatory molecules, pointing to the essential role of MT1-MMP in G-CSF induced mobilization.

Prevention of diabetes in NOD mice by embryonic stem cell transplant-derived haematopoietic chimerism R.K. Burt, L. Verda, D.A. Kim, L. Statkute, Y. Oyama, C. Link, S. Ikehara Northwestern University Medical School (Chicago, USA) ; NewLink Genetics (Ames, USA); Kansai Medical University (Osaka, JP) We have recently demonstrated that embryonic stem cellderived hematopoietic stem cell transplantation (ESC-derived HSCT) results in successful hematopoietic engraftment in irradiated mice across MHC barriers without any symptoms of graft versus host disease (GvHD). Herein, we report a new ESC-derived HSCT approach for the prevention of autoimmune diabetes in NOD mice.

Female six week old NOD/LtJ mice were sublethally irradiated and transplanted with ESC-derived HSC. To induce differentiation, R1 ESC were cultured methylcellulose-based medium supplemented with SCF, IL-3 and IL-6. An enriched c-kit+ ESC-derived cell population was injected intra bone marrow (IBM) or IV. Mice were followed by blood glucose measurements and chimerism analyses until onset of diabetes or until 40 weeks after transplantation. Nine NOD mice were held as controls. Peripheral blood donor (H2b) versus recipient (H2Kd) chimerism was measured by flow cytometry. Nine out of 10 mice the from IBM group and 5 out of 8 from IV group did not become hyperglycemic in contrast to control group where 8 out of 9 mice were euthanized because of diabetes (Graph). The level of chimerism achieved after transplantation was 9.1% ± 6.71% in the IBM group and 2.5% ± 2.78% in the IV group. Histological examination showed that most of islets were replaced by lymphocytic infiltration or fibrous tissue in controls (even in case of a mouse without clinical evidence of diabetes). In 78% (14/18) of animals from ESC-derived hematopoiesis, remission was confirmed by histology revealing the absence of insulitis and normal immunohistochemical staining of islet cells for insulin. Prevention of diabetes / insulitis was predicted by the percentage ESC-derived hematopoietic chimerism. All mice with > 5% ESC-derived chimerism remained free of diabetes and insulitis. High concentration of IFNg was detected only in culture containing GAD65 and splenocytes from NOD control mice. Proliferative response of splenocytes derived from ESCT-NOD chimeric mice were diminished toward recipient and host lymphocytes compared sustained response to third party antigens. It is possible to establish mixed allogeneic hematopoietic chimerism in sublethally irradiated NOD mice without subsequent GVHD by using ESC-derived hematopoietic stem cell transplantation. Mixed allogeneic chimerism achieved with ESCT prevents diabetes in NOD mice when performed before onset of diabetes.

Neuroblastomas: 2903 pts are registered, 60% are male, median age: 3.8 yrs. The OS [EFS] is 37% [33%] with a median observation period (MOP) of 8.5 yrs. The OS for pts (n=2353) in first remission is 38% (CR1/VGPR/PR/SD/PRD) and 28% for relapse (CR2/SR/RR) pts (n=248). The OS rates in compliance with the status at MGT are 42% for CR1 (1071 pts), 36% for VGPR/PR (1175 pts), 23% for PRD (55 pts), 35% for SD (52 pts), 45% for CR2 (109 pts), 18% for SR (94 pts) and 5% for RR (23 pts) and 6% for UR (19 pts). The Busulfan-Melphalan MGT approach produced statistically superior OS rates during first remission: OS 48% vs. OS 36% with other regimens (p<0.001) and is currently under randomised investigation in the European HR-NBL-1/ESIOP study. Ewing tumours: 1175 pts up to 18 yrs., 55% are male, median age: 12.7 yrs. The OS (EFS) rates are 40% [36%]; MOP: 6.7 yrs. The OS for 756 first remission pts is 46% and 30% for 290 relapse pts

Haematopoietic stem cell transplantation in childhood: report from the Paediatric Diseases Working Party of the European Bone Marrow Transplantation Group M. Miano, M. Labopin, O. Hartmann, E. Angelucci, J. Cornish, E. Gluckman, F. Locatelli, A. Fischer, M. Egeler, O. Reuven, C. Peters, J. Ortega, P. Veys, G. Michel, A. Iori, D. Niethammer, G. Dini on behalf of the PDs WP of the EBMT During the last 35 years, the number of SCTs in children who have been registered in the EBMT data base has tremendously increased, and the number of patients who were reported between May 1996 and 2002 is similar to the total number reported over the previous 28 years. The aim of this study is to provide the full picture regarding all children given SCTs between 1970 and 2002, and who were reported to the EBMT registry. In the studied period 31,713 SCTs (18,803 allogeneic and 12,910 autologous) in patients < 18 years of age were registered by 420 centres belonging to 29 European and 14 extra-European countries; 110 of the 420 centres performed paediatric transplantation alone. The remaining 310 centres provided "combined" programmes whereby children represented more (n=23) or less (n=287) than 25% of treated patients. Following May 1996 the number of transplants that were reported by exclusively paediatric centres increased, while the number of transplants reported by combined centres decreased. Moreover, the number of countries involved in this programme has increased, with increased activity mostly in Eastern and extra-European Countries. The number of alternative SCTs, including Volunteer Unrelated Donor (VUD), Haploidentical Family Donor (HFD), and CB SCT progressively increased and reached the percentage of 61%