S60 Posters

form in macrophages to toxic forms in parenchymal cells,and that intestinal iron absorption as well as transfusionaliron could contribute to their iron overload.

P035 Significance of bone marrow storage iron inpatients with myelodysplastic syndromes

L. Wong, L. Go, W. Koss, T. Wajima°.Hematology/Oncology, Texas A&M University HealthScience Center and Scott and White Clinic, USA*E-mail: wajimaty@aol.com

Introduction: The myelodysplastic syndromes(MDS) areclonal neoplastic disorders of hematopoietic stem cells.Most patients present with anemia and a majority willeventually develop transfusion-dependency(TD). In MDS,the increase activity of inflammatory cytokines suppresseserythropoiesis and accumulates iron in the bone marrow.Significance of bone marrow storage iron(BMSI) in MDShas not been evaluated. The study aims to find whetherBMSI is a significant biological factor in MDS and apredictor for TD.Materials and Methods: Sixty-five MDS patients werestudied retrospectively: median age 63 (36−89), 42 male,23 female; 33 RA, 8 RARS, 17 RAEB, 7 RAEB-T. Medianfollow up time was 5.5 years (1.2−12). BMSI is evaluatedby Prussian blue stain. Iron storage is graded from 0 to 4+.The stained BMSI was divided into low group 0~+1, normalgroup +1~+2, and high group +3~+4. BMSI was comparedwith hemoglobin (Hgb), FAB classification, IPSS, karyotypeanalysis and TD. TD was defined as a minimum one unitof packed red cell transfusion per month for symptomaticanemia.Results: Thirty eight patients (59%) had high BMSI,twenty-five (38%) had normal BMSI and two patients(3%) had low BMSI. High iron group had median Hgb8.8 g/dl (6.3−11.3), normal and low-group had medianHgb 11.2 (9−13.3). Incidence of high BMSI was seen45% in RA patients, 50% RARS, 70% RAEB, and100% RAEB-t. Ninety-two per-cent of patients with highBMSI became TD. Two patients with normal BMSI andringed sideloblasts(RSB) became TD. Two RA patients hadhigh BMSI, but did not develop TD. Among high BMSIgroup, 16 (42%) had a normal karyotype and 22 (52%)showed clonal aberrations (12 intermediate, 10 poor). Highiron group had higher IPSS, whereas low/normal group hadlower IPSS. All RAEB-t had high BMSI, lower Hgb, TD,higher IPSS and poor karyotype.Discussion: In MDS, inflammatory cytokines are increasedwhich suppress erythropoiesis, interfer iron metabolismand accumulate iron in bone marrow and/or may induceRSB. Both high BMSI and RSB are related to impairmentof iron utilization, free radical formation that result indyserythespoiesis and anemia. High BMSI correlated withanemia, FAB classification, karyotype, IPSS, and TD.

Conclusions: BMSI has a significant biological factor forMDS, which needs further investigation. High BMSI is animportant clinical predictor for TD.

P036 The oral iron chelator ICL670 is a potentinhibitor of NF-kB and this activity isindependent from iron overload in MDS cells

D. Cilloni °, V. Rosso, E. Messa, F. Messa, F. Arruga,I. Defilippi, R. Catalano, S. Carturan, C. Bittoto, C. Boveri,P. Nicoli, E. Bracco, G. Saglio. Dept. of Clinical andBiological Sciences, University of Turin, Turin, Italy*E-mail: daniela.cilloni@unito.it

Background: Patients affected by MDS undergo ironoverload due to blood transfusions. Recently oral chelationis under evaluation to reduce iron induced organ damage.It was reported that iron chelation is able to reduce the Hband platelets transfusion requirement. Iiron activates NF-kBthrough TNFa release. It was demonstrated that NF-kB isabnormally activated not only in AML but also in MDSpatients The aim of the study was to evaluate the effects ofthe oral chelator ICL670 (Novartis) on NK-kB activity toidentify a possible mechanism responsible for the observedreduced transfusion requirements during chelation therapy.Methods: 20 BM samples were collected from MDSpatients. 8 were RA, 8 RAEB, and 4 AML secondary toMDS (s-AML). 12 of the patients presented iron overload(by SQUID biomagnetic liver susceptometry) and highferritin levels. The remaining 8 patients were collectedbefore transfusions and they presented normal liver ironconcentration (LIC) and ferritin levels. MNC cells wereincubated with 100mm ICL670 for 3 hrs. K562 and HL60cells were analyzed as control. Incubated and control cellswere evaluated for NF-kB activity using both EMSA andELISA method.Results: We detected an increased activation of NF-kBas compared to healthy subjects in 4 out of 8 RA 6out of 8 RAEB, in all the cases of s-AML and in celllines. No significant difference was detected in NF-kBactivity comparing patients with or without iron overload(p = 0.5). The levels of NF-kB activity increase duringdisease progression being higher in RAEB and s-AML ascompared to RA (p = 0.003). We demonstrated a correlationbetween NF-kB activity and blast percentage (r = 0.75) butnot between NF-kB and ferritin levels (r = 0.5). Amongpatients with increased NF-kB (n = 14) the incubation withICL670 induced a reduction of NF-kB activity (p = 0.0002).No difference was detected in NF-kB inhibition comparingpatients with or without iron overload. In addition, ICL670also inhibits NF-kB activity in HL60 and K562 cells.Discussion: NF-kB is abnormally activated in MDS patientsand this is not apparently related to iron overload beingpresent in many patients before transfusion with normal

2. Molecular mechanisms and pathophysiology I – Stromal cells, cytokines and apoptosis S61

ferritin levels and in cell lines. ICL670 acts as a potent NF-kB inhibitor and this property could explain the activity onBM cells which results in the improvement of the Hb andplatelets levels. This latter effect seems to be independentfrom the reduction of iron storage induced by oral chelation.

P037 Treatment with Epo/G-CSF of patientswith low/int-I risk myelodysplasticsyndrome diminishes relative CLIP(class II-associated invariant chain peptide)amount on hematopoietic precursor cells

M.E.D. Chamuleau°, A. Zevenbergen, L. van Dreunen,T.M. Westers, G.J. Ossenkoppele, A.A. van de Loosdrecht.VU Institute for Cancer and Immunology, VU UniversityMedical Center, Amsterdam, The Netherlands*E-mail: m.chamuleau@vumc.nl

Introduction: Effective tumor antigen presentation byMHC class II antigens to CD4+ T helper cells is crucialfor anti-tumor immune responses. MHC class II antigenscan be presented by tumor cells themselves resulting inhigh immunogenicity. However, if the antigen binding siteof MHC class II molecules is occupied by CLIP, whichis a remnant of the MHC class II chaperone molecule,survival of patients with acute myeloid leukemia (AML) issignificantly shortened [1]. Moreover, high CLIP expressionon minimal residual disease cells of AML patients iscorrelated to high relapse rate. We hypothesize that lossof immunogenicity of pre-leukemic hematopoietic precursorcells of low/int-I risk MDS patients could play an importantrole in the progression to AML.Methods: We evaluated 22 patients treated with astandardized regimen of epoetin-beta (NeoRecormon) andfilgrastim (Epo/G-CSF). Bone-marrow (BM) samples weretaken before treatment and after 12 months and analyzed byflow-cytometry for the extracellular expression of HLA-DRand CLIP on hematopoietic precursor cells en myeloid blasts(both defined by CD34+/CD45dim expression). RelativeCLIP amount was determined by taking into account boththe total number of DR and CLIP positive cells as well as themean fluorescence intensity of these cells. For comparisonBM samples of 10 healthy donors and 99 AML patientswere analyzed.Results: The mean relative CLIP amount on hematopoieticprecursor cells of low/int-I risk MDS patients was 0.58. Thisis significantly higher than on normal precursor cells (meanrelative CLIP amount 0.011 (p = 0.006 Mann Whitney U(MWU)) and comparable to AML leukemic blasts (meanrelative CLIP amount 0.16 (p = 0.188 (MWU)). Treatmentwith Epo/G-CSF increased HLA-DR expression anddiminished CLIP expression and consequently diminishedmean relative CLIP amount to 0.11 (significant differenceas compared to start of treatment (p = 0.024 (Wilcoxon)).Highest relative CLIP amount was found on precursor cells

of non-treated low/int-I risk MDS patients. After 1 year ofEpo/G-CSF treatment, relative CLIP levels were lower thanon AML blasts but still significantly higher than on normalhematopoietic precursor cells (p = 0.03 (MWU)).Discussion: Besides hematological improvement, Epo/G-CSF treatment induces a lower level of relative CLIP onhematopoietic precursor cells of low/int-I risk MDS patientswhich may have impact on immunogenicity. The increasedimmunogenicity could enable the immune system to controlthe disease and prohibit progression to AML.

References

[1] Chamuleau, M.E., et al. Class II-associated invariantchain peptide expression on myeloid leukemic blastspredicts poor clinical outcome. Cancer Res. 64, 5546–5550 (2004).

P038 Inhibition of poly ADP ribose polymerase(PARP) induces cell death in leukaemiccell lines alone or synergistically withhistone deacetylase inhibitors: potential foranti-leukaemia therapy

T.J. Gaymes°, S. Shall, F. Farzaneh, G.J. Mufti. Dept.of Haematological Medicine, Leukaemia Sciences Labs,The Rayne Institute, GKT School of Medicine, DenmarkHill campus, London, United Kingdom*E-mail: terry.gaymes@kcl.ac.uk

It has recently been reported that cells defective fordouble strand break (dsb) DNA repair can be selectivelytargeted for apoptosis through abrogation of their PARPactivity. It has also been reported that leukaemic cellshave inherent defects in dsb DNA repair. Exploitationof DNA repair defects using PARP inhibitors (PI) thusrepresents a specific and less toxic form of therapy for anumber of haematological malignancies. In order to testthe efficacy of PI therapy we analysed myelodysplasticsyndrome (MDS) and acute myeloid leukaemia (AML)cell lines and the potential for combination therapywith inhibitors of DNA methyltransferase (DNMTi) orhistone deacetylase inhibitors (HDACi). We report thatfrom a panel of MDS and leukaemic cell lines, themyelomonocytoid leukaemic/myelodysplastic cell line, P39,the T-lymphoblastoid cell line, Jurkat, and the AML celllines U937 and KG-1 displayed abnormal cell cycle profilesand apoptosis in response to the PI KU-0058948 (1M). Incontrast, normal control cells displayed standard cell cycleprofiles and no apoptosis in response to PI. Clonogeniccytotoxicity assays showed that these cell lines exhibitedbetween 45−70% cell survival compared with 100% cellsurvival in control cells (p< 0.05) in response to PI. Wenext explored the use of PI in combination with DNMTi orHDACi. Whilst KU-0058948 offered only additive effectson DNMTi cytotoxicity, a non-cytotoxic concentration of