P509 to 8 of 10 possible MM occurred: No MM (4%), 1 MM (10%), 2 Cord Blood Bank of the Czech Republic, six month check MM (15%), 3 MM (22%), 4 MM (25%), 5 MM (12%), 6 MM I. Fales, S. Rahmatova, P. Kobylka, S. Matejckova, L. (6%), 7 MM (3%), 8 MM (2%). There was no significant Zahlavova, A. Hruba Institute of Hematology and Blood association between the number of MM (also analysed in Transfusion (Prague, CZ) GvHD or rejection direction) using HR-level for both HLA-A, -B and -DRB1 and HLA-A, B, C, DRB1 and DQB1 and the Cord Blood Bank of the Czech Republic (CCB CR) was incidence of aGvHD grade II-IV. There was a trend that MM in established in the year 1996. But the first collection of the cord class I HR were associated with neutrophil recovery; HLA-A blood (for the related part) in this project was performed in locus typing analysed in rejection direction as well as 1 or 2 A- 1994. It was planned collection for sibling suffering with LAD locus HLA disparities were associated with reduced CI of syndrome. The transplantation of the fully matched graft was engraftment (p= 0.04). The same was observed for the HLA- performed in the same year and it was first transplantation of C-KIR epitopes in rejection (HvG) direction (p=0.01). No the patient with this diagnosis by cord blood on the world. significant correlation was found between numbers of HLA- Actually 3 processing and 28 collection centres are MM on HR level with 2-year survival. Despite the cooperating in this project under management of the Cell heterogeneity and number of patients analysed, it shows that therapy department of the Institute of haematology and blood the actual degree of mismatching is even higher than transfusion. In the related part of the CBB CR 130 graft units previously expected. It also reveals that subtyping for HLA-A, - are stored and 6 graft from them were transplanted. In B, -C and –DQ does not improve the 2-year survival. Thus in unrelated part of the CBB more than 2500 units are stored. CBT most likely subtyping of HLA alleles should not be Information about more than 1600 grafts was submited to the required on a routine basis. Czech Bone Marrow Donors register and by it to the BMDW. From this part of the CBB CR also 6 grafts were transplanted (4 children, 2 adults). From the year 2004 is CBB CR fully P511 valuable NETCORD member. CBB CB consistently performs The chances of finding a 10/10 HLA allele-matched six month check. Without this check the graft is not released unrelated donor are highly predictable for the register. Thanks to this policy has CBB CR delay in J.-M. Tiercy, B. Chapuis, U. Schanz, R. Seger, P. Schneider, releasing of the grafts to register database, but on the other G. Nicoloso, R. Schwabe, A. Gratwohl University Hospital side the quality of the CBB is guranted. For this reason before (Geneva, CH); University Hospital (Zurich, CH); Kindersspital mentioned disproportion between figures of stored and (Zurich, CH); Blood Transfusion Center (Lausanne, CH); released grafts exists. Due to small area of our country and Swiss Bone Marrow Donor Registry (Berne, CH); University still conservative attitude of population to the migration for Hospital (Basel, CH) work, is this six month check very succesfull, more than 93% and probably will be even better. Six month check has two Histocompatibility testing for the identification of a matched part, pediatric evaluation of the child´s health and mother´s unrelated stem cell donor is a costful and timely procedure. blood re-testing for infectious diseases. After six month check Waiting in vain for an ideal donor can delay alternative 0,1 % of the grafts were discarded due to infection of the strategies, e.g. haploidentical HSC transplantation or non- mother, and 0,89% due to pediatric examination of the baby. transplant. In order to improve donor search strategy and So 1% of the grafts were discarded after six month check all to efficiency we introduced in 2002 an estimation of the gether. This check cannot be performed in 3% cases. In 2% probability to identify a 10/10 matched donor, i.e. compatible the mothers were not contacted and in 1% the mothers at the allele-level for HLA-A/B/C/DRB1/B3/B5/DQB1. The refused cooperation. probabilities were classified at initiation of donor search as low (no 10/10 match is expected in the 6-months framework of the search), intermediate (50% chance), or high (100% chance). Of 175 consecutive searches, 32 patients were classified as Stem cell donor having a high, 61 an intermediate, and 82 a low probability to find a 10/10 matched donor. The prediction was correct for

97% (high), 61% (intermediate), and 89% (low) of the patients.

The overall positive (high/intermediate probability) and P510 negative (low probability) predictive values were 73% and High-resolution HLA typing by sequencing for HLA-A, B, 89%, respectively. For the 73 patients with a low probability, C, DR, DQ in 122 unrelated cord blood/patient pairs. Does incompatibilities were linked to HLA-C (40%), HLA-DRB1 it improve long-term clinical outcome? (26%, mainly DR4, DR13), and HLA-B (18%) alleles. The B18, G. Kögler, J. Enczmann, V. Rocha, E. Gluckman, P. Wernet B35, B44 and B51 haplotypes accounted for 73% of HLA-C University of Duesseldorf (Duesseldorf, D); Eurocord, Hopital mismatches. The average number of ABDR-identical Saint-Louis (Paris, F) donors/patient in BMDW Registry for each category was 316

(high), 56 (intermediate), and 60 (low). Hence, the number of The CB Bank Düsseldorf has provided to date (November donors was not a reliable parameter to discriminate between 2004) 240 CB units (232 unrelated and 8 related) to transplant low and intermediate probability. The most relevant criteria centers worldwide. To determine the impact of HLA high were individual allele frequencies, B18/B51 haplotypes, resolution (HR) typing with outcomes after unrelated cord phenotypes with <= 2 ABDR antigen-matched donors in blood transplantation (UCBT), DNA samples of 122 CB BMDW Registry, as well as the experience of the HLA recipients and their unrelated CB grafts were analysed for laboratory with unusual B-Cw and DRB1-DRB3 associations. HLA-class I (A, B, C) and -class II (DRB1 and DQB1) – Of the 73 patients with a low probability, 18 were transplanted mismatches based on HR typing. When mismatches (MM) early after diagnosis with a mismatched donor (12 with a 9/10 were analysed for HLA-A, -B based on low resolution-typing donor), and 8 with a haploidentical related donor. These data and -DRB1 based on HR-typing, the following MM situation show that the chance to find or not find a 10/10 allele-matched resulted: No MM (13%), one MM (40%), two MM (36%), three donor is highly predictable. Such a step should be integrated MM (8%), four MM (3%). If the MM for -A and -B alleles early on in the donor search process and in the therapeutic detected by HR-typing were included, the situation was as algorithm. follows: 0 MM (6%), 1 MM (28%), 2 MM (48%), 3MM (11%), 4

MM (6%), 5 MM (1%). When A, B, C, DR and DQ based on

HR typing were analysed a higher additional frequency of up

S125 P512 P513 Granulocyte donation - yield and donor safety. A In vivo time course of plasmatic levels of retrospective study of 115 collections myeloperoxidase, a biomarker of oxidative stress, after U. Axdorph, A. Sollén-Nilsson, A. Svensson, M. Sjödahl, M. granulocyte colony stimulating factor-induced stem cell Hansson Karolinska University Hospital, Solna (Stockholm, S) mobilisation: profile of safety for the donors F. Morabito, A. Saija, M. Martino, G. Console, A. Taimano, P. Objective: Granulocyte (PMN) transfusions are given to Minciullo, M. Cuzzola, P. Iacopino, S. Gangemi Azienda patients with life-threatening infections, refractory to Ospedaliera (Reggio Calabria, I); University of Messina antimicrobial treatment. The objective was to evaluate yield (Messina, I) and complications in granulocyte donation during a 10 year observation time. When stimulated, neutrophils undergo an oxidative burst Methods: PMN donors were identified through a registry for resulting in the generation of large amounts of superoxide and blood donors. Data concerning G-CSF, blood values, yield, in the release of the heme enzyme MPO, which utilizes H2O2 complications and recent status of the donors were evaluated. to convert chloride to hypochlorous acid. Sustained levels of From 1997, a check-up was done 3 weeks post collection: this oxidant are associated with inflammatory side effects, Hemoglobin (Hb), white blood cell counts (WBC), platelets such as cell injury and tissue damage. Since the (Plt), sodium, potassium, calcium, creatinine. administration of rHuG-CSF followed by the leukapheresis Results: 71 consecutive donors, aged 39 years (median; procedure could induce a condition of oxidative stress, it might range 24-59) were included. 6-7 L of blood was processed. be of interest for the donor to verify whether and when this During the first years, HES 450/0.7 was used as health injurious process is triggered off. The aim of this study sedimentation agent, later replaced by HES 200/0.5. was to assess the in vivo time course of plasmatic MPO levels Stimulation and yield (see Table): Series I. 61 donors were in a group of donors mobilized with rHuG-CSF. Plasmatic given G-CSF 300 ug sc the evening before donation and MPO levels were measured before starting rHuG-CSF hydrocortisone 100 mg iv 15 minutes before collection. 10 administration, on day 5 of rHuG-CSF administration, on the donors were stimulated with hydrocortisone solely. 5 same day soon after the end of the first leukapheresis collections were cancelled after giving G-CSF. 41 donors were procedure and one week after rHuG-CSF withdrawal. Twenty- harvested for 2 consecutive days. Series II. 5 donors donated consecutive normal donors (14 male and 6 female; median PMN twice with an interval of 16 months (median; range 13- age, 46 years; age range, 16 to 56 years) were included in this 47) between the collections. Thus, a total of 115 collections study. rHuG-CSF was given at 10 µg/kg/day in two half doses were performed. every 12 hours. Plasma levels of MPO were determined by an Safety: Events during harvest: headache and nausea (n=1), EIA method. When compared with baseline values, plasma hypotension (n=1), ventricular extrasystoles (n=1). All of 40 MPO levels increased significantly in donors after 5 days of tested donors at check-up had normal values except 3: Hb rHuG-CSF treatment (Figure), mantaining comparable 113 g/L (n=1), WBC 2.9 x 109/L and Plt 109 x 109/L (n=1) and concentration following leukapheresis procedure. Finally, as Plt 464 x 109 (n=1). Follow-up was 40.5 months (median; measured in 9 subjects, plasma MPO returned to the baseline range 1-119 months). 6 of 71 donors are excluded from further values within 7 days following rHuG-CSF withdrawal. The donations due to disease: hypertension (n=3), anaphylaxis results of our study show a significant increase of plasma (n=1), cancer mammae (n=1), mesenterial thrombosis (n=1, 4 MPO levels, a biomarker of oxidative stress, after 5 days of years after G-CSF stimulated donation. This donor has a rHuG-CSF administration. Noteworthy, leukapheresis heterozygous factor V mutation). procedure failed to significantly influence plasmatic MPO Conclusions: This study confirms that G-CSF stimulated levels, since similar plasma MPO concentrations were donors give higher yield than donors stimulated with obtained at the same day 5 of rHuG-CSF administration both corticosteroids only. Events during harvest were infrequent before and immediately after leukapheresis. Importantly, and mild. So far, no late complications are likely to be related plasma MPO levels returned to baseline values after 7 days of to the stimulated donations. rHuG-CSF withdrawal. Interestingly, in patients with septic shock, G-CSF peaks are followed, in most cases, by an increase of polymorphonuclear cell burst activity, which returns to baseline levels within 4 days. In conclusion, here we report the first in vivo study on the time course of MPO, a biomarker of oxidative stress, after rHuG-CSF-induced stem cell mobilization. These findings may contribute to a better understanding of the oxidative reactions and of the safety profile in stem cell donors.

S126 P514 to transport of the product to its final destination of a transplant The successful mobilisation of peripheral blood stem centre. During the study period to date, ANT had received 342 cells using recombinant human stem cell factor in heavily work-up requests and recorded 26 serious events. Of the 26 pretreated patients who had failed a previous attempt events, 18 (69%) were as a result of donor deferrals at the using a granulocye colony-stimulating factor based time of medical assessment. In view of the comprehensive 3- regimen page medical questionnaire that donors are required to M.A. Dawson, A.P. Schwarer, J.L. Muirhead, A. Spencer, G.M. complete when they join the ANT Register and at every Bollard, M.J. Bailey Alfred Hospital (Melbourne, AUS) subsequent stage of testing, it was not unexpected that the problems detected were largely unpredictable. They included Patients with hematological malignancies who are heavily abnormal test results from the haematology and biochemistry pretreated are poor mobilizers of peripheral blood stem cells performed at medical, in particular, liver function tests. (PBSC). Up to 30% of these patients fail to collect sufficient However, high blood pressure was a problem that prior PBSC for an autologous transplant (> 2.0 x 106 CD34+ screening may have detected. Seven (27%) donors declined cells/kg). In this study we assessed the efficacy of to donate when selected. All of these donors had been on the recombinant human stem cell factor (rHuSCF) to successfully Register for many years (range 6-20 years) and had been remobilise patients who had already failed a G-CSF-based involved in tests for previous patients but had not previously mobilisation regimen. Patients who had failed mobilisation with donated. Serious events monitored after collection include G-CSF (10 µg/kg twice daily) with-or-without concomitant those occurring to the product at the collection centre, chemotherapy were remobilised a minimum of 3 weeks later occurring during transit either to the product or to the courier, with the addition of rHuSCF (20 µg/kg/day). Cytokine therapies and notification of any deficiencies in the product after were commenced concurrently and leukaphereses was delivery. During the study period, no serious adverse events initiated on day 5 of cytokine therapy. A maximum of 5 occurred in these categories. leukaphereses was scheduled with the aim of collecting sufficient PBSC for transplantation. A total of 48 patients were assessed in this study, with 29/48 (60%) reaching a P516 cumulative total of > 2.0 x 106 CD34+ cells/kg following Assessment of histocompatible siblings as bone marrow remobilisation. The inclusion of chemotherapy in the or PBSC donors: what do we do about ''borderline mobilisation regimen was associated with a higher yield of cases''? PBSC for both the initial G-CSF-based and subsequent K.W. Douglas, J.E. Sinclair, R. Green S.N.B.T.S. Clinical rHuSCF-based regimens (0.90 vs 0.54 (x 106 CD34+ cells/kg) Apheresis Unit (Glasgow, UK) P <0.01; and 2.36 vs 1.34 (x 106 CD34+ cells/kg) P <0.01, respectively). The variables on univariate analysis that were The medical assessment of potential sibling bone marrow or significantly associated with a successful remobilisation PBSC donors is always a complex process, with potential included the total yield of PBSC from the G-CSF-based hazards of the procedure to the donor, and the potential of regimen, performance status, baseline platelet count and disease transmission from donor to recipient, having to be albumin. Patients with multiple myeloma were also more likely weighed against the danger to the recipient of not proceeding than patients in other disease categories to successfully to transplant. JACIE, the Accreditation Body for bone marrow remobilize. There was no threshold of total PBSC collected and PBSC collection and transplantation facilities, has issued from the failed G-CSF-based regimen below which successful detailed recommendations regarding allogeneic donor remobilisation with the rHuSCF-based regimen was not assessment. These are that there must be written protocols for possible. We therefore propose a predictive model [PBSC donor evaluation; that these protocols must assess the expected = 0.6 + (G-CSF-based total collection) + 2 (rHuSCF- potential risk of [malignant, autoimmune or infectious] disease based day 1 collection)] to calculate the cumulative total of transmission from donor to recipient; and that the risks to the PBSC expected if harvesting were to continue to a maximum donor from the bone marrow or PBSC collection process must of 5 leukaphereses. This algorithm will allow the early be addressed. In the event of a donor being selected who identification of patients who are unlikely to reach the does not meet the normal donor selection protocol of the local threshold of PBSC required for transplantation. It will therefore institution, JACIE recommends ''documentation of the allow physicians to direct the resources involved in PBSC rationale [for donor selection] and of the informed consent of collection in a more appropriate and economical manner. the donor and of the recipient''. Our unit carries out assessment of histocompatible sibling bone marrow or PBSC donors for the West of Scotland area. As part of a review of P515 our own detailed donor selection protocol, retrospective audit Serious event monitoring was performed on the case records of all histocompatible S. Cleaver, A. Greenwood, D. Hatton, J. O'Connor, A. siblings selected as donors over the past three years, to Madrigal The Royal Free Hospital (London, UK) determine how many selected donors fell outwith our normal donor selection criteria. Data were available for 43 sibling The Anthony Nolan Trust (ANT) participates in the World donors who have either already proceeded to bone marrow / Marrow Donor Association Serious Events and Adverse PBSC harvest, or who are scheduled for harvest in the next Effects Registry (SEAR), the focus of which is serious events three months. A total of 5 donors (11.6%) fell outwith normal occurring to unrelated donors during or after donating. selection criteria. Three of these donors would normally have However, there are several factors that can prevent a patient been rejected due to a potential risk of disease transmission: receiving an adequate haematopoietic stem cell donation that one donor was Hepatitis B core antibody positive but PCR can occur between the time a definitive donor work-up request negative; another was Hepatitis C seropositive but PCR is received by the donor registry to the time that the donated negative; and the third donor had psoriasis with psoriatic product arrives at and is evaluated by the transplant centre. At arthropathy. In all three cases, the risk to the recipient of not the beginning of 2004, ANT set up a scheme to document and proceeding to transplant, and the lack of a suitable alternative review the incidence and nature of serious events occurring donor, was felt to outweigh the risk of disease transmission. during donor work-up that either resulted in adverse effects for Two donors would normally have been rejected due to a the intended recipient, or had the potential to do so. The work- relatively higher risk to the donor from the collection up period covers the time from selection of the definitive procedure, due to pre-existing medical problems. One donor donor, through donor medical assessment, harvesting or had significant hypertension, left ventricular hypertrophy and collection of the bone marrow or peripheral blood stem cells, mild learning difficulties; the second donor had significant

S127 alcohol problems with abnormal LFTs. Both of these donors P518 were happy to proceed with donation after full explanation of Comparison of CD34+ enumeration results in unrelated the potential risks. Our data would suggest that a significant peripheral stem cells products between collection and minority of potential histocompatible sibling bone marrow or transplant centres PBSC donors are likely to fail standard donor selection D. Lysák, J. Navrátilová, V. Koza Czech National Marrow procedures, but may still be acceptable as donors under Donor Registry (Pilsen, CZ) particular circumstances. We are now investigating the best procedure to be followed for the documentation of full informed Peripheral blood stem cells from unrelated volunteer donors consent from both donor and recipient in such cases. are increasingly being used as a stem cell source for allogeneic transplantation. The international exchange of hematopoietic stem cells results in increasing requirements on P517 precise graft evaluation. Allogeneic peripheral blood stem cell mobilisation and Objectives: The aim of the study was to compare selected collection and follow-up of donors: a single-centre quality control measurements on apheresis products and to experience assess the inter-centre variability of the results obtained by the N. Worel, G. Leitner, M. Horvath, K. Gerhartl, H.T. Greinix, P. collection and the transplantation centres. Kalhs, A. Rosenmayr, B. Pelzmann, P. Höcker University Donors and methods: 84 apheresis products from unrelated Hospital (Vienna, A); Austrian Bone Marrow Donors (Vienna, donors were analyzed in our centre between 4/03 and 10/04. A) 47 products (56 %) were collected by our registry and exported, 37 (44 %) were imported from foreign registries. The Allogeneic mobilized peripheral blood stem cells (PBSCs) are percentage, absolute CD34+ and nucleated cell counts for our increasingly used as graft source instead of bone marrow. laboratory were compared with the results provided by partner Although the short-term safety profile of granulocyte colony- registries in a simple survey. 59 products (70 %) were stimulated factor (rhG-CSF) seems acceptable, minimal data evaluable. In remaining cases no response or incomplete data exist regarding long-term safety. We reviewed data of 185 were recorded. The simple linear regression and unpaired T- allogeneic donors (siblings n=156, unrelated donors, URD n= test were used for statistical analysis. Our data were obtained 29) on side effects of rhG-CSF administration and adverse by ISHAGE gating strategy, dual platform approach and no- events of leukapheresis procedure. For follow-up we assessed lyse no-wash sample preparation. The laboratory participates peripheral blood counts, electrolytes, lactic dehydrogenase in CD34 stem cell EQA programme. Queries on sample (LDH), alkaline phosphatase (AP), total protein and albumin, processing in foreign laboratories were not included in the coagulation parameters and medical history on days survey. 1,7,30,100,365 and than yearly for 5 years. Results: The median CD34+ cell percent on apheresis The main adverse events related to rhG-CSF were bone-pain products was 0.54 (0.18- 1.54) according to our laboratory vs. (74%), headache (52%), myalgia (41%), fatigue (40%), sleep 0.46 (0.10- 2.06) for the others (p=0.57, r=0.8). The difference disturbance (30%) and were rated as moderate to severe by over 20 % in CD34+ percent measurement was found in 30 % 35% of the donors. PBSC collection was performed using a products. The absolute CD34+ count reached 3.76 vs. 3.78 continuous flow blood cell separator, processing 3 to 3.5 times x108 (p=0.45, r=0.9) and absolute nucleated cell count 655 vs. total blood volume. In 2 (1%) donors a central venous catheter 649 x108 (p=0.47, r=0.9) respectively. There were no was necessary. Citrate was used as anticoagulation fluid and statistically significant differences in all observed parameters a continuous calcium infusion was given to reduce citrate between the data groups. toxicity. Reinfusion of autologous platelet-rich plasma was Conclusions: The variation in results of CD34+ enumeration is performed in donors with post-donation thrombocytopenia (< determined by a large array of factors including the sample 80-100 G/L). Two donors (1%) were hospitalized within 1 to 2 processing, gating strategy, data analysis etc. Other possibly weeks after PBSC collection due to severe enteritis and influencing factors may be the sample condition or the sample subarachnoid hemorrhage, respectively. Ninety-two donors alteration during transport and storage. Our analysis of (50%) were lost for follow up. From the remaining 93 donors, simultaneous assessment of CD34+ cell content in apheresis URD’s (n=24) had a median of 4 check-up’s (range, 1-8) products revealed a good correlation between the centres, during the first 100 days (median, range 1-1095) after PBSC which implies an acceptable inter-laboratory variation and a collection, whereas siblings had a median of 3 (range, 1-7) good level of standardisation. during the first 30 days (median, range 1-1825). Follow-up of blood parameter showed a loss of platelets due to rhG-CSF mobilization and leukapheresis, but values returned to normal P519 within 30 days after donation. Compared to baseline values, Confirmatory typing results as an indicator of quality of white blood cells showed a decrease 7 days after collection, the HLA typing of the donor pool: Czech National Marrow they returned to normal between day 30 and 100. LDH and AP Donor Registry experience showed a significant increase during PBSC mobilization but P. Jindra, V. Koza, H. Pittrova, J. Navratilova, F. Mrazek, Z. returned to normal within 1 week after donation. The other Ambruzova for the Czech National Marrow Donor Registry blood parameters remained unaffected. No anomaly was found in medical history during a median follow-up period of The accuracy of HLA types is necessary condition for the 100 days (range, 1-1825). With the particular focus on donor efficient operations of unrelated bone marrow donor registries. safety a standardized approach to data collection of follow- However, since R usually rely (1) on HLA data supplied by up’s to monitor short- and long-term effects in all centers outside laboratories and (2) the serology for HLA Class I is still should be established. prevailing, the system for external quality control (EQC) is necessary. The standard approach is EQC exercises, i.e. blind samples regularly sent to HLA labs. Hovewer, more objective estimation of the HLA typing quality of the donor pool could be deduced from the analysis of confirmatory typing (CT) results - to compute the overall rate of discordant typing and the nature of discrepancies. We tested this program in order to evaluate quality of HLA typing of the Czech National Marrow Donors Registry (CNMDR). CNMDR has a pool of 28 000 donors typed by serology for HLA-AB, with 75 % also HLA-DR DNA

S128 typed and CT results information is continuously collected 450). She was treated with anti-convulsant and discontinued separately for HLA-AB (serology) and DRB1 (DNA) since CsA. FLAIR and brain MRI showed high signal intensity 1998. During period of 1998-2003 altogether 394 CT results lesions in left occipital area and compatible with posterior were analysed for HLA-A,B and 527 for DRB1. From a total of reversible encephalopathy syndrome. 394 CTs, serological HLA-A/B phenotypes were confirmed in Conclusions or Comments: The mechanism causing 369 (93,7 %) donors in whom 1550/1576 (98,4%) serologically neurotoxicity of tacrolimus and CsA may be similar assigned HLA-A/B antigens (ag) were correct. The overall rate pathophysiological. In present case, leukoencephalopathy had of inaccurately HLA-AB serotyped donors is thus 6,3 %. no precipitating factor and could occur even in normal drug However in 16/25 (64%) of incorrectly typed donors the concentration. Although neurological symptoms improved and discrepancies were caused by HLA-A or -B serologically the lesions on MRI disappeared completely after withdrawal of missed ag (false homozygosity). Majority of misassigned as tacrolimus and cyclospoine, careful caution should be well as missed ag were less frequent or rare ones with necessary for repeated occurrence. relatively high proportion of ag (10/25 of discrepant CTs) belonging to A19 and A28 group. Discrepancy rate was higher for donors recruited during 1993-1998 than for donors recruited later (60% vs. 40%) that could indicate continuous Graft versus host disease improvement of the HLA typing quality of CNMDR donor pool, probably by using better typing reagents. The DRB1 accuracy of CNMDR was excellent; only 5/527 (0,9%) retyped donors P521 were unconfirmed, with 2 of them being the clerical type TGF-beta and allogeneic stem cell transplantation: friend errors. Conclusion: While the HLA accuracy for Class II in or foe? CNMDR is about 99 %, for Class I the overall rate of the T. Banovic, K. MacDonald, E. Morris, V. Rowe, R. Kuns, A. incorrectly typed donors is about 6%. However within some Don, S. Ledbetter, A. Clouston, G. Hill Queensland Institute of specific groups of donors – especially the HLA-AB Medical Research (Brisbane, AUS); Genzyme Corporation homozygous ones- the overall error rate is clearly higher. The (Framingham, USA); University of Queensland (Brisbane, systematic DNA retyping of HLA-AB homozygous samples will AUS) thus improve the reliability of HLA record of the CNMDR and decrease the ratio of donors possibly inappropriately called up Objectives: Donor treatment with granulocyte–colony for CT stimulating factor (G-CSF) attenuates ability of donor T cells to

induce acute graft-versus-host-disease (aGVHD) after

experimental allogeneic stem cell transplantation (SCT) Regulatory issues although paradoxically increases the severity of chronic GVHD (cGVHD). We investigated the role of the regulatory cytokine transforming growth factor beta (TGF-beta)in this paradox. P520 Methods: Two well-established murine models of acute and Repetitive leukoencepalopathy due to an alternative use chronic GVHD where recipients are transplanted with of tacrolimus (FK506) and cyclosporine after unrelated splenocytes from donors treated with G-CSF and following allogeneic bone marrow transplantation SCT administered control or TGF-beta neutralising antibody. D.H. Yang, J.J. Lee, Y.K. Kim, H. Kook, S.H. Cho, I.J. Chung, Results: Neutralization of TGF-beta after SCT significantly H.J. Kim Chonnam National University (Jeollanam-do, KOR) increased aGVHD (survival: 17% v 83%, anti- TGF-beta Ab v control; P<0.01) and the concurrent prevention of IL-10 Background & Significance: Tacrolimus (FK506) and production further exaggerated this effect (median survival 17 cyclosporine (CsA) are widely used in immunosuppressive v 30 days, P<0.01). Early following SCT, donor T cells agent after bone marrow transplantation. Several neurologic preferentially produced TGF-beta and were able to regulate side effects were reported previously associated with aGVHD induced by T cells from control treated donors tacrolimus or cyclosporine, however, repetitive (survival: 30% v 5%, P<0.02). Whilst the neutralization of TGF- leukoencephalopathy is a rare manifestation after alternative beta augmented the proliferation and expansion of donor T use of tacrolimus and followed by cyclosporine for prevention cells after SCT, it paradoxically impaired cellular cytotoxicity to of graft-versus-host disease (GVHD). host antigens and associated graft-versus-leukaemia (GVL) Case: A 19-year-old girl with acute myelogenous leukemia effects. In the B10.D2-->Balb/c model of cGVHD, delayed underwent allogeneic bone marrow transplantation form an neutralization of TGF-beta (from day 14 after SCT) attenuated HLA-matched unrelated donor. She was treated with the severity of GVHD as determined by semi-quantitative cyclophosphamide (60mg/kg) and total body irradiation (1200 histopathology (P<0.05) while neutralization from day 0 was cGy) for conditioning and received intravenous tacrolimus ineffective. During cGVHD, TGF-beta was produced by (0.03 mg/kg) on the day before transplantation and short-term infiltrating CD11b+ mononuclear cells rather than from donor T methotrexate. On day 20, she developed disorientation and cells. memory impairment while serum concentration of tacrolimus Conclusions: While the production of TGF-beta by donor T was a 19.7 ng/mL (normal range: 7-20). She had a no clinical cells early after transplantation attenuates aGVHD and manifestation of hypertension, infection and organ dysfunction. improves GVL, the production of TGF-beta late after SCT is On neurologic examination, definite memory impairment was from mononuclear cells and mediates cGVHD. The detected. Brain magnetic resonance imaging (MRI) including therapeutic neutralisation of TGF-beta late after allogeneic diffusion weighted and FLAIR imaging revealed abnormalities SCT may attenuate the severity of cGVHD following in the bilateral hippocampus and around white matter. After transplantation of G-CSF mobilised stem cell allografts whilst discontinuation of tacrolimus, neurologic symptoms were maintaining the beneficial early regulatory effects of TGF-beta gradually improved and bilateral hippocampal lesions were on aGVHD and GVL. disappeared on follow-up MR imaging. We changed and restarted a immunosuppressive agent, CsA (10mg/kg p.o. daily), six days after cessation of tacrolimus because of her phobia for tacrolimus. On twelve days after medication of CsA, she suddenly developed tonic-clonic seizure and serum concentration of CsA was a 293.2 ng/mL (normal range: 100-

S129 P522 received either 100 mg (n=40) or 50 mg (n=61) Alemtuzumab Transplant associated microangiopathy in recipients of in combination with ciclosporin (3 mg/kg/day IV). Both cohorts allogeneic stem cell transplants were comparable in terms of underlying diseases as well as M. Martinez, C. Bucher, G. Stuessi, D. Heim, A. Buser, D. patient, donor and treatment characteristics (advanced stages Tsakiris, A. Tichelli, A. Gratwohl, J. Passweg Basel University of hematologic malignancies n=83, median age 44 yrs, Hospital (Basel, CH) median donor age 37 yrs, female to male transplants n=19, matched unrelated donors n=67, sibling donors n=34, Objectives: Transplant associated microangiopathy (TAM) is myeloablative conditioning regimen n=101). Alemtuzumab observed frequently after allogeneic stem cell transplantation was applied as a two-hour infusion on days -6 to -2. No (HSCT). Because of the impression of increasing frequency serious acute adverse effects were noted during a total of 505 we studied occurrence, severity, risk factors and outcome of antibody infusions. The clinical grades of severity of acute patients with TAM after HSCT. GvHD were nearly identical between the two Alemtuzumab Patients and Methods: We studied 221 consecutive patients dose levels (50 mg/100 mg): grades 0: 51%/48%, I: 31%/30%, receiving allogeneic HSCT in a single center between 1995 II: 15%/17%, III-IV: 3%/5%. Consequently, transplant-related and 2002. Occurrence of TAM was defined as LDH >300 U/L, mortality (TRM) at day 100 was comparatively low with 3% ± bilirubine >25 ymol/L a decrease in hemoglobine >10g/L, and 2% (59/61 survivors) after 50 mg and 10% ± 5% (36/40 shistocytes >2-5/hpf in the first 100 days posttransplant. Risk survivors) after 100 mg. The 1-yrs survival estimate for pts in factors examined included donor type, donor-recipient sex early disease stages is 86% ± 9% (16/18 survivors [89%]). match, ABO incompatibility, the occurrence and severity of This estimate is 44% ± 14% (54/83 survivors [65%]) in acute GvHD and stem cell source. Outcomes analyzed were advanced disease stages. Causes of TRM were TAM, and overall survival. The Kaplan-Meier estimator, bacterial/mykotic infections (9%), with multiorgan failure (4%), cumulative incidence and Cox proportional hazards regression EBV-LPD, acute cardiac arrest, and leukencephalopathy (3%). models were used to analyze time to TAM onset and mortality. 16 pts have died from disease recurrence (16%), the majority Results: 68/221 patients met the criteria for TAM. The after day 100. In conclusion, both investigated total dose cumulative incidence of TAM was 31 (25-38)% at 100 days. levels of Alemtuzumab are comparatively effective in Patients with TAM had higher LDH (TAM 1261 vs no TAM 635 preventing acute GvHD and are associated with a reasonable U/L) higher bilirubine (TAM 75 vs no TAM 21 ymol/L) and short-term outcome in a predominantly unfavorable patient higher creatinine (TAM 192 vs 137 ymol/L). Patients with TAM population. Further, our experience indirectly supports that the had more often neurologic symptoms (TAM 27/68 vs no TAM inactivation of host APCs by Alemtzumab is the dominant 37/153 p=0.02). mechanism in preventing acute GvHD. The occurrence of TAM did not change over time (33% before vs 28% after 2000). TAM was not more frequent after peripheral stem cells than after bone marrow (peripheral blood P524 30% bone marrow 32% p=0.7) and not more frequent in Allogeneic transplantation with identical MHC: clinic- patients with high cyclosporine vs. low levels (33% vs 30% pronostic value of discrepances of microsatellite DNA p=0.7). TAM was significantly but not closely associated with regions between recipient and donor aGvHD; 38% in patients with grade II-IV aGvHD versus 23% M. Alcoceba, M. Díez-Campelo, P. Martín-Jiménez, L. in patients without aGvHD (p=0.01). In multivariate analysis, Blázquez, A. Armellini, M.E. Sarasquete, C. Fernández, C. risk factors for TAM included unrelated donor (RR: 3.5 (2-6); Chillón, A. Balanzategui, M.V. Mateos, R. García-Sanz, D. p<0.0001), age (RR: 1,02; p=0.06), female sex (RR: 1.6 (1- Caballero, M. González, J.F. San Miguel Hospital Clínico of 2.6); p=0.06) and ABO-incompatibility (major incompatibility Salamanca (Salamanca, E) RR: 1.9 (1.2-3.2) p 0.01). One year-survival in patients with TAM (43 +- 12%) was much Introduction: Detection of disparity in microsatellite DNA lower than in patients without TAM (78 +- 7%) p<0.0001. In a regions between recipient and donor allows for monitorization multivariate model of survival TAM was independently of the degree of haematopoietic chimerism. It is known that associated with mortality after adjustment for donor type, age disparities between donor and recipient in various polymorphic and GvHD occurrence and severity. systems(mainly MHC) are associated with an increased Conclusions: TAM is observed frequently after allogeneic incidence of graft-versus-host disease (GVHD).However, it is HSCT and is associated with considerable mortality. Risk still unknown if STR disparities could have a similar biological factors of TAM are similar to acute GvHD but occurrence of effect. TAM does not correlate closely with clinical grade of aGvHD. Aim: To study the relationship between STR disparities and frequency of GVHD, overall survival and event free survival in patients who have received allogeneic stem cell P523 transplantation(SCT). Acute graft-versus-host disease prophylaxis using a Patients:163 consecutive SCT with peripheral blood stem cells combination of alemtuzumab and ciclosporin: from identical MHC sibling donor at a single center were comparison between two dose levels of alemtzumab included in the study. Their characteristics were: median age H. Biersack, R. Peceny, A. Elmaagacli, R. Trenschel, D.W. 44(17-69);Male/Female:95/68;Sex disparity:46%; Beelen University Clinic Essen (Essen, D) Diagnosis:40AML, 26ALL, 24MDS, 20MM, 17CML, 14NHL, 10CLL, 9HD, 1CLL+HD, 1CMPD, and 1Hypereosinophilic Alemtuzumab (Campath-1H), a humanized monoclonal Syndrome. The conditioning regimen was reduced intensity in antibody directed against the CD52 antigen, has demonstrated 80 patients and myeloablative in 83. All 163 transplants particular high efficacy in the prevention of acute graft-versus- engrafted and were valuable for acute GVHD(aGVHD), while host disease (GvHD). It is generally thought that its major 134 were included in the analysis of chronic GVHD(cGVHD), mechanism is in-vivo donor T-cell depletion (TCD). In addition, according to the available follow-up. Alemtuzumab can act on host antigen-presenting cells (APCs) Methods: After genomic DNA extraction, PowerPlex®16 and may thereby inhibit the induction phase of acute GvHD. At System kit(Promega Corporation,Madison,WI) was used to the commonly applied total dose level of 100 mg amplify 16 STR regions(15 plus gender marker,Amilogenin). Alemtuzumab, both mechanisms appear to be involved, while The amplified products were analysed using GeneScan after low-dose Alemtuzumab (i.e. 50 mg total dose) its effect 2.1(Applied Biosystems,Foster City,CA) after electrophoresis on host APCs appears to prevail. To evaluate the clinical in the ABIPrism 377(Applied Biosystems).The chi-square and efficacy of the two dose levels, two cohorts of patients (pts)

S130 t-Student tests were used for statistical analysis. Log-rank P526 analysis was used for comparing differences in survival. Role of ICOS: ICOSL interaction in allogeneic bone Results: The number of STR disparities ranged from 4 to marrow transplantation 15(median: 9). The number of loci discrepancies was not G. Richter, A. Mollweide, S. Burdach Munich University of related to any clinical parameter included in the analysis Technology (Munich, D) (aGVHD, cGVHD, EFS y OS). However, when patients were grouped according to STR mismatches(<= 10 disparities and Graft versus host disease (GVHD) remains a major >10,n=112 and 35, respectively), shorter OS was associated complication after allogeneic bone marrow transplantation. in patients with >10 disparities(p=0.0077). Matched in TPOX Acute GVHD is initiated by alloreactive donor T cells that was associated with relapse(p=0.0423). Discordances in recognize HLA disparities on host cells as well as peptides D18S51 was associated with higher grades of aGVHD presented by them. The infiltration of several target organs severity(p=0.033). Disparities in D16S539 was associated with such as gut, liver, and skin by donor leukocytes including T cGVHD(p=0.023). cells is thought to be one of the key processes in the early Conclusions: STR discrepances can have a marginal influence phase of a GVHD. The activation and expansion of the donor on the evolution after allogeneic hematopoietic SCT. Thus, the T cells, leading to the secretion of proinflammatory cytokines presence >10 disparities is associated with poor survival. In and the recruitment of additional inflammatory effector cells to addition, there were differences in the post-transplant these sites, further damages the affected tissues. behaviour according to the discrepant marker.That fact could Costimulatory receptors on T cells have a key role in this T cell point out the presence of genetic complexes neighbour to the receptor/CD3 dependent activation process. The inducible DNA regions of this markers involved in the immune response. costimulator (ICOS) is a new member of the CD28/CD152 receptor family that regulates T cell activation and function. The apparent involvement of ICOS/ICOSL interaction in P525 several T cell effector functions and the abundant expression Extracorporeal photopheresis in graft-versus-host of ICOSL in a variety of tissues also affected in a GVHD disease treatment. Single-centre experience in 41 provided the rationale to investigate its role in GVHD refractory chronic GvHD patients development. C57Bl/6 mice were lethally irradiated and S. Guidi, P. Guglielmelli, G. D'Ascenzo, L. Lombardini, C. reconstituted with allogeneic spleen cells (sc) in the Nozzoli, R. Saccardi, A.M. Vannucchi, A. Bosi University of absence/presence of a blocking anti-ICOS-Ligand (ICOSL) Florence (Florence, I) mAb. Controls were reconstituted with syngeneic sc. Reconstituted mice were monitored for weight loss and Chronic GvHD affects more than 50 % of long term survival. 3x107 sc were sufficient to reconstitute irradiated hematopoietic stem cells transplantation recipients survivors mice. Mice reconstituted with allogeneic sc experienced 12- and is a major cause of morbidity and mortality. Standard 16% weight loss around day 4 after transplantation and died of GvHD treatment is unsatisfactory because poor results and acute GVHD within 7-10 days after transplantation. Mice toxicity. ECP mechanism of action is unknown but recently it reconstituted with allogeneic sc together with an anti-ICOSL has been suggested that ECP acts modulating NK cells and mAb showed increased signs of GVHD as restricted movability ACP cells and this treatment modality is reported safe and and morbidity. However, weight loss and overall survival was efficacious. We studied 41 cGvHD steroid refractory patients. identical to isotype treatment groups. In contrast. mice injected Stem cells source was 29 identical sibling and 12 unrelated at day 3 after transplantation with an anti-ICOSL mAb gained donors, 27 bone marrow, 14 peripheral blood stem cell. 19 pts weight again and survived for additional 18 days although were early and 22 advanced stage. Diagnosis were CML 8, antibody treatment was already stopped at day 11 after AML/SMD 15, ALL 10, MM 4, CLL 2 and NHL 2.Transplant transplantation. Analysis of potential mechanisms based on procedure was performed as elsewhere described. 19 de novo these different findings will be presented. cGvHD, 4 quiescent form and 18 evolution from aGvHD. (skin 31/41 pts, liver 17/41 pts,oral sicca 19/41 pts,ocular sicca 13/41 pts, bowel 9/41 pts, bronchiolitis 10/41 pts, myositis 4/41 P527 pts, thrombocytopenia 5/41 pts, serositis 2/41 pts and joint First controlled phase II trial of the interleukin-2 receptor contractures 2/41 pts). After a mean of 17 cycles of ECP antagonist basiliximab in steroid-refractory acute graft- (range 1-46), cGvHD resolved completely in 12/41 (29%) and versus-host disease partially in 21/41 (51%). 9/42 patients failed,two experienced M. Schmidt-Hieber, W. Knauf, T. Fietz, H. Schrezenmeier, E. stable disease, two died for disease relapse. Best results were Thiel, L. Uharek, I.W. Blau Charite-Campus Benjamin Franklin observed in skin, liver, bowel, lung and thrombocytopenia. All (Berlin, D); Hämatologische/Onkologische Schwerpunktpraxis responding pts were able to reduce conventional (Frankfurt, D); University Hospital Ulm (Ulm, D) immunosuppression, that has been discontinued in 4. PS improved from 66% (range 20-90 %) to 84 % (range 60- Background: Acute graft-versus-host (aGVHD) disease occurs 100%). We have also compared the results of pts who started in 30-80% of patients who undergo allogeneic stem cell ECP early versus those who started treatment later. Our study transplantation (SCT) and contributes significantly to demonstrate a 50% of complete response if the ECP was transplant-related mortality. We conducted a controlled phase started within six months from cGvHD onset, even if we have II trial to assess the efficacy and feasibility of treatment with obtained a 67% of partial response within 12-58 months. basiliximab, a chimeric monoclonal antibody directed against No severe side effects were documented. ECP is a safe the alpha-chain of the interleukin-2 (Il-2) receptor in aGVHD. therapy for extensive cGvHD resistant to conventional Patients and Methods: Eligibility criteria included prior treatment, that it must be started early and continued until best allogeneic SCT and at least grade 2 steroid-refractory aGVHD response. of one target organ. The primary endpoint of the trial was to determine the number of patients who achieved a complete or partial response of aGVHD; secondary endpoints included assessing the toxicity and duration of remission. Basiliximab was administered intravenously at a dose of 20 mg on days 1 and 4. Twenty-two patients were enrolled between January 2000 and October 2004. The median age was 52 years (range 31-65 years). Nine pts. had an HLA-identical sibling graft, one pt. had a locus B mismatch, while the others had a matched

S131 (10 pts.) or mismatched unrelated donor (2 pts.). All patients Acknowledgements. Supported by a Research Grant of the underwent peripheral blood stem cell transplantation. SCT Ministry of Health of the Czech Republic No: MZ was performed in 9 pts. with AML, 4 pts. with ALL, 4 pts. with 00065269705. multiple myeloma, 3 pts. with B-CLL, one pt. with severe aplastic anemia and one pt. with CML. Prophylaxis against aGVHD consisted of CSA and MTX or MMF; in the case of an P529 unrelated donor, we added ATG. Four pts. had grade 2 Extracorporeal photoimmune therapy with UVADEX for aGVHD (18%), while 9 pts. each had grade 3 and grade 4 the prevention of acute graft-versus-host disease in aGVHD (41% each). patients undergoing standard myeloablative conditioning Results: A complete response of aGVHD was achieved in 8 and allogeneic haematopoietic stem cell transplantation pts. (36%) and a good partial response in 12 pts. (55%). With S. Abhyankar, P. Shaughnessy, B. Bolwell, K. van Besien, M. exception of 2 pts. with refractory aGVHD we found a primary Mistrik, O. Ilhan, A. Grigg, H. Prince, A. Dodds, A. Machado, J. response rate of 90%. Six pts. (27%) had a recurrence of Wang, U. Thienel, C. Peters, F. Foss, J. Apperley Kansas City aGVHD and were again treated successfully with an Il2- Cancer Center (Kansas City, USA); Texas Transplant Institute receptor antagonist (basiliximab or daclizumab). Unfortunately, (San Antonio, USA); The Cleveland Clinic Foundation 4 pts. (18%) died from complications of refractory aGVHD. (Cleveland, USA); University of Chicago (Chicago, USA); Basiliximab-related toxicity was not observed in any of the Klinika Hematologie a Transfuziologie (Bratislavia, SVK); patients. The rate of severe infections following Ankara University Medical School (Ankara, TR); Royal immunosuppression with basiliximab is comparable to that Melbourne Hospital (Melbourne, AUS); Peter MacCallum found with other treatment modalities for aGVHD. Cancer Institute (East Melbourne, AUS); St. Vincents Hospital Conclusion: We conclude that basiliximab is efficient and (Sydney, AUS); Instituto Portugues de Oncologia de Francisco feasible for steroid-refractory aGVHD and should be evaluated Gentil (Lisbon, P); Therakos, Inc (Exton, USA); New England in further trials. Medical Center (Boston, USA); Hammersmith Hospital Imperial College School of Medicine (London, UK)

P528 Host antigen presenting cells (APCs) may be important for Pulse cyclophosphamide in the treatment of GvHD alloantigen presentation and stimulation of donor T cells in unresponsive to steroids acute graft versus host disease (aGvHD). Extracorporeal J. Mayer, M. Krejci, M. Doubek, Z. Pospisil, Y. Brychtova photoimmune therapy (ECP) has shown benefit in some University Hospital Brno (Brno, CZ); Masaryk University Brno patients (pts) with aGvHD and is associated with a decrease in (Brno, CZ) host APCs. We report preliminary results of the first multi- institutional phase II study examining ECP with a standard Objectives: Corticosteroid-resistant GvHD remains extremely myeloablative preparative regimen and allogeneic stem cell difficult to manage and is associated with high morbidity and transplantation. ECP was given on 2 consecutive days from D- mortality. We hypothesized that there are logical reasons for 10 to D-6, followed by cyclophosphamide 60 mg/kg/d for 2 the usage of pulse cyclophosphamide (CY) in this setting: (1) days and TBI 1200 cGy delivered over 3 days. Prophylaxis for the efficacy of CY for the treatment of many autoimmune aGvHD was cyclosporine 3-5 mg/kg daily beginning D-1 and disorders and for immunosuppression before allogeneic then adjusted to keep levels between 200-600 ng/ml and hematopoietic stem cell transplantation (BU+CY or CY+TBI); methotrexate 10 mg/m² on D1, 3, 6, and 11 for pts who had (2) the described efficacy of high-dose CY (200 mg/kg) matched unrelated donors (MUD) or HLA class I one-antigen supported by autologous or pseudoautologous hematopoietic mismatched related donors (MMRD); or 10 mg/m² on D1 and stem cell transplantation for the treatment of severe GvHD. 5 mg/m² on D3, 6, and 11 for pts who had matched related Methods: For steroid-refractory GvHD, CY was administered donors (MRD). Sixty-six pts have been enrolled in the study at the dose of 1000 mg/m2 as an iv. infusion under vigorous and data are available on 61 (40 male) pts with a median age hydration in 17 patients (11 aGvHD, 3 post-DLI GvHD, and 3 of 39 (range 20-60) years. Diagnoses included: ALL (n=14), cGvHD progressing from aGvHD). Continuous administration AML (n=17), CLL (n=3), CML (n=16), MDS (n=3), NHL (n=5) of cyclosporine A was not interrupted during the CY treatment and other (n=3). The 28 pts who received bone marrow and steroids were slowly tapered. engrafted at a median of 21 (range 9-30) days and the 33 pts Results: Pulse CY (20 administrations in total) showed high who received peripheral blood engrafted at a median of 15 efficacy in the treatment of GvHD of the skin (100 % response (range 10-32) days. Grade II-IV and III/IV aGvHD developed in rate, RR=CR+PR), of the liver (73 % RR), and oral cavity (100 13 (45%) and 2 (7%) pts, respectively, who received MRD % RR). However, its ability to control severe intestinal GvHD transplants (n=29), and in 21 (68%) and 10 (32%) pts, was much lower (only 29 % PR). Pulse CY, except for respectively, who received MUD transplants (n=31). No manageable myelosuppression of short duration (usually 1-4 aGvHD occurred in 1 pt who received an MMRD transplant. days) in some patients, exerts a remarkably good toxicity Seven pts relapsed, 3 with ALL, 2 with CML, 1 with NHL, and profile and does not appear to increase the risk of 1 other. Overall, 43 pts (70%) are alive with a median follow opportunistic infections, mixed chimerism, relapses, or up of 280 (range 23-560) days. Thirteen pts who received posttransplant lymphoproliferative disease. Leukopenia with MUD transplants (3 pts each with relapse, MOSF, and WBC below 1.109/L developed after 11 out of the 20 CY aGvHD; and 1 pt each with pseudomonas infection, CMV administrations (55 %) and thrombocytopenia under 50.109/L pneumonia, neurotoxicity, and sepsis/pneumonia) and 5 pts developed seven-times (35 %). Response to Cy pulse did not who received MRD transplants (2 pts with relapse and 1 pt significantly depend on the development of leukopenia. Seven each with pneumonia, arterial thrombosis, and sepsis) have patients died. In five of them the direct cause of death was died. Preliminary results of this study reveal no adverse effects therapy-refractory intestinal GvHD. Overall survival is 57 % of ECP on regimen related toxicity or engraftment after a with longest follow-up of 43 months. standard myeloablative preparative regimen and ASCT. The Conclusions: Pulse CY seems to be cheep, effective, and non- overall survival of patients in this trial is encouraging and toxic treatment of steroid-refractory GvHD of the skin and the warrants further study. liver, and produces long-term control of this posttransplant complication. Its ability to control intestinal GvHD is much lower, however. In future trials, this pulse CY might also be tested in combination with other drugs with a different mechanism of action.

S132 P530 given IP at the time of onset of aGVHD and every forth day (a Serum levels of soluble fas ligand and TRAIL are total of four doses). associated with acute and chronic graft-versus-host Results: All animals receiving native HSA developed lethal disease after allogeneic stem cell transplantation aGVHD. MTX-HSA prevented aGVHD in 18 of 21 evaluable C. Wirth, C. Schmid, H. Adler, H. Kolb Ludwig-Maximilians- animals, whereas 5 of 7 evaluable rats receiving free MTX University (Munich, D); GSF Research Center for Environment died due to aGVHD. Survival in the prophylactic MTX-HSA and Health (Munich, D) group was significantly better (p<0,05) than in the MTX or control groups. Two of eight rats treated with MTX-HSA for Introduction: Induction of apoptosis represents an important established aGVHD became long term survivors, while 6 of 8 effector mechanism of cytotoxic T lymphocytes (CTL), that is rats died due to aGVHD. While toxicity of MTX-HSA was not mediated by several receptor/ligand pairs, as the detectable, MTX at a dose of 0,5mg/kg was associated with Fas/FasLigand and the TRAIL/TRAIL-Receptor system. temporary weight loss and lethargy. In addition MTX-HSA Experimental and clinical data suggest that these effector suppressed proliferation of alloreactive T-lymphocytes in vitro systems may also play a role in the pathogenesis of Graft- in a dose dependent manner. versus-Host diseases (GvHD), and in the Graft-versus- Discussion: MTX-HSA effectively prevents experimental Leukemia (GvL) reaction. This killing mechanism may be aGVHD and is associated with a superior efficacy and less accompanied by the the release of soluble FasLigand (sFASL) toxicity than MTX. and TRAIL into the circulation. Material and Methods: To further investigate this association we determined the serum sFASL and TRAIL levels of P532 allogeneic stem cell transplantation recipients with and without Prognostic significance of platelet recovery pattern after acute and chronic GvHD, using an enzyme linked allogeneic HLA-identical sibling transplantation and its immunosorbent assay. Detection levels were 9 pg/ml for the association with severe acute GvHD sFAL and 14 pg/ml for the TRAIL assay. D.H. Kim, S.K. Sohn, J.H. Baek, J.G. Kim, K.S. Lee, J.S. Suh, 89 samples from 68 patients (male and female, 34 each) were K.B. Lee Kyungpook National University Hospital (Daegu, available. Patients patients were transplanted for AML (n=24)., KOR) ALL (n=11). CML (n=13), multiple myeloma (n=10), myelodysplastic syndrome (n=4), non-hodgkin lymphoma Background: Thrombocytopenia (TP) is a frequent (n=4) and non-malignant hematological diseases (n=2). 39 complication after allogeneic stem cell transplantation (SCT) had an HLA identical family donor, 29 had a matched and regarded as a poor prognostic factor when assessed unrelated donor. At time of sample collection, clinical signs of beyond day 100. However, little was known on the clinical acute GvHD were present in 59 and of chronic GvHD in 30 significance of platelet recovery pattern before chronic GVHD cases. Six transplant recipients did not have GvHD and were develops. used as controls, as were 20 healthy volunteer donors. Methods and Materials: Eighty-five patients receiving HLA- Samples were taken at a median of 29 and 328 days from identical sibling SCT were stratified according to their platelet transplantation in acute and chronic GvHD, respectively. recovery pattern between day +30 and +90, and analyzed Results: Serum levels below the detection level were found in their transplant outcomes and its association with each all but two control samples. In contrast, median levels of component of acute GVHD grading system. sFASL were 21 (range: <9–76) pg/ml in acute and 49 (range: Results: Fifteen patients (18%) were stratified in persistent TP, <9-195) pg/ml in chronic GvHD (p<.001, multigroup 33 patients (39%) in unstable TP, and 37 patients (43%) in comparism). For TRAIL, median leves were 30 (<14-153) non-TP. The persistent TP which was strongly associated with pg/ml for acute and 45 (<14-110) pg/ml for chronic GvHD (p < severe acute GVHD (p<0.001), showed worst 2-year OS 0.001 as compared to control for aGvHD and for cGvHD). (p<0.0001) and highest NRM rates (p<0.0001) with highest Conclusion: The presence of significantly elevated serum opportunistic infection rate (p<0.0001). In multivariate levels confirm previously reported smaller studies. Both analyses, the platelet recovery pattern was identified as an receptor/ligand systems may be associated with GvHD independent prognostic factor (p=0.02) together with the pathogenesis, in particular in chronic GvHD. Further disease risk (p=0.02) in terms of OS, and as the only investigations are warranted for a better insight into the role of independent prognostic factor in terms of NRM (p=0.005) and apoptosis inducing systems in GvHD. the incidence of infectious events (p<0.001). Persistent TP was identified as a significant prognostic factor for OS (p<0.001), NRM (p<0.001), and strongly associated with the P531 development of extensive chronic GVHD (p=0.03). Methotrexate-albumin is effective in prophylaxis and Conclusions: The platelet recovery pattern between day +30 treatment of experimental acute GvHD and +90 could predict the prognosis of recipients in terms of N. Hofmeister, E. Frei, B. Steiner, K. Sievert, H. Terpe, H. OS and NRMs after HLA-identical sibling SCT, and was Schrenk, M. Freund, G. Hartung, D. Wolff University of strongly associated with the severity of acute GVHD. Rostock (Rostock, D); DKFZ (Heidelberg, D) During the last years major progress has been made in the development of targeted chemotherapy using cytotoxic drugs covalently bound to human serum albumin (HSA). We explored the immunosuppressive activity of methotrexate (MTX) covalently bound to HSA in vitro as well as in prophylaxis and treatment of experimental acute GVHD (aGVHD).

Methods: Seventy five female F1 hybrid BN/Lew-rats were

irradiated with 8.2Gy (n=65) or 9.9Gy (n=10). One day after irradiation each rat received 4X107 bone marrow cells and 1.5x107 spleen T-cells from female Lew-rats. Prophylactic treatment consisted of MTX-HSA 2mg (based on MTX)/kg body weight (n=26), MTX 0.5mg/kg (n=10), or native HSA (n=31) given intraperitonealy (IP) on days 0, 4, 8 and 12. Treatment of aGVHD consisted of MTX-HSA 2mg/kg (n=8)

S133 P534 Possible involvement of CCL28 in the localisation of CCR3+ T-cells in intestinal GvHD C.M.J.M. Faaij, N.E. Annels, K. Zuidwijk, A.C. Lankester, T. Revesz, M. Bierings, R.M. Egeler, M.J.D. van Tol Leiden University Medical Centre (Leiden, NL); University Medical Centre (Utrecht, NL)

Acute graft-versus-host disease (GvHD) is one of the major complications of allogeneic stem cell transplantation and is caused by donor T lymphocytes activated by alloantigens on host APCs. The main target organs are skin, gut and liver, with GvHD of the gut being one of the most severe and potentially

lethal forms. In order to affect distinct organs, donor T cells must first selectively home to these target sites. In the present P533 study we used the gut-homing integrin CD103 to identify and IL-23, IL-6 and BCL-2 are most important activating agents further characterise T cells in the blood of paediatric SCT of early acute GvHD patients suffering from gut GvHD. We found that T cells of M. Zaraysky, D. Butlitskiy, A. Sipol, M. Vladovskaia, A. these patients highly expressed CD103 as compared to their Vitrischak, N. Mikhailova, L. Zubarovskaia, B. Afanasiev, N. healthy donors and patients with only skin GvHD. Flow Serebrianaya, S. Ketlinskiy SPb State Pavlov Medical cytometric analysis revealed a distinct chemokine receptor University (St. Petersburg, RUS); Institute of Highly Pure profile of the CD103+ T cells, with expression of both the Th1- Biopreparations (St. Petersburg, RUS) associated chemokine receptors CCR5 and CXCR3 as well as the Th2-associated chemokine receptor CCR3. By triple colour Acute ''Graft-versus-Host-Disease'' (GvHD) is the major immunofluorescent staining of gut biopsies, T cells expressing complication of allogeneic haematopoietic stem cells these receptors were also found infiltrating the intestinal transplantations (aHSCT). Some cytokines which produced by mucosa. The significance of CCR3+ T cells in the gut GvHD donor and recipient cells play a crucial role in difficult aGvHD biopsies was further supported by the high expression of the pathogenesis. ligand for CCR3, CCL28, which was expressed by both the Methods: Total RNA from seventeen oncohaematologic epithelial cells as well as by the intraepithelial lymphocytes. In patients treated with aHSCT was collected before conditioning contrast, CCL28 was only weakly and selectively expressed starting, at the haematopoietic recovery day and on D+30 after by the epithelial cells of healthy gut. As Th1 and Th2 HSCT. Using an original reverse-transcriptase polymerase lymphocytes are distinguished by their cytokine production chain reaction (RT-PCR) we have explored the BCL-2, profile, the production of Th1 versus Th2 cytokines by the interleukin-1beta (IL-1b), IL-2, IL-6, IL-10 (semiquantative CCR3+ T cells of the blood after stimulation with PMA and protocol), and IL-23, IL-27-receptor (IL-27R) (quality protocol) ionomycin was investigated. CCR3+ T cells of gut GvHD mRNA genes expression in patients with vs. without aGvHD. patients only produced the Th1 cytokines, IFN-gamma and Results: In our study we demonstrated that aGvHD developed TNF-alpha but not the Th2-associated cytokine IL-4. Thus more frequently in patients who had increasing expression of although CD103 is responsible for the homing of GvHD IL-10 before conditioning starting (P=0,009). Increased levels reactive T cells to the gut, the interaction between CCR3 and of IL-6, IL-23 and BCL-2 mRNAs in patients, who developed its ligand CCL28 may play a role in the specific localisation aGvHD before white blood cells level normalizing, also and trafficking in the intestinal mucosa and in the effect of presented clinician significance (P=0,001, P=0,046 and these cells on the course of GvHD. P=0,004, respectively). The appearing of aGvHD signs on day 30 after HSCT correlated with increased IL-23 mRNA expression (P=0,042). The level of IL-2 mRNA in patients with P535 aGvHD at D+30 was increased, but not significantly (P=0,06), Identification of a skin-homing population of CD4+ T cells probably due to immunosuppressive therapy. We also showed in patients with skin GvHD that mRNA coexpression of BCL-2 and IL-23 in the same N.E. Annels, C.M.J.M. Faaij, A.C. Lankester, E. Spierings, patients was significant at each of assessment periods after M.W. Schilham, E.P. Bowman, M. Bierings, T. Revesz, R.M. aHSCT (P=0,006 – recovery of hematopoiesis and P=0,013 at Egeler, M.J.D. van Tol Leiden University Medical Centre D+30). In other hand, the mRNA coexpression of BCL-2 and (Leiden, NL); DNAX Research Inc. (Palo Alto, USA); IL-27R was revealed only at the early post-HSCT period University Medical Centre (Utrecht, NL) (P=0,001). Conclusion: Proposed original method of semiquantitive Chemokines and their receptors are known to play a crucial assessment of main cytokines expression could be used as role in inflammatory diseases. Graft-versus-host disease additional early prognostic approach for aGvHD development (GVHD), a potentially fatal complication of allogeneic stem cell prediction and assessment of suppressive therapy transplantation (SCT), is a disease in which distinct tissue effectiveness for immunology conflict, developed after aHSCT. sites, namely skin, liver and intestines can be affected. Since We also suggest that role of B-cells as main producers of IL- the mechanism of this specific tissue involvement is not well 23 and BCL-2 in the development of early aGvHD should not understood, we hypothesised that the expression of tissue- be underestimated. specific homing molecules on allogeneic activated T cells may be directing these cells to the GVHD target organs. To investigate this we have carried out four-colour flowcytometry to study the expression of chemokine receptors and adhesion molecules on CD4 and CD8 peripheral blood T cells taken from twelve paediatric allogeneic SCT recipients at regular intervals following transplant. In addition, triple-colour immunofluorescent staining of cryosections from skin biopsies was performed to characterise the T cells infiltrating this tissue in those patients with GVHD.

S134 In the ten patients who suffered from skin GVHD, a significant indirectly support the previous observations about lower population of CD4+CCR10+ T cells temporarily appeared in efficacy of DLI in eradication of EMR. the peripheral blood some weeks after skin GVHD became apparent. This coincided with an increase in CD4+CLA+ T cells. In contrast, there was no increase in these skin homing P537 molecules on the CD8+ T cells of any of the GVHD patients Glucocerebroside, a known modulator of natural-killer-T- studied. The SCT patients without GVHD and one patient who cells, alleviates chronic graft-versus-host disease in a developed only liver GVHD did not show such an increase of murine model CD4+CCR10+ T cells. Immunofluorescent staining of A. Nagler, M. Margalit, M. Ohana, R. Alper, O. Pappo, E. cryosections from GVHD skin biopsies revealed that all the Rabbani, D. Engelhardt, Y. Ilan Chaim Sheba Medical Center infiltrating CD4+ T cells expressed CCR10 whereas the (Tel Hashomer, IL); Hadassah Hebrew University Medical infiltrating CD8+ T cells did not. Center (Jerusalem, IL); ENZO Biochem Inc. (New York, USA) Thus we can conclude that there is an increase of a CD4+CCR10+ T cell population specific to those SCT patients Background: Chronic graft-versus-host-disease (cGVHD) is a who developed skin GVHD. These cells could represent either major obstacle to successful bone marrow transplantation. an inflammatory or indeed an anti-inflammatory population. Natural-Killer-T (NKT) lymphocytes may have a role in The latter possibility is suggested by their appearance after regulation of the immune attack in cGVHD. Glucocerebroside the onset of skin GVHD and their disappearance after (GC) is a naturally occurring glycolipid that plays a role in the resolvement of skin GVHD. We are currently performing immune modulation of NKT lymphocytes. studies to determine which of these possibilities is correct. Objectives: To determine the immune modulatory effect of GC in a murine model of cGVHD. Methods: cGVHD was generated by infusion of 2x107 P536 splenocytes from B10.D2 donor mice into Balb/c recipient Leukaemia relapse after allogeneic bone marrow mice, which received 7Gy 60Co total body irradiation (TBI) transplantation - how does chronic GvHD influence the prior to transplantation. Recipient mice were followed for 55 pattern and onset of relapse? days. Clinical and histological parameters of cGVHD- A. Al Beihany, E. Sahovic, N. Chaudhri, F. Al Mohareb, F. Al associated cutaneous, liver and bowel injury were assessed. Sharif, H. Al Zahrani, H. Al Omar, A. Al Shanqeeti, P. Seth, S. To determine the mechanism of GC-mediated immune Zaidi, M. Morshed, K. Al Anazi, M. Aljurf King Faisal Specialist modulation, intrahepatic and intrasplenic lymphocytes were Hospital&Research (Riyadh, SA) isolated and analyzed by FACS for CD4+, CD8+,CD56+ and NKT subpopulations. Serum cytokine levels were determined. Allogeneic hematopoietic cell transplantation (HCT) is a Results: Treatment with GC significantly alleviated cGVHD. standard treatment for pts with hematologic malignancies. GC-treated mice gained weight and manifested a significant Disease relapse remains the most frequent cause of treatment decrease in skin cGVHD clinical score (0.91 vs. 1.66 in GC failure. It usually involves the bone marrow, but in a smaller treated vs. control mice, respectively). Similarly, improvement subset of pts it occurs in extramedullary site(s), either alone or in the GVHD mediated liver, bowel and cutaneous tissue injury concurrently with marrow involvement. was observed in the GC treated mice compared to controls. Between 7/1985 and 10/2003, a total of 601 related allogeneic The beneficial effect of GC was associated with a 58% and a HCT were performed at our institution for various 31% reduction of the intrasplenic and the intrahepatic NKT cell hematological malignancies. We performed analysis of our number, respectively, accompanied by a 27% decrease of prospectively collected database to assess the occurrence, intrasplenic CD3+CD4+ T cells. Intrasplenic (but not mode of relapse, natural history, contributing factors and intrahepatic) decrease in NK cell number was also observed in outcome of pts experiencing relapse after related allogeneic GC treated animals (48% vs. 7%). Administration of GC led to HCT. After a median follow up of 41.3 (0.6-120) months, 143 increased serum IFN-gamma and TNF-alpha levels in GC (23.8%) pts experienced disease relapse. Primary diagnosis treated compared to untreated controls (46 vs. 27, and 92 vs. was AML in 58, ALL 38, CML 45 and MDS in two pts. Median 52 pg/ml, respectively), while IL10 levels decreased (61 vs. 83 age was 22.8 (14.1-49.36) years. There were 82 males and 61 pg/ml, respectively). females. 80 pts had conditioning with BU/CY while the Conclusions: Administration of GC led to significant remaining received TBI based conditioning. GVHD prophylaxis amelioration of chronic GVHD. This effect was associated with consisted of short course MTX and CSA in the majority of pts an altered NKT lymphocyte distribution, decreased numbers of (90.2%), the remaining pts received additional steroid effectors T lymphocytes and a shift towards a Th1-type prophylaxis.Out of 143 initial relapses, 40 (28%) occurred in cytokine profile. Glucocerebroside may serve as a new extramedullary site(s), 31 with and 9 without bone marrow therapeutic measure for amelioration of cGVHD. involvement. 43 (30%) pts had a history of acute GVHD (aGVHD) and 56 (39.2%) pts had chronic GVHD (cGVHD), extensive in 27 (48 %). In a univariant analysis of potentially P538 relevant factors, we found that isolated extra medullary Impact of HLA class I and class II DNA high-resolution relapse (EMR) was associated with the presence of cGVHD in HLA typing on graft-versus-host disease and survival in 7/9 (77.8%) compared to 34/103 (33%) for medullary and adult unrelated stem cell transplantation after in vivo T- 15/31 (48%) for concurrent medullary and extramedullary cell depletion with CAMPATH: a single transplant centre relapse (p=0.015). When revised Seattle classification for experience limited and extensive cGVHD was applied, cGVHD was J. Perz, R. Sergeant, R. Szydlo, D. O`Shea, T. Khan, N. extensive in 6/9 (66.7%) with isolated EMR, in 14/103 (13.6%) Davey, E. Olavarria, J. Apperley Hammersmith Hospital of medullary and in 7/31 (22.6%) of concurrent relapses (London, UK) (p=0.002). At the time of relapse 7/9 (77%) with isolated EMR were found to have full donor chimerism. Median time to Aim: Outcome of unrelated donor stem cell transplantation relapse for isolated EMR was longer (11.4 months) compared (SCT) is influenced by matching for HLAclassI and II. We to (8.16) for medullary and (8.53) for concurrent relapses but investigated the effects of DNA high-resolution HLAtyping this was not statistically significant (p=0.306). Our data (HR) on acute and chronic graft-versus-host disease (aGvHD suggests that the expected GVL effect in pts with cGVHD may and cGvHD) and overall survival (OS) in adult patients/donors preferentially maintain marrow remission without preventing typed for HLA-A,B,Cw,DRB1,DPB1 by HR and low resolution relapse in extramedullary sites.The findings of this study also (LR) and for DQ by LR.

S135 Patients/methods: 70patients (pts.)(median age32y,18–54) As expected, T cells subsets, B cells and NK cells numbers were treated with allogenic SCT from unrelated donors for were significantly higher in PBSC grafts (versus MSC) up to diseases: 45 Ph-positive chronic myeloid leukaemia (CML)in 30 fields higher. The proportions of CD4+ and CD8+ T-cell 1.chronic phase (1.CP), 11 CML in advanced phase (AP),11 subsets were comparable in the 2 types of graft and similar to acute lymphoid (ALL) and myeloid (AML)leukaemia,2 multiple normal values observed with peripheral blood in healthy myeloma (MM) and 1 Ph-negative CML. Conditioning adults. The incidence of acute GVHD was proportional to the consisted of total body irradiation (TBI) in 68 pts. combined (CD4+CCR7+/CD4+CCR7neg) ratio in the graft. Thus, with cyclophosphamide(63) or etoposide(5) and of patients who developed acute GVHD had received a cyclophosphamide/busulphan in 2 pts. In all pts. GvHD (CD4+CCR7+/CD4+CCR7neg) ratio > 2,79 than those who prophylaxis was provided by cyclosporin A and short-term did not develop acute GVHD (p=0.006) suggesting a methotrexate with in vivo T-cell depletion (TCD) by CAMPATH detrimental role of these CD4+CCR7+ subsets in term of (Anti CD52) in 69pts.and ATG in 1. acute GVHD. Conversely, a high incidence of early death was Results: With LR typing 49 patients/donors(69%) were fully observed in patients receiving a matched but 21 had classI mismatch (MisM). HR typing (CD4+CCR7+/CD4+CCR7neg) ratio <1.93 (p<0.03)suggesting revealed 7 additional pts. with classI or IIMisM a protective role of these subsets in term of early transplant (1A,6B,6DRB1,4Cw,in some pts.>1), thus 42 related mortality. patients/donors(59%) were fully matched. AGvHDgradeIII-IV Conclusion: this report contributes to the establishment of occured in 7pts.(10%) whilst extensive cGvHD was seen in reference values regarding the distribution of CD4+ and CD8+ 10of57(18%). There was no statistical difference in the T cell subsets within PBSC and MSC grafts. Although, the occurence of aGvHD or cGvHD between fully matched and quantity of infused T cell does not seem to influence early mismatched pts./donors at LR or HR: 2of21 with LRMisM(9%) complications after allo-SCT, the qualitative composition of and in 4of28 with HRMisM(14%) presented with aGvHD and graft regarding T cell subsets, especially the 1of17(6%) with LRMisM and 2of22(9%) with HRMisM with (CD4+CCR7+/CD4+CCR7neg) Ratio, appears to be of great cGvHD. The probability of transplant-related mortality (TRM) importance in acute GVHD occurence. for the whole cohort was 16% at day100 and 23% at day180. 1patient failed to engraft. 3-years survival probabilities were significantly different(p=0.004) for pts. with CML1.CP(63%, P540 good risk) when compared with all other diseases(23%,poor Inolimomab as front-line therapy for acute graft-versus- risk). Comparisons of survival according to LR/HR matching host disease: a phase I/II study did not show any differences, neither in the whole cohort, nor M. Michallet, C. Faucher, D. Blaise, I. Yakoub-Agha, J.P. for CML1.CP(45pts.) nor for poor risk diseases(25pts.). Jouet, N. Milpied, F. Nicolini, A. Thiebaut, Q.H. Le, S. Maury, Conclusion: In 70pts.(64%CML1.CP) treated with in vivo TCD C. Vigouroux, E. Gadolet, C. Vermot-Desroches, O. Subiger, I. with CAMPATH HRtyping did not reveal additional pts. at risk Darlavoix, A. Zinai, J.C. Bay Hôpital Edouard Herriot (Lyon, F); of severe aGvHD or extensive cGvHD and it did not have an Institut Paoli Calmettes (Marseille, F); CHU (Lille, F); CHU impact on OS after unrelated SCT. The overall rates of aGvHD (Nantes, F); CHU (Creteil, F); OPI (Limonest, F); CHU and cGvHD were low compared to published data in nonTCD- (Clermont-Ferrand, F) SCT. Further investigations on larger cohorts are needed to elucidate the impact of HRtyping in this setting. Background: The second line therapeutic strategies for acute graft versus host disease (aGvHD) remain unsatisfactory with high rates of failure. Inolimomab, a murine anti-IL-2 receptor P539 (anti-CD25), administered early enough in patients with steroid Naive and non-naive CD4+ and CD8+ T-cell subsets refractory aGvHD, demonstrated interesting potentialities on distribution in blood and marrow grafts and their impact response rates and survival (Dhédin N. et al. EBMT 2003, on patients' outcome after allogeneic stem cell abstract no 851). The introduction of inolimomab as first-line transplantation therapy in combination with steroids in the management of I. Yakoub-Agha, P. Saule, S. Depil, C. Grutzmacher, L. Magro, aGvHD is assessed. P. Cracco, F. Bauters, F. Dufossé, F. Villard, J.P. Jouet, J.P. Study design: We performed a pharmacokinetic study of Dessaint, M. Labalette Hôpital Claude Huriez (Lille, F); inolimomab in patients developing a grade II-IV aGvHD Laboratoire d'immunologie (Lille, F); ETS-Nord (Lille, F) receiving 2mg/kg/day of Methylprednisolone in combination with an 8-day induction treatment of inolimomab (0.1, 0.2 and Peripheral blood stem cells (PBSC), an alternative to marrow 0.4mg/kg/day: 5 patients at each dose level, 0.3mg/kg/day: 6 stem cell (MSC), is associated with enhanced engraftment and patients). Additional inolimomab sequences depended on accelerated recovery after allo-SCT. However, despite an aGvHD status on Day 9. GvHD was assessed according to increased number of infused donor T cells, the incidence of Glucksberg’s classification. Patients who achieved CR acute GVHD with PBSC appears to be identical than with received a maintenance therapy with inolinomab at the same MSC. Recent works on the heterogeneity of the human CD4+ daily dose, thrice a week for 3 weeks, and patients with PR and CD8+ T cells have individualized 4 subsets: namely, naive received a 2nd course of induction regimen, followed by a 2 (CCR7+CD45RA+), central memory TCM week-maintenance regimen. Patients with NR, MR and PD (CCR7+CD45RAneg), effector memory TEM, were left at investigator’s decision. The final response was (CCR7negCD45RAneg), and CD45RA+ effector memory cells assessed at Day 28 after the initiation of inolinomab treatment. TEMRA, (CCR7negCD45RA+). To our knowledge, the T cell Preliminary results: Twenty one patients were enrolled [12 M, subsets proportions in MSC and PBCSP grafts remain 9 F; median age: 51 years (29-61)] harboring a severe unknown. On the other hand, the impact of the donor T cell aGvHD. Primary diseases were hematological diseases for 16 subsets on patients’ outcome is still to be investigated. patients (5 AML, 2 NHL, 2 MDS, 2 CLL, 1 ALL, 1 CML, 1 MM, Between Sept 2003 and Sept 2004, 33 consecutive 1 MPS, 1 HD) and solid tumors for 5 patients. All patients hematopoietic grafts (17 MSC and 16 PBSC) were considered underwent an allogeneic HSCT (15 PBSCT, 6 BMT from 17 for this study. Multiple combinations of immunophenotyping HLA matched related, 2 HLA matched unrelated and 2 HLA analysis were used to prospectively examine immune mismatched unrelated donors), after either myeloablative (8) parameters related to graft as well as to early reconstitution. or reduced intensity (13) conditioning regimens. The objective CD45, CD3, CD4, CD8/ CD3, CD8, CD16, CD56, CD19, CD4, response rate of aGvHD at Day 28 was 52.4% (9 CR, 2 PR). CD28, CCR7, CD45RA. Early post-transplant complications At Day 100, 8 patients died mainly from aGVHD with or including acute GVHD, relapse and infections were assessed. without infection. The three-month survival rate was 61.9% (CI

S136 95%: 38.4%-81.9%). Pharmacokinetic data are still under missed on clinical examination. We plan a prospective analysis and will be presented. evaluation of patients with an abdominal CT scan pre- However, it seems that inolimomab serum concentrations transplant and on day +30. above 5µg/mL at Day 3 induce early, good and sustained responses. Conclusion: These results suggest that inolinomab, in P542 combination with steroids, is efficient and safe as initial Expansion of CD94+ NK-like T-cells after haematopoietic therapy of severe aGvHD. stem cell transplantation. Excess of CD94+ T-cells after alternative as compared to MHC identical donor transplantation B. Grzywacz, K. Bogunia-Kubik, D. Dlubek, A. Lange Lower Silesian Centre for Cellular Transplantation (Wroclaw, PL); Institute of Immunology and Experimental Therapy (Wroclaw, PL)

The lectin-like molecule CD94 is expressed on NK cells and a subset of T cells. The expression of CD94 on antigen- experienced T cells is a consequence of CD8+T cell activation induced by infectious or allogeneic stimulation. The CD94 ''bright'' T cells are inhibited, whereas CD94 ''dim'' T cells are activated upon CD94 triggering due to differential signal transduction via associated NKG2A or NKG2C co-receptors respectively. In healthy individuals CD94+ constitute a minor subset (± 5%) of T cells. The ligands for CD94 are HLA-E and P541 MHC encoded heat shock protein HSP70. HSP-70 triggers NK Abdominal computer tomography severity score - in cells via CD94 by it's C-terminal portion. Two allelic patients with intestinal graft-versus-host disease: dimorphisms (G/A and C/T) of the HSP70 gene at nucleotide correlation with outcome positions 2763 and 2437 result in Glu - Lys and Met-Thr E. Biscaldi, F. Gualandi, S. Luchetti, M.T. van Lint, G.A. alterations at aminoacid positions 602 and 493 respectively. Rollandi, A. Bacigalupo S.Martino's Hospital (Genoa, I) The aim of the study was to investigate the prevalence of CD94 expression on T cells in patients after Hematopoietic Background: We have therefore been carrying out over the Stem Cell Transplantation (HSCT). past years, sequential Computerized Tomography (CT) scans The lymphocytes of HSCT patients were studied by flow of the abdomen in these patients , to evaluate parenchimal cytometry, 39 patients were transplanted from HLA matched and intestinal abnormalities. siblings, 36 from alternative donors. HSP70 polimorphisms Aim of the study: to test whether Computerized Tomography were investigated using ARMS-PCR or PCR-RFLP for 2763 or (CT) would (a) pick up features characteristic of intestinal 2437 polymorphic positions, respectively. GvHD and (b) would be predictive of mortality In the early post-transplant period (100d.) CD94+ represented Patients, 32 patients with clinical evidence of gut GvHD higher percentage of T cells in patients transplanted from enhanced CT at various times after onset of GvHD. There alternative donors than in those transplanted from HLA- were 18 males and 14 females, median age was 35 (22-60) . identical siblings (Mean: 26.4 Vs 17.8; p=0.04). The difference The donor was an HLA identical sibling in 20, unrelated in 10, was more pronounced in the CD94 ''dim'' (p=0.04) than in and a family mismatched donor in 2 cases. At the time of CT CD94 ''bright'' (p=0.11) subsets. At later time points (100 - scan 24 patients had clinical grade I gut GvHD , 6 had grade II 1000 d.) alternative- and sibling-donor transplanted groups did and 2 grade III gut GvHD. no longer differ (24 Vs 24,2). CT scan: CT study was performed after a distension of We tested if the polymorphism of HSP70 may contribute to the intestinal wall with almost 500-700 cc of tap water. This allows difference. Among alternative donor transplant pairs 42% and an ideal identification of the focal bowel wall anomalies 27% were mismatched at HSP-70 positions 2763 and 2437 (multilayering, focal enhancement, thickening). respectively. Incompatibility at neither one of the positions led CT was performed with a single slice scanner, with following to higher CD94+ T cells expansion. technical parameters: 3 mm slice thick, 0,7 s per scan, 2 mm In the vulnerable to aGvHD early period after HSCT the subset index, pitch 1,3, 120 KV, 300 mAs, 70 s of delay time, 2 cc/Kg of CD94 expressing NK- like T cells expanded to a higher of body weight of iodinated c.m. (Iopamidol), injected with a extent after transplantations from alternative donors than from flow rate of 2,5 ml/s. HLA identical siblings. The excess of CD94+ ''dim'' cells, which CT severity score. Patients were scored for the presence or are activated upon CD94 triggering, may contribute to the absence of the following radiologic signs: (1) thickening of the higher occurence of aGvHD seen after alternative donor antral gastric wall; (2) mesenteric hyperemia, (3) thickening transplantation. The recipient-donor mismatches at HSP70 C- and multilayering of the small bowel wall and (4) ascites. terminal residues did not facilitate the excess of CD94+ T Results: We could not find a correlation between CT severity cells. score and GvHD at the time of CT scan (p=0.2). This is due to the fact that 13 patients had CT severity score of 3-4, whereas only 2 patients had gut GvHD grade III. P543 Of the 14 patients with CT severity score of 0-1, two died of HLA allele associations with graft-versus-host disease transplant related complications (TRM) (14%). Of the 18 following allogeneic haematopoietic stem cell patients with CT severity scores of 2-4 , ten (55%) died of transplantation TRM. The correlation between CT signs and TRM is V. Kitra, O. Moraloglou, F. Petra, K. Spanou, I. Peristeri, E. significant (p=0.01). Gousetis, S. Delikou, N. Constantinidou, S. Graphakos St. Conclusions: We have described radiologic signs of Sophia Children Hospital (Athens, GR) enteropathy in patients with intestinal GvHD. A CT scan based score has been devised and shows strong correlation with Introduction: Acute and chronic graft versus host disease transplant related complications. The CT severity score (aGVHD, cGVHD) remains a serious complication after appears to identify objective enteropathy, which may be

S137 allogeneic hematopoietic stem cell transplantation (allo- of group2 (mean time=199 d). 15/21 (71%) pts are alive in HSCT). group1 and 15/24 (62%) in group2. Deaths were relapse- Pathogenetic mechanisms involved in GVHD are not fully related in 5/6 cases in group1 and in 5/9 cases in group2. investigated, although considerable progress has been made CMV reactivation and disease were noted in 1 (group1) and 6 in the disease research and treatment. (group2) pts but 8 CMV positive recipients in group2 were Previous studies have shown the involvement of HLA alleles in noted vs 1 (group1). Preemptive therapy for EBV-induced aGVHD and/or cGVHD occurrence after HLA identical allo- lymphoproliferative disorder was given in 4 pts of group1 vs 1 HSCTs. pt (group2). Adenovirus (ADV) infection and disease were Patients and methods: In this retrospective study we seen in 9 (group1) and 1 (group2) pts. investigated the correlation of HLA class I and II alleles with Conclusions: overall survival (66%) is interesting for UHSCT the occurrence of aGVHD, in 100 children, aged 1-18years but follow-up is still short. There’s no statistical difference for who were transplantated with HLA identical sibling donors for aGVHD but perhaps for cGVHD. Difference between the 2 genetic or malignant diseases. Patients were grouped groups for relapse is not significant. There are more EBV and according to GVHD severity: aGVHD O-I: 66%, II-IV: 34% and ADV reactivations in group1 without related mortality. We can cGVHD: 12%. The frequencies of HLA A, B, DR alleles were think that we need more pts to define clearly optimal ATG estimated for each patient group separately and were dose in UHSCT. correlated with the severity of aGVHD and the development of cGVHD. Statistical analysis was performed by the use of x² test and Fischer’s exact test and the estimation of relative risk P545 (RR). C0 Cy-A monitoring as predictor of aGVHD and adverse Results: HLA-A, B and DR analysis did not present any effects: would two-hour post dose Cy-A (C2) level be statistically significant association with the severity of a GVHD. better? A significant increase of HLA-B22 was found in cGVHD A. Ibatici, A. Nocera, B. Bruno, M.T. Van Lint, A. Bacigalupo (p=0,007, RR=11). It is interesting to be noted that in cGVHD Ospedale San Martino (Genoa, I) group HLA A10, B8 and DR3 antigens were completely absent, although this finding did not reach statistical Introduction Acute graft versus host disease (aGVHD) remains significance. a major cause of treatment failure after allogeneic stem cell Conclusion: To conclude, in this study an association of HLA- transplantation (ASCT). Using HLA-identical sibling grafts, the B22 with the development of cGVHD was shown, with incidence of severe aGVHD (grade III-IV) has been reduced potential biological significance. Associations between HLA down to 10-15%. Cyclosporine-A (CyA) is the most widely alleles and aGVHD reported in previous similar studies were used immunosuppressive agent for ASCT and trough serum not confirmed, most probably due to differences in the level (C0) monitoring has been recommended to prevent immunogenetic profiles of the studied populations. aGVHD and minimize toxic effects. Since low C0 CyA showed to correlate with the risk of developing aGVHD (Wee et al NEJM 1986), C0 monitoring P544 has been used to measure drug exposure. However, it has Antithymocyte globulin-based preparative regimen for been recently demonstrated in solid organ transplantation that unrelated donor stem cell transplantation: is there an single point C2 monitoring is a better predictor than C0 level of optimal dose? acute rejection and of adverse effects. L. Clement, A. Salmon, D. Bensoussan, C. Andre-Botte, P. Aim of study 1) to test the correlation between C0 CyA in the Bordigoni CHU Nancy-Brabois (Vandoeuvre, F) first 2 weeks after ASCT and the risk of GVHD; 2) to test differences between C0 and C2 in patients on oral CyA. Unrelated haemopoietic stem cell transplantations (UHSCT) Material and Methods: 1) We analyzed 565 consecutive pts have an increased risk of severe graft- versus-host disease with hematologic disorders, undergone myeloablative ASCT (GVHD) and transplant-related mortality. In vivo pretransplant from HLA-id sib (1992-2003), using CyA + methotrexate, as depletion of host immune cells by Antithymocyte globulin GVHD prophylaxis. C0 monitoring was evaluated twice a week (ATG) showed efficacy as prophylaxis (Px) of GVHD but until day+100 (Abbott AxSYM, range 150-300ng/ml). The optimal dose of ATG is unknown. correlation between median values C0 (days 1-10, 11-20) and Aim of study: evaluate toxicity and efficacy of 2 doses of ATG the development of aGVHD (subgroups were 0-I, II, III-IV) was (Fresenius; 90 mg/kg (HD) vs 15-60 mg/kg (LD)) in 45 analyzed using the chi-square test. consecutive UHSCT. 2) We measured a 12-hour CyA pharmacokinetic profile in 7 Patients and Methods: 21 pts received HDATG(group1)(sex HLA-id sib recipients taking oral CyA (Neoral, Novartis) at ratio H/F=18/3; mean age=16.8y; 17 for haematologic doses ranging between 75-125mg BID/day. The blood malignancies). 24 pts were given LDATG(group2)(sex ratio samples were hourly drawn from a peripheral vein. Data H/F=14/10; mean age=17.6y; 22 for haematologic distribution between C0 and C2 were analyzed using F-ratio malignancies). Grafts were respectively in group1 and 2: testing. marrow in 14 and 13 pts, peripheral blood stem cells in 6 and Results: 1) Severe aGVHD developed in 8 (1%) and 32 (6%) 11 pts, cord blood(CB) cells in 1 pt of group1. All pt and pts within d+30 and d+100, respectively. C0 median value donor(P/D) pairs were HLA-typed with high resolution PCR- were 147ng/ml (dd 1-10)(p=0.08) and 163ng/ml (dd11- SSP (except the single CB graft): 14 (group1) and 15 (group2) 20)(p=0.04). 2) The 12-hour profile showed the time- P/D pairs were HLA-A,-B,-C,-DR,-DQ compatible; respectively distribution of Neoral with most of Cmax ranging between 1-3 3 and 9 received a 1 mismatched transplant; 3 P/D pairs of hours after oral CyA dose (dotted curve). C2 Median values group1 received a 2 mismatched transplant; P/D pair of CB were 500±193, whereas C0 were 187±57.8 (F-test p=0.002). transplant was 4/6-compatible. CSA and MTX were given as Conclusion: As shown by our results, the C0 point can be GVHD Px in 15 and 17 pts; 5 and 7 pts received CSA and predictive of aGVHD within d+20 after ASCT. By measuring MMF; CSA and steroids were given as GVHD Px in CB 12-hour CyA profile in this cohort, it seems that C0 does not transplant. accurately reflect drug exposure, according to data from solid Results: engraftment was achieved in all but 1 pt in each organ transplants. We open a prospective trial to assess group (95%).Incidence of aGVHD (grade II-IV) at D100 was whether C2 monitoring may result more appropriate than C0 to 63% in group1 and 55% in group2. cGVHD was seen in 9 pts minimize the risk of GVHD and CyA-related toxicity after of group1 (50%) and in 14 pts of group2 (66%). Relapse was ASCT. diagnosed in 6 pts of group1 (mean time=206 d) and in 5 pts

S138 P547 Rapamycine (Sirolimus) as salvage therapy for chronic graft-versus-host disease M. Jurado, C. Vallejo, J. Pérez-Simón, S. Brunet, P. Balsalobre, J. Pérez-Oteyza, I. Espigado, A. Romero, D. Caballero, J. Díez, L. Vázquez Hospital Virgen de las Nieves (Granada, E); Morales Meseguer (Murcia, E); Hospital Clínico (Salamanca, E); Hospital Sant Pau (Barcelona, E); Gregorio Marañón (Madrid, E); Hospital Ramón y Cajal (Madrid, E); Hospital Virgen del Rocío (Sevilla, E); Hospital Gregorio Marañón (Madrid, E)

Introduction: Patients with refractory chronic graft versus-host- disease (GVHD) are a challenging for therapy, and, to date, we have no specific and established therapeutic options for P546 them. A new immunosuppressive agent, rapamycin, has been Influence of the intensity of the conditioning regimen on used in combination with calcineurin-inhibitors in both the characteristics of acute and chronic graft-versus-host prophylaxis and acute GVHD, with interesting results. We disease after allogeneic transplantation present a retrospective study in which we analyze the M. Díez-Campelo, J. A. Pérez-Simón, R. Martino, S. Brunet, effectiveness of rapamycin in patients with unresponsive A. Urbano, M. D. Caballero, A. León, D. Valcarcel, E. chronic GVHD. Carreras, M. C. Cañizo, J. López-Fidalgo, J. Sierra, E. M. Patients and methods: Thirty three patients with Ocio, C. Castilla, M. V. Mateos, J. F. San Miguel Hospital oncohematological malignancies ( 12 CML, 10 lymphomas, 8 Clínico Salamanca (Salamanca, E); Hospital de la Santa Cruz acute leukemia, 1 severe aplasia, 1 myelodisplastic syndrome, y San Pablo (Barcelona, E); Hospital Clínico Barcelona 1 chronic myelomonocitic leukemia), received transplants from (Barcelona, E); Hospital General del SAS (Jeréz de la related (n=26) or unrelated donors (n=7); the source of HLA Frontera, E); Facultad de Estadística Universidad de identical hematopoietic stem cells was marrow in 4 patients Salamanca (Salamanca, E) and peripheral blood in 29 patients. Before starting rapamycin, patients had been treated with one (n=6), two (n=12), three Although increasing numbers of allogeneic transplants are (n=10) and four or more (n=5) lines of therapy, and the status being performed using reduced intensity conditioning for GVHD were 4 patients in relapse, 10 patients in partial regimens (allo-RIC), most of the current experience on graft- response and the remaining 19 patients were refractory or versus-host disease (GVHD), comes from the use of progressed from other immunosuppressive therapy. Forty two myeloablative conditioning regimens (MCR).We analyzed the percent of patients presented sclerodermia and 25 % of them characteristics of GVHD among 150 consecutive patients showed platelet lower than 1x105/L. Patients received undergoing allo-RIC as compared to 88 concomitant patients rapamycin in combination with CSP (16), FK-506(9), MMF (4) undergoing MCR. All patients received the same GVHD or PRD (4) as salvage therapy. prophylaxis (cyclosporine-methotrexate) and peripheral blood Results: We observed 25 patients with clinical response (8 stem cells from an HLA identical sibling.In the MCR and allo- complete and 17 partial remission) and 4 whom failed for RIC groups, incidence of acute GVHD (aGVHD) were 69% therapy. Four patients were not evaluable, one because of and 47%, respectively (p<0.001) and incidence of grades 2-4 early death and 3 because of not enough time to see aGVHD were 51% vs 33%, (p=0.0009). Only conditioning type response. Toxicity was unseen in 12 and life threatening in 3 (MCR vs allo-RIC) significantly influenced the incidence of patients, two because of hemolytic uremic syndrome and one aGVHD in multivariate analysis: HR= 2.16 (95 % CI: 1.52- with renal failure. Statistical analysis revealed non significant 3.07), p<0.0001. Cumulative incidences of chronic GVHD impact for number of lines of therapy previous to rapamycin, (cGVHD) were 68% and 76% among MCR and allo-RIC patients with showed sclerodermia and GVHD clinical status patients (p=0.06). Cox univariate analysis confirmed that this pre-Rapa. However those patients with platelets less than trend was due not to a higher incidence of extensive but of 1x105/L had a lower overall survival. The Kaplan-Meier limited cGVHD among allo-RIC patients [HR=3.3 (95% CI: estimate of survival for the entire group was 84% at 30 1.42-8.08), p=0.0017]. Morevoer, among patients who months. developed cGVHD, incidence of extensive cGVHD was higher Conclusions: Given these interesting results, and the lack of a in the MCR than it was in the allo-RIC group (88 vs 64%, gold standard therapy for unresponsive chronic GVHD p=0.01) and there was a higher incidence of severe skin patients, should be explored with new clinical trials the role of cGVHD (39.4% vs 9.6%, p=0.008). Duration of rapamycin on the control of chronic GVHD. immunosuppression was shorter among allo-RIC patients with 35.5% requiring systemic immunosuppression 36 months after transplant compared to 68.8% in MCR patients (p=0.028).The P548 use of allo-RIC modifies the incidence and characteristics of Low plating density boosts mesenchymal stem cell both acute and chronic GVHD and decreases the proliferation immunosuppression requirements in the long-term follow up. C. Malischnika, E. Rohde, D. Thaler, B. Vogel, W. Linkesch, G. Lanzer, H. Schaider, D. Strunk StemCellCluster (Graz, A); Pharmaceutical Sciences (Graz, A)

Bone marrow (BM) mesenchymal stem cells (MSC) are multipotent cells with a fascinating spectrum of functions reaching from support of hematopoietic SC to induction of immune tolerance in addition to their inherent capability to generate osteo-, chondro-, and adipocyte progeny. Ex vivo expanded human MSC are currently evaluated in studies for the prevention and treatment of graft-versus-host disease (GvHD) and for HSC engraftment support. This study was performed to define optimized expansion conditions to

S139 generate the requested quantity of >106 MSC/kg/patient for We conclude that a simple laboratory evaluation is highly clinical purposes and to analyze their influence on MSC predictive for the risk of NRM in patients surviving 100 days phenotype and function. after alloHCT. The prognosis is particularily poor for patients Based on contradictory published evidence we tested the with plasma protein <60 g/L and bilirubin >1 mg/dL that may influence of log fold decreased cell seeding density (2500; reflect the impaired function of the liver and intestines due to 250; 25; 2.5 MSC/cm²) in two defined standard media (LG- the conditioning-related toxicity, GvHD, or infectious DMEM, alpha-MEM) using selected FCS. The MSC surface complications. phenotype was analyzed by flow cytometry. Chemokine receptor and ligand profiling by RT-PCR and multiplex protein array were employed to search for indicators of autoregulation P550 of cell proliferation and homing. Maintenance of SC The influence of administration order of busulphan and characteristics was tested in CFU-F assays and by osteo-, cyclophosphamide on dendritic cells in stem cell chondro-, and adipogenic differentiation. transplantation mouse model Best MSC multiplication could be achieved in alpha-MEM at C. Nilsson, J. Forsman, E. Cussen, Z. Hassan, M. Abedi- seeding densities below 250/cm². Densities between 25- Valugerdi, H. Concha, M. Jansson, R. Tehranchi, M. Hassan 250/cm² almost guaranty to obtain 108 MSC from starting Karolinska Institute (Stockholm, S) 10mL BM aspiration within <4 weeks compared to >6 weeks under standard conditions (2500/cm2). Using this protocol Busulphan (Bu) in combination with cyclophosphamide (Cy) is >1010 MSC for repeated applications can be generated within used frequently as a preparative regimen for patients 3 months. Amplified cells displayed the common undergoing stem cell transplantation (SCT) for both malignant CD73+/90+/105+/29+/13+/34-/45-/133-/146+/HLA-AB+ and non-malignant disorders. However, the treatment related phenotype and retained SC potential. Lowest cell yield was toxicity is still the dose-limiting factor. The order in which obtained with 2500 MSC/cm2 in DMEM. Preliminary data on busulphan and cyclophosphamide is administered may chemokine receptor pattern indicate precise pathways for the influence both the adverse effects and the therapeutic efficacy MSC autoregulation of proliferation and mobility. Provided that of Bu/Cy regimen and also the amount of dendritic cells. Host the immunosuppressive and HSC supporting functions are dendritic cells can stimulate CD4+ donor cells and cause unaffected by culture conditions as indicated in the literature GVHD. In the present study, we investigated the effect of the low density MSC expansion should be strongly recommended. administration order of Bu/Cy on the myeloablative-, Better insight into the behavior of MSC with respect to immunosuppressive effect and the recovery of dendritic cells proliferation, homing efficiency and regulatory function is and their origin in a mouse model. crucial to assess evolving therapeutic strategies. Female balb-C mice were divided in two groups, the first group received liposomal busulphan (15mg/kg/dayx4) followed by Cy (100mg/kg/dayx2) while the second group was given Bu/Cy in P549 reversed order. At the day of transplantation (day-0) donor (8- The prediction of non-relapse mortality for patients 10 weeks old males) marrow cells (sca1) were isolated from surviving 100 days after alloHCT femurs, separated and infused via the tail to the recipients S. Giebel, J. Holowiecki, J. Wojnar, M. Krawczyk-Kulis, M. (6x105cells per mouse). Kopera, L. Kachel, M. Markiewicz, I. Wylezol, A. Wnuk Mice were sacrificed during the conditioning and up to 30 days Silesian Medical University (Katowice, PL) after the treatment. No significant difference was observed in the myeloablative effect between both treatment groups. The Extensive chronic GvHD is a major cause of non-relapse immunosuppressive activity as expressed as CD3+/CD19+ mortality (NRM) for patients surviving 100 days after and CD4+/CD8+ was similar for both treatment schedules. allogeneic hematopoietic cell transplantation (alloHCT). The cytokines (IL-2, TNF-alfa and IFN-gamma) detected at However, it may develop at different time post-transplant. In day 0 were lower when Cy was given first compared to that this study we searched for laboratory parameters that let observed when Bu was given first. CD11C+ from donor origin predict the risk of NRM evaluated exactly on day +100. was significantly higher from day +1 until day + 9. The We analysed 255 patients, aged 29 (10-56) years, who administration of Cy prior to Bu resulted in significantly higher remained alive and disease-free on day +100 after alloHCT CD 86+ cells between day 0 and day + 5 compared to that from an HLA-identical sibling (n=177) or matched unrelated found in mice received Bu prior to Cy. Dendritic cell origin was volunteer (n=78) perforemed in a single institution (Katowice, detected using fluorescence in situ hydridization (FISH) by Poland) between 1992-2003. The diagnosis was as follows: labelling Y-chromosome (from the donor). The labelled cells CML -99, AML -70, ALL -55, SAA- 10, NHL -10, MDS -7, other were counted as the percent of 500 cells from each animal. -4. All patients were treated with myeloablative conditioning In summary, we conclude that Cy-Bu treatment has resulted in based on chemotherapy (n=215) or TBI (n=40). Bone marrow lower levels of cytokines that most probably can be less served as a source of stem cells in 224 cases, peripheral harmful for the new stem cells, faster engraftment and higher blood – in 31 patients. ratio of donor dendritic cells. This treatment strategy may be a In an univariate analysis, the following laboratory parameters promising strategy to reduce transplantation related were associated with increased incidence of NRM: ANC <1.5 complications. vs. >=1.5 G/L (30% vs. 15%, p=0.006), PLT >=100 vs. <100 G/L (10% vs. 29%, p<0.0001), Hb <11 vs. >=11 g/dL (25% vs. 6%, p=0.0001), total protein <60 g/L vs. >=60 g/L (39% vs. P551 6%, p<0.0001), AspAT elevated vs. within normal limit (21% HLA-DPB1 specific responses between unrelated vs. 14%, p=0.04), Alkaline phosphatase elevated vs. within individuals can be demonstrated by the production of normal limit (33% vs. 15%, p=0.003), bilirubin <1 vs. >=1 interferon gamma in an ELISpot assay mg/dL (11% vs. 45%, p<0.0001). In a multivariate analysis, B.E. Shaw, L.D. Barber, N.P. Mayor, S.G.E. Marsh, J.A. only decreased protein (HR=6.97 (3.3-14.7), p<0.0001) and Madrigal Nottingham City Hospital (Nottingham, UK); Anthony elevated bilirubin (HR=3.52 (1.91-6.48), p<0.0001) Nolan Research Institute (London, UK); The Royal Free independently influenced the risk of NRM. The cumulative Hospital (London, UK) incidence of NRM equaled 6% if none of the above factors was present, 10% - for elevated bilirubin alone, 22% - for HLA-DPB1 has been shown in numerous studies to be a hypoproteinaemia alone, and 70% for elevated bilirubin and molecule with immunogenic potential. In unrelated donor hypoproteinaemia, both present. haematopoietic stem cell transplantation (HSCT) an impact

S140 due to the matching status at this molecule has been shown. CD4fitc/CD25pe/CD45percp/CD3apc; This effect is due not only to the presence of a mismatch, but CD8fitc/CD25pe/CD45percp/CD3apc. also to the particular type of mismatch (at allele, or even Instead, the monitoring of the chimerism is performed on epitope, level). There is evidence that certain mismatches may marrow and peripheral blood from the 30th day post- be better tolerated than others. The mechanism for these transplant, and when requested by the physicians, in FISH (if effects is incompletely understood. Functional studies the donor's sex is different from the recipient) or molecular considering the impact of DPB1 in unrelated donor pairs have analysis of VNTR (9-100 bp: vWF2, D1S80, YNZ22, vWF1, 3'- often been hampered by the lack of high resolution tissue HVR) and STR (1-6 pb: HumARA, HumTH01, S33). typing, which has made the results difficult to interpret (due to In this way we realize, as reported from other works, that all potential 'hidden mismatches'). patients without a rapid engraftment of the donor T-cells reject Our aim was to ascertain whether DPB1 specific responses the graft (see Fig.1), while those patients with a fast recovery could be detected in a highly sensitive functional assay, using of the donor T-cells are at risk of acute GvHD. With these unrelated donor pairs, when all other HLA mismatches were methods we are able to give quick information about the donor excluded. An ELISpot assay was used with the production of T-engraftment so the medical team could provide with the IFN-gamma as a readout. Volunteer donors were chosen who immunosuppression therapy, or the infusion of the donor were not only matched for HLA-A, -B, -C, -DRB1, -DQB1, but lymphocytes, needed to allow the graft to gain a foothold. also homozygous for the chosen haplotype, A*0101, Cw*0701, B*0801, DRB1*0301, DQB1*0201. This allowed for an homogenous background on which to test the effects due to differences in a single HLA-DP molecule. One-way interferon gamma ELISpot assays were performed, using serial dilutions of responder PBMCs (starting at 0.3 x 106 cells /ml) stimulated with irradiated DP mismatched PBMC (0.3 x 106/ml) for 72 hours. Background responses were determined using unstimulated PBMC and auto stimulated cultures. A positive PHA activated PBMC control was included in each assay. Spot forming cells at frequencies ranging from 0 to 800/million cells were detected. Responses were shown to be DP specific by inhibition of SFC when the MLR was performed in the presence of an anti-DP specific monoclonal antibody (B7/21.2). To our knowledge, this is the first demonstration that the ELISpot assay can be used to detect low frequencies of DP alloreactive T cells, after brief in vitro stimulation, in the P553 context of an ELISpot assay between unrelated donor pairs. Immunosuppressive effects of polydeoxyribonucleotides This furthers the evidence for the immunogenicity of this R. Buhmann, T. Yang, M. Svihla, H.J. Kolb GSF (Munich, D); molecule. Klinikum Grosshadern Medical Center (Munich, D)

Objective: Defibrotide, the sodium salt of a single-stranded P552 polydeoxyribonucleotide was shown to mediate Simple methods to study immune reconstitution in immunosuppressive effects. In the current study we patients who underwent allogeneic transplant investigated whether randomly synthesized single-stranded G. Pricolo, L. Stani, A. Prudenzano, M. Strusi, F. Spedicato, P. polydeoxyribonucleotides of different length could provide Mazza Ospedale Moscati (Taranto, I) similar effects. Methods: Proliferation of peripheral blood mononuclear cells In HLA-matched transplants, engraftment is the result of donor (PBMC) or purified T-lymphocyte subsets in response to T cells mounting a successfull alloresponse against residual allogeneic PBMC´s, B-cells or dendritic cells (DC) was recipient T cells, causing the eradication of the recipient assessed in the presence or absence of dNTP´s and single- immune system. The process is dynamically counterbalanced stranded polydeoxyribonucleotides of 20, 40 and 80 base- by destructive graft rejection mechanisms of residual recipient length. After 72h or 120h incubation, 3H-thymidine (3H-TdR) T cells and NK cells on the transplant. Experience with highly incorporation was measured. Cell viability was measured by immunosuppressive but non-myeloablative fludarabine and trypan blue exclusion. cyclophosphamide transplant regimens illustrates the Results: Polydeoxyribonucleotides inhibit in a dose-dependent importance of T-cell engraftment in establishing long-term manner. Moreover, longer polydeoxyribonucleotides provide a hematopoiesis. In the first few weeks after transplant 100% of better inhibitory capacity than shorter one. Cytotoxic effects of the T cells are of donor origin, while recovering hematopoiesis polydeoxyribonucleotides, even in high concentrations could is predominantly recipient. Weeks to months later, recipient not be observed. hematopoiesis is replaced by that of the donor, following what Conclusion: Randomly synthesized polydeoxyribonucleotides is believed to be a graft-versus-marrow effect of the transplant. inhibit T-cell proliferation in vitro in a dose- and length- In contrast, recipients who do not achieve early donor T-cell dependent manner, and so suggest to mediate or to potentiate engraftment are at risk for graft rejection from residual host immunosuppressive effects in vivo. immune cells. In our centre we usually start to study the immune recovery from the early phases of the engraftment subsequent the CD34+ stem cells infusion (leucocytes > 500/ul) and every time the clinical condition of the patient needed it. In order to examinate the T-cells we apply a simple method in CFM to study the peripheral blood of the recipient: CD3fitc/CD16-56pe/CD45percp/CD19apc; CD3fitc/CD8pe/CD45percp/CD4apc ; CD4fitc/CD8pe/HLA-DR percp/CD3apc; CD57fitc/CD8pe/CD45percp/CD3apc; CD45ra fitc/CD45 ro pe/CD3 percp/CD4apc; CD45ra fitc/CD45rope/CD3percp/CD8apc;

S141 P554 severe acute (n=3 grade III; n=1 grade IV) or extensive Patients with GvHD demonstrate an impaired epithelial chronic GVHD (cGVHD) (n=7) following HSCT from matched barrier function and a significant reduction in the bacterial sibling (n=9) or partially matched related (n=2) donors diversity suggesting a role of the intestinal microflora in between march 2003 and november 2004. GVHD prophylaxis the pathogenesis of the disease consisted of cyclosporine (CSA) and MTX (n=6) and CSA and K. Rieger, O. Goldenberg, A. Fischer, H. Troeger, J.D. mycophenolate (MMF) (n=5). Etanercept 25 mg was given Schulzke, T. Fietz, A. Muessig, C. Gentilini, D. Sommer, K. subcutaneously twice weekly for 4 weeks followed by 25 mg Freyberg, E. Thiel, U. Goebel, L. Uharek Charite Campus weekly for 4 weeks. Concurrent immunosuppressive agents Benjamin Franklin (Berlin, D); Charite Campus Mitte (Berlin, included corticosteroids (n=9), MMF (n=7), CSA (n=5), D) tacrolimus (n=4) and rapamycin (n=2). Nine patients had steroid refractory acute/chronic GVHD (median duration of Objectives: The translocation of bacteria over the intestinal steroids: 43 days; range 10-632), 1 had steroid dependent epithelium, the processing of bacterial antigens by host GVHD and 1 was intolerant to CSA and tacrolimus. At the time antigen presenting cells (APCs) and their presentation to of initiation of Etanercept, 9 patients had skin, 7 had GI, 3 had donor lymphocytes is crucial for the development of intestinal liver, 3 had pulmonary and 3 had oral involvement; 10 patients GvHD. Since most of the predominant bacteria in human feces had >= 2 organs involved. do not grow in culture, investigation of microbiological Results: Etanercept was started at a median of 38 days (range pathogens and their capacity to promote GvHD was so far 0-1499) after the diagnosis of GVHD. Patients received hindered by methodical restrictions. Etanercept for a median of 7 doses (range 5-12). There were Methods: Denaturating gradient gel electrophoresis: We no infusion related side effects. Steroid taper was initiated in 6 analyzed bacterial communities in feces of twelve allograft patients (55%) as early as 5 days after the start of Etanercept. recipients (1) before transplantation, (2) during conditioning Overall response was 64% (CR=37%; PR=27%). Responses regimen, (3) immediately after engraftment and (4) on the were observed in 3/4 patients with aGVHD and 4/7 patients onset of intestinal GvHD or when gastrointestinal symptoms with cGVHD. Most responses were seen in liver (3/3) and GI- occurred. Probes were instantly frozen at –20°C until analysis. GVHD (6/7). Infections were observed in 7 (64%) patients and After homogenization, proteinase-digestion and mechanical included viral (n=3), fungal (n=2) and bacterial (n=1) disruption of bacterial cells, 16S rDNA was amplified and pathogens. KM survival since Etanercept initiation was 45%. separated by gel electrophoresis. High-frequency transmural One patient died of infection and 2 of disease progression. impedance analysis: Intestinal biopsies derived from patients Conclusions: our preliminary data indicate that Etanercept is with suspected GvHD were inserted into a Ussing chamber by well tolerated and can induce a high response rate in patients means of a container system. Epithelial resistance was with steroid-refractory aGVHD and cGVHD, particularly in the measured by impedance analyses. setting of GI involvement. Further investigation is warranted in Results: In addition to a reduction of the microbial diversity larger number of patients. after administration of broad-spectrum antibiotics we found striking differences in patients with cutaneous and intestinal GvHD: While in faeces samples collected before P556 transplantation 17 to 25 bands were detected (n=12), a Role of cGvHD on TRM and relapse after allogeneic dramatically reduction to 1 to 5 bands appeared in patients sibling myeloablative peripheral stem cell transplantation with histological proven intestinal GvHD (n=9). In four patients F. Bonifazi, G. Bandini, S. Falcioni, F. Palandri, M. Stanzani, with cutaneous GvHD four to 17 bands were detectable. B. Giannini, M. Giovannini, M. Arpinati, M. Baccarani Inst. of The epithelial resistance in patients with GvHD was reduced Haematology "Seràgnoli" (Bologna, I) severily impaired in patients with GvHD. Beeing based on a normal intestinal epithelial resistance of 40-50 ohm.cm² , Even if it’s largely recognized that PBSC transplants are patients with GvHD showed a significant decrease leading associated with higher cGVHD incidence, the true benefit of from 20-36 ohm.cm² in GvHD I° to 0-3 ohm.cm² in GvHD IV cGVHD on transplant outcome (relapse and TRM) is still under indicating a severe loss of epithelial barrier function. debate. Here we report on a large series of 104 consecutive Conclusion: Patients with GvHD demonstrate a severily PBSC myeloablative allogeneic sibling transplants for impaired epithelial barrier function and a significant reduction haematological malignancies, with an homogeneous GVHD in the bacterial diversity suggesting a role of the intestinal prophylaxis with CsA and methotrexate. Since the current microflora in the pathogenesis of the disease. classification of cGVHD is unsuitable to describe the severity The reduction of bacterial diversity in intestinal GvHD strongly of cGVHD and its impact on outcome, we subgrouped pts with suggests the use of probiotic treatment in allogeneic stem cell extensive cGVHD into moderate (less than 4 organs involved transplantation. and/or Karnofsky index equal/more than 70%) or severe. Cases were defined as standard risk pts if the diagnosis was acute leukaemia (AL) in first remission, chronic myeloid P555 leukaemia in first chronic phase and chemosensitive multiple Salvage therapy with etanercept for patients with severe myeloma. All the remaining pts were considered as high risk acute and chronic GvHD patients. The overall incidence of cGVHD was 62.5%; 61.5% F. Locatelli, A. Busca, A. Dall'Omo, F. Sizzano, C. Ceretto, S. of such cases developed the extensive type. Median lines of Aliberti, E. Lovisone, M. Falda Ospedale San Giovanni Battista therapy were 1 for limited, 2 for extensive-moderate and 4 for (Turin, I) severe cGVHD; however, failure rate has been 78% and the probability of immunosuppression discontinuation has been GVHD is a major concern in allogeneic HSCT and the major less than 30% at 5 yrs for extensive cGVHD pts. Survival was risk factor of transplant-associated morbidity and mortality. affected by cGVHD (5yrs OS has been 92% for limited Etanercept is a recombinant human soluble tumor necrosis cGVHD, 76% for cGVHD extensive-moderate, 70% for no factor (TNFa) receptor fusion protein that inhibits TNFa, a cGVHD, 53% for extensive severe). major mediator in the pathogenesis of GVHD. TRM probability for the entire series was 25.7%, resulting Purpose: to evaluate the safety and efficacy of salvage lower, although not statistically, in the standard risk group Etanercept therapy in patients who have failed multiple (19% vs 27%, p=0.106). TRM was 50% at 5 yrs in pts with combinations of immunosuppressive drugs. extensive-severe cGVHD whereas it was lower than 10% in all Methods: a retrospective evaluation of 11 patients who the remaining patients. received Etanercept as salvage therapy for treatment of

S142 On the contrary, the actuarial relapse probability was clinical GvHD and has been used to investigate the significantly influenced by the phase of the disease (14.5% at pathophysiology of the disease. This model remains unique as 3 years for standard risk vs 50.5% for high risk, p=0.0029) and a highly predictive assay utilising only human tissue. The no significant differences were found among pts with or model involves sensitising donor lymphocytes with recipient without GVHD. lymphocytes in vitro in a primary mixed lymphocyte reaction We conclude that severe cGVHD negatively affects outcome and then evaluating the secondary response on recipient skin after PBSC transplantation because it has more impact on biopsies by grading the graft-versus-host reactivity (grades I- TRM than on relapse. IV) histopathologically using the Lerner grading system for GvHD. Treatment of responder cells with PUVA has shown down-regulation of graft-versus-host reaction in 26 out of 28 P557 cases (p<0.001). PUVA treatment of the responder cells Decreased graft-versus-host disease in cord blood induces high rates of apoptosis and inhibits proliferation that is transplantation is due to both decreased stimulatory also seen in patient’s cells treated with ECP. GvHD is a T cell effects and deviation of response by dendritic cells mediated disease. ECP has been found to have marked N. Naderi, A. Pourfathollah, K. Alimoghadam, A. clinical responses despite less than 5% of circulating Ghavamzadeh, S. Moazeni Hormozgan University of Medical lymphocytes being treated at each visit, and thus ECP is Sciences (Tehran, IR); Tarbiat Modares University (Tehran, postulated to have immunomodulatory effects. Similarly in the IR); Tehran University of Medical Science (Tehran, IR) skin explant model, downregulation of the GvHR was seen in 6 out of 9 experiments when PUVA treated donor responder Dendritic cells (DCs) are the most potent stimulators of cells were combined with untreated responder cells (p=0.026). primary T cell responses and have a pivotal role in eliciting This unique human model will allow further investigation of distinct T helper cell responses. Following allogenic cord blood cytokine and cell mediated changes that may help us to (CB) transplantation, the incidence and severity of acute graft understand the mechanism of ECP and develop this exciting versus host disease (GVHD) is low. To determine the new therapy. mechanism, both CB and adult peripheral blood (PB) Fig1 – Skin explant – No treatment of responder cells (grade mononuclear cells (MNC) and DCs were compared. III GvHR – characterised by clefts at the dermo-epidermal Mononuclear cells (MNC) were separated from PB and CB junction) and fresh peripheral blood DCs (PBDCs) were enriched as a Fig2 – Skin explant – PUVA treatment of responder cells HLA-DR+ cell population, lacking the CD3, CD11b, CD14, (grade I GvHR – very little keratinocyte damage of the CD16, CD19, CD56 by magnetic bead depletion. For cord epidermal basal layer with no other discernable abnormality) blood DCs (CBDCs) enrichment, CD34 and CD66b monoclonal antibodies were added to the above cocktail. Irradiated MNC and linage- enriched PB/CB dendritic cells were co-cultured with allogeneic adult T lymphocytes for five days and T cell proliferation was measured using 3H- thymidine incorporation and ELISA method was used for cytokine measurements. The obtained results show that both cord blood MNCs and DCs were significantly poor stimulators of the mixed leukocyte reaction compared with PBMCs and PBDCs (P<5%). CB MNCs and DCs both induced significantly less IFN-gamma production than PB MNCs and DCs (P<0.05). Although T cells co-cultured with CBDCs were produced more IL-4 but the differences were not significant. These results show that CBDCs predominantly resemble DC2 in stimulating T cells and induce a biased response against Th1, which could be an important element in low incidence as well as milder severity of acute GVHD after CB transplantation.

P558 Development of the human skin explant model of graft- versus-host disease for the investigation of the mechanism of action of extracorporeal phototherapy S.R. Marshall, X.N. Wang, C.J.J. McCarthy, G.J. Jackson, A.M. Dickinson University of Newcastle Upon Tyne (Newcastle Upon Tyne, UK)

Graft-versus-Host disease (GvHD) remains the most serious complication following haemopoietic stem cell transplantation, with an incidence of 40-60% and can be fatal in up to 50% of cases. Extracorporeal phototherapy (ECP) is a novel treatment of both acute and chronic GvHD involving psoralen and UVA (PUVA) treatment of peripheral blood cells with high reported success even in those resistant to conventional immunosuppressive treatments. ECP appears to induce selective immune suppression without increased rates of infection or disease relapse. It is well tolerated by patients with minimal side effects and therefore is emerging as a highly desirable therapy. However, its mechanism of action remains poorly understood. In our department the human skin explant model for GvHD has been shown to be highly predictive of

S143 P559 SPECTRA. Flow Cytometry determined the CD3+ and CD34+ Down-regulation of interleukin-13 by PUVA treatment in a cell content of products. Indications for HSCT included Acute skin explant model of graft-versus-host disease may Leukemia (5 ALL, 13 AML), Chronic Leukemia (15 CML, 4 highlight the importance of this cytokine in the CLL), Lymphoma (3 Hodgkin's, 27 NHL), MDS (14), Multiple mechanism of extracorporeal phototherapy Myeloma (8) and Non-Malignant Diseases (9). All received S.R. Marshall, X.N. Wang, C.J.J. McCarthy, G.J. Jackson, myeloablative preparative regimens containing busulfan A.M. Dickinson University of Newcastle Upon Tyne (Newcastle combined with either cyclophosphamide or fludarabine and Upon Tyne, UK) ATG. GVHD prophylaxis was CSA beginning on day -3 (2.5mg/kg iv q12hr adjusted to target trough level of 250ng/dl) Graft-versus-Host disease (GvHD) remains the most serious and methotrexate (10mg/m² d1 and 5mg/m² d3,6,16 and Qwk complication following haemopoietic stem cell transplantation, through d100 or until onset of GVHD). Methylprednisolone with an incidence of 40-60% and can be fatal in up to 50% of was substituted for methotrexate in 7 pts and FK 506 for CSA cases. Extracorporeal phototherapy (ECP) is a novel in 9 pts at some point in their course. Rounding T cell doses to treatment of both acute and chronic GvHD involving psoralen the nearest integer x108, the modal T cell dose was 2x108 and UVA (PUVA) treatment of peripheral blood cells with high CD3+ /kg with a distribution skewed toward higher doses. reported success even in those resistant to conventional Outcomes comparison of high versus low T cell dose pts used immunosuppressive treatments. ECP appears to induce a cut-off of 4x108 CD3+/kg. The high dose pts were aged 19- selective immune suppression without increased rates of 65 (med 48) with 34/64 mis-matched donors, 17 of which were infection or disease relapse. It is well tolerated by patients with female to male. The low dose pts were aged 19-68 (med 44) minimal side effects and therefore is emerging as a highly with 15/34 mis-matched donors, 9 female to male. ASBMT desirable therapy. However, its mechanism of action remains relapse risk was significantly higher (p=0.004) in the high T poorly understood. An in vitro human skin explant model for cell dose pts. The low dose pts had a median survival of 474 GvHD has been used to study the role of cytokine changes days as compared to 162 days for the high dose pts (p=.04) induced by ECP. The model involves sensitising donor due higher early relapse rates(p=.03) in the high dose pts. lymphocytes with recipient lymphocytes in vitro in a primary Over all, the G-CSF/DEX mobilized products appeared to mixed lymphocyte reaction and then evaluating the secondary cause less than expected aGVHD. With follow up for survivors response on recipient skin biopsies by grading the graft- from 6-84 mo (med 34), the cumulative probability of versus-host reactivity (grades I-IV) histopathologically using developing aGVHD and cGVHD is shown below. There was the Lerner grading system for GvHD. The assay has been no difference in the incidence of cGVHD between groups while proved to be superior to other assays used to predict clinical the difference in aGVHD between the high and low dose pts GvHD and has been used to investigate the pathophysiology trended toward significance (p=.156,logrank). High T cell dose of GvHD. Skin explant supernatants were collected and stored products may increase aGVHD but appear to have no effect at –80 C and measured in batches using a fastquant multiplex on cGVHD rates. Th1/Th2 cytokine assay. In 9 experiments where PUVA treatment of responder cells caused down regulation of GvHR in the skin explant model, statistically significant downregulation of IL-13 (p=0.05) was demonstrated. Downregulation of IFN-g (p=0.068) and IL-5 (p=0.185) was also noted but did not reach statistical significance. No changes of IL-6, IL-1b, IL-10, IL-2, IL-4 or TNFalpha levels were demonstrated. Further evidence for the importance of IL- 13 was highlighted in 3 skin explant assays where no downregulation of GvHR was seen with PUVA treatment and IL-13 levels remained high. Jordon et Al (Blood Jan2004) have P561 documented the association between IL-13 production and the The presence of CCR5delta32 allele in the recipients and a incidence of acute GvHD. Our data adds further evidence to lower frequency of CD26 positive cells decrease the risk challenge the notion that acute GvHD is purely a Th1-type of aGvHD after allogeneic haematopoietic stem cell cytokine response and that there may be a significant link transplantation between the Th2-type cytokine IL-13 and acute GvHD. K. Bogunia-Kubik, D. Dlubek, A. Lange Institute of Furthermore, downregulation of IL-13 by PUVA may be Immunology and Experimental Therapy (Wroclaw, PL); Lower important in the control of GvHD. More skin explant assays will Silesian Center for Cellular Transplantation (Wroclaw, PL) be performed to substantiate this provisional finding. The recent literature data have suggested a potential role of chemokine receptor CCR5 in a mouse model of GvHR. These P560 prompted us to analyse if there is any association between the Impact of G-CSF plus dexamethasone mobilisation and T- polymorphism of CCR5 encoding gene and aGVHD in the cell dose on outcomes in MRD blood stem cell transplants recipients of allogeneic haematopoietic stem cell transplants A. Tilden, S. Gupta, W. Vaughan, K. Ashraf, L. Lamb, R. Katz, (HSCT). CCR5delta32 polymorphism (associated with the D. Salzman, M. Carabasi UAB (Birmingham, USA); USC (Los expression of a disrupt receptor) was analysed in 73 recipients Angeles, USA) of allogeneic sibling HSCT. In addition the proportions of CD26+ cells in blood were studied in 21 patients at various The use of blood stem cell (BSC) products for allogeneic time-points after transplantation. CD26 is a dipeptidyl transplant may increase the risk of GVHD due to the high peptidase IV that modifies the activity of CCR5 ligands number of T cells in these products. We previously showed including CCL5. Cleavage by CD26 converts CCL5 into a that dexamethasone (DEX) can safely be used to reduce the shorter form with a higher affinity to CCR5. number of T cells collected from BSC donors without effecting The proportions of CD26+ cells were higher during aGVHD the CD34+ cell content. We examined the impact of DEX manifestation at 4 (11.8+3.4% vs 25.0+4.9%, p=0.04) and 6- administration to donors on outcomes in 98 patients (pts) who week after transplantation check-points (15.1+4.2% vs received HLA-identical sibling BSC transplants between 6/97 27.6+5.4%, p=0.04 for CD26+ in patients lacking and having and 12/03. Donors were mobilized with GCSF (10- aGvHD II-IV grades). 16mg/kg/day x5) and DEX 10mg/m²/day x3 (day -2 and -1 po, and day 0 iv) followed by BSC collection on a COBE

S144 We also found, that patients with a defective gene, carrying demonstrate an immature dendritic cell (DC) phenotype and the CCR5delta32 allele had less frequently aGVHD as avidly phagocytose the apoptotic lymphocytes. The compared to patients lacking this complication (2/11 vs 30/62, subsequent antigen-processing mechanism is believed p=0.06). responsible for generating the cytotoxic anti-clonal response, These results may suggest an association between the CD26 which targets other clonal T cells. These observations have expression, CCR5 gene polymorphisms and the risk of led to a suggestion that ECP may be more effective if treated aGvHD. Taking together, higher expression of dipeptidyl cells were incubated overnight prior to re-infusion. However, peptidase IV - CD26 associates with an increased risk of significant levels of T cells rapidly become apoptotic when aGvHD while the presence of a defective CCR5 lowers tested ex-vivo and, independent of apoptosis induction, down- susceptibility to this complication. regulate pro-inflammatory cytokine production. The aim of this This work was partially supported by ALLOSTEM grant. study was to determine the immediate effects of ECP on some significant markers of activation and co-stimulation on both monocytes and T cells. P562 Pre and post ECP samples from 14 patients, receiving ECP Vitamin D receptor polymorphism associates with the therapy, were tested for various activation and co-stimulatory development of acute GvHD after allogeneic markers. The monocytes were evaluated for CD91 and CD36 haematopoietic stem cell transplantation expression, whilst the expression of CD25, CD28, CD62L, K. Bogunia-Kubik, P. Middleton, J. Norden, A. Dickinson, A. CD152 and CD154 were determined on T cells. The pre and Lange Institute of Immunology and Experimental Therapy post samples were compared statistically to determine change (Wroclaw, PL); University of Newcastle Medical School and both the pre and post samples were compared to 14 (Newcastle upon Tyne, UK); Lower Silesian Center for Cellular age/sex-matched controls. Monocyte expression of CD36 Transplantation (Wroclaw, PL) increased immediately post ECP (p=0.027), whilst the percentage of CD25+ (p=0.021) and CD28+ (p=0.036) T cells Recently, Middleton et al. (2002) reported the association fell significantly. between VDR polymorphisms and aGvHD and survival after CD36 is an important receptor in the uptake of apoptotic allogeneic sibling HSCT. In the present study the association material and its up-regulation post ECP may be beneficial in of VDR polymorphisms in patients and donors of allogeneic the immunomodulatory mechanism proposed for ECP. HSCT (36 sibling and 19 alternative transplants) was revisited However, the lack of any increase in activation or co- in order to assess whether the relationship could be also stimulatory markers would indicate that ECP-treated T cells observed in Polish patients treated with vit. D. VDR ApaI, TaqI play no role in a subsequent immunomodulatory role, other and FokI alleles were typed using SSCP. than that of immunogenic material. In addition, the reduction of Patients with ApaI aa more frequently developed grades II-IV CD25 and CD28 would be greatly beneficial in other T cell aGvHD as compared to those carrying A allele (0.60 vs 0.24, mediated conditions treated by ECP, such as Graft versus p=0.033). This association was seen in patients treated with Host Disease (GvHD). These conditions are perpetuated by CsA alone (0.50 vs 0.10, p=0.033) with a non-significant excess T cell activation. prevalence in those receiving multi-agent GvHD prophylaxis (0.29 vs 0.18) and transplanted with sibling (0.29 vs 0.08) or alternative (0.50 vs 0.22) donors. A strong tendency was P564 observed towards the higher risk of aGvHD among patients Cyclosporine A (CsA) 2 hour-concentration after oral with FokI FF (0.36 vs 0.13, p=0.074) in both sibling (0.25 vs intake varies between patients without correlation to 0.07) and alternative (0.50 vs 0.25) settings. No significant graft-versus-host disease after allogeneic haematopoietic correlation was observed for VDR polymorphisms and the stem cell transplantation overall incidence of chronic disease or survival. Logistic L. Barkholt, Y. Böttiger, H. Bodegård, M. Elbander, J. Aschan, regression analyses were performed to assess the impact of Z. Hassan, K. LeBlanc, J. Mattsson, O. Ringdén Karolinska VDR alleles. The following factors were considered: recipient University Hospital (Stockholm, S) age, F-M transplantation, conditioning regimen, diagnosis, transplant material, GvHD prophylaxis, and recipient VDR Background: Allogeneic hematopoietic stem cell ApaI aa or FokI FF genotypes. Multivariate analyses proved transplantation (HSCT) is still burden by graft-vs-host disease the associations of F-M transplantation (OR=6.21, p=0.039) (GVHD) and infectious complications due to initial immature and ApaI aa (OR=4.43, p=0.077) (analysis I), and immunity and prophylactic immunosuppression against GVHD. myeloablative treatment (OR=7.84, p=0.080) and FokI FF In kidney, heart and liver transplant patients, the risk of acute (5.76, p=0.032) (analysis II), with an increased risk of aGvHD. graft rejection was decreased by dosing CsA based on Thus our present study confirmed the previously reported concentrations obtained 2 hours after oral intake (C2). relationship between the ApaI A allele (associated with low Objective: To study correlation between the incidence of acute VDR activity) and the decreased risk of aGvHD. In variance to GVHD (aGVHD) and C2 concentrations. the former study aa homozygosity but not single a allele Material and methods: Sixty patients were monitored biweekly constituted a risk factor. Moreover, a novel association of between Jan 2001 and June 2004 for through level of CsA recipient FokI FF genotype and the increased aGvHD risk was (C0) and C2 during the first admission of HSCT. Patients with found. minimum 2 C0 and C2 pairs (n=47; 14 with HLA identical and 33 with matched unrelated donors (MUD)) were further evaluated for correlation with acute GVHD <=3 months P563 postHSCT. AUC (area under curve) concentrations were Extracorporeal photopheresis up-regulates CD36 analysed in 25 patients. CsA was given to reach the C0- expression on monocytes and reduces the number of T- through level of 100 and 250-300 ng/mL in patients with HLA cells positive for CD25 and CD28 identical donor and MUD, respectively. J. Bladon, P.C Taylor Rotherham General Hospital Results: The incidence of aGVHD >=grade 2 was 4/14 (29%) (Rotherham, UK) and 6/33 (18%) in patients with HLA donor or MUD, respectively (NS). The median C2 was 688 (114-1546) ng/mL The beneficial effects of Extracorporeal Photopheresis (ECP) and 696 (range 121-2512) ng/mL in patients with aGVHD in clonal T cell disorders are thought to involve the activation >=grade 2 and those with GVHD=grade 2 and HLA

S145 donor or MUD, respectively. The median C2 was 566 (range ATG binding epitopes has been identified. The mechanisms of 121-1217) ng/mL and 565 (135-2512) ng/mL in patients with cytotoxic activity induced by ATG include complement aGVHD=grade 2 when C0 guided the CsA dosage. subpopulations of PBMNC by PCD phenotype and inhibition Interindividual variations in CsA uptake and metabolism may patterns. explain the wide variation of C2. Methods and Results: Subpopulation of PBMNC were sorted by magnetic antibody cell sorting. For T-cells we used anti- CD3, for B-cells anti-CD19, for NK cells anti-CD56 and for P565 monocytes anti-CD14. For binding studies cells were New clinical grading system for chronic GVHD predicts incubated with ATG Fresenius at 20µg/ml, then labeled with duration of systemic immunosuppressive treatment, FITC anti rabbit IgG. Binding was detected by fluorometrie. GVHD-specific and overall survival For biological activity cells were incubated with ATG in S. Kim, K. Kim, J. Rhee, S. Lee, J. Kang, K. Park, J. Lee, W. increasing doses for 24 hours. Cell death was then assessed Kim, C. Jung, M. Lee, K. Park Samsung Medical Center, by Annexin/PI doublestaining. Caspase 3 activity was Sungkyunkwan University (Seoul, KOR) assessed by intracytoplasmatic staining for active Caspase 3 using a fluorochrome coupled antibody against human active We investigated outcomes according to a new clinical grading Caspase 3 fragment. system for chronic GVHD in 38 patients who suffered chronic Furthermore, a wide variety of caspase and serine protease GVHD after an allogeneic hematopoietic stem cell inhibitors was added to sort for candidate protesases transplantation (HSCT). Thirty-four out of 125 patients who mediating the cell death. underwent allogeneic bone marrow transplantation and 4 out We found that all subpopulations strongly bind ATG. Further of 8 patients who underwent allogeneic peripheral blood all subpopulations undergo PCD in a dose dependent manner. transplantation suffered chronic GVHD. The phenotype of PCD was mainly Annexin/PI double positive, We reviewed medical records for the identification of three risk thus resembling secondary necrosis. Caspase 3 activity was factors (RFs): extensive skin involvement (ESI), low, and inhibition had no effect on cell death. Neither thrombocytopenia (TP), and progressive type of onset (PTO). granzyme B nor broad serine protease inhibition could We categorized the patients into three grade groups: grade 1 abrogate cell death. (0 RF), grade 2 (1 RF or TP+PTO), grade 3 (2-3 RFs). Sixteen Discussion: We found a strong binding capability of ATG to all patients were classified into grade 1, 19 into grade 2 and 3 PMNC subpopulations. Further, when looking at biological into grade 3. activity we found ATG to induce PCD in all subpopulation The median onset date of chronic GVHD was 186.5 d (range independent of complement. The phenotype of cell death was 100-635), and the median duration of systemic of secondary necrosis. Neither caspase nor serine protease immunosuppressant (IST) for chronic GVHD was 270 d (range inhibition could abrogate cell death. This implicates a different 16-1714). The probability of withdrawal of systemic IST at 1, 2 mechanism than apoptosis being involved. It has to be further and 3 years was 61%, 76% and 87%, respectively. The investigated if recently described mechanisms of autophagy patients with grade 2 or 3 chronic GVHD had prolonged play a role in ATG induced PCD. duration of systemic IST (p=0.034). Analysis based on a In conclusion the anti GvHD activity of ATG might not be multivariate model showed that chronic GVHD grade was an limited to T-cell depletion but also to depletion of NK-cells and independent factor in the duration of IST (p=0.043). antigene presenting cells by programmed cell death. The probability of GVHD specific survival (GSS) at 5 years was 52%. Analysis based on a multivariate model showed that prior occurrence of acute GVHD (p=0.001), chronic GVHD P567 grade (p=0.004), serum bilirubin over 1.5mg/dl (p=0.049) and Correlation between chimerism analysis with STR-PCR transplantation-risk factor (p=0.003) were independent method and acute GVHD after haematopoietic cells prognostic factors in GSS. transplantation Twenty-two of 38 patients with chronic GVHD were still alive A. Bassi, A. Mousavi, H. Ghaffari, K. Alimoghaddam, A.A. and the estimated 3-year overall survival (OS) rate for the Hedayatiasl, B. Chardouvali, A.R. Shamshiri, M. Iravani, B. whole chronic GVHD patients was 60%, while that for the Bahar, F. Toutounchian, M. Jahani, A. Ghavamzadeh, H. group with chronic GVHD grade 1 and grade 2+3 was 64 and Ghaffari, A.R. Shamshiri Tehran University of Medical 48%. Analysis based on a multivariate model showed that Sciences (Tehran, IR) prior occurrence of acute GVHD (p=0.001), chronic GVHD grade (p=0.002), serum bilirubin over 1.5mg/dl (p=0.015) and The development of GVHD represents a major determinant of transplantation-risk factor (p=0.019) were independent outcome after Allo-HCT. Donor T cells that recognize recipient prognostic factors in OS. alloantigen initiate this complication. Thus percent of donor T- In conclusion, new clinical grading system for chronic GVHD cell in recipient should be correlate with incidence and severity predicts duration of systemic immunosuppressive treatment, of GVHD. Molecular monitoring of host/donor GVHD-specific and overall survival. lymphohematopoietic cells in the post transplant period could be useful in predicting the occurrence of GVHD. Patients who candidate for Allo-HCT were included in this P566 study. Preparative regimen and GVHD prophylaxis Rabbit anti-thymocyte globulin induces cell death in administered according to disease specific protocol in PBMNC subpopulations by secondary necrosis transplant ward. Patients were evaluated on days +15, +30 C. Grüllich, C. Ziegler, J. Finke University of Freiburg and +60 after HCT for chimerism analysis with STR-PCR (Freiburg, D) method and observed for GVHD manifestation until day +100 after HCT. Correlation of incidence and severity of acute Introduction: Rabbit Anti-Thymocyte Globulin (ATG) is widely GVHD with chimerism analysis (Whole blood, T-Cell , PMN) used as GvHD prophylacsis in allogenic bone marrow on days +15, +30 and +60 after HCT was assessed with transplantation. Cytotoxic activity of ATG against T- statistical method. lymphocytes leads to a in vivo T-cell depletion. From 58 patients analysed,21 patients (36.2%) were female ATG is generated by immunizing rabbits with Jurkat proteins and 37 patients(63.8%) were male. Median age was 19.5 resulting in a polyclonal antibody. Hence, a wide variety of years (range 2-51years). Patients were 18 thalassemia, 15

S146 AML, 10ALL, 4CML, 6AA, 2 breast cancer, 1 CLL, 1 group. The difference was not statistically significant (chi2 test: histiocytosis x and 1 renal cell carcinoma. The most common p = 0,52; OR: 0,58; 95% CI: 0,11-3,06). regimens for HCT were cyclophosphamide + busulphan and Conclusion: Our data do not confirm any statistical association fludarabin + busulphan. Thirty-six (62%) patients had aGVHD of acute or chronic GVHD with TNF-alpha-308 polymorphism in first 100 days after HCT. Patients with full chimerism of in this 37 children study group. whole blood in day 30 after HCT had more aGVHD (p.value:0.003) and more severe aGVHD (p.value:0.01) than patients with mixed chimerism. Patients with full chimerism of P569 T-cells in day 30 after HCT had more aGVHD (p.value:0.03) Mycophenolate mofetil AUC targeting for GvHD than patients with mixed chimerism. Patients with full prophylaxis after allogeneic haematopoetic stem cell chimerism of whole blood in day 60 after HCT had more transplantation aGVHD (p.value:0.039) than patients with mixed chimerism. J. Freiberg-Richter, A. Jenke, S. Pursche, M. Bonin, E. Patients with full chimerism of T-cells in day 60 after HCT had Schleyer, G. Ehninger, M. Bornhäuser University Hospital more aGVHD (p.value:0.03) and more severe aGVHD (Dresden, D); KH Merseburg (Merseburg, D) (p.value:0.038) than patients with mixed chimerism. After multivariate analysis with binary logistic regression in our Purpose: Mycophenolate mofetil (MMF) is an effective study we find that chimerism is affected with fludarabine in immunosuppressive drug and well established to prevent graft conditioning regimen. Incidence and severity of aGVHD is rejection in solid organ transplantation. Studies in recipients of affected by both whole blood Chimerism in day 30 and type of solid organ grafts have shown that MPA trough plasma levels underlying disease. are not sufficient to predict the effectiveness of therapy. With univariate and multivariate analysis we conclude that Further investigations provided some evidence that a MPA- occurrence of acute GVHD after HCT is affected by patterns of AUC0-12 (area under concentration-time curve) between 30- whole blood and T-cells chimerism in day 30and 60, type of 60 µg/ml*h are necessary for effective immunosuppression in conditioning regimen and type of underlying disease. renal transplantation. In contrast, studies by our group demonstrate low MPA plasma levels in the early phase after conditioning therapy and HCT. Most likely as a result of this a P568 high rate of acute and hyperacute GvHD was observed. TNF-alpha-308 single nucleotide polymorphism was not Methods: We launched a study for patients with MDS or AML associated with graft-versus-host disease after allogeneic receiving conditioning with fludarabine and busulfan and haematopoietic stem cell transplantation in children hematopoetic stem cells from HLA-compatible siblings or I. Bodova, J. Lukac, J. Horakova, S. Sufliarska, K. unrelated donors using a combination of tacrolimus and MPA- Jancovicova, L. Kovacs Comenius University Medical School AUC targeted iv MMF as GvHD prophylaxis. The target range (Bratislava, SVK) of MPA-AUC0-12 was 30-45µg/ml*h. 12 h AUC measurements were performed once a week on three Introduction: GVHD remains the most common complication occasions. For detection of MMF and MPA we used an after allogeneic bone marrow transplantation, which increases isocratic RP-HPLC-system with Fluorescence-Spectroscopy. its morbidity and mortality. TNF-alpha is one cytokine known MMF doses were adjusted in steps of 500 mg/d calculated by to play a major role in the early phase of acute GVHD. It has taking into account the relative difference between measured been reported that elevated TNF-alpha levels during pre- and target MPA-AUC0-12. transplant conditioning are associated with acute GVHD. Results: Preliminary data in eight patients indicate that Cytokine gene polymorphisms associated with increased targeted dose adjustment (up to 5g/d) is feasible to achieve production of this cytokine have been identified. There is a therapeutic MPA-AUC (median MPA-AUC0-12 after second single base polymorphism TNF in position-308 (G/A). dose adjustment = 39 µg/ml*h).No severe side-effects were Patients and methods: In the present study we evaluated the observed with maximum doses of up to 4.5 g/day. 3 pts had association of TNF-alpha single nucleotide polymorphism grade II-III (CTC) gastrointestinal symptoms. Acute (G/A) and acute or chronic GVHD in 37 patients, median age extramedullary toxicity seems to be less compared to standard 11 years (range 0,8-17,6), after BMT for malignant (19 pts.) prophylaxis with CSA/MTX. After Tx with related donor the and non-malignant (18 pts.)diseases. Twenty-four male and rate of significant GvHD (>=grade II) is 35% and in the 13 female patients underwent non-T-cell depleted allogeneic unrelated setting the rate of GvHD (>=grade II) is 55%. stem cell transplantation from HLA-identical sibling (32 pts.) or Conclusion: MMF adjusted according to MPA-AUC seems to 10/10 matched unrelated donor (5 pts.), median age 10,1 be a feasible way to achieve therapeutic drug levels, which years (range 0-56,7), after myeloablative conditioning might result in effective control of GvHD and are tolerated well regimen. All but one patient (a syngeneic donor) received by the patients. Further accrural and follow-up is necessary to GVHD prophylaxis: CS-A alone in 12 patients, CS-A + MTX in confirm the feasibility and efficacy of this approach. 20 patients and CS-A + MTX + ATG in 4 patients. TNF polymorphism was analysed by PCR amplification followed by digestion with Ncol restriction enzyme. There were 120 volunteers in the control group. Results: The cumulative incidence of grades II-IV acute and chronic GVHD was 32% and 29% respectively. Incidence of risk cytokine gene genotypes TNF-alpha-308 (G/A) were similar in control group and in patients (33% vs. 22%), chi2 test: p = 0,26. In 32 sibling transplants there were 26 D/R pairs with TNF-308 G/G and 6 D/R pairs with G/A TNF genotype. From all 37 patients TNF-308 genotype G/A was present in 8 pts. (22%) and wild type G/G genotype in 29 pts. (78%). Acute grade II-IV GVHD developed in 3 pts. (38%) in G/A group while in 9 pts. (31%) in G/G group. The difference was not statistically significant (chi2 test: p = 0,72; OR: 0,75; 95% CI: 0,14-3,82). Three patients died before day 100. For chronic GVHD 35 pts. had been evaluated. Three patients (38%) developed chronic GVHD in G/A group vs. 7 (26%) in G/G

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P570 Patients: All adult pts with lymphomas, acute and chronic Measurement of circulating endothelial cells in patients leukemias, in CR after auto-SCT or allo-SCT performed 1986 with chronic graft-versus-host disease to 2002 in Lund and Göteborg, see table. S. Buchholz, A. Woywodt, M. Stadler, T. Weihkopf, E. Methods: A questionnaire was filled out by all allo- and auto- Dammann, H. Haller, M. Haubitz, A. Ganser, B. Hertenstein SCT pts. 88 % of allo-SCT pts and 90 % of auto-SCT pts Hannover Medical School (Hannover, D) agreed to participate. Their dystonic symptoms were classified in three categories: 1. “The dystonia syndrome”, defined as muscular dystonias or cramps in >1 locations, at least weekly, Chronic GVHD (cGVHD) is a major cause of transplant-related not provoked by strenuous physical activity; 2. Unspecific morbidity after allogeneic stem cell transplantation. Vascular dystonias, including nocturnal muscle cramps, 3. No dystonic injury and changes in vascular endothelial cells have been symptoms. described in patients with cGVHD. Recently we demonstrated In addition, standardised telephone interviews were carried out that circulating endothelial cells (CEC) are an indicator for in 138 allografted pts for further evaluation. endothelial damage in correlation to the conditioning regimen Results: The prevalence of any sort of cramp was similar in in 39 patients (Woywodt et al., Blood, 2004 May 1; 103 (9): the allo- and auto-transplanted group (73% vs 63%, p=.09). 3603-5). However, there was significantly more pts with “the dystonia In this study CEC were measured in 20 patients with cGVHD syndrome” in the allo-transplanted group (52% vs 26%, (16 male, 4 female; median age 42.5, range 19-62). Onset of p<.0001). The following factors were associated with “the cGVHD was in median 297 days after transplantation (range dystonia syndrome” in the allo-transplanted group; occurrence 103-851) (17 patients), 3 patients developed cGVHD (day 101, of chronic GvHD (p=.0009), ongoing immunosuppression at 278, 473) after donor lymphocyte infusion. CEC were isolated time of survey (p=.009), previous acute GvHD grade II or more from peripheral blood by use of Dynabeads coated with (p=.02) and female gender (p=.05). antibodies against CD 146, were stained for UEA-1 and Conclusion: Our results suggest that dystonias after allo- counted. transplants are surprisingly prevalent manifestations of GvHD. The skin was involved in cGVHD in 19 patients, 9 patients additionally had signs of fasciitis. 11 patients showed elevated liver enzymes as sign of liver involvement. 5 patients suffered from pulmonary GVHD (bronchiolitis obliterans) when CEC were measured. At time of measurement of CEC patients were treated with steroids (17 patients), in combination with CsA (9 patients), tacrolimus (1 patient) or rapamycine (7 patients), 1 patient additionally received MMF. Numbers of CEC were elevated in patients with cGVHD (median 40 CEC/mL), but showed a wide range (8-1000 CEC/mL). CEC were significantly elevated compared to 17 age-matched healthy controls (median 8/mL, range 4-24/mL, p<0.001; Mann-Whitney-U-test). In 8 patients with active cGVHD numbers of CEC were within normal range (8-20/mL), but 5 of them still underwent therapy with CsA at this time. There was no significant correlation between number of platelets, transaminases, gGT or AP and number of CEC (t- test) and patients with pulmonary involvement of cGVHD showed higher numbers of CEC (median 84/mL, range 48- 1000/mL) compared to patients with localized skin involvement or hepatic dysfunction (median 20/mL, range 8-254/mL, p=0.007). P572 Our data indicate that CEC may serve as a novel marker of The incidence of acute GvHD is diagnosis-dependent and microvascular endothelial damage in patients with cGVHD, may reflect the intensity of preceding chemotherapy especially in patients with pulmonary cGVHD. Further J. Wojnar, J. Holowiecki, S. Giebel, M. Krawczyk-Kulis, M. prospective studies will be required to investigate larger Markiewicz, B. Stella-Holowiecka, T. Kruzel, I. Wylezol, K. numbers of patients and to evaluate potential correlations Nowak Silesian Medical University (Katowice, PL) between numbers of CEC and manifestations and intensity of cGVHD. The goal of this study was to determine factors associated with the incidence of acuteGvHD taking into account patient and donor characteristics, disease, and factors associated with the procedure. P571 330 recipents of alloHCT from an HLA-identical sibling Dystonias after allogeneic stem cell transplantation are (n=223) or matched unrelated volunteer (n=107) performed in associated with GvHD a single institution (Katowice, Poland) between 1992-2003 P. Wernstedt, A. Kisch, S. Lenhoff, A. Lindmark, J. Dykes, M. were included in the analysis. Median recipient age equaled Hardling, M. Brune Sahlgrenska University Hospital 29 (10-56) years. The diagnosis was as follows: CML -136, (Gothenburg, S); Lund University Hospital (Lund, S) AML -82, ALL -70, SAA- 13, MDS -12, NHL -11, other -6. All patients were treated with myeloablative conditioning based Background: We have previously reported the occurrence of on chemotherapy (n=279) or TBI (n=51). Bone marrow served muscular dystonias - painful, sometimes incapacitating as a source of stem cells in 290 cases, peripheral blood – in skeletal muscle contractions in various locations - in 40 patients. GvHD prophylaxis consisted of cyclosporin A 3 allografted patients (pts). This survey intended to further mg/kg, short-course methotrexate, prednisolone 0.5 mg/kg on investigate these symptoms. days 1-28 (244/330 patients), and ATG (URD-HCT recipients Aims. To describe symptoms and prevalence of dystonias only). after allogeneic stem cell transplantation (allo-SCT) and to The cumulative incidence of acute GvHD grade II-IV and III-IV elucidate if there is a “dystonia syndrome” associated to varied and depended on the diagnosis: MDS - grade II-IV - chronic Graft-versus-host disease (GvHD). 58%, grade III-IV -33%, CML -47% and 29%, AML -15% and

S148 7%, ALL -22% and 9%. Results of the multivariate analyses GVHD. Although the present results do not demonstrate any are listed in Tables 1 and 2. statistically significant difference, they show a trend towards a higher frequency of oral cGVHD among patients undergoing nonmyeloablative vs conventional SCT.

P574 Minimal GvHD after myeloablative unrelated donor stem cell transplantation using both in vitro and in vivo T-cell depletion with Campath-1H P.A. von dem Borne, F. Beaumont, I. Starrenburg, M.A. Oudshoorn, G. Hale, J.H.F. Falkenburg, W.E. Fibbe, R.

Willemze, R.M.Y. Barge LUMC (Leiden, NL); University of Conclusions: 1) Patients with MDS and CML are at higher risk Oxford (Oxford, UK) for development of acute GvHD of both grade II-IV and grade

III-IV. These subgroups are characterised by less intense T-cell depletion reduces transplant related mortality by preceding chemotherapy. In contrast, acute leukemia patients diminishing GVHD. For effective T-cell depletion we employ a receive induction-consoliadation regimens including myeloablative regimen for matched unrelated donor SCT antracyclines and high-dose cytarabine. We speculate that this using both in vivo and in vitro Campath. DLI is used after at a kind of treatment may reduce the number of residual dendritic later time point for graft versus tumor effect if necessary. cells of the host that, in turns, are essential for the initiation of Thirty patients (median age 33 years, range 18-48) were GvHD reaction. 2) Prophylactic use of prednisolone does not transplanted from 1997 to 2002. Diagnoses were: CML prevent aGVHD. Moreover, it increases the risk of severe form chronic phase (n=8), CML accelerated phase (n=2), CML blast of this complication, probably – by inducing steroid-resistance. crise (n=2), AML (n=7), MDS (n=2), ALL (n=7), NHL (n=1) and

Fanconi anemia (n=1). Six patients had one HLA mismatch,

the others were identical. Conditioning consisted of Campath P573 i.v. with TBI and cyclofosfamide. In vitro T cell depletion was Oral chronic graft-versus-host disease following performed by adding Campath to the graft (Campath in the allogeneic conventional myeloablative versus non- bag). Post-transplant GVHD prophylaxis consisted of myeloablative stem cell transplantation ciclosporin and methotrexate. The stem cell source was bone F. Demarosi, D. Soligo, A. Carrassi, G. Lambertenghi Deliliers, marrow in 19 patients and peripheral blood in 11 patients. I. Manusè, C. Minazzi, A. Sardella Università degli Studi di One graft failure was observed, all other patients had Milano (Milan, I) sustained donor engraftment. Acute GVHD was observed in

12 patients (maximally grade I-II skin), no severe acute GVHD Over the past decade, nonmyeloablative conditioning was experienced. Limited chronic GVHD developed in 2 regimens have been designed to reduce toxicity and allow patients, resolving after treatment. Only in one patient stem cell transplantation (SCT) in elderly and medically extensive chronic GVHD developed, which did not resolve. compromised patients. Graft-versus-host disease (GVHD) is CMV reactivation occurred in 23% of patients, one patient the most common complication of allogeneic SCT. Oral developed CMV disease. No EBV disease was observed. Ten involvement occurs in 80% of patients suffering from chronic patients received DLI at a median of 17.4 months after SCT (9 GVHD (cGVHD). Actually, it is unknown whether GVHD may patients with relapsed CML, one patient with relapsed ALL). be different after nonmyeloablative as compared with After DLI acute GVHD grade I-II developed in 4 patients, and myeloablative SCT. Aim of the study was to prospectively GVHD grade III-IV in 3. Chronic GVHD developed in 5 analyzed the occurrence and clinical aspects of oral cGVHD in patients, of which 2 extensive, resolving in all except one patients who underwent allogeneic conventional myeloablative patient. With a median follow up of 37 months(range 21-84) 17 and nonmyeloablative HSCT. patients are alive (57%). One CML patient has persistent This prospective study consecutively enrolled adult patients molecular disease unresponsive to increasing doses of DLI. with haematological diseases undergoing HSCT between All other patients are in CR with the CML patients in molecular January 2000 and July 2004. Before the hospitalization period remission. Five patients (17%) died because of relapsed all patients received an oral examination to exclude the disease (2 AML/MDS and 3 ALL). Treatment related mortality presence of oral mucosa lesions. After transplantation, all was 26% (1 rejection, 2 GVHD, 1 myocardial infarction, 4 patients received an oral examination starting the day +100. In infections). all patients an oral biopsy specimen was obtained. Patients In conclusion, matched unrelated donor SCT following were divided in two groups based on transplant type: myeloablative conditioning using T-cell depletion with conventional myeloablative (Group 1) and nonmyeloablative Campath in vivo as well as in vitro results in good engraftment, HSCT (Group 2). The study involved 106 adult patients. We minimal GVHD and a survival of 57%. Relapse rate was not considered 34 (32.1%) and 72 patients (67.9%) were excluded increased with this strategy. This regimen appears to be for early death (+100) or because failed to attend the follow up successful for young adults with high-risk malignancies. visit. Seventeen out of 34 patients received conventional myeloablative HSCT (Group 1) and 17 underwent nonmyeloablative HSCT (Group 2). The follow-up period P575 ranged from 100 to 805 days (mean: 337.6 days). The Cyclosporine-methotrexate versus cyclosporin- preliminary results show that the cumulative incidence of oral mycophenolate in the prevention of GvHD in stem cell cGVHD during the first 805 days from transplantation was transplantation using myeloablative regimens 70.6%, but it was lower among patients in the Group 1 D. Pohlreich, A. Vitek, V. Valkova, M. Markova, J. Maalouf, M. (58.8%) than in the Group 2 (82.3%) (p = 0.25 Fisher’s exact Kouba, P. Soukup, P. Cetkovsky Institute of Hematology and test). There was no significant difference between the two Blood Transfusion (Prague, CZ) groups in the clinical aspects of oral cGVHD, such as localization, lesion type and symptoms. Cyclosporine A (CSA) in combination with methotrexate (MTX) Conclusions. Few reports have systematically analyzed the is the standard combination used for GvHD prevention in incidence, severity, quality, and timing of GVHD after patients undergoing allogeneic stem cell transplantation (allo- nonmyeloablative SCT. No reports are available about oral SCT). The known side effects of methotrexate and especially

S149 its acute gastrointestinal toxicity may play a significant role in P577 increasing peri-transplant mortality. Based on the results of a Pre-engraftment syndrome following haematopoietic stem published study (Bornhauser, Blood 2000) describing the cell transplantation successful use of CSA in combination with Mycophenolate Y.-H. Lee, S.-W. Lee, K.-T. Noh, S.-H. Kim, H.-C. Kwon, J.-S. (MMF) in GVHD prevention following non-myeloablative Kim, H.-J. Kim Dong-A University Medical Center (Busan, transplant regimens, we analysed retrospectively patients KOR) undergoing matched related or unrelated allo-SCT using myeloablative regimens. We evaluated the effect of Characteristic clinical findings of fever, skin rash with or exchanging MTX for MMF (in combination with CSA) on the without the evidence of fluid retention, mimicking engraftment development of GIT toxicity and engraftment. We also syndrome, have been observed during pre-engraftment period evaluated the incidence of acute GvHD. In both groups, total in patients with hematopoietic stem cell transplantation body irradiation was used in half the patients (MMF 50% vs. (HSCT). Fifty patients were analysed retrospectively to MTX 55%). The group receiving MMF (n=26) had a establish the clinical entity, to characterize the clinical course significantly lower incidence of mucositis (30% vs. 70%) of the syndrome. Seven patients (14%) were shown to have compared to the group receiving MTX (n= 20). In the MMF non-infectious neutropenic fever accompanied by skin rash group, no severe mucositis was noted, while in the MTX with or without the evidence of fluid retention in the pre- group, grade III-IV (WHO) mucositis was recorded in 30% of engraftment period, which were not associated with patients. Duration of hospitalisation, opiate use for pain control engraftment syndrome. In 3 (23.1%) out of 14 patients with and duration of total parenteral nutrition were longer in the cord blood stem cell transplantation, non-infectious fever, skin MTX group. The average time to granulocyte engraftment and rash and tachypnea developed on 7-8 days post-transplant, the incidence of acute GvHD (MMF 32% vs. MTX 40%) did not which were 5-14 days prior to neutrophil engraftment. The differ between the two groups. pathologic findings of skin biopsy showed the metaphase This study shows that the use of MMF in combination with dermatitis, similar with the findings of acute graft versus host CSA in the prevention of GvHD in patients undergoing disease. Two of them spontaneously recovered just with fluid myeloablative allo-SCT is associated with a significantly lower restriction and oxygen inhalation, however, one patient expired incidence of mucositis and surprisingly has no effect on with complicated pulmonary hemorrhage in spite of aggressive engraftment, and is as effective in the prevention of GvHD as supportive therapy including fluid restriction and steroid standard prevention using MTX. treatment. In 4 (17.4%) out of 23 patients with allogeneic bone marrow transplantation, non-infectious fever and skin rash developed on 5-7 days post-transplant, which were 4-5 days P576 prior to neutrophil engraftment. All of them recovered with Use of sirolimus (rapamune) in the treatment of steroid- steroid treatment only. These characteristic findings were not refractory GvHD found in the patients with autologous peripheral blood stem D. Pohlreich, A. Vitek, V. Valkova, M. Markova, M. Kouba, M. cell transplantation. We could not find any risk factors for this Soukup, M. Zajickova, J. Maalouf, P. Cetkovsky Institute of syndrome, such as sex, age, body weight, incompatibility of Hematology and Blood Transfusion (Prague, CZ) blood type and HLA type, number of cells infused, use of G- CSF. However, the speed of neutrophil engraftment was Acute GvHD not responding to standard first-line therapy, so- significantly faster in the patients with pre-engraftment called steroid-refractory GvHD, is a serious problem in syndrome. The 5 year overall survival rate was not patients undergoing allogeneic stem cell transplantation (allo- significantly different in the patients with and without pre- SCT). Therapeutic second-line modalities (ATG, monoclonal engraftment syndrome. We first report the pre-engraftment antibodies) are often unsatisfactory and steroid-refractory syndrome consisting of non-infectious fever, skin rash with or GVHD remains associated with high peri-transplant mortality. without fluid retention during pre-engraftment period in Promising results have been recently published relating to the patients with HSCT. The disease entity and diagnostic criteria use of sirolimus. of pre-engraftment syndrome are obscure yet, thus more close Study outline: Sirolimus (Rapamune) was administered to 13 observation and pathophysiological research would be patients undergoing allo-SCT on adequate required. immunosuppression (CS-A, FK 506, +/- MMF) who developed acute GvHD that was resistant to steroid therapy (3x persistence, 8x progression). The initial dose of sirolimus was P578 2mg/day, with a target serum concentration of 8-12 ug/l. Impact of day +11 methotrexate on the incidence of acute Duration of administration was usually 6 weeks. and chronic graft-versus-host disease after HLA-identical Results: A therapeutic response was achieved in 6 (46%) and allogeneic peripheral blood stem cell transplantation a complete remission in 5 (38%) patients. Clinical responses S.K. Sohn, D.H. Kim, J.H. Baek, Y.R. Do, K.S. Lee, J.S. Suh, were seen in 5 (3-6) days, with a complete response by day K.B. Lee Kyungpook National University Hospital (Daegu, 14 (9-14) of treatment in patients with GvHD: skin grade. I-II KOR); Keimyung University, Dongsan Medical Center (Daegu, (4x), liver grade II (1x), GIT grade II (1x). 7 patients were KOR) resistant (54%), with GIT grade III-IV (6x) and liver grade III (1x) GvHD. 2 patients with GIT GvHD were resistant despite Background: Cyclosporine (CSA) plus 4 doses of effective plasma levels of sirolimus, 4 patients did not attain methotrexate (MTX) is the most commonly used regimen for any measurable plasma concentration. The main adverse GVHD prophylaxis. It has been previously found that the event noted was the development of a TTP-like syndrome with omission of day +11 dose of MTX was associated with dominant severe thrombocytopenia in 3 (23%) patients increasing risk of severe acute GVHD in BMT setting. requiring treatment termination in 2 patients. 4 patients died (2 However, little is known on its impact in peripheral blood stem of infection, 1 of GvHD progression, 1 of disease progression). cell transplantation (PBSCT) setting. 9 patients are alive (1.5-18 months of follow-up). Methods: In the current study, we attempted to evaluate the Conclusion: Sirolimus represents an alternative treatment of role of day +11 MTX on the incidence of acute and chronic steroid-refractory GvHD. The remaining problem is its GVHD after HLA-identical PBSCTs. The cases who died probable ineffectiveness in the treatment of GvHD affecting before day +11 were excluded. Of the 68 eligible patients, 30 the GIT. Another limitation of its use is the availability of only patients received 4 doses of MTX (MTX4), while 38 patients an oral form and its limited intestinal absorption. received less than 3 doses (MTX3) because of severe

S150 mucositis (n=34), hepatic dysfunction (n=3) or renal failure treated with several immunosuppressive medications and with (n=1). MTX. Four died (3 because of progression of scleroderma). Results: The cumulative incidence of acute grade 2-4 GVHD Eosinphilia and increased levels of LDH 1-4 months before the was 60% in MTX4 and 86% in MTX3 (p=0.04), while that of onset of the skin sclerosis, younger age, infused CD3 cell severe grade 3, 4 GVHD was 7% in MTX4 and 39% in MTX3 dose, previous extensive chronic GVHD and presence of (p=0.02). Of the 61 patients evaluated for chronic GVHD, the autoimmune markers are significantly associated with the cumulative incidence of chronic GVHD was 54% in MTX4 and development of sclerodermatous GVHD in our experience. 97% in MTX3 (p=0.001), while that of extensive chronic GVHD Early onset time, generalized skin sclerosis and higher skin was 26% in MTX4 and 63% in MTX3 (p=0.004). There were score seem to be risk factors for resistance to no differences in the overall survival (p=0.12) or relapse immunosuppressive therapy and poor outcome. incidence (p=0.83) between MTX4 and MTX3 groups. A better definition of risk factors for sclerodermatous chronic Conclusion: The current study suggested that day +11 MTX GVHD, also based on the knowledge about seemed to be associated with the development of acute and immunopathogenesis, should allow early diagnosis and better chronic GVHD. However, cautious interpretation on the result therapeutic strategies. of current study is needed because patients' clinical situation that did not allow to administering day +11 MTX in MTX3 group might predispose to the development of GVHD. P580 Cytokines dysregulation and chronic graft-versus-host disease after allogeneic haematopoietic stem cells transplantation with reduced-intensity conditioning C. Skert, D. Damiani, A. Michelutti, R. Stocchi, E. Calistri, F. Patriarca, M. Cerno, R. Fanin Ospedale Università (Udine, I)

Chronic graft-versus-host disease (cGVHD) is an important cause of late morbidity and mortality after allogeneic haematopoietic stem cells transplantation (HSCT). The unbalance of immune response toward Th1-type or Th2-type with cytokines dysregulation seems to play a key role in its pathogenesis. We assessed the serum levels of tumor necrosis factor-alfa (TNF-alfa), interferon-gamma (IFN-gamma), IL-4, IL-10, IL-6 and soluble tumor necrosis factor receptor I and II (sTNFR) in 8 healthy donors and in 12 patients undergoing allogeneic HSCT with reduced intensity conditioning. Serum levels were assessed before transplantation, weekly during the first month and monthly from second to twelfth month after HSCT. Cyclosporine A was used as immunosuppressive prophylaxis for all patients but one (mycophenolate) from third month until its tapering or until the beginning of the therapy for extensive cGVHD. Nine patients developed cGVHD (6 extensive) at a median time of 6 months (5-10). The levels of cytokines did P579 not differ significantly in patients pre-HSCT and healthy Sclerodermatous chronic graft-versus-host disease after donors. From third to seventh month, TNF-alfa levels were allogeneic haematopoietic stem cell transplantation: significantly higher in patients with cGVHD than in patients incidence, outcome and risk factors without cGVHD; the latter had significant lower TNF-alfa levels C. Skert, F. Patriarca, D. Damiani, M. Cerno, C. Filì, A. than healthy controls in the same period. One month before Geromin, A. Sperotto, R. Fanin Chair and Division of the onset of cGVHD, patients with extensive cGVHD had Haematology (Udine, I) significant higher levels of TNF-alfa, IFN-gamma and IL-4 than patients with limited cGVHD. At fourth month, IL-10 levels Sclerodermatous chronic graft-versus-host disease (GVHD) is were significantly higher in patients with than in patients often refractory to standard immunosuppressive therapy and without cGVHD and in patients with extensive than in patients life-threatening, especially in an advanced phase of disease. with limited cGVHD. From third to seventh month, sTNFR-I We evaluated its incidence, clinical characteristics, outcome and sTNFR-II levels were significantly higher in patients with and risk factors in 174 patients who underwent allogeneic than in patients without cGVHD. From sixth to eighth month, haematopoietic stem cell transplantation from related and sTNFR-II levels were significantly higher in patients with unrelated donors. extensive than in patients with limited cGVHD. Conditioning regimen included total body irradiation and These results suggest that: 1) the different cytokines could cyclophosphamide or busulfan and cyclophosphamide. have different kinetics after HSCT in relation to the Cyclosporine A and a short course of methotrexate (MTX) development of cGVHD; 2) different patterns of cytokines were given for GVHD prophylaxis. Of the 133 patients could be related to the onset and severity of cGVHD. Further surviving more than 4 months, 14 (10.5 %) showed chronic studies involving a larger number of patients in different GVHD with sclerodermatous features after a median time of transplant settings are required to confirm our results. 15 months (5-54). Previously, 12 (86%) of them had extensive chronic GVHD, 10 with skin involvement (leopard-skin eruption or lichenoid lesions); 8 patients presented myalgias and arthralgias 1-2 months before the onset of scleroderma. Skin was evaluated by the modified scleroderma skin scoring method (maximum score: 66). The median skin score was 16 (5-63). Joint contractures developed in 7 patients. Eight patients had a complete or partial remission and one a steady- state disease after first or second line therapy, which included MTX. Five patients were non responsive. Two of them were

S151 P581 ATG and complement, the mean number of myeloid and Bronchiolitis obliterans in recipients of haematopoietic plasmocytoid dendritic cells decreases from 3168 to 739 x stem cell transplantation: report of 16 cases 103/ml (p=0.004) and from 5314 to 790 x 103/ml (p=0.01), O. Arce, A. Bermudez, L. Yañez, A. Insunza, R. Perez respectively. For in vivo investigation, patients receiving pre- Montes, A. Iriondo Hospital U. Marques de Valdecilla transplant ATG given at a dose of 60 mg were included and (Santander, E) compared to those without ATG. ATG given as part of the conditioning regimen prior to allogeneic stem cell Background: We evaluated the characteristics of bronchiolitis transplantation induces a stronger reduction of circulating obliterans after undergoing bone marrow transplantation. MDC and PDC than chemotherapy alone (100% vs 80%). Methods: 16 cases of bronchiolitis obliterans (BO) in recipients These data show that ATG induces depletion of circulating of hematopoietic stem cell transplant between 1994 and 2004 myeloid and plasmacytoid dendritic cells, which might be – were retrospectively analysed (14 allogenic and 2 autologous) beside the T-cell depletion - a further mechanisms to reduce Haematological diseases were chronic myeloid leukaemia (7), graft-versus-host disease after allogeneic stem cell Hodgkin lymphoma (3), acute lymphoblastic leukaemia (2) and transplantation others (4). BO was diagnosed on transbronchial biopsy (8), open lung biopsy (4) and autopsy findings (4). The relationship male/ female was 11/ 5 and the median age at transplant was 36.6 years (range 15-57). The chemotherapy lines median Graft versus malignancy received previously transplant was 2 (range 0-4); 6 patients received drugs related with pulmonary toxicity and 7 patients radiotherapy (2 localized and 5 total body irradiation). Results: The median time between transplantation and P583 diagnosis of BO was 10,68 months (range 1-36) and the Increased risk of relapse for patients undergoing HLA-C median survival was 34,3 months (range 1-107). In all cases matched URD-HSCT and lacking ligands for KIR2DL2/3 predominant respiratory symptoms consisted of non- S. Giebel, F. Locatelli, J. Wojnar, M. Zecca, M. Krawczyk- productive cough and dyspnea; The chest radiology and Kulis, G. Giorgiani, M. Markiewicz, I. Wylezol, A. Holowiecka- computed tomography scan showed non-specific findings. Goral, J. Holowiecki Silesian Medical University (Katowice, Two broad histopathological patterns are characteristic: PL); IRCCS Policlinico San Matteo (Pavia, I) constrictive bronchiolitis (7/16) and proliferative bronchiolitis (9/16). Constrictive bronchiolitis tends to be progressive and HLA-C molecules serve as ligands for inhibitory killer patients have less response to therapy. In 40% of all cases immunoglobulin-like receptors (KIRs) and may be subdivided coexistent infectious were seen and Aspergillus sp. was the into two groups, namely C(1) and C(2), based on the KIR- most frequent organism. ligand specificity (for KIR2DL2/3 and KIR2DL1, respectively). Among 14 allogenic recipients, 7 patients developed acute In a previous study, we demonstrated that KIR-ligand graft-versus-host disease (GVHD) grades II-IV and they were incompatibility has beneficial impact on outcome following treated with high dose steroids and 5 patients developed unrelated donor-hematopoietic stem cell transplantation (URD- extensive chronic GVHD. 8 cases were diagnosed during HSCT) suggesting a role of NK cell-mediated graft-vs.- immunosuppressive treatment (cyclosporin +/- steroids) and 4 leukaemia effect (Blood 2003, 102: 814-9). More recently, after supression (median 4 months). Earlier introduction of Cook et al. observed that also in a setting of HLA-identical immunosupression therapy was the treatment more frequent. sibling HSCT, HLA-C genotype may influence outcome (Blood Conclusions: The risk factors for bronchiolitis obliterans, such 2004, 103: 1521-6) (HLA-C(2) homozygous patients had as irradiation, chemotherapy with pulmonary toxicity drugs, reduced probability of survival). graft-versus-host disease and coexistent infectious are The goal of the current study was to analyze the impact of presents in the most of our cases. The earliest diagnosis and HLA-C genotype on long-term outcome of URD-HSCT the intensification of the immunosupressive treatment are recipients for whom donors were prospectively searched using related with best survival. high-resolution DNA typing and were fully matched for the HLA-C loci. To this purpose, we analyzed 102 patients (median age 20 years, range 0.5-50) with haematological P582 malignancies (CML 39, ALL 27, AML 22, MDS 11, NHL 2, MM ATG induces ex vivo and in vivo depletion of circulating 1), given transplantation in two Institutions (Katowice, Poland myeloid and plasmacytoid dendritic cells and Pavia, Italy) between 1999 and 2004. Patients were L. Fang, M. Engel, B. Fehse, A. Zander, N. Kröger University divided into three subgroups: HLA-C(1),C(2) heterozygous Hospital Hamburg (Hamburg, D) (n=44), HLA-C(1) homozygous (n=42), and HLA-C(2) homozygous (n=16). Anti-thymocyte globulin (ATG, Fresenius) as part of the We found that patients lacking HLA-C(1) alleles (i.e. HLA-C(2) conditioning regimen prior to allogeneic stem cell homozygous) had lower probability of disease-free survival transplantation has been shown to be effective in preventing compared with the remaining subgroups (32% vs. 47%, acute and chronic GvHD without an increased risk of graft- p=0.03). The difference resulted from higher incidence of rejection. The effect of ATG on dendritic cells which represent relapse (36% vs. 15%, p=0.005) whereas the incidence of a population of bone marrow-derived cells specialized in non-relapse mortality was comparable (32% vs. 38%, p=0.47). antigen capture, processing and presentation to After adjusting for other risk factors, namely diagnosis (acute immunocompetent cells is still poorly understood. A major role leukaemias vs. others), disease status (early vs. advanced), of donor-derived dendritic cells for the development of acute recipient’s age, source of stem cells (BM vs. PB), and type of GvHD has recently been shown in a murine allogeneic bone conditioning (TBI- vs. Cht.-based), the negative impact of marrow transplantation model We investigate the in-vivo and HLA-C (2) homozygosity on the incidence of relapse remained ex-vivo effect of rabbit anti-thymocyte globulin (ATG) on significant (RR 4.12 (1.32-12.87), p=0.01). myeloid (MDC) and plasmacytoid dendritic (PDC) cells. We conclude that patients lacking HLA-C(1) alleles are at Myeloid dendritic cells were stained with a combination of higher risk of relapse following URD-HSCT. We speculate that antibodies against CD14, CD16, CD85k (ILT3) and CD33, the effect may result from the compromised repertoire of while plasmacytoid dendritic cells were identified with accompanying activatory receptors that are believed to antibodies to CD14, CD16, CD85k (ILT3), and CD123 (IOTest, mediate NK cell-dependent graft-vs.-leukaemia reaction. Beckman & Coulter, Marseille, France). After incubation with Further studies are warranted to determine disease-specificity

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