key: cord-0033713-22o7u46e
authors: nan
title: Publication Only
date: 2004-03-21
journal: Bone Marrow Transplant
DOI: 10.1038/sj.bmt.1704503
sha: 371fb4ba22e791bef830b7c75b23aff6152d3455
doc_id: 33713
cord_uid: 22o7u46e

nan

The therapeutic options for patients whose Acute Lymphocytic Leukaemia (ALL) relapses following bone marrow transplantation are limited. We are currently developing an adoptive immunotherapy protocol for these patients based on the in vitro expansion of leukaemia-specific T cells by dendritic cells (DC) derived from CD34+ haematopoietic stem cells. In these experiments we used a cytokine-enriched serum-free medium to expand DC from purified CD34+ stem cells from cord blood, a medium which can also be used to generate DC from bone marrow or mobilised peripheral blood CD34+ cells. Following optimisation of the medium we investigated conditions for RNA loading of the DC as this has been shown to be an effective means of generating immune responses to tumour antigens. In vitro-transcribed (IVT) enhanced green fluorescent protein (eGFP) mRNA was used as a model protein to optimise variables affecting transfection efficiency by electroporation. Flow cytometry analysis of eGFP expression in the whole culture and in the CD11c+DR+ fraction (designated as the DC fraction in these cultures) allowed comparison of variables (cuvette size, cell concentration, voltage, capacitance and electroporation medium) affecting cell viability and transfection efficiency. We further investigated pre-and post-electroporation conditions including incubation times and exposure of cells to different temperature environments. Using our optimal conditions we achieved 91% eGFP transfection efficiency with 90% cell viability of the whole culture, and 93% of the DC expressing eGFP. Cord blood CD34+derived DC electroporated with flu matrix protein mRNA under the optimal conditions were able to present antigen and be killed by flu specific CD8 CTL clone. Adult monocytederived DC similarly transfected with the flu protein were able to amplify tetramer-positive autologous CD8 CTL in vitro. These findings will now be extended to expand leukaemia-specific CTL for adoptive immunotherapy.

Haematopoietic stem cell transplantation after high-dose therapy. A single-centre experience G. Mihaylov, B. Avramova, L. Gartcheva, M. Jordanova, University Pediatric Oncohaematology Hospital (Sofia, BG) Between October 1997 and November 2003, 81 stem cell transplantations (71 Autologous and 10 Allogenic) following high dosage chemotherapy were performed in 71patients with hematological malignancy. The type of diseases, treated with autologous stem cell transplantations were: M. Hodgkin (MH) -13, Non Hodgkin lymphoma (NHL) -11, RhabdomyoSa (RMS) -4, Ewing Sa -4, Lymphoepithelioma -6, Neuroblastoma -3, Germ cell Tu -3, ANLL -5, Myeloma multiplex (MM)-4, ALL -2, Ca mammae -3, Multiple Sclerosis (MS)-2, Pulmoblastoma -1. The median age was 22, 4 years (3 yrs -59 yrs). The number of cells infused ranged from 1, 27 to 8, 34 x106 CD34/kg. At the time of SCT 19 patients (31,1%) were in complete remission (CR) while 34 (55,7%) had a partial remission (PR) and 8 (13,2%) had a progression. 30 pts after autologous PBSCT are alive, 17 in CR, 7 with stable disease and 6 with relapse. Median follow up is 34, 5 months (2 -67 months). There was 2 cases (3%) therapy related death. The remainder died from relapse or tumor progression. Time to relapse was 9,5 months (2 -17 mo). Time to neutrophil recovery was from +8 to +22 day. Based on our experience almost 46% of patients with indication for HDT as defined by national protocols remain alive and 26% with NED. In the group of allogeneic transplanted patients ANLL -3, ALL -4, MDS-1, ASAA-1, CML -1; the median age was 15,1 years (10 yrs -39 yrs). The number of cells infused ranged from 4, 27 to 9, 34 x106 CD34/kg. At the time of SCT 7 patients (70%) were in complete remission (CR) while 3 (30%) had a progression. 5 pts after allogeneic PBSCT are alive, 4 in CR, 1 with graft rejection 8 months after transplantation. Median follow up is 12 months (2 -42 months). There was 3 cases with transplantation related death. The remainder died from relapse or disease progression. Time to relapse was 3 months (2 -4 mo). Time to neutrophil recovery was from +8 to +18 day. The quantity of allogeneic group does not permit to define a conclusion.

Donor lymphocyte infusion from HLA-matched siblings outside of the context of a haematopoietic stem cell transplantation: a pilot study J. Peccatori, M. Bernardi, A. Pescarollo, C. Corti, P. Servida, E. Guggiari, F. Lunghi, E. Poggiali, C. Bonini, F. Ciceri, M. Bregni, Istituto Scientifico H.S. Raffaele (Milan, I) Graft-versus-tumor effect following HSCT is a pivotal form of immunotherapy. Preliminary data provide evidence for a GVT effect in renal, breast, ovarian and other tumors. However, the onset of GVT effect is delayed by months from the time of the transplant, and usually occurres following cyclosporine withdrawal, indeed most patients show a tumor progression soon after the transplant and patients with rapidly progressive tumors are not eligible to HSCT. We thought to avoid the prolonged immunosuppression needed in HSCT and directly infuse cells from a HLA-matched sibling in patients with advanced tumors without any GVHD prophylaxis but only an immunosuppressive chemotherapy to facilitate engraftment of donor cells. We evaluated this strategy in 11 patients with metastatic solid tumors (8 renal cell, 1 ovarian, 1 colon cancer and 1 Ewing sarcoma). All the patients had a full HLA-matched sibling but were ineligible to allogeneic transplant because of a life expectancy of less then three months. The first 8 patients were treated by fludarabine 30 mg/m2/d (days -8 to -6) and cyclophosphamide 300 mg/m2/d (days -8 to -6), starting from day 0 an escalating dose DLI (3, 5 and 10x10e7 CD3/kg) was performed at two weekly intervals, in the absence of aGVHD. In the last 3 patients the dose of fludarabine was 25 mg/m2/d (days -8 to -4) and the DLI started from 5x10e7 CD3/kg. The primary end-point of the study was the achievement of transient engraftment by cytogenetic analysis (or by PCR-based analysis of VNTR polymorphism in gendermatched pairs) in PBMC. Three patients achieved a prevalent donor chimerism at day 7 and/or 14 after the first infusion; one of them developed a grade IV aGVHD at day +20 and died because of GVHD at day +40. Our results point out how heavily pretreated patients are more likely to achieve a mixed chimerism (3/3) compared to chemotherapy-naive patients with renal cell carcinoma (0/8). When prevalent donor chimerism was achieved, we observed a graft-versus-host effect, indicating that donor cells are active and could be mediators of a graft-versus-tumor effect too. The use of DLI outside of the context of HSCT needs further studies: future trials should consider prior chemotherapy exposure for patients selection and try to enhance tumor response while reducing GVHD. For this purpose we are evaluating the selection of allogeneic T or NK cells or the use of engineered T lymphocytes with a suicide gene to be able to abrogate GVHD if it occurs.

D. Decaudin, J.M. Vantelon, A. Turhan, S. Assari, J.H. Bourhis, F. Némati, J. Michon, W. Vainchenker, C. Boccaccio, Institut Curie, Institut Gustave Roussy (Paris, Villejuif, F) Thrombocytopenia after high-dose chemotherapy and autologous stem cell transplantation (ASCT) is a major complication that can be life-threatening. We therefore conducted a phase II study to evaluate the impact of ex vivo expanded megakaryocyte precursor cells on the nadir and duration of thrombocytopenia in nine patients with relapsed non-Hodgkin's lymphoma. All patients underwent Peripheral Blood Progenitor Cell (PBPC) mobilization with standard chemotherapy and G-CSF, and one to three apheresis procedures were performed to obtain at least 4.106 CD34+ cells/kg. Two fractions were cryopreserved: a first fraction of at least 2.106 CD34+ cells/kg as non-manipulated PBPC, and a second fraction that was obtained after CD34 positive selection (Isolex 300i, Baxter) of 1 to 2.106 CD34+ cells/kg. Ten days before ASCT, this fraction was thawed and cultured in X-Vivo medium with 10 ng/ml of MGDF and 25 ng/ml of SCF. CD34+ cells were kept at 37°C in a 5% CO2 atmosphere for 10 days, then washed. The patients received high-dose BEAM chemotherapy (BCNU, cyclophosphamide, cytarabine, and melphalan) and were reinfused on day 0 with standard PBPC and ex vivo expanded progenitor cells. Due to reinfusion of cells cultured in the presence of SCF, patients received cetirizine and ranitidine from day -1 to day 1. No toxicity was observed after reinfusion. One patient was not infused with cultured cells because of a cardiac contra-indication. At day 10 of culture, the mean fold expansion was 9.27x (range: 4.85-16.77) for nuclear cells, 2x (range: 0.94-6.33) for CD34+ cells, and 676x (range: 142-1,183) for CD41positive cells. The mean number of nuclear and CD34 cultured cells reinfused was 14.25.108/kg (range: 2.96-18.8) and 2.79.106/kg (range: 0.77-5.7), respectively. The median date of platelet transfusion independence (> 15.000/µl) was day 8 (range 7-12). The patients' platelet recovery is represented in the figure.

All patients received at least one platelet transfusion. Patients recovered absolute neutrophil counts > 500/µl on a median day 12 (range: 10-15). One patient died suddenly at home on day 35; no autopsy was performed, but no relationship could be established between this patient's death and ex vivo cultured PBPC.

In conclusion, ex vivo expansion of megakaryocyte progenitor cells was safely reinfused into patients, but failed to prevent thrombocytopenia < 15.000/µl. This result could be attributed to a lower PBPC count collected for ex vivo cultures.

A. Carotti, F. Falcinelli, A. Terenzi, F. Falzetti, T. Aloisi, E. Lucia, M. Capponi, J. Saab, R. Iacucci Ostini, T. Zei, F. Aversa, University of Perugia (Perugia, I) Objective: This study assessed the efficacy and safety of DLI as therapy for relapse of hematological malignancies after T cell depleted haematological stem cell transplant (HSCT). Patients and Methods: 38 patients with chronic myeloid leukaemia (CML) (n=24), acute myeloid leukaemia (AML) (n=9), acute lymphoid leukaemia (ALL) (n=2) or Multiple Myeloma (n=3) received matched (30) or mismatched (8) T cell depleted HSCT. Upon molecular (12), cytogenetic (15) and/or haematological (11) relapse at a median of 12 months (range:1-132) after transplant, all patients received DLI. DLI ranged from 1x10e4/Kg to 3x10e7/Kg according to disease, type of, and time to, relapse, HLA disparity betwen donor and recipient and response. Results: DLI infusions ranged from 1-30 in number (median 9). Dose escalation was required for response in 23 patients. CML (24 patients): 19 patients responded (10 molecular, 9 cytogenetic remissions) . Eight year probability of remission was 83%, with a significant difference (p<0.0038) according to time to relapse after transplant (90% late vs 60% early). All 24 patients survive at a median follow-up of 5 years (range 10-110 months). AML (9 patients): 3 patients achieved haematological and molecular remission; 2 achieved mixed chimerism with no disease progression. ALL (2 patients): 1 patient responded. Myeloma (3 patients): 1 patient responded. No DLI-related aplasia was observed. Graft versus Host Disease (GvHD): 1 case of acute Grade II resolved with anti-thympcyte globulin (ATG), steroids and cyclosporine; 3 cases of acute Grade I resolved with steroids alone; 4 cases of limited chronic GvHD controlled with low dose steroids. Conclusions: Patients who relapse after a T cell depleted matched or mismatched HSCT tolerate dose escalation DLI. Many, particularly patients with CML, respond and achieve sustained remission.

Post-thawing PBPCs washing protocol using the SEPAX System R. Saccardi, P. Bufano, R. Caporale, A. Carraresi, L. Lombardini, F. Pagliai, P. Pizzano, S. Urbani, A. Bosi, S. Azzalin, J. Mizeret, A. Tichelli, S. Meyer, L. Landi, C. Elssner, Ospedale di Careggi, Biosafe, Kantonsspital Basel (Florence, I; Eysins, Basel, CH) The SEPAX cell separation system (Biosafe SA, Eysins, Switzerland) is a new type of cell centrifugation device for the automated processing of blood or blood components in a closed and sterile environment. A specific protocol and disposable kit for the automatic washing of thawed leukapheresis products (PBSCs for autologous transplantation) was developed, allowing removal of most of DMSO and hemolyzed plasma before the infusion in the patient. The procedure was based on the manual method proposed by the New York Cord Blood Bank for thawed Cord Blood Units. The trial was carried out with PBSC products excluded from clinical use, mainly due to disease progression, in two independent laboratories. Thawed cell were slowly diluted in an isotonic buffer solution including Dextran40 5% and Albumin 2.5%, then centrifuged and resuspended in the same solution. The following parameters were analyzed before and at different time points (0, 0.5, 3, 6, 24 hrs) after the procedure: Volume; TNC Count; Plt Count; Hematocrit; CD34+ absolute count; MNC Count; viability (7-AAD assay); clonogenic potential; sterility test and DMSO concentration. The procedure was shown as both feasible and reproducible; initial S305 cell recovery after washing was satisfactory and major differences with control (unmanipulated) samples were detected at 24 hrs. Cell recoveries are calculated as percentage of pre-washing values and summarized in the table. Furthermore cell washing prevented clotting/clumping events occasionally reported in unmanipulated samples; DMSO removal was always higher than one log. As the machine works in a closed system connected to the collection and cryopreservation bags, the risk of bacterial contamination during processing is minimal. The Sepax system allows a simple and automated washing procedure with no user dependence; a prospective pilot clinical trial is under development.

A. Perez-Corral, M. Casanova, M. Ballesteros, G. De la Rosa, D. Blanco, J. Anguita, N. Herández de Leon, P. Sánchez-Mateos, J.L. Diez-Martin, T. Pintado, A. Gomez-Pineda, Gregorio Marañon Hospital (Madrid, E) Introduction: It is well known the use of human peripheral blood mononuclear cells (PBMC) as dendritic cell (DC) precursors for immunotherapy. Typically PBMC are obtained from buffy coats, but this process is being abandoned, so an alternative source of monocytic cells is required. One possibility is the use of the PBMC retained by the leucorreduction filters (LRF), though there is insufficient information about the quality of these cells. We present our experience with the generation of DC from LRFmonocytes compared to fresh blood donation monocytes. Material and methods: We analysed 4 samples (S1,S2,S3,S4) from healthy people: S1-2 were obtained by direct vein puncture and S3-4 from 2 LRF(ATS LPL Pall Medicall), once separated from the donation bag. The erythrocytes were discarded by Ficoll gradient. Collected cells were purified by CD14 magnetic beads (Milthenyi Biotech) to obtain monocytic cells. To generate DC, monocytes were cultured in RPMI 10% Fetal Calf Serum, adding 1000 UI/ml GM-CSF and 5 ngr/ml IL-4 on days 0, 2 and 4. To analyse DC differentiation, cells were incubated with CD14 FITC and CD1a CYCrome (BD) and analysed in FACSCan flow cytometer. Cell viability was assessed with Annexin V FITC (early apoptosis) and 7AAD (late apoptosis-cell death) (BD). Results: S1-S2 (fresh blood) were, on day 6, 100% CD14-and 90.1% and 98% CD1a+ respectively, indicating a complete differentiation. S3-S4 from LRF showed, on day 6, 2 different cell population(A,B): The A population (47% of S3 and 38% of S4), either had only partially differentiated (100% CD14+; 60% CD1a+ in S3) or presented monocytic phenotype (100% CD14+; 0% CD1a+ in S4). Only the B population (53% of S3 and 62% of S4), had a proper DC phenotype (100% CD14-; 100% CD1a+). The percentage of cell loss between days 0 and 6 was 40% and 19% in S1-2 (fresh blood) and 92% and 83% in S3-4 (LRF). Cell viability on day 0 was assessed in 3 other random samples of LRF showing 70.3%, 84.4% and 75.3% Annexin V FITC positivity and 44.9%, 62.9% and 51.1% 7AAD positivity respectively. Conclusions: In our experience, the use of LRF to obtain DC precursors presents some inconvenients. Despite being a rich source of mononuclear cells, while monocytes purified from fresh blood donor samples reach a complete differentiation, cells extracted from LRF only partially differentiate, always remaining immunophenotipically a monocytic population. The cause of this phenomenon could be the low viability of our samples. A.S. Michallet, F. Nicolini, J.P. Tremisi, V. Dubois, S. Hayette, C. Vigouroux, J.P. Bourgeot, X. Thomas, M. Michallet, Hopital E. Herriot, Laboratoire d'Histocompatibilité, EFS (Lyon, F) We retrospectively analyzed the results of DLI given for relapse after HLA sibling-donor conventional allogeneic HSCT in 30 patients [19 males and 11 females; median age of 34 y (17-47)] performed in our center. The median follow-up of this series is 107. 5 months (69-184) . The diagnosis before transplantation were: 14 CML, 9 AML, 4 ALL, 1 NHL, 1MDS, 1 MM. All patients received bone marrow as source of stem cells. GVHD prophylaxis consisted in classical association of CsA+MTX. The status pre-DLI showed 26 hematological relapses, 1 extramedullar relapse and 3 cytogenetical relapses. The median interval between DLI and relapse was 1.9 months (0-52). Ten patients have received a single dose which varied from 0.1 to 2.64x108 CD3+/kg, among 5 patients have received a Bulk Dose (BD). The 20 other patients have received Escalating Doses (ED) which ranged from 0.01 to 3x108 CD3+/kg. Fifteen patients (50%) achieved a Complete Response (CR) of their disease. Among these 15 DLI responders, 13 showed full donor chimerism (FDC) and 2 mixed chimerism (MC) despite full disease response and with a median follow-up of 107 months after transplant and 58 months after DLI, 10 patients remained in FDC and 1 in MC. The median number of DLI necessary to obtain these CR was 2 (1-5) with a median interval between 2 DLI of 3.5 months (0.25-13). Three patients (10%) developed after DLI a grade III acute GVHD, 1 grade IV and the 2 acute GVHD developed an extensive chronic GVHD. Among patients who developed acute or chronic GVHD only CML patient was in continuous complete cytogenetical response. With a maximal follow-up of 10 years, 19 (63%) patients died and 11 (37%) are alive. The 3-year probability of survival was 60% (95%CI ). The 11 long-term survivors were CML patients: 6 in complete molecular response (CMR) and 2 in complete cytogenetical response after DLI without any other intervention, 3 patients have relapsed in chronic phase (CP) after DLI: 1 achieved CMR after Imatinib mesylate, 1 achieved hematological response after Imatinib mesylate and second allo-transplantation after reduced intensity conditioning and 1 remained in CP dispite Imatinib mesylate therapy . When restricting the analyzis only to CML patients, the 3-year probability of survival was 92.8% (95) (96) (97) (98) (99) (100) ). This long-term analyzis underlines the need for prospective trials comparing imatinib mesylate alone either combined with DLI or followed by DLI for relapse of CML after allogeneic HSCT.

Cytokine production during myeloablative and reducedintensity conditioning in allogeneic stem cell transplantation patients M. Remberger, B. Sundberg, Karolinska Institutet (Stockholm, S) We analysed serum cytokine levels in 178 allogeneic stem-cell transplantation patients during the pre-transplant conditioning. Samples were drawn daily during the conditioning and serum levels of TNF-alpha were analysed by automated chemoluminescence immunoassay. Conventional myeloablative conditioning was given to 119 patients, while 59 patients received reduced intensity conditioning (RIC). Most patients had a haematological malignancy with a median age of 37 years (range 1-67). Anti-thymocyte globulin (ATG) was given to 126 S306 patients as part of the conditioning. The use of ATG significantly increased the TNF-a levels the last four days before transplantation. We found significantly higher TNF-a levels, days -4 to -2 before transplant, in the RIC group compared to myeloablative conditioning independently of ATG treatment. No effect of age and disease stage was found. In patients not given ATG, we found a correlation between high TNF-a levels on day -2 and acute GVHD grades II-IV. To conclude, during conditioning for HSCT, patients conditioned with RIC and those treated with ATG had increased levels of TNF-a.

The effect of cytokines on ex vivo expansion of megakaryocyte progenitor cells from essential thrombocythaemia K. Kwon, H. Song, H. Kim, H. Lee, M. Hyun, Dongsan Medical Center, Youngnam University Medical Center (Daegu, KOR) Essential thrombocythemia (ET) is a member of myeloproliferative disorders, characterized by regulatory defect in stem cells and it is possible that ET can produce autonomous megakaryocyte (MK) growth. We had examined the ability of spontaneous production of MK colony forming unit (CFU-MK) and compare the sentitivty of cytokines between ET and normal control. We had measured the degree of CFU-MK expansion under condition of single and combination of cytokines on ex vivo expansion of MK progenitor cells. Bone marrow mononuclear cells were aspirated from patients with ET and normal persons and then cultured in serum-free medium (MegaCultTM) supplemented with SCF, G-CSF, TPO, IL-11 and 2 types of cytokine combination. In the absence of cytokines, CFU-MK of ET expanded significantly than normal (p<.05), which represented spontaneous formation of CFU-MK. There were increased generation of CFU-MK in cytokine added well in both groups and significantly increased CFU-MK of ET with G-CSF, TPO, IL-11 and SCF + G-CSF + TPO + IL-11, respectively (p<.05). In summary, our results demonstrated autonomous MK growth characteristics of ET and most of cytokines we used can be helpful in ex vivo expansion of CFU-MK. Further investigation will be needed to find out the most effective culture system and cytokine combination for ex vivo MK progenitor expansion.

Erythropoietin receptor on AR42J cells can be induced for proliferation signal by erythropoietin C. Bose, K. B. Udupa, University of Arkansas for Medical Sciences (Little Rock, USA) It has been reported earlier that breast cancer cell lines express erythropoietin receptors (EPOr) and incubation of these cells with erythropoietin (EPO) enhances their proliferation (Acs et al, Cancer Res. 161, 3561, 2001) . We wanted to investigate whether EPOr were present in other transformed cell lines and also whether EPO can induce proliferation signals in these cells. We selected a transformed pancreatic cell line, AR42J, to investigate this aspect. AR42J cells were grown and cell extracts were prepared. Extracts were separated by gel electrophoresis and transferred on to nitrocellulose membranes. Using antibody to EPOr, Western blots were performed. We observed a band corresponding to EPOr on these membranes, the intensity of which was 41.8% of the intensity of bands corresponding to same quantity of protein prepared from bone marrow cells. In further studies AR42J cells were incubated with 1 U EPO and Western blots were performed on the cell extracts for proliferation signal indicator of mitogen-activated protein kinase, extra-cellular regulated kinase 1/2 (ERK-1/2). We could see an increase in the intensity of ERK-1/2 bands with time, reaching the maximum of 3.3 fold by 5 minutes of incubation. Hence we could show the presence of EPOr in AR42J cells and its presence induced signal for cellular proliferation in presence of EPO. It is very important to understand the role of EPO in the proliferation of transformed cells since EPO is often supplemented after bone marrow transplantation to enhance the recovery.

Peripheral blood stem cell infusions are being utilized with increasing frequency as a source of marrow repopulating cells following the administration of myeloablative chemoradiotherapy for the treatment of many malignant and benign conditions. Appropriate cryopreservation protocols are needed to reduce the cost, time and enhance safety ad effectiveness of these cryopreservation methods. The current study is the first study from the only bone marrow transplantation center in Iraq, it was conducted on 9 healthy male donors their median age was 34.8 years (range 21-46) . The plan of the study was as follows: 1-mobilization of PBSC by injecting the patients with G-CSF 5mic/kg for 2days. 2-stem cell collection was done by pheresis machine (Haemonetics MCS+). 3-PBSC processing in clean room using safety cabinet class II. PBSC was mixed with the following solutions: A-sol -1-10% DMSO + Autolugus plasma B-sol-2-10% DMSO + Autolugus plasma + Hanks solution C-sol-3 -5% DMSO + Autolugus plasma + PBS 4-each one of the solutions was cryopreserved in 3 methods -Freeze A CONTROLLED RATES FREEZING and stored in liquid nitrogen.

-Freeze B directly to liquid nitrogen without CRF.

-Freeze C blood bank mechanical freezer -60 degree. Storage for 1 month viability testing was done by trypan blue dye exclusion test after thawing at day 1-3 7-30. Results: Showed sol -3 with 5% DMSO rather than 10% had the same results in the viability so it can be used instead of sol-1 to minimize DMSO toxicity. We can use sol-2 and to be put directly in liquid nitrogen without controlled rate freezing. In case we apply blood bank mechanical freeze as a method of cryopreservation solution 3 is the best in such cases. Table: Showing the percentage of stem cell loss in different freezing methods.

Opioids induce a reversible inhibition of haemopoietic progenitor cell growth H. Dimitriou, M. Kampa, G. Notas, E. Kouroumalis, E. Castanas, M. Kalmanti, University of Crete Medical School (Heraklion, GR) Hemopoietic stem/progenitor cells self renew and differentiate under the influence of a number of growth factors. Opioid agonists have been shown to induce a decrease of cell growth in a number of malignant and non-malignant cell types. In the present study we examined if opioids could interfere with the development and growth of primitive hemopoietic cells expressing the CD133 antigen. Cord blood samples (n=6) were subjected to CD133 immunomagnetic column selection with subsequent exposure to opioid agonists (as1-casomorphin, as1, 10-6M) and antagonists (diprenorphine, 10-5M) or both, in semisolid cultures for the development of CFU-GM colonies. Colonies developed after 7 days of culture were replated in fresh medium in the absence of opioids (replating capacity, AUC). RT-PCR was performed for the detection of opioid or somatostatin receptors in order to elucidate the mechanism involved. Our results showed that these agents, at low concentrations, could induce an inhibition of CFU-GM development (day 14 CFU-GM colonies: no addition of opioids 279 +/-45.4, in the presence of as1 216.2 +/-44.9, in the presence of diprenorphine 171 +/-61.8 and in the presence of both 191.9 +/-62.7 ). Furthermore this inhibition was reversible as it was shown by the AUC (from cultures: without addition of opioids 193.12 +/-56.34, in the presence of as1 240.06 +/-74.89, in the presence of diprenorphine 223.25 +/-60.35 in the presence of both 233.09 +/-67.89. No opioid or somatostatin receptors were detected by RT-PCR suggesting that opioids exert an inhibitory effect in hemopoietic progenitors development which is not mediated by classical opioid receptors. As opioids are abundantly found in bone marrow and are administered in patients undergoing transplantation, our evidence suggests a possible therapeutic intervention that could become available for stem cell transplantation.

A flow cytometry-based approach to ABCG2 function suggests that the transporter differentially handles the influx and efflux of mitoxantrone M. Garcia-Escarp, V. Martinez-Muñoz, I. Sales, J. Barquinero, J.C. Domingo, P. Marin, J. Petriz, Vall d'Hebron, IDIBAPS, Universitat de Barcelona (Barcelona, E) To better characterize the function of the ABCG2 transporter in vitro, we generated three cell lines (MXRA, MXRG and MXRT) stably expressing ABCG2 after transfection of wild type ABCG2 and two mutants (R482G and R482T), respectively. ABCG2 expression and function were analyzed by flow cytometry using monoclonal antibodies, a variety of fluorescent substrates and a series of potential inhibitors of the transporter. ABCG2 expression was detected in all cell lines. The cell lines effluxed mitoxantrone (MXR), but only the mutants effluxed rhodamine 123 (rh123), SYTO13, doxorubicin and daunorubicin. After incubation with MXR, intracellular accumulation was 9-and 22fold higher in MXRA than in MXRT and MXRG cells, respectively, suggesting that ABCG2 also modulates the influx rate of the drug. Flow cytometry kinetic studies of MXR efflux revealed that MXRG cells effluxed 50% of the drug at a faster rate than MXRA and MXRT cells (t50: 15.3 vs. 27.8 and 44.5 min, respectively) . MXRG cells also extruded rh123 and SYTO13 at a faster rate than MXRT cells. ABCG2-mediated transport was inhibited by fumitremorgin C, cyclosporine A and PSC-833, but not by verapamil or probenecid, two well-known ABCB1 inhibitors. Finally, MXRG cells displayed the highest level of resistance to MXR, doxorubicin and daunorubicin in the cytotoxicity assays. In summary, glycine mutations at position 482 have a significant impact on ABCG2 function, by modifying its substrate specificity and its influx/efflux rates. This study also demonstrates that flow cytometry constitutes a powerful tool for the kinetic analysis of ABC transporters expressed in primitive hematopoietic precursors. Grant #SAF2002-02618 (MCYT).

The effect of administration order of busulphan and cyclophosphamide on the myeloblative and immunosuppressive properties of the conditioning regimen C. Nilsson, J. Forsman, C. O´Connor, H. Concha, M. Hassan, Huddinge University Hospital (Stockholm, S) Summary: Busulphan (Bu) in combination with cyclophosphamide (Cy) has become a highly used conditioning regimen in SCT during the last years. However, the treatment related-toxicity is still the dose-limiting factor. In the present paper, we investigated the influence of the administration sequence of busulphan and cyclophosphamide on both the myeloablative and the immunosuppressive effect of the conditioning regimen. Balb-C mice were divided into three groups. Bu-Cy group received Bu followed by Cy. Cy-Bu group received Cy followed by Bu. Busulphan dose was 8.75mg/kg/day for four days and cyclophosphamide dose was 100mg/kg/day for two days. Control consisted of untreated animals. Bone marrow and spleen were harvested during conditioning regimen and up to 19 days after the treatment. CFU-GM assay was performed on marrow cells. Immunological analyses were performed using spleen cells. Liver status was determined using AST, ALT and bilirubin before, during and after conditioning. No significant difference in the myeloblative effect was found between both treatments schedules. Both treatment schedules have resulted in a decreased capacity of the bone marrow cells to form colonies by 60 %. The suppression observed in CFU-GM maintained at the same level for both treatments from day -2 and to day +19.

The immunosuppressive effect expressed as CD3+/CD19+ and CD4+/CD8+ was also similar. The levels of cytokines, IL-2, TNFalfa and IFN-gamma at day 0 were lower in Cy-Bu regimen. The most substantial difference between the two groups was observed on TNF-alpha that was significantly (p<0.05) decreased during day -3 to day +6 when Cy was administered first compared to that when Bu was given first. The value of TNFalpha was normalized at day + 6 and no difference between the two groups was observed to day + 19. The liver enzymes (AST and ALT) were higher in Bu-Cy regimen. Significant (p<0.05) increase in ALT values were observed in mice treated with Bu first from day + 3 until day + 19. The lower cytokine levels on day 0 may be less harmful for transplanted stem cells and the lower AST and ALT values may indicate less liver toxicity. This investigation may have clinical impact in SCT setting.

A new method for optimal purity and recovery of peripheral blood stem cells F. Zinno, G. Peluso Cassese, A. Lanti, F. Messina, R. Massarelli, G. Isacchi, Tor Vergata University (Rome, I) Objectives: The treatment of peripheral blood stem cells (PBSC) is a procedure that requires a standardized method that assures an effective recovery after cell manipulation without any damage to cell's sort. Methods : In four cases a new method (DACS SC-Charter Medical-U.S.A.) to obtain the restriction of volume of target cells and depletion of unwanted cells with concomitant high purity of CD34+ progenitor cells was used for leukapheretic concentrates manipulation .The principle of the system is based on the use of the Buoyant Density Solution 60 (BDS60); the density of the solution has been formulated to separate lower density cells ,including CD34+ cells from higher density cells after centrifugation. Four patients (48,24,14 and 13 months old) affected by Burkitt' s Lymphoma, Retinoblastoma, Neuroblastoma and Medulloblastoma respectively, were included in this study. Results: All data are plotted in the table below.

The median restriction of WBC was 28.7% , the recovery of CD34+ cells was 88% and cell viability was always higher than 95%. All patients achieved the graft's threshold. Conclusions: Although the preliminary results and the few number of the case, in our experience this new method has determined an excellent recovery of CD34+ cell, a remarkable depletion of the other cell lines and a consistent reduction of the volume.

Immunophenotypic characterisation of circulating progenitor cells after peripheral blood stem cell transplantation C. Albo, J. de la Fuente, C. Ares, E. Feteira, M.C. Alonso, Xeral-Cies Hospital (Vigo, E) Introduction: Hematopoietic progenitor cells (HPC) are circulating in the peripheral blood (PB) before engraftment following auto or allo-PBSCT. It is known that the CD34 protein is a reliable marker for the identification of a small fraction of PB mononuclear cells in which HPC are contained. The CD34+ cells represent a heterogeneous cell population consisting of both pluripotent progenitors as well as stem cells committed. The aim S309 of this study was to investigate the immunophenotypic characteristics of these postransplant circulating progenitor cells. Patients: Forty seven auto and nine allo-PBSCT recipients were consecutively selected from January 2000 to August 2002 for this study. The median age was 46.5 years (17-69) . 20 patients had non-Hodgkin lymphoma, 16 myeloma, 8 Hodgkin lymphoma, 7 acute leukemia, 4 solid neoplasms and 1 patient chronic myeloid leukemia. CELL PREPARATION: 20 ml of PB in heparin was obtained on days 4, 9, 11, 14, 16 y 18 after infusion. ANALYSIS: Cells were incubated with the following combinations of monoclonal antibodies (MoAbs): a) anti CD34-FITC/anti CD90-PE/anti CD38-CyCrome; b) CD34-FITC/CD117-PE/HLA-DR-PerCP; c) CD34-FITC/ CD13-PE/CD33-CyCrome. Samples were analysed by FACscan flow cytometer (BD, San Jose, CA) equipped with Cell Quest and Paint-A-Gate software (BD). STATISTICAL ANALYSIS: Routines within SPSS (Statistical Package for the Social Sciences) were used for the estimates shown. Results: CD34+ cells were undetectable on day 4 after transplantation. A CD34+ population reappeared from day 9 to day 18 after transplantation, depending on the patient, along with neutrophil and platelet recovery. Table 1 show the reactivity of CD34+ and the other markers explored in this study.

ND: not detectable Conclision: Subsets of CD34+ HPC enriched in pluripotent stem cells (CD90+ or HLADR-) were hardly detected during the very early posttransplant period, then these HPC diminished on days 14 to 18. Whereas, HPC that expressed panmyeloid associated antigens (CD33, CD13, CD117) increased after engraftment and they occupied the major part of hematopoietic progenitor cells.

Thermally induced catastrophic cryocyte breaks M.J. Watts, S.J. Ings, C. Balsa, C. Penn, D.C. Linch, University College London Hospitals (London, UK) Shattered or "catastrophic" frozen stem cell bag breaks can cause distress to patients and staff, lead to the loss of stem cells or if salvageable, the risks of a non-sterile infusion. Since 1995 at UCLH, stem cells have been frozen in cryocyte bags (Baxter Healthcare) and no breaks have occurred in our storage system of -140degC freezers. All of our breaks are associated with the frozen delivery of the cells. In November 2001, the cells were shipped in plastic Dewars, half-filled with liquid nitrogen. Over a 7-month audit period, 9 cryocyte breaks occurred affecting 9/68(13%) of patients and 305 bags issued (3% breakage). This system was replaced by shipping the frozen bags in individual cardboard boxes in a purpose-made vapour phase shipper (-190degC) and over the next 8 months there were no cryocyte breaks for 91 patients and 310 bags delivered. From April to November 2003 however, we had a series of 20 catastrophic cryocyte breaks affecting 14/64(22%) of patients and 282 cryocyte bags delivered (7% breakage). Four key observations were made during this period; 1) No breaks were recorded at issue of the cells from the -140degC freezers or when the bags were inspected on delivery to the ward in the shipper about 30min later. All of the breaks were reported over 1 hour after this when bags were found broken in the shipper before thawing. 2) In two instances on the ward, bags were heard to break undisturbed in the shipper. 3) Fifteen of the 20 bag breaks were of one particular cryocyte lot H02G015050, for which 142 bags had been issued (11% breaks). 4) During the same period 90 bags of another lot H02J17036 had been used and issued without breaks. We therefore filled 10 bags of lot H02G015050 with thawed stem cells from a deceased patient, refroze the cells and placed 5 bags in each of two vapour phase shippers. Earlier experiments had shown that the air temperature within the shipper was -140degC when the stem cells were first transferred from the mechanical freezer and took over 1 hour to fall to -190degC. After 3 hours in the shipper the cryocyte bags were examined and one of the ten bags had broken catastrophically without ever leaving the room. It therefore appears that thermal stresses alone at -190degC can break a proportion of cryocyte bags previously stored at -140degC, in the absence of liquid nitrogen contact or significant mechanical trauma. We have since delivered 23 frozen stem cell bags for the last six patients in dry ice without incident.

CFU-GM culture of the stem cell graft is not predictive for bone marrow recovery and outcome after autologous peripheral blood stem cell transplantation J. van der Lelie, B.M.J.M. Odijk, A. de Vries-Van Rossen, C. Voermans, Acad Ziekenhuis bij de Universiteit van Amsterdam, Sanquin (Amsterdam, NL) Introduction: For a successful autologous peripheral blood stem cell transplantation (autopbsct) a minimum number of CD34+ cells is required. In our centre as in many others also GFU-GM cultures are performed routinely. We studied whether these offer prognostic information on bone marrow recovery and outcome. Patients and methods: Between 1994 and 2003 131 autopbsct's were performed in 125 patients. Diagnoses were: lymphoma 54, multiple myeloma 39, acute leukaemia 24, solid tumor 7 and auto-immune disease 1. The number of CD34 cells harvested was counted and CFU-GM cultures were performed before freezing and after thawing. Results: 91% of the patients received 4.106 CD34+ cells/kg or more. The number of CFU-GM before freezing ranged from 12-1126.104/kg (median 140.104/kg). The recovery after thawing was highly variable varying from 0,16-97% (median 37%). The GFU-GM dose infused ranged from 0,3-299.104/kg (median 43.104/kg). The estimated overall survival at 5 years for all patients was 60%. There were no differences in survival between patients receiving a dose of CD34 or CFU-GM higher or lower than average. All patients had neutrophil recovery. The median number of days till a neutrophil count >0,1.109/l and > 0,5.109/l in patients who had received a lower than average number of CFU-GM's was 13 and 15 days. In patients receiving a higher dose this was 12 and 14 days, not a statistically or clinically significant difference. Six patients had a delayed platelet recovery. Three of them had received a CFU-GM dose lower than average, three above. Relapse might have been responsible as four of these patients died of relapse 2-6 months after transplantation. No differences were found between patients receiving a higher or lower dose of CFU-GM and length of hospital stay or transfusion requirements. CFU-GM number before freezing and recovery after thawing were not predictive either. Conclusion: CFU-GM is not predictive for bone marrow recovery and outcome after autopbsct. The decision whether a graft is adequate for transplantation can be made on CD34 number.

Polymopphism of human platelet antigens by HLA-identical siblings L.L. Golovkina, R.M. Kutyina, Scientific Center of Hematology (Moscow, RUS) Single nucleotide polymorphism (SNP) is a source of human genom^s diversity. SNP-identification is used for the difference of HLA-identical individuals. It is known that syntesis of platelet^s glycoproteins with alloantigens, based on them, has a code by several pairs of chromosomes. Different localization of gens HPA (human platelet antigens) and HLA in chromosomes determines their independent assortment with transmition of genetic information. The purpose of research is to study the distribution of genes HPA by HLA-identical siblings and to estimate allogenic bone marrow^s survival. Method of research: polymarese chain reaction with allele-specific primers (ASP-PCR) by PROTRANS firm. We identified 8 allele gens "a" and "b" of locuses HPA-1,-2,-3 and -5. DNA was given off the leucocytes founded in bone marrow and peripheric blood. We investigoded 13 pairs of HLAidentical siblings cosisted of patients with hematological malignancies and bone marrow donors. Each pair could have differencies in alleles of one or more gens. 5 pairs os siblings had differencies in allele gens of locus HPA-1, 6 pairs -in locus HPA-3, 4 pairs -in locus HPA-5 and 1 pair -in locus HPA-2. 3 pairs of siblings had no differences in HPA-gens. So more often these differences were stipulated on one section of the 17-th chromosome and by gens HPA-5, located on the long branch of the 5-th chromosome. According to our facts, these gens are polymorphal for Russian population. All the above mentioned differences worked as a mark for estimation of the bone marrow survival in the patients with hematological malignancies. We followed the appearance of cells with donor genotype (marker) and/or disappearance of cells with recipient^s genotype. We have estimated the bone marrow survival on the 30-th, the 60-th and on the 90-th day after BMT. As we could see, for +30 days after BMT 3 persons had only cells of donor genotype, that testified about donor type of hemopoiesis. For +30 and +60 days after BMT 2 persones have possessed cells of both genotypes, that allowed to make a conclusion about mixed type of hemopoiesis. For +90 days after BMT these patients have not had cells with own markers. We could see recovery of donor type of hemopoiesis. So the conclusion is it is possible to use new mollecular mark of allele-specific locus of HPA-gens for estimation of bone marrow survival in patients with hemotological malignancies.

Role of IL-18 and IL-2R evaluation in prediction of acute graft-versus-host-disease in allogeneic bone marrow transplantation M. Iravani, A. Ghavamzadeh, M. Shaiegan, G. Babaee, F. Tarabadi, A. Deihim, M. Aghaii-pour, B. Bahar, A. Talebian, Z. Nasri Moghadam, Shariati Hospital (Teheran, IR) Background: Cytokines released by type 1 T -helper cells are thought to play a pivotal role in acute graft-versus-host disease. The ability to predict the likely occurrence of graft-versus-hostdisease (GVHD) after bone marrow transplantation (BMT) would be extremely valuable. By serially measuring serum levels of soluble IL-2 receptor (sIL-2R), IL-18 following allogeneic BMT, we tried to define their relationship to aGVHD as complication of the transplantation and determine useful markers for aGVHD predictors. Sampels and methods: Serum sIL-2R, IL-18, levels were measured by sandwich ELISA in 219 sera samples from 39 patients (with haematological disorders before and after allogeneic BMT) and 28 controls. All patients received BMT from HLA-identical siblings. Results: 25 patients developed aGVHD and serum levels, of sIL-2 Rand IL-18, in sera drawn before transplantation, in patients with acute graft-versus-host disease (aGVHD +), were increased in comparison of patients without acute graft-versus-host disease (aGVHD -) and control group and there wasn't any significant differences in serum levels of sIL-2 Rand IL-18 in aGVHDpatients and controls. Serum level of IL-18, in aGVHD+ patients, was increased during day 3 -24 after BMT, and there was a significant difference in patients with GVHD 0 -GVHD III. In majority of patients with acute GVHD (60%), the peak levels of IL-18 and IL-2R was achieved on day 10 after BMT and the rise in sIL-2R and IL-18 preceded of clinical signs of GVHD (mean day 15 after BMT). Level of IL-18 in patients with aGVHD had strongly correlated with the severity of aGVHD on Day 10 after BMT. Conclusion: Our data concluded that IL-18 plays an important role in the development of aGVHD and IL-18 level might be an indicator for aGVHD, reflecting the severity of the disease. These findings suggest that IL-18 may play important roles in the pathogenesis of aGVHD and that measurement of serum IL-18 levels can be useful.

Mycophenolate mofetil for treatment of graft-versus-host disease following stem cell transplantation when cyclosporine is contraindicated P. Rzepecki, B. Batiuk, J. Zolnierek, T. Sarosiek, E. Bartczak, C. Szczylik, Military Medical Institute (Warsaw, PL) Graft versus host disease /GVHD/ is the main cause of morbidity and mortality after the allogeneic stem cell transplantation and decreases the therapeutic benefit of this method. Current agents for the prevention and treatment of GVHD have limited efficacy and often toxic side effects are observed. Cyclosporine /CsA/ and steroids are treatment of choice for acute and chronic GVHD. There is no established treatment when cyclosporine is contraindicated.

Mycophenolate mofetil is a new immunosuppressant with a selective mechanism of action. CellCept significantly decreased the intracellular pool of guanosine nucleotides in T and B lymphocytes leading to inhibition the production of antibodies and the generation of cytotoxic T cells. We treated 10 patients with contraindication to cyclosporine because of its renal toxicity-8 and CsA induced TTP-2. Six of them had acute GVHD, four chronic GHVD. All patients received initial dose of mycophenolate mofetil 2 g daily and steroids 2mg/kg/daily for acute and 1mg/kg/daily for chronic GVHD. When response was observed doses of drugs were slowly reduced. All patients with chronic GVHD /skin, mucosa, liver involvement/ responded and live without any immunosuppressive treatment. Three patients with acute graft versus host disease /liver and skin involvement, stage-3/ improved. The last patient with intensive gut involvement didn't response and received campath as salvage therapy. One patient with improvement during therapy died because of viral pneumonia leading to massive hemorrhage into lung tissue. Out of this case none of our patients treated with mycophenolate mofetil experienced severe adverse events. Results from this experience suggest that mycophenolate mofetil may have clinical benefit in the treatment of GVHD when CsA is contraindicated. Future studies are needed to confirm this observation.

Molecular monitoring of mixed chimerism early after allogeneic stem cell transplantation may predict the occurrence of acute GvHD M. Jaksch, M. Uzunel, M. Remberger, B. Sundberg, J. Mattsson, Clinical Immunology, Center for Allogeneic Stem Cell Transplantation (Stockholm, S) Early diagnosis of acute graft-versus-host disease (GVHD) greatly improves outcome for patients after allogeneic hematopoietic stem cell transplantation (SCT). In the present study, we investigated whether chimerism patterns during the early posttransplantation period may be used to predict the occurrence of severe acute GVHD after SCT. Thirty-three patients with various hematological malignancies (n=26), non-malignant disorders (n=4) and solid tumors (n=3)-underwent allogeneic SCT at Huddinge University Hospital. Median age of the 21 male and 12 female was 30 years (range 0,5-61). Cell source was mobilized peripheral blood stem cells (n=17) or bone marrow (n=16) from HLA-identical siblings (n=14), matched unrelated donors (n=15) or mismatched donors (n=4). Conditioning regimens were fTBI+Cy (n=11), Bu+Cy (n=14) or reduced intensity conditioning (n=8). Twenty-two patients also received ATG. The vast majority of patients received MTX combined with CyA as GVHD prophylaxis. The kinetics of donor/recipient mixed chimerism (MC) within the T-cell lineage was evaluated by PCR amplification of variable numbers of tandem repeats (VNTRs) loci. Peripheral blood samples were analyzed on day 7 and 10 after SCT for all patients included. Acute GVHD was diagnosed in 22 patients within the first three months after SCT, 15 of these patients developed acute GVHD grades II to IV. Although the number of lymphocytes present during the first week after transplantation is limited, blood samples provided adequate cell material for the method used. Interestingly, 13 patients showed complete donor chimerism already on day 7 after SCT. The difference in the clearance rate of host T-cells between day 7 and day 10 was compared. We found that there was a significant higher risk (p=0,025) of developing acute GVHD grades II-IV in patients with complete donor T-cell chimerism day 7 after SCT together with patients who increased 20 percentage units or more in donor T-cells between day 7 and 10 after SCT. Our data suggest that molecular monitoring of mixed chimerism early after transplantation may be useful in predicting the occurrence of moderate to severe acute GVHD after SCT.

Low-dose rabbit ATG prevents GvHD in sibling allogeneic peripheral blood stem cell transplantation for chronic myeloid leukaemia F. Bonifazi, G. Bandini, S. Falcioni, F. Palandri, M. Stanzani, M. Amabile, G. Martinelli, M. R. Motta, S. Rizzi, J. El-Cheick, B. Giannini, O. Farese, M. Rovito, G. Rosti, M. Baccarani, Bologna University (Bologna, I) Our initial experience (Nov 1995 -Sept 1999 with peripheral blood stem cell (PBSC) transplants in 21 Chronic Myeloid Leukemia (CML) patients resulted in an unacceptable rate of extensive chronic graft-versus-host disease (cGVHD). Due to that reason, since October 1999 we have added low dose rabbit ATG in the conditioning regimen on a group of 10 CML patients. We present here a comparison between the two groups. A total of 31 CML patients underwent an allogeneic peripheral blood Stem Cell Transplants (SCT) from HLA identical sibling donors. Median age was 41 years (range 19-53), 21 were males and 10 females. The median interval diagnosis-transplant was 14 months (range 4-59). Conditioning treatment consisted mainly of BU-CY and the GVHD prophylaxis was short term MTX+CsA. 10 patients received low dose rabbit ATG (15 mg/kg, Fresenius, Bad Hamburg, Germany). All patients engrafted and no rejection occurred. Acute and chronic GVHD were graded according to the standard criteria in patients surviving longer than 30 and 90 days after allogeneic SCT. Acute GVHD grade II-IV developed in 7/21 patients who didn't receive ATG and 2/10 patients who received ATG. Chronic GVHD occurred in 15/21 patients without ATG and in 4/10 patients with ATG. In particular we observed extensive cGVHD in 10/21 patients in the former cohort and in 4/10 patients in the latter. Five hematological relapses occurred in the no-ATG group (2 in chronic phase, 3 in advanced phase) and no relapses in the other group. The patients were strictly studied at molecular level for the bcr-abl rearrangment after transplant; the incidence of isolated positivity was similar in the two groups. In conclusion, even if the number of patients is small and this is not a randomized study, we suggest low dose rabbit ATG is a promising agent in decreasing the rate of GVHD, in particular extensive cGVHD, without increasing the risk of relapse.

Clinical and prognostic evaluation of cGvHD after sibling allogeneic peripheral blood stem cell transplantation. A large, single centre analysis F. Bonifazi, G. Bandini, S. Falcioni, F. Palandri, B. Giannini, M. Stanzani, M. Arpinati, B. Urbini, S. Rizzi, O. Farese, J. El-Cheick, M. R. Motta, M. Baccarani, Bologna University (Bologna, I) From 1995 to 2002, 104 allogeneic peripheral blood Stem Cell Transplants (SCT) have been performed from a sibling HLA fully matched donor after a myeloablative preparative regimen. All patients received the same graft-versus host disease (GVHD) prophylaxis with CsA and short term methotrexate (15-10-10-10 mg/sm). Median age was 41 years (range 19-57), 76 were males and 28 females. The diagnoses were Acute Leukemia or MyeloDisplastic Syndrome in 43 patients, Chronic Myeloid Leukemia in 20, Multiple Myeloma in 32, Lymphoma in 8, Myelofibrosis in 1. Conditioning treatment consisted mainly of BU-CY (70) or unfractioned single-dose TBI (34). All patients engrafted and no rejection occurred. Acute and chronic GVHD were graded according to the standard criteria in patients surviving longer than 21 and 90 days respectively. Thirty-one patients (29.8%) experienced aGVHD gr II-IV and 16 (15.4%) gr III-IV. Chronic GVHD occurred in 52/92 (55,5%) evaluable patients and it was extensive in 32/52. The median number of CD34+ cells infused was 6.3 x 106/kg (range: 0.9-19.5 x 106/kg). No effect of the number of CD34+ was observed both on survival and on cGVHD occurrence. Univariate analysis showed that cGVHD occurrence had a statistically significant correlation with sex mismatch (F donor, M recipient), patients' age ( older than 41 years), blood compatibility and prior aGVHD grade II-IV. In multivariate analysis the prognostic factors for cGvHD occurrence were patients' age, sex mismatch and prior GvHD. The clinical characteristics of cGVHD were also analysed in terms of topography of organ involvement, duration of immunosuppressive therapy, probability of withdrawal of immunosuppression, relationship with tapering of CsA, response to therapy and impact on survival. Briefly, 70% of patients developed cGvHD independently from CsA tapering. The most frequent organs involved were skin (77% of patients), oral cavity (75%), eyes (61,5%), liver (42%) and genital tract (19,2%). The median number of organs involved was 4. Even though multiple courses of therapy were administered, unfortunately extensive cGvHD didn't respond in 78% of cases. Chronic GvHD showed a non significant effect on relapse, while it was statistically associated with an increased transplant-related mortality.

Frequent presence of donor-derived epithelial cells in buccal mucosa smears following allogeneic haematopoietic stem cell transplantation in 54 patients: level not related to procedure, time after transplantation, or graft-versus-host disease G. Juliusson, U. Frödin, G. Bäckström, K. Karlsson, C. Malm, University Hospital (Linkoping, S) Following sex-mismatched SCT donor-derived epithelial cells have been found in various tissues (NEJM 2003; 349:570) , including liver and buccal mucosa (Lancet 2003; 361:1084) . Donor cell chimerism is quantifiable with molecular detection of short tandem repeats on minimal tissue samples. Buccal mucosa is a target of chronic graft-versus-host disease, and epithelial cells are readily available for repeated non-traumatic sampling through mouth swabs. To evaluate a possible association to GvH we evaluated donor cell chimerism in buccal smears from 54 patients at various times after SCT (27-1476 days, median 587 days). Mean age was 45 years. The diagnoses were AML 18, CML 6, ALL 4, Myeloma 16, NHL 5, CLL 3, AA 2. Blood SC from siblings were used in 32, from unrelated donors in 16, and unrelated BM in 6 patients, with a median CD34+ cell dose of 5x10(6)/kg. Melphalan-based adjusted conditioning was used. Blood cell chimerism was measured biweekly, and 25 patients had DLI due to mixed blood cell chimerism and/or residual/progressive disease. Microscopy identified pure epithelial samples in buccal smears, and contamination of blood did not occur. In 15 patients repeated monthly buccal samplings were performed during the first 3-5 months after SCT. In 10 patients simultaneous sampling from each bucca was analysed, showing a high correlation, excluding patchy distribution. There was a median of 16% donor-derived cells in the buccal smears (range 1-92%). Forty-two (78%) of the patients retained a mostly recipient-cell derived buccal mucosa (median 87%, range 60-99%), whereas 12 patients (22%) had 52-92% donor-derived cells (median 59%). All four ALL-patients had high proportion of donor-derived buccal cells (median 56%, range 53-74%). There was no correlation between the level of buccal chimerism and type of procedure, disease stage and risk group, time from SCT or outcome as regards engraftment, need for supportive care, or GvH. In the longitudinal analyses, all but two had stable levels: one myeloma patient with full donor blood cell chimerism and relapse had decreasing proportions of donor-derived mucosa cells, and one AML patient had increasing proportion donorderived mucosa cells after treatment with DLI for mixed blood cell chimerism. Most patients had significant proportion of donorderived buccal cells, some had a dominance of donor-derived buccal cells, but there was no clear correlation between level of chimerism and clinical situation.

Replacement of calcineurin inhibitors with daclizumab as treatment for acute graft-versus-host disease associated thrombotic thrombocytopenic purpura S. Wilhelm, D. Wolff, B. Steiner, C. Junghanss, J. Casper, M. Freund, University of Rostock (Rostock, D) Thrombotic thrombocytopenic purpura (TTP) in association with GVHD after allogeneic haematopoietic stem cell transplantation (alloHSCT) is associated with high morbidity and mortality. Since calcineurin inhibitors (CI) may be part of pathophysiology of TTP, we evaluated the efficiency of replacement of CI by daclizumab in GVHD associated TTP. Patients and methods: Of 133 adult patients who underwent alloHSCT at our institution from 1/98-11/03, 10 (7,56 %) developed TTP defined by intravascular hemolysis with red cell fragmentation and de novo thrombocytopenia. Nine patients received an alloHSCT from a matched unrelated donor and one received an alloHSCT from a HLA matched sibling for haematologic malignancies. Conditioning regimens consisted of treosulfan/fludarabin/ATG (n=4), TBI/cytarabine (n=3), treosulfan/cyclophosphamide (n=1), TBI/cyclophosphamide (n=1) and TBI/ cyclophosphamide/VP16/campath-1H (n=1). The median time from transplant to onset of TTP was 116 days post transplantation (d33-348). All patients had acute GVHD at the time of onset of TTP (grade I n=1, grade II n=3, grade III n=3, and grade IV n=3). Treatment of TTP consisted of daclizumab 1mg/kg weekly IV and discontinuation of treatment with CI in 8 of 10 patients. Treatment of acute GVHD consisted of methylprednisolone 2mg/kg/d (n=3), additional etanercept (n= 5), or additional sirolimus (n=2). Results: In patients receiving daclizumab TTP resolved completely in 2/8 patients and 5/8 patients improved with hemolysis and/or thrombocytopenia. In one of the partial responders TTP resolved completely in combination with defibrotide. No progression of TTP and GVHD was observed following replacement of CI by daclizumab. Complete remission of GVHD was achieved in 3/8 patients, 1/8 patient showed improvement of GVHD by one grade and 4/8 patients showed no improvement of GVHD. Two patients not receiving daclizumab died due to progression of TTP (n=1) and sepsis (n=1), while 3/8 patients receiving daclizumab survived with a median follow up of 66 days post TTP (d35-88). 5/8 patients died at a median of 26 days (d15-116) after developing TTP due to infection (n=4) and relapse of Hodgkins disease (n=1). Discussion: The results demonstrate that replacement of CI by daclizumab results in improvement of TTP in a significant proportion of patients and does not lead to progression of GVHD. Nevertheless mortality remains high due to a high infection rate caused by GVHD and associated immunodeficiency.

Risk factors for acute GvHD after allogeneic haematopoetic stem cell transplantation in children A. Vitrischak, E. Semenova, M. Ovsyannikova, A. Pugachev, E. Morozova, N. Mikhailova, L. Zubarovskaya, B. Afanasyev, SPb State I. Pavlov Medical University (St. Petersburg, RUS) Allo-HSCT is a treatment of choice for different hematological diseases. Acute graft-versus-host disease (aGVHD) is a main complication leading to significant morbidity and mortality. The aim of the study was to evaluate aGVHD in depending on age, stem cells (SC) source, prophylaxis regimen, ABO/ sex match of donor-recipient. This study analyzed data of 50 patients (pts) <=21 years old (yo) after matched related (33) & unrelated (17) allo-HSCT from 1991 to 2003 in comparing with 25 pts > 21 yo. The pts <=21yo were with acute lymphoblastic leukemia (ALL)-28, acute myeloblastic leukemia (AML)-13, chronic myeloid leukemia-8, myelodisplastic syndrome-1. Source of SC was bone marrow (BM) -31 pts, peripheral blood (PB) -19 pts. Sex matching of donor/recipient were male (m)/m-20, female (f)/f -8, m/f -14, f/m -8. Major ABO-mismatch was in 9 pairs, minor ABO-mismatch -14 pairs, ABO-matched -21 pairs, bidirection mismatch-6 pairs. Conditioning regimens were Bu+Cy±VP-16±ALG. GVHD prophylaxis regimens were CsA+MTX, CsA+Pred, CsA+MTX+Pred. Results. aGVHD occurred in 25(50%) pts <=21yo, in 13(52%)pts >21yo.aGVHD I-II was higher in pts<=21yo 85, CI=1, 89, p=0, 015) , III-IV was higher in pts>21yo 65, CI=1, 56, p=0, 025) . aGVHD caused death in 18,7% pts <=21yo, in 35,0% pts>21yo. In pts <=21yo after related-vs-unrelated allo-HSCT aGVHD occurred in 15(45%)pts and 12(70%)pts, [I-II-10(66%), III-IV -5(33%)] and [I-II-4(33%), III-IV -8(66%],resp. Prophylaxis of aGVHD I-II and III-IV were: CsA+MTX -4(26%) and 3(20%), CsA+Pred -1(16%) and 2(33%), CsA+MTX+Pred -10(41%) and 7(29%),resp. In BM group aGVHD I-II-10(32%)pts, III-IV-3(10%)pts, in PB group -5(26%) and (31%), resp. aGVHD was in 4(44%) pts with major ABO-mismatch [I-II-3 (33%), III-IV -1(11%)], in 10(71%)pts with minor ABO-mismatch [I-II -5(36%), III-IV -5(36%)], in 11(52%)pts with ABO-matched donors , III-IV -7(33%)], in 3(50%)pts with bidirection ABOmismatch [I-II -2(33%), III-IV -1(16%)]. Depending on sex match aGVHD were in 12(60%) m/m pairs [I-II -7(35%), III-IV -5(25%)], in 6(75%) f/f pairs[I-II -5(62%), III-IV -1(12%)], in 5(35%)m/f pairs[I-II -1(7%), III-IV -4(28%)], in 4(50%)f/m pairs[I-II -2(25%), III-IV -2(25%)]. The risk of haemorrhagic cystitis was higher after unrelated allo-HSCT vs related allo-HSCT in 7, 7 time (p=0, 001) and increased in 3 times (p=0,015) in pts with aGVHD. aGVHD risk was in 7 times higher in pts with CMV-infection(p=0,023). Thus, the risk of aGVHD is increased in older pts, PB transplant, minor ABO-mismatching, in pts with CMV-infection. aGVHD increased risk of hemorrhagic cystitis.

Low incidence of toxicity and acute GvHD in patients transplanted from HLA-matched unrelated donor conditioned with fractionated-TBI (FTBI), cytoxan and ATG Fresenius: a single-centre report F. Benedetti, M. Sorio, M. Krampera, O. Perbellini, M. Colosio, M. Gottardi, F. Mosna, Policlinico G.B. Rossi (Verona, I) A major limitation to unrelated hematopoietic cell transplantation (HCT) has been the toxicity related to conditioning regimens. For this it has been restricted to pts younger than 50 years, with a good performance status (PS). Patients and methods: Since January 1997 and December 2002, 21consecutive pts (AML 9, ALL 2, CML 6, MDS 4), median age 36 years (range 12-54), M/F=13/8, underwent HCT form HLAidentical unrelated donor. As conditioning regimen they received FTBI (200Gy in 6 doses over 3 days), CTX 120 mg/Kg, and ATG Fresenius 40 mg/kg over 4 days starting on day -4. All pts received un-manipulated bone marrow on day 0. GVHD prophylaxis consisted of CSA 3 mg/Kg i.v. starting on day -1, and MTX 15 mg/mq i.v. on day +1, 10 mg/mq i.v. on day +3 and +6. This group of pts was compared to a group of 53 pts matched for age (median 41, range 16-58), diagnosis (AML 17, CML 13, MDS 12, NHL 8, ALL 2, SAA 1) and PS, transplanted from HLAidentical siblings in the same unit and period. The control group was conditioned with FTBI and CTX, without ATG. Results: All pts, in both groups, engrafted. The only side effects to be reported was fever around 38,5C° the first day of infusion. Transplant related mortality (TxRM) occurred in 4/21 (19%) unrelated transplants, as compared to 12/53 (22%) in the control group. The incidence of acute GVHD grade >= II in the unrelated HCT was 42% (7 pts grade II, and 2 pts grade III), and 42% in the sibling group (23/53, 18 grade II, 4 grade III and 1 grade IV). The incidence of relapse was 19% (4/21 pts) and 15% (8/53 pts), respectively. The number of bacterial infections was the same in the 2 groups. Is to be noted 3 cases of persistent CMV reactivation. Relapses occurred in 4/21 (19%) unrelated HCT and in 8/53 (15%) in the control group. At present, with a median follow-up of 28 months (range 6-44 ), 12/21 (57%) pts are alive and free from disease in the unrelated group, 33/53 (62%) in the second group. The quality of life is good for all the 12 alive pts: only 2 of these have extended c-GVHD (2/12, 16%). In the sibling transplant group the incidence of c-GVHD is 33% (11/33 alive). Conclusions: The total dose and the type of ATG is still under investigation in many centers. Our data seem to demonstrate that 40 mg/Kg ATG Fresenius is well tolerated with no side effects. Engraftment, toxicity, TxRM, relapse rate and incidence of acute GVHD were comparable to those observed in pts transplanted with HLA identical sibling.

Pulmonary failure after stem cell transplantation -a symptom of transplant-associated thrombotic microangiopathy?

K. Kentouche, H.J. Mentzel, D. Fuchs, J. Hermann, B. Gruhn, F. Zintl, Friedrich-Schiller-University (Jena, D) Pulmonary failure (PF) after stem cell transplantation (SCT) is a severe complication comprising a mortality up to 90 %. It is usually caused by viral, fungal and to a less extent bacterial infections. However there is a considerable number of patients in whom no infectious aetiology can be found. This entity is described as idiopathic pneumonia syndrome or diffuse alveolar haemorrhage. Regimen related lung injury is found in patients receiving a SCT-preparation containing busulphan, resulting in a chronic deterioration of lung function. We present four patients who developed PF while treated for transplantation associatedthrombotic microangiopathy (TA-TMA). For patients details see the table below.

All patients were treated for graft vs. host disease. When TA-TMA was diagnosed there was concomitant acute renal failure in all of them. Patients 1,2, 4 had assisted ventilation while patient 3 received CPAP-ventilation. Discussion: The lungs are not regarded as an organ involved in thrombotic microangiopathy usually. However TMA is a systemic disease and the pulmonary microvasculature may be highly susceptible in SCT-patients. Unlike to chronic busulphan-lung disease, PF in our patients was rapidly progressive. A review of TA-TMA literature reveals a high incidence of PF as the primary cause of death in these patients. The patients presented here developed PF while on PE-treatment or plasmainfusion for TA-TMA. Therefore it is not to distinguish whether PF is a manifestation of TA-TMA itself or triggered by plasmatherapy. A positive correlation has been described between circulating, oligoclonal T-cell subsets and clinical responses to ECP, in SS and cGVHD patients. Molecular analysis of the VBeta TCR repertoire of TCR beta genes represents the golden standard technique. Recently, a quantitative VBeta domains analysis can be performed using monoclonal antibodies directed against the VBeta domains. In particular, the IOTest Beta Mark (Beckman-Coulter), is constituted of 8 vials containing 3 separated, labeled monoclonal antibodies that enable to stain more frequently detected VBeta domains. A clonal T-cell expansion can be suspected when the VBeta subset represents more than 20% of the T-population. Aims of the present study were: 1-analyze the VBeta repertoire in patients candidate and submitted to ECP to search a correlation between disease and ECP responsiveness; 2-investigate if oligoclonal subsets are disease-exclusive in cGVHD and SS patients. From February 2003, 2 cGVHD and 2 SS patients at the onset of disease were enrolled. ECP protocol was designed according to published protocols treatment for cGVHD and SS and to date, only patient number 4, completed ECP treatment. Before starting, and after every ECP cycle, PBL samples were submitted to cytometry quantitative VBeta domains detection, in addition every samples was stained with anti CD2 or CD4 or CD8. Cytometry analysis disclosed repeated oligoclonal CD2+ subsets, CD4+ in SS patients CD8+ in cGVHD: VBeta16, Vbeta12, VBeta14, VBeta20 and VBeta 7.2. VBeta14 and VBeta20 represented more frequently >20% of CD2+ Tcells. All patients but n°2, showed clinical improvement after 6-8 ECP courses, at least. PBL phenotype analysis disclosed a progressive decreasing in absolute lymphocyte counts; VBeta domains analysis showed a dramatic, progressive decrement (mean reduction assessed at 80%), of clonal expanded VBeta14 and VBeta20 in three responsive patients. In conclusion, these preliminary data seem to confirm positive correlation between clinical/ECP response and beta oligoclonal subsets, though the limited number of patients to date studied. Flow cytometry makes quantitative VBeta domain analysis faster and reliable, however molecular studies are ongoing to confirm VBeta clonal expansion in the studied patients.

Does stem cell source could predict frequency and intensity of acute and chronic graft-versus-host disease?

L. Ristic, D. Stamatovic, L. Tukic, O. Tarabar, M. Elez, S. Marjanovic, L. Simic, M. Malesevic, Military Medical Academy (Belgrade, CS) Introduction: Acute(a)and chronic(c)graft versus host disease (GvHD) still represent frequent and severe complication of allogeneic stem cell transplantation (SCT), either if stem cell source is peripheral blood (PBSCT) or bone marrow (BMT). In many studies comparative analysis were done to evaluate frequency and intensity of acute and chronic GvHD after BMT and PBSCT. AIMS: Retrospectively we have compared frequency and clinical characteristics of acute and chronic GvHD in the two groups of patients (pts) treated with allogeneic PBSCT and BMT. METHODS: We have analized 71 pts, average age was 29 years (9-52), M/F 48/23, with different hematological diseases (14-AA, 23-CML, 17-AML, 16-ALL, 1-MM). All patients had HLA-DR compatible related donor and received unmanipulated suspensions of stem cells. There were 49 pts treated with BMT and 22 pts treated with PBSCT. Conditioning regimen was adjusted to the primary disease, and GvHD prophylaxis was combination of CsA+ Mtx+ MPDN. Results: Pts in the group with PBSCT have got significantly richer sample of mononuclear cells-MNC than the other group (8,7 vs. 2,46x 108 /kg BW, p<0,001). aGvHD (2-4o) was registered in 70,6% pts treated with PBSCT, with no statistical difference between the groups. Skin and buccal mucosis are usually target tissues; rarely there are involvements of other organs. aGvHD occur after ƒy 14 days (9-21) in the group with PBSCT, and after +18d (14-27) following BMT. There were not statistically significant difference in incidence of lower grade aGvHD (0-20) in compared groups (59,2%-BMT vs. 44,8% in PBSCT). However, intensive aGvHD (3-40) were significantly more often reported in the group of pts with PBSCT (35, 8% vs. 11, 9%, p<0, 05) , and about 10% of pts had a fatal outcome. Frequency of cGvHD was significantly greater in the group PBSCT (79,6% vs. 41,2%, p<0,05). We have noticed that pts with peripheral blood as a source of stem cells had more often atypical forms of cGvHD, which was similar to aGvHD in due to involvement of gastrointestinal organs, aproximately 5 months following SCT. Conclusion: Stem cell source has not significant impact on frequency of aGvHD, but intensive forms (grade 3 and 4) of aGvHD are more often in pts with PBSCT. Pts with PBSCT had more often cGvHD, and frequently an extensive and atypical form. For detailed estimate of stem cell source influence on frequency, intensity and outcome, we need analyses on the homologous groups of pts.

Extracorporeal photochemotheraphy in the treatment of extensive chronic graft -versus-host disease M. Soli, R. Raimondi, M. Lunghi, R. Baricordi, F. Rodeghiero, M. Belloni, Vicenza Immunohaematology and Transfusion Unit, Medical Genetics (Vicenza, Ferrara, I) Fifteen patients affected by haematological malignancies transplanted with HLA matched allogeneic stem cells have been treated with extracorporeal photochemotheraphy (ECP). They have been treated twice (consecutive days) weekly for three times, then twice every fifteen days for three times and then twice a month as maintenance. All of them had extensive cutaneous chronic graft versus host disease (cGVHD) and were in treatment with immunosuppressive agents. Four patients had oral ulcerations. Two patients had gastrointestinal symptoms. 361 leukapheresis have been done using Cobe Spectra cell separators. We used mononuclear (MNC) program with a processing target of two blood volumes. The collected highly enriched MNC concentrates (mean 86%, range 52.1-98.7) were always collected in small volumes (mean 125 mL, range 44-160). The collected cells haematocrit was always less than 5.5% (mean 2.5, range 0.7-5.5). A mean of 6.3 x 10e9 MNC cells (range 0.2-20.8) per procedure have been UV-A irradiated using a PUVA Combi Light photoirradiator. Just before UV-A irradiation the concentrate volumes were adjusted to 300 mL with 0.9% saline solution and added with 8-methoxypsoralen to get a psoralen final concentration of 200 ng/mL. The required radiation level (2 joule /cm2) was supplied in about two minutes. The success of irradiation was evaluated by removing the lymphocyte proliferation response to phytohaemagglutinin. The drops of Stimulation Index (methyl-3H-thymidine radioactivity counts per minutes stimulated cultures/counts per minutes unstimulated cultures) were higher than 80% (mean 88.3, median 94.2, range 12.9-98.1). All patients well tolerated the procedure. Fourteen patients improved (response higher than 50%) or stabilized (one patient) in skin disease. Three patients of four improved in oral ulcerations, one patient of two reduced diarrhoea episodes. Ten patients were able to taper steroid dose, one patient stopped steroid therapy, one patient stopped mycophenolato mofetil without worsening of cGVHD symptoms. We indicate that ECP performed with Cobe Spectra plus PUVA Combi Light is helpful to cGVHD affected patients.

Engraftment characteristics and incidence of graft-versushost disease using G-CSF stimulated marrow for allogeneic stem cell transplant S.M. Tan, T.C. Ong, S. Jameela, A. Yong, K.M. Chang, V. Purushotaman, Hospital Kuala Lumpur (Kuala Lumpur, MY) Peripheral blood stem cell transplant has been a preferred option in many transplant centres today. It results in early engraftment as well as lower transplant-related morbidity and mortality. Although the incidence of acute GVHD is similar to marrow source, there is an increased incidence of chronic GVHD. In view of this, our institution has preferred to use G-CSF primed marrow for our allogeneic transplant programme. A total of 56 patients received HLA-matched allogeneic stem cell transplantation between May 1999 and September 2003. Fifty one patients received only G-CSF stimulated marrow. Four patients required top-up peripheral blood stem cells because of insufficient marrow cells after red cell depletion. One patient received peripheral blood stem cells only because of major ABOmismatch. We report the engraftment characteristics as well as acute and chronic GVHD for those patients who received only G-CSF stimulated marrow. All patients received a myeloablative conditioning and a T-cell replete HLA-matched graft. Cyclosporine and methotrexate were given as GVHD prophylaxis. Patients received a median of 5.08 x 108/kg nucleated cells (1.56 to 11.5 x 108/kg). Neutrophil engraftment was noted on day +18 (day +13 to +27). A sustained platelet count above 20 x 109/L(day +10 to +41) was similarly observed. The incidence of acute GVHD was 51%. Majority were grade I to II. Only 7% and 11% had grade III and grade IV GVHD respectively. The incidence of chronic GVHD was 38%. Less than half (16%) were limited. The rest were extensive of which two were due to the use of donor lymphocytes for disease relapse. Early transplant mortality within 100 days was seen in 16% (8 patients). Three died of sepsis, one of which had chronic severe aplastic anaemia who had failed numerous immunosuppressive regimens. Four patients died of severe grade IV GVHD, one of whom had concomitant CMV enteritis, one had idiopathic pneumonia syndrome. In conclusion, G-CSF primed marrow is a viable alternative to peripheral blood stem cells. It results in early engraftment and a low incidence of transplant-related mortality. The incidence of acute and chronic GVHD is similar to that of non-primed marrow source.

Clinical benefit in treating sclerodermic chronic GvHD with iloprost in two patients after allogeneic BMT from HLAidentical sibling donors M. Sorio, F. Mosna, M. Krampera, M. Gottardi, H. Aqel, F. Benedetti, Policlinico G.B. Rossi (Verona, I) Scleroderma is a possible feature of extensive cGVHD, occuring as skin changes (tightness, thickening, non-pitting induration of the face, neck, hands and trunk -thorax and abdomen), sclerodactyly, digital pitting scars, abnormal finger or hand edema, abnormal skin pigmentation, Raynaud's phenomenon. Although it is a well-known possible complication in allogeneic BMT recipients, there is at present no general agreement about treatment.

Here we describe two cases affected by extensive cGVHD presenting with scleroderma after allogeneic BMT from HLAidentical sibling. In both cases, fractioned TBI and Cytoxan were used as transplant conditioning. Short MTX and CSA were used as GVHD prophylaxis. Scleroderma appeared when the two patients were at +15 and +39 months from transplant, respectively. Both patients presented with extensive chronic skin induration of the neck, shoulders, upper and lower limbs and the trunk, with pain and sense of tension in common movements. Face and hands were less involved by sclerodermic process. There were no other organs involved. Both patients were first treated for more than one year with mofetil mycophenilate and photoapheresis with no clinical benefit.

In an effort to improve the results, we decided on the basis of clinical trials used in idiopathic systemic sclerosis, to treat the two patients with repeated courses of synthetic PGI-2 (Iloprost), given at 50 microgr a day, over 8 hours iv continuous infusion for 5 days in our Outpatient Department. Iloprost was well-tolerated with little or no side effects: hypotension and cardiac arythmias, considered the main side effects of the therapy, have not been observed. This treatment was successful in reverting the process and reducing the extension of the disease, with gaining of skin tenderness in the surfaces affected and disappearance of pain. This benefit started after the 5th course. Clinical improvement continued thorughout the next courses. The first six courses were given with a monthly schedale and then every 3 months as mainteinance therapy. At present, the two patients are alive and well, with scleroderma reduced to less than 20% of the skin surface, and with no functinal joint impairment. In our two cases, Iloprost has proved itself a valid alternative choice for the treament of refractory scleroderma in the context of chronic GVHD.

Ethnic difference in the incidence and severity of graftversus-host disease in Malaysia L. Wong, S.M. Tan, T.C. Ong, S. Jameela, A. Yong, K.M. Chang, V. Purushotaman, H. Roshida, Kuala Lumpur Hospital (Kuala Lumpur, MY) Ethnic difference in the incidence and severity of GVHD is not well documented in multi-ethnic societies. Japanese and Thai groups have reported comparatively lower incidence of GVHD compared to the West. Malaysia has a multiracial composition of Malays, Chinese, Indians and "natives"(indigenous non-Malays). The purpose of this study was to assess the characteristics and significance of this difference. We retrospectively analysed all patients who underwent sibling HLA-matched HSCT in our institution between May 1999 and September 2003. The HLA typing were done at Institute of Medical Research, Kuala Lumpur; Class I HLA typing was done by serology method and class II HLA typing by Polymerase chain reaction (PCR) method. Fifty-six patients received a myeloablative regimen and T-cell replete G-CSF primed marrow or blood stem cell graft. All patients had cyclosporine and methotrexate as GVHD prophylaxis. The diagnosis of GVHD was based on clinical evidence with histological confirmation where possible and was graded according to the consensus criteria. Fifty-four patients were evaluable for acute GVHD and 45 patients who survived beyond 100 days were analyzable for chronic GVHD. The incidence of grade II to !V acute GVHD was 40.7%. The incidence was 54% in Malays, 27% in Chinese, 50% in Indians and 14% in the native group. The difference was statistically significant between the Malays and Chinese (p<0.01). The incidence of extensive chronic GVHD was 20% and this was similarly higher amongst the Malays (26%) and Indians (50%) than the Chinese (8%). Apart from the ethnic difference, our study also showed that the other possible causes for an increased incidence of severe GVHD were higher recipient age, donor-recipient mismatch and higher donor parity amongst the Malay patients. The interpretation from our study is limited by the small data pool. To address this issue of GVHD incidence and severity ,perhaps for the purpose of intensifying GVHD prophylaxis in our patients, larger cohorts involving all the transplant centres in Malaysia will be required.

Allogeneic stem cell transplantation after reduced intensity conditioning in the treatment of advanced haematological malignancies M. Malesevic, D. Stamatovic, L. Tukic, B. Balint, M. Elez, V. Glavicic, B. Todoric-Zivanovic, Military Medical Academy (Belgrade, CS) Introduction: Myeloablative conditioning regimen is treatment of choice for patients younger than 50 years in good vital condition in due to high transplant related mortality-TRM (> 30%). Reduced intensity conditioning (RIC), as a new regimen of less myeloablative intensity, reduces TRM and allows faster reconstitution of donor lymphocytes, which are responsible for the antitumor effect. Aims: Evaluation of efficacy (with respect to engraftment, frequency of remissions, donor chimerism) and safety (TRM) of stem cell transplantation (SCT) with RIC in our Center. Methods: 14 patients (pts), average age 22 years (9-52), 4 females and 10 males, were treated with allogeneic SCT with RIC: 7 pts were with initial disease of »high risk« or with resistent disease (5 with ALL, 1 with AML and 1 with MM), while 7 pts were relapsed after the first SCT (6 after allogeneic-3 CML, 1 ALL and 2 AML, and 1 after autologous-ALL). Donors were HLA compatible sibling in all the cases. Stem cell source in 6 pts was bone marrow and in the others was peripheral blood. Basic drug in the conditioning regimens was Fludarabin (Flu) and we have combined them with other agents depending on the primary disease. All pts have recieved unmanipulated suspension of mononuclear cells. GvHD prophylaxis were maintained with the combination of CsA and MMF (up to 30 days). Chimerism (mixed and donor) was analized at the day +30, +60 and +90. Results: Engraftment were registrated in 12/14 pts (85,6%); Three pts (21,4%) have died in first 100 days (early death). Two of them had sepsis and one had intensive aGvHD grade 4 (d+39.). Eleven pts (78,5%) have achieved complete clinical and hematological remission, but three of them (21,4%) have relapsed (d+40., d+180 and d+210.). Cytogenetic analysis, chimerism and RBC phenotype on the d+90. have detected 100% donors chimerism in 10/11 (90,9%) pts. Acute GvHD had 5 (41,6%) and cGvHD 6 (50%) pts, with an extensive form in 3 of them. Discussion: Despite our heterogeneous group of pts. (variety in the primary diseases, stage of its progressiveness, stem cell source), our results of using allogeneic SCT after RIC in the treatment of advanced hematological malignancies could be encouraging according to TRM, engraftment, achieving remissions and donor chimerism and they are keeping with other studies published up to now.

D. Kata, J. Holowiecki, B. Stella-Holowiecka, J. Wojnar, S. Krzemieñ, J. Najda, G. Helbig, Silesian Medical Academy (Katowice, PL) We recently demonstrated that patients with haematologic malignancies receiving haematopoietic stem cell transplants (HSCTs) from killer immunoglobulin-like receptor (KIR) ligand incompatible donors show tendency for lower marrow relapse rate and have higher probability of overall survival than those who do not ( Blood. 2003,102:814-9) . However, there are no available data on the frequency of possible extramedullary (EM) disease in these patients. We report for the first time a case of unusual isolated leukaemic involvement of multiple EM sites after HSC transplantation from unrelated KIR-ligand disparate donor in a 28-year-old man. The patient was diagnosed with Ph-positive, bcr/abl-positive CML in

Oktober 2000 and was primarly treated with hydroxyurea. 7 months after the diagnosis he developed blastic crisis associated with additional chromosomal aberration t(3;21)(q34;q11). The patient was put on the treatment with imatinib and returned to the accelerated phase of the disease. In April 2002 HSC transplantation from unrelated mismatched in HLA-C donor was performed. The preparative regimen included busulphane, cyclophosphamide, Ara-C and ATG. A total of 3,851 x 10^8/kg WBC, 3,211 x 10^6/kg CD34+ cells and 7,099 x 10^7/kg CD3+ cells was transplanted. The patient engrafted very well showing trilineage reconstitution within 31 days. An episode of grade II acute intestine GVHD was observed, followed by moderate chronic mucosal, hepatic and pulmonary form of this disease. 15 months after HSC transplantation the patient presented with a 6 x 8 cm firm tumor of the left testicle and paraspinal masses of the Th2-Th5 region, penetrating the vertebral canal and causing paraparesis. The biopsy of the mass revealed dense infiltration of immature cells expressing myeloperoxidase and CD15 antigen. Bone marrow aspirate showed no evidence of leukemia, confirmed by normal karyotype and the absence of bcr/abl transcripts. The patient was treated neurosurgically and with local radiotherapy to the involved fields achieving substantial regression of the pathologic infiltrates, however without improvement in neurologic deficits. He finally died on interstitial pneumonia caused by cytomegalovirus reactivation. On the basis of this case we hypothesise that putative natural killer (NK) cells-mediated GVL effect after HSC transplantation from KIR-ligand incompatible unrelated donor may be ineffective against EM disease in high-risk patients, while still being potent in medullary site. The mechanisms underlying this phenomenon need futher studies; one may speculate that -for example -NK cell alloreactivity decreases during the later period after transplantation below the level required for effective immunological control of peripheral tissues.

Cytomegalovirus surveillance in paediatric patients under allogeneic stem cell transplantation: a single centre experience S. Bravo, V. Bordon, L. Van Renterghem, G. Laureys, C. Dhooge, Y. Benoit, Ghent University Hospital (Ghent, B) Objective: to study the incidence of early and late CMV infection, and the impact of recipient and donor CMV serostatus on CMV reactivation, in our paediatric population. Methods: We performed a retrospective analysis of 49 consecutive paediatric patients undergoing allogeneic-HSCT between April 1997 and December 2002, with a minimum followup of 12 months. Of these 49 transplants 24 were identical siblings donors, 24 were MUD and mismatch-related donors and 2 were haploidentical transplants. 37 were transplanted for a malignant disease and 12 patients received HSCT for nonmalignant pathologies, median age was 6.8 years (3 months to 15 years). In all patients the presence of CMV DNAemia was determined by a qualitative PCR, performed weekly till day +100, every 14 days till 6 months and monthly until 1 year post-HSCT. CMV infection was considered if 2 consecutive blood samples were positive for CMV DNA. All our patients received irradiated and leucodepleted blood products. Prophylaxis consisted in Acyclovir and standard immunoglobulines, in case of recipient and/or donor CMV seropositivity status CMV-immune globulines (Ivegam-CMV) were used. Patients with 2 consecutive positive samples for CMV DNA or one positive test with suggestive clinical symptoms received gancyclovir. Results: The CMV positive (+) and negative (-) serological status of recipient (R) and donor (D) at transplant was: R -/ D -: 29/49 (59%); R -/ D +: 5/49 (10%); R + / D +: 6/49 (12%); R + / D -: 9/49 (18%) Of our 49 patients 5 (10%), showed significant CMV-DNAemia before day+100 (between day +12 and day +72). From this 5 patients 2 developed a CMV-disease (1 enteritis and 1 pneumonitis), the others were asymtomatic. One patient had a CMV-primary infection 9 months after transplant. Conclusion: In this study, the recipient CMV seropositive status (R+) is a significant risk factor for development of CMV reactivation: 5/15 (30%) as compared with the seronegative receptor (R-): 1/34 (2,9%) P0.008 The CMV reactivation was also significantly higher in seropositive recipients with seronegative donors 44% (4/9) vs 16%(1/6) seropositive receptors with seropositive donors. P0.008 We observed a low incidence of significant CMV-DNAemia (10%) and CMV-disease after HSCT in our population, compared with other studies. This could be explained by the use of CMVimmune globulines, but a prospective study is needed to confirm our results.

Management of pulmonary aspergillosis in children following allogeneic stem cell transplantation using caspofungin and/or voriconazole P. Sedlacek, P. Keslova, R. Formankova, L. Sramkova, Y. Jabali, P. Timr, J. Stary, University Hospital Motol, University Hospital Ceske Budejovice (Prague, Ceske Budejovice, CZ) Aspergillus infection is associated with unacceptably high mortality and is the primary cause of infectious death following stem cell transplantation (SCT). Therefore until recently patients with history of invasive fungal infection were often not considered as candidates for allogeneic SCT. Here we report four children (7, 8, 17 and 18 years old) with proven or probable pulmonary aspergillosis (PA) diagnosed pretransplant in three patients (4 months, 1 month, 1 week) and in one 6 months after allogeneic SCT. Transplantation from unrelated donor was indicated in three patients (first partial remission of MLL/AF6 positive AML, second complete remission of Ph+ ALL, AML/MDS with 5q-). One patient was transplanted using matched sibling as frontline therapy for severe aplastic anemia (SAA). Diagnosis of PA was based on clinical symptoms and typical CT scans in four, positive culture from bronchoalveolar lavage in two (Aspergillus species, Aspergillus fumigatus). Abelcet was primary used to succesfully treat patient with proven PA diagnosed 4 months before SCT from matched unrelated donor while on chemotherapy. Over transplantation period voriconazole was used (40 days) and despite later manifestation of acute graft versus host disease (GVHD) grade II no reactivation of aspergillosis has occurred. In patient with SAA PA was diagnosed at the time he was treated for grade IV GVHD. Frontline therapy with voriconazole was initiated, for clinical progression combined with caspofungin for 50 days, than again treated with voriconazole only (total 6 months). Over next 6 months with no signs of further reactivation of PA immunosuppression was gradually discontinued and finally voriconazole was stopped. Two patients in long-lasting pretransplant marrow aplasia were succesfully treated for probable PA (concomitant Candida crusei sepsis in one) for 28 days with caspofungin overlaping with voriconazole for one week and continue on voriconazole since throughout period of acute GVHD grade II in both. CT scans showed resolution of PA in all four patients with no reactivation 13, 4, 2 and 2 months following SCT despite all patients being treated for grade II-IV GVHD. To prove fungal infection is not easy. In immunocompromised patients therapy must often be initiated as soon as the diagnosis is suspected. Both caspofungin and voriconazole alone or in combination seem to be tolerable and efficious tools and are promising alternatives to amphotericin B lipid formulations.

Microbiologically documented bacterial and fungal infections following peripheral blood progenitor cell transplantation for haematological malignancies F. Altuntas, O. Yildiz, B. Eser, E. Alp, M. Özkan, I. Sari, E. Ekici, N. Koc, M. Cetin, A. Ünal, Erciyes University (Kayseri, TR) Background: This study was carried out to assess the isolation rate of bacterial and fungal causative agents in the early and late infections in patients who underwent peripheral blood stem cell transplantation (PBSCT). Methods: Infections are a major cause of morbidity and mortality in patients undergoing high-dose therapy and subsequent autologous or allogeneic haemopoietic stem cell transplantation. Conditioning and the pre-engraftment period were defined as the early period; the post-engraftment period until one year was defined as the late period. This study was performed to evaluate early and late infections in 114 patients who underwent PBSCT (84 autologous, 30 allogeneic) in a single institution in 1997 until 2003. All the patients received antibiotic prophylaxis and hematopoietic growth factors during neutropenia. Antimicrobial prophylaxis was mainly ciprofloxacin, acyclovir, fluconazole and TMP/SMX orally. Febrile patients received i.v. imipenem or cefepime plus amikacin or ceftazidime plus amikacin to which vancomycin were added if fever persisted for 2-3 and 5 days, respectively. Amphotericin B was further given on day 7 or 8 of fever. Result: A total of 117 episodes with microbiologically documented infections were seen 90 of 114 patients. Seventynine percent of the patients experienced at least one febrile episode with microbiologically documented infections during their post-transplant course. Of these episodes, 69 (59%) were in the early period and 48 (41%) were in the late period. In the early period, 38.8% of causative organisms were gram-positive, 51.5% were gram-negative and 7.7% were fungi. The most common pathogens were Coagulase-negative Staphylococcus (CoNS) and E. coli in the early period. In the late period, 44.6% of causative organisms were gram-positive , 44.6% were gramnegative and 6.8% were fungi. CoNS and E. coli were also the most commonly isolated agents in this period. Totals of 19 microbiologically documented catheter infections were seen; 11 were in the early period and 8 were in the late period. The most common pathogen was CoNS in catheter related infections. Resistance to methicillin was detected 47.4% of S. aureus and 86.5% of CoNS isolates. Conclusions: The isolation rate was in accordance with previous reports; similar percentages of gram-positive and gram-negative isolates were found in patients with underwent PBSCT in the early and late period. A remarkably low rate of viridans group streptococci and fungi were observed. Besides, Mycobacterium tuberculosis was isolated in one patient and Salmonella typhi in two patients. The spectrum of pathogens detected in these case serves as the basis for recommendations on the choice of empiric antimicrobial treatment regimens. Therefore, studies reporting local microbiological findings as well as their susceptibility profiles are necessary. We suggest that local microbiologic surveillance should be known before empiric antimicrobial therapy is started in each institution. Purpose: Empirical antimicrobial combinations of cefepime plus isepamicin (CI), imipenem plus amikacin (IA), cefepime plus amikacin (CA), meropenem plus isepamicin (MI) in posttransplant neutropenic fever were compared. Materials and methods: Totally 145 patients, who underwent to autologous peripheral blood stem cell transplantation (PBSCT) between January 1998 and June 2003 at our BMT Center, were enrolled in the study. In four separate cohort studies, 20 patients received CI combination (cefepime 2 gr / 8 h, isepamicin 15 mg/kg once daily), 26 patients IA (imipenem 500 mg /6 h, amikacin 15 mg/kg once daily), 31 patients CA (cefepime 2 gr / 8 h, amikacin 15 mg/kg once daily) and 68 patients MA combination (meropenem 1gr / 8 h, amikacin 1 gr once daily). vancomycin 1 gr/12h or teicoplanin 200 mg/12h were added in patients with uncontrolled fever despite therapy. Neutropenic fever was defined as any temperature elevation over 38,3 º C or duration of fever over 38,0 º C for at least one hour. Comparisons were made between CA and CI, IA and MA, CA and MA groups. Results: Between groups there was no statistical significant difference regarding age, gender, number of solid tumors and hematological malignancies, preparative regimens, number of total nucleated cells and CD34+ cells, number of febrile attacks and antimicrobial therapies before autologous PBSCT. Mortality rates due to infections (CA: 6,5%, CI: 5%, IA: 3,8%, MA: 5,9%), rates of serious organ specific infections (CA: 3,2%, CI: 5%, IA: 3,8%, MA: 2,9%) and rates of hemoculture positivity (CA: 32,3%, CI: 30%, IA: 50%, MA: 42,6%) were not significantly different. Rate of teicoplanin, vancomycin, fluconazole and amfotericin B administration were not different too. Moreever leucocyte engraftment duration, hospitalization duration, number of days with fever and days on intravenous antibiotherapy were not different between groups. Conclusion: Empirical antimicrobial combinations of CA, CI, IA and MA after autologous PBSCT do not have any superiority over each other. We describe a case of Plasmodium falciparum infection in a 25year-old male patient with a myelodysplastic syndrome (refractory anemia with excess blasts), who underwent allogeneic peripheral blood stem cell transplantation (PBSCT) in September 2003. Before PBSCT, the patient and his HLAidentical brother had no clinically evidence of malaria infection S318 and blood smears were negative in both the donor and recipient. Conditioning regimen consisted of total body irradiation (9 Gy) and cyclophosphamide 120 mg/kg for 2 days. GvHD prophylaxis consisted of CsA and MTX. Engraftment was well documented on day 17 post-transplantation. Spiking fevers occured on days 19 and 21, associated with a pancytopenia, splenomegaly, and neurologic signs. There was no evidence of GvHD, CMV infection, or thrombotic microangiopathy. Plasmodium falciparum parasites were found on the peripheral blood smear (parasitemia=23%). Quinine 30 mg/kg three times a day for 10 days was given. The fever subsided within 3 days, and pancytopenia vanished in 14 days. Parasitemia cleared in 6 days. The patient left the unit on day 46 with no further complications. In the case we describe, the screening of donors showed that infection was acquired from 1 blood unit given 5 days before transplantation. The blood donor was a native of an endemic area. We suggest that malaria, even in non endemic areas, should be considered as a possible cause of pancytopenia in the posttransplant period.

Background: Presumed or proven pulmonary mould infections are frequent causes of infectious morbidity in patients with acute leukemia. Although no longer considered as contraindications for subsequent allogeneic stem cell transplantation, the management of affected patients during granulocytopenia and immunosuppression remains challenging. Methods: In the absence of an evidence-based approach to secondary antifungal prophylaxis, six adolescents (14 to 18 years) with acute leukemia (AML, 4; biphenotypic leukemia, 1; ALL, 1. Matched related, 3, and mismatched/unrelated donor, 3) and possible (5) or probable (1; published EORTC/MSG criteria) invasive pulmonary mould infections received liposomal amphotericin B (1 to 3 mg/kg/d) from the start of conditioning until engraftment and ability to take PO medication, followed by oral voriconazole (200 mg BID) until day +180. Five patients (pts) had a good partial (>50% reduction in pulmonary infiltrates), and 1 a complete response to standard antifungal agents prior to admission. Results: Engraftment occurred after a median of 16 days (R, 13-21) . In addition to standard immunosuppression with methotrexate and cyclosporin A, 3 pts received a short (<14 days), and 2 prolonged courses (48 and 60 days) of corticosteroids for reversible acute grade I GVHD of the skin (3) or other (2) reasons. The median duration of IV amphotericin B treatment was 31 days (R, 23-38), followed by a median of 158 days (R, 39-180) of PO voriconazole. Liposomal amphotericin B was well-tolerated by all patients. CTC/NCI grade III increases in hepatic transaminases possibly related to voriconazole treatment occurred in 2 pts, leading to its transient or permanent discontinuation in 1 pt each. At a median follow-up of 180 days (R, 141-180), 3 pts have a near complete, and 2 pts a complete resolution of pulmonary infiltrates. All five pts are alive, and 4 are in continuous hematological CR. One pt with recurrent leukemia had to be taken off voriconazole on day +91 due to disseminated tuberculosis; he died on day +135 from refractory leukemia with newly arisen possible pulmonary mould infection. Conclusions: This limited series supports the notion that successful secondary prophylaxis for possible or probable invasive pulmonary mould infections can be achieved during and following allogeneic stem cell transplantation using standard agents with broadspectrum antifungal activity against opportunistic filamentous fungi.

A. Tartarone, R. Matera, G. Romano, G. Iodice, M. Vigliotti, G. Mele, N. Di Renzo, Centro di Riferimento Oncologico di Basilicata (Rionero in Vulture, I) Background: oral mucositis is a severe problem for patients (pts) undergoing high dose chemotherapy (HDC) followed by peripheral blood progenitor cells transplantation. Once mucositis has occurred, treatment consists of measures to palliate symptoms. Multiple agents have been used prophylactically to reduce this toxicity with discordant results. Oral cryotherapy, through vasoconstriction, can decrease the mucosal damage induced by agents with short half-lives, such as melphalan (L-PAM). Aim of this study was to evaluate the effect of local cryotherapy on oral mucositis in pts treated with high-dose (HD) L-PAM. Materials and methods: from December 2001, 23 pts median age: 45 years (range 38-63), 5 with multiple myeloma, 9 with non Hodgkin lymphoma, 7 with breast cancer and 1 with ovarian cancer underwent HDC including L-PAM. Five pts received HD L-PAM (140-160 mg/m2) alone and 17 pts a L-PAM including conditioning regimen. All these pts were asked to suck on ice chips for 30 minutes, starting 5 minutes prior to the L-PAM infusion. These pts received also antimycotic and antibiotical prophylaxis with fluconazole 200 mg/d and ciprofloxacin 500 mgx2/d, respectively. Results: we observed oral mucositis (NCI-CTC) G0 2/22 pts (9%), G1 12/22 pts (55%), G2 3/22 pts (13%), G3 5/22 (23%) pts. Median duration of hospitalization was: 22 days (range 18-30). Conclusions: in our experience, the incidence of severe mucositis was significantly lower respect to historical data; so, the prophylaxis with cryotherapy should be considered in pts receiving high dose L-PAM.

I. Panovska-Stavridis, D. Efremov, L. Cevreska, N. Siljanovski, M. Ivanovski, Z. Stojanovski, A. Pivkova, S. Krstveska-Balkanov, B. Georgievski, Medical Faculty-Skopje (Skopje, MK) Acute hepatitis B virus (HBV) infection is a frequent complication after blood stem cell transplantation (SCT). It may occur as a result of a reactivation in previously seropositive patients or as de novo infection transmitted trough blood products. Until the introduction of Lamivudine, a highly effective nucleoside analogue that inhibits HBV replication, the management of HBV infection in immunocompromised hepatitis B-infected patients has been mainly supportive. Recently, contradictory observations have been reported about the effectivenes and safety of Lamivudine treatment of acute HBV infection following SCT. To further address this issue we evaluated 56 patients who underwent 60 SCT; 17 allogeneic and 43 autologous; from September, 2000 to November 2003, at the Clinic for Hematology, Medical Faculty-Skopje. Serological screening for HBV, before the transplant showed that five patients (4 with autologous and one with allogeneic SCT) are HBsAg asymptomatic carriers and one patient that underwent allogenic SCT has acquired immunity to HBV. After the SCT, one asymptomatic HbsAg carrier and the patient with immunity to HBV developed acute HBV infection. Also, in two more patients, who underwent allogeneic SCT and previously were seronegative for HBV, after transplantation, de novo acute HBV infection occurred. In all of them oral Lamivudine at a dose of 100mg/day was administrated. After the median follow-up period of 5.4 months, in all four patients the clinical signs and symptoms of acute B hepatitis, resolved. Seroconversion was documented in two patients, and two become asymptomatic HBsAg carriers. Antiviral therapy was well tolerated and no serious side effects occurred. Our experience indicates that Lamivudine is a safe and effective drug for treatment of acute HBV infection in immunocompromised patient and should be considered as prophylaxis agent against HBV reactivation in carriers of HBV who undergoes SCT.

Successful treatment of multiple episodes of EBV-LPD after SCT. A case report E. Haastrup, K. Mueller, S. Rosthoej, B. Laub-Pedersen, L. Vindeloev, N. Jakobsen, C. Heilmann, Rigshospitalet, Aalborg Sygehus (Copenhagen, Aalborg, DK) Epstein-Barr virus lymphoproliferative disease (EBV-LPD) occurs after SCT in the setting of impaired T-cell function. EBV-LPD is found in up to 24% of the high-risk patients and is often a fatal complication. We here report a case of EBV-LPD relapsing several times, which was treated successfully with a combination of Rituximab, surgery and chemotherapy. A 9-y-old girl with ALL in CR3 underwent allogeneic BMT with an unmanipulated MUD marrow graft after standard preconditioning with TBI, cyclophosphamide and ATG followed by GvHD prophylaxis with MTX and ciclosporin. The course was complicated with grade III GvHD responding well to treatment with ATG and steroid. Day 125 post transplant the girl had polyneuropathy and respirator treatment was necessary. The condition improved after treatment with plasmapheresis and i.v. immunglobulin. Day 402 post transplant the girl developed fever and abdominal pain. An M-component was detected and EBV PCR was positive in the plasma. A CT scan demonstrated a large abdominal tumour and pericardial effusion. Biopsy from the tumour revealed a biclonal plasmacytoma, which was EBV PCR positive. Although CD 20 could not be detected on the EBV transformed cells, the patient responded well to treatment with Rituximab. Day 460 post transplant two new plasmablastic lymphomas emerged in the subcutis and these were successfully treated with Vincristine and dexamethazon. Day 580 post transplant focal activity was observed in the abdomen by a PET scan. A tumour in the left colon flexure was removed surgically. Due to tumour adherences splenectomy was necessary. The histology revealed plasmacytoma as previously. At Day 791 post transplant a PET-scan showed multiple new foci in the lungs. The patient was again treated with Rituximab and the following PET scan showed complete clearance of the lung foci and no other signs of pathological activity. The patient is currently well 2014 days after SCT with no signs of EBV-LPD or relapse. This case illustrates that it is possible to treat successfully multiple episodes of EBV-LPD. The case is interesting because of the histological manifestation of the EBV-LPD as a plasmacytoma. Furthermore it is remarkable that the tumour could be effectively treated with Rituximab despite the fact that the tumours were all CD 20 negative histologically. Cytomegalovirus (CMV) infection and disease are major complication of allogeneic HSCT. In TCD haplo-HSCT setting, the delayed immune recovery results in prolonged risk of posttransplant CMV reactivation. Pre-emptive therapy based on detection of CMV before disease develops, is effective to reduce the risk of CMV-associated sickness and death. Prolonged ganciclovir treatment is limited by myelotoxicity, particularly after CD34+ selected HSCT. In view of this, we performed a study to evaluate the efficacy of foscarnet as single agent pre-emptive treatment of CMV reactivation. We treated with foscarnet 9 patients with a median age 53 (range 38-59), affected by AML (8 patients), ALL (1 patient). Six patients were recipients of CD34+ selected allogeneic HSCT from haploidentical donor; 3 patients received allogeneic HSCT from mis-matched related or unrelated donor with in vivo T-cell depletion with ATG. All patients were CMV seropositive; one out of 9 donors was CMV seronegative. All patients received foscarnet at standard dose of 90 mg/kg twice daily after single detection of pp65 antigenemia in peripheral blood samples. Median time of pre-emptive treatment from transplant was 56 days (range 35-148), median CD3+ lymphocyte count at treatment was 16/mcl. Patients were monitored for CMV quantitative pp65 antigenemia twice weekly; response to foscavir was defined as negativization of pp65 antigenemia for at least 2 consecutive determinations under treatment. Failure to foscavir was defined as the persistence of pp65 antigenemia after at least 10 days of treatment. We collected a total of 19 foscavir treatments in 9 patients; response was detected in 15/19 episodes (78%). Before starting foscavir, the median pp65 CMV antigenemia level was 6 nuclei /105 MNC (range 1-387). A shift to ganciclovir treatment was performed in 3 patients who failed to respond to single-agent with foscarnet and in 1 patient with severe cerebellar neurologic toxicity. Duration time of pre-emptive treatment was 15 days (range 8-44). Overall, mild urinary tract complication with/without haematuria was detected in 5 patients; mild renal failure requiring dose adjustment was detected in 10/19 patients. No significant myelotoxicity from foscavir was registered. Our data encourage the use of foscarnet in larger studies to prove the efficacy of this anti-viral agent as single-agent preemptive treatment of CMV reactivation in T-cell depleted allogeneic HSCT recipients.

Early infectious complications after high-dose chemotherapy and stem cell transplantation for haematological malignancies Z. Stojanoski, A. Pivkova, L. Cevreska, N. Siljanoski, O. Karanfilski, I. Panovska, M. Ivanovski, B. Georgievski, Clinical Center (Skopje, MK) From September 2000 to October 2003, 56 patients (30 with AML, 2 ALL, 4 CML, 8 HD, 8 NHL, 7 MM and 1 SAA), underwent 60 cycles of high-dose chemotherapy and stem cell transplantation. 43 patients were threated with autologous and 17 with allogeneic stem cell transplantation from HLA identical siblings. We retrospectively analyzed the infective complications that occurred during an aplastic phase after stem cell transplantation. All patient were treated in sterile room, conditioned with HEPA filters. Median duration of neutropenia ( granulocyte count < 0,5 x 109/L) was 11 days. Growth factor was administered to all patients until neutrophil engraftment. All patients received oral antimicrobial prophylaxis consisting of ciprofloxacin, trimetoprim-sulfamethoxazole, acyclovir and fluconazole or fluconazole plus itraconazole.The overall incidence of fever requiring antimicrobial treatment was 50%. 10% of patients have fever of unknown origin (FUO), whereas primary bacteriemia occurred in 33,3%, pneumonia in 10%, severe skin infection in 3,33%, neutropenic enterocolitis in 10%, and acute viral B-hepatitis in 1 patient. Infecton was fatal in one (1,66%) patient, due to septic shock. The most frequently isolated were Gram-pozitive cocci from central venous line and sputum. Non-Albicans Candida were predominantly isolated fungi (Non-Albicans Candida vs Candida Albicans = 60% vs 40%).

First-line therapy consisting third generation cephalosporine plus amycacin was succesful in 66% of patients. Infection-related mortality in our as other studies is very low. Confirming other study reports we detected no significant differences between patients threated with autologous or allogeneic stem cell transplantation with respect to infectious complications. Despite the advent of novel antifungal agents, response rates to single light therapy of patients (pts) with proven fungal infections are between 40-60%, and may be lower for allo SCT pts. In-vitro studies show the combination of amphotericin B lipid complex (ABLC) and caspofungin (C) is synergistic. Human studies are needed to confirm the safety and efficacy of the combination. Objectives: To assess tolerability and safety of concomitant ABLC and C in empiric antifungal therapy of febrile neutropenic (FN) pts. Methods: Primary endpoint: tolerability and safety of combined therapy (Rx). Secondary endpoint: apyrexia during Rx. Inclusion criteria: pyrexic >72 hours (h) prior to Rx, refractory to antibiotics, neutropenic (neutrophils <0.5 x 10(9)/L). Pts received C 70mg (Day 1) then 50mg/day and ABLC 5mg/kg/day. Results: Of 26 enrolled, 15 pts (26-70 years) are evaluable to date; 2 withdrew consent, 3 discontinued Rx, 1 died from septic shock. Pts were FN due to treatment for haematological malignancies. Pts received 8-29 days of Rx. Fever resolved in all pts at least transiently, 7 were long-term apyrexic. Only 1 patient had a serious adverse event (SAE) pneumonia/respiratory failure unrelated to Rx. Renal function remained normal except for 3 pts with renal insufficiency. Only these 3 pts had dose reduced. Non-serious AEs included 6 cases of mild/moderate renal insufficiency, 3 were Rx related (2 with relevant renal history, 1 overdosed). Other AEs were nausea/vomiting (7), diarrhoea (6), hypokalaemia (6), chills (5), headache (3). Conclusions: Combination antifungal therapy of ABLC and C was effective in resolving fever and well tolerated over 8-29 days of therapy and up to 39 days of follow up. This study lays the basis for further investigation of the synergistic effects of this combination therapy. Keywords: Amphotericin, caspofungin, combination

Methods: A prospective survey was carried out in Turkey from October 1997 till January 2003 . The objective was to evaluate safety and efficacy information on the use of AmBisome in patients with fungal infections where conventional amphotericin B cannot be used. Fifty six sites actively participated and 550 case record forms were collected. Information on demographics, underlying disease, indications for use of AmBisome together with the dose, duration of therapy, evaluation of safety and treatment outcome have been collected. Results: Forty-one percent of the study population were female. The average age was 14.6 yrs (0.02 -78.5). This included 260 adults, 242 children (<15years) and 43 neonates (< 2 months) with a median weight of 43,0 kg, 21.5 kg and 2. kg respectively. In 78.2% of the patients haematological malignancy was the underlying disease. BMT was described in 33 patients (6.4%). Two hundred and seventy seven patients (50.4%) a fungal infection had been diagnosed. At baseline 210 patients (43.6%) had FUO, pulmonary symptoms in 170 patients ( 35.3%), haematological symptoms in 123 patients ( 25.5%), hepatic disease in 21 patients (4.4%) and CNS symptoms in 10 (2.1%). 52% of the patients were pre-treated with antifungal therapy: 147 patients received CAB, 162 patients fluconazole. The median dose of AmBisome at start was 1 mg/kg/day, for an median daily dose of 1.16 mg/kg for a median duration of 14 days ( 2 to 185 days). The median highest dose was 1.25 mg/kg/day and given for 13 days. The median of the highest dose in neonates was 2 mg/kg/day and given for a median of 16 days (3-28). Side effects were described in 100 patients (18.2%) The most frequent adverse event was hypokalaemia in 49 patients ( 8.9%). In 33 cases this was considered mild (18 patients) or moderate (25 patients). Fever was reported in 26 patients (4.7%). For 540 patients information on the outcome of therapy was available: 56.48% were cured, and clinical improvement was reported for 20.9% of patients (CR + PR= 77.41%). Ninety-eight Patients died, in 86 of these patients the underlying disease was the cause of death. Treatment failure was described for 31 patients (22 fungal deaths, 4%). Conclusion: AmBisome is considered as an effective and safe treatment with few side effects, as described in the literature.

Between March 1998 and August 2003 -143 pts underwent 154 HDT procedures supported by autologous stem cell transplantation (11 double HDT+auto-SCT). These data were analyzed to compare incidence, severity, type of early infectious complications and possible correlations of infections with short term recovery after autologous stem cell transplantation in two groups of pts according to diagnosis of acute leukemia (AML,ALL) and lymphoma (NHL,HD,MM). Results: Median duration of neutropenia was similar in both pts groups. In acute leukemia group 33 pts (66%) developed fever, with fever of unknown origin (FUO) accounting for 27% of febrile episodes. Microbiologically documented infection (MDI) occurred in 19 of 36 pts diagnosed with any infection. Results of positive blood cultures revealed 18 bacteriemias, in 9 pts associated with CVC infection. Among the 8 clinically documented infections (CDI) there were 5 pulmonary infiltrates, 2 soft tissue and skin infection and 1 enterocolitis. In lymphoma group -83 pts (80%) developed fever with FUO in 33 pts (40%). In 44 cases (53%) of infection microbial evaluation revealed pathogen responsible for infection (MDI). Among the MDI, positive blood cultures were found in 30 cases. Bacteriemia was connected with CVCinfection in 12 pts. CDI were diagnosed in 6 pts. Life threatening, severe infection complications were observed in 1 leukemia patient and in 8 lymphoma patients. The incidence of fever, FUO and MDI were similar in both groups of pts. Median duration of intravenous antibiotic use was longer in acute leukemia pts than in lymphoma pts (21 vs 16 days, p<0,001). In acute leukemia occurrence of FUO did not affect duration of short term engraftment. Correlation between duration of neutropenia and MDI was found (p=0,03). In the group of pts with lymphoma occurrence of FUO was connected with delayed erythrocyte recovery after transplantation (p=0,03). Delayed erythrocyte and platelet engraftment were observed in pts diagnosed with MDI (p=0,02 and p=0,046, respectively). Any type of CDI did not affect engraftment kinetics. Occurrence of clinically documented infections (CDI) had no impact to the rate engraftment in both groups of pts. Conclusions: Patients with different hematological malignancies have similar rates of early infectious complications after HDT+autoSCT. The correlations of any particular type of infection with engraftment kinetics are different in both groups of patients. We diagnosed early onset of CMV infection (before day +100) in 14/17 patients and late onset (after day +100) in 3/17 patients. Conclusions: 1.The risk factors of CMV infection in analised group of children are: type of transplant; presence of GVHD and intensity of its prophylaxis and treatment, recipient seropositivity before transplantation. 2. In most cases reactivation of CMV infection was diagnosed. 3. CMV infection can also occur in late posttransplantation period (> 100 days). 4. CMV reactivation can occur in patients after autologous HSCT.

Is control of cryptosporidiosis following stem cell transplantation due to specific CD4 T-cells?

Objectives: To analyse the development of Cryptosporidium parvum (CP) specific T-cell responses in patients with symptomatic cryptosporidiosis undergoing stem cell transplantation (SCT). Methods: T-cell responses were studied by analysing both CPspecific cytokine production and lymphocyte proliferation. CP antigens used were the recombinant proteins Cp17 and P23. Four children with diarrhoea due to CP were studied sequentially before and after SCT. T-cell responses were analysed in conjunction with clinical symptoms and with detection of CP in stool samples by light microscopy, polymerase chain reaction and antigen detection by enzyme linked immunoassay. Results: CP-specific CD4 T-cell responses developed in all four patients after SCT. This development was associated with clearance of CP-DNA or -antigen from stools and recovery from symptomatic disease. Conclusion: Recovery from cryptosporidiosis following SCT is associated with the detection of CP-specific CD4 T-cell responses. The recombinant antigens Cp17 and P23 are immunodominant antigens and according to our limited data are useful tools in monitoring CP-specific T-cell responses in immunocompromised hosts.

A.N. Békássy, E. Holst, S. Lenhoff, H. Norrgren, C. Svensson, University Hospital (Lund, S) We will draw attention to Actinomyces spp. as causative agents for invasive disease seen in two children with atypical clinical presentation. In both children there was an abrupt onset of symptoms and no evidence of infection elsewhere. Actinomycotic diseases are endogenous infections arising from the mucous membranes, primarily involving the cervicofacial regions, the chest, abdomen, and pelvis but extremely rarely causing central nervous system (CNS) infection. We isolated Actinomyces spp.: A. israelii and A. odontolyticus, respectively, from the paranasal sinuses following hematopoietic stem cell transplantation (HSCT) in two children, leading in one of them to CNS-involvement most probably due to haematogenous dissemination. Patient FD is a 12 yrs-old-boy with acute Ph' ALL grafted with sib-PBSC after RIC in CR1. One year later, while receiving high dose corticosteroids for GvHD, he developed bilateral chronic pyogeneic sinusitis. Cultures revealed A. israelii and surgical drainage was necessitated on 4 occasions (endoscopic antrostomy, ethmoidectomy and sphenoidectomy). This infection could be cured with iv Benzyl-Penicillin after 20 months' treatment, but pulmonary complications occurred caused by Candida rugosa and Aspergillus fumigatus. The boy is a/w. Patient MO is a 10 yrs-old-boy with Dyskeratosis Congenita, a syndrome of late onset congenital bone marrow failure. Bilateral endoscopic maxillar antrostomy was performed in order to drain the obstructed sinuses 4 weeks after grafting while on CsA immunosuppression following RIC and allogeneic MUD HSCT. He developed meningoencephalitis two weeks later on and A. odontolyticus was identified as causative agent both from the sinuses and cerebrospinal fluid. An appropriate, but far too short antimicrobial coverage most probably facilitated a hematogenous spread of the infection to the CNS. Immunosuppression is probably playing a major role in the etiology of these infections. The proper diagnosis will be overlooked unless pathological specimens are routinely examined for the organism. Actinomyces seems an emerging problem in HSCT patients and further studies would be appropriate to evaluate its incidence as well as possible mechanisms of invasive infection. 

Cytomegalovirus infection in allogenic stem cell recipientsa retrospective analysis of 64 patients R. Serventi Seiwerth, V Bogdanic, B. Maksimovic, M. Mikulic, M. Mrsic, D. Nemet, D. Nemet, D. Sertic, N. Beader, B. Labar, University Hospital C (Zagreb, HR) Allogenic stem cell recipients are at great risk to develop CMV infection and CMV related complications resulting in high mortality rate when CMV infection appears. Therefore efforts are being directed towards prevention, early diagnosis and treatment of CMV after allogenic stem cell transplant. We have made a retrospective analysis of 64 patients who underwent allogenic stem cell transplantation in our transplant center from January 2000 to October 2003. Median age of our patients was 23,8 years (4 -51 years) 38 males and 26 females. 41/64 patients were transplanted for acute leukemia ( 64,05%), 6/64 (9,37 %) for CML, 7/64 (10,93%) for severe aplastic anemia , and 10/64 for other malignant hemoblastosis. 52 pts( 81,25 %) received transplant from a sibling , and 12 patients received SCT from HLA identical unrelated donor. Before the transplantation CMV serology was performed and 59/64 pts (92,18 %) were CMV IgG positive, and 1 patient was anti CMV anti IgM positive. Only 7/64 donors (10,93 %) were anti CMV IgG negative. CMV pp 65 antigenemia in the leukocytes in peripheral blood was performed once weekly after the SCT. 31/64 ( 48,43 %) of our patients developed CMV pp 65 antigenemia -22 sibling transplant recipients (42,30 %) and 9/12 unrelated transplant recipients (75 %). In 22 out of 31 CMV pp 65 positive patients acute GvHD gr II -IV was diagnosed at the same time. CMV pp 65 antigenemia was observed 3 to 21 weeks posttransplantly with the highest incidence in the weeks 4 and 5 after transplantation. Patients were treated with gancyclovir 5 mg / kg body weight bid until CMV pp65 antigenemia dissapeared.8 of our patients received CMV prophylaxis, 31 preemptive therapy and 6 were treated for CMV infection with gancyclovir and high dose immunoglobulins. 20/31 pts become CMV pp 65 Ag negative, while 11/ 31 remained CMV pp65 Ag positive but only 6 patients developed CMV infection : 4 pts with CMV pneumonia and 1 pt with CMV enteritis died, and 1 pts with CMV cystitis recovered. Conclusion : CMV infection is a serious complication following allogenic SCT. Permanent monitoring for CMV antigenemia is mandatory, and prophylaxis and preemptive therapy for CMV reduces the incidence of CMV infection and mortality.

L. Fouillard, L. Serfaty, F. Isnard, J.P. Laporte, P. Coppo, D. Jeaulmes, N.C. Gorin, J. Gozlan, Hopital Saint Antoine (Paris, F) There is a risk of fatal hepatitis B virus (HBV) liver disease among patients who are persistently HBs Ag positive after haematopoietic stem cell transplantation (HSCT). However the availability of HBV suppressive agents can modify the natural history of HBV infection during the profound immunosuppression and immune recovery following HSCT. We report a 21 year old asian woman patient with refractory acute myeloid leukemia (AML) who relapsed 7 months after an autologous transplantation and 12 grays total body irradiation. The patient received an allogeneic unrelated mismatch transplantation after a reduced intensity conditioning (RIC) regimen as salvage therapy. RIC regimen was Busulfan 4mg/kg, fludarabine 120mg/m2 and Anti Thymocyte Globulin (5 mg/kg). Graft versus host disease (GVHD) prophylaxis combined Ciclosporine A and Mycophenolate Mofetil. A grade II GVHD (skin and digestive) occured on day 45 post HSCT and was resolutive under anti R/IL2. Chimerism was 100% donor. 18 months post HSCT the patient is currently alive and well in complete haematological and molecular remission. At time of AML diagnosis, the patient was diagnosed a B chronic hepatitis confirmed by the presence of HBs Ag, anti HBc and anti HBe antibodies. Anti HBs antibody and HBe Ag were negative. The level of HBV DNA was 362.000 copies/ml. The serological status (Hbe Ag negative) suggested a pre C mutation of HBV. B chronic hepatitis was controlled under the nucleoside analogue Lamivudin (100 mg/day). Before RIC regimen and allogeneic SCT the level of HBV-DNA was 119.415 copies/ml. On day 71 post SCT a reactivation of B chronic hepatitis was assessed by the level of HBV-DNA which dramatically increased up to 200x106 copies/ml with elevated aminotransferase. The liver biopsy showed an active hepatitis (METAVIR score: A1F0). Because of this escape from lamivudin, the nucleotide analogue adefovir (10mg/day) was introduced. On day 97 post SCT the level of HBV-DNA dropped to 3.3x106 copies/ml. The last control on day 419 post NST showed a level of HBV-DNA of 4700 copies/ml under adefovir. No side effect of adefovir was observed. This observation indicates that B chronic hepatitis with high levels of HBV-DNA is not an absolute contraindication for a profound immunossuppession therapy such a RIC regimen providing a control of the level of HBV DNA under nucleotide analogue. Background: Cardiac complications are limiting factors regarding the success of high-dose chemotherapy supported by blood stem cell transplantation. Aim: The assessment of the presence of late cardiac impairment in patients after allogeneic peripheral blood stem cell transplantation for hematological malignancy. Patients and methods: 37 patients (20female/17male) aged 48+10 years (median 47) surviving in long-term remission of the disease 45,5+11,4 months (median 43) after transplantation were enrolled in the study. We measured ejection fraction of the left ventricle (EF), diastolic function and index of global left ventricular performance (TEI index) by means of echocardiography, pVO2 analysis during spiroergometry. We performed short-term spectral analysis of heart rate variability (HRV) during tilt and supine position and calculated LF (low frequency), HF (high frequency), LF/HF ratio and TP (total power) parameters. The dispersion of QTc interval was assessed from 12-leads rest ECG record. Results: The presence of cyclosporine-induced hypertension was diagnosed in 30% of patients. Two patients (5,4%) had asymptomatic decline of EF below 50%. Diastolic dysfunction of the left ventricle was diagnosed in 32% and pathologically increased TEI index (>0,5) in 41% of patients. Decreased exercise tolerance and pVO2 (< 80% of reference value) at spiroergometry were found in 21% of patients. Seventeen patients (46%) revealed significant prolongation of QTc dispersion (> 44 ms) on ECG record. The most frequent finding was the presence of impaired sympathetic-vagal tonus. The impaired vagal and inappropriate sympathetic tonuses were found at HRV examination in 32% of patients during tilt and in 54% of patients during supine position. Conclusions: Our results suggest, that high-dose chemotherapy supported by allogeneic peripheral blood stem cell transplantation gives rise to adverse cardiac effects. However, the described disturbances are subtle in most cases without clinical relevance. To exclude progressive cardiotoxicity, longterm follow-up is required.

Neurologic complications after haemopoietic stem cell transplantation. Single-centre experience

Background. The frequency and type of neurologic complications (NC) following hemopoietic stem cell transplantation (HSCT) are not well assessed. We have analysed the NC in 125 consecutive patients who underwent HSCT in a period of 3.5 years in a single HSCT Unit. Patients and results. Between February 2000 and November 2003, 125 consecutive patients with different hematologic malignancies were submitted to HSCT (101 autologous-AUTO-, 24 allogeneic-ALLO-). Twelve (9.6%) developed NC. BEAM (5) was the conditioning regimen most used in these patients, followed by Cy/TBI (2), BuMel (1), FludaBu (1), FludaMel (1), Mel200 (1) and Cy+VP16+carboplatin (1). All of ALLO-HSCT patients (4) received MTX+CsA as prophylaxis of GVHD. The median time of the appearance of NC was day 13 (range day -4 to 2,5 years). Seizures were the most frequent neurological symptom (7 cases, 58%), followed by confusion (2, 20%), cerebellum syndrome (1, 8%), hallucinations (1, 8%) and headache (1, 8%). Cerebral MR imaging was abnormal in 3 cases: diffuse encephalopathy in cerebellar area (due to intrathecal metotrexate prior and during AUTO-HSCT), subdural hematoma (due to thrombocytopenia) and thrombosis of cortical sinus (due to CsA). Seizures were related with imipenem in 3 cases, with busulfan in 2 and with cefepime (in the setting of renal failure) in one. One out of the 2 episodes of confusion was due to acyclovir and the remaining (2.5 years after ALLO-HSCT) had an unknown cause. All of drug-related NC had a good evolution after removal of the drug (imipenem, acyclovir, CsA). Only the patient with subdural hematoma needed surgery and the case with thrombosis received treatment with heparin without further complicactions. Its of note that up to now any NC has been caused by infections involving CNS. Conclusions. In our series NC (specially seizures) were frequent and drug toxicity was the most relevant cause. Our results contrast with past studies in which infections and hemorrhage were the most frequent causes of NC after HSCT. Supported by Grant P-EF-03 from Fundación Internacional José Carreras para la Lucha Contra la Leucemia.

Quality of life assessment of haematopoietic stem cell transplantation patients in Istanbul: a pilot study M. Çetiner, S. Kalaça, S. Ratip, Ç. Yazgan, C. Adigüzel, S. Kalayoglu-Besisik, D. Argon, I. Kaygusuz, G. Aktuglu, M. Bayik, Marmara University, Acibadem Hospital, Istanbul University, Sisli Etfal Hospital (Istanbul, TR) The number of patients with haematopoietic stem cell transplantation (HSCT) are increasing and a significant number of patients survival has been prolonged. These issues make it imperative to assess the quality of life of the HSCT patients. Twenty eight allogeneic and autologous stem cell transplants from three major university hospitals in Istanbul, Turkey, were recruited into this pilot study. Center on Outcomes, Research, and Education (CORE) functional assessment of chronic ilness therapy -bone marrow transplantation (FACT-BMT) Version-4 (Turkish FACT-BMT) assessment questionnaire was employed for quality of life assessment. Mean age of the patients was 34.2 ± 11.8 (17-67) years, and mean time that elapsed from the time of transplant to the quality of health assessment was 4.3 ± 3.5 (1-14) years. The questionnaire analysed the main psychosocial topics including physical well being, social/family well being, functional well being, emotional well being and other main psychosocial topics. Cronbach alpha results were 0.73, 0.75, 0.78, 0.62, 0.41 respectively. The mean values of physical well being, social/family well being, functional well being and emotional well being items were 0.93±0.6, 2.70±0.8, 2.65 ± 0.74, 1.69±0.71 respectively. There was a positive correlation between the the good performance scores and affirmative answers to the physical well being questions (p=0.002). "Physical well being" items were positively correlated with "emotional well being" items (r=0.48, p=0.017). The results showed that patients younger than 35 years had lower mean values for functional well being and satisfaction with life items compare to older age group (p=0.02). Fear of inability to having children (p=0.04) and loss of concentration (p=0.04) were more prominent in patients younger than 35 years. Patients with a better performance status had better apetite (p=0.01) and were more satisfied with their physical appearance (p=0.02). The mean score of regret having a tranplant was decreasing by time (r=-0.42, p= 0.02) while scores of having better apetite (r=0.41, p=0.03) and being more independent in terms of mobility (r=0.43, p=0.02) were increasing. In conclusion, "physical well being" is the most important parameter affecting the emotional well being of the transplant patients, functional well being got better with time, a good performance status and length of post transplant period had a positive effect on the psychosocial adaptability of the patients.

New approaches for reduction of oral mucositis in patients undergoing high-dose chemotherapy with haematopoietic stem cell support

Oral mucositis (OM) is a dose-limiting toxicity in cancer patients (pts) undergoing chemotherapy (CT). It can limit oral intake, increase the risk of local and systemic infections and decrease the patient's quality of life and compliance to the treatment. Estimate CT-related mucositis incidence range from 40% for standard CT to 100% among "high risk" protocols (i.e. high-dose CT programs). Currently the clinical approach to OM in cancer pts is not well established and the results of many studies based on both prophylaxis and therapy are heterogeneous (Bone Marrow Transplant 2003;31:1-10). The range of local approaches includes oral cryotherapy, topical antimicrobials (Chlorhexidine, Iseganan) and laser treatment. Multiple studies have been performed to asses the effects of these agents on mucosal injury in cancer pts receiving high-dose chemotherapy (HDCT) resulting in a trend toward improved oral candidiasis without real therapeutic advantage in reducing mucositis. The recently introduced association of Benzydamine and jaluronate shows promising activity in both prevention and treatment of severe mucositis. The advent of new systemic agents, including keratinocyte growth factor, transforming growth factor beta, interleukin-11 and the cytoprotectant Amifostine, provides interesting perspectives in this field. In 2001, a multicenter phase II study was designed to evaluate the effectiveness of Amifostine in the reduction of oral mucositis induced by HDCT. To date, 100 patients with both solid tumors and hematological malignancies undergoing HDCT with autologous pheripheral blood stem cell transplantation, were randomized to receive either placebo or Amifostine 740 mg/mq by intravenous bolus within 30 minutes prior to Melphalan-based conditioning regimen. Preliminary data on feasibility and toxicity of the drug indicate that Amifostine treatment was well tolerated at this dose level, without important side effects. Only in 10 pts (10%) was documented grade 1 hypotension and grade 1-2 nausea during Amifostine, but the interruption of infusion was never required. The availability of new agents for the prevention of severe CTrelated mucositis promises to substantially reduce treatmentrelated morbidity and improve quality of life in HDCT setting. Further trials, including both local and systemic approaches are warranted.

This study was tried to observe the STRs patterns in blood, buccal mucosa, and hair of the persons undergone allogeneic bone marrow transplantation (BMT) and to speculate the most appropriate sample for proper forensic identification and paternity testing in the persons undergone allogeneic BMT. Four socially active persons undergone HLA identical sibling allogeneic BMT (duration after BMT: 18 to 60 months) and their families(total 16 persons) has been enrolled in this study. Blood, buccal swab, and hair samples have been used to calculate allele frequencies of the twenty STR loci, D5S818, D13S317, D7S820, D8S1179, D21S11, D18S51, D3S1358, vWA, FGA, F13A1, ACTBP2, FES/FPS, D1S1656, GABA, D10S2325, D12S391, D18S535, THO1, TPOX, CSF1PO with Amp/F/STR Profiler Plus PCR amplification kit. All STRs of blood in the persons undergone allogeneic BMT has been changed to the those in the donors. STRs of oral mucosa in the persons undergone allogeneic BMT has shown the diverse patterns. STRs of oral mucosa in the persons undergone allogeneic BMT showed the tendency of which STRs of donors which were initially predominant had been decreased with the progress of time after allogeneic BMT. STRs of hair in the persons undergone allogeneic BMT has showed mostly those of themselves. Consequently, in the persons undergone allogeneic BMT, STRs examination of hair seems to be more useful for forensic identification and paternity testing than those of blood or buccal mucosa.

Autologous transplant in over 60 patients with lymphoproliferative disorders: a single-centre experience in 63 patients D. Capelli, A. Olivieri, A. Scortechini, M. Montanari, M. Lucesole, M. Brunori, E. Troiani, G. Gini, M. Masia, R. Pasquini, F. Balducci, P. Leoni, Clinica di Ematologia (Torrette, I) Between May 1994 and December 2002, we performed 76 autotransplants in 63 consecutive patients, aged over 60 years (range: 60-78, median: 64), 20 of whom aged over 65 years, affected by lymphoproliferative disorders. Twentyseven were females and 36 males; 33 patients were affected by MM and 13 received a double autotransplant, 24 had HG-NHL, 5 had LG-NHL, 1 had HD. The pretransplant disease status was CR in 7 (21.2%), PR in 22 (66.7%), and progression in 4 (12.1%) MM patients; we also transplanted 11 CR (36.7%), 15 PR (50%), and 4 progressive (13.3%) lymphomas. The conditioning regimen included Melphalan alone in 24 MM patients (35 procedures) or associated with other drugs in 8 MM (10 procedures) and 11 Lymphoma patients; BEAM in 18 Lymphomas and TBI in 1 MM and 1 Lymphoma patients. A median of 5.8 x10e6/Kg CD34+ cells (range 1.2-28.6) was reinfused. Engraftment kinetics was similar to that observed in younger patients with a median of 11 (range: 4-25) days for neutrophils >500/microl, 13 (range: 10-83) days for platelets >20,000/microl and 19 (range: 11-67) days for platelets >50,000/microl. A median of 2 RBC (range: 0-11) and 2 platelet (range 0-9) units were transfused. Extrahematological toxicity (WHO), mainly consisted in infections and mucositis: we observed 13 grade III-IV infectious episodes (17.1%) and 15 cases of severe mucositis (19.7%) which required TPN infusion only in 4 patients. Median duration of hospitalisation was 21 days (range: 4-61). Toxicity, hospital stay and supportive care did not differ from those of a cohort of younger patients. At present, 38 patients are alive (14 in CR, 13 in PR, 5 in PD and 6 in relapse) with a median follow-up of 27 months (1-107); 19 patients died from PD, 1 died in CR for a CMV pneumonitis post-allogeneic transplant and 4 died from transplant-related toxicity (5%), 3 of whom aged over 65 years (TRM=15%). Median OS and PFS were respectively 69 and 34 months in MM and 64 and 35 months in Lymphoma patients. Quality of life, evaluated using a QLQ-C30 questionnaire in 16 patients at day 0, +15, +30, +60, +90, was substantially comparable to that of a similar group of younger patients. In our experience PBPC transplantation is feasible in patients aged 60 years and the toxicity of this procedure is acceptable, even though patients over 65 years still show a not neglegible TRM; therefore patients with hematological malignancies potentially curable with high-dose therapy should also be candidates for HDT.

Preliminary results of psycho-diagnostic evaluation in adolescents who received SCT in paediatric age M. Canepa, B. Cappelli, E. Lanino, P. Levrero, G. Dini, M. Faraci, Institute G. Gaslini (Genoa, I) Our aim was to evaluate the psycho-diagnostic areas in a heterogeneous group of adolescents receiving stem cell transplantation (SCT) between 1987 and 2000 at the Gaslini Hosp.We present the preliminary results of testing administrated to recipients during a scheduled follow-up.Cognitive Behavioural Assessment (CBA-FormaG) was used.Normal values were obtained by testing a large group of healthy Italian adolescents by CBA-FormaG.We analysed 9 patients transplanted at a median age of 9 yrs,4 females (F),5 males (M).Median age at CBA-test was 19 yrs,average 8 yrs after SCT.Five patients had received allogeneic SCT, while 4 underwent autologous SCT.Acute (5/5) and chronic (4/5) graft versus host disease(GvHD)occurred in all the allogeneic recipients. Two of 9 patients underwent orthopaedic surgery for avascular necrosis, 5 M:0.94± 1.22SD), psycho-social adaptation (n.v.= F:12.06 ±4.59SD; M:12.93±4.88SD), and wellness (n.v.=F:5.02 ±3SD; M:6.75±3.66SD). Levels of anxiety and of self-disclosure were within normal range in all pts.Psychiatric risk was above normal in one female (value 9).High levels of emotional instability were observed in two females (values 15-16). One female had had unusual experiences (value 2). No sensation seeking was reported. One patient (F) showed hostility levels above normal (value 10). Two females had abnormal psycho-physiological disturbances (values 3 in both). Five pts (3 F; 2 M) had low psycho-social adaptation (value 14 in 1 M and 1 F; value 18 in 2 M; value 20 in 1 F).One female showed low levels of wellness (value 1).This preliminary study shows that most pts who received SCT in paediatric age have a all-round good cognitive behavioural assessment. Females recipients were more frequently subject to psychological disturbances. Low psycho-social adaptation was observed in both genders. This study suggests that CBA-FormaG could be a valid instrument for identifying critical areas during follow-up of SCTpts.

The objective of the study is evaluation and treatment of late effects on thyroid function and glucose metabolism in pediatric long-term survivors of hematopoietic cell transplantation (HCT). Nineteen patients (11 girls and 8 boys) aged 2-20 years (median 14 years) were evaluated prospectively post transplant. The reason for HCT were: AML (n=7), ALL (n=4), NHL (n=2), CML (n=2), JMML (n=1), SCID (n=1), MDS (n=1) and HES (n=1). The patients underwent autologous HCT (n=7), allogeneic MSD-HCT (n=6), MUD-HCT (n=4) and HLA-mismatched related HCT (n=2). In three children two transplants were performed. Conditioning regimen consisted of high-dose chemotherapy, incl. busulfan in all but 2 cases. TBI was not applied, however pretransplant CNS prophylactic radiotherapy was performed in 9 children. Posttransplant period was complicated by GvHD in 10 patients, which required steroid administration. Endocrine function was evaluated from 4 to 84 months (median 18 months) after cessation of steroid therapy. Analysis of TSH, fT3, fT4, aTPO, TRH test, HbA1c and oral glucose tolerance test (OGGT -with the measurement of glycemia and insulinemia) was performed. Hypothyroidism was found in 4 pts, 2 pts had abnormal aTPO and thyroid hormone substitution was applied. Patients with hypothyroidism underwent CNS radiotherapy pretransplant and developed acute GvHD grade II-III. Time from steroid therapy cessation did not influence the onset of abnormal thyroid function Impaired OGGT was found in 7 children, whereas hyperinsulinism was detected in 11 pts. Mean glycemia (OGGT) was 26.88± 4,83 mmol/l, insulinemia 131.49± 81,35 mIU/ml. Abnormal insulin-glycemic index I>0,3 (mean 0.43 ± 0.15) was detected in 8 patients, in 3 children HbA1c was increased (incl. 2 with abnormal insulin-glycemic index). BMI was 17.54± 2.67 in the analysed group and remained within normal range. Hyperinsulinemia in OGTT was accompanied by higher BMI: negative correlation between BMI nad hyperinsulinemia was found (r= -0,18, p<0,05) . No correlation was found between time from steroid therapy cessation and hyperglycemia/hyperinsulinemia in OGTT and conditioning regimen and GvHD occurrence. In conclusion: Children require standardised endocrine follow-up after HCT, because thyroid and glucose metabolism dysfunctions may occur frequently and worsen the quality of life posttransplant. The advent of new therapeutic approaches to multiple myeloma made necessary the introduction of novel methods for detection of minimal residual disease. Among others approaches residual disease can be detected by using flow cytometry. We have examined the co-expression of CD19, CD38, CD45, CD56, and CD138 molecules in cells of bone marrow aspirates in patients with multiple myeloma by multi-color flow cytometry. For the detection and characterization of multiple myeloma cells, combinations of following antibodies were used: anti-CD19 FITC, anti-CD38 FITC, anti-CD56 PE-Cy5, anti-CD45 PE, anti-CD45 PE-Cy5 and anti CD138 PE. The samples were analyzed using EPICS XL (Coulter) flow cytometer, and the analysis was based on at least 50,000 events. Samples from 15 patients were analyzed of whom 2 smoldering myeloma, 2 plasmocytoma and 11 multiple myeloma. The percentage of multiple myeloma cells ranged between 2.5% and 90% in bone marrow aspirates. The expression of CD138, CD38, CD45, CD19 and CD56 molecules was found in 73%, 80%, 93%, 13% and 26% of examined cases, respectively. In our opinion, multiple myeloma cells are best characterized by following combinations of antibodies: CD38+ /CD138+/CD45+. The identification of a malignant clone is the first and the most important step in the characterization of the disease, determination of its prognosis and the detection of residual disease after treatment. Multi-color flow cytometry represents a method which can meet these goals.

Monitoring of minimal residual disease using a quantitative polymerase chain reaction in chronic myeloid leukaemia patients after allogeneic haematopoietic stem cell transplantation

Allogeneic hematopoietic stem cell transplantation (HSCT) is an effective therapy for chronic myeloid leukemia (CML), and allowed to obtained hematologic, cytogenetic and molecular remission in most of the patients (pts). However, disease relapse still remains a frequent cause of treatment failure. Following grafting relapse presumably results from the inability of the conditioning regimen and graft versus leukemia reaction to eliminate leukemic cells. The persistence of these cells after HSCT is known as minimal residual disease (MRD). The BCR/ABL measurement allowed to find residual disease in CML pts. We studied 70 pts (mean age 38, range 17 -52 years) in complete hematological and cytogenetic remission after myeloablative (55) and non-myeloablative (15) HSCT. Myeloablative regimen consisted of busulfan 16 mg/kg and cyclophosphamide in a dose of 120 mg/kg (39 pts) or 200 mg/kg (16 pts). Pts in non-myeloablative group were conditioned with fludarabine 150mg/m², busulfan 8mg/kg (9) or fludarabine 150mg/m², and melphalan 140mg/m² (6). After engraftment, MRD was assessed with a help of RT -PCR using mRNA obtained form peripheral blood leukocytes (Hughes et al., 1991) . Serial dilutions of cDNA was used to determine BCR/ABL expression. Molecular monitoring of MDR was conducted for a mean of 40 (3 -141) months, according to the following scheme: initially 3 months after HSCT and subsequently every 3 months during the first year, in the second year every 4, and thereafter every 6 months. Pts were divided according the results of BCR/ABL status into three following groups: high -risk (HR, 22 pts) PCR (+) with increasing levels of oncogene, intermediaterisk (IR, 17 pts) with fluctuations of PCR positivity / negativity or constant expression of BCR/ABL and low -risk (LR, 31 pts) with PCR negativity or decreasing level of oncogene. In the study group after a mean 42 (3 -71) months of the follow-up 14 pts relapsed (11 pts in HR: 6 cytogenetic, 5 hematologic; 2 pts in IR: 1 cytogenetic, 1 hematologic; and 1 in LR: cytogenetic). Conclusion: qualitative PCR technique allowed to detect MRD many months after allografting, even in pts without clinical relapse. Therefore, to predict disease reoccurrence the quantitative PCR assay should be recommended.

A. Leather, J. Murray, M. Angelica, H. Lee, R. Chopra, J. Cavet, K. Branson, Christie Hospital (Manchester, UK) Introduction: Variable Tandem Repeat Monitoring (VNTR) markers are increasingly used for monitoring the engraftment of donor cells after stem cell transplantation. We undertook this study to evaluate the efficacy of VNTR analysis in predicting clinical relapse. Method: We analysed 126 consecutive transplants undertaken on our adult leukaemia unit, over a period of 42 months; of these 58 were sibling allografts and 68 were matched unrelated donor transplants.

Post transplant VNTR's were performed on peripheral blood or bone marrow samples at 6 weeks, 3, 6, 9, 12, 15 and 18 months and then six monthly for the next 2 years. Long term monitoring continued yearly until the patient was 5 years post transplant. We assessed the correlation between VNTR and remission status via conventional monitoring, eg bone marrow analysis or radiological imaging. Results: VNTR data was collected on 110 of the 126 transplants during the study period. Of the remaining 16 cases, 9 died prior to the first VNTR test at 6 weeks, 4 patients did not have a VNTR taken and 3 patients had non-informative markers and were therefore unable to be analysed by this method. Clinical monitoring showed that 46 from 110 patients had relapsed, at a median time of 6.5 months post transplant, however, in only 15 (33%) of these cases was an abnormal VNTR obtained. The VNTR abnormality was detected first in 4 of these cases prompting further action to be taken. In the 64 patients remaining in remission 8 cases showed one abnormal VNTR reading (range 0 -95% donor) at a median of 10.5 months post transplant, however, subsequent VNTR's reverted to 100%. In no cases did the abnormal VNTR alone directly change the management for the patient. In 56 patients VNTR have remained at 100% donor and the patient remained in remission. Conclusion: In 67% of relapsing cases VNTR remained 100% donor although the patient had demonstrated clinical relapse. Additionally even when an abnormal VNTR correlated with bone marrow analysis indicating mixed chimerism or frank relapse, in only 9% of cases was the VNTR the first indicator of mixed chimerism. Therefore in the majority of cases it failed to provide an early warning of future relapse. Conventional monitoring by bone marrow analysis remains essential.

M. Zagrivnaja, B. Fehse, A. Zander, N. Kröger, University Hospital Eppendorf (Hamburg, D) Achieving molecular remission after treatment of multiple myeloma is associated with long term freedom of disease and possible cure. Because molecular remission seems to be the ultimative goal for achieving cure, we performed donor lymphocyte infusion (DLI) in two patients with persistent positive immunofixation after withdrawl of all immunosuppression Both male patients were treated with an auto-allo tandem approach using fludarabine (150 mg/m²), melphalan (140 mg/m²) and antithymocyte globulin (ATG: 3 x 10-20mg/kg) three months after a cytoreductive autograft (melphalan 200 mg/m²). Both patients achieved a near complete remission with only positive immunofixation after allografting. Allele-specific oligonucleotides (ASO) based upon the clonal rearrangement of immunoglobulin heavy chain genes and ASO -polymerase chain reaction (ASO-PCR) were developed for each patient. The specificity of the ASO-PCR was tested using patient or control DNA (healthy donors). Patient DNA isolated from bone marrow (BM) samples which was obtained at diagnostic and after allografting at the variable time points was used for a further molecular analysis. In the first 51 year old patient (IgG kappa, IIIA) cyclosporin A was discontinuated on day 111 after dose-reduced allografting from his HLA identical sibling and two months later he received 1 x 106CD3+cells/kg from his donor due to still positive immunofixation. Six weeks later he developed a mild GvHD (grade I) of the skin which resolved completely after a short course of prednisone. The immunofixation has become negative and nested PCR with patient specific primers did not show a specific rearrangement ( 15 months+). In the second 39 year old patient with del 13 (IgG lambda, IIIA) cyclosporin A was discontinuated on day 134 after allograft and 3 months later he received 1 x106 CD3+ cells/kg due to persistent positive immunofixation. Immunofixation remains positive and 13 weeks after the first DLI the patient received a second DLI (1 x 106/CD3+cells/kg). Without GvHD the immunfixation become negative and molecular remission was noted 7 weeks after DLI and remains negative for now 4 months. Our results suggest that molecular remission can be induced by DLI after allografting in those patients who have still a small amount of paraprotein after withdrawl of all immunosuppression. To define the currently unknown optimal timing of DLI, quantitative real-time PCR should be used as guide for treatment Chimerism analysis has become a routine method for the documentation of engraftment and also for detection of minimal residual disease. During the last years, molecular methods, mostly based on PCR analysis of highly informative repetitive DNA elements, have become particularly important. Several recent reports have focussed on the use of commercial multiplex STR systems. However, these assays have been optimized for forensic purposes and do not necessarily fulfill all needs for chimerism analysis. As a first step to find an optimized solution, knowledge on the level of informativity of different STR-systems in the context of chimerism analysis would be helpful. Thus, the aim of the current analysis was to compare different STR markers for their informativity in a large group of patients undergoing transplantation from HLA-matched related donors. These markers were part of different commercially available STR-kits (Powerplex 16, AmpFlSTR SGM or Triplex AFS amplification kit) or selected from the literature or different databases according to reported informativity. A total of 27 STR markers were analyzed in 203 HLA-matched related patient/donor pairs (406 individuals). The STR constellations between donor and recipient were grouped from type 1 (= not informative) to type 5 (best suited for chimerism analysis). Results: Analyses of almost 11.000 single STRs were included. According to the ranking criteria, the informativity of the STR systems was found highly variable, ranging from 5% to 49% type 5 STRs. Informativity of an STR was signifcantly correlated with the degree of heterozygosity (r = 0.86; P < .0001), but not with the total number of alleles present. Among the most informative STRs were Penta E, SE33, D2S1338 and D18S51. In addition, in spite of the fact that 27 markers were used, there was still one individual in whom no highly informative marker was found. Conclusions: These data indicate that selection of appropriate STR markers can help to improve diagnostics based on STR analysis, but this will still not allow optimal analysis in all patients. The frequency of early complications (before 100 days) of HSCT was measured on 97 patients with hemoblastosis, underwent allogeneic HSCT (n=33, median age 24±2,0) and autologous HSCT (n=64, median age 25,5±1,6). Conditioning regimens in alloHSCT were myeloablative in 26 patients (76,4%) and consist of Bu+Cy, and non-myeloablative in 8 patients (23,6%), consist of Flu+Ms+ALG or Flu+Bu+Alk. In group of MA HSCT 11 patients had CML, 8 -ALL, 5 -AML, 2 -MDS. In group of NMA HSCT 5 patients had CML, 2 -ALL, 1 -AML. The frequency of infectious complications in NMA HSCT group was less than in MA HSCT group: 57,1% and 88,4% respectively. The median duration of febrile neutropenia was significantly lower in NMA HSCT group: 7±1,5 è 12±1,7 days (p<0,05). Hemorragic complications were observed in 9 patients with MA HSCT (34,6%) and in 3 with NMA HSCT (42.8%) (p>0,05). Acute GVHD grade 2-4 was observed in 5 patients with MA HSCT (19,2%) and was not found in group of NMA HSCT (p>0,05). The toxicity of conditioning regimens for cardiovascular system, respiratory system, digestive and urogenital system (only grade 3 or more toxicities were considered) were in MA HSCT group 38.4% and 28% in NMA HSCT. The engraftment of stem cells was achieved in 100% cases as in MA as NMA HSCT groups and was significant faster in NMA HSCT (18±4,1 and 24±2,5 days respectively, p<0,05). Conditioning regimen in autologous HSCT MA (Bu+Cy) was in 25 cases (39%), in NMA 39 cases(61%) ( LACE, BEAM). In group of MA HSCT 15 patients had AML, 10 -ALL; in group of NMA 13 had AML, 16 -ALL, 10 -MM. The number of infectious complications were smaller in NMA HSCT (87,1% and 92%, respectively, p>0,05). Toxicity complications grade 3-4 were some frequently in MA HSCT, than in NMA HSCT (40% and 30,7% respectively, p>0,05). Hemorragic complications were in 10 cases of auto HSCT (15,6%) and there was no significant differences between them after MA and NMA HSCT (ÌÀ-16%, NÌÀ-15,3%). The recovery of absolute neutrophil count and platelets were significant faster in NMA HSCT (22±2,0 and 31±2,5, p<0,05; 17±1,7 and 29±3,0, p<0,05, respectively). In conclusions, the use of NMA conditioning regimens as well in alloHSCT, as in auto HSCT accompany mote faster engraftment; the frequency of hemorragic complications was significantly lower, while the frequency of infectious and toxicity complications do not had significant differences. Objectives: The main objectives of the study were: 1/investigation the feasibility and efficacy of NST, and 2/evaluation for engraftment and analysis of chimerism. Patients and methods: Sixteen patients, median age 44 (range 18-61) suffered from various rHM (2MDS, 1CML, 1ALL, 3CLL, 3MM, 1NHL, 1HD) and certain aST (2RCC, 2CRC) were enrolled into the study. At the moment of NST the disease status was: 11PD, 3SD, 1PR and 1CR. All patients received fludarabinebased conditioning regimen. Stem cells were obtained from PB of HLA-matched siblings by standard techniques. The median number of CD34+ cells infused on day 0 was 3,27 x 106/kg. Prophylaxis against GvHD included CsA±MMF or CsA+MTX. Chimerism was assessed by the microsatelite VNTR-PCR analysis once per month. Results: Three patients (1RCC, 1ALL, 1MM) died during transplant related period either due to disease progression, acute GvHD or infections. Four additional patients developed acute GvHD -degree 1 to 3. There were no other grade 3 or 4 transplant related complications. Hematological recovery was prompt with neutrophils > 1.0 G/L at median 13 days and platelets > 50.0 G/L at median 17 days. No graft rejection was observed. Either mixed or complete donor chimerism was found in all the cases between day +30 and +210. In eight patients DLI were applied and encouraging influences of disease extent and chimerism status were observed in 6 of them. Chronic GvHD was found in 7 cases (6 limited and 1 extensive). Results at +100 day were as follows: 3 CR, 4 PR, 6 SD. Four patients died over 12 months after the transplantation: 3 due to progression of their diseases (1MDS, 1HD, 1RRC) and 1 because of heart infarct (CLL in PR). Nine patients have been still living: 4CR, 2PR, 2SD, 1PD. Non-relapse mortality at +100 day was 12.5% and at 1-year 18.5%. Median time to progression was 382 days and median overall survival was 415 days. Conclusions: These results indicate that the NST procedure is feasible and allows consistent engraftment with minimal transplant related complications and stable donor chimerism. This approach seems to be promising even for patients with resistant hematological malignancies and certain advanced solid tumors. Non-myeloablative stem cell transplantation can prolong overall survival of patients who have no chance of being cured with any standard therapeutic modality or are ineligible for conventional hematopoietic stem cell transplantation.

Recombinant erythropoietin after reduced-intensity conditioning for allogeneic stem cell transplantation D. Blaise, V. Ivanov, K. Bilger, C. Faucher, M. Mohty, P. Ladaique, D. Sainty, C. Chabannon, N. Vey, D. Maraninchi, Institut Paoli Calmettes (Marseille, F) We reported that erythropoiesis reconstitution after ASCT prepared with RIC was different than after standard myeloablative regimens (Ivanov, EBMT, 2003) . For this reason we investigated whether pts treated with RIC might benefit from early stimulation of erythropoiesis with recombinant erythropoietin (EPO). In a pilot study, EPO was administered to 21 patients treated with ASCT prepared with a RIC (Fluda (30 mg/m²/d x 5), Bus (2mg/kg/dx2) and ATG (Thymoglobuline: 2.5 mg/kg/d 4, 3 or 1 day) without post graft G-CSF. Starting on day 1, pts received epoetine beta (Neorecormion ®, Roche) (10000 IU, IV thrice a week) (N=10) or darbepoetin alpha (Aranesp ®, Amgen) (150 mcg IV once a week) (N=11). Change to Aranesp ® was motivated to allow weekly treatment in pts discharged after graft infusion. Age: 49 (27-61); M/F: 14/7 with myeloid (4), lymphoid (12) or Solid (5) malignancies. 10 and 11 pts had an Hb count prior to CDT respectively < 11 or = 11 g/dl. These pts (EPO+) were compared with a series of 26 pts treated with the same RIC but without EPO post ASCT (EPO-) and matched on age, diagnosis and pre graft Hb count (age: 49(27-60); M/F: 12/14 with myeloid (11), Lymphoid (9) or Solid (6) malignancies. 15 and 11 pts had an Hb count prior to CDT < 11 or = 11 g/dl. No side effect have been so far reported. The number of transfused RBC units were lower in EPO+ group (RBC units: EPO+: 2 (0-4); EPO-: 2 (0-12); p=0.05). 81% of the pts in EPO+ group received no or 1 transfusion as compared to 54% in EPO-group (p=0.05). EPO effect was major in pts with an initial Hb value < 11g/dl (EPO+: 2 (0-4); EPO-: 4 (2-12); p=0.01). The major difference occurred in Hb recovery with a significant difference between the 2 groups at each time point after day 15. Respectively 86% and 90% of the patients EPO+ presented an Hb level <= 11 g/dl on day 30 and 60 as compared with 31% (p=0.0002) and 35% (p=0.0001) of the patients EPO-. We conclude that EPO stimulation after ASCT prepared with RIC is safe, is likely to allow faster Hb recovery and lower transfusion needs and invites prospective comparative investigation including economical and quality of life evaluation. Objective: Conventional allogeneic blood and marrow transplantation with myeloablative conditioning regimen is associated with high morbidity and mortality in old patients with coexisting diseases. So non myeloablative stem cell transplantation (NMSCT) with less toxic and more immunosuppressive conditioning regimen is an alternative for conventional transplantation. Our aim is to evaluate the result of this kind of transplantation. Methods and patients: Twenty patients (M= 14, F=6; median age 43 years, range 8-55 y.) received NMSCT from related donors. Diagnoses: AML= 7, CML=6, Lymphoma=2, MDS=2, ALL=1, Fanconi anemia=2. Conditioning regimen consisted combination of Fludarabine, ATG, Busulfan in respect of disease. GVHD prophylaxis regimen was cyclosporine A ± methotraxate± MMF. DLI were given as available after NMSCT according to evaluation of chimerism. Results: 19/20 patients engrafted. One patient had no engraftment even after DLI. 13/20 were in CR, 4/20 had disease progression, 3/20 relapsed. Full chimerism was observed in 12 patients after transplantation. Six patients had mixed chimersim and DLI were given to 4 pts, but only one of them converted to full chimerism. After a median follow up of 7 months (1-29 months) 11 patients are alive (55%), 10/11 patients in CR (10%); 3/11 have acute and/or chronic GVHD. 9/20 died (45%), 3/9 due to GVHD, second malignancy and infection and 6/9 due to relapse and primary disease. Disease free survival in 14 months was 50 %and Overall Survival was 55%. Conclusion: According to this study NMSCT may be curable for patients who can not afford conventional toxic and potentially lethal myeloablative conditioning regimen. Better assessment of chimerism and dose adjustment of immunosuppressive drugs and DLI in appropriate time may improve the results.

Non-myeloablative stem cell transplantation for the treatment of haematologic malignancies and metastatic solid tumours: a single-centre experience M. Cetin, F. Altuntas, I. Sari, B. Eser, O. Er, O. Canoz, B. Kaplan, M. Ozkan, E. Albayrak, S. Bahar, Y. Ozkul, T. Patiroglu, A. Unal, Erciyes University (Kayseri, TR) Background: The rationale behind non-myeloblative stem cell transplantation (NST) is to induce optimal graft-versus-Tumor effects for elimination of all malignant cells by donor alloreactive immunocompetent cells as an alternative to standard high-dose myeloablative chemoradiotherapy. Patients and methods: In our 21 patients [the median patient age was 35 years (range, 19-52 years)] with relapsing acute leukemia (n = 4); chronic myeloid leukemia (n = 14), non-Hodgkin's lymphoma (resistant Burkitt Lymphoma and Lymhoplasmocyte Lymphoma: n = 2), and metastatic Ewing sarcoma (n =1) suggest that nonmyeloablative conditioning including fludarabine, anti-T-lymphocyte globulin, and low-dose busulfan is extremely well tolerated. Graft-versus-host disease (GVHD) prophylaxis consisted of low-dose cyclosporine (Cs A), some with low-dose methotrexate. Result: Twenty patients showed rapid 3-lineage engraftment. Three patients experienced no acute GVHD at all; grade I-II aGVHD was seen in 14 patients (66%) ( 6 grade I, 8 grade II) and severe grade III/IV GVHD was observed in 4 patients (19 %). Nine patients (45%) developed chronic GVHD, but only 3 patients (18%) experienced extensive chronic GVHD. Patients without significant acute (>grade 1) and/or chronic GVHD were scheduled to be treated with DLI (3 patients). Clinical responses were observed in 18 of 21 patients (86%) who had active disease at transplantation. After a median follow-up of 332 days (range 60-670) 9 patients are alive. One patient (4.7%) [relapsing AML] died of treatment-related complications and 2 patients (9.5%) [rezistant Burkitt Lymphoma and metastatic Ewing sarcoma ] died of progressive disease. Conclusion: Our data suggests that NST may provide an option for cure for malign diseases, while minimizing procedure-related toxicity and mortality.

Autologous stem cell transplantation followed by nonmyeloablative allogeneic procedure for oncohaematologic patients G. Salas, M. Jurado-Chacón, A. Romero, M. Almagro, P. Lopez, A. Moratalla, P. Navarro, J. De Pablos, Hospital University Virgen de las Nieves (Granada, E) Background: A new nonmyeloablative stem cell transplantation (NSCT) appears as a new technique for some oncohematologic patients. Generally a low non relapse mortality without any loss of antitumoral effect make this approach very attractive. However, the incidence of GVHD is still a common complication which causes some deaths. Objectives: to evaluate the toxicity of 11 patients in whom a NSCT was performed , preceded (n=2) or no by autologous SCT (n=9). Methods: from June 01 through October 03, 11 patients (range age 21 to 63) with oncohematologic diseases (1 non-Hodgkin's lymphoma, 4 Hodgkin's disease, 4 Multyple Myeloma, 1 acute myeloblastic leukemia, 1 chronic lymphocitic leukemia) underwent a NSCT. In nine of them were also performed an autologous SCT ( 45 to 120 days before) because of their advanced disease situation. As conditioning for autologous SCT were given Melfalan 200 mg/m2 (4) and BEAM (5) and for NSCT consisted of fludarabine (90 mg/m2) and TBI of 2 Gy, followed by infusion of peripheral blood from sibling donors. Results: Seven patients developed neutropenic fever during the NSCT while only 2 of them had positive CMV antigenemia. At day 84 after NSCT, 90 % and 70% of patients showed a complete myeloid and lymphoid chimerism. Acute grade II-IV GVHD was present in four patients. Among 11 patients, 4 had chronic GVHD and 3 patients are still at risk of developing this complication. At day 100, non relapse mortality was 0%. To date 7 patients are alive (6 in complete remission and one with minimal residual disease) while 3 died because of progression disease and one of respiratory failure (pulmonary aspergillosis and obliterans bronchiolatis). Conclusion: We think these results are satisfactory and as other authors we would have to explore this new approach in some kind of hematologic patients to put in perspective this new procedure.

Treatment of high-risk acute lymphoblastic leukaemia relapse after allogeneic stem cell transplantation with secondary reduced-intensity conditioning regimen allogeneic transplantation or immunotherapy B. Piatkowska-Jakubas, P. Mensah, D. Hawrylecka, J. Krawczyk, A.B. Skotnicki, Jagiellonian University (Cracow, PL) Relapse of leukemia is the more disappointing cause of failure after allo-SCT for ALL. We report four patients aged 18-31 years (2 females and 2 males) with high-risk ALL defined as t(9;22), t(1;19) and hiperleukocytosis who relapsed after allo-SCT. The disease status before first allo-SCT was as following: CR2 -2 pts, PR -2 pts, conditioning regimen consisted of Cy-TBI (n=3) and Bu-Cy2 (n=1). All pts achieved complete donor chimerism. Relapse after first allo-SCT occurred at a median time of day 87 (60-110). 3 pts received as frontline a second allo-SCT from the same donor with FLAG-Ida reduced-intensity conditioning (RIC) regimen. GVHD prophylaxis was low dose methotrexate and early tapered cyclosporine in 2 pts, Ph+ ALL patient received no GVHD prophylaxis. One patient developed severe cardiac (WHO grade 3) and mucosal (WHO grade 4) toxicity. In 1 patient rapid withdrawing of immunosuppresion was able do reestablish donor hematopoiesis without GVHD symptoms. Outcome: all transplanted patients engrafted and achieved complete remission with full donor chimerism, Ph+ ALL patient become negative in PCR. One patient died on day +88 because of septic shock and MOF. After a median follow up 330 days (100-679) three patients are alive and disease free: one developed extensive chronic GVHD, 2 pts are doing well. We conclude that second allo-SCT with FLAG-Ida reducedintensity conditioning (RIC) regimen seems to be an alternative treatment option for leukemic relapse after allo-SCT in high-risk ALL patients with possibility of achieving a complete remission.

Allogeneic reduced intensity conditioning regimen haematopoietic stem cell transplantation S. Fürst, V. Dubois, A.S. Michallet, F. Nicolini, X. Thomas, J. Troncy, H. Rafii, L. Gebuhrer, M. Michallet, Hôpital E. Herriot, Laboratoire d´Histocompatibilité, Laboratoire d'Histocompatibilité (Lyon, F) We reporte here a patient with relapsed AML after allogeneic RICT who was treated by two doses of Gemtuzumab ozogamicin (MylotargTM) (MO). It was a 46-year-old woman who presented a secondary AML-M2 2 years after surgery, radiation and chemotherapy (6 cycles of FEC) of a breast cancer (pT2, N(X), M0, negative receptors) in CR. Bone marrow aspiration found 90% of blast cells and immunophenotyping showed a strong positivity for CD 33, CD 13, CD 117, CD 65, CD 64, MPO, HLA-DR and less for CD 34. Cytogenetic and molecular studies showed the presence of a t (9;11) i (10) (q 11) [20] with rearrangement of the MLL gene (11q23). Induction chemotherapy with high dose aracytine and idarubicine resulted in a PR with persistant cytogenetic abnormalities. After consolidation chemotherapy with high dose aracytine, a morphological and molecular CR was documented. The patient received an additional cycle of high dose aracytine before allogeneic RICT from a matched unrelated female donor. The stem cell source selected was peripheral blood with 2.3 x 106 CD 34/kg and 3.1 x 108 CNT/kg. RIC consisted of Fludarabine and 2 Gy total body irradiation. Graft-versus-host disease (GVHD) prophylaxis consisted of cyclosporine (CsA) and mycophenolate mofetil (MMF). Chimerism analysis showed a mixed profile with 22% of recipient cells on day +20. At day +37 she developed a grade III GVHD. On day + 42 we observed an hematological relapse. In parallel, chimerism analysis at day +47 showed 93% of recipient profile. The immunosuppressive therapy had been stopped and hydroxyurea was given. Later on, aGVHD developed to grade IV (liver III, skin IV) requiring corticosteroids given at 2 mg/kg/d. Therefore, because of leukaemia progression, MO was given at a dose of 3 mg/m2 at day +67 and day +70 without any further treatment. Major toxicity was grade 4 myelosuppression. At day +91 posttransplant and day 24 after MO, the patient achieved complete hematological and molecular remission documented in the bone marrow. Chimerism analysis showed a full donor chimerism (FDC) profile. Despite preexisting liver GVHD the patient did not develop venoocclusive disease. At last follow-up, 9 months after RICT and 14 months after AML diagnosis the patient is still alive and in CR, molecular remission and in FDC. This report demonstrates the feasibility and efficacy at the molecular level of MO given for relapsed AML after allogeneic RICT. The application of DLI, however, is associated with a profound risk of GvHD. Depletion of CD8+ lymphocytes from either the allotransplant or from DLI has proven feasible to reduce the incidence of GvHD (Nimer et al. Transplantation 1994, 57:82-7; Giralt et al. Blood 1995, 86:4337-43) . We set up an approved phase-I allotransplantation study which combines the well-established Campath-1H (5x20mg)/Fludarabine (5x30mg/m2)/Melphalan (1x140mg/m2) preparative regimen with the preemptive administration of CD8depleted DLI in patients with haematological malignancies not eligible for myeloablative conditioning. After withdrawal of Cyclosporine A immunosuppression, CD8-depleted DLIs are scheduled in a dose-escalating regimen starting on day +60 after HLA-identical sibling transplantation and on day +120 after HLAmatched unrelated donor transplantation, respectively. For CD8depletion a new CliniMACS(TM)-based depletion protocol using clinical grade CD8 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) is used. Up to now, we obtained experience in the depletion procedure on buffy coat-derived peripheral blood mononuclear cells of healthy donors. The depletion protocol was easy to handle and was efficient in reducing the content of CD8-positive cells to levels undetectable by conventional flow cytometry. Thereby, we fulfil the requirements for an approval of the procedure by the local authorities. The first patients will be enrolled in December 2003. Results of depletion efficiency in donor lymphocyte products and the early clinical course of treated patients will be presented.

Thrombotic microangiopathy after non-myeloablative conditioning for allogeneic stem cell transplantation -report of four cases M. Kornacker, T. Luft, A.D. Ho, H.-J. Schaefer, University of Heidelberg (Heidelberg, D) Thrombotic microangiopathy (TMA) has been described as a complication of bone marrow or stem cell transplantation. It is usually associated with high dose therapy for autologous transplantation or myeloablative conditioning in the allogeneic setting. TMA after bone marrow or stem cell transplantation is thought to be caused by endothelial damage due to total body irradiation, high dose chemotherapy, graft vs. host disease, cyclosporine A therapy and systemic infections. At our institution, patients with reduced intensity conditioning (RIC) 6-12 weeks after autologous transplantation (combined autograftingallografting) were at higher risk of developing TMA than patients after myeloablative conditioning regimens. 74 patients were transplanted after RIC (see table) . Of these, 4 muliple myloma (MM) patients conditioned with 2 Gy total body irradiation and fludarabine suffered from TMA. The cause for the higher incidence of TMA after RIC is not clear. GVHD was present in only one patient before onset of TMA, occured later in the course of TMA in another patient and never developed in the remaining 2 patients. Cyclosporine A levels were in the expected range (mean concentration 221 ug/l (median 217 ug/l). High dose therapy or myeloablative radiation was not part of the conditioning regimen, but 3 of 4 patients received high dose therapy with Melphalan (200 mg/m2) 6-12 weeks before conditioning therapy as part of an combined autograftingallografting procedure. Overall, 3 patients received 2 and one patient 3 high dose therapies during the course of their disease. All patients had infectious complications at the time of TMA onset. One patient suffered from CMV reactivation, 2 patients from CMV reactivation and diarrhoe positive for adenovirus and one patient from diarrhoe positive for rotavirus. 3 of 4 patients died after a median interval of 16 weeks, 2 from invasive aspergillosis during active TMA and one patient from relapse of multiple myeloma without evidence of active TMA. One patient is alive without evidence of TMA but suffering from renal failure. We conclude that combined autografting -allografting increases the risk for TMA after allogeneic stem cell transplantation Evidence for the existence of GvT effect in several malignant diseases has brought about a revolution in the perception of stem cell allotransplant to confer GvT from alloreacting immune cells. From January 2001 to November 2003 12 patients, median age 53 years (range 37-66 years). with solid tumors (5 renal, 5 colorectal and 2 lung cancer) underwent allogeneic Non-Myeloablative Stem Cell (NST) from a family donor. Following pre-medication with Ethyol 740 mg/m2 pts were conditioned with Cyclophosphamide 60 mg/kg (days 1and 2), and Fludarabine 25mg/m2 for 5 days. On day 0 a mean of 5.7 x 106 /kg CD34+ and 3.0 x 108/kg CD3+ cells were infused. GVHD prophylaxis consisted of Cyclosporine 3mg/Kg C.I... Mean time to ANC >1000/mm3 was 7 days and to platelets > 50x103 was 8.5 days. Two pts required 1 DLI (D +42 and D+ 72 respectively), 8 patients obtained a full chimerism at a median of 80.5 days (range 15-184); 2 pts required DLI for PD. Two pts died during the conditioning regimen, 7 pts died from PD at 36, 41, 83, 141, 247, 456 e 568. At a median follow up of 83 days (range 6-764 mos).6 patients are alive, 4 in PD and 2 too early. One pt had an acute cutaneous GVHD G III (68 days) and two pts showed limited chronic GVHD (oral mucosa). From this preliminary experience on a limited number of pts we can only draw the conclusion that NST in solid tumor is a safe and feasible procedure also in elderly patients. We cannot confirm previous published experience of tumor regression nor we had a clinical evidence of an evident GvT effect. Longer follow up and greater number of patients are needed to analyze the survival curves of patients submitted to NST as compared to patients treated with standard therapy. Nasopharyngeal carcinoma (NPC) is a chemotherapy sensitive cancer. Allogeneic stem cell transplantation may produce graftversus-tumor effect, so this is a logical approach to treat NPC. We reported a patient with stage IV NPC treated by allogeneic stem cell transplantation with reduced intensity preparative regimen. A 40-year-old man presented in Oct 2002 with left cervical lymphadenopathy. He was diagnosed NPC with multiple spinal metastasis at stage IV (T4N3bM1). He was given four cycles of chemotherapy. The chemotherapy comprised a 3-week cycle of mitomycin, epirubicin, and cisplatin on day 1 and fluorouracil and leucovorin on day 8 (MEPFL). Partial remission was initially achieved, but tumor subsequently progressed. Allogeneic PSCT from an HLA-identical sibling was done in Aug 2003 using a reduced intensity preparative regimen of fludarabine 30 mg/m2/day for 5 days and cyclophosphamide 60 mg/m2/day for 2 days. He received 6.77x10^6 CD34 cells per kilogram and 9.59x10^8 CD3 T cells per kilogram. The neutrophil count fell to less than 100 per cubic millimeter at day 8 and rose to more than 500 per cubic millimeter 12 days after transplantation. It took 18 days for the platelet count to exceed 50,000 per cubic millimeter post transplantation. Regression of left cervical lymphadenopathy has been observed since day 14. He had complete donor-T-cell chimerism without donor lymphocyte infusion on day 85. Grade II graft-versus-host disease with skin involvement developed on day 75, which was controlled by low dose steroid and cyclosporin. Radiologic evaluation at day 85 revealed complete remission of cervical lymphadenopathy and partial remission of spinal metastasis. Although the data are preliminary, allogeneic transplantation with reduced intensity preparative regimen is evaluated as one of the favorable treatment approaches for some selected patients with NPC. We reporte here a patient with relapsed AML after allogeneic RICT who was treated by two doses of Gemtuzumab ozogamicin (MylotargTM) (MO). It was a 46-year-old woman who presented a secondary AML-M2 2 years after surgery, radiation and chemotherapy (6 cycles of FEC) of a breast cancer (pT2, N(X), M0, negative receptors) in CR. Bone marrow aspiration found 90% of blast cells and immunophenotyping showed a strong positivity for CD 33, CD 13, CD 117, CD 65, CD 64, MPO, HLA-DR and less for CD 34. Cytogenetic and molecular studies showed the presence of a t (9;11) i (10) (q 11) [20] with rearrangement of the MLL gene (11q23). Induction chemotherapy with high dose aracytine and idarubicine resulted in a PR with persistant cytogenetic abnormalities. After consolidation chemotherapy with high dose aracytine, a morphological and molecular CR was documented. The patient received an additional cycle of high dose aracytine before allogeneic RICT from a matched unrelated female donor. The stem cell source selected was peripheral blood with 2.3 x 106 CD 34/kg and 3.1 x 108 CNT/kg. RIC consisted of Fludarabine and 2 Gy total body irradiation. Graft-versus-host disease (GVHD) prophylaxis consisted of cyclosporine (CsA) and mycophenolate mofetil (MMF). Chimerism analysis showed a mixed profile with 22% of recipient cells on day +20. At day +37 she developed a grade III GVHD. On day + 42 we observed an hematological relapse. In parallel, chimerism analysis at day +47 showed 93% of recipient profile. The immunosuppressive therapy had been stopped and hydroxyurea was given. Later on, aGVHD developed to grade IV (liver III, skin IV) requiring corticosteroids given at 2 mg/kg/d. Therefore, because of leukaemia progression, MO was given at a dose of 3 mg/m2 at day +67 and day +70 without any further treatment. Major toxicity was grade 4 myelosuppression. At day +91 posttransplant and day 24 after MO, the patient achieved complete hematological and molecular remission documented in the bone marrow. Chimerism analysis showed a full donor chimerism (FDC) profile. Despite preexisting liver GVHD the patient did not develop venoocclusive disease. At last follow-up, 9 months after RICT and 14 months after AML diagnosis the patient is still alive and in CR, molecular remission and in FDC. This report demonstrates the feasibility and efficacy at the molecular level of MO given for relapsed AML after allogeneic RICT. Forty patients were with ANLL, 2 -ALL, 8 -NHL, 3 -HD, 9 with solid tumors. All patients had normal lung function tests, measured before SCT. Stem cells source were: G-CSF mobilized peripheral blood stem cells (PBSC) -in 51 patients, bone marrow (BM) -in 4, combined PBSC+BM -in 8. The median number of infused CD 34+ cells was 6.8x106 cells/kg (range 0.14-17.7x106). Forty-five patients received HD-L-PAMbased preparative regimens before SCT, 8 carboplatin-based regimens, and 10 busulphan-based regimens. Median time to achieve neutrophils >0.5x109/L was 18 days (range 8-54 days). Results: Seven patients (11%) showed clinically relevant PC at median + 11 day after stem cell infusion (range 4-17 days). Five cases were acute respiratory distress syndrome (ARDS) attributable to engraftment syndrome: all patients were successfully treated with methylprednisolone 5-20 mg/kg body weight, one case of diffuse alveolar hemorrhage: mechanical ventilation for 10 days required, successfully treated with methylprednisolone 15 mg/kg body weight and platelet transfusions, and one case of idiopathic pneumonia syndrome without isolation of infectious organism. There were no deaths attributable to the PC within first 100 days after SCT. In multivariable analysis we identified rinse of neutrophils counts as the only significant variable predicting for PT (p=0.01). Introduction: Erdheim-Chester disease is a rare multisytemic non-Langerhans form of histiocytosis that usually affects adults. It´s cause is unknown and prognosis is poor, especially in case of extraosseous involvement. Treatment forms are similar to those used in Langerhans cell histiocytosis (LCH), but are known to have little effect. Case report: We report on a meanwhile 7 year old boy who presented at the age of four with exophthalmus and bone pain. Radiography showed symmetrical bilateral osteosclerosis of long tubular bones. In the cerebral magnetic resonance (MRI) we found a voluminous mass involving both orbits and cerebral sinuses and abdominal sonography showed involvement of the pancreas. Diagnosis of Erdheim-Chester disease was confirmed by bone biopsy, showing foamy histiocytes negative for CD1a and S-100. The disease was refractory to therapy (modified LCH protocol with Prednisolon, Vinblastin and 6-MP, reinduction with Etoposid) and even progressed involving the mediastinum and both kidneys. Treatment was continued with corticoids in changing dosis, Etanercept and Infliximab-but failed. So we decided to perform double high dose chemotherapy with stem cell rescue relying on a single case report in an adult patient. Conditioning consisted of Etoposid (60mg/kg) and Melphalan (140mg/m2) the first time and Thiotepa (400mg/m2), Etopsid (60mg/kg) and Melphalan (100mg/m2) for the second course two months later. Results: Now six months after autologous SCT the patient is fine, painless and without drugs for the first time-supposing stable disease. Cerebral MRI shows a slightly reduction of intracerebral and orbital mass. Conclusion: The diagnosis of Erdheim Chester disease is a curiosity in childhood with marginal chemosensitivity and poor outcome. SCT with Etoposide as part of the cytotoxic treatment, which is known to be effective on neoplastic cells of the monocytic lineages, offers a useful treatment modality in refractory disease. IVIG has been reported to reduce the incidence of chronic GvHD (cGvHD). A role in the treatment of severe viral infections has also been advocated. Its use in paediatric HPT, where severe GvHD and CMV-related disease are less common, is unclear. The aim of this study is to analyse the influence of prophylactic IVIG on GvHD and viral infections in children receiving allogeneic HPT. Patients and methods: From 1996 to date, 108 children (median age 11± 4.6years) received allogeneic HPT at our centre. Indications for transplant included ALL (75), AML (12), MDS (6), CML (10), undifferentiated (3),Lymphoblastic NHL (1), HLH (1). 60 received unrelated donor (UD) and 48 related donor (RD) grafts. Endpoints to this study were the influence of IVIG on the incidence and severity of acute (aGvHD) and cGvHD and on the incidence of viral infections. Results: Data on aGvHD were available in 104 patients: 31 had no aGvHD and 73 had aGvHD (12 with severe grades III and IV aGvHD), treatment being necessary in 55 cases. Data on cGvHD were available in 97 patients: 75 with no cGvHD and 22 with cGvHD (20 limited, 2 extended disease), 17 of which required treatment. 30 patients developed viral infections: CMV antigenemia (17, 2 with disease, over a total of 41 pairs with positive pre-HPT status), HZV (7), respiratory viruses (4), rotavirus(1), adenovirus (4). In children receiving RD HPT, a non significant tendency for a lower incidence of aGvHD was found (8/15 vs. 24/31). No influence on cGvHD was found. Children receiving UD HPT, also had a non significant lower incidence of severe forms of aGvHD (3/41 vs. 4/16) and a lower incidence of cGvHD (9/39 vs. 5/13, p<0.06). Interestingly, they did not suffer extended forms of the disease. These parameters were not altered after considering patients treated with Campath. Overall, IVIG did not seem to influence the development of viral infections. In the specific case of CMV, no influence was seen in the development of positive antigenemia, relapse or development of organ disease. Conclusion: IVIG seems to provide protection against the development of cGvHD and severe forms of aGvHD in children receiving UD HPT. No other beneficial effects have been found in our series. The precise role of IVIG in paediatric transplantation should be analysed in prospective studies. Recent reports on adult haemopoietic progenitors transplants (HPT) suggest the existence of a graft versus leukaemia (GvL) effect derived from ABO incompatibility with no increased acute or chronic graft versus host disease (aGvHD, cGvHD). In order to assess whether ABO incompatibility has an effect on outcome in paediatric HPT (where GvHD and GvL are known to be less intense) we have conducted a retrospective study where all the allogeneic paediatric HPT performed at our centre between 1996 and 2003 (n=108, median age 11 ± 4.6 years) were included. 60 patients received grafts from unrelated donors and 48 from related donors. Indications for HPT included ALL (75 cases), AML (12), MDS (6), CML (10), undifferentiated (3), Lymphoblastic NHL (1), HLH (1). Data on ABO match were available in 81 cases of which 26 had ABO mismatch. These cases were disclosed according to the direction of the alloimmune reaction: donor against recipient (12), recipient against donor (11) and bi-directional (3). Main outcome measures were aGVHD and cGvHD, final outcome (alive/dead) and cause of death (relapse/toxic). Data on the development of aGvHD were available in 77 cases. ABO mismatch was present in 5/21 cases without aGvHD and 19/56 cases with aGvHD. These differences were not significant. However, the only cases with grade IV were seen in the mismatched group. Data on cGvHD were available in 74 cases. 17/56 cases without cGvHD and 6/18 with cGvHD had ABO mismatch. These differences were not significant, although a tendency to experience extended disease was seen in patients with ABO mismatch. When examining the direction in which the mismatch was orientated, a non significant tendency to experience aGVHD in cases with donor against recipient reactivity was found (9/10 vs. 48/67). Although no relation was found between mismatch against donor and the development of cGvHD, a significant relation (p<0.03) was found between experiencing severe forms of cGvHD and the presence of alloreactivity orientated against recipient (2/10 vs 0/64). No relation was found between the presence of ABO mismatch or the orientation of the alloreactivity and the final outcome or the cause of death. Overall, in paediatric HPT, ABO mismatch does not seem to increase the incidence of GvHD, although it appears to increase its clinical severity, specially in cases where the alloreactivity follows a donor vs. recipient orientation. No antitumoural effect seems to be associated to this phenomenon.

Long-term remission in a child with ALL relapse after allogeneic stem cell transplantation using chemoimmunotherapy U. Gross-Wieltsch, P. Bader, P. Lang, T. Klingebiel, D. Niethammer, J. Treuner, E. Koscielniak, Olgahospital, University Children`s Hospital (Stuttgart, Tuebingen, Frankfurt, D) Introduction: The prognosis of children with subsequent relapse after allogeneic stem cell transplantation (allo-SCT) is very poor. A further SCT, by which long term remission could be reachieved, is mainly associated with high rate of morbidity and mortality. Immunotherapy by donor lymphocyte infusion (DLI) has become a convincing treatment option in relapsed patients with CML. However, the benefit of this treatment option is very limited in relapsed patients with ALL.

Here we present the case report of an infant with ALL, who relapsed after allo-SCT from a matched unrelated donor (MUD) in whom a further remission could be achieved by a combination of low intensive chemotherapy and DLI as consolidation. Case report: At the age of 2months, a pro-B-ALL with bone marrow (BM) and central nervous system (CNS) involvement was diagnosed. A 1st remission was achieved by chemotherapy (Interfant Pilot 98 protocol). After 6 months on maintenance therapy the patient developed a combined CNS and BM relapse. 2nd remission was achieved again by chemotherapy ( ALL-BFM 95 and 99P protocol). Then a MUD-SCT was performed with Tcell depleted peripheral blood stem cells. Post transplant, the patient developed increasing mixed chimerism and received two doses of DLI (day +87 and +117;25,000 CD3+/kg) . However, open hematological BM relapse could not be avoided. After 2months of induction chemotherapy a 3rd remission was achieved.

Then oral maintenance therapy with mercaptopurin/methotrexate and triple ith.-chemotherapy every 4-8 weeks was given until 24 months post relapse. 4 months later, reinduction with dexamthason/vincristin pulses for 7 days was started, over a period of 8 months due to increasing minimal residual disease (MRD). Intermittently, additionally DLI with increasing doses (25,000/kg-71,538/kg CD3+) and intervals (1-5 months) were given up to 37 months post relapse when the patient finally became MRD negative.

Conclusion: Long term remission can be achieved by combined prolonged, low intensive chemoimmunotherapy in children with high risk leukemia relapsing after MUD SCT. The course of MRD and chimerism is shown in table. Grants: Deutsche Krebshilfe (PB) and Wilhelm Sander Foundation (PB)

Objectives: To calculate the individual dose requirements of oral busulfan (BU) needed to achieve therapeutic plasma levels, to study the factors related to the variability of BU pharmacokinetics in pediatrics and to follow-up the clinical evolution of the patients. Patients and methods: Eleven patients (7M/4F), with mean age of 7 years (0.5-17), diagnosed of AML (6), ALL (1), CML (1), CNS tumour

(1), adrenoleukodystrophy (1) and congenital dyserythropoietic anaemia (1), undergoing allogeneic (6) or autologous (5) hematopoietic cell transplantation were included. They received an initial dose of 1 mg/Kg/6h (total 16 doses) of oral BU as a part of the conditioning regimen. Blood samples were taken 0, 1.5, 3, 4 and 6 h. after the first BU dose. For the pharmacokinetic analysis a one-compartment model was fitted to the available concentration-time data by non-linear regression (ADAPT-PC). BU dosage was adjusted, if needed, to achieve BU steady-state plasma levels (Css) between 600-900 ng/mL. Regimen-related toxicity and actuarial event-free-survival were recorded. Results: The Css BU estimated for the standard dose was 855±550 ng/mL (X±SD). The dose was reduced (21%) in 4 patients, increased (9%) in 1 and maintained in the rest. The total dose given was 14.3±5.1 mg/Kg vs 16 mg/Kg (standad dose). Mean BU clearance (Cl/F) was 4.07 mL/min/Kg with a coefficient of variation of 47.2%. A significant correlation between age and BU Cl/F was found. As a consequence, the mean total dose required in patients =< 3 y was 16.1 mg/Kg and for those > 10 y was 11.9 mg/Kg. All patients developed mucositis grade II/III. One patient presented neurologic and severe hepatic toxicity (veno-occlusive disease). All patients engrafted. Mean time to recovery granulocyte was 15±8 days and platelets 21±9 days. Three patients died due to leukaemia relapse, acute graft versus host disease and hepatic veno-occlusive disease. Eight patients remain now in continuous complete remission. Actuarial eventfree-survival is 0.72 ± 0.13 at 4 years.

Conclusion: The variability in BU pharmacokinetic parameters shows the necessity of individualising the doses of BU in pediatrics. Age seems to be the most important factor affecting this variability.

S. Martínez-Nadal, I. Alcorta, J. Estella, S. Rives, T. Toll, E. Tuset, Hospital Universitari Sant Joan de Déu (Barcelona, E) Background: Cryopreservation at cell concentration greater than 200 x 10e6/mL in 5% dimethylsulfoxide (DMSO) permits the infusion of a reduced amount of DMSO, minimising the adverse effects. We evaluate the long term hematological recovery of patients transplanted with this approach. Methods: The apheresis products of 27 consecutive autologous PBSC transplant patients were cryopreserved with 5% DMSO in autologous plasma at a final concentration equal or greater than 200 x 10e6/ml. The product was frozen and preserved at -80°C in a mechanical freezer. The patients were mobilized with G-CSF 24 mcg/kg for 3-5 days (n=23) or a combination of chemotherapy and G-CSF (n=4). All patients were submitted to large volume leukapheresis with the Cobe Spectra AutoPBSC procedure. Long term hematological recovery was assessed by blood counts 12 months post transplant. Results: The patients were 16 boys and 11 girls with a median age of 10.7 y (range 0.6 to 19). Diagnosis were CNS tumour in 7, Ewing's sarcoma in 6, ALL in 6, Hodgkin's disease in 2, neuroblastoma in 2, AML in 2, Burkitt's lymphoma in 1 and osteosarcoma in 1 patient. Median (range) volume of the cryopreserved products and their cellular concentration were 200 mL (70 to 700) and 263 x 10e6/mL (192 to 572) respectively, with a median CD34 content of 3.66 x 10e6/kg (range 1,89 to 25). Median time from cryopreservation to transplantation was 10 days (range:9 to 108). Median amount of DMSO infused was 0,34 ml/Kg (range 0.11 to 0.63). One patient died of transplant related complications 10 days after infusion, 5 patients relapsed and died of their underlying diseases between 1 and 10 months post transplant, and 2 additional patients relapsed after 8 and 10 months and were receiving intensive salvage chemotherapy one year posttransplant. Thus, 19 patients are available for analysis at least 12 months post transplant. Hematological recovery was complete with normal hematological counts 1 year post-transplant in 11/19 evaluable patients. One patient with Ewing's sarcoma and congenital spherocytosis had an Hb 9,8 g/dL. Four other patients had Hb values ranging from 10,7 to 12,5 g/dL, slightly under normal for age. Three patients mantained platelet counts below normal levels (86, 118 and 138 x 10e9/L). Conclusions: PBSC cryopreservation at cell concentration greater than 200 x 10e6/mL in 5 % DMSO results in a sustained long term hematological recovery and permits the infusion of a reduced amount of DMSO.

Severe combined immunodeficiency is a true pediatric emergency; children with SCID were the first patients with immunodeficiencies to be successfully transplanted with unrelated and T-cell-depleted, haploidentical bone marrow. The pattern of inheritance of SCID is X-linked and autosomal recessive (ADA def, Jak3, RAG1, RAG2, IL 7R alpha). In this report, we describe a patient 1y/old boy with B T SCID that underwent HLA 4/6 matched unrelated cord blood transplantation from Menhaiem Cord Blood Bank (Germany).Conditioning regimen was Busulfan 1 mg/kg/day for 2 days and Cyclophosphamide 10mg/kg/day for 2 days. GVHD prophylaxis consisted of Cyclosporine A and Methotrexate. The post transplant course was complicated by skin GVHD at day +7 and GI GVHD at day +9 then at day +14 arrhythmia, hypotension and rising of central venous pressure happened with LDH, SGOT, and CPK increment. The patient treated with Dopamine, Diuretics, Prednisolon and IVIG .Severe GI GVHD developed at day +50 which was initially treated with prednisolone 2mg/kg/day.Despite that GI GVHD progressed and Mycophenolate mofetile 600mg/m²/day added to prednisolone. Chimerism assessment with STR_PCR method revealed 65% donor engraftment. He is now experiencing catch_up growth and development 11months after UCB transplant. Unrelated cord blood is a stem cell source in SCID patients and unrelated cord blood transplantation is a curative treatment in patients without appropriate donor.

Relapse of acute myeloid leukemia (AML) after allogeneic transplant is always associated with poor prognosis with no clear treatment options. Donor lymphocyte infusions (DLIs) showed to be effective in treating the relapse of chronic myeloid leukemia (CML) while the use of DLI in case of AML relapse revealed far lower response rates. We tried to use salvage chemotherapy regimen for malignant clone burden reduction followed by DLI in two pediatric cases of early hematological relapses after allogeneic PBSC transplants from HLA identical siblings for the treatment of AML in the I complete remission (CR). The first patient was two years old boy with acute megakaryocytic leukemia (AMgL) without any underlying diseases and the other of the age of four with AML, FAB-M2. Both received unmanipulated PBSC, CD34+ dose 12,3x106/kg and 6,65x106/kg respectively. GVHD prophylaxis consisted in standard scheme of cyclosporine and methotrexate. Mixed chimerism has been achieved with no signs of GVHD in both cases. Withdrawal of immunosuppression was performed because of the increase in recipients' cell burden. Early hematological relapse was documented at day +79 for AMgL and at +134 for AML, M2. For the relaps treatment we started with myelosuppresive regimen as reported by Levine, et al (J Clin Oncol, 2002 20:405-412 ) that consisted of cytarabine 100 mg/m2 a day for 7 days and daunorubicin 30 mg/m2 a day for 3 days. Both patients received G-CSF-primed DLIs from the same identical siblings with CD3+ 1x108/kg in nadir phase and the second dose of 3x108/kg 28 days later for AMgL and 1x108/kg in nadir and 4,2x108/kg 14 days later for AML, M2. Complete chimerism (CC)was acchieved at day 28 following the first DLI for AMgL and at day 21 for AML, M2. No post-DLI GVHD prophylaxis was used with the development of isolated hepatic acute GVHD (confirmed by liver biopsy) for AMgL patient and with no signs of GVHD for AML, M2. Patients are in CR with follow up of 3 months after the first dose of DL infusion for AMgL patient and 1 month after the first DLI for AML, M2. Conclusion: We suggest that myelosuppresive, but not myeloablative chemotherapy before G-CSF-primed DLI may be a good option for management of early relapse of AML in children. The role of stem cells, infused with DLs must be appreciated as well in possibly preventing post DLI aplasia. Objectives: T-cell reconstitution is of great importance for the success of autologous or allogeneic stem cell transplantation (SCT). Therefore longitudinal analysis of T-cell reconstitution should increase the understanding of immunological reconstitution and lead to improved treatment strategies. Patients and Methods: Peripheral blood samples were obtained twice weekly from three children starting at day +1 after SCT. FACS analysis was performed using antibodies directed against CD3,CD4,CD8,CD19,CD62L, CD45,CD14 and CD45RA. For TREC analysis real time polymerase chain reaction was used. One child was diagnosed with severe aplastic anemia (SAA) and transplanted with parental CD34-selected PBSCs, one child suffered from AML and was transplanted in CR1 with HLAmatched MUD-BM using a non myeloablative conditioning regimen and one child had severe combined immunodeficiency syndrome (SCID) and was transplanted with BM of a HLAidentical sibling. Results: All children showed complete three lineage engraftment. T-cell recovery was delayed in the patient receiving parental CD34-selected PBSCs, in contrast TREC-content was lower in the child with AML grafted with BM after non myeloablative conditioning. Details are depicted in the table below. Results: Patient 1 (AML) Patient 2 (SAA) Patient 3 (SCID) CD3/4/8/+cells/mcl peripheral blood Conclusion: The longitutinal analysis of lymphocyte subsets together with the analysis of the TREC content provides precise information on immunological recovery as well as of thymic function and therefore provides additional information necessary for patient-specific immunmodulation (e.g. Donor lymphocyte infusion, immunosuppressive treatment).

Background: The use of erithropoietin alpha (EPO) at the conventional dose of 10.000 u.i. three times a week is often ineffective in the treatment of non-haemolytic and non haemorragic anaemia after allogeneic bone marrow transplantation (BMT). Patients and methods: We tested the use of high dose of erithropoietin after allogeneic BMT in 18 patients grafted for CGL (8), ALL (1), AML (7), HD (1), MDS (1), from HLA identical sibling donor (7), HLA mismatched family donor (1) and HLA matched unrelated donor (10). The conditioning regimen was TBI in 9 patients. EPO was administered in 15 patients with haemoglobin level <9 gr/dl and in 3 patients with haemoglobin level <10gr/dl. Patients were treated with erithropoietin alpha at the dose of 40.000 u.i. twice a week for two consecutive weeks, then 40.000 u.i. weekly for two weeks and then 40.000 u.i. twice a months for 1 month. Seven patients (39%) were transfusion dependent at the beginning of the treatment. The interval between BMT and EPO treatment was 104 dd (range 45-300). Results: The increase of Hb levels was 2.3 gr/dl at 15 days (range 1-4), 3.6 g/dl at 30 days (range 2-7) and 1.7 g/dl at 60 days (range 1-4); there was no response in 2 patients: one patient relapsed after BMT and the other one developed persistent CMV infection. We didn't observe decrease in WBC and Platelets count and other side effects. All the patients became transfusion independent. Conclusions: The use of high dose of erithropoietin alpha after allogeneic transplantation improves the haemoglobin levels, reduces the transfusion requirement and improves the quality of life.

Anxiety and depression during bone marrow transplantation. Preliminary data of a randomised study C. Leon, R. Laborda, E. Gris, M. Palao, N. Llacer, R. Martino, J. Briones, A. Sureda, S. Brunet, J. Sierra, Hospital Santa Creu i Sant Pau (Barcelona, E) Introduction: Mental disorders may occur at different stages of BMT. Previous research examining psychological distress reported that the period of isolation influenced on anxiety and/or depression in 41% of the patients. Among psychosocial interventions for reducing treatment-related stress, relaxation and imagery were most useful. Purpose of the study: The aim of this study was to gather information about the effectiveness of a specific behavioral intervention, as relaxation, in reducing psychological distress in patients undergoing BMT. Description of the project: A randomized controlled trial is being used with repeated measure assessment. Participants are recruited from the Clinical Hematology Division from Hospital de la Sta. Creu i Sant Pau. Both allogeneic and autologous transplant recipients are included in the study. The patients are randomized into an intervention group and a control group. Psychological intervention of a modified form of Jacobson's method for Progressive Muscle Relaxation and guided imagery, is taught to the patients of the intervention group. Clinical adverse effects during hematopoietic progenitors cells infusion, and psychological status, according to the Hospital Anxiety and Depression Scale (HADS), at baseline and during the inpatient period (day -1, +1, +10 and at discharge), are evaluated.

Results: Sixty patients will be included in the study, thirty on each arm. Preliminary results in 20-30 patients will be presented by March 2004. This study was supported by a grant from the Michael Wright Leukemia Research and Facilities Foundation.

Central venous access for peripheral blood stem cell harvest: single-centre experience A. Vale, C. Ramírez, R. Varela, P. Torres, G. Deben, Hospital Juan Canalejo (La Coruña, E) An appropiate venous access is essential for the collection of peripheral blood stem cells (PBSC). In our institution 231 patients with hematological diseases underwent PBSC collection for autologous transplant since 1995 until november 2003. Most of them had bad peripheral venous access and was necessary use a central venous catheter (CVC). At first we used a Hickman CVC (until 14 french of diameter) inserted in subclavian vein but the complications were frequent, specially low blood inlet flow and problems with the insertion (besides other as infection and thrombosis) and we had to look for other type of catheter. Patienst and Methods: In the middle of 2001 we started to use a double lumen CVC rigid of 12 french of diameter, inserted in the jugular vein under fluoroscopic guidance by radiologists immediately before the first apheresis and removed the day after the last apheresis procedure. The thick walls and the large lumen are ideal to keep a high blood flow without wall collapse under high collection pressure. On the other hand these catheters have a limited durability (less than 4 weeks) so they are used only for the collection and other type of catheter, implantable, type Porta-cath, is inserted in subclavian vein for chemotherapy, supportive care and for the transplant procedure. Results: More than 200 apheresis in 58 patients were done with this type of catheter, using a Fenwal CS-3000 (Baxter) separator and processing 3 blood volume. The median CD34 collected were 5.2 x 10e6 CD34/kg. The median number of apheresis was 3. The median flow rate was 65 ml/min and the median time that the CVC was inserted was 4 days (range 3-24). No signs of thrombosis, bleeding or bacteriemia were observed in any patient. Although some patients have had venous thrombosis in relation with previous catheter there were no problems during insertion of these catheters (probably due to radiology guidance). In 2 patients who had the catheter inserted for more than 10 days local signs of infection in the insertion site were observed and treated locally. The most frequent secondary effect was minor discomfort at the puncture site. Conclusions: The use of a large lumen and thick wall catheter inserted under fluoroscopic guidance specifically for apheresis sessions had allowed to reduce the complications related with the catheter during PBSC collection.

A. Rüther, W. Brugger, W. Willenbacher, German Agency for HTA at DIMDI, Villingen Hospital (Cologne, Villingen, D) Objectives: Health Technology Assessment (HTA) provides up to date information on medical efficacy, effectiveness, economic efficiency and on ethical implications of medical procedures and processes. Its validity and high quality leads to growing importance of HTA as base for decision making in Health Care all over Europe. The necessity to transport scientific results to the political decision makers is obvious especially regarding costly procedures like allogenic or autologous stem cell therapy (SCT). HTA could serve as a perfect tool, but is the community of transplantation performers (e.g. the EBMT) aware of it? Methods: A thoroughly literature search for assessments on bone-marrow and peripheral stem cell transplantation was performed on the main databases for primary research (Medline, Embase, Scisearch) and on the main databases for HTA and systematic review (Cochrane Library, INAHTA, ECRI, DAHTA, HTAi) over the last 10 years. The results had been handsearched by two independent scientists for qualifying Health Technology Assessment in SCT. Health Technology assessment was qualified by regarding efficacy, effectiveness and efficiency of a procedure, providing structure, transparency (methods, i.e. included/excluded trials) and recommendations. Results: 100 articles could be identified as qualified as "assessment" by authors. Handsearching showed that out of this 100 articles 11 were narrative reviews, 18 systematic reviews and only 15 Health Technology Assessment reports. Conclusion: HTA as a mean for transporting scientific results to political and economical decision makers is not used to a sufficient degree in SCT up to now. The potential of HTA, which has been clearly shown in other medical specialities, should also be used more extensively in the setting of SCT in the future.

The use of low molecular weight heparins (dalteparin) for prophylaxis against thromboembolic complications during allogeneic haematopoetic stem cell transplantation M. Ozcan, E. Soydan, C. Ustun, H. Celebi, O. Arslan, M. Arat, G. Gurman, O. Ilhan, H. Koc, N. Konuk, A. Uysal, M. Beksac, H. Akan, Ankara University Faculty of Medicine (Ankara, TR) The efficacy of low moleculer weight (LMW) heparins for preventing thromboembolic complications after allogeneic hematopoetic stem cell transplantation (AHSCT) was investigated by a prospective randomized single center study. Thirty eight patients with leukemia who underwent AHSCT enrolled to the study after they signed the informed consent form after local commitee approval. Following stratification of patients according to their diagnosis (acute and chronic leukemia), sex, ALT level and stem cell source (peripheral and bone marrow) they were randomized to dalteparin and control arm. Patinets in the dalteparin group (n=17) received the drug 2500 Ü/gün subcutaneously from day -9 to +100 post transplant. Dalteparin administration was stopped in case of high risk or clinical evidence of major bleeding. Patients were observed for veno occlusive disease (VOD) by weight gain, hepatomegaly, presence of ascites and liver function tests; for cathater trombosis and other thromboembolic complications by clinical manifestations and suitable laboratory tests. Both groups were comparable according to their age, time from diagnosis to transplantation, donor age, ABO and sex mismatched cases and number of infused mononuclear and CD34+ cells. None of the patients in both groups experienced VOD. Catheter related trombosis was detected only in one patient in the dalteparin group. Nine out of 17 patients in the study group received the drug till posttransplant 100th day as intended. The drug was stoppped due to high risk of bleeding in nine of the patients. There was no life threatining bleeding and severe adverse event related to dalteparin use. The incidence of aGVHD and transplant related early mortality was similar and aGVHD was the only cause of death for both groups. During a median follow up period of 38.7 (1.6-51.3) months 9 patients in the dalteparin group and 13 patients in the control group developed cGVHD. Relapse rates were similar in both groups (p=0.6). Estimated DFS for four years were 56% vs 65% (p=0.4) for dalteparin and control group respectively and OS was 65% for both groups. The sides of this study group did not allow us to make a firm conclusion about dalteparin prophylaxis against thromboembolic complications so this study should be further planned in a different cohort of patients in which VOD is more prevelant. But dalteparin was found to be a safe drug with acceptable adverse event profile in our study cohort of transplant patients.

S. Sohn, D. Kim, J. Kim, J. Suh, K. Lee, K. Lee, Kyungpook National University Hospital (Daegu, KOR) Although the use of peripheral blood stem cells instead of bone marrow is still under debate in transplantation from HLA-identical sibling donors, an allogeneic peripheral blood stem cell transplantation (PBSCT) with a stronger graft-versus-leukemia (GVL) effect may be preferable as the source of stem cells especially in advanced hematological diseases. We reported the outcome for 27 consecutive patients with high-risk hematological diseases treated with allogeneic PBSCT. The median dose of CD34+, CD3+ cells, and MNC infused was 8.18x106/kg (range, 2.78-72.89x106/kg), 1.50x108/kg (range, 0.06-4.25x108/kg), and 7.17x108/kg (range, 0.95-15.85x108/kg), respectively. The median time taken for the ANC and platelets to reach 500 and 20,000x106/ul was 15 days (range, 9-25 days) and 16 days (range, 10-56 days), respectively. Three patients (11.1%) experienced transplant-related mortality within 90 days after transplantation. Fifteen (64.0%) out of evaluable 24 patients developed chronic GVHD (6 limited, 9 extensive). There was a significant difference of OS between group with chronic GVHD and without chronic GVHD (p=0.0253). The causes of death included relapse (6 cases) and non-relapse mortality (infection; 4 cases, chronic GVHD-related death; 3 cases). The 4-year overall survival rate and disease-free survival rate was 43.3% and 35.8%, respectively. The positive role of chronic GVHD was found in patients with high-risk hematological diseases receiving an allogeneic PBSCT.

M. Mohyeddin, Z. Goliaei, K. Alimogaddam, A. Ghavamzadeh, Shriati Hospital (Tehran, IR) Objectives: The number of nucleated cells (NC) infused into the recipient is highly correlated with the probability and speed of cord blood (C.B) hematopoietic stem cell transplantation, therefore it is necessary to obtain CB units with sufficient NCs. Material and methods: 190 CB collected from normal vaginal and cesarean deliveries with placenta in uterus and immediately were processed and assessed for volume, NC count and CD34+ cell count. Then these parameters were correlated with maternal and neonatal characteristics such as mother's age and weight, parity, type of delivery, gestational age, baby's birth weight and height, and sex. Result: In univariate analysis total nucleated cell (TNC) count was significantly increased in: vaginal deliveries (p=0.001), women older than 25 years (p=0.02), women with 1 or 2 parities (p=0.03) and babies heavier than 3100 gr (p=0.001). In bivariate correlations, significant increase was seen in mother's age (p=0.001, r =0.024) longer gestational age (p=0.03, r =0.16) and in heavier babies (p=0.07, r =0.018). In multiple regression analysis significant increase was observed only in vaginal deliveries and heavier babies (p=0.003, r2 =0.07). Conclusion: Our data indicates, vaginal delivery and the heavier babies showed significant increase inTNC by multiple regression analysis. However, the r2=0.07; and even when all factors were considered r2=0.16, which means that considering these factors for CB screening is not a valuable indicators, therefore such screening seems unnecessary.

The cord blood programme of the Florence Cord Blood Bank L. Lombardini, R. Saccardi, S. Urbani, R. Caporale, P. Pizzano, A. Carraresi, P. Bufano, F. Pagliai, A. Bosi, Ospedale di Careggi (Florence, I) The Florence Cord Blood Bank (CBB) started its activity in 01/1996. The Florence CBB is Vision 2000 certified and it is included in the Italian GRACE network (Hemopoietic Cells Amplification Collection Group).GRACE coordinates the activity of most Italian Banks, connecting them to the international network NETCORD. Several collection centers located throughout Tuscany, partecipate in the program. At present 13 collection sites are operative and 6 in training. Cord blood unit (UCB) is collected by specifically trained personnel. After collection UCB is transferred to the laboratory and processed within 44 hours after delivery. As to reduce the storage space, the Unit is concentrated before cryopreservation. Tests are performed to assess the biological characteristics. All valitaded UCB are registered in a local database and included in GRACE and NETCORD. From 1996, 4 ,511 UCB were collected; 822 of these have been cryopreserved and validated for unrelated while 21 for related transplants. Median volume and total nucleated cells (TNC) at collection and after separation, were respectively 86.9mL(34.2-202.2)and 1,460x10e6(510-3,448),and 52mL(13.4-122)and 1,180x10e6 (430-3,308).The most frequent exclusion reason is low TNC count (42%). A total of 271 searches have been performed by transplant centers from Europe(47%), USA(42%), Australia(9%) and other countries (Canada, Israel, Cina, Cile, Argentina). Twenty-eight UCBs have been shipped to Italy(46%),USA(34%),Europe(17%)and

Canada(3%). The median time between search and shipment was 49 days (7-124).Twenty-six patients, 15 female and 12 male, underwent transplantation:the underlying disease was ALL(9), AML(4), CML(2), SCID(2), NHL(2), SWA(2), thalassemia(1) and osteopethrosis(1). Median age and weight were respectively 15 years (0.3-58) and 44Kg (4-88). Two UCBs were HLA-matched, 16 mismatched for one antigen, 9 for two and one for three antigens. Median infused TNC was 4x10e7/Kg (1.2-45). The median time to achieve ANC>500/microL was 23 days (13-144) in 22/25 evaluable pts. Three pts did not engraft (1pt with osteopethrosis,2 adult pts). Acute GVHD was observed in 13/25 evaluable pts. Relapse was observed in 4/25 evaluable pts. After a median follow-up of 124 days (17-1,429) overall and disease free survival were respectively 48% and 46%. TRM was 35%. The aim of CBB should be the storage of high quality products according to banking accreditation programs. FACT accreditation is the new aim of Florence CBB. Kim, H. Jung, Y.H. Kim, J.S. Kim, J. Oh, Y.T. Kim, S.H. Kim, S. Shin, Korea University Medical College (Seoul, KOR) Background: This study was designed to verify the best culture method and condition for the human hematopoietic stem cells differentiation from SNU 3 embryonic stem cell lines developed from Department of Obstetrics & Gynecology, College of Medicine, Seoul National University, Seoul, Korea.. Methods: Cultured embryonic stem cells were classified to control and study groups. Control group was that of which embryonic stem cell mass itself were cultured to LTC-IC assay. Study group was that of which embryonic body manufactured from embryonic stem cell mass were cultured to LTC-IC assay. The effectiveness of hematopoietic stem cell production were compared between control and study groups. To count the number of hematopoietic stem cells differentiated from embryonic stem cells according to culture period, CD34+ cells were separated from LTC-IC assay using MACS method at 1, 3, 5, 7, 10, 14,, 21 days of LTC-IC assay. And then, Separated CD34+ cell were cultured to semisolid methyl-cellulose media to confirm the ability of differentiation capacity to progenitors. Results: No CD34+ cells were separated in control group. Contrarily, CD34+ cells were separated in study group in 3-7 days of LTC-IC assay. CD34+ cells were counted as 0.4-1.2X103, 1.4-2.6X103, 0.5-0.6X103 at 3, 5, 7 days of LTC-IC assay, respectively. CFU-GM colony count was 0.4-1.2X103, 1.4-2.6X103, 0.5-0.6X103, 0.6 X 103 at 3, 5, 7 days of LTC-IC assay, respectively. CD34+ cells were not separated in day 1 and 10 days after of LTC-IC assay in control group. Conclusion: In this study, we can confirm that the differentiation of hematopoietic stem cells from embryonic stem cells is possible in the condition of which embryonic body manufactured from embryonic stem cell mass is cultured to LTC-IC assay and the period of which embryonic stem cells differentiate to hematopoietic stem cells is 3 to 7 day of LTC-IC assay.

B.S.

A. Bhattacharya, M.A. Slatter, C.E. Chapman, D. Barge, T.J. Flood, M. Abinun, A.J. Cant, A.R. Gennery, Newcastle General Hospital, Regional BloodTransfusion Centre (Newcastle upon Tyne, UK) A 6 week male with purpura, thrombocytopenia and reduced platelet volume was confirmed with Wiskott-Aldrich syndrome (WAS) on genetic analysis. In view of the uncertain longterm outlook for WAS, stem cell transplantation was performed. A donor search found 2 umbilical cord stem cell (UCSC) units matched at 10/10 and 6/6 HLA loci, respectively. Cytoreductive conditioning according to EBMT Inborn Errors Working Party guidelines, of rabbit anti-thymocyte globulin 7.5mg/kg, busulphan 16mg/kg and cyclophosphamide 200mg/kg with cyclosporin and methotrexate as graft versus host disease (GvHD) prophylaxis, was given. Fever, dry cough, and clear nasal discharge developed On D -4. On D+1 adenovirus type 1 was isolated from nasopharyngeal secretions. He received the blood group O UCSC unit providing 5.78x10^7/kg mononuclear cells and 0.9x10^5/kg CD34+ stem cells. Because of concerns that a CD34+ stem cell dose of < 1.7x10^5 was associated with markedly inferior rate and likelihood of engraftment, and about potential disseminated adenoviral infection, the second cord unit was requested. Eight days after the first transplant, the blood group AB UCSC was infused without further chemotherapy providing 4.1x10^7/kg mononuclear cells and 0.17x10^5/kg CD34+ stem cells. He was treated with ribavirin and cidofovir after blood became adenovirus PCR positive. Neutrophil engraftment was achieved 34 days after the first cord infusion. Grade I skin GvHD resolved with topical steroid treatment. On D+50, donor 1 and 2 and recipient alleles were detected in the mononuclear cell fraction. The adenoviral infection cleared: he was discharged on D+58. At 9 months post transplant, there was 27% donor 2 erythroid chimerism on blood group analysis with normal platelet volume (consistent with donor platelet chimerism),and count of 104x10^9/L. On separated leukocyte chimerism studies, T cells showed predominance of donor 2, with a trace of donor 1 and recipient; other cell lines showed both donors and recipient. Vaccine antigen responses are currently being assessed. Conclusion: The nucleated cell dose is said to correlate with the CD34+ stem cell dose, but both cord units for this patient contained low CD34+ stem cell numbers despite acceptable nucleated cell counts. A stable trimeric state has been achieved following infusion of two HLA-matched unrelated cord blood units. Immune function is still being assessed but seems normal to date.

L. Salvaneschi, P. Bergamaschi, C. Perotti, C. Del Fante, G.L. Viarengo, L. Bellotti, A. Marchesi, C. Parisi, IRCCS Policlinico S. Matteo (Pavia, I) Introduction: In parallel with large-scale banking of umbilical cord blood (UCB), cost effectiveness and saving of storage space in liquid nitrogen become major issues in UCB Banks policy. Our institution developed a UCB processing method consisting in volume reduction by plasma depletion and cryopreservation in autologous plasma plus 10% DMSO. Material and Methods: Assuming an mean loss of 15% nucleated cells (NC) after processing, we performed volume reduction if NC were >= 940 x 10^6 in the unprocessed UCB. If NC were < 940 x 10^6 but > 800 x 10^6 NC before processing, we cryopreserved UCB as whole blood. Volume reduction was obtained by centrifugation, as shown in figure 1 . Plasma was first removed and than added to volume reduced UCB (VR-UCB), in order to reach a uniform volume, suitable for a unique cryobag. The final volume after addition of cryoprotectant medium (DMSO representing 10%) was also standardised and suitable for storage in a unique canister.

Results: From June 2003, we have cryopreserved 88 VR-UCB units (59,5%) and 60 unprocessed ones (40%). For storage in liquid nitrogen, each VR-UCB needed one canister instead of two on average, as estimated in case of no volume reduction. So we were able to save 50% of storage space. On the contrary, 72% of unmanipulated units were banked in 2 or more bags. Mean NC recovery pre and post processing was 94%. VR-UCB viability, CD34+ cells, CFU-GM and total CFU were compatible with previous data in our Bank (see table 1). Moreover (see figure 2 ), no albumin was required for cryopreservation medium, thus saving money and avoiding risk of infectious disease transmission. A consistent decrease in DMSO utilization was also produced, thereby reducing its toxicity and further improving cost saving. Finally, time of manipulation and charge of work was lower for VR-UCB. Conclusion: In routine activity, our UCB processing strategy revealed to be feasible and time saving. Storage space requirement was greatly reduced and might be further minimised, by lowering the threshold of >= 940 x 10^6 NC and allowing part of unprocessed units to undergo volume reduction. Quantity and quality were preserved in VR-UCB. Neither exogenous substances addition nor expensive devices were employed as well as the use of cryoprotectant medium reagents resulted consistently decreased, thus diminishing DMSO related toxicity and contributing to reduce costs.

S. Davey, S. Armitage, V. Rocha, F. Garnier, J. Brown, C.J. Brown, R. Warwick, D. Fehily, S. Watt, E. Gluckman, A. Vora, M. Contreras, C. Navarrete, The London Cord Blood Bank, National Blood Service, Hopital Saint Louis, Sheffield Children's Hospital (London, UK; Paris, F; Sheffield, UK) Cord blood transplantation has been associated with a lower severity of GVHD compared to that seen following bone marrow transplantation whereas the rate of relapses seems to be unaltered. We have analysed the characteristics of 5500 cord blood units stored at the London Cord Blood Bank, including 59 units transplanted into a high risk and heterogeneous group of patients. Over 40% of the collected units are from ethnic minority origins with a median volume of 79ml (range 40-240) and a median TNC of 85.3 x 107 (range 40-595). Transplant outcome data was analysed in 44 patients with a median clinical follow up of 14 months (range 3-44). The average patient's weight was 28Kg (range 5-80) and the median age was 8 years (range 0.7-40). The median number of nucleated cells infused was 4 x 107/Kg (range 1.10-16). Neutrophil engraftment of 500/mm3 was observed in 33 (74+/-%) patients with an average time of 28 days (range 11-60). The Kaplan-Meier estimate of acute GVHD (grade II or higher) at day 100 was 37+/-7% and in 27 (62%) patients, it was grade I or absent. The overall survival and disease free survival at 2 years was 49+/_8% and 41+/-8 respectively. Furthermore 2 years after transplantation the survival rate was 69% and 54% for patients receiving a 6/6 or 5/6 HLA matched units respectively. Infection was the main cause of transplanted related mortality in these patients. These results indicate that banked cord blood units are a good alternative source of haematopoietic stem cells for transplantation.

Haploidentical allogeneic haematopoietic cell transplantation with CD34 selected stem cells for the treatment of haematologic malignancies: high survival and low incidence of GVHD W. A. Bethge, C. Faul, R. Handgretinger, P. Lang, L. Kanz, H. Einsele, University of Tuebingen, University of Tubingen, St. Jude Children's Research Hospital (Tuebingen, Tubingen, D; Memphis, USA) Introduction: Availability of allogeneic hematopoietic cell transplantation (HCT) is limited by the lack of suitable matched related or unrelated donors. As virtually every patient has a haploidentical family donor, strategies to make haploidentical HCT feasible are being developed. Transplantation of megadoses of hematopoietic CD34+ stem cells have been shown to allow allogeneic HCT across major HLA-barriers. Methods: We report a retrospective study of eleven haploidentical HCTs performed sequentially at a single institution using CD34 selected stem cells. The median age of the patients was 30 (range, 19-47) years. Underlying diagnosis of the patients were acute myeloid leukemia in 3, acute lymphoblastic leukemia in 3, chronic myeloid leukemia in 2, and one patient each with Non-Hodgkin's lymphoma, myelodysplastic syndrome and paroxysmal nocturnal hemoglobinuria. All patients received a conditioning regimen containing 12 Gy total body irradiation, cyclosphosphamide (120 mg/kg) and anti-thymocyte globuline. Etoposide (40 mg/kg) was added in 5 and thiotepa (10 mg/kg) in one patient. All patients received granulocyte colony stimulating factor mobilized peripheral blood stem cells which were T-cell depleted by CD34 enrichment using the CliniMACS device (Miltenyi Biotech, Bergisch-Gladbach). A median of 9.7 x 10E6 (range, 4.12-14.38x10E6) CD34+ cells/kg with a median of 2.2x10E4 (range, 0.42-4.2x10E4) CD3+ T-cells/kg were transplanted. None of the patients received prophylaxis for graftversus-host-disease (GVHD). Results: Engraftment with a median granulocytic take (>0.5 granulocytes/nL) on day 19 (range, 12-42) and full donor chimerism was achieved in all patients. Seven patients received 1-2 doses of donor lymphocytes with 2.5x10E4 CD3+ cells/kg 30-60 days after HCT. Seven of the 11 patients are alive with a median follow-up of 14 (range, 2-72) months. Four patients died, one of relapse, one with multiorgan failure, and two patients with infection. Eight patients achieved complete remission (CR) following HCT of which 6 are still in CR and alive. Three of these patients were transplanted in CR. Acute GVHD°II was seen in one and chronic GVHD (2 limited, 2 extensive) in 4 patients. Conclusion: This study demonstrates that successful engraftment of haploidentical HCT in adults is feasible and safe with a low incidence of GVHD and low treatment related mortality. To confirm these promising results larger prospective studies are ongoing.

Age-related differencies in healthy donors PBSC mobilisation. A single-centre experience D. Lysák, K. Cerná, S. Vokurka, V. Koza, Charles University Hospital (Plzen, CZ) Mobilized peripheral blood progenitor cells (PBSC) are increasingly being used for allogeneic transplantation. The age of the donors increases according to reduced-intensity conditioning transplantation expansion. The aim of the study was to analyze efficacy and safety of mobilization in older compared to younger PBSC donors. Donors and methods: From 1/00 to 10/03, a total of 49 donors received G-CSF to mobilize PBSC for allogeneic transplantation. The median age was 26 (16-30) years for group A (25 donors) and 55 (51-75) years for group B (24 donors). Donors were HLA-matched siblings or unrelated donors. There was no significant difference between the groups concerning sex or weight distribution. G-CSF (10 ug/kg) was administered once daily for 5-6 days (d). CBC, CD34+ cells count were monitored at d 4-6. A total of 84 leukapheresis (LP) were performed using peripheral (67%) or central (33%) vein access, about 3 blood volumes per run were processed. The LP products were analysed for the number of WBC, CD34+ and CD3+ cells. A target for collection was 3x106 CD34+ cells/kg b.w. Results: WBC kinetics was similar in both groups peaking at day 5 (median 43.1 x109/l group A vs 37x 109/l group B, ns). Peak level of CD34+ cells in the peripheral blood was observed at d 5 of G-CSF and was significantly higher for group A (med. 84 CD34+/ul vs 57, p=0.048). Aphereses led to decrease in platelet count at d 6 (med. 159 x109/l group A, 122 x109/l group B, ns). The median number of CD34+ cells in the leukapheresis product per kg b.w. of the donor was: A 5.9 (2.2-11.6) vs B 4.1 (0.9-9.1) x 106/kg (p=0.0095). The median number of CD3+ cells x 108/kg was slightly higher in group B (2.5) than in group A (2.1, ns). To reach the target, younger donors had to undergo less LP (1LP 48%) than the older group (1LP 17%). Target values were reached in 88% (group A) and 83% (group B) of the donors. The side effects of G-CSF treatment and aphereses were minimal in all donors (bone pain, mild citrate toxicity). Conclusions: PBSC can be efficiently mobilized in all donors, regardeless of their age. Older donors compared to younger seem to have a lower response to G-CSF; to obtain a sufficient graft two aphereses are more frequently necessary. Although mobilization was well tolerated in both groups, it still remains to verify complication rate in larger population of older donors and maybe appoint a specific collection criterions.

Alleviation of citrate-related side effects during leukapheresis procedures P. Andersen, L. Vistisen, J. Mikkelsen, E. Dickmeiss, Rigshospitalet (Copenhagen, DK) Objective: Citrate is the anticoagulant of choice in leucapheresis procedures and citrate related side effects caused by the lowering of ionized Ca in the circulating blood are well known. They may -especially during prolonged procedures-be quite intolerable. As suggested by Bolan et al., this may be managed by Ca-infusion. We describe a simple prophylactic procedure, which completely alleviates symptoms of neuromuscular hyperexcitability in the stem cell donors. Materials and methods: Leukapheresis was performed in 36 stem cell donors (15 allogenic and 21 autologeuos) with the Cobe Spectra, and ionized calcium in plasma (piCa) was monitored prior to and at the end of the collection. In 21 of the collections continuously infusion of saline substituted with 43µmol Ca++/ml (125 ml Ca-Sandoz added to 500 ml. saline) was given in the return line. This solution was administered by an infusion pump (imed Gemini PC-1) at a rate of 0.3 times the rate of ACD-A infusion (ml/min) as read by the Cobe Spectra machine. 1 ml. infused ACD-A (containing 126 mmol Citrate) was compensated by the return of 0.3 ml Ca-solution (containing 13.2 mmol Ca). A group of 15 donors had no Ca-infusion. Results: In the group of donors substituted with Ca-solution the piCa concentration prior to collection was 1,25mmol/l (median, range 1,13 -1,35). In the group of donors not receiving Ca, the piCa was1,23 mmol/l ( median, range 1,16 -1,52). The median values for the piCa at the termination of collection were in the two groups 1,1 (median, range 0,95 -1,33), and 0,91 mmol/l (median, range 0,7 -1,22), respectively. The piCa concentration was significant higher (p < 0,001) in the group of donors substituted with Ca during collection, compared to the group not substituted. Conclusion: We found a significant decrease in piCa among 15 donors not receiving calcium substitution, compared to 21 donors receiving calcium substitution. Normal individuals can metabolise approx. 10 mmol citrate/kg. BW/min. Citrate is infused at a rate of 8.9 mmol/kg.BW/min. in a standard leucapheresis by the Cobe Spectra. This delicate balance can seemingly be tipped to the safe side by the administration of Ca in an amount corresponding molarwise to approx. 10% of the infused citrate. Ca substitution is now the standard in all our leucapheresis procedures and since earlier experienced symptoms of increased neuromuscular hyperexcitability , especially among healthy allogenic donors has been avoided.

Kinetic of G-CSF and CD34+ cell mobilisation after once or twice daily stimulation with recombinant human granulocyte stimulating factor (lenograstim) in healthy volunteers. An intra-individual cross-over study N. Kröger, S. Sonnenberg, L. Cortes-Dericks, P. Freiberger, H. Mollnau, A. Zander, University Hospital Eppendorf (Hamburg, D) G-CSF given as splitted dose increased the harvested number of CD34+ cells in comparison to a once-daily schedule but the mechanism is poorly understood. To investigate the schedule dependency of granulocyte colony stimulating factor (G-CSF) in healthy volunteers with respect to CD34+ cell mobilization, we applied the same dose of G-CSF in four healthy volunteers in two different schedules (once-daily versus splitted doses twice daily) in a cross-over design after a wash-out period of three months. CD34+ cell kinetic in serum was performed as well as G-CSF serum kinetic on day 1 and 4 after stimulation. In all volunteers the twice daily schedule leads to a higher CD34+ cell count after four days of G-CSF stimulation (median 94.5 vs 47 /µl; p=0.05). On day one and four, there was a higher Cmax of G-CSF serum level after the once-daily application (15175 vs 6859 pg/ml and 7440 vs 2388 pg/ml, respectively) than after the twice-daily schedule. In contrast, after the once daily application the Cmin of G-CSF serum level was lower than after the twice-daily schedule (663 vs 1361 pg/ml and 246 vs 441 pg/ml, respectively). No difference of area under the curve (AUC) for G-CSF was seen on day 1 and day 4 after G-CSF stimulation. We suggest that application of G-CSF twice-daily leads to a higher CD34+ cell mobilization due to a higher Cmin and therefore to a more continuous serum baseline level resulting in a more efficient CD34+ cell mobilization.

Heterogeneous loss of the Y chromosome in leukocyte lineages after SCT from a non-HLA-identical family donor I. Buño, P. Nava, A. Simón, M.A. Muñoz, P. Balsalobre, R. Carrión, D. Serrano, A. Gómez-Pineda, J.L. Díez-Martín, Hospital Gregorio Marañón (Madrid, E) Introduction: Loss of the Y chromosome is a known phenomenon considered physiological in males of advanced age. Recent advances in the biology of stem cell transplantation (SCT) have favored the use of donors of increasing age. Objective: To evaluate the influence of the loss of the Y chromosome in a 56 years old male donor on the outcome of SCT. Material and methods: A 33 years old female with AML in relapse after autologous SCT underwent allogeneic SCT from a non HLA-identical family donor (NIFD, father). Conditioning regimen consisted in Tio/Flu/Mel+ATG. The PB graft was T-cell depleted by CD34+ immunomagnetic selection (CliniMACS, Miltenyi Biotec) to contain 7.3 CD34+ and 0.092 CD3+ cells x10e6/Kg. Chimaerism quantification was performed by FISH for the sex chromosomes (CEP XY, Vysis) on PB and BM smears as well as on leukocyte lineages (CD3+, CD4+, CD8+, CD19+ and CD15+) purified from PB by immunomagnetic selection (MiniMACS, Miltenyi Biotec). Results: FISH analysis of donor PB showed loss of the Y chromosome (X0 cells) in 30% of the cells. The patient showed complete chimaerism (XY/X0 cells) in all samples analyzed post-SCT excepting for the PB sample obtain on day +13 (2% XX cells). XY/X0 cell ratio showed different values and evolution throughout the post-SCT period, in the different sample types analyzed. XY/X0 cell ratio was constant around 60/40 in BM, CD8+, CD19+ and CD15+ cells. However, it varied from 55/45 to 70/30 in PB and CD3+, at the expense of CD4+ T-cells (from 70/30 to 95/5). The relative increase of XY T-cells (mostly CD4+ cells) during the post-SCT period could be due to a reduced rate of generation/maturation or to an increased rate of elimination of X0 T-cells. Moreover, the patient received immunosuppressive therapy (CsA, 3 mg/Kg/d x days -1 to +27) as GVHD prophylaxis and high dose of I.V. steroids (from day +60 to death) for the treatment of neuropatic toxicity. It is possible that X0 lymphocytes show an increased sensitivity to immunosuppression, which would compromise the immune capacity of the patient, who in fact died from generalized sepsis on day +201 post-SCT. Conclusion: Male donors older than 50 years of age should be studied by FISH on PB samples in order to rule out the loss of the Y chromosome. If this is detected, a close follow up of the recipient patient must be performed to gain insight into its implication in the outcome of SCT, and eventually validate or censure the use of such donors.

N. Maillard, F. Audat, L. Dal Cortivo, M.C. Quarre, M. Rachid, A. Fisher, B. Varet, M. Cavazzana-Calvo, F. Lefrère, Hôpital Necker-Enfants Malades (Paris, F) We report the harvests of 103 consecutive allogenic adult stem cell donors between 1996 to 2003. G-CSF ( 10 microg/kg/d) was administered s/c over 5 consecutive days. Donors were seen on day 4 of G-CSF to research side effects and perform peripheral WBC and CD34+ cells counts. If donors WBCcount was >50 x 10e9/l, and/or suspicion of splenomegaly, G-CSF was reduced (5 microg/kg/d). LK were performed on day5.1 or 2 LK were performed until the target CD34+ cell dose was collected (3 to 8 x 10e6 x CD34+ cells/kg of the intended recipient). The median age was 37 year (19 to 67), 8 of them were > or= 60 years. The median WBC count, was 41 x 10e9/l on day4. 10% of donors exhibited a WBC count above 50 x 10e9/l. For all these donors, G-CSF dose was reduced to 5 microg/kg. None of donors presented clinical increase in spleen size. Only 6 donors need a femoral venous catheter. The median PB CD34+cells count before LK, was 43/microl and 64/microl on day4 and day5. 90 (88%) and 11 (10%) donors satisfied the minimal count 3 x 10e6 CD34+ cell/kg of recipient's body weight with one and two LK, respectively. 2 additional donors reached only 1.58 and 1.9 x 10e6 CD34+ cell/kg collected after two consecutive LK.The hematologic reconstitution was effective for the 2 recipients. None of the donor needed a rescue by bone marrow aspiration. All donors with 1 LK insufficient on day5, presented a PB CD34+cell count <20/microl on day 4 while all donors with a PB CD34+cell count >20/microl on day 4, were successfully collected on day 5 with one LK. Higher donors'age was associated with a reduced PBSC mobilization. 18% of donors older than 40 years old had a PB CD34+cell count below 20/microl on day 5 while only 4% of younger donors exhibited such similar poor PBSC mobilization. 10 donors presented a post LK platelet count <50 x 10e9/l. The nadir varied according the number of blood masses processed during the LK. A total of 40 donors had a second LK on day 6 with a median platelet count before and after PBSC collection of 119 x 10e9/l (39-220) and 56 x 10e9/l (18-139). 15 out of this 40 donors presented a platelet count below 50 x 10e9/l after the procedure. None of our donors presented hemorragic complication. Every tested donor presented a normal blood cell count one month after the PBSC collection.

The structure and function of the Czech Bone Marrow Donor Registry and Cord Blood Bank E. Ivaskova, L. Kupkova, I. Kolarikova, D. Vagnerova, P. Kobylka, I. Striz, Czech Bone Marrow Donor Registry (Prague, CZ) Introduction: The Czech Bone Marrow Donor Registry (CBMD -CS1), since the beginning of its establishment in 1991, has become a member of the World Marrow Donor Association (WMDA) and Bone Marrow Donors Worldwide (BMDW), further more, in 1997, an agreement with the National Marrow Donor Program (NMDP) USA was made. Methods: The CBMD is a coordinating center which provides the following activities: -Recruitment, record-keeping -Searching unrelated donors for Czech patients -Examination and preparation of potential unrelated donors of haematopoietic stem cells for bone marrow or peripheral cells collection (PBSC) -Cord blood units searches in umbilical blood banks -Organizing haematopoietic stem cell transportation from international registries and coordinating all these activities with other institutions Results: The CBMD database includes 16751 unrelated donors and 1044 cord blood units until November 2003. The Prague Cord Blood Bank is the first cord blood stem cell registry in the Central Europe. From 1994 to November 2003, the CBMD found and identified matched unrelated donors for 178 Czech patients.There are about 20-42 unrelated stem cell transplants per year in the Czech Republic. A prevalent manifestation, also in the Czech Republic, is an increasing number of PBSC transplantations. In the last 5 years, the transplantations using PBSC have reached the number of 53. During the last 9 years, the Czech donors provided their haematopoietic stem cells totally for 40 national and international patients. From 1994 to 11/2003, there have been transplanted 119 bone marrow products, 53 PBSC grafts and 6 cord blood units. The transplantation rate of donors from different registries is as follows: 44% -Germany, 15% -CBMD, 12% -NMDP, 9% -UK, 5% -France, 2% -Italy and 1% -Belgium, Switzerland, The Netherlands, Wales, Sweden, Spain, Canada, Australia. Conclusion: CBMD is a HUB registry in the Czech Republic and is closely connected with the Cord Blood Bank of Prague. Since its foundation, the CBMD is connected with international registries through the mediation of BMDW, later on through the NMDP and ZKRD-EMDIS. During 12 years of its existence the CBMD registry has found unrelated donors for 178 patients . 40 CBMD donors have donated the haematopoietic cells both for national and international patients.

Rituximab for pure red cell aplasia after AB0 incompatible allogeneic stem cell transplantation S. De Matteis, F. Sorà, L. Laurenti, P. Chiusolo, G. Zini, N. Piccirillo, G. Farina, M. Tarnani, G. Leone, S. Sica, Universita Cattolica S. Cuore (Rome, I) Pure red cell aplasia is a rare complication of allogeneic stem cell transplantation (SCT) performed across the ABO complex. This complication is thought to be due to the persistence of recipient isohemagglutinins produced by residual B cells with the result of a profound inhibition of erythroid precursors in the bone marrow. Treatment relies on steroids with or without erythropoietin (Epo) extended immunosuppression, plasma exchange or donor stem cell boosting. Recovery of erythropoiesis is difficult and often incomplete and treatment itself may induce further complications. A recent case report has been published in the successful treatment of PRCA after SCT in a child treated with a single dose of rituximab. On the basis of these results we describe here a young female with PRCA after ABO incompatible (donor AB Rh+; recipient 0 Rh+ ) allogeneic SCT for secondary leukemia. SCT was performed using G-CSF mobilized peripheral blood stem cell (CD34+ 6.3x106/kg) from her HLA-identical donor after standard myeoablative conditioning regimen BuCy2.GvHD prevention included cyclosporine A and MTX. The post-transplantation course was unremarkable and although the reticulocyte count did not recover, the patient was discharged at day +41. Bone marrow was normal unless for erythroid precursors which were 1-2% of total cellularity. During follow-up the patient remained anemic with extremely low reticulocytes and requiring at least 1 unit of RBC per week. Epo 10.000u/every other day was added by day+55 but was not effective in reducing the transfusion requirements. Considering the long-standing PRCA and the absence of response to steroids, cyclosporine and Epo a 2nd stem cell donation with CD34+ cell selection was planned. In the mean time the temporary unavailability of the donor prompted us to administer rituximab at the single dose of 400mg on day+122. After 3 weeks no response was observed then a 2nd dose of rituximab was administered. Reticulocytes count increased slowly, reaching a value of 2.4% before the 3rd dose and 1 week later reticulocytes showed a striking increase by day +171 when a 4th dose was administered. Hgb level normalized by day +171 when complete switch to blood donor type was observed. The patient is in CR with limited cGvHD 12 months after transplantation. This report represent to the best of our knowledge the successful treatment of post-transplantation PRCA with rituximab in an adult patient. The graft-versus-leukemia (GVL) effect in the treatment of acute leukemia has been extended by the introduction of nonmyeloablative allogeneic stem cell transplants (SCT) which have made it possible to perform SCT in patients who could otherwise not have undergone myeloablative conditioning. The aggressive use of the GVL treatment mode is demonstrated by the following patient history. After 2 years with an untreated idiopathic myelofibrosis, a 57 year old patient was diagnosed with secundary acute myeloid leukemia with a deletion of chromosome 20 and trisomy 8, both associated with poor prognosis. The leukemia proved refractory to two remission induction courses with idarubicin, cytarabin and etoposide. Because of his age and relatively poor general condition we opted to perform a non-myeloablative ´mini-allogeneic´ sibling SCT after conditioning with fludarabin (30mg/m2/d , 4days), cytarabin (2g/m2/d, 4days) and rabbit ATG (10mg/kg/d, 2days). Anti-GVHD prevention consisted of Cyclosporin A (5mg/kg/d) which was tapered off after 3 months. Peripheral blood (PB) and bone marrow showed persisting leukemia. Donor lymphocyte infusions (DLI) were started at day +92 after transplant (1x107 CD3+/kg) and repeated on days +121 (2x107 CD3+/kg), +135 (1x108 CD3+/kg), +165 (2x108 CD3+/kg) and +198 (1.4x108 CD3+/kg). In total 5 DLI were administered with increasing dosages. At no time did the patient show evidence of acute or chronic GVHD. Chimerism analysis showed 36% donor at day +29, with decreasing presence of donor cells till >95% acceptor was reached at day +177. At day +267 patient underwent a second ´mini-allogeneic´ SCT after conditioning with fludarabin (30mg/m2/d , 4days), cyclophosphamide (1g/m2/d, 3days) and rabbit ATG (10mg/kg/d, 2days). Anti-GVHD prevention consisted of cyclosporin A (5mg/kg/d) which was stopped already at day +16 in the presence of 44% blasts in the PB. In the absence of acute GVHD DLI was administered on day +27 (1.2x108 CD3+/kg) resulting in grade II GVHD (predominantly skin) and a decrease in the number of circulating blasts, and at day +48 (3.5x108 CD3+/kg) resulting in grade II GVHD (skin and gut) which was treated with steroids and cyclosporin A. The patient is currently in persisting complete remission with a >95% donor chimerism at day +260. This case demonstrates the value of a second non-myeloablative SCT and an aggresive strategy of DLI in order to maximally stimulate the GVL effect in a refractory leukemia patient. (1992) (1993) (1994) (1995) (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) Y. Hakobyan, Hematological Center (Yerevan, ARM) Objective: In Armenia, patients with acute leukemia (AL) made up 30-45% of all hematological cases (over 200 cases in year), applied in 1992-2003 for medical care to the Hematology Center. The aim of our work is investigation of AL epidemiological and diagnostics problems that are very actual questions in practical hematology. Design and Methods: We heve analyzied over 2150 hematological cases from 1992 to 2003 and about 710 were AL patients cases. Results: Absolute number of patients with malignant hematological disorders, over the period has increased. The specific weight of the AL within the overall group of hematological malignancies has gradually decreased from 55% in 1992 to 28% in 2003. The proportion of the so-called "non-differentiated" acute leukemia incidence was also considerably decreased. In 1992, at least 15 cases of the "non-differentiated" AL were diagnosed (25% of all AL), versus no such a case, identified in 2003. Analyzing the epidemiology of the registered AL morbidity, we come to a conclusion, that over the years it was noticed to come most of all from the following regions of Armenia: city of Yerevan, Armavir, Shirak, Kotayk, Aragatsotn, Ararat, compared with just few patients, applied from those regions (marzes) of Syuniq, Vayq, Tavush, Lory and Gegarkhuniq. Thanks to the development and advancement of the diagnostic methods and tools, a significant re-arrangement of morbidity has taken place in the composition of both blood tumors in general and AL, in particular. Such a reduced demand for the care could also be interpreted as a result of limited information, usually communities get on advances in cancer management and patients' opportunities to receive qualified clinical care at the specialized institutions, such as Hematology Center. Conclusions: Detailed program which include cancer public awareness, health promotion and professional education about advances in blood disease management and other fatalities is necessary and should be perceived in those regions of Armenia, where registered AL incidence has found low or not detected at all. In those regions with documented AL morbidity, more intensive efforts and departure consultations should be implemented for early diagnosis and referring all potential cases to the specialized institutions.

S. Vokurka, V. Koza, P. Jindra, K. Cerna, T. Karvunidis, P. Skopek, M. Svojgrova, E. Bystricka, A. Volfova, L. Novak, Charles University Hospital (Pilsen, CZ) Introduction: As precise treatment strategy of acute leukemia (AL) depends on its prognostic stratification, risk factors determination is a basic procedure at the AL diagnose. The whole strategy, however, is also influenced by presence of HSC donor. In that point of view, Core family search (CFS) as a basic search procedure should be realised as soon as possible and its duration carefully watched. We have set this analysis to check our capability of managing CFS in AL pts. Objectives: To analyse number, duration and outcomes of CFS in newly diagnosed AL pts, with the age of up to 65, treated by induction chemotherapy in our dept. in 2000-02. Methods: Retrospective analysis. Pts were analysed for presence and number of HLA-typed siblings, CFS start, duration, outcomes, complications and reasons for unaccomplished CFS. Results: n=86 pts (66 AML, 20 ALL) with median age 47 (19-65) were analysed. 64/86 pts (74%) had at least one sibling and CFS was started in 55/64 (85%) of them. Median number of days from diagnose to CFS was 5 (1-95), resp. 7 in 2000, 2 in 2001, 3 in 2002. In 40 pts (72%) the CFS was started by day 7 since diagnose. Median CFS duration was 12 (2-64) days. In 44 (80%) pts the CFS outcomes were available by day 28 since diagnose. Reasons for 11 (20%) prolonged CFS: weak family cooperation 7/11 (all pts were older then 50), delayed indication to CFS 6/11 (all pts older then 60). HLA-identical donor was found for 21 (38%) pts. Reasons for unaccomplished CFS in 9/64 (15%) pts: a sibling´s bad health status -4/9, a patienst´s status contraindicating SCT -2/9, unknowen -3/9. 90% of these pts were older then 55, with median age 62. Conclusions: Our management of CFS in AL pts appears to be satisfactory. Chance for finding a sibling-donor was relevant and majority of pts were indicated for CFS early after diagnose with CFS results available by the end of induction chemotherapy. Some more improvement, however, could be done. Delay in CFS indication, duration and cooperation was predominantly observed in elderly pts. CFS must be iniciated and finished as soon as possible, without discriminating elderly pts enrolled in an induction therapy. CFS results must be known by the end of induction therapy in order to help manage further treatment strategy, and if needed, other searches -extended family and unrelated -were not postponed.

Outcome of autologous stem cell transplantation using TBIcontaining or non-TBI-containing conditioning regimens as one of the post-remission treatments in patients with acute myeloid leukaemia in first complete remission W. Min, H. Kim, K. Eom, S. Park, Y. Park, Y. Kim, S. Lee, D. Kim, J. Lee, C. Park, C. Kim, Catholic HSCT Center (Seoul, KOR) A total of 177 newly diagnosed adult AML patients were treated in first CR(CR1) with conditionings using a TBI-containing or combination of triple-alkylating agents followed by autologous transplantation(AT). Cytogenetic risk groups were classified and most patients received mobilized peripheral blood stem/progenitor cells (PBSCs) and some received harvested bone marrow cells or bone marrow plus PBSCs in CR1 after conditioning. The infused cell dose consisted of a median of 4.2+/-2 (range, 1.2-17.1) x 106/kg CD34+ cells. With the median follow-up of 46 months (range, 2-125 months) after CR1, the estimated 5-year disease-free survival (DFS) rate overall was 66%(95% confidence interval[CI], 61%-71%) and the event-free survival rate at 5 years were 58%(95% CI, 53%-63%). According to the cytogenetics for patients with AML other than M3 subtype, the long-term DFS were 71.4%, 25% in order of favourable, unfavourable risk groups. Forty three patients were relapsed (43/177, 24%) at the median 9 months post-transplant(range, 1.5-60 months). Overall 7 patients(4%) died associated with AT. Infused CD34+ cell dose(p=0.0392), percentages of the initial peripheral blood leukemic blasts(p=0.0958), and chromosomal abnormalities(0.0617) were significantly associated with survival. In conclusion, our two kinds of conditioning regimens in AT are feasible for adults with variable risk AML followed for over a 10year period. Further, the exact causes of higher relapse rate associated with higher CD34+ content in the mobilized PBSCs are warranted.

Matched unrelated haematopoietic stem cell transplantation in a paediatric patient with Down's syndrome and acute lymphoblastic leukaemia K. Ehlert, A.H. Groll, S. Bielack, H. Juergens, J. Vormoor, University Children´s Hospital Muenster (Muenster, D) Children with Down syndrome (DS) have an increased risk for leukemia. Conventional high-dose chemotherapy and HSCT are often described to result in higher therapy-related toxicities as in non-DS patients, especially severe mucositis, infections and airway problems. These side effects are partially attributed to altered pharmacokinetics in DS patients, leading to increased sensitivity to drugs such as MTX and cytosine arabinoside. We describe the case of a 16-year-old boy with ALL and DS, undergoing peripheral blood SCT from a matched unrelated donor (MUD) in first CR. According to treatment protocol ALL-BFM 2000 delegation to HSCT resulted from prednisone-poorresponse on day 7 with more than 1000leukemic blasts/mikroliter in peripheral blood and more than 25% leukemic blasts in bone marrow on day 33 of induction therapy. The preparative conditioning therapy (TBI 12Gy, Etoposide 60mg/kg)was tolerated well. Peripheral blood stem cells were transfused at a dose of 10,75x10E6/kg CD34+ cells. GvHD prophylaxis consisted of ATG (40mg/kg), CsA and MTX (10mg/m2 day +1 and +3). Engraftment was documented on day +12. Early posttransplant complications included moderate, acute GvHD of the gastrointestinal tract, grade 2, reflux esophagitis grade III, suspected VOD, CTC grade 1, and severe mucositis, CTC grade 4. He quickly recovered from these complications and could be discharged from hospital on day +25. Complications after day +100 included a blood stream infection with Enterococcus faecalis and autoimmune thyreoiditis, which was clinically asymptomatic. The boy relapsed 15 months post-transplant, but up to that point he had been in an excellent condition with a good quality of life and no signs of chronic GvHD. In conclusion, even in DS patients hematopoietic stem cell transplantation from unrelated donors and a myeloablative conditioning regimen can be performed with tolerable toxicity.

Isolated ocular relapse in a patient with PH+ acute lymphoblastic leukaemia in second complete haematological remission with imatinib J. Martínez, J. de la Rubia, M. Hurtado, F. Moscardó, G. Sanz, L. Senent, M. Sanz, Hospital Universitario La Fe (Valencia, E) Introduction: Although involvement of the iris and anterior chamber of the eye is a recognized but unusual manifestation of leukemia in patients with acute lymphoblastic leukemia (ALL), it has never been described in the setting of Ph+ ALL. We report a 55-year old female diagnosed of Ph+ ALL in second complete remission (CR) who developed an isolated ocular relapse on imatinib therapy. Case Report: ALL was diagnosed in September 1999 and CR was achieved with first-line induction therapy. On February 2000, she underwent allogeneic bone marrow transplantation from an HLA-identical sibling donor. Mild acute (grade I) and extensive chronic GvHD was observed after transplant. On May 2001, 15 months after transplantation, first hematologic relapse was detected. At that time, she was included in a phase II study with Imatinib (600 mg/day). Second hematological and molecular CR (BCR/ABL negative by RT-PCR) was achieved two months later. On March 2003, while in second CR, she was programmed to undergo cataract surgery of the right eye. At that time, ophtalmic examination of the opposite eye was carried out before surgery showing uveitis with hypopyon. Anterior chamber paracentesis was decided and cytological and molecular survey of the aqueous humor revealed BCR/ABL positive leukemic cells. Results of chimeric studies of blasts cells showed receptor origin. At this time bone marrow was still in CR and BCR/ABL rearrangement was negative. Topic steroids were administered and Imatinib dose was increased to 800 mg/day. However, two months later (June 2003) bone marrow molecular relapse was detected and mixed chimera (98% of cells from donor origin and 2% of receptor) was present in bone marrow. Donor lymphocyte infusion and local radiotherapy was planned, by the patient died due to septic shock shortly thereafter. Conclusion: Our finding illustrates a delayed leukemic ocular recurrence despite continued treatment with Imatinib and hematological and molecular CR. This fact suggests that Imatinib may not penetrate into the eye that may become a sanctuary. Besides, longer survival observed with Imatinib therapy in the subset of high-risk patients, raises the possibility of the appearance of previously not described extramedullary leukemic recurrences in these cases.

S. Machherndl-Spandl, O. Krieger, H. Kasparu, M. Girschikofsky, J. Koenig, R. Koplmueller, O. Zach, P. Oppitz, M. Fridrik, D. Lutz, Elisabethinen Hospital, BH Schwestern, AKH (Linz, Wels, A) Since the coincidence of Ph+ ALL with other hematologic malignancies is a rare event, we report on two female patients suffering from Ph+ acute lymphoblastic leukemia with the coincidently appearance of a B-cell-chronic lymphatic leukemia or a Myelodyspastic syndrome (RAEB), respectively. Both patients received an ALL induction protocol (GM-ALL) and achieved complete hematologic remission before transplantation. The first case is that of a 47 year old woman diagnosed with Ph+ ALL and a B-chronic lymphatic leukemia (bone marrow infiltration with 20% B-CLL cells, CD5/CD19 pos.). She was transplanted in August 1999 with bone marrow from her HLAidentical brother and acquired an acute GvHD Grade III (Skin III, Liver 0, GI III) followed by an extensive chronic GvHD of the skin (sclerodermiformal type). At the time of transplantation a persisting bcr-abl positive clone as well as CD5/CD19 positive cells were detectable in the bone marrow. A cytogenetic remission was achieved 6 months after TX. Negativity of bcr-abl rearrangement in the bone marrow was demonstrated 6 months (day + 161) after TX. 52 months later the patient is alive in CCR with a complete donor hematopoesis and without any sign of a malignancy. In the second patient, a 43 year old woman, a Ph+ acute lymphoblastic leukemia and a Myelodysplastic syndrome (RAEB) with an infiltration of the marrow by 10% myeloid blasts were diagnosed (Cytogenetics: 44-46,XX,t(9;22), complex aberrations i.e. der(1),t(1;?),-13,+1-2mar).

After induction therapy a cytogenetic complete remission was achieved while dysplastic changes and a bcr-abl positive clone persisted. In April 2002 she was transplanted with peripheral stem cells from her HLAidentical brother and suffered from an acute GvHD Grade III (Liver), which turned into a limited chronic GvHD (sicca syndrome). One month after transplantation a complete moleculargenetic and hematologic remission without myelodysplasia could be demonstrated in the bone marrow. Until now (19 months after TX) the patient is alive in CCR. Conclusion: ALL-induction treatment followed by allogeneic stem cell transplantation in first CR is a curative treatment for patients with high-risk ALL and coincident hematologic disorders. A graft versus leukemia effect may be essential for persistent elimination of the Philadelphia chromosome-positive cell clone together with a second clonal hematologic disease.

Stem cell transplantation for haematological malignancesthree year single-centre experience B. Georgievski, L. Cevreska, N. Siljanoski, A. Stojanovik, O. Karanfilski, Z. Stojanoski, I. Panovska, A. Pivkova, R. Dukovski, P. Gerasimova, V. Milenkov, Medical Faculty (Skopje, MK) Between September 2000 and October 2003, 60 SCT were preformed in 56 patients with hematological malignances, 43 autologous SCT (PBSC 21, BM 21, BM+PBSC 1) and 17 allogeneic sibling matched transplantations (PBSC 13, BM 4). All patients were isolated with facilities of HEPA filtration and reverse barrier nursing. Diagnosis of patients undergoing SCT were AML 30 (50%); ALL 2 (3,3%); CML 4 (6,6%); AA 1 (1,6%); HD 8 (13,3%); NHL 8 (13,3%); MM 7(11,6%). The age of transplant recipients was between 22-63 years (mean 41) in autologous SCT and 20-54 years (mean 34) in allogeneic SCT. The number of infused mononuclear cells in the autologous setting ranged from 1,63-8,0x108/kg (mean 3,41) with median CD 34+ cell dose 3,2x106/kg (1,2-6,8) and 1,71-6,0 x108/kg (mean 3,85) with CD34+cell dose 3,9x106/kg (2,1-7,2) in allogeneic SCT. Disease status prior autologous SCT revealed 31 (72%) patients in complete remission (CR) and 16 (28%) with refractory disease. In allogeneic recipients only 4 (24%) patients were in CR, while in the rest of patients 13 (76%) SCT was preformed in active disease as only possible therapeutic option. Conditioning regimens consisted of Bu-Cy 2+M for patients with acute leukemia; BEAM and ICE for patients with HD, NHL; high dose Melphalan for MM patients and nonmyeloabative regimen with Flag/Ida. In the presented group 2 patients with AML had double and 1 patient with NHL received triple transplant. The median interval from initial diagnosis until SCT was 3-24 months (mean 12). Growth factor was administered to all patients until neutrophil engraftment. Median time to ANC>0,5x109/L and Plt >20 x109/L was +11days (9-21) and +15 days (8-32) for autologous SCT and +12 days (10-24) and +14 day (9-24) for allogeneic SCT respectively. One patient died of early transplant complication on day +10 due to pneumonia. Median follow up for autologous recipients was 12 months (1-31) and 35 (80,49%) patients are alive in CR and 8 (19,50%) patients died due to relapse of disease or tumor progression. In the group of allogeneic recipients median follow up was 12 months (2-30) and 11(63%) patients are in CR still alive and 6 (37%) died due to acute GVHD (3), chronic GVHD(1) and infective complications (2). Hematopioetic stem cell transplantation can be carried out in Republic of Macedonia in a specialized BMT unit. Preliminary results show a survival rate over 80% in autologous group and over 60% in allogeneic group of patients.

Successful peripheral stem cell mobilisation and effective in vivo purging with gemtuzumab ozogamicin in a patient with secondary acute myeloid leukaemia S. Lueff, M. Réti, P. Reményi, Á. Bátai, A. Tremmel, G. Kriván, T. Masszi, St. László Hospital (Budapest, HUN) Patient: A 52-year-old female patient was diagnosed in August 2002 with secondary acute myeloid leukaemia. Three lineage dysplasia, myelomonocyter immature cell morphology and 98 % CD 33 positivity of blast cells on flow cytometry were observed. She received low dose fractionated spinal irradiation 8 years before the diagnosis because of non-malignant bone disorder. The metaphase cytogenetics revealed 47,XX, +8 karyotypes. The patient was treated with high dose cyosine-arabinoside + idarubicin+ etoposide as an induction therapy. Complete morphologic remission was achieved after one course but 1/20 47,XX+8 karyotype remained at the same time, which disappeared after the first consolidation therapy. The postremission therapy consisted of three courses of high dose cytosine arabinoside + two cycles of gemtuzumab ozogamicin. The patient had no HLA identical sibling donor. The quality of remission was examined by metaphases cytogenetic and chromosome 8 centromer fluorescent in situ hybridisation (FISH) on 1000 cells and all of those were negative. Gemtuzumab ozogamicin therapy was decided because unfavourable prognostic features. The patient is still in complete remission for 9 months following the cessation of therapy. Mobilisation: Six mg/m2 gemtuzumab ozogamicin +G-CSF was given (5 microgram/kg/day) followed by 18 days period of agranulocytosis, without significant diminishing of platelet and red blood cell number. The peripheral blood CD 34 + concentration was measured on day 16 (34/microliter) and peripheral leukapheresis was done. The amount of CD 34 + cells collected by 2 consecutive apheresis days apheresis were 4,28x106/kg. The FISH examination revealed a 3/1000-cell positivity in the collected specimen. At the same time 0/2000 cells showed 8 trisomy by FISH in the bone marrow. One month later a second course of gemtuzumab ozogamicin was given. At that time 5,15x 106/bwkg CD 34+ cells were collected. The interphases FISH examination of the second collected specimen showed no trisomy 8 positive cells between 2000 cells. Conclusion: Gemtuzumab ozogamicin 6mg/m2 iv plus G-CSF (5mikrogram/kg/day) mobilised sufficient number of CD 34+ cells repetitively following 4 courses of high dose cytarabine containing regimen. Consecutive doses of gemtuzumab ozogamicin can effectively purge minimal residual disease to the level undetectable by FISH.

Allogenic stem cell transplantation in a patient with acute myeloid leukaemia and HIV, hepatitis C infections S. Shamansky, I. Fedotova, J. Popkov, A. Burya, O. Rukavicin, Main Military Clinical Hospital (Moscow, RUS) We report on the first case in Russia of allogenic stem cell transplantation (allo-SCT) in patient with AML. The leukemia was complicated by HIV and hepatitis C infections. Our patient is 47 years old, male. The diagnosis of AML (FAB-M2 with t(8;21) was made on July 2002. The treatment included two induction remission courses with conventional doses ("7+3" -Ara-C + Dauno). The hematological remission was achieved after the first induction course. On September 2002 we diagnosed the acute hepatitis C with severe cytolytic syndrome (positive PCR results: HCV-3,4 x 10^8copy/ml). On December 2002 HIV-infection IIB study was diagnosed (positive PCR results: HIV1 -3,76 x 10^5copy/ml). The patient didn't recieve chemotherapy. So, allo-SCT was chosen the treatment of AML. 26.03.2003 the patient received a SCT (MNC -6,05 x 10^8/ kg, CD34+ -2,27 x 10^6/kg, CD3+ -1,75 x 10^8/kg) from HLAidentical sibling after conditioning with fludarabin 150 mg/m2 and melphalan 140 mg/m2. GvHD prophylaxis consisted of cyclosporin and a short course of methotrexat. The time of neutrophil recovery was 16 days. Patient was treated with antiviral therapy. On D+60 positive PCR results: HCV-1,1 x 10^8copy/ml, HIV1-4,23 x 10^5copy/ml. On D+180 positive PCR results: HCV-1,0 x 10^8copy/ml, HIV1-1,47 x 10^5copy/ml. PCR analyses on D+34, +60, +160 showed complete molecular remission with full donor chimerism. Patient had acute GvHD (grade II), local skin chronic GvHD from D+140 and CMVenteritis (positive PCR). At presents (D+250) our patient is alive and is receiving antiviral, anti-cGvHD therapy. Conclusion: We didn't recive the data for the activation of HIV after allo-SCT over the follow up. HIV-infection isn't an absolute contrindication to allo-SCT in patients with AML.

Cytokine profiles as a response predictor for immunosuppressive therapy in patients with aplastic anaemia W. Min, H. Kim, J. Lee, Y. Park, Y. Kim, K. Han, C. Kim, Catholic HSCT Center, Dept. of Clinical Pathology (Seoul, KOR) Objective: We tried to reveal the direct evidence for which factor is useful as a predictive marker of response to the conventional immunosuppressive therapy in patients with aplastic anemia. Methods: Thirty-four consecutive patients with moderate or severe idiopathic aplastic anemia patients were studied. Antithymocyte globulin(ATG) was administered for 5 days in combination with methylprednisolone in addition to short-term G-CSF. Cyclosporin A was administered for at least 6 months. Hematologic responses were also monitored through at least 6 months after the intiation of treatment. For an assay of levels of various cytokines, we used flow cytometry method at each time point of pre-ATG and post-ATG at day 7 in addition to at 2 months. Results: Overall response rate to the ATG therapy was 38.2% (13 of 34). We found very close clinical associations in the serum levels of IL-10 and IL-6 at day 7 post-ATG (p=0.0190, p=0.0026 in order) and also of IL-4 and IL-6 at pre-ATG (p=0.0011, p=0.0217 in order). Conclusion: We suggest a predictive value of some cytokines as prognostic factors in patients with aplastic anemia pre-and post-ATG.

M. Koldehoff, A. Elmaagacli, R. Trenschel, M. Ditschkowski, N. Steckel, M. Hlinka, R. Peceny, D. Beelen, University Hospital of Essen (Essen, D) PNH is a rare acquired clonal hematological stem cell disorder characterized by intravascular hemolysis and is associated with bone marrow failure syndromes including aplastic anemia, myelodyplasia and leukemia. The only potenially curative therapy is an allogeneic HSCT, which can restore normal bone marrow function. Between October 1984 and July 2002, six patients (pts) (3 male, 3 female) with PNH underwent allogeneic HSCT in our center. The median age at transplant was 33.4 (17-46) years. Two pts received TNI (4 x 3.2 Gy), one pt TBI (4 x 2,5 Gy), one pt BU/CY, one pt BU/ATG/TNI (4 x 3.2 Gy) and one pt TBI (4 x 2.5 Gy)/CY/TT/ATG for conditioning therapy. Five pts received graft from HLA-identical donors, one from a partially HLAidentical donor. Four pts had sibling donors and two pts had MUD. Graft source were bone marrow in five cases and CD34 enriched PBSCT in one case. Two pts died: one pt who was conditioned with TNI at post-transplant day +4 from septicemia, the other pt who was conditioned with BU/CY at post-transplant day + 783 from aspergillus infection. The other four pts showed three lineage engraftment and normal expression of CD55 and CD59 antigen by flow cytometric analysis. Four of six pts are alive with stable engraftment and full donor chimerism between day + 428 to day + 6924. Acute GvHD of grade "d II occurred in one pt and chronic GvHD in two pts (one limited and one extensive), respectively. Allogeneic HSCT using related or unrelated donor could eradicate PNH clones and may cure pts with the disease.

Intense immunosuppression and stem cell transplantation in severe autoimmune diseases I. Lisukov, S. Sizikova, A. Kulagin, I. Kruchkova, A. Gilevich, L. Konenkova, E. Zonova, E. Chernykh, O. Leplina, T. Sentyakova, A. Demin, V. Kozlov, Institute of Clinical Immunology (Novosibirsk, RUS) Intense immunosuppression and stem cell transplantation (SCT) has been proposed as an investigational therapy for patients with severe refractory autoimmune diseases. Ten patients (9 female and 1 male, ages 15-39 years) with primary SAD were included in the study from 1998 to 2003. Six patients with systemic lupus erythematosus (SLE), three with primary or secondary progressive multiple sclerosis (MS), and one patient with idiopathic thrombocytopenic purpura (ITP) have undergone intense immunosuppression and SCT. All SLE patients were seriously ill, with SLE disease activity indices (SLEDAI) of 6-30, including 2 case with central nervous system lupus, 1 case with lung vasculitis and 3 cases with nephritis and nephrotic syndrome. Patients with MS were severely disabled, with EDSS 6 (n=2) and 6,5 (n=1). Previous immunosuppression included pulse cyclophosphamide (Cy) intravenous, prednisolone (standard doses and pulse therapy), oral Cy and azathioprine for SLE patients, and dexamethasone and mitoxantrone for MS patients with little or no effect on disease progression. ITP patient relapsed after splenectomy and had nonresponder severe thrombocytopenia. Autologous hematopoietic stem cells were collected from bone marrow (n=4) or mobilized from peripheral blood with either granulocyte colony-stimulating factor (G-CSF) (n=1) or Cy and G-CSF (n=5). Pretransplant conditioning regimens included BEAM (n=3), Melphalan 140 mg/m2 + Etoposid 1600 mg/m2 (n=2), Cy 200 mg/kg (n=2) and Melphalan 140 mg/m2 alone (n=3). On day 1 and 2, antithymocyte globulin (ATG; 60 mg/kg) was given for in vivo T cell-depletion (n=4). Mean time to engraftment of an absolute neutrophil count greater than 500/ml and a nontransfused platelet count greater than 20,000/ml occurred respectively on day 14 and 16 for 3 SLE and all MS patients, and on day 50 and 68 for ITP patient. Three SLE patients died on days 11, 22 and 63 due to transplant-related severe infections. Four patients with follow-up of 9 to 66 months achieved a drug free complete remission (2 SLE, 1 MS, ITP). Three patients showed stabilization with median follow-up 35 months (1 SLE, 2 MS). We conclude that intense immunosuppressive conditioning and autologous SCT may induce a sustained remission in a fraction of patients with severe refractory autoimmune diseases.

M. Musso, F. Porretto, A. Crescimanno, V. Polizzi, R. Scalone, Ospedale La Maddalena (Palermo, I) Pure Red Cell Aplasia (PRCA) is characterized by selective marrow aplasia of erythroid compartment. Immunosuppressive therapy achieves good results in about 25% of cases. Autologous or Allogeneic stem cell transplantation (HSTC) could represent a valuable strategy in selected patients. We report a 29-year-old woman treated successfully by DLI infusion after sibling HSTC for acquired refractory relapsed PRCA. Non myeloablative conditioning regimen consisted in Cyclophosphamide 60mg/Kg/day for 2 days, Fludarabin 30mg/m2 x 4 days. Complete recipinet hematopoiesis was noted at one month after allo HSCT, but it reverted to full donor engraftment after three escalating DLI infusions. The potential role of graft-versus-autoimmunity (GVA) effect induced by Allogeneic HSCT followed by DLI infusions in the treatment of the disease is discussed, and physiopathogenetic impluication are analysed.

Three strategies of high-dose chemotherapy +autologous stem cell transplantation in autoimmune diseases Y. Shevchenko, A. Novik, T. Ionova, K. Lyadov, G. Gorodokin, V. Melnichenko, National Pirogov's Medical Surgical Center, Multinational Center of Quality of Life Research, Russian Cooperative Group for Cellular Therapy (Moscow, St. Petersburg, RUS) Objectives: Among a number of unclear questions for HDCT+ASCT in patients with autoimmune diseases is the indications for conducting HDCT+ASCT. We developed the concept of HDCT+ASCT which includes three strategies: early, conventional and salvage ASCT. The aim of the study was to demonstrate the feasibility of the concept strategies in patients with multiple sclerosis (MS) undergoing HDCT+ASCT. Methods: Three case reports have been analyzed out of the database of ten patients with MS after HDCT+ASCT (EBMT protocols). Patient K. underwent early ASCT (male, 25 years old; spinal cerebral remittent subtype; base-line EDSS -1.5; followup 9 months); patient F. -conventional ASCT (female, 35 years old; secondary progredient subtype; base-line -EDSS 5.0; followup 54 months); patient P. -salvage ASCT (female, 48 years old; spinal primary progredient subtype; base-line EDSS 7.5; followup 42 months). Clinical and QoL evaluation was provided at baseline, at discharge, at 3, 6, 8, 12 months and later every half a year after ASCT. MRI was conducted at baseline, in 6, 12 months and at the end of follow-up. QoL was assessed by FACT-BMT and FAMS. Integral QoL index was assessed by the method of integral profiles. Results: Clinical examination and MRI revealed disease stabilization in patient K. and clinical remission in patients F. and P. EDSS decreased from 1.5 to 1.0 in patient K.; from 5.0 to 4.5 in patient F. and from 7.5 to 6.5 in patient P. at the end of followup. Integral QoL index increased dramatically as compared to base-line: in patient K. -from 80.0 to 110.5 (FACT-BMT); in patient F. -from 46.5 to 87.0 (FACT-BMT); in patient P. -from 95.5 to 158.0 (FAMS). Definite QoL improvement was documented one year after ASCT and remained stable throughout the later follow-up. Conclusion: Three case reports on early, conventional and salvage ASCT demonstrated its feasibility in MS patients. Three strategies of ASCT are suggested at different stages of the disease and have different pathogenetic and overall goals. To support the evidence of three strategies of HDCT+ ASCT further studies are worthwhile.

Allogeneic BMT for refractory Behçet's disease having relapsed following ABMT F. Gualandi, A.M. Raiola, A. Dominietto, V. Galbusera, S. Bregante, A. Bacigalupo, A.M. Marmont, Ospedale San Martino (Genoa, I) (BD) of 10 years duration in a 30 year old lady who was treated with HDIS according to the BEAM protocol, and then reconstituted with unmanipulated ASC (CD34+ 3.2/Kg). There was a complete abrogation of all clinical symptoms, and a notable improvement of brain perfusion by SPECT analysis (CD34+ 3x10^6/Kg). However this remission was short, other 2 remissions following Infliximab were similarly transient, and on May, 2003 she received an unmanipulated BMT from her HLA-identical brother after RIC with TT 10 mg/Kg and CY 100 mg/Kg. Hematologic reconstitution was rapid, and complete cytogenetic chimerism ascertained on day +70. On day +30 grade III gastrointestinal and skin GVHD appeared, which responded to a combination of ATG, CyA and photopheresis. However liver GVHD also appeared with peaks of bilirubin 33.5 mg%, FA 2460 UI/L and GGT 2900 UI/L. She was treated with CY 3 gr and donor MSC (5x10^7), in addition to the aforementioned protocol. She responded favourably, with bilirubin 2.4 mg% on day +190. No recurrence of aphtosis and headache has occurred up to how (+200). There is a case report from Japan where a cord blood SCT was given for coincident diseases (MDS and intestinal BD), with CR of both (3). This is the first case in which a regular BMT has been performed for BD alone. Prolonged FU is obviously necessary. We still believe that intense IS and the switch from patient to donor immune system suppresses the autoimmune response which mediates much of the diverse organ damage in BD, but may not affect the etiologic trigger(s). 

M. Mitterbauer, P. Kalhs, F. Keil, W. Rabitsch, K. Dieckmann, P. Hoecker, A. Rosenmayr, G. Mitterbauer, K. Lechner, C. Mannhalter, U. Jaeger, H. Greinix, University Hospital of Vienna (Vienna, A) Between 1995 and 2003 nineteen patients with poor risk chronic lymphoid leukemia (CLL) stage B or C (n=18) or A with risk factors (n=1) with a median age of 51 (range, 27-62) years underwent autologous (n=11) or allogeneic (n=8) hematopoietic stem cell transplantation (HSCT) at the University Hospital of Vienna. Median time from diagnosis to HSCT was 30 (range, 6-77) months. All patients given autologous stem cell transplantation (ASCT) had chemosensitive disease with partial remission (n=9) or complete remission (CR) (n=2) and received B-cell depleted peripheral blood stem cell (PBSC) grafts. Eight patients with refractory disease (n=5) or chemosensitive relapse (n= 3) underwent allogeneic HSCT with unmanipulated bone marrow (n= 3) or PBSC (n= 5) from sibling (n=7) or unrelated donors (n=1). In the majority (n= 14) conditioning therapy consisted of total body irradiation (TBI) of 12-13.2 Gy and cyclophosphamide. Three patients underwent nonmyeloablative (NMA) conditioning with fludarabine and TBI of 2 Gy. In the majority, graft-versus-host disease prophylaxis consisted of cyclosporine (CsA) with methotrexate. No patient died of treatment-related causes. Complete clinical remission was attained in 10 patients after ASCT and in 6 after allogeneic HSCT. Molecular remission of the immunoglobulin heavy chain and/or light chain gene rearrangement was attained in 7 patients after ASCT and in 3 after allogeneic HSCT. Three patients died of disease progression/relapse 1-17 month after transplantation. After a median follow-up of 39 (range, 3-105) months, 10 autologous and 6 allogeneic graft recipients are alive and 12 (ASCT: n=8, allogeneic HSCT: n=4) are in continuous CR. Probability of overall survival is 90% after ASCT and 70% after allogeneic HSCT. In summary, none of our patients died of transplant-related causes. CLL patients undergoing ASCT in CR and PR can attain long-term survival. Moleclar remission seems to be a prerequisite for long-term disease-free survival. However, longer follow-ups and larger patient numbers are necessary to draw conclusions about cure. Selected patients with refractory disease can be rescued by allogeneic HSCT.

Cytogenetic resistance to imatinib: analysis in a series of 20 patients A. Bermudez, L. Yañez, O. Arce, A. Insunza, M. Lopez, R. Perez-Montes, E. Bureo, M. Albajar, E. Conde, C. Richard, J. Baro, M. Colorado, M. Ruiz, A. Batlle, M. Gutierrez, A. Iriondo, HU. Valdecilla (Santander, E) Introduction: Imatinib Mesylate (STI571), a selective inhibitor of BCR-ABL tyrosin kinase, induces cytogenetic responses in most patients with chronic myeloid leukemia (CML). We analyzed our experience in patients with CML treated with imatinib at a starting dose of 400 mg daily. Patients: We studied 20 patients (16 males and 4 females) with CML in chronic phase. The median age was 60 years (28 to79) with a mean time from diagnosis to imatinib therapy of 45 months (1 to 180). At the time of diagnosis, the mean white blood cell count was 127x109/L and 7 patients had palpable splenomegaly greater than 5cm. Interferon-alpha was used previously in 19 patients with a therapy mean duration of 12 months. 10 patients had cytogenetic resistance to interferon and 9 had intolerance. Results: At the time of analysis after a median follow up of 24 months, 18 patients had a complete hematologic response during first month of treatment. Six patients developed severe myelosuppresion during the course of therapy and treatment interruption or adjustment was necessary. Eleven patients achieved cytogenetic response: 1 patient with minor cytogenetic response, 3 patients with major cytogenetic response and 7 patients with complete cytogenetic response including 5 patients with molecular remission. Nine patients (45%) showed cytogenetic resistance to STI, 2 patients had primary resistance and 7 patients had secondary resistance. In this last group, 4 patients showed new cytogenetic abnormalities in the Ph positive clone, one patient developed resistance associated to BCR-ABL kinase domain mutation and two patients had late hematological relapse without cytogenetic study because lost to follow up. Three patients with clonal evolution showed severe hematological toxicity during the first three months of treatment, with STI interruption during more 8 weeks. Considering non response group respect response group, we observed statical significance in evolution CML time (76.5 vs 27.3 months), previous chemotherapy lines (2.5 vs 1.7) and STI interruption time due to toxicity during first three months (11.1 vs 0.54 weeks). Conclusion: Our study showed high level of cytogenetic resistance to STI. Clonal evolution was related with severe and longer duration of hematological toxicity when therapy was started.

Mini-ICE protocol as mobilisation therapy before autologous peripheral blood progenitor cell transplantation for chronic myelogenous leukaemia M. Cioch, J. Manko, D. Jawniak, A. Dmoszynska, M. Malek, Medical University of Lublin (Lublin, PL) The role of Autologous Peripheral Blood Progenitor Cell Transplantation (APBPCT) in chronic myelogenous lekaemia (CML) is still controversial but this method presents very interesting therapeutic option for patients lacking matched related or unrelated donors for allogeneic transplantation. We have investigated toxicity and efficacy of mini-ICE regimen (idarubicin, cytarabine, etoposide) according to Carella et al. protocol in mobilisation of peripheral blood progenitor cells in this disease. Eleven patients (5 female and 6 male; median age 34.5 years) with first chronic phase of Ph-positive chronic myelogenous leukemia were treated with mini-ICE regimen followed by G-CSF to mobilise and collect peripheral blood stem cells. Mobilisation of CD34+cells was ineffective in 3 patients. One patient died because of septic shock. The side-effects in other patients were mild-moderate. Eight patients underwent leukaphereses (from 2 to 3) and the mean 5.54 x 10^6/kg of CD34+ cells (range: 0.29 -12.46 x 10^6/kg), after the mean 18.8 days (range: 7 -36 days) of G-CSF application were obtained. Cytogenetic study of peripheral blood stem cells products using FISH technique revealed complete bcr/abl negativity in 6 patients (54.5%), one patient had a minimal cytogenetic respone (75% bcr/abl positive cells), and one had no response (100 bcr/abl positive cells). The statistical analysis (Spearman test) showed correlation (p < 0.05) between the shorter time from diagnosis and bcr/abl gene eradication. Two patients were autografted; one of them still remains in CR (53 months after transplantation). We suppose, that the importance of APBPCT in CML will be more, when imatinib will be applied to reduce minimal residual disease.

Life-threatening hepatic complications in myeloablative stem cell transplantation in advanced phase CML patients receiving imatinib immediately prior to conditioning Á. Bátai, S. Lueff, P. Reményi, G. Kriván, M. Réti, A. Tremmel, I. Bodó, T. Masszi, St. László Hospital (Budapest, HUN) Imatinib is an effective therapeutic option in advanced phase CML. However, most patients treated with imatinib in the blast phase develop progressive disease rapidly. Thus, treatment with imatinib has to be followed by other treatment strategies including stem cell transplantation. The mild to moderate liver toxicity of imatinib might therefore be aggravated by the conditionig regimen. We report two patients transplanted immediately after imatinib therapy because of accelerated phase Ph+ CML. Two young male patients (pt1: 25y and pt2: 27y ) received 1 and 1,5 months imatinib 600-800 mg daily dose in progressive disease. Both patients had significant splenomegaly resistant to imatinib therapy. Therefore, they underwent bone marrow / peripheral stem cell transplantation from HLA identical siblings following Bu/Cy conditioning regimen with standard short Seattle GVHD prophylaxis. Patient 1 after early engraftment and without GVHD has shown typical clinical signs of VOD from day +40. The liver abnormalities perfectly resolved after two months of prociclide, heparin and diuretic therapy. From day +120 haematological relapse with mixed chimerism developed, and he died on day +201 in relapse not having responded to DLI and subsequent chemotherapy. Patient 2 experienced progressive elevation of serum bilirubin, gamma glutamil transferase and alkaline phosphatase from day +1, that reached 731 microM/l se-Bi and 551 U/l alkaline phosphatase on day +26. Hepatorenal syndrome developed characterised by se-creatinin 560 microM/l . Repeated duplex ultrasonography of the portal and hepatic veins excluded VOD. On day +28 non-take was diagnosed and retransplantation has been initiated. Conclusion: Both of our accelerated phase CML patients treated with standard Bu/Cy conditioning transplantation immediately following imatinib therapy experienced severe, life threatening hepatic injury. Caution should be taken in the above setting and the observation should be confirmed in a prospective manner.

DLI in relapsed hyperoesinophilic syndrome after allogeneic, CD34+ highly enriched peripheral blood stem cell transplantation: proof of a graft-versus-HES effect R. Peceny, A.H. Elmaagacli, M. Ditschkowski, R. Trenschel, H. Ottinger, D.W. Beelen, University of Essen (Essen, D) Hypereosinophilic syndrome (HES) is a heterogeneous disease entity with a marked, possibly T-cell induced eosinophilia leading to end organ damage without clear evidence of clonal evolution or other causes of eosinophilia. Allogeneic stem cell transplantation was performed in few, mostly young adult patients worldwide. A clear proof of a graft-versus-HES effect is missing so far. Our patient was diagnosed with hypereosinophilic syndrome in August 2001 at the age of 39. Consecutive therapy with corticosteroids, hydroxyurea, cyclophosphamide and alphainterferon did not induce any remission. He suffered from deteriorating end organ function: endomyocardial fibrosis with restrictive cardiomyopathy and a ventricular thrombus, severe vasculitis with dry gangrene of 9/10 toes, CNS involvement with deterioration of cognitive function and chronic renal insufficiency, ECOG performance status 2 -3. In September 2002, he was transplanted with CD34+ highly enriched peripheral blood stem cells from his HLA-identical brother without any immunosuppression post transplant. GvHD did not occur. End organ damage remitted quickly with normalization of cognitive function, complete resolution of vasculitis and improvement in cardiac and renal function. In January 2003, a relapse had to be diagnosed: eosinophil count up to 10,500 /µl in peripheral blood, loss of chimerism overall and in the CD34+ compartment. Deteriorating renal function was a sign of reoccurrence of end organ damage. Donor lymphocyte infusions (DLI) were given in two increasing doses of 1 x 10^6 and 3.3 x 10^6 CD3+ T-cells per kg body weight without any further change in treatment. Five weeks after the 2nd DLI, serum creatinine nearly normalized. Chimerism became complete in the T-cell compartment, overall and in the CD34+ compartment seven weeks after the 2nd DLI. Acute GvHD of the skin and liver, overall grade III, was rapidly controlled by prednisone in a dose of 0.5 mg/kg resulting in a limited, secondary chronic GvHD of the oral mucosa. Prednisone could be tapered to 0.2 mg/kg up to now. The ECOG performance status has improved to 1. The ventricular thrombus is resolving slowly, the patient being in an ongoing complete remission and fully chimeric. For the first time, a graft-versus-HES effect could clearly be shown. Thus allogeneic stem cell transplantation should be considered for patients failing classical treatment options and low dose imatinib before irreversible organ damage occurs.

Continuous molecular response after discontinuation of imatinib mesylate following relapse after non-myeloablative allogeneic stem cell transplantation for CML W. Zinke-Cerwenka, M. Eibl, H. Sill, G. Höfler, P. Staber, W. Linkesch, University Graz (Graz, A) Background: In case of relapse after allogeneic stem cell transplantation (SCT) DLI has been the favoured frontline therapy. After the introduction of imatinib mesylate in the initial treatment of CML it is now also used in patients relapsing after allogeneic SCT with encouraging results. But there exist no literature data if the drug can be stopped after achieving a molecular response. Patient: A 59-year-old Caucasian female with CML in chronic phase diagnosed in March 2001 received imatinib mesylate starting in June 2001 after therapy failure with interferon-alpha combined with Ara-C and hydroxyurea. She achieved a complete hematologic and cytogenetic response in September 2001 but because of hepatotoxicity the drug had to be discontinued for several times and a cytogenetic relapse occurred in August 2002. The patient had an HLA-matched sibling donor and underwent nonmyeloablative SCT (conditioning: fludarabine (150mg /m2 and TBI with 200 Gy) in November 2002. Evaluation on day +28 showed an inconspicuous bone marrow and a persistent cytogenetic relapse (53% Philadelphia positive metaphases by fluorescence in situ hybridisation analysis). Bone marrow biopsy in May 2003 revealed relapse of CML and a decrease in donor chimerism was found in the peripheral blood. Imatinib mesylate was started at the end of May with 400 mg by day. Thirteen days later the patient showed a pancytopenia which was thought to be due to imatinib mesylate or sulfonamide which has been given by the family doctor because of an infection and both drugs have been stopped. The bone marrow examination indicated a toxic damage compatible with the use of sulfonamide. Results: Because of the slow regeneration of the bone marrow imatinib mesylate could not be restored immediately but the reevaluation one month later when the patient was still off drug, showed a complete donor chimerism and complete cytogenetic and molecular remission. This results could be achieved after a imatinib mesylate administration of only 13 days. Later on the drug could be restarted but the patient refused to take it again. At the end of October the 2-step reverse transcription-polymerase chain reaction (RT-PCR) for BCR-ABL transcripts was still negative indicating a continuous molecular response four and a half months after the discontinuation of therapy.

M. Ramzi, H. Noorani, M. Zakernia, M. Haghshenas, Shiraz University of Medical Sciences (Shiraz, IR) Allogeneic stem cell transplantation as a curative treatment for disorders of hematopoietic system and chemo-sensitive malignancies was established in Shiraz university of medical sciences in 1993. In this report we will describe 10-year experience of allogeneic stem cell transplantation in our center in south of Iran. From June 1993 to May 1993, 181 patients underwent allogeneic marrow transplantation from HLA identical or one antigen mismatch related donor in our center. Main Indications for HSCT in our center were Thalassemia Major (n=112), Acute Myelogenous Leukemia (n=27), Chronic Myelogenous Leukemia (n=18), Acute lymphoblastic Leukemia (n=10) and Aplastic Anemia (n=8). Conditioning chemotherapy regimen were busulfan 14-15 mg/kg and cyclophoshamide 200mg/kg"b anti thymocyte globulin (ATG) 40 mg/kg for thalassemic patients, busulfan 16 mg/kg and cyclophosphamide 200 mg/kg for leukemia patients and cyclosphophamid 200 mg/kg + ATG 90 mg/kg for patients with aplastic anemia. Graft versus host disease prophylaxis consisted of cyclosporine A, prednisolon and methotrexate. In thalassemic group the average of age was 9.5 years with range of 2-20 years and 87 of 112 patients (77.6%) are living with full engraftment with median follow up of about 6 years (range 6-120 month). In leukemia group average of age was 30 year (range 6-45). Thirty seven of 55 patients (67%) in long-term follow up are disease free and were cured. The best result was obtained in AML with overall cure rate of 74%. The most important major complication was severe acute GVHD, 10 patients with sever acute GVHD expired. According to results and experiences in patients with thalassemia major HSCT is our recommendation as treatment of choice and only curative methods in class I to III according to Lucarelli risk classification. In addition these data reflect the important role of HSCT hematopoetic in improving of survival for a variety of other hematopoietic system disorders in our center.

M. Morey, A. Sampol, B. Galmés, A. Novo, A. Galmés, M.A. Durán, J. Besalduch, Hospital Universitario Son Dureta (Palma de Mallorca, E) Allogeneic Bone Marrow transplantation is the only curative treatment for Thalassemia Major(TM) but is associated with a relatively unacceptable level of transplant related mortality (8-15%) mainly due to acute and chronic graft versus host disease (GVHD). Cord Blood Transplant has some advantages respect bone marrow source, basically less GVHD incidence, but also has some disadvantages, mainly the scanty volume of blood and number of stem-cells collected. In children cases likes this, it is a minor problem. We described an 8 years old girl with TM who was transplanted with cord blood from her identical sibling sister with Thalassemia Minor (Tm) and remains, 22 months later, with Tm without transfusion requirements. The patient began classic treatment of TM with periodical transfusions at few months of life She received blood transfusions every 21 days in aim to maintain the Hemoglobin (Hb) level > 9 g/dl. She achieved normal growth without organic defects. When she was 8, her mother became pregnant of a female fetus with Tm. Delivery was on November 2001 and 140 ml of cord blood were collected and cryopreserved at -80ºC. The sister was HLA full compatible with the patient. Two months later the patient was conditioned with Busulfan, Cyclophosphamide and ATG regimen. CsA and MTX were used as GVHD prophilaxis. At that moment, the patient´s weight was 33 kg. Infusion of 4.9x107/kg total nucleated cells and 0.29x106/kg CD34 was done. Hematologic neutrophil and platelet recovery was achieved in 22 and 30 days respectively. Thirty two days after infusion the level of Hb was 7 g/dl; since then she did not received any transfusions. At present, 22 months later, she maintain Hb levels between 9 and 10 g/dl, She didn´t present acute or chronic GVHD signs nor infectious complications and she is maintaining an estable mixed chimerism. Cord blood is a good source of stem cells to perform an allogeneic transplant between identical siblings in childhood. It is described that the use of Thiotepa in the conditioning regimen is associated with better outcome. In our case, despite a little delayed engraftment, there wasn´t treatment failure and transplant related complications.We also confirm that mixed chimerism is enough to avoid transfusions in TM patients.

Objective: Transplantation of bone marrow is a frequently discussed therapeutic option in the treatment of malignant lymphoma. By analyzing the quality of life(QOL) in the lymphoma patients undergoing our own BMT program we tried to evaluate indications for transplantation. Methods: Data from survival lymphoma patients treated at the Nanfang hospital between 1993 and 2003 were analyzed retrospectively. 63 patients (non-Hodgkin's lymphoma n=41; Hodgkin's disease n=22) were included. Median age was 41 years. 29 patients received autologous bone marrow transplantation (ABMT). 15 patients received allo-BMT from HLA two or three loci mismatched related donors and 19 patients received HLA-identical sibling unrelated donor marrow. Patients enrolled received the Psychosocial Adjustment to Illness Scale (PAIS) questionnaire assessing psychological and social adjustment to chronic illness or its sequelae. The questionnaire was administered two days prior to the BMT and 10, 30 and 80 days following the BMT. The date were analyzed using descriptive statistics and an ANOVA for repeated measures. Results: The median overall PAIS score for all patients was 49 (range 19-74). The patients received BMT from haplotypematched related donor scored highest and the patients received HLA identical unrelated donor marrow got the lowest score. Older patients had significantly poorer QOL compared with younger patients. Mild to moderate anxiety and depression were present throughout the hospitalization. Conclusions: These findings indicate that factors associated with poor QOL are the older age, presence of long-term sequelae, chronic GVHD and relapse. A longitudinal study is ongoing for clinicians and nurses to develop treatment strategies to prove the quality of life in the patients undergoing BMT.

Prospective randomised study comparing 12-week vacop-b regimen versus 6 -week vacop-b plus high-dose sequential followed by autologous bone marrow transplantation in high-risk IPI aggressive non-Hodgkin lymphoma. A Brazilian GEMOH report Despite the improvement of treatment of aggressive non-Hodgkin's lymphomas in the past 20 years, no differences have been observed in terms of overall results using protocols of 1st, 2nd or 3rd generations. Autologous bone marrow transplantation (ABMT) seems to be the salvage treatment of choice in sensitive aggressive NHL. However, the role of ABMT as front line therapy is not clear in the literature. French and Italian randomized studies, in a retrospective analysis, suggested that ABMT had better results than conventional CT for high-risk NHL, at diagnosis. However, prospective studies did not confirme this observation. From September 1998 to June 2003, a multicenter phase III prospective randomized study was carried out comparing: VACOP-B 12 weeks (group A) and 6-week VACOP-B + high dose sequential therapy (CY, 4g/m2; VP-16, 2g/m2) followed by ABMT (group B), focusing on overall survival, DFS, EFS and mortality rate. Valuable patients were 27 (A) and 29 (B). Male gender 41 (73.2%) and female 15 (26.8%). Median age was 38.3 years (17-60). At the diagnosis, the presented histology/WF was: F 3.6% (2 patients); G 78.5% (44 patients); H/K 17.9% (10 patients); 83% phenotype B (47 patients) 12.5% T (7 patients) and not done 3.6%. With a median observation time of 275 days (8-1670), the overall survival was 37% and 35% (NS), DFS (pts=27) was 90% and 40% (P=0.03) and EFS was 42% and 26% (NS) in the group A and B, respectively. Twentynine (51%) patients died with a median of 162 (8-1139) days and most of them (51.7%) died of infection complications and 20.7% (6 patients) of progressive disease. Thirteen patients died during VACOP-B with a median of 40 days most of them due to progressive disease. These data are preliminary; but it seems that high-dose therapy followed autologous bone marrow transplantation do not promote benefit as front line therapy in patients presenting IPI high risk. The IPI demonstrated a high sensitivity to detect the poorest patients, as demonstrated by high mortality rate in this category of patients.

A.L. Van de Velde, A.P. Gadisseur, E. Steel, S. Van Steenweghen, W. Schroyens, Z.N 

The term mantle cell lymphoma (MCL) describes a subtype of Bcell non-Hodgkins lymphoma (NHL) that has a distinct morphologic, immunologic, and genetic phenotype. They represent a difficult problem, to establish the diagnosis but mostly because of their refractoriness to standard lymphoma treatments. One approach to improve outcome is high-dose chemotherapy (HDCT) and autologous stem cell transplantation (ASCT). Combined immunochemotherapy increases complete and overall response rates. We retrospectively analysed the clinical features and outcome of 37 patients (29 males; 8 females) with MCL diagnosed and treated from 1991 to 2003 at two institutions in the same geographic area. Patient characteristics were: mean age 62years (range 34-82), B symptoms in 73 %, 100 % stages III-IV and 44 % with elevated LDH. Blastoid variant was present in 2 cases. Eleven patients received HDCT as consolidation treatment after first-line chemotherapy; 8 after a relapse or progression and 18 were treated with chemotherapy alone. Anti-CD20 monoclonal antibodies were given in three patients before, in 2 after and in 5 without transplant. The 2-year overall and relapse-free survival were73 % and 63 % respectively in the transplant group and 67 % and 44 % in the non-transplant group. Estimated median overal survival was 49 months (51 in the transplant group; 40 in the non-transplant group). Median time to relapse was 43 months. Seventy % of patients not transplanted in first complete remission relapsed or died. Three patients received an allogeneic stem cell transplantation (aSCT) in second or thirth remission. Our data confirm the unfavorable clinical course of MCL and the need for alternative therapeutic strategies for this NHL category.

Integration of rituximab with chemotherapy for the collection of lymphoma-free peripheral blood stem cells in non-Hodgkin lymphoma E. Tothova, M. Fricova, A. Kafkova, N. Stecova, T. Guman, S. Raffac, E. Svorcova, M. Hlebaskova, E. Kaclikova, V. Takac, Medical Faculty Hospital and UPJS (Kosice, SK) Background: Autotransplantation is effective in the long-term control of non-Hodgkin´s lymphoma (NHL), but lymphoma cells have been proved to contaminate stem cell harvests and may contribute to relaps. Several in vitro methods have been examinated in an attempt to abolish graft contamination. These usually produce loss of cells, are time-consuming and expensive, and neoplastic depletion is often partial with residual PCRpositive cells. We investigated the in vivo purging potential of Rituximab in patientns (pts) with progressive NHL enrolled in a programme of immunochemotherapy and autotransplant. Methods: Our study was conducted in 27 consecutive pts with untreated CD 20 and bcl 2 positive NHL (Follicular lymphoma-7, chronic lymphocytic leukemia-13 and the other NHL in leukemic phase-7), 14 pts received Rituximab. Patients received 4 courses of standard-dose chemotherapy (CHOP or FLU-CY), followed one course of high-dose cyclophosphamid (5g/m/2) plus G-CSF (CHT). Patients allocated to Rituximab received iv infusions of 375mg/m/2 48 hours before stem cell collection and in 3 weekly doses after transplantatin (R-CHT). Results: Clinical response (CR) after transplantation was evalluated in 26 pts who comleted the treatment. The complete response rate was obtained in 100% in the Rituximab group (PCR negative in 79%) versus 50% of controls (p<0.01) (CHT). Yield of purged CD 34+ cells was with median 5.23x10/6/kg in CHT and 8.76x10/6/kg in R-CHT pts (p<0.01). Toxicity in both arms was acceptated (no difference). No significant difference was observed between CHT and R-CHT group in the mean number of days spent with neutropenia and trombocytopenia. Two-years progression-free survival was 86% in R-CHT and 58% in CHT group. Conclusions: Aside from providing PCR-negative harvests, the chemoimmunotherapy treatment produced complete clinical (100%) and molecular remission in 79% of evaluable pts. The preliminary results of this study show that the addition Rituximab to transplantation regimen is effective in pts with advanced stage NHL, with low toxicity, no adverse effects on stem cell harvesting, haematological recovery, or incidence of infections. Background: according to some studies patients (pts) with DLBCL in 1.PR have not good prognosis treated with a conventional therapy and these pts are recommended for highdose therapy (HDT) and autologous transplantation (ASCT). But there are some controversies and published data are relatively insufficient. Therefore we retrospectively analysed outcome of 19 pts with DLBCL in 1.PR treated with HDT and ASCT and we compared their outcome with a control group of pts with DLBCL in 1.CR treated identically. Patients and methods: since 1993 19 pts (group 1) with median of age 43 (range:20-65) yrs with DLBCL in 1.PR after anthracycline-based therapy (IPI at dg.: low/low-intermediate 63%pts, high-intermediate/high 37%pts) have been treated with HDT (BEAM) and ASCT (median of time from dg. to ASCT: 4 months). The control group (group 2) of 20 pts with DLBCL in 1.CR treated identically as 1st line treatment does not differ for any significant variables such as age (p=0.69), IPI at dg. (p=0.74), time from dg. to ASCT (p=0.89) and follow-up (p=0.85). Also 1st line treatment, mobilizing and conditioning regimens were the same for both groups. CR and PR status was retrospectively analysed and re-evaluated according to published criteria (Cheson et al.,1999) . Results: in group 1 at median of follow-up of 70 (range:7-103) months 14 pts (73%) are alive in 1.CR, 5 pts (27%) have relapsed and 3 of them died. The estimated probabilities of 8years EFS and OS are 72% and 83%.In group 2 at median of follow-up of 64 (range:2-97) months 15 pts (75%) are alive in 1.CR, 5 pts (25%) have relapsed and 4 of them died. The estimated probabilities of 8-years EFS and OS are 72% and 72%.We observed no apperant difference in 8-years EFS (p=0.972) and OS (p=0.84) between both groups. Conclusion: 1) our data suggest that HDT and ASCT for pts with DLBCL in 1.PR is well-tolerated (no treatment related death) and can improve their outcome which is comparable with outcome of pts with DLBCL transplanted in 1.CR. 2) of course the surprisingly good results could be partially influenced by relatively lower number of evaluated pts and by the fact that in the absence of a surgical procedure we cannot exclude that a residual mass on CT scan in some pts in PR could be only fibrotic or necrotic tissue rather than residual disease. 3) in spite of mentioned partially controversial facts we suppose that HDT and ASCT is the most convenient treatment strategy for pts with DLBCL in 1.PR.

Autologous stem cell transplantation for newly diagnosed follicular lymphomas: low toxicity and extended progression free survival F. Narni, A. Donelli, A. Cuoghi, P. Bresciani, S. Pozzi, R. Marasca, G. Leonardi, M. Morselli, M. Luppi, G. Longo, G. Torelli, Uni. Modena, Policlinico (Modena, I) In spite of an indolent course, advanced stage follicular lymphomas (FL) are incurable with conventional chemotherapy. High dose therapy could be investigated in selected patients, provided toxicity were acceptable. Improvements in support therapy and the widespread use of peripheral blood stem cells (SC) have made autologous transplantation a relatively safe procedure. This prompted us to start a pilot study and, since 1995, 23 patients have been enrolled at our center. Median age was 49 (26 -65). Most patients (80%) had stage IV disease and bone marrow involvement. For patients who achieved a complete response or a good partial response after CHOP, SC were mobilized with cyclophosphamide or DHAP plus G-CSF, and high dose therapy consisted of BEAM. High risk patients or poor responders (residual high tumor burden or bone marrow infiltration >50%) switched to a program of high dose sequential therapy. Nine patients received not-manipulated hematopoietic SC, 7 received ''ex vivo'' purged SC (6 by CD34+ positive selection; 1 by positive+negative selection), and 8 patients underwent ''in vivo'' purging with anti-CD20 monoclonal antibody (rituximab). Residual disease was monitored at different steps by nested PCR. Treatment was well tolerated, with a median time to engrafment of 9 days (7-12), and a median stay in hospital of 22 days (15-35). RBC and platelet transfusions were necessary, respectively, in 52% and 100% of the patients. The mean number of RBC and platelet units given to transfused patients was, respectively, 2.2 (1-5) and 3.7 (1-7). Fever occurred in 52% of the patients (mean 2,8 days in febrile patients) and mucositis in 85%. We did not observe, so far, any case of myelodysplasia or secondary cancer. With a median FU of 58 (8-95) months from transplant, all patients are alive, 5 have relapsed, and 5years estimated PFS is 70%. CD34+ selection had no impact on relapse. In one patient, molecular response could be restored and maintained by rituximab alone. Only a few data have been published regarding autologous transplantation in FL patients at diagnosis. Although the FU is relatively short and the number of patients still small, the present data suggest that autologous transplant is a safe and effective therapy for selected, not heavily pretreated young patients with advanced stage disease. Especially since ''in vivo'' purging strategies can easily be carried out, autologous SC transplantation should be investigated in randomized trials.

Autologous stem cell transplantation for high-risk lymphoma -a single-centre experience N. Theorin, A. Radlund, A. Seger-Mollén, G. Juliusson, C. Malm, University Hospital (Linkoping, S) Autologous stem cell transplantation (ASCT) has been shown superior to conventional chemotherapy in the treatment of relapsed, high-risk or refractory but chemosensitive lymphomas. However, there have been reports of a high incidence of transplant-related mortality (TRM) with many patients developing myelodysplastic syndrome (MDS)or secondary acute myeloid leukemia (sAML), possibly related to pre-ASCT treatment. We present our experience with 92 patients undergoing ASCT in 1991-2003. Diagnoses included Hodgkin's disease (HD, n=23), high-grade Non-Hodgkin´s lymphoma (HG-NHL, n=50) and (transformed) low-grade NHL (LG-NHL, n=19). The median age at ASCT was 45 years (18-68) and time from diagnosis 23 months (4->100). ASCT was performed in 1st remission in 32 patients, 2nd remission in 45 patients, >= 3rd remission in ten patients and progressive disease (PD) in five patients. Conditioning was with the BEAM (BCNU, etoposide, cytosine arabinoside and melphalan) regimen in 85 patients and the stem cell source was mobilized peripheral blood stem cells (PBSC) in 74 cases, bone marrow (BM) in 14 cases and BM+PBSC in four cases, with PBSC being the only stem cell source since 1995. 18 patients with B-cell-NHL have received rituximab as part of the conditioning since 2001. 87 patients (95%) were treated with antibiotics due to fever/infection during hospitalisation and positive (bacterial) cultures were seen in 38 (42%) cases. There were no toxic deaths. With a median follow-up of five years (range 3 months->12 years) the estimated 5-year over all survival (OS) and progression-free survival (PFS) are 69 and 59% respectively. No difference is seen between the different forms of lymphoma, although there is a trend towards a lower PFS in LG-NHL. All patients transplanted with PD have had progression after ASCT, but no other statistical differences related to disease status at SCT were seen. Due to the relatively short follow up no comparison regarding the possible additional effect of rituximab has been made, although there may be a positive effect on OS/DFS. 24/28 deaths were attributable to progression. Six of 36 patients with relapse have achieved a new remission with subsequent treatment. Three patients (3 %) have developed MDS/sAML. We conclude that ASCT is a well-tolerated and effective treatment for high-risk lymphoma. In this material we have a low incidence of TRM.

Survival of patients with progressive disease following autologous transplant for lymphoma O. Tarabar, L. Tukic, D. Stamatovic, L. Ristic, V. Glavicic, M. Elez, M. Malesevic, Military Medical Academy (Belgrade, CS) Introduction: Relapse after autologous stem cell transplantation (auto SCT) for lymphoid malignancy is associated with a poor prognosis, with median survivals of 10,5 to 25 and 3 to 7,5 months reported for patients (pts) with Hodgkin's disease (HD) and non-Hodgkin's lymphoma (NHL), respectively. A further remission may be attained with conventional-dose chemo and radiotherapy, but is rarely durable, particularly if the time interval between transplantation and relapse is shorter than 12 months. Aim: To assess survival of pts with HD and NHL whose disease had progressed after auto SCT. Patients and methods: Between January '97. and May '03, a total of 36 pts with lymphoma, 16 with HD and 20 with NHL underwent auto SCT in our center. Twenty percentage pts with NHL and 25% with HD were transplanted with active disease. In all pts source of stem cells was mobilized peripheral blood. The conditioning regimen was BEAM in HD and CVB in NHL pts. Results: In all, 9 of the 16 HD pts (56%) relapsed or progressed post-transplant, while for NHL the proportion was 9 of the 20 (45%) pts. The mean time to relapse was 13 and 11 months for HD and NHL pts, respectively. The median survival from relapse/disease progression was 18 months for HD and 6 months for NHL (not statistically significant by the log-rank test). In NHL pts, longer postrelapse survival was associated with indolent histologies. The median survival post-transplant was 28 and 16 months for realpsed pts with HD and NHL, respectively. Among relapsed pts, 6 (3 NHL; 3 MH) of them are still living more than 40 months. Conclusion: The short-term prognosis for patients with disease progression postautologous transplant were somewhat better for HD compared to NHL. Long-term survival is poor in both disease. The optimal therapy for relapsed pts with lymphoma after autologous transplant is unclear; whether such pts have more benefit from second autologous transplant or from nonmyeloablative allogeneic transplant.

Which patient with aggressive non-Hodgkin lymphoma should be transplanted in first remission (single-centre experience)

S. Genadieva-Stavrik, B. Georgievski, N. Siljanovski, Z. Stojanoski, G. Zografski, L. Ivkovski, L. Cevreska, Clinical Center (Skopje, MK) The therapy available nowadays makes this group of patients with aggressive Non-Hodgkin's lymphoma curable. To make the right and optimal therapeutic approach we need to stratify those patients in subgroups of patients with "high" and "low" risk, which was achieved with this study. This study comprises 148 patients with histopathology diagnosis of aggressive Non-Hodgkin's lymphoma treated at the Department of Hematology in the period 1989-2000, which gave us the observation period of 15 to 159 months. There were 88 male patients, median age 53 years and 60 female, median age 52 years. Most of the patients were in the advanced clinical stage at the disease on the initial presentation of the disease, 24% of the patients in the third stage and 43% in the fourth stage. B symptoms were noted in 44% of the patients. Bone marrow infiltration was found in 29%. Bcl-2 positively was confirmed in 26 cases by imunohistochemistry and there was proliferative index Ki-67 above 60% in 32 patients. Imunophenotipisation suggested that 80% of the cases are B-cell lymphoma. The patients were treated with antracicilin included regiments.After initial chemotherapy complete remission was achieved in 60%, partial response in 4% and there was no response in 32% with early deaths in 4%. At the end of the study 32% of the patients were alive and well, 32% were deceased and the reminder had unknown status.According to the univariante analysis the following parameters had significantly influence the overall survival in the patients with aggressive Non-Hodgkin's lymphoma: initial anemia, initial LDH, the stage of the disease, ECOG score, bone marrow infiltration, number of sites of extranodal infiltration, lymphoma subgroup according to various classification systems, morphology of the lymphoma cell, imunophenotype profile, percent of Ki-67 positively, bcl-2 positively, time to complete remission. The multiply progression analysis produce mathematical model through which we can anticipate the expected survival in each patients individually based on the statistically most influential prognostic markers, those achieving stratification of the patients in risk groups. In our study 32% of the patients with "high" risk are alive and "low risk patients have 70% 5-years overall survival. The patients with "high" risk are group of patients where use of agressive chemotherapy and haematopoetic stem cell transplantation would improve therapeutic results and overall survival.

High-dose consolidation chemotherapy with autologous transplantation after intensive sequential induction therapy improves complete remission rate in patients with poor prognosis non-Hodgkin lymphomas T. Papajík, L. Raida, E. Faber, J. Vondráková, R. Urbanová, Z. Pikalová, S. Vlachová, M. Jarosová, V. Kajaba, J. Zapletalová, K. Indrák, University Hospital Olomouc (Olomouc, CZ) At the beginning of the 21st century, non-Hodgkin´s lymphomas represent one of the most curable common neoplasms. Unfortunately, the majority of patients with high-risk and advanced disease either fail to achieve complete remission (CR) or ultimately relapse. The combination of well-defined prognostic indicators can quite accurately delimitates a subgroup of poorprognosis patients with high-probability of conventional chemotherapy refractory/recurrent disease and short overall survival. Several studies have suggested that the first-line therapeutic intensification can lead to a better prognosis and longer overall survival of such selected patients. 64 untreated patients under the age of 65 with poor prognosis lymphoma (31 pts. with diffuse large B-cell lymphoma, 8 with T-cell lymphoma, 10 with mantle cell lymphoma, 15 with follicular lymphoma) were treated with intensive induction therapy (regimens PACEBO, IVAM, HAM; including high-dose of methotrexat and cytarabine). Among the 54 pts. evaluated after the induction therapy, 20 pts. (38%) achieved CR and 31 (56%) obtained partial response (PR), most of them with more than 75% regression of tumor size. 3 pts. (6%) failed to response. 49 of 54 stimulated pts. (91%) successfully collected peripheral blood stem cells (PBSC) and 46 of them underwent autologous transplantation (AT) after BEAM conditioning regimen. 38 pts. were evaluated at day +100 after AT and 32 pts. (84%) reached CR/CRu, 3 pts. (8%) very good PR, 3 pts. (8%) relapsed or progressed. At the time of this analysis 16 pts. failed to respond, relapsed or progressed, and 11 of them died. The 48 pts. are alive without any evidence of active disease with a median follow-up of 22 months (range, 4-55 months). Multivariate analysis identified the following pretreatment characteristics as significantly correlated with poorer survival: age, beta-2-microglobulin and serum thymidine-kinase values, histological diagnosis of mantle cell and T-cell lymphoma. The probability of overall survival (OS) between pts. achieved CR or PR after intensive induction therapy was not statistically different and their probability of post-transplant OS was very similar. We conclude that the intensive sequential induction therapy leads to a significant tumor regression and subsequent autologous transplantation allows similar tumor control and may prolong OS in both the CR or PR pretransplant groups of patients.

T. Cobo, R. De Paz, M. Canales, M. Martin, A. Lopez, D. Hernandez, J. Garcia-Bustos, F. Hernandez-Navarro, Hospital La Paz (Madrid, E) Objective: Primary mediastinal large B-cell lymphoma (PMLBL) is a distinct clinicopathological entity with characteristic features and a more aggressive course than other diffuse large-B cell lymphoma. Some authors have previously reported that patients with PMLBL can achieve better results following high-dose chemotherapy and autologous stem cell transplantation (ASCT). The aim of our study was to analyse the outcomes with ASCT in this group of patients. Patients and methods: We have included in this study 10 patients treated in our centre, with mediastinal mass and histopathological diagnosis of diffuse large B-cell lymphoma according to REAL/WHO classification. The mean age was 31 years and there was a clear female predominance (male/female ratio 1/9) and stage II at diagnosis (8 out of 10 patients) whereas B symptoms and bone marrow involvement were exceptionally noted (0 and 1 patient respectively). IPI score was 0 or 1 in 8 out 10 patients. At moment 7 patients have undergone ASCT and 6 of them had receive more than 1 lines of chemotherapy prior to transplant, the another patient had also received prior mediastinal radiation therapy. Three out of 10 patients analysed are actually following the initial regimen of chemotherapy usually administered in our centre. Results: At transplant only 3 out of 7 patients had achieved a complete remission and 4 patients were in partial remission. Following ASCT all patients attained a complete remission. With a maximal follow-up of 84 months only a patient have relapsed, the progression-free survival has not yet been reached and the median overall survival is 30 months. Conclusions: The clinical features are similar to the previously reported in this entity including young female predominance, intrathoracic involvement and low risk IPI. Despite this favorable prognosis only 3 patients were in complete remission before ASCT. Bearing in mind this issue, the results attained in our series seems to confirm the relevant role of ASCT in this group of patients and should be considered as part of primary therapy for patients with PMLBL.

H. Pohl, A. Shimoni, N. Kroeger, H. Martin, V. Vucinic, N. Basara, A. Nagler, A. Zander, A. Fauser, M. Kiehl, Clinic for BMT and Haematology/Oncology, Chaim Sheba Medical Center, University Clinic (Idar-Oberstein, D; Tel-Hashomer, IL; Hamburg, Frankfurt, D) Due to dose intensified standard combined radio-and chemotherapy regimens a substantial proportion of patients will be cured. In patients relapsing therapy of choice is an autologous stem cell transplantation with high remission rates. Despite these progress in therapeutic modalities some patients suffer from refractory disease or relapse after autologous transplantation. As graft versus Hodgkin`s lymphoma effects have been described already allogeneic stem cell transplantation might be an therapeutic effort in these high risk patients. We herein analysed the course of 14 patients receiving an allogeneic transplant between 07/99 and 11/03. Median age was 23 years (range 19 -41) and patients received a median of 4.5 chemotherapies (range 2 -8) before transplant. All but one received previously an autologuos transplant. Disease status at transplant was CR in 2, PR in 1, SD in 5, and PD in 6 patients. Eight patients received a transplant from a related HLA-identical donor and 6 from an unrelated donor (4 matched, 2 DRB1 mismatch). All but one received peripheral blood stem cell grafts. Different conditioning regimens were used (Fludarabine (Flu)/busulfan (Bu) "b ATG n=6, Flu/Melphalan (Mel) "b Campath n=5, Bu/Cy n=1, TBI/Cy n=1, Mel/Thiotepa/ATG n=1). Four patients received donor lymphocyte infusions in escalating dosage for SD (n=2), PD (n=1), or mixed chimerism (n=1), respectively. Overall survival is 43% and disease free survival 28.6% after a median follow up of 25 months (1 to 52 months). Transplant related mortality is 14.3 %. Five patients died due to progressive disease and one due to refractory GVHD. Taking into account that all patients were heavily pre-treated and that only two patients were transplanted in CR these data argue for a strong graft versus Hodgkin effect. Especially a melphalan/fludarabin "b ATG reduced conditioning seems to be very effective in this setting. Furthermore, it`s speculative but very likely that the results of allogeneic stem cell transplantation will improve significantly if patients receive the allo transplant earlier during their disease. Thus, risk factors should be defined and those patients with a high risk feature should be scheduled for allo transplant within a clinical trial.

Evidence for a graft-versus-lymphoma effect in hairy cell leukaemia H. Schieder, F. Ayuk, A. Kratochwille, N. Kröger, T. Zabelina, H. Renges, N. Stute, A.R. Zander, University Hospital Hamburg Eppendorf (Hamburg, D) A graft-versus-lymphoma (GVL)-effect after allogenic stem cell transplantation (ASCT) has been described especially in low grade lymphomas.We present the case of a patient with relapsed hairy cell leukemia after ASCT with reduced intensity conditioning ( RIC) who achieved a complete remission ( CR) by donor lymphocyte infusion ( DLI). Case report:

A 33 year old woman with refractory hairy cell leukemia was transplanted at our BMT-unit in july 2002. Bone marrow biopsy bevor ASCT showed a diffuse infiltration with hairy cells. Donor of peripheral blood stem cells was her HLA-identical father. The conditioning regimen consisted of fludarabin ( 3x30mg/qm), melphalan( 150mg/qm) and ATG ( 3x10mg/kgKG). After transplantation the patient didn`t develop acute graft versus host disease (aGVHD). In october 2002 bone marrow biopsy and FACS-analysis both showed a complete remission, which was confirmed in april 2003. Immunosuppression was withdrawn at day +170 after ASCT. One year after ASCT bone marrow biopsy showed again a 5-10% marrow infiltration by hairy cells as well as a positive FACSanalysis. The patient now received a donor lymphocyte infusion with 1x10 7 CD3+ cells/kgKG . In the follow up she developed. mild GVHD of the skin and liver. Bone marrow biopsie two month after DLI showed a complete remission. Conclusion: A complete remission after RIC in refractory disease and especially remission induction by DLI support the existence of a GVL-effect a in hairy cell leukemia.

Total therapy for mantle cell lymphoma W.J. Jurczak, A. Zaluska, D. Krochmalczyk, P. Mansah, B. Piatkowska-Jakubas, A.B. Skotnicki, Collegium Medicum UJ (Cracow, PL) Bad prognosis of Mantle cell Lymphoma (MCL) is caused by inevitable development of resistance to chemotherapy. Therefore achieving a complete clinical response and eradication of all neoplastic cells is a condition for prolonged EFS. The plateau on survival curves is not seen even after most efficient chemotherapy regimens (i.e. hyperCVAD -MA) or consolidating the first CR by high dose chemotherapy with auto-SCT. Similarly relapses are observed after combining monoclonal antibodies with the first line chemotherapy. Most of the MCL patients seen at our Department are in IVB clinical stage, with massive bone marrow infiltration, marked leukocytosis (in 1/3 of them above 150 000 per micro liter) and splenomegaly (in ¼ of them over 25 cm in imaging studies). MCL was initially diagnosed in 10% of the cases (26 out of 255 patients) where bone marrow aspirate or peripheral blood was immunophenotyped for suspected chronic lymphocytic leukemia. The results achieved before introducing monoclonal antibodies were poor, all 16 patients died within the first 18 months. Our current strategy is to reduce the tumor burden by the first 2 chemotherapy cycles (hyper-CVAD regimen), and later combine chemotherapy with Rituximab (R) (hyper-CVAD -R, MA -R, hyper-CVAD -R, MA -R). Subsequent chemotherapy cycles are given at 21 day intervals, the last MA-R is used to mobilize the stem cells for a transplant procedure. TBI or BEAM conditioned auto-SCT is followed after engraftment, by Rituximab maintenance (4 doses at 3 week intervals). Out of 10 patients included 1 developed resistance despite the combination chemoimmunotherapy, 1 was lost in transplant related mortality, the rest of them are alive, with the average EFS of 1 year (range 0.2 -2.5 years). Patients in clinical remission (confirmed by imaging studies and bone marrow cytology) are monitored for minimal residual disease by: 3 color phenotype analysis, FISH and cytogenetic studies and trephine biopsies. Although time and randomized studies are necessary to draw any firm conclusions on survival, we feel, that one might defend such an aggressive therapy protocol on clinical ground

Autologous peripheral blood stem cell transplantation in 158 multiple myeloma patients: a single-centre experience M. Krejci, R. Hajek, A. Svobodnik, A. Krivanova, J. Mayer, J. Vorlicek, University Hospital Brno-Bohunice (Brno, CZ) Background:

Autologous peripheral blood stem cell transplantation (PBSCT) has an established role in the treatment of symptomatic eligible multiple myeloma (MM) patients (pts). It is not yet completely clear whether some subgroup of MM pts has more benefit from this procedure than other one. Methods: We have retrospectively analyzed 158 pts with MM undergoing PBSCT in our centre between 1995 and 2002. The conditioning regimen was high dose melphalan (200 mg/m2) in all pts. At diagnosis of MM 104 (66%) pts were stage III according Durie-Salmon, 32 (20%) II and 22 (14%) I. Median age at transplantation was 55 years (range: 28-69). We evaluated the influence of many clinical important parameters (age, gender, type of MM, stage of MM, responses before and after PBSCT, some laboratory parameters at diagnosis /beta2microglobulin, hemoglobin, calcium, albumin, C-reactive protein/, time of PBSCT from diagnosis, number of lines of previous regimens and others) on both PFS and OS after transplant to define pts with better prognosis. The median follow up from transplant was 50 months. Results: At the time of transplant, 12 (8%) pts were in complete remission (CR), 102 (65%) were in partial remission (PR), 24 (15%) achieved only minimal response (MR), 14 (9%) were non responding (NR) and 6 (3%) had progression (PG). The median number of CD34+ cells infused was 4.7x10E6/kg (range: 0.9-22.3). The median time to platelet recovery (> 50x10E9/l) was 13.0 days, while the neutrophil engraftment (> 0.5x10E9/l) was achieved at a median time of 13.0 days. Following PBSCT, 50 (32%) pts were in CR and 95 (60%) in PR. Median OS and PFS from transplantation were 65 and 33 months. Transplant related mortality (TRM) was 3%. Age at transplant under 60 years and good response (CR+PR) after transplant correlated with significantly longer OS (p = 0.002, p < 0.001). Pts with stage III and IgA type had shorter OS than others (p = 0.009, p = 0.046). The status of disease prior to autograft, time of PBSCT from diagnosis, number of lines of previous regimens and some laboratory parameters at diagnosis (hemoglobin, calcium, albumin, C-reactive protein) did not significantly affect PFS and OS after transplant. Details will be presented. Conclusion: PBSCT is safe and effective procedure in MM patients with low TRM and high number of responses (CR+PR=92%). In our group of MM pts the outcome of PBSCT was dependent of age, response to transplant, type of MM and stage of MM.

Vinorelbine and cyclophosphamide plus G-CSF as mobilisation regimen for patients with multiple myeloma S. Palmieri, B. Pocali, C. Copia, E.M. Schiavone, M. Annunziata, A. Viola, F. Ferrara, Cardarelli Hospital (Naples, I) High dose cyclophosphamide (HD-CTX) is the most commonly used mobilizing regimen for patients with multiple myeloma (MM); however, hematologic and extra-hematologic toxicity related to HD-CTX is substantial and hospitalization is usually needed, resulting in increasing pressure on bed availability. Different attempts have been made in order to investigate the efficacy and toxicity of alternative approaches aiming at collection of peripheral blood stem cells (PBSC) with reduction of side effects and costs of the procedure. Accordingly, we designed a novel mobilization regimen based on the combination of CTX and vinorelbine (VNB) for patients with MM. The following schedule was adopted: VNB 30 mg/sqm on day 1, CTX 1500 mg/sqm on day 2, G-CSF 10 microg/kg/die from day 3 to CD34+ cells peak. All patients were programmed to receive the regimen in an outpatient setting. Eleven patients with a median age of 57 years (37-73) were enrolled in the study. Myeloma subtype was IgG in 5 cases, IgA in 4, light chain in 1, and solitary plasmocytoma refractory to previous radiotherapy in the last patient. In all cases VNB-CTX was part of first line therapy and was given after three courses of VAD with (n=6) or without (n=5) thalidomide. Collection was successful in 11/11 cases (100%), with a median of 10 days (range 8-11) to reach the peak value of 59x10E6 CD34+cells (range 33-229). Following mobilization a median of 9.6x10E6 CD34+ cells (range 4.2-15.35) were collected with a median of 2 apheresis (range 1-2). There were no episodes of grade 3-4 neutropenia or thrombocytopenia, while one patient required 1 packed RBC unit. Fever or hemorrages did never occur and there was no extra-hematologic toxicity. Nine patients received autologous stem cell transplantation (ASCT), while two are waiting for the procedure. In autografted patients hematopoietic recovery was fast with a median time to neutrophil and platelet recovery of 12 and 14 days, respectively. As compared to a previous series of 30 patients in which HD-CTX was used, the decrease in costs, mainly related to absence of hospitalization during chemotherapy and reduction of supportive treatment, was of 2250 euro for patient. We conclude that VNB-CTX is a highly efficient and well tolerated regimen for patients with MM programmed to receive ASCT. In addition, the regimen allows precise timing of harvesting PBSCs and may represent an attractive alternative to HD-CTX in this setting.

Autologous stem cell transplantation in patients with plasmacytoma: the Finnish experience E. Jantunen, E. Koivunen, M. Putkonen, T. Siitonen, E. Juvonen, T. Nousiainen, Kuopio University Hospital, Tampere University Hospital, Turku University Hospital, Oulu University Hospital, Helsinki University Hospital (Kuopio, Tampere, Turku, Oulu, Helsinki, FIN) Although ASCT is considered standard treatment in multiple myeloma (MM), limited experience is available on this approach in patients with plasmocytoma (PC) but without overt MM. Altogether twelve patients with PC received ASCT in Finland in 1994-2002. There were nine males and three females with a median age of 50 years (32-64). Ten patients had plasmocytoma of bone, whereas two patients had extramedullary plasmocytomas. The median time from the diagnosis to ASCT was nine months . Nine patients (69 %) had received radiotherapy prior to the progenitor cell mobilisation. At the time of ASCT seven patients were in Ist CR or PR, two patients had primarily refractory disease and three patients had relapsed or progressed after primary therapy. HDT consisted of MEL200 (N=7), MEL200 x 2 (N=3) or TBI-MEL (N=2). Eleven patients received PB grafts (CD34+ selected in two patients) and one patient BM+PB. No transplant-related deaths occurred. With a median follow-up of 37 months (9-95) from ASCT, eleven patients (92 %) are alive. To date five patients (42 %) have progressed 6-81 months from ASCT. Two patients have received second ASCT as a part of relapse treatment. ASCT is feasible in this patient population resulting in promising progression free and overall survival. Randomised trials are needed to assess the real value of ASCT when compared to other treatment options in this patient population. Introduction and Objectives: Autologous PBSC transplantation is the treatment of choice for symptomatic MM patients under 70 years of age as intensification after induction treatment. However, the best method to mobilizing and collecting progenitor cells is not well defined the collections results could be influenzed by several variables related with disease status and with the technical procedures used. Patients and Methods: We present our single Centre experience comparing four methods used for PBSC mobilization and collection cell for autologous PBSCT in MM. From 1989 we have included 125 patients with MM (58M/57F). median of age was 50 years (28-70): 6 stage I, 24 stage II and 95 stage III . Median

Maintenance treatment with bisphosphonates + interferon + dexamethasone + thalidomide (quadruple maintenance) after autologous peripheral blood stem cell transplantion in multiple myeloma: preliminary results A. Alegre, C. Martínez-Chamorro, A. Escudero, B. Aguado, S. Osorio, S. Nistal, R. Córdoba, J.J. Gil-Fernández, P. Sánchez-Godoy, C. Burgaleta, F. Casado, J.M. Fernández-Rañada, Hospital de la Princesa, Clínica Rúber, Hospital Gregorio Marañón, Clínica Moncloa, Hospital Severo Ochoa, Hospital Príncipe de Asturias, Hospital Virgen de la Salud (Madrid, Toledo, E) Introduction: The favourable results of APBSCT in MM, are counteracted by the fact that relapses and progressions are inevitables. Different strategies must be designed aiming to prevent these relapses including different maintenance treatment. We present our preliminary experience with a cuadruple maintenance therapy including low dose of thalidomide. Objectives: 1) Evaluate the toxicity and applicability of a prolonged cuadruple maintenance therapy post-APBSCT in MM including low dose of oral thalidomide Patient and Methods: Criteria of inclusion:1) MM with previous APBSCT with stable hematopoiesis and CR or PR postransplant. We also included patients rescued with standard dose of thalidomide for initial signs of progresion during previous triple maintenance treatment . Patients included: 12 patients with symptomatic MM (5 stage II and 7 stage III), median of age 58 years ,42-69; 7 M, 5 F, have been included in the study. 3 patients received intensification with BUMEL (Busulphan 12 mg/Kg orally and Melphalan 140 mg/m2 i.v.) and 9 with Melphalan 200 mg/m2 iv. Cuadruple Maintenance Tretament: Bisfosfonates: Pamidornate 90 mgs iv or Zolendronate 4 mg iv /month, minimum x 12 months post transplant + INFa2b 3 MU s.c.3 x w until relapse or progression + Dexamethasone 20 mg orally x 4 days every 6 weeks, minimum x 12 months postransplant + Thalidomide 50-100 mg/d, according tolerance, until relapse or progression. Results: Maintenance treatment including low dose oral thalidomide was initiated at a median of 46 days post-transplant (range 23-158 days) in the nine patients without previous treatment after transplant. No patient failed to tolerate this dose of thalidomide. The most common adverse effects were constipation, dry skin and somnolence No grade 3-4 adverse effects were documented. We have observed only one case of progression, although the median follow up is still short (10 months; range 5-30 months). Conclusions and Comments: Cuadruple maintenance treatment presents an acceptable tolerance.. Longer follow up and future randomized trials will be needed to validate the role of thalidomide and its long-term effect when used as maintenance therapy in the post-transplant setting.

Unselected autologous followed by CD34+ selected peripheral blood stem cell transplantation for multiple myeloma F. Sorà, V. De Stefano, P. Chiusolo, L. Laurenti, E. Rossi, N. Piccirillo, A. Di Mario, M. Palladino, G. Leone, S. Sica, Universita Cattolica S. Cuore (Rome, I) We report the feasibility and outcome of 21 consecutive patients with multiple myeloma (MM) less than 60 years of age treated with double autotransplant using unselected followed by CD34+ selected peripheral blood stem cells (SC). Median age was 53, 7 were females and 14 males. Disease status was available in 14 patients (66.6%) and included stage III in 9 patients (64.3%) II in 5 patients (35.7%). M-protein was IgG (52.3%), IgA (28.5%) and light chain (19.2%). Previous therapy included 2 courses of classical VAD (range 2-3) followed by cyclophosphamide 4g/m2 +G-CSF and a 1st round of SC collection. Then VP-16 (2g/m2) + G-CSF was followed by a 2nd round of SC collection and CD34+immunoselection (Miltenyi CliniMacs). Patients were then conditioned with busulphan 16mg/kg and melphalan 90mg/m2 (11 patients) or 140mg/m2 (6 patients) for the 1st transplant receiving a median dose of 6x106/kg (range 4-24.3) CD34+ cells and with high dose melphalan (200mg/m2) in the 2nd transplantation receiving a median dose of 3.85x106/kg (range 1.3-15.9) CD34+ cells. G-CSF 5µg/kg was administered after the 2nd transplantation only. When complete hemopoietic recovery was obtained all patients were started on alpha-interferon 3x106 every other day until relapse or progression. Response to chemotherapy was: 8 partial remission (PR) (38%), 9 disease progression (DP) (43%), 3 minimal response(MR)(14%), 1 complete remission (CR)1 (5%). CD34+ immunoselection was possible in 19/21(90%). All but 4 patients proceeded to double autotransplant with an interval of 6 months. After the 1st and 2nd transplantation, median time to PMN>0.5, >1x109/l, plts>20, >50x109/l and Hgb>10g/dl was 14 and 11 (p0.004), 15 and 12 (p=0.002), 11 and 12, 13 and 16 (p=0.03) 16 and 31 respectively. Length of hospitalization was similar. No patients died from transplant-related complications. Response to 1st transplant was: PR 48%, CR24%, DP 14%, MR 9%, no change 5%. After the 2nd transplantation there were 8 patients in CR (47%), 7 patients in PR (41%), 2 patients were in DP (12%). With a median follow up of 32 months of 17 patients completing the protocol, 11 patients are alive, 2 in relapse, 4 in PR and 5 in CR according to EBMT criteria. Six patients died at a median of 31 months (range 1-41) from MM relapse or progression. One patient in relapse at 12 months was lost to follow-up. OS at median of 32 months is 58% with an EFS of 51%, for patients achieving CR DFS was 62% at a median of 20 months.

Successful treatment for relapse of multiple myeloma after allogeneic peripheral blood stem cell transplantation with Velcade® as exemplified in two cases W.I. Blau, W.U. Knauf, O. Marinets, E. Thiel, Charite', Campus Benjamin Franklin (Berlin, D) A 35-years-old male underwent related allogeneic haematopoietic stem cell transplantation (HSCT) to treat IgA-Multiple Myeloma (MM) after standard chemotherapy and after autologous PBSCT in year 2000. Conditioning started with total body irradiation 12 Gy day-6 to day-4 and cyclophoshamide 60mg/kg BW day-3 and day-2. Immunosuppressive treatment comprised a short course methotrexate and cyclosporine A. Leukocyte engraftment was shown at day +12, platelet recovery at day +21. Full donor chimerism was achieved at day +30. The patient developed acute GVHD of skin grad 1, only. He relapsed 2 years later and became successfully treated by disruption of immunosuppressive therapy and DLI. He achieved a complete remission with normalisation of monoclonal IgA and peripheral blood counts for one year. Clinical symptoms of anemia and increasing paraproteinemia were recorded in August of this year. The patient received two courses of Velcade® 1,3mg/m² IV during one month, and there after all laboratory parameters normalized. For the last three months the patient had no signs of disease, no paraprotein detected and a very good clinical conditions. A 62 years old male was treated due to IgA-MM by standard chemotherapy and autologous PBSCT, but he did not achieve complete remission. One year ago after reduced toxicity conditioning regimen (fludarabin/treosulfan) he received allogeneic HSCT from HLA-identical sibling. Immunosuppression was administred with a short course methotrexate and cyclosporine A. Leukocyte engraftment was shown at day +12, platelet recovery at day +12. Full donor chimerism was not achieved at all time of observation. The patient developed acute GVHD of skin grad II. IgA paraproteinaemia decreased four S357 times without normalisation up to day +100. Due to relapse immunosuppressive therapy was disrupted without any clinical or laboratory effects. Nine month after HSCT patient was treated with Velcade® 1,3mg/m² IV. After start of therapy IgA paraproteinaemia decreased rapidly from 50g/l to 8g/l (N<4g/l). As shown by these case reports, relapse of MM after allogeneic HSCT can be successfully treated by the new proteasom inhibitor bortezomib (Velcade®).

Autologous stem cell transplantation for multiple myeloma in elderly patients M. Eibl, W. Zinke-Cerwenka, P. Neumeister, A. Wölfler, H. Sill, W. Linkesch, Division of Hematology (Graz, A) Introduction: Patients over 65 years of age are usually excluded from autologous stem cell transplantation (ASCT) due to a postulated high transplant related mortality. The issue of age limits is important because the median age of multiple myeloma (MM) patients is currently over 65 years. We retrospectively analysed the outcome of patients at the age of 65 years and older, after ASCT for MM. Patients and methods: From September 1999 to June 2003, 12 patients (7 men and 5 women) with a median age of 68.5 years (range: 65-70) underwent one (n=7) or two (n=5) ASCTs for MM. Pretransplant staging revealed IIIA in 8 patients and IIIB in 4 patients. The median time from diagnosis to ASCT was 9 months (range: 3-76). Eight patients received one therapy line (3 cycles of VAD) before stem cell priming and ASCT, the remaining patients got two (n=2) or three (n=2) different cytostatic regimens. All but two patients suffered from at least two concomitant disorders. An elevated beta-2-microglobuline above 3 mg/l was measured in 5 patients. Conditioning consisted of melphalan 100 mg/m2 in one patient, 140 mg/m2 in two patients, 200 mg/m2 in four patients, 2 x 100 mg/m2 in one patient, and 2 x 200 mg/m2 in four patients. A median of 3.75 x 106 CD34+ peripheral blood progenitor cells/kg b.w. (range: 1.88-8) were transplanted. Results: Engraftment occurred in all patients. The median times to achieve a sustained absolute neutrophil count above 0.5 x 109/l and a sustained platelet count above 20 x 109/l were 11 (9-13) and 11.5 days (10-25), respectively. The median time to the first discharge from hospital was 16 days (13-27). Response to ASCT was observed in nine cases: complete remission in 4 patients and partial remission in 5 patients. Now after a median follow up of 17.5 months (11-49), seven patients (58%) are alive and progression free. The patients who died, had an elevated beta-2-microglobuline and their disease stage was IIIB pre transplant. Cause of death were transplant related complications in one patient (8.3%) and disease progression in the remaining four. Conclusion: ASCT for MM is a safe and effective procedure also in elderly patients with normal renal function. Even tandem transplants are well tolerated. The use of peripheral blood stem cells and conditioning without total body irradiation might have improved the outcome in selected elderly patients during the last years

Prognostic value of prame gene in multiple myeloma I. Abramenko, I. Kryachok, N. Belous, N. Kostukova, V. Chomenko, V. Bebeshko, Research Center for Radiation Medicine, Kiev Bone Marrow Transplantation Center (Kiev, UKR) Background: The expression of the cancer/testis gene PRAME recently was found in patients with acute leukemia, chronic myeloid leukemia and multiple myeloma (MM). The aim of our study was to evaluate the significance of PRAME gene as a prognostic marker and a marker for detection of minimal residual disease in multiple myeloma patients.

Methods: reverse transcription-nested PCR and original set of specific primers were used. 41 MM patients were enrolled in this study. The MM was diagnosed according to standard criteria and patients were staged according to Durie and Salmon classification. MM patients younger than 65 years were treated with 3-4 VAD cycles, 2-4 g\m2 cyclophosphamide and high dose chemotherapy with 200 mg/m2 melphalan and peripheral blood stem cell transplantation (PBSCT). PRAME gene expression was evaluate in all patients before treatment, in 21 pts on day +100 and in 10 pts -1 year after transplantation . Results: We found that the majority of MM patients were PRAME-positive (60,97%), no correlation with stage, type of monoclonal protein and the durability of disease was found. The only high level LDH was associated with PRAME expression. On day +100 gene PRAME disappeared in 7 out of 17 initially PRAME positive patients and was found again in 2 patients in 1-7 months before the clinical relapse, 5 other patients are in remission. The only one patient with PRAME expression still has complete remission 1 year after PBSCT. Conclusion: these results suggest that the detection of PRAME gene may be used as a marker of molecular response and as a prognostic factor for multiple myeloma, but the study needs to be continued possibly with quantitative PCR analysis.

Pattern of relapse and outcome in chemosensitive patients with multiple myeloma relapsing after HDT/autotransplantation intensification E. Giné, J. Bladé, J. Esteve, L. Rosiñol, F. Fernández-Avilés, C. Martínez, M. Rovira, A. Urbano-Ispizua, P. Marin, E. Carreras, E. Montserrat, Hospital Clinic (Barcelona, E) Background:

Combination chemotherapy followed by intensification with autotransplantation is part of the up-front therapeutic approach in newly diagnosed younger patients with multiple myeloma. There is scarce information on the pattern of relapse and outcome post transplant in patients relapsing from HDT. Aim: To report the pattern of relapse as well as the outcome of patients with multiple myeloma responding to initial chemotherapy and intensified with HDT. Patients and Methods: From May 1991 to December 2002, 75 chemosensitive patients underwent HDT intensification at our institution as a part of the initial treatment. Median age was 54 years (range 38-69) and there were 46 males and 29 females. The median time from the initial treatment to intensification was 8.7 months. Results: Response rate achieved after HDT was: CR 34 %, PR 64 % and MR 2 %. The median overall survival and event-free survival for all the patients were of 68.2 and 37.3 months, respectively. So far, 37 out of 75 patients have relapsed. The relapse rate was significantly lower in patients who were in CR after HDT (36% vs. 57 %, p= 0.007). The pattern of relapse consisted of an increase in the M-protein size with typical features of symptomatic MM in 93 % of the patients (including extramedullary plasmocytomas in 24 %), while only 7 % showed progressive disease with no increase in the M-protein.The median time from relapse to the need for rescue treatment was only 1.5 months. Median survival from relapse was 25.6 months. No difference was observed between patients relapsing from CR versus partial or minimal response. Conclusion: The pattern relapse after HDT does not seem to be different from that observed after conventional chemotherapy. The time from relapse to the need of treatment is very short. The survival from relapse is two years, irrespective of the previous degree of response to HDT.

Despite generally high cure rates in patients with metastatic germ cell cancer, patients with progressive disease on first-line cisplatin-based chemotherapy following high-dose salvage therapy exhibit poor prognosis. The evaluation of new drugs in testicular cancer patients with absolute cisplatin-refractory disease has demonstrated that paclitaxel is one of the few agents with antitumor activity in these patients. Docetaxel, the semi-synthetic paclitaxel analogue, showed at least the same efficacy in some solid tumors including advanced breast and non-small-cell lung cancer. In order to evaluate tumor response, toxicity and mobilizing capacity we have started internal protocol with docetaxel inclusion in pretransplant salvage therapy. Therapy schedule consists of 2 or 3 cycles of docetaxel at a dose 100 mg/m2 in combination with ifosfamide 6 g/m2 + cisplatin (TI) followed by two sequential cycles of high-dose etoposide 1500 mg/m2 and carboplatin 24 AUC with autologous stem cell support and 3 weeks intervals between all chemotherapy elements. At this time, only five patients had been included in the protocol. All patients were absolutely refractory to cisplatin and received a median of 5 platinum-containing treatment cycles (range 3-7) prior to docetaxel administration. Salvage chemotherapy was well tolerated in all patients. Toxicity consisted mainly of hematological side effects with grade 3 or 4 leukocytopenia. No non-hematological toxicity was observed. Stem cells were successfully collected after first or second cycle of docetaxel and G-CSF in all cases. The median number of collected CD34(+) cells was 8,86 x 106/kg (range 3,69-14,6). First two patients received one high dose regimen with carboplatin, etoposide and ifosfamide 10 g/m2. One of these patients died because of renal toxicity. After this case, we have included only two drugs in high dose regimen. Response to whole therapy: 4 patients are alive, with complete remission in 3 patients (one after resection of residual tumor) and partial response in one. Median observation time was 15 months (range 9-28). Conclusion. We hope that treatment strategies, such as "dose dense" chemotherapy and the use of new cytotoxic agents with application of high-dose chemotherapy with peripheral stem cell rescue may by a key to improvement therapy results.

M. Maur, R. Sabbatini, A. Donelli, P. Bresciani, L. Cafarelli, F. Narni, P.F. Conte, University of Modena (Modena, I) Ewing's Sarcoma (ES) and peripheral primitive neuroectodermal tumors (PNETs) arise from bone or soft tissue, predominantly affect children and young adults. We present results achieved with HDC consolidation in 4 patients (pts) with ES/PNET diagnosed on 2002. Four pts were diagnosed as ES/PNET, 2 pts were c-kit negative and 1 pt was c-kit positive. Median age was

Leucomax®-Schering Plough); Group 2: Cyclophosphamide 2-4 g/m2 iv + G-CSF(filgrastim, Neupogen®,Amgen) sc; Group 3: G-CSF alone 10 mcrg/Kg/d sc x 5 (Group 3A) y 20 mcrg/Kg/d sc x 5 (Group 3b)

Results: ( Patients characteristics included in each group did not show significant differences

Graft > 20x10e9/Lplat: Global: 12(7-71)

The main variables that influence the results, apart from mobilization scheme, were previous treatment with melphalant and the number of lines of previous chemotherapy.These two variables showed the major negative impact. Comments and Conclusions: The methods with best results regarding rates of CD34+/kg harvesting include chemotherapy followed by the cytokine G-CSF. However, the use of G-CSF alone is enough to reach the minimal CD34/kg targetted rate of 2x10e6/Kg. This method simplifes the collections, reducing the morbidity and the cost of the global procedure. Patients with previous treatment including alkilants as melphalan may have compromised the hemopoietic reserve for PBSC mobilization

Second autologous transplantation for multiple myeloma patients relapsing after the first autograft and a pilot study for the evaluation of experimental therapy

Design of study: The principle is repeated AT with testing different experimental approaches in MM pts relapsing/ progressing after the 1st AT. The pts are treated with the same + something more -same or very similar induction and reinduction, same myeloablative regimen in the 1st and the 2nd AT and a different maintenance, experimental th.after the 2nd AT. The evaluation of the results of each therapeutic approach uses intraindividual analysis -the comparison of event free survival I (EFS I) (after the 1st AT) and EFS II (after the 2nd AT) in one pt. Both EFS I and EFS II are compared in one pt., therefore the interindividual differences are excluded. Subjects: 32 pts with relapsing/progressing MM after the 1st AT were included in the pilot study between 1/97 and 1/03, median follow-up was 75,2 months

IRL) Objective: The most common mobilising regimen in myeloma is the combination of cyclophosphamide (doses ranging from 1.5-7g/m2) and granulocyte colony-stimulating factor (G-CSF) at a standard dose (5mcg/kg). The optimal doses in this combination remain unclear. We performed a retrospective single-centre audit of 38 patients who received cyclophosphamide (2g/m2) and

The median peripheral blood CD34+ count on Day 1 of leucapheresis was 92/microlitre (mcl) (range: 24-290) and the median Day 1 yield was 5.16 (0.8-12.5) x106 CD34+ cells/kg. The median total yield of 6.21x106 CD34+ cells/kg was reached after a median of 1 day (range: 1-3) of leucapheresis. A minimum Day 1 yield of >2x 106 CD34+ cells/kg was harvested in 34 (92%) patients. Only 1 patient (3%) failed to mobilise. The target total yield of >4.5x106 CD34+ cells/kg, sufficient to allow for 2 highdose treatments, was achieved in a single leucapheresis in 22 (59%) patients. In this group (>4.5), the median peripheral blood CD34+ count on Day 1 was 139/mcl, the median peripheral blood White Cell Count 11x109/l and the median Day 1 yield 7.9x106 CD34+ cells/kg. In the 16 patients who did not achieve this target Day 1 yield, the respective Day 1 values were 46/mcl, 9.2x109/l and 2.92x106 CD34+ cells/kg. 32 (86%) of 37 patients commenced leucapheresis on either the 8th or 9th day after chemotherapy. Fever (>38OC) developed in 5 (13%) patients. Intravenous antibiotics were required in 6 (16%) patients. Conclusion: In summary, cyclophosphamide (2g/m2) and 'highdose' G-CSF (10mcg/kg) is an effective mobilisation regimen

Low incidence of relapse in patients submitted to allogeneic stem cell transplantation for myelodysplastic syndromes or secondary acute myeloid leukaemia. A single-centre retrospective study C. Finelli, J. El-Cheikh, F. Bonifazi, G. Bandini, M. Arpinati, S. Falcioni, M. Stanzani, E. Vigna, C. Bosi, M. Malagola, M. Rondoni, S. Gaitani, P. Piccaluga, G. Martinelli, N. Testoni, M. Baccarani, Institute of Hematology (Bologna, I) 32 patients (pts) referred to our Institution because of high-risk Myelodysplastic Syndromes (MDS) or Secondary Acute Myeloid Leukemia (2ary AML) were submitted to Allogeneic Stem Cell Transplantation (SCT) from HLA-identical siblings or Voluntary Unrelated Donors (VUD) between March 1995 and February 2003 (minimum follow.up: 9 months) . 2ary AML was diagnosed on the basis of: a) a previous history (> 3 months) of MDS, or b) previous chemotherapy or radiotherapy, or c) "high risk" cytogenetic abnormalities. The clinical and haematological features of the pts were: 22 males, 10 females, median age: 40 (16-61) yrs; diagnosis: MDS: 10 pts; 2ary AML: 22 pts; interval from 1st diagnosis to months. Among the 10 MDS pts, SCT was performed as 1st line treatment in 9 cases, while 1 pt underwent SCT after chemotherapy, with a refractory disease. Among the 22 pts with 2ary AML, 7 were transplanted in Complete Remission (CR) after chemotherapy, 3 were in Partial remission (PR), 10 were transplanted in haematological relapse or with refractory disease, and 2 received 1st line SCT. The donors were HLA-identical siblings in 21 cases, VUD in 11; the source of haemopoietic stem cells was the bone marrow (BM) in 18 cases, and the peripheral blood in 14. The conditioning regimen included Total Body Irradiation (TBI) in 8 pts, and was busulphan-based in the other 24 subjects. In 6 pts a Reduced Intensity Conditioning Regimen (RIC) was administered. Cyclosporin A (Cs-A) and Methotrexate (Mtx) were given to all pts as GVHD prophylaxis, and Antithymocyte Globulin (ATG) (Fresenius) (15 mg/Kg) was added in VUD transplants. In 16 patient acute GVHD was diagnosed ( grade I: 5 pts; grade II: 2 pts; grade III: 7 pts; grade IV: 2 pts) and, among 24 evaluable pts, 12 subjects developed chronic GVHD, extended in 5 pts, limited in the remaining 7 cases. 23 pts (71.5%) died, 4 (12.5%) because of relapse , 10 (31%) for GVHD or infections, and 8 for other causes related to SCT, (1 death was not related to SCT), with a overall transplant-related mortality (TRM) of 56.2%. None of the MDS pts and none of the pts who received 1st line SCT relapsed. TRM was not reduced (50%) in pts submitted to RIC SCT. Overall survival of our series was 47.8 % at 12 months, 39.1 % at 2 years, and 22.3 % at 4 years.

A comprehensive diagnostic surveillance protocol for idiopathic (de novo) and secondary myelodysplastic syndromes in childhood L. Farinasso, L. Garbarini, F. Timeus, M.E. Basso, S. Aschero, A. Iavarone, A. Linari, E. Gottardi, U. Ramenghi, P. Saracco, University of Turin, Regina Margherita Hospital (Turin, Orbassano, I) Introduction: Myelodysplastic syndromes (MDS) in childhood are rare and heterogeneous clonal disorders of hematopoiesis with possible evolution to acute myeloid leukemia (AML). MDS can occur de novo (idiopathic) or as a complication of both preexisting bone marrow disorder (secondary MDS) or previous therapy (t-MDS). In childhood MDS frequency is underestimated. Case study: Since 1996, a diagnostic surveillance protocol for diagnosis of de novo and secondary MDS has been applied at our Institution. Morphologycal analysis on peripheral blood, marrow aspirate and biopsy, immunophenotypic clustering by flow cytometry, standard cytogenetic test, FISH and biomolecular analysis (AML translocations, WT1 gene), hemopoietic progenitors assay (with/without T-depletion and sequential dose of growth factors), GPI-linked proteins, circulating CD34+, bone RMN for evaluation of fibro-adipose tissue were performed. Results: Secondary MDS: patients (pt) with bone marrow failure syndromes, both congenital (14 FA, 1 Kostmann syndrome, 2 dyskeratosis congenital) and acquired (36 Aplastic Anemia, AA) were monitored for clonal evolution every 6-12 months. Children with Down syndrome and those with malignancies receiving chemotherapy, especially alkylating agents, were investigated at time of unexplained cytopenia. Among AA, 1 pt developed RAEB-t 27 months after diagnosis, a second patient showed a cytogenetic anomaly with marrow myeloid hyperplasia 4 years after diagnosis. 3 out of 14 FA developed MDS/AML while G-CSF treatment. t-MDS: 1 out of 6 APL t(15;17) pt developed CMML 36 m from diagnosis and 1 out of 59 Hodgkin disease developed RAEB-t 16 m after auto-BMT. Among patients with unexplained refractory single/multilineage cytopenia with/without organomegaly, 7 were diagnosed as idiopathic MDS (age 1 months -7.5 yrs); 1 rapidly progressed to AML-M7; 1 JMML. The most frequent cytogenetic abnormality was monosomy 7. Conclusions: In our experience, association of cytogenetic test, marrow biopsy and circulating CD34+ cells evaluation is very sensitive in predicting MDS.

Between January 1995 and March 2000 eleven patients with inflammatory breast cancer (IBC) and 24 patients with high risk breast cancer (HRBC) stage II. and III. were consolidated with HDC followed by reinfusion of CD34+ positively sellected SC (CellPro, Baxter) and G-CSF support. The patients with IBC were with a median age of 42 years (range 32-50 years) and median positive lymph nodes (LN) 8.5 (range 4-33). The patients with HRBC were with a median age of 43.5 years (28-55 years), and median positive LN 9 (5-15). The type of induction chemotherapy and HD CHT was not strictly defined. The most used induction regimen was FAC (Adriamycine, 5-FU, Cyclophosphamide) or AC ( Adriamycine, Cyclophosphamide).HD CHT regimen was CTC (Cyclophosphamide, Thiotepa, Cycloplatin). Thirty six months overall survival and event free survival was 72.7% and 54.6% respectively in IBC, or 66.6% and 45.8% respectively in HRBC. The patients reached leukocyte count > 1.0 x 109/l with a median on day +16.5 and on day+18 respectively . One patient died from treatment related complication. The main toxicity was neutropenic fever in 9 patients (82%) with IBC and in 20 patients (83%) with HRBC. Nausea or vomiting G 3-4 were present in 1 and 7 patients, leak sy in 1 and 1 patient, veno-occlusive disease in 1 and none patient, for IBC and HRBC respectively. Conclusion: HD chemotherapy followed by autologous CD34+ positively sellected SC support is in our hands a feasible and safe method in the treatment of patients with IBC and HRBC.

High dose chemotherapy followed by stem cell reinfusion in breast cancer (BC), is still an area of controversies in terms of DFS and OS. We retrospectively analyzed the outcome of patients with high risk (n: 60), or metastatic bresat cancer (n: 17) treated with highdose chemotherapy (HDC) and peripheral blood stem cell (PBSC) transplantation between March 1996 and July 2003 with a median follow up of 53 months (9-179). The median age of patients was 46 years (range: 25-63 ). Among metastatic patients 2/17 underwent to HDS in first CR, 10/17 in partial remission, 5/10 in SD. PBPC mobilization consisted in Cyclophosphamide (Cy) 7g/m2 (n: 43), Cy 4 g/m2 (n: 12), Epirubicin 150 mg/m2 (n: 12 ), FEC (n: 4) followed by G-csf 5µg/Kg/day or G-csf alone without chemoterapy (n: 5). Fourteen leukapheresis neoplastic contaminated products were purged by Max sep device (Baxter) using murine antihuman monoclonal antibodies vs breast cancer antigens. Conditioning regimen used consisting in Idarubicin+L-PAM (n: 42), Thiotepa+Cyclophosphamide (n: 3) Mitox+L-pam (8) Thiotepa+L-pam (11) other (13). All patients engrafted. ANC count > 0.5 x109/L and a PLT count > 20 x109/L were registered on day 9 (7-11) and 14 (8-19) respectively. Grade III-IV mucositis occurred in % of patients. No other severe extrahaematologica toxicity was registered. The TRM was 2,5% (2/77). 34/60 (67%) of HR patients are alive with a median follow up of 51 months with a median DFS of 55%. The median OS of metastatic patiants was 24 months (12-24) Autologous peripheral blood progenitor cell rescue was provided in our center, from January 1998 to April 2003, to 34 men with relapsed or refractory germ-cell tumors and to 14 women with breast cancer (6 for relapsed or metastatic desease, 8 for high risk primary desease). The average age of men was 29 years and of women 47 years. Stem cell mobilization was performed after the 3rd cycle of VeIP in men and after the 3rd or 4th cycle of conventional treatment in women in combination with G-CSF. The amount of CD34+ cell/kg b.w. was between 1.8-10.3x10^6. High-dose conditioning regimen CARBOPEC was used in men. Women with breast cancer had usually conditioning regimen ICE, CTCb. The treatment was well tolerated without transplantrelated mortality. Engraftment was rapid, recovery of hematopoiesis in neutrofils over 1.0x10^9/l and platelets over 50x10^9/l was reached an average on days +10 and +13 respectively in men and +9.5 and 12.0 resp. in women using filgrastim 5 /ug/kg beginning the day +6. Using WHO criteria, hematological toxicity was grade 4, non -hematological toxicity of the therapy was predominantly of grade 2 -3.The follow -up period range from 3 to 64 months in patiens with germ cell tumors, but at present 23 (67.6 %) out of 34 men-patients are alive (18 pts -52,9% are in CR), 11 (32.4 %) pts died. In patiens with breast cancer the follow -up period range 10-47 months, 7 (50.0%) out of 14 women are alive in CR, 7 died. In conclusion, high-dose chemotherapy with autologous PBPC rescue in patients with advanced germ-cell tumors and breast cancer is effective strategy for the treatment. According to the latest news the best results were obtained during the first -line treatment for poor prognosis germ cell tumors and in salvage setting in good risk ones and in high risk patiens with breast cancer in adjutant setting ( younger patients less than 45 years, high risk, low positivity of c-erb B2). The future will evaluate the role of this method in the therapy of patiens with germ-cell tumors and breast cancer.

Docetaxel as part of combination pre-transplant therapy in patients with germ cell tumours I. Korenkova, E. Karamanescht, V. Sakalo, V. Khomenko, Kiev City BMT Center (Kiev, UKR) (Askin-Rosai tumor) localized to chest wall at the diagnosis, after complete surgical resection, received 3 courses of doxorubicin (ADM) and cyclophosphamide (EDX) followed by 3 courses of ifosphamide (IFO) and etoposide (VP16) plus radiotherapy. After a relapse free interval (RFI) of 10 months, he had a bone relapse and received salvage chemotherapy according to CEVAIE schedule (EDX, ADM, VP16, IFOS, VCR) followed by HDC and autologous stem cell transplantation (ASCT). Other 3 pts (2 ES metastatic to bone and bone marrow and 1 PNET with pelvic recurrence) received CEVAIE regimen followed by HDCT plus ASCT. For all pts, HDC consisted of melphalan plus busulphan (3 pts) and thiotepa (1pt) followed by ASCT (mean CD34+ 5 x 10E6/Kg). After conventional chemotherapy, 2 pts were in complete remission (CR), 1 had a very good partial response (VGPR) and 1 was in partial response (PR). After HDC consolidation the pt in VGPR achieved CR, while pt in PR obtained only a transient clinical benefit. Among the 3 pts in CR (two c-kit negative, while for the other one data is not valuable) and after HDC consolidation two relapse after 3 months and are presently alive. One is receiving salvage conventional chemotherapy while the other one is undergoing a reduced intensity alloSCT from a HLA identical sibling, update data will be presented. The pt in PR, rapidly progress and died 3 months after ASCT. The induction regimen can achieve a minimal disease status in ES/PNET but remissions are short lasting in spite of HDC. As suggested by other c-kit positivity may predict a more aggressiveness phenotype and chemoresistance. In conclusion HDC with ASCT after conventional chemotherapy does not assure long term control of ES/PNET in adults. New strategies included adoptive immunotherapy are under development.