Abstracts

1. Young Investigators A1 Novel protocols for the detection and characterization of putative disseminating breast cancer stem cells J. Szkandera1, M. Balic1, N. Rapp1, J. Strutz1, R. J. Cote2, H. Samonigg1 1

Division of Oncology, Department of Internal Medicine, Medical University Graz, Graz, Austria 2 Department of Pathology, University of Miami Miller School of Medicine, Miami, USA

Breast cancer is one of the most common causes of death in women of all ages, mainly due to the tendency of primary breast carcinoma to metastasize. Breast tumours are composed of phenotypically and functionally different breast cancer cells. A small population of tumour cells has the capability of self-renewal, extensive proliferation and possible capability to metastasize to regional and distinct locations. These putative breast cancer stem cells have been identified as CD44þCD24
=low or ALDHþ. The presence of these cells within disseminated tumour cells may be the reason for the failure of systemic adjuvant chemotherapies. For the development of new therapeutic strategies the identification and characterization of disseminating putative breast cancer stem cells is needed. This requires the use of multiple antibodies (CK, CD44, CD24 and ALDH) labelled with fluorochroms of different colours and spectral image analysis to separate the different colour spectra. We have developed an optimized method for multi-marker analysis of disseminating putative breast cancer stem cells by using Quantum Dot and DyLight Technology, in which secondary antibodies are coupled to nanometre-sized fluorescent dyes. Two of the markers, CD44 and CK, have been successfully conjugated with the labelled secondary antibody. Since the used CD24 was an IgM, it had to be sequentially labelled with a secondary antibody coupled with a dye. With this novel protocol it is now possible to detect disseminating tumour cells and distinguish distinct subpopulations. Future studies will be performed to determine the expression of specific therapeutic targets on the putative disseminating breast cancer stem cells.

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Division of Hematology and Oncology, Tumor Biology and Angiogenesis Laboratory, Innsbruck Medical University, Innsbruck, Austria 2 Division of Hepatology, Innsbruck Medical University, Innsbruck, Austria

Bortezomib used for medical treatment of multiple myeloma exerts profound antitumoural and anti-angiogenic effects by inhibiting the 26S proteasome. The aim of this study was to analyze the molecular targets in arrested and growing endothelial cells in vitro and the impact of bortezomib on growth and vascularization of solid tumour xenografts in vivo. Application of bortezomib induced cell-cycle arrest in endothelial cells due to upregulation of the cycline-dependent kinase inhibitors p21CIP1, p27KIP1 and the tumour suppressor p53. Apoptosis was only observed in proliferating, but not in growth-arrested endothelial cells by upregulation of the pro-apoptotic Bok and Noxa proteins. Angiogenic activities based primarily on cell proliferation, such as sprouting of endothelial spheroids in vitro and blood vessel formation in the chicken chorioallantoic membrane assay (CAM) in vivo were strongly inhibited by bortezomib. In contrast, vascularization of the CAM in the presence of certain solid tumour xenografts was not inhibited after application of bortezomib. A secreted factor of tumour cells inhibited the anti-angiogenic action of bortezomib. Size exclusion, ion-exchange chromatography and mass spectroscopy identified GRP-78 a chaperone protein of the unfolded protein response as responsible factor. A variety of solid tumour cell lines (PC-3, HRT-18) but not myeloma cell lines (U266, OPM-2) secreted GRP-78, a protein normally expressed and retained in the endoplasmatic reticulum. Based on our data we conclude that a variety of solid tumours, but not myeloma cells have these hitherto unknown mechanism to generate resistance against the anti-angiogenic activity of bortezomib.

A3 Making functional Endothelial progenitors: animal serum-free humanized large-scale propagated adult blood-derived Endothelial colony-forming cells assemble stable perfused vessel in vivo A. Reinisch1,2, N. A. Hofmann1,2, A. C. Obenauf3, K. Kashofer4, E. Rohde1,5, K. Schallmoser1,5, D. Thaler1,2, M. Fruehwirth1,2, W. Linkesch1,2, M. R. Speicher3, D. Strunk1,2 1

A2 GRP-78 secreted by tumour cells blocks the anti-angiogenic activity of bortezomib J. Kern1, G. Untergasser1, H. Zoller2, G. Gastl1, E. Gunsilius1, M. Steurer1 memo Suppl 2/09

Stem Cell Research Unit, Medical University of Graz, Graz, Austria 2 Department of Hematology and Stem Cell Transplantation, University Clinic of Internal Medicine, Medical University of Graz, Graz, Austria 3 Institute of Humen Genetics, Medical University of Graz, Graz, Austria 4 Institute of Pathology, Medical University of Graz, Graz, Austria

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University Clinic of Transfusion Medicine and Blood Group Serology, Medical University of Graz, Graz, Austria

Endothelial colony-forming cells (ECFCs) have been described as the prototype of blood- and vessel-derived endothelial progenitor cells (EPCs). Their vessel-forming capacity makes ECFCs a promising tool to study vascular homeostasis, regeneration and tumour-angiogenesis. This study was initiated to develop the first animal protein-free large-scale expansion system for adult human blood-derived ECFCs and to test their functionality in vitro and in vivo. We isolated ECFCs directly from whole blood with a novel recovery strategy. ECFC propagation was done under animal-protein-free culture conditions with pooled human platelet lysate (pHPL). ECFC long-term proliferation potential was monitored and phenotype was analyzed by flow-cytometry and immune-cytochemistry. Functionality was studied during vascular network assembly in vitro and in two models for human vessel formation in immune-deficient mice in vivo. Genomic stability was assayed with chromosome G-banding and array-comparative genomic hybridization (array-CGH). A mean of four ECFC colonies=mL peripheral blood could be recovered. The cells could be expanded to mean 1.5  0.5 108 ECFCs within 11–25 days. Consecutive analysis confirmed ECFC purity, immune-phenotype and sustained proliferation potential. Genomic stability was shown by karyotyping and array-CGH. Large-scale expanded ECFCs functioned to form vascular networks in vitro and assembled stable human vessels connected to murine circulation in vivo. This demonstrates that proliferating functional, storable and genomically stable human ECFCs can be expanded to clinical quantity in an animal protein-free system. This procedure for large-scale ECFC propagation should help to set a new standard to study therapeutic applicability and risk profile of vesselforming EPC-based investigational new drugs.

A4 FTY720 inhibits Treg expansion in vitro and in vivo A. M. Wolf1, K. Hochegger2, R. Zeiser3, C. Duerr3, M. Sixt4, G. Gastl1, A. Rosenkranz2, D. Wolf1 1

Internal Medicine V, Hematology and Oncology, MUI Innsbruck, Innsbruck, Austria 2 Internal Medicine IV, Nephrology and Hypertension, MUI Innsbruck, Innsbruck, Austria 3 Division of Hematology and Oncology, Department of Medicine, Medical University Freiburg, Freiburg, Germany 4 Max Planck Institute for Biochemistry, Munich, Germany

CD4þCD25þ regulatory T-cell (Treg) entry into secondary lymphoid organs (SLO) and local expansion upon activation are critical for their immunosuppressive action. Trapping of adoptively transferred Treg in SLO therefore represents an attractive strategy to tip the balance towards a more immunosuppressive milieu. Systemic ad-

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ministration of the sphingosine-phosphate receptor agonist FTY720 has been proven to trap harmful effector T-cells in SLO, thereby inhibiting their migration towards target tissues. We now provide first evidence that selective trapping of adoptively transferred Treg in inflammatory lymph nodes can be achieved by ex vivo exposure of Treg to FTY720. Despite selective FTY720 treatment of Treg allowed for their proper localization within the T-cell areas of SLO, it abrogated their protective effect after transfer in murine models of acute experimental glomerulonephritis and acute graft-versus-host disease. This was due to FTY720-induced blockade of Treg proliferation in vivo and in vitro. On a cellular level FTY720 abolished IL-2 induced phosphorylation of STAT-5, which was paralleled by a loss of FoxP3 expression during Treg expansion. Thus, selective exposure of Treg to FTY720 favours their accumulation in inflammatory SLO but in parallel abrogates their in vivo immunosuppressive potential by blocking IL-2 induced expansion, which is a prerequisite for their full immunosuppressive function.

A5 Evaluation of high-resolution melting analysis as a diagnostic tool to detect the BRAF V600E mutation in colorectal tumours M. Pichler1, M. Balic1, E. Stadlemeyer1, C. Ausch2, M. Wild3, C. Gülly4, T. Bauernhofer1, H. Samonigg1, G. Hoefler3, N. Dandachi1 1

Division of Oncology, Department of Internal Medicine, Medical University of Graz, Graz, Austria 2 Department of Surgery, Ludwig Boltzmann Research Institute of Surgical Oncology, Danube Hospital, Vienna, Austria 3 Institute of Pathology, Medical University of Graz, Graz, Austria 4 Center for Medical Research, Medical University of Graz, Graz, Austria

BRAF V600E is the predominantly occurring mutation of the cytoplasmic kinase BRAF, and, in colorectal cancer, its determination provides a diagnostic exclusion criterion for hereditary non-polyposis colorectal cancer. The aim of our study was to develop a sensitive BRAF V600E high-resolution melting (HRM) assay. We first established and optimized the BRAF HRM assay using a cell line dilution model, enabling us to detect 1% mutant DNA in a background of wild-type DNA. In a comparison, DNA sequencing and real-time allele-specific PCR in the cell line dilution model HRM assay proved to be more sensitive than DNA sequencing and denaturing highperformance liquid chromatography, retaining the same sensitivity as real-time allele-specific PCR. In a learning set of 13 patients with known BRAF V600 status, the mutation was detected with high concordance by all four methods. Finally, we validated the HRM assay on 60 formalin-fixed, paraffin-embedded colorectal cancer samples. Although all mutated samples were correctly memo Suppl 2/09

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identified by HRM, the detection limit of the HRM assay decreased when using low-quality DNA derived from formalin-fixed, paraffin-embedded samples. In conclusion, HRM analysis is a powerful diagnostic tool for detection of BRAF V600E mutation with a high sensitivity and highthroughput capability. Despite the expected decrease in sensitivity, HRM can reliably be applied in archival formalin-fixed, paraffin-embedded samples tissues.

A6 Oxygen sensing of somatic endothelial progenitor cells N. A. Hofmann1, A. Reinisch1,2, K. Schallmoser1,3, E. Rohde1,3, S. Chatterjee1, R. Birner-Gruenberger4, D. Strunk1,2 1

Stem Cell Research Unit Graz, Medical University of Graz, Graz, Austria 2 Department of Hematology, Medical University of Graz, Graz, Austria 3 Clinic of Blood Group Serology and Transfusion Medicine, Medical University of Graz, Graz, Austria 4 Centre for Medical Research, Medical University of Graz, Graz, Austria

Endothelial colony-forming cells (ECFCs) are endothelial progenitor cells (EPCs) with robust proliferative potential and profound vessel-forming capacity. This makes ECFCs a promising tool to study vascular homeostasis and regeneration. We hypothesized that arterial as compared to venous oxygen levels differentially regulate ECFC proliferation and functionality. Adult human ECFCs were isolated directly from the whole blood with a novel recovery strategy and propagated under animal proteinfree conditions. Phenotype was analyzed by flow-cytometry and immune-cytochemistry. EPC hierarchy, long-term proliferation, wound-repair, as well as migratory and vasculogenic functions were monitored under defined oxygen levels. Molecular responses to different levels of oxygenation were assessed by proteomic profiling. Results revealed reduced colony size under venous (50.9  1.2 mmHg) compared to arterial (75.2  0.9 mmHg) oxygen concentration. Hyperoxic conditions (149.5  1.0 mmHg) resulted in a significantly elevated number of high proliferative potential (HPP) colonies (60  18% of total colonies) compared to arterial (9  6%) and venous (0%) oxygen conditions. The absolute colony number in the culture (3.2  0.2 colonies=cm2) was unchanged independent of oxygenation. Increased oxygen levels resulted in an augmentation of ECFC proliferation in primary and long-term cultures. Vascular wound repair was increased with escalating oxygen supply in scratch-assays and was promoted mainly through improved migration. In vitro Matrigel+-vascular network formation showed similar results. Utilizing proteomic analysis we identified several stress proteins involved in oxygen sensing. This demonstrates that migratory and proliferative functions of memo Suppl 2/09

ECFCs are regulated by stress-induced proteins in an oxygen-dependent manner. Cellular differences due to oxygen availability may be key factors influencing vascular homeostasis and repair in situ and during therapeutic vasculogenesis.

A7 MicroRNA-34a expression correlates with MDM2 SNP309 polymorphism and treatmentfree survival in chronic lymphocytic leukaemia D. Asslaber, J. D. Pinon, I. Seyfried, P. Desch, M. Stöcher, A. Egle, O. Merkel, R. Greil Laboratory for Immunological and Molecular Cancer Research, Paracelsus Private Medical University, Salzburg, Austria

MicroRNAs (miRNAs) have emerged as important protagonists in cancer research by virtue of their ability to regulate the expression of numerous genes on a posttranscriptional level. MiR-34a has recently been described as a well-defined downstream target of p53. Here we demonstrate that miR-34a levels correlate with the MDM2 polymorphism SNP309 in patients without overt p53 aberrations. Moreover, miR-34a levels are shown to act as surrogate marker for p53 activity and could predict treatment-free survival in CLL. In knockdown and over expression experiments we establish physiological functions of miR-34a in a B-CLL context. Finally, we aimed at studying miR-34a levels in the Tcl1 mouse model for CLL where we found a striking miR-34a increase during the malignant but not during the premalignant phase of disease development.

A8 Phenotypic characterization of CD341==CD38
AML stem cells and their response to anti-leukaemic drugs H. Herrmann1,2, C. Baumgartner1, W. R. Sperr1, S. Holmes3, P. Valent1,2 1

Division of Hematology and Hemostaseology, Department of Internal Medicine I, Medical University of Vienna, Vienna, Austria 2 Ludwig Boltzmann Cluster Oncology, Vienna, Austria 3 Domantis Ltd., Cambridge, UK

In acute myeloid leukaemia (AML), the clone is organized hierarchically with mature cells and stem cells that self-renew and repopulate NOD=SCID mice with leukaemias. AML stem cells (SC) supposedly reside within the CD34þ=CD38
fraction of the clone. We examined the expression of cytokine receptors (SCFR=KIT, IL-3Ra, GM-CSFRa, IL-3=GM-CSFRß, G-CSFR, M-CSFR, TGFßR=

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endoglin, EPOR, TPOR=MPL, FLT3 and VEGFR=KDR) and other markers on CD34þ=CD38
cells in 30 patients with AML, and determined responses to cytokines and antileukaemic drugs. AML SC were found to display a variable pattern of cytokine receptors and other surface antigens. The IL-3R and CD44 were expressed consistently on AML SC in all donors. In most patients, at least a subset of SC co-expressed KIT, G-CSFR, TGFßR, FLT3, CD33 and CD133. By contrast, AML SC usually did not express GMCSFR, M-CSFR, EPOR, TPOR and KDR. Exposure of AML stem cells to SCF, IL-3 or G-CSF prevented apoptosis, whereas EPO showed no effect. In most donors, ARA-C, fludarabine, clofarabine and Mylotarg were found to promote apoptosis in AML SC in a dose-dependent manner. Moreover, ARA-C was found to cooperate with fludarabine and clofarabine in producing apoptosis in AML SC. 3H-thymidine uptake experiments confirmed growth-inhibitory drug effects on purified CD34þ=CD38
AML SC. In particular, ARA-C, fludarabine and clofarabine were found to inhibit cytokineinduced 3H-thymidine-incorporation in sorted AML SC in all donors. Together, our data show that multicolour flow cytometry and combined staining for surface markers and AnnexinV is a valuable approach to determine apoptosis-rescuing effects of cytokines and apoptosis-inducing drug effects in AML SC.

A9 Loss of the tumour suppressor RKIP in acute myeloid leukaemia A. Zebisch1,2, M. Haller3, K. Hiden1, O. Rath2, B. Doyle2, A. Wölfler1,4, R. Delwel4, W. Kolch2, J. Troppmair3, H. Sill1 1

Division of Hematology, Medical University of Graz, Graz, Austria 2 The Beatson Institute for Cancer Research, Glasgow, UK 3 Daniel Swarovski Research Laboratory, Innsbruck Medical University, Innsbruck, Austria 4 Department of Hematology, Erasmus University MC, Rotterdam, The Netherlands

RAF kinase inhibitor protein (RKIP) has recently been described as a negative regulator of the MAPK signalling cascade. As constitutive activation of this pathway frequently occurs in acute myeloid leukaemia (AML), we investigated a possible involvement of RKIP in the pathogenesis of this disorder. Analysis of RKIP by Western Blot was performed in 101 AML patient samples, 19 AML cell lines and purified CD34þ haematopoietic stem and precursor cells of six healthy individuals. Twenty-one of 101 (21%) patient samples and 5=19 (26%) cell lines exhibited complete or partial loss of RKIP, which was significantly associated with a myelomonocytic or monocytic subtype (p < 0.0001). These results could be corroborated in an independent cohort of 285 AML patients by gene expression profiling data using Affymetrix U133A GeneChips.

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Significant downregulation of RKIP was found in a distinct cluster almost exclusively comprising AML samples of the M4 and M5 subtypes (p < 0.0001). To test for a causative role of RKIP silencing in the development of a monocytic phenotype, we performed cell differentiation assays of the immature AML cell line HL-60. Its monocytic differentiation, which was induced by treatment with vitamin D3, was paralleled by a significant downregulation of RKIP. Concomitant knockdown of RKIP with siRNA further increased differentiation, supporting a critical role for RKIP in this process. To evaluate the effects of RKIP loss on malignant transformation, we performed colony-formation assays in NIH3T3 cells transformed by oncogenic C-RAFS427G, which was characterized in t-AML previously. The growth potential of these cells was significantly increased by RKIP knockdown with siRNA and, conversely, significantly decreased by RKIP overexpression. In conclusion, these data demonstrate that loss of the tumour suppressor RKIP is a frequent event in AML, which may be linked to the development of a monocytic phenotype.

A10 Clonal CD4 T cells in B-chronic lymphocytic leukaemia (B-CLL): evidence for a potential pathologic immune reaction driving CLL D. Trapin, C. Holler, T. Kocher, U. Denk, J. Pinon, R. Greil, A. Egle 3rd Medical Department of Oncology, Hematology, Hemostaseology, Rheumatology and Infectiology, Paracelsus Private Medical University, Salzburg, Austria

Several lines of evidence underscore the importance of antigenic selection in the pathobiology of B-CLL. The VH3-21 heavy chain rearrangement represents a separate prognostically severe subtype, and the incidence of stereotyped B-cell receptors (BCR) is a further indication of an antigenic drive. Ongoing antigen receptor signalling may also contribute to CLL progression. However, the role of other components of an immune reaction is less clear. Whilst we have clear indications that specific T-cell subsets are associated with advanced disease, and that these support CLL survival, their immunologic identity is unclear. We report 91 CLL patients analyzed for molecular immunologic features of the CLL clone together with analysis of T-cell clonalities. In our previously untreated subcohort T-cell clones were observed in 49% of cases. The presence of clones correlated with a significantly shorter time to first treatment. This suggests that an expanded T-cell clone may drive CLL progression, and that the interaction of CLL T cells with CLL may be clonally restricted. Indeed we found patients with identical TCR sequence in T-cell clones in conjunction with a stereotyped BCR. These patients also shared a common HLA class II haplotype, suggesting a completely stereotyped memo Suppl 2/09

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CLL immune reaction. In immunochromatography experiments we found significant physical interaction of CLL cells with a limited number of T-cell clones in all patients analysed. Thus, our data support the idea of an antigen-specific CD4 T-cell interaction with mature CLL cells, and suggest a specific form of T-cell help may be clinically relevant for CLL progression.

kinases. As expected, both drugs were found to bind to wt-ABL, SRC-kinases and TEC-family kinases including BTK. Targets preferentially bound and inhibited by bosutinib were STE20s, the FES=FER family, PYK2 and TBK1. Together, bosutinib and dasatinib synergize with each other in producing anti-leukaemic effects on CML cells expressing BCR=ABL-T315I.

A12

2. Myeloid Malignancy

SOCS1, SOCS3 and PTPN6 methylation in patients with Philadelphia-negative chronic myeloproliferative diseases (CMPD)

A11

M. Födermayr, M. Huber, H. Hauser, A. Weltermann, D. Lutz, O. Zach

Cooperative anti-neoplastic effects of bosutinib and dasatinib on CML cells carrying the T315I mutant of BCR=ABL 1

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1st Medical Department, Hospital Elisabethinen, Linz, Austria

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K. Gleixner , R. L. Remsing , U. Rix , R. Meyer , H. Herrmann1, K. Schuch3, W. F. Pickl3, M. Augustin4, J. H. Till4, C. Sillaber1, G. Superti-Furga2, P. Valent2 1

Department of Internal Medicine I, Division of Hematology and Hemostaseology, Medical University of Vienna, Vienna, Austria 2 Center for Molecular Medicine, Vienna, Austria 3 Institute of Immunology, Medical University of Vienna, Vienna, Austria 4 Millipore UK Ltd., Dundee, UK

In patients with chronic myeloid leukaemia (CML), imatinib-resistance is usually associated with BCR=ABL mutations. For these patients, novel tyrosine kinase inhibitors (TKI) represent alternative treatment options. However, not all patients respond to these drugs, especially when leukaemic cells bear the BCR=ABL-mutant T315I. Bosutinib is a novel TKI that has been described to act growth-inhibitory in BCR=ABL-transformed cells. We examined the effects of bosutinib alone or in combination with dasatinib on growth and survival of primary CML cells (n ¼ 6) and various CML cell lines. Bosutinib was found to counteract proliferation, cell-cycle progression and viability of imatinib-sensitive and imatinib-resistant K562 cells, primary CML cells and Ba=F3 cells bearing various imatinib-resistant mutants of BCR=ABL, except T315I (IC50 > 1 mM). Dasatinib showed similar effects but again did not block growth of leukaemic cells bearing BCR=ABL-T315I. Unexpectedly, however, we found that bosutinib and dasatinib synergize with each other in producing growth-inhibition in primary CML cells exhibiting BCR=ABL-T315I at pharmacologic concentrations (0.01– 1 mM). Clear synergistic effects were also observed with Ba=F3 cells bearing BCR=ABL-T315I. We also performed multiplexed kinase assays, chemical proteomics and mass spectrometry using K562 and primary CML cells. In these experiments, dasatinib and bosutinib were found to express an overlapping but non-identical profile of targetmemo Suppl 2/09

The Jak2 V617F mutation can be detected in the majority of CMPD patients and leads to constitutively activated Jak2 proteins. SOCS1, SOCS3 and PTPN6 are interacting with and inhibiting Jak2 kinase activity. Epigenetic gene silencing of these proteins by DNA methylation activates the Jak-STAT pathway and might be an alternative mechanism in patients without any activating mutation. DNA was prepared from blood of 21 patients with PV, 19 patients with ET, 8 patients with MF and 22 healthy controls. Methylation-specific PCR (MSP) was performed for SOCS1 exon2, SOCS3 exon2, SOCS3 intron and PTPN6 promoter. SOCS1 exon2 was methylated in 46, 62, 32 and 13% of healthy controls, PV, ET and MF, respectively. SOCS3 exon2 was methylated in all patients and controls, whereas SOCS3 intron and PTPN6 methylation was only detected by increasing the number of PCR cycles to 40. Accordingly, SOCS3 intron methylation was detected in 36% of healthy controls and in 19% PV, 26% ET and 38% MF, whilst PTPN6 promoter methylation in 9, 19, 0 and 13%, respectively. Between Jak2 V617F positive and negative CMPDs no significant differences in the frequency of methylation of these four genomic regions were observed so far. Methylation of CpG islands in the examined Jak2 pathway suppressing genes were found in the peripheral blood from CMPD patients and healthy controls. The observed methylation status joins the conflicting results from previous publications. However, whether differences between PV, ET and MF patients observed is of any clinical relevance should be analyzed further on.

A13 Clinical significance of serum CD44 levels in myelodysplastic syndromes (MDS) J. Löffler1, U. Germing2, W. R. Sperr3, P. Valent3, H. Zwierzina1, H. Ulmer4, R. Stauder5

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Department of Internal Medicine I, Medical University, Innsbruck, Austria 2 Department of Hematology, Oncology and Clinical Immunology, Heinrich-Heine-University, Düsseldorf, Germany 3 Department of Internal Medicine I, Division of Hematology and Hemostaseology, Medical University of Vienna, Vienna, Austria 4 Department of Biostatistics and Documentation, Innsbruck Medical University, Innsbruck, Austria 5 Department of Internal Medicine V (Hematology and Oncology), Medical University, Innsbruck, Austria

Aberrant expression of the adhesion molecule CD44 correlates with poor prognosis in various neoplasms. To evaluate the prognostic impact of CD44 in myelodysplastic syndromes (MDS) serum levels of soluble CD44 (sCD44) were measured in 130 MDS patients using an enzyme-linked immunosorbent assay (ELISA). sCD44 levels were significantly elevated in MDS patients compared to healthy donors (p < 0.05), and were found to correlate with distinct FAB subtypes. The highest levels of sCD44 were found in patients with CMML and in patients with MDS transfornmed into acute myeloid leukaemia (AML). In univariate analysis elevated levels of sCD44s were significantly correlated with shorter overall survival in MDS-patients (9 vs. 37 months; p < 0.000). In multivariate analysis sCD44s displayed prognostic significance for survival independent from the International Prognosis Scoring System (IPSS). To test for refined prognostication, each IPSS risk group was split into two separate categories based on sCD44s levels. Using this approach, MDS patients with a shorter survival were identified both in the IPSS low-risk (p ¼ 0.014) and in the IPSS Int-1 group (p ¼ 0.031). The CD44sadjusted IPSS defines a cohort of MDS patients with unfavourable prognosis within the low and intermediate-1 IPSS groups, which might be helpful in risk stratification and in therapeutic algorithms.

In mast cell (MC) neoplasms, clinical problems requiring therapy include i) the local aggressive and sometimes devastating growth of MC and ii) mediatorrelated symptoms. A key mediator of MC responsible for clinical symptoms is histamine. Therefore, the use of histamine-receptor (HR) antagonists is an established approach to block histamine-effects in these patients. We screened for additional beneficial effects of HR-antagonists on primary neoplastic human MC obtained from patients with systemic mastocytosis (SM), primary canine mastocytoma cells, the human MC line HMC-1 and the canine MC line C2. In particular, we asked whether any of the HR antagonists would exert growth-inhibitory effects on neoplastic MC. We found that the HR1 antagonists terfenadine and loratadine suppress the spontaneous growth of neoplastic MC in all donors tested (human patients, n ¼ 3; canine patients, n ¼ 7) as well as growth of HMC-1 cells and C2 cells. The effects of both drugs were found to be dose-dependent (IC50; terfenadine, 1– 10 mM; loratadine, 10–50 mM) and occurred in HMC-1.1 cells (subclone lacking KIT D816V) as well as in HMC1.2 cells expressing the imatinib-resistant mutant KIT D816V. Both terfenadine and loratadine also produced apoptosis in neoplastic MC (HMC-1, C2) as determined by morphology, flow cytometry (Annexin V staining) and in a Tunel-assay. The other HR1 antagonists tested i.e. fexofenadine and diphenhydramine, and the HR2 antagonists famotidine, cimetidine and ranitidine showed no significant effects on growth or survival of MC. To explore the mechanism of terfenadine and loratadine-induced growth inhibition, we examined the effects of these agents on KIT expression in HMC-1 cells. In these experiments we found that both drugs inhibit expression of KIT in HMC-1 cells. In summary, our data show that the HR1 antagonists terfenadine and loratadine, apart from their anti-mediator activity, exert growth-inhibitory effects on neoplastic MC and inhibit KIT expression. Whether these drugs also block growth of neoplastic MC in vivo remains to be determined.

P01 The H1 receptor antagonists terfenadine and loratadine inhibit growth and induce apoptosis in neoplastic mast cells

Evaluation of anti-neoplastic effects of INNO-406 on human and canine neoplastic mast cells

E. Hadzijusufovic1,2, K. V. Gleixner1, B. Peter1,2, K. Schuch3, W. F. Pickl3, M. Willmann2, P. Valent1,4

B. Peter1,2, E. Hadzijusufovic1,2, K. Schuch3, K. Blatt1, K. V. Gleixner1, W. F. Pickl3, M. Willmann2, P. Valent1,4

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Division of Hematology and Hemostaseology, Department of Internal Medicine I, Medical University of Vienna, Vienna, Austria 2 Department of Companion Animals and Horses, Clinic for Internal Medicine and Infectious Diseases, University of Veterinary Medicine Vienna, Vienna, Austria 3 Institute of Immunology, Medical University of Vienna, Vienna, Austria 4 Ludwig Boltzmann Cluster Oncology, Vienna, Austria

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Division of Hematology and Hemostaseology, Department of Internal Medicine I, Medical University of Vienna, Vienna, Austria 2 Department of Companion Animals and Horses, Clinic for Internal Medicine and Infectious Diseases, University of Veterinary Medicine Vienna, Vienna, Austria 3 Institute of Immunology, Medical University of Vienna, Vienna, Austria 4 Ludwig Boltzmann Cluster Oncology, Vienna, Austria memo Suppl 2/09

Abstracts

Advanced systemic mastocytosis (SM) is an aggressive neoplasm defined by uncontrolled growth of neoplastic mast cells (MC) in multiple organs. In these patients, neoplastic MC are usually resistant against conventional anti-neoplastic drugs. The tyrosine kinase receptor KIT is frequently mutated in neoplastic MC in SM and supposedly contributes to abnormal MC growth. The D816V-mutated variant of KIT is most commonly detected. Therefore, KITtargeting drugs are currently tested for their ability to block malignant growth in SM. We determined growth-inhibitory effects of the multikinase inhibitor INNO-406 that interacts with KIT and Lyn, on the human MC lines HMC-1.1 (KITD816V-) and HMC-1.2 (KIT-D816Vþ), primary neoplastic MC (human and canine) as well as the canine mastocytoma cell line C2. As assessed by 3H-thymidine incorporation, INNO-406 dose-dependently inhibited the proliferation of HMC-1.1 cells (IC50: 10–100 nM) and C2 cells (IC50: 50 nM), but did not inhibit the growth of HMC1.2 cells. In primary neoplastic MC, INNO-406 was effective in canine patients, but not in human KIT D816Vþ patients. The growth-inhibitory effects of INNO-406 on HMC-1.1 and C2 cells were found to be associated with apoptosis as assessed by microscopy, flow cytometry and Tunel assay. INNO-406 was found to downregulate the expression of phosphorylated (p)KIT in HMC-1.1 and C2 cells, but did not downregulate pKIT in HMC-1.2 cells. By contrast, Lyn was dephosphorylated by INNO-406 in both HMC-1 subclones. Together, we show that depending on the presence and type of KIT mutation, INNO-406 exhibits growth-inhibitory effects on neoplastic MC, presumably through targeting of the KIT kinase.

mTOR inhibitor NVP-BEZ235 (Novartis, Basel) on IgEdependent mediator release in human BA and cultured cord blood cell-derived MC as well as on growth of neoplastic BA (KU812) and MC (HMC-1). NVP-BEZ235 inhibited IgE-dependent histamine release in BA in a dose-dependent manner (IC50 0.5–1 mM). In addition, NVP-BEZ235 inhibited anti-IgE-induced upregulation of CD63 and CD203c on BA, although responses were not seen in all donors. In cultured MC, NVP-BEZ235 decreased IgE-dependent upregulation of CD63 as well as release of beta-hexosaminidase, but did not block upregulation of CD203c. As assessed by 3H-thymidine uptake, NVP-BEZ235 inhibited the growth of KU812 cells (IC50: 0.01–0.05 mM) and HMC-1 cells (IC50: 0.005–0.01 mM). Apoptosis-inducing effects of NVP-BEZ235 were also observed in both cell lines, although the concentrations required to induce clear effects were higher compared to proliferation. Together, NVP-BEZ235 inhibits spontaneous growth of neoplastic BA and MC as well as IgEdependent activation and mediator secretion in BA and MC. Whether these effects of NVP-BEZ235 have clinical implications remains at present unknown.

P04 Quantification of mutated NPM1 in comparison to WT1 in the follow up of adult AML patients O. Zach, M. Födermayr, S. Machherndl-Spandl, H. Kasparu, O. Krieger, M. Girschikofsky

P03 Effects of the PI3 kinase==mTOR inhibitor NVP-BEZ235 on growth and IgE-dependent mediator release in human basophils and mast cells K. Blatt1, H. Herrmann1,2, I. Mirkina1,2, M. Klauser1, P. Valent1,2 1

Division of Hematology and Hemostaseology, Department of Internal Medicine I, Medical University of Vienna, Vienna, Austria 2 Ludwig Boltzmann Cluster Oncology, Vienna, Austria

Growth and function of basophils (BA) and mast cells (MC) are triggered by various cytokines=ligands and signal transduction pathways. In allergic reactions, cross-linking of high affinity IgE-binding sites leads to activation and mediator secretion. In MC, the KIT-ligand SCF promotes IgE-dependent mediator secretion. In mastocytosis, KIT is mutated at codon 816 and expressed as constitutively activated target in neoplastic MC. The PI3kinase is a key signalling molecule in IgE receptor-dependent activation and KIT D816V-dependent activation in MC. We examined the effects of the dual PI3-kinase= memo Suppl 2/09

1st Medical Department, Hospital Elisabethinen, Linz, Austria

The expression of Wilms’ tumour gene (WT1) is a reliable marker for monitoring minimal residual disease (MRD) in peripheral blood (pB) and bone marrow (BM) of AML patients. However, due to low WT1 expression in normal haematopoietic cells, an artificial cut-off limit defining WT1 overexpression has to be introduced into real-time RT-PCR assays. Mutations in the nucleophosmin-gene (NPM1) are present in about 50% of AML patients with normal karyotype and are a feasible MRD marker for these patients, too. For quantification of WT1 transcripts, a TaqMan RT-PCR assay was used. NPM1 mutations were screened via a melting curve based LightCycler assay and quantified with TaqMan RT-PCR. In 20 AML patients (13 female, 7 male, median age 59 years [35–84]) NPM1 mutations were detected and in 19 of them transcripts were quantifiable with TaqMan RT-PCR. During follow up, 212 pB and BM samples of these patients were tested for WT1 and mutated NPM1 in parallel. A total of 204 samples yielded concordant results: NPM1 was positive when WT1 was overexpressed and negative in samples with normal WT1 expression. However, three NPM1 negative samples (1 pB, 2 BM) expressed WT1 at high levels at that time, conversely five NPM1 positive samples (4 pB,

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Abstracts

1 BM) had normal WT1 expression. Quantification of WT1 and NPM1 mutations yields highly concordant results in the follow up of AML patients. NPM1 facilitates the interpretation of WT1 results, especially in samples with WT1 expression near cut-off limits and is a valuable supplement to routine MRD monitoring.

P06 In vitro effects of tyrosine kinase inhibitors on natural killer cells from CML patients and healthy controls A. Krimbacher1, B. Kircher2 1

P05 Array CGH als potente Methode in der Diagnostik akuter Leukämien ohne typische genetische Veränderungen S. Breitenfellner1, R. Marschon1, G. Tschurtschenthaler2, H.-C. Duba3, G. Webersinke1 1

Labor für Molekularbiologie und Tumorzytogenetik, Krankehaus Der Barmherzigen Schwestern Linz, Linz, Österreich 2 I. Interne Abteilung, Krankehaus Der Barmherzigen Schwestern Linz, Linz, Österreich 3 Humangenetische Untersuchungs- und Beratungsstelle Landesfrauen und Kinderklinik Linz, Linz, Österreich

Einleitung. Genetische Vera¨nderungen dienen der Klassifikation, als prognostische Marker und beeinflussen die Therapie von akuten Leuka¨mien, Fa¨lle ohne typische molekularbiologische und zytogenetische Vera¨nderungen sind jedoch nicht selten. Derartige AML und ALL Fa¨lle wurden mittels arrayCGH genomweit auf Aberrationen analysiert, um einen mo¨glichen genetischen Hintergrund der Erkrankung aufzuzeigen. Methoden: Zytologisch und immunpha¨notypisch besta¨tigte AML- und ALL-Fa¨lle wurden mittels konventioneller Zytogenetik sowie FISH und molekularbiologischen Verfahren auf typische Aberrationen der WHO-Klassifikation 2008 hin untersucht. Unauffa¨llige Proben wurden fu¨r die Hybridisierung auf Affymetrix SNP 6.0 Arrays, welche 1.8 Millionen Marker aufweisen, verwendet. Genomische Gewinne und Verluste außerhalb bekannter Regionen mit Copy Number Variations (CNV) wurden na¨her analysiert. Ergebnisse. AML Proben zeigten kleine Aberrationen einzelner Gene, welche zytogenetisch aufgrund der Gro¨ße nicht erfasst werden konnten. Einige dieser Gene ko¨nnten in direkten Zusammenhang mit der Leuka¨mie stehen, wie zum Beispiel ZBTB16, IL1R2 oder EPS8, die eine Rolle in ¨bertragung und Apoptose spielen. Die Auswertung Signalu der ALL Proben zeigte unterschiedliche Resultate. Zum einen fand man auch dort kleine Aberrationen, beispielsweise einen Verlust des AF9 Gens. Zum anderen fand man große Deletionen und Zugewinne bis hin zu Aneuploidien. Zusammenfassung. Die vorgestellten Ergebnisse unterstreichen die diagnostischen Mo¨glichkeiten der ArrayCGH bei akuten Leuka¨mien ohne typische genetische Vera¨nderungen. Neben der Erfassung potenzieller tumorassoziierter Aberrationen auf Genniveau bietet die arrayCGH eine wertvolle Erga¨nzung in der Diagnostik von Neoplasien, die aufgrund schlechter Kultureigenschaften oftmals keine spezifische Metaphasenanalyse zulassen.

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Division of Clinical Pharmacology, Department of Biochemical Pharmacology, Innsbruck Medical University, Innsbruck Austria 2 Department of Internal Medicine V (Hematology and Oncology), Innsbruck Medical University, Innsbruck, Austria

The introduction of orally available tyrosine kinase inhibitors (TKIs) for the treatment of chronic myelogenous leukaemia (CML) had an enormous impact on the outcome of this disease. However, there is growing evidence that TKIs can display immunomodulatory or even immunosuppressive activities. Natural killer (NK) cells are involved in leukaemia cell killing. To date, the knowledge regarding the effect of TKIs on natural killer cells is limited. Thus, we have analyzed the in vitro effect of imatinib mesylate and nilotinib on NK cells from five CML patients and three healthy controls. CD56 expression of NK cells from CML patients and healthy controls remained unchanged after exposure to imatinib and nilotinib at various concentrations. Further, CD16 expression of NK cells was not influenced by nilotinib and low-dose imatinib mesylate, but exposure to higher concentrations of imatinib mesylate (25, 50 mM) significantly diminished CD16 expression of normal and leukaemic NK cells. After a 24-hour incubation with imatinib mesylate (10 mM) and nilotinib (5 mM) the number of viable NK cells decreased: This cytotoxic effect was more pronounced for leukaemic NK cells compared to normal NK cells. Natural killing capacity and interferongamma production of NK cells decreased after treatment with imatinib mesylate to a similar degree in CML-derived and normal NK cells. Notably, the suppressive effect of nilotinib on natural cytotoxicity was less pronounced. In summary, imatinib mesylate and nilotinib at higher concentrations can exert negative in vitro effects on the viability and cytotoxicity of NK cells from CML patients and healthy controls.

P07 Impaired base excision repair in acute myeloid leukaemia N. Scheer1, W. Olipitz1, F. Quehenberger2, K. Hiden1, J. Rankl1, D. Schlembach3, H. Sill1 1

Division of Hematology, Medical University Graz, Graz, Austria 2 Institute for Medical Informatics, Statistics and Documentation, Medical University Graz, Graz, Austria 3 Department of Obstetrics and Gynecology, Medical University of Graz, Graz, Austria memo Suppl 2/09

Abstracts

Impaired DNA repair mechanisms are increasingly recognized as contributors to genomic instability in acute myeloid leukaemia (AML). In this study we sought to determine the role of base excision repair (BER) in the pathogenesis of AML. BER is the main mechanism for the repair of DNA base lesions. Oxidative DNA lesions were induced by hydrogen peroxide (H2O2) in blast cells from 30 patients with therapy-related AML (t-AML). Single strand breaks as BER intermediates were analyzed using the Comet Assay. Peripheral blood mononuclear cells (MNCs) from 30 age- and sex-matched healthy individuals as well as purified CD34 þ umbilical cord blood haematopoietic stem and progenitor cells (HSPCs) served as control. A significant difference in DNA repair capacity was found between normal MNCs, HSPCs and AML blasts. Whereas all MNCs and nearly all HSPCs (30=31, 97%) showed DNA repair, 15=30 (50%) t-AML samples exhibited markedly decreased or absent DNA repair. This phenomenon was also observed in 13=35 (37%) samples from patients with de novo AML, 14=26 (54%) with AML following myelodysplastic syndromes as well as in 5=10 (50%) AML cell lines. Importantly, differences in apoptosis or intracellular ROS concentrations did not account for this finding. We next tested the functionality of BER enzymes. Cleavage assays with oligonucleotides harbouring either the oxidative DNA lesion 8-oxoguanine (8-oxo-G) or the abasic site analogue furan were incubated with whole cell extracts of AML cell lines. Whereas cleavage of furan was not affected, diminished cleavage of 8-oxo-G was observed in those cell lines with decreased DNA repair. In conclusion, we provide evidence that initial steps in the BER pathway are impaired in a proportion of AML cells which could potentially contribute to the pathogenesis of this disorder.

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Purpose. As the course of disease in MDS is very variable, there have been attempts to define very low-risk subgroups  with close-to-normal life expectancy based on the standardized mortality ratio (SMR). This retrospective multicentre analysis of 897 primary MDS patients aimed to scrutinise this finding by considering also excess of mortality (EoM). Methods To define possible no risk  subgroups in MDS, SMRs and EoM (excess mortality attributable to MDS per patient-year) were computed. Risk-groups of the IPSS, its cytogenetic subgroups, ¨sseldorf-score, the Spanish-Sanz-score, the German-Du the French-Lille-score, the Bornemouth-score, the Lausanne-Bournemouth-score and the PI-score, subdivided by age and sex were investigated. Results. Median age was 68 years (16–99), median survival 47 months. The total SMR was 4.72, with 378 of 480 observed deaths attributable to MDS; EoM ¼ 0.14 (additional deaths per patient-year). Males (SMR ¼ 4.83) had a slightly higher SMR than females (SMR ¼ 4.55). Younger patients had a significantly increased SMR of 10.10, but lower excess mortality (EoM ¼ 0.12) than the elderly (SMR ¼ 3.52, EoM ¼ 0.16) and 90% vs. 72% disease-related deaths. SMRs for low-risk-groups of all scores ranged between 2.03 and 3.7, EoMs between 0.04 and 0.52. Additionally splitting risk-groups by age and sex, the lowest SMRs were around 1.5 and all differed significantly from 1, except ¨ sseldorf-low-risk-female  66 years (SMR ¼ 1.7) and for Du Du¨sseldorf-low-risk-female > 66 years (SMR ¼ 1.5). Conclusion. We, like others found small subsamples with a non-significant SMR around 1.5, due to high general mortality in older age. Excess in mortality in older age groups is at least as high as in younger patients.

P08 Can subgroups without disease-related deaths be defined in MDS, categorizing by age, sex and well-known scoring systems?

Department of Medicine II, St. Johannes Hospital Duisburg, Duisburg, Germany

P09 Evaluation of in vivo anti-neoplastic effects of rapamycin in patients with refractory AML: a pilot study

T. Nösslinger1,2, H. Tüchler2, A. Makrai1,2, U. Germing3, W. Sperr4, O. Krieger5, D. Haase6, M. Lübbert7, R. Stauder8, A. Giagounidis9, P. Valent4, M. Pfeilstöcker1,2

A. Böhm1,2, M. Mayerhofer3, S. Herndlhofer1, P. Knöbl1, C. Sillaber1, W. R. Sperr1,4, U. Jäger1,4, P. Valent1,4

1

1

3rd Med. Dept for Hematology and Oncology, Hanusch KH Ludwig Boltzmann Institute for Leukemia Research and Hematology, Hanusch KH 3 Heinrich-Heine-University Düsseldorf 4 Department of Internal Medicine I, Division of Hematology and Hemostaseology, Medical University of Vienna, Vienna, Austria 5 Elisabethinen Hospital Linz, Linz, Austria 6 Department of Hematology and Oncology, University of Göttingen, Göttingen, Germany 7 Division of Haematology and Oncology, University of Freiburg Medical Centre, Freiburg, Germany 8 Department of Internal Medicine V (Hematology and Oncology), Medical University of Innsbruck, Innsbruck, Austria 2

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Department of Internal Medicine I, Division of Hematology and Hemostaseology, Medical University of Vienna, Austria 2 Bone Marrow Transplantation Unit, Medical University of Vienna, Vienna, Austria 2 Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Vienna, Vienna, Austria 4 Ludwig Boltzmann Cluster Oncology, Vienna, Austria

The mammalian target of rapamycin (mTOR) has recently been identified as a potential target in acute myeloid leukaemia (AML). We treated five patients with chemotherapy-refractory AML with the mTOR-inhibitor rapamycin at 2 mg per os daily for 14 days, with dose adjustment allowed to reach a target serum rapamycin

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concentration of 10–20 ng=ml. Four of five patients received additional hydroxyurea at constant dose during treatment with rapamycin. Two patients achieved a leucocyte response during rapamycin therapy. In one of these patients, a prolonged response was seen. In the other patients, blast cell counts remained stable or increased during treatment with rapamycin. We did not observe any severe haematologic or non-haematologic side effects of rapamycin in this study. In summary, rapamycin at 2 mg per day acts cytoreductive in a subgroup of patients with chemotherapy-refractory AML. We assume that higher doses and drug combinations are required to obtain long lasting anti-leukaemic effects in these patients.

3. Lymphatic Malignancy A14 Heat shock protein 32-targeting drugs induce growth arrest and apoptosis in leukaemic cells in acute lymphoblastic leukaemia (ALL) R. A. Meyer1, H. Herrmann1, K. V. Gleixner1, S. Cerny-Reiterer1, A. Cruze2, R. Hubmann1, M. Shehata1, M. Mayerhofer3, W. R. Sperr1, W. F. Pickl2, J. V. Melo4, H. Nakamura5, U. Jäger1, H. Maeda5, P. Valent1 1

Department of Internal Medicine I, Division of Hematology and Hemostaseology, Medical University of Vienna, Vienna, Austria 2 Institute of Immunology, Medical University of Vienna, Vienna, Austria 3 Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Vienna, Vienna, Austria 4 Hematology Division, IMVS, University of Adelaide, Adelaide, Australia 5 Laborarory of Microbiology and Oncology, Department of Pharmaceutical Sciences, Sojo University, Kumamoto, Japan

Hsp32 has recently been described to act as a target in various leukaemias. We examined the expression and functional role of Hsp32 in acute lymphoblastic leukaemia (ALL). Leukaemic cells were obtained from patients with Ph þ ALL (n ¼ 8) and Ph
ALL (n ¼ 12). In addition, Ph þ ALL cell lines (Z-119, BV-173, TOM-1 and NALM-1) and Ph
cell lines (RAJI, RAMOS, REH and BL-41) were examined. As assessed by immunostaining and qRT-PCR, primary ALL cells and all ALL cell lines were found to express Hsp32. An siRNA against Hsp32 was found to induce growth arrest and apoptosis in RAJI cells and TOM-1 cells. We then examined the effects of two pharmacologic Hsp32-inhibitors, PEG-ZnPP and SMA-ZnPP. As assessed by 3H-thymidine uptake, both agents were found to inhibit proliferation in BCR-ABL þ and BCR-

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ABL-negative ALL cell lines. The effects of PEG-ZnPP and SMA-ZnPP were dose-dependent (IC50 1–10 mM) and associated with apoptosis as determined by microscopy and AnnexinV-staining. In NALM-1 cells, PEG-ZnPP and SMA-ZnPP also produced growth arrest, but with slightly higher IC50 values (20 mM). Hsp32-targeting drugs were also effective in producing growth-inhibition in primary (Ph þ and Ph
) ALL cells (IC50 1–10 mM). No major differences were found when comparing results in imatinib-sensitive and imatinib-resistant patients. In drug combination experiments, Hsp32-targeting drugs were found to cooperate with imatinib and nilotinib in producing growth inhibition and apoptosis in Ph þ ALL cell lines. Moreover, cooperative anti-leukaemic effects were obtained when Hsp32-targeting drugs were combined with bendamustine. Overall, our results suggest that Hsp32 is a novel interesting target in ALL.

P10 Free light chain (rFLC, dFLC, iFLC) monitoring indicates early relapse E. Willenbacher, D. Nachbaur, S. Gasser, G. Gastl, W. Willenbacher Department of Internal Medicine V (Hematology and Oncology), Medical University of Innsbruck, Innsbruck, Austria

Rationale. FLC are parameters used for monitoring MM pts. We retrospectively analyzed our transplanted MM pts. with regard to early diagnosis of relapse compared to standard M-Protein measurements (PP). FLC ratio (rFLC), value of the involved light chain (iFLC) and difference of involved=uninvolved light chain (dFLC) were analyzed separately. Patient characteristics between MAR 2005 and OCT 2008, 73 pts. were either alive after, or undergoing HSCT for MM. For 50 evaluable pts. at least two consecutive FLC were documented. Results. A total of 1008 FLC measurements were performed (median 18=pt.) (9 S; 9 U), range: 0–36 (S: 2– 36, U: 0–28). Whilst only 84% of the pts. were evaluable by PP, dFLC, iFLC and rFLC were uniformly applicable. CR was achieved in 40% of pts. by PP, 32%, 30% and 30% of pts. by dFLC, iFLC and rFLC . Accordingly the percentage of pts. never in CR was much higher by FLC techniques vs. only 2% by PP. Early response prediction was feasible by the FLC measurements in 10% of pts. Remissions duration and time to loss of CR were nearly halfed using dFLC, iFLC and rFLC strategies. Eluding to a median advancement of relapse diagnosis of 367 (dFLC), 357 (iFLC) and 217 (rFLC) days, thus opening the way to early implementation of disease modifying treatment approaches. A differential use of the three FLC calculation methods needs further data. This process continues as well as analysis of urine FLC parametres and analysis of the biochemical relapse course of non-transplanted myeloma pts.

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P11 Fludarabine modulates composition and functionality of the T-cell pool in patients with chronic B-cell lymphocytic leukaemia F. Gassner, L. Weiss, R. Geisberger, R. Greil, I. Tinhofer Laboratory for Immunological and Molecular Cancer Research, 3rd Medical Department, Paracelsus Private Medical University, Salzburg, Austria

The combination of cytotoxic treatment with strategies for immune activation represents an attractive strategy for tumour therapy. Following reduction of high tumour burden by effective cytotoxic agents two major immunestimulating approaches are being pursued. First, innate immunity can be activated by monoclonal antibodies triggering antibody-dependent cellular cytotoxicity (ADCC). Second, tumour-specific T-cell responses can be generated by immunization of patients with peptides derived from tumour antigens and infused in soluble form or loaded on to dendritic cells (DC). The choice of cytotoxic agents for such combinatory regimens is crucial since most substances such as fludarabine are considered immunosuppressive by themselves thus hindering the intended immune activation whilst others such as cyclophosphamide can have immunostimulatory activity. We tested in this study whether fludarabine and=or cyclophosphamide which, when combined, represent a very effective treatment regimen for B-chronic lymphocytic leukaemia (BCLL) would interfere with a therapeutic strategy of T-cell activation. Analysis of peripheral blood samples from B-CLL patients prior and during fludarabine=cyclophosphamide (FC) therapy revealed rapid and sustained reduction not only of tumour cells but also of CD4þ and CD8þ T-cells. This correlated with a significant cytotoxic activity of FC in T-cells in vitro. Unexpectedly, T-cells surviving fludarabine treatment in vitro had a more mature phenotype, were significantly more responsive to allogeneic stimulation by DC and showed a shift towards TH1 cytokine secretion after stimulation with PMA. In conclusion, FC therapy though inducing significant and relevant T-cell depletion seems to generate a micromilieu suitable for subsequent T-cell activation and might represent a useful conditioning for subsequent tumour vaccination.

P12 Molecular cytogenetic analysis of plasma cell malignancies (cIg-FISH): towards efficient algorithms in MM and MGUS

Introduction. The employment of cIg-FISH markedly improved the sensitivity of interphase FISH in cytogenetic analysis of plasma cell (PC) diseases. This method facilitates the identification of clonal PCs by simultaneous cytoplasmic light chain immunostaining, and thus provides an important prerequisite for detecting prognostically relevant genetic aberrations in multiple myeloma (MM) and monoclonal gammopathy of unknown significance (MGUS) even in samples with very low PC count. Methods. Mononuclear cells isolated from bone marrow samples by density gradient centrifugation were immobilized on to cytospin slides. Cytoplasmic Ig light chain immunofluorescent staining was performed according to the method of Fonseca (Blood 2002, 100 (4):1417). A panel of probes detecting the most important recurrent aberrations with prognostic impact i.e. -13=13q-, t(11;14)(q13;q32), t(4;14)(p16;q32), -p53 was used for initial analysis. Results. At least one chromosome abnormality was detected in 56 of 78 patients (71.8%), two or more were seen in 30 individuals (53.6%). In numerous cases, a remarkable amount of additional information was rendered by variant signal patterns observed with the probe panel used: for example, supernumerary signals indicating hyperdiploidy, missing spots reveiling deletion or loss of chromosomes and split IgH-signals presumably caused by rearrangements different from those specifically tested for. Conclusions. Molecular cytogenetic analysis of malignant PCs with simultaneous cytoplasmic light chain immunostaining provides an essential diagnostic tool for detecting prognostically significant clonal aberrations in MM and MGUS. Based on our own observations and data from the literature, an efficient approach to first (and occasionally second) line FISH analysis will be discussed with regard to both economic and diagnostic considerations. The application of well-designed diagnostic algorithms may help to obtain timely and clinically relevant information on genetic abnormalities with unfavourable [t(4;14), t(14;16), -p53] and favourable [t(11;14), hyperdiploidy] prognostic impact.

P13 B-Chronic lymphocytic leukaemia cells are highly sensitive towards inhibition of the hedgehog signalling pathway P. Desch1, D. Asslaber1, H. Schnidar2, I. Tinhofer1, G. Regl2, F. Aberger2, T. N. Hartmann1, R. Greil1 1

E. Krömer-Holzinger, M. König, T. Lion

Laboratory For Immunological and Molecular Cancer Research, III Medical Department, University Hospital Salzburg, Salzburg, Austria 2 Department of Molecular Biology, University of Salzburg, Salzburg, Austria

Children’s Cancer Research Institute, St. Anna Children’s Hospital, Vienna, Austria

The Hedgehog (Hh) signalling pathway directs patterning during early development and regulates cell pro-

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liferation and survival. Aberrant cell autonomous or ligand-dependent activation of Hh signalling contributes to tumourigenesis and malignant cell survival in several tumour types. We investigated the effect of inhibition of two key members of Hh signalling, Smoothened and Gli1, on cell survival of B-cell chronic lymphocytic leukaemia (B-CLL) cells. Whilst transcribing lower levels of Smoothened and Gli1 compared to normal B lymphocytes, B-CLL subgroups with prognostically unfavourable cytogenetic aberrations and an unmutated immunoglobulin VH gene mutational status displayed increased expression levels of several pathway members. Treatment of B-CLL cells with the Smoothened antagonist cyclopamine and the Gli antagonist GANT61 specifically decreased target gene expression and induced apoptosis in primary CLL cells. However, cyclopamine and GANT61 treatment of normal B and T lymphocytes had little impact on the viability of these cells. Combination of GANT61 and fludarabine treatment as well as GANT61 treatment alone was able to reduce protective effects of stromal cells cocultured with CLL cells. In summary, B-CLL cells displayed high sensitivity towards Smoothened and Gli1 inhibition and thus Hh molecules might be promising novel therapeutic targets in combination with conventional chemotherapeutics in this malignancy.

P14 Assoziation von Polymorphismen in DNA-Reparaturgenen und Chromosomenaberrationen mit der chronisch lymphatischen B-Zell leukämie (CLL) C. Ganster1, J. Neesen1, A. Gaiger2, U. Jäger2, H. Esterbauer3, C. Mannhalter 3, B. Kluge1, C. Fonatsch1

Genotypen von rs13181 im Nukleotidaustausch-Reparaturgen Xeroderma pigmentosum D (XPD) und rs25487 im Basenaustausch-Reparaturgen X-ray repair complementing defective repair in Chinese hamster cells 1 (XRCC1) kommen in Patienten mit ungu¨nstiger Prognose signifikant ha¨ufiger vor als in Kontrollen: Bei den Genotypen von rs13181 zeigten sich signifikante Unterschiede unter dem co-dominanten Modell (A=A vs. G=G: OR ¼ 2,66, p ¼ 0,024) und bei den Genotypen von rs25487 unter dem dominanten Modell (A=C and C=C vs. A=A: OR ¼ 2,44, p ¼ 0,01). Signifikante Unterschiede in der Verteilung der Genotypen konnten bei rs13181 auch zwischen allen CLL Patienten und der Kontrollgruppe gefunden werden (A=C and C=C vs. A=A: OR ¼ 1,37, p ¼ 0,03). Diese angeborenen genetischen ¨ nstigen cytogenPolymorphismen, die vor allem mit ungu etischen Aberrationen assoziiert sind, ko¨nnten mo¨glicherweise helfen, den Verlauf einer CLL vorherzusagen. In weiterer Folge soll der Einfluss dieser Polymorphismen mit Hilfe klinischer Daten wie Krankheitsverlauf und ¨ berlebenszeit verifiziert werden. U

P15 Tolerability of novel myeloma therapies in the clinical routine setting at University Hospital Graz S. Sormann1, S. Reitter1, M. Pichler1, T. Stranzl2, D. Dekic3, W. Linkesch1 1

Division of Hematology and Oncology, Department of Internal Medicine, Medical University Graz, Graz, Austria 2 Celgene GmbH, Vienna, Austria 3 CTM GmbH, Vienna, Austria

1

Department für Medizinische Genetik, Medizinische Universität Wien, Wien, Österreich 2 Klinische Abteilung für Hämatologie und Hämostaseologie, Universitätsklinik für Innere Medizin I, Medizinische Universität Wien, Wien, Österreich 3 Klinisches Institut für Medizinische und Chemische Labordiagnostik, Medizinische Universität Wien, Wien, Österreich

Sequenzvariationen in DNA-Reparaturgenen ko¨nnen fu¨r verschiedene Krebsformen disponieren oder deren Krankheitsverlauf beeinflussen. Daher wurden sieben Polymorphismen mit potenziellem Einfluss auf die Funktion der Genprodukte in fu¨nf DNA-Reparaturgenen ausgewa¨hlt und deren Einfluss auf die Inzidenz der chronisch lymphatischen Leuka¨mie (CLL) in 461 CLL Patienten und einer gleich großen in Alter=Geschlecht entsprechenden Kontrollgruppe analysiert. Da cytogenetische Aberrationen wichtige Prognosefaktoren fu¨r die CLL darstellen, wurden die Ha¨ufigkeiten der Genotypen auch in 133 Patienten mit aus cytogenetischer Sicht guter Prognose-del(13q)-und in 69 Patienten mit eher schlech¨berpru ¨ ft. Die selteneren ter Prognose-del(17p), del(11q)-u

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Methods. The aim of our analysis is to review data from a random sample of 30 patient of 120 myeloma patients in our MM-registry to assess the safety and tolerability of myeloma therapies in the era of the novel agents. Statistical analyses were descriptive and exploratory. For continuous variables, descriptive statistics were presented, for categorical and dichotomous variables the number and percentage in each category using R version 2.7.2. Results. The median age at the time of admission was 64 years. Eleven of twenty one (52%) patients with available karyotype analyses presented with cytogenetic abnormalities. Nineteen patients had IgG myeloma. Renal insufficiency was present at diagnosis in 9=30 patients. Fifteen patients underwent transplantantion with PAD as the most common induction regimen. Maintenance therapy after SCT was considered beneficial in the majority of patients (10=15). Seventeen of thirty patients have received  1 novel agent. Most of the non-SCT patients received novel drugs as upfront-therapy. Therapy in second line setting was dominated by novel therapies (82%). In total, 37% of the patients experienced clinically relevant drug-related toxicities, in 7 cases related to Thal, memo Suppl 2/09

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Bor or Len. Five patients received Thalidomide-based regimens, two of these with clinically relevant toxicities. Twelve patients received Bortezomib-containing therapies, six with toxicities (e.g. 5 PNPs). Seven patients were exposed to Lenalidomide with neutropenia in one patient. Conclusions. In majority of patients side effects of Lenalidomide are limited to cytopenia which presents a well-controllable problem. Many patients treated with Bortezomib or Thalidomide suffer from polyneuropathy. This side effect often presents difficulties in treatment and causes loss of quality of life for patients. Thus Lenalidomide constitutes an advantage in patients pretreated with PAD to maintain remission as well as in patients with certain risk for polyneuropathy due to other comorbidities.

P16 Novel drugs for high-risk B-CLL patients substance library screening N. Wacht, F. Hamacher, T. Melchardt, I. Tinhofer, O. Merkel, R. Greil Laboratory for Immunological and Molecular Cancer Research, 3rd Medical Department, Paracelsus Private Medical University, Salzburg, Austria

To identify drugs, which preferentially kill B-CLL cells of high-risk patients (CD38 high, p53del, IgVH unmut) from a set of preselected (size, solubility, diversity) substances we performed a screen with 1496 natural compounds. The model system was a T-ALL cell line (CEMC7H2), which mimics the genetic background of a p53 deleted patient. The T-ALL screen and the screen with patient derived B-CLL cells identified 53 substances which were active in both cell systems. These compounds were then tested in B-CLL cells of 9 high-risk patients. Only substances that were active in >80% of those patients were selected for further development (46). After toxicity tests in both cancer and non-cancer human cell lines derived from different tissues we have identified nine substances which were effective in B-CLL cells and show minimal toxicity the cell lines tested (HepG2, SHP77, WS1 HEK-293 and HUVECs). B- and T-cells of healthy donors and also T-cells of B-CLL patients were considerably less affected. In B-CLL cells of fludarabine resistant and del17p13 patients the selected substances could induce apoptosis effectively. These substances are potentially novel drugs for the treatment of B-CLL high-risk patients. Patent applications are being evaluated. In future studies, myelotoxicity tests and tests with Tcl1 transgenic mice are planed to show in vivo efficacy.

P17 A microRNA signature for NPM-ALK lymphoma O. Merkel1, D. Laimer2, F. Hamacher1, M. Scheideler3, Z. Trajanoski3, R. Greil1, L. Kenner2 1

Laboratory for Immunological and Molecular Cancer Research, Salzburg, Salzburg, Austria 2 Ludwig Boltzmann Institute for Cancer Research, Vienna, Austria 3 Institute for Genomics and Bioinformatics, Graz University of Technology, Graz, Austria

About half of the patients with Anaplastic Large Cell Lymphoma (ALCL) bear the t(2;5) (p23;q35) translocation which results in the expression of the chimeric NPM-ALK (Nucleophosmin-Anaplastic Lymphoma Kinase) protein. The ALK-kinase is constitutively activated and autophosphorylated through NPM-induced dimerization and sends its signals to many different growth promoting and antiapoptotic pathways including PI3K=Akt1 and Jak=Stat. NPM-ALK induces regulatory T-cell characteristics and has been reported to enhance AP1 family members cJun and JunB. Recently, miRNAs have emerged as tiny but potent molecules to regulate cell differentiation and proliferation. To elucidate the potential role of miRNAs in this disease we used the mouse model which expresses the human NPM-ALK fusion protein in T-cells. First we compared the miRNA expression of healthy spleen tissue with the NPM-ALK lymphomas of littermate controls. Interestingly, we found a massive deregulation of 61 miRNAs. We then studied miRNA expression in three human ALCL cell lines (Karpas 299, SR-786 and SU-DHL-1) bearing the NPM-ALK translocation and compared them to a pool of purified T-cells from healthy donors. Also in this case about 60 miRNAs were deregulated. When we compared the deregulated murine miRNA group to the group derived from human cell lines we found an overlap of more than 31%. In this common NPM-ALK signature we found miR-17, miR-18a, mir20a=b and miR-106a to be upregulated and miR-22, miR-26a, miR-29a=b, miR-30a, miR-101, miR-125a=b, miR-146a, miR-146b-5p, miR-191, miR-195, miR-342-3p and miR-374 to be downregulated. Interestingly, all upregulated genes belong to the miR-17-92 cluster which has been shown to be c-myc regulated.

P18 The Austrian myeloma registry (AMR): report from the 1st year E. Willenbacher1, J. Drach2, H. Ludwig3, G. Gastl1, M. Verdino4, W. Willenbacher1 1

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Department of Internal Medicine V (Hematology and Oncology), Medical University of Innsbruck, Innsbruck, Austria

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2

Division of Oncology, Department of Medicine I, Medical University of Vienna, Vienna, Austria 3 1st Medical Department, Center for Haematology and Medical Oncology, Wilhelminenspital, Vienna, Austria 4 Verdino.com, Vienna, Austria

In July 2008 the first IRB approval for the AMR, an Internet based, anonymized clinical registry devoted to Multiple Myeloma (MM), intending to collect real life data covering all aspects of treatment and the natural course of MM in Austria was secured. After a single centre pilot optimization phase, further roll-out of the system is under way in multiple austrian haematology centres of different sizes and structure (academic, non-academic) since Jan. 2009 and up to now (Feb. 2009) c. 120 pts. have been accrued. At reference centres patient data way back to the year 2000 is being collected to compile a data set for historical control group generation, facilitation of matched pair analysis and longitudinal study approaches. This data set will be open to interested researchers on application to the AMR scientific advisory board. Statistical tools enabling free combination of parametres beyond purely descriptive statistics (e.g. putative predictive or prospective risk factors combined to course of disease, or different treatment modalities and schedules) as well as data export facilities to standards statistical software applications (e.g. SPSS and EXCEL) are established just now and will serve individual centres to monitor their MM pts. quite effectively and furthermore enable anonymized benchmark analysis. Perspectives and putative applications of the system: like linking with clinical trial data bases of national study groups or a nationwide virtual myeloma biobank which could lead to a win-win situation for all interested clinicians and basic researchers in the field will be discussed.

P19

outcome has been described. We report on 2 pts. with IgM MM with alternative features. Methods. Cytogenetic analysis was performed using interphase FISH covering major loci of known prognostic significance. Results. In pt. 1 (60y, m) (IgM MM, ISS II, Durie IIIA) FISH for IGH rearrangements was negative, whereas loss of 13q and 17p loci was deteced (monosomy 13). Treatment according to the Richardson protocol was initiated. The pt. achieved sCR after escalation to VTD. In pt. 2 (67y, f) (IgM MM with ISS II, Durie IIIA) a t(11;14) negative IGH rearrangement was detectable. After 6 þ 2 cycles of chemotherapy according to the Richardson protocol a PR according to EBMT criteria and a complete cytogenetic remission were achieved. Conclusions. We report on two patients with IgM MM and distinct cytogenetic abnormalities. In contrast to a recent report, the cytogenetic aberrations detected in these 2 pts. were, not a t(11;14) or a deletion 13q, but an IGH rearrangement not involving CCND1, BCL6 and MALT1, as well as a combination of a monosomy 13 and a p53 deletion. Based on these high-risk features the BD regimen was chosen for the 1st-line therapy, and proved to be effective in the short term. An update on the clinical course of these pts. will be presented and discussed.

P20 Final results of oral fludarabine with concomitant subcutaneous alemtuzumab in relapsed=refractory B-chronic lymphocytic leukaemia (B-CLL): the FLUSALEM study A. Egle1, T. Melchardt1, L. Pleyer1, I. Tinhofer2, A. Lang3, F. Keil4, J. Thaler5, E. Gunsilius6, M. Fridrik7, R. Greil1 1

Bortezomib (B) based therapy in two cases of IgM myeloma: an effective therapeutic option for a rare disease with distinct cytogenetic features E. Willenbacher1, M. Erdel2, U. Strasser3, G. Gastl1, S. Schmidt1, W. Willenbacher1 1

Department of Internal Medicine V (Hematology and Oncology), Medical University of Innsbruck, Innsbruck, Austria 2 Division of Clinical Genetics, Department of Medical Genetics, Molecular and Clinical Pharmacology, Medical University of Innsbruck, Innsbruck, Austria 3 Institute of Pathology, Medical University of Innsbruck, Innsbruck, Austria

Introduction. IgM myeloma is a rare disease (<0.5% of all myelomas=0–2% of all IgM paraproteins). An association with the t(11;14) translocation, as well as with deletion 13 known to be associated with poor clinical

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Laboratory for Immunological and Molecular Cancer Research, 3rd Medical Department, Paracelsus Private Medical University, Salzburg, Austria 2 University Medical Center Charité, Berlin-Buch, Germany 3 Department of Medicine, Hospital Feldkirch, Feldkirch, Austria 4 Department of Hematology and Oncology, Hospital Leoben, Leoben, Austria 5 Department of Medicine IV, Klinikum Wels-Grieskirchen, Austria 6 Department of Internal Medicine I, Center of Internal Medicine, Medical University of Innsbruck, Innsbruck Austria 7 Department of Internal Medicine 3, Oncology, General Hospital Linz, Linz, Austria

Relapse of B-CLL presents a crucial problem to patients and clinicians. Using an alemtuzumab=fludarabine combination the German study group showed a high 83% ORR in 36 relapsed=refractory patients, but did not report risk factors or MRD analysis. Also a greatly reduced dose of three times 30 mg of intravenous alemtuzumab per cycle (FluCam regimen; Elter, JCO 2005) was used. Our FLUSALEM protocol combines four cycles of oral memo Suppl 2/09

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fludarabine (30 mg=m2 3d) with continuous alemtuzumab therapy (30 mg s.c. 3  weekly for 16 weeks), increasing the cumulative alemtuzumab dose and allowing an out-patient mangement. Sixteen patients are evaluable, median age was 60 years, 50% of patients had stage Rai III=IV and median follow up was 18 months. Overall response was 87.5% (CR rate 56%, compared to 30% in the FluCam Study). Median PFS was 16 months (compared to 12 months in the dose-reduced FluCam) and 5 of 16 patients show continued remission after 500–1300 days. Therefore, 20% of our patients have a longer PFS than the longest reported from the FluCam study. Grade III infection was observed in four patients (25%, 3 CMV reactivations and 1 PJP in a trimethoprim-intolerant patient on pentamidine inhalation prophylaxis). No fatal infections were observed. CD4 depletion was profound and prolonged (3=4 with CD4 < 200=ml after 6 months). Both, CD38 risk and mutation status were not predictive of PFS or response quality. MRD analysis by four colour flow cytometry (Rawstron, Blood 2001) was performed. MRD negativity at 2 months after the end of therapy predicted a trend in PFS (p ¼ 0.09) and identified all but one patient with a PFS above the median. In conclusion, oral fludarabine combined with 16 weeks of subcutaneous alemtuzumab was feasible in an out-patient setting. Infectious complications were manageable and response rates were high for previously treated patients. High remission qualities irrespective of risk factors as well as long-term progression-free survival in MRD negative patients was observed.

P21 Secretome analyses of primary bone marrow fibroblasts isolated from MGUS and multiple myeloma patients support the role of the microenvironment for myeloma pathogenesis J. Drach1, A. Slany2, T. Mohr2, J. Griss2, J. Ackermann1, C. Zielinski1, C. Gerner2 1

Division of Oncology, Department of Medicine I, Medical University of Vienna, Vienna, Austria 2 Institute for Cancer Research, Department of Medicine I, Medical University of Vienna, Vienna, Austria

Background. The microenvironment of tumour cells in the bone marrow may be actively involved in the progression of malignant diseases. In multiple myeloma (MM), interactions of bone marrow stromal cells with the malignant plasma cells have gained significant importance as targets for novel therapeutic agents. Aims. Based upon these observations, we aimed at analyzing in detail the secretory capacity of bone marrow (BM) fibroblasts obtained from patients with MGUS and MM to better understand their contribution to disease progression. Methods. We analyzed the secretome of primary bone marrow fibroblasts of patients with MGUS and memo Suppl 2/09

MM by proteome profiling based on highly sensitive mass spectrometry. Results were compared to those obtained with normal BM fibroblasts. Results. The secretome profile of normal BM fibroblasts included various extracellular matrix (ECM) proteins including fibronectin, collagens and laminins, in addition to some chemokines and cytokines including CXCL12, follistatin-like 1, insulin-like growth factor binding proteins 4, 5 and 7; and SPARC. In contrast, the secretion profile of BM-derived fibroblasts from MGUS patients was altered: This included secretion of CSF-1, chitinase-3-like protein 1, insulin-like growth factor II (IGF-II) and insulin-like growth factor binding protein 6. All proteins specific for the MGUS background were also found in BM fibroblasts from MM patients. In addition to those we found specific secretion of stem cell growth factor, stanniocalcin-1, hamartin and matrix metalloproteinase 28 as well as increased amounts of alpha-fetoprotein. Co-culture of primary MM cells with these fibroblasts further stimulated the secretion of ECM proteins. Conclusion. BM fibroblasts from MGUS and MM background display a specifically altered secretion profile, and BM fibroblasts from MGUS background already show tumour-promoting activities as indicated by the expression of IGF-II. Proteome profiling of secreted proteins may thus help to identify relevant tumour-associated proteins, to increase our understanding of cell cooperativity and thereby increase our understanding of progression events in monoclonal gammopathies.

P22 Analysis of the T-cell compartment in the El-TCL1 transgenic mouse model for B chronic lymphocytic leukaemia J. Pinon, C. Heyder, T. Kocher, C. Holler, U. Denk, R. Greil, A. Egle Laboratory for Immunological and Molecular Cancer Research, Paracelsus Private Medical University, Salzburg, Austria

B-cell chronic lymphocytic leukaemia is characterized by a clonal expansion of CD19=CD5 B cells. However, B-CLL patients also exhibit many T-cell abnormalities including increased absolute T-cell numbers, and a relative increase in effector and memory CD4þ T-cells in patients with ‘unmutated’ B-CLL. To further investigate the contribution of T-cell dysfunction on the establishment and progression of B-CLL, we monitored the changes in the T-cell compartment in Em-TCL1 transgenic mice. Similar to human B-CLL, leukaemic TCL1 transgenic mice have increased absolute numbers of T-cells. We also observed a marked increase in the number of memory and effector T-cells compared to naı¨ve T-cells, predominantly in the CD8þ subset. The relative shift from naı¨ve to central memory T-cells parallels the incidence of CD19=CD5 hyperplasia in the TCL1 mice. Furthermore,

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transplantation of tumour cells into congenic recipients could direct similar changes in the T-cell compartment of recipient mice. Because memory and effector T-cells represent antigen-experienced cells, we analyzed the complementarity determining region three of the T-cell receptor of purified CD4þ and CD8þ populations to determine the degree of clonality of the various T-cell families. More clonal T-cells were observed in the infiltrated organs of leukaemic mice. Interestingly, more mono- and oligoclonal CD4þ T-cells have also been observed in the peripheral blood of patients with unmutated B-CLL. Thus, in many aspects, the TCL1 mouse recapitulates many of the T-cell abnormalities observed in CLL patients, particularly those with the unmutated subtype.

P23 Akt inhibition by the novel Akt inhibitor induces B-CLL apoptosis in primary human B-CLL cells with unmutated IGVH and CD38 high expression S. Hofbauer, J. Pinon, I. Tinhofer, T. Hartmann, R. Greil Laboratory for Immunological and Molecular Cancer Research, Paracelsus Private Medical University, Salzburg, Austria

Akt is a key factor in cell survival and is implicated in the progression of many tumour types. In CLL, pharmacological interference with the PI3K=Akt signalling pathway has demonstrated a central role for this axis in CLL cell survival; however, direct targeting of Akt has not been extensively addressed so far. We studied the efficacy of the novel and specific Akt inhibitor AiX in inducing cell death in vitro in primary human CLL cells. In vitro, AiX treatment of primary human CLL cells induced apoptosis and in all tested CLL samples but exhibited a preference for high-risk CLL samples with an unmutated immunoglobulin status or high CD38 expression. CLL cell death was accompanied by increases in the pro-apoptotic members Bim, and decreases in the pro-survival members Mcl-1 and XIAP. Interestingly, Akt inhibition lead to stabilization of p53 by functional blockage of MDM2. In a more complex in vitro co-culture model, treatment with AiX was able to overcome the protective effects mediated by primary stromal cells without interfering with the stromal cell survival. Moreover, AiX was effective in inducing apoptosis in B-CLL cells continuously stimulated with CD40L, a model for CLL lymph node proliferation centres that is also associated with an increased resistance of these cells to various cytotoxic agents. In future we will investigate the effect of Akt inhibition in vivo. Therefore, we will treat mice transplanted with CLL cells from the TCL1A-transgenic mouse model and monitor for reduction in tumour load and delay in disease progression.

P24 Tcl1a expression directly influences Akt signalling and Akt-dependent survival in B-chronic lymphocytic leukaemia cells S. Hofbauer, J. Pinon, I. Tinhofer, T. Hartmann, R. Greil Laboratory for Immunological and Molecular Cancer Research, Paracelsus Private Medical University, Salzburg, Austria

The leukaemic proto-oncogene Tcl1a is a modulator of the cell survival kinase Akt interacting with this kinase in a complex as assessed by overexpression studies thereby regulating the Akt kinase activity. Tcl1a overexpressing mice were shown to develop a CLL similar disease. Furthermore, Akt signalling was shown to play a central role in the survival of human CLL B lymphocytes integrating numerous upstream signals. The possibility of Tcl1a and Akt to build a complex and the role of Tcl1a in the tumourigenesis of CLL, lead us to investigate the potential connection of Tcl1a and Akt activation in human CLL cells. We found dramatically enhanced Tcl1a expression in the CLL cell line MEC-2 compared to the closely related CLL cell line MEC-1 and this corresponded with enhanced Akt phosphorylation as well as with activation of Akt downstream targets. Tcl1a could be co-immunoprecipitated with the Akt isoforms 1 and 2 in MEC2 cells confirming the direct protein–protein interactions of Tcl1a and Akt isoforms. Furthermore, Tcl1a expression levels affected Akt activation as knocking down Tcl1a in MEC-2 cells by siRNA abrogated Akt phosphorylation and Tcl1a overexpression in MEC-1 concomitantly increased Akt phosphorylation. Intriguingly, Tcl1a knockdown in MEC-2 cells sensitized these cells towards fludarabine treatment suggesting a role of the Tcl1a=Akt axis in chemoresponsiveness of CLL cells. In summary, we showed the direct interaction of Tcl1a and Akt isoforms is important for Akt activation and Akt-dependent cell survival in B-CLL. Therefore, interruption of the Tcl1a-Akt interaction or blocking Akt kinase activity is an important strategy in finding a novel treatment regimen for B-CLL.

P25 The role of the VLA-4 integrin in adhesion and survival of chronic lymphocytic leukaemia cells K. Sahakyan, W. Wang, G. Rubenzer, P. Desch, S. Hofbauer, R. Greil, T. Hartmann Laboratory for Immunological and Molecular Cancer Research, Paracelsus Private Medical University, Salzburg, Austria

Growth and survival of chronic lymphocytic leukaemia (CLL) B cells are favoured by microenvironmental

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interactions between CLL and non-tumoural accessory cells. The integrin VLA-4 is an important retention factor for haematopoietic stem cells in the bone marrow microenvironment. Our earlier results furthermore demonstrate a key function of this integrin in the bone marrow homing of CLL cells in murine adaptive transfer models. We now aimed to dissect the role of VLA-4 for cell adhesion and cell adhesion-mediated drug resistance of CLL cells. VLA4 expression on CLL cells was strongly associated with CD38 expression and with unmutated immunoglobulin variable heavy chain (IGVH) gene status. In addition, VLA-4 expression was significantly higher in patients with prognostic unfavourable cytogenetic aberrations, including 17p-, 11q- and trisomy 12. CLL cells that were co-cultured with bone marrow stroma cells were protected against spontaneous and fludarabine-induced apoptosis and this protection was dependent on the direct cell–cell contact. VLA-4 positive CLL cells had a significant higher potential to adhere to stromal cells than VLA-4 negative cells. Concordantly, VLA-4 positive cells were more susceptible towards stromal cell adhesion-mediated fludarabine resistance. Targeting the CLL-stromal cell interactions with a specific inhibitory antibody reduced the adhesion of VLA-4 positive CLL cells to the stroma and antagonized cell adhesion-mediated drug resistance. Our findings provide a rationale to further investigate the therapeutic potential of VLA-4 blockage in high-risk CLL subgroups.

P26 Circulating B-cell chronic lymphocytic leukaemia cells display impaired migration to lymph nodes and bone marrow

to transmigrate across VCAM-1, ICAM-1 and CXCL12 expressing endothelium whereas when LFA-1 expression was regained in subsets of CLL cells, these lymphocytes rapidly transmigrated the endothelium. Furthermore, when injected into tail veins of immunodeficient mice, normal B cells rapidly homed to lymph nodes (LNs) in a LFA-1 dependent manner whereas CLL cells did not. Nevertheless, only residual CLL subsets could re-enter BM whereas, both, normal and CLL cells homed to the mice spleen in a LFA-1- and VLA-4-independent manner. Our results suggest that CLL cells have a reduced capacity to adhere and transmigrate through multiple vascular endothelial beds and poorly home to lymphoid organs other than spleen. Integrin blocking could thus be an efficient strategy to prevent circulating CLL cells from reaching prosurvival niches in LNs and BM but not in spleen.

4. Stem Cells and Transplantation A15 C==EBPa expression marks multipotent haematopoietic and thymic progenitor cell specification towards the myeloid lineage A. Wölfler1,2, A. Danen-van Oorschot1, J. Haanstra1, M. Valkhof1, P. van Strien1, E. Vroegindewij1, T. Cupedo1, I. P. Touw1 1

T. Hartmann1, V. Grabovsky2, W. Wang1, P. Desch1, G. Rubenzer1, S. Wollner3, I. Binsky2, A. Vallon-Eberhard2, A. Sapoznikov2, M. Burger3, I. Shachar2, M. Haran4, M. Honczarenko5, R. Greil1, R. Alon2 1

Laboratory for Immunological and Molecular Cancer Research, Paracelsus Private Medical University, Salzburg, Austria 2 Weizmann Institute of Science, Weizmann, Israel 3 Department of Internal Medicine, Freiburg University Clinic, Freiburg, Germany 4 Kaplan Medical Center, Hematology Institute, Rehovot, Israel 5 BiogenIdec Inc., Cambridge, MA, UK

Homing to secondary lymphoid organs and bone marrow (BM) are central aspects of leukaemic pathophysiology. We investigated the roles of the two major lymphocyte integrins LFA-1 and VLA-4 on B-cell chronic lymphocytic leukaemia (CLL) cells in these processes. We found that the majority of CLL cells expressed significantly reduced LFA-1 due to low b2 integrin transcripts. VLA-4 expression was heterogenous but underwent rapid activation by the BM chemokine CXCL12. CLL cells failed memo Suppl 2/09

Department of Hematology, Rotterdam, Erasmus Medical Center Rotterdam, The Netherlands 2 Division of Hematology, Medical University of Graz, Graz, Austria

Transcription factors control lineage specification and differentiation of stem and progenitor cells. To study a role of C=EBPa, a factor indispensable for the production of granulocytes, in myeloid lineage specification of haematopoietic stem and multipotent progenitor cells (defined as Lin-Sca-1 þ c-Kit þ , LSK), we generated a knock-in mouse model expressing Cre recombinase under the regulation of the Cebpa promoter. Crossing these mice with EYFP reporter mice enabled us to trace Cebpa-driven Cre=EYFP expression in single cells and their progeny. Whilst only 3% of long-term haematopoietic stem cells (CD150 þ CD48-CD34- LSK cells) expressed EYFP, 20% of multipotent progenitors (CD34 þ LSK cells) were EYFP þ indicating an upregulation of C=EBPa already in this early compartment. When cultured in a multilineage cytokine cocktail, EYFP þ LSK cells gave predominantly rise to GM colonies, whereas EYFP- cells formed multiple types of colonies. The specification of EYFP þ LSK cells towards GM lineages was further sup-

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ported in GM-CSF-supplemented colony assays. Surprisingly, a substantial fraction of early thymic progenitors was also positive for C=EBPa (22%), whereas the percentage decreased during T-cell maturation resulting in the lowest EYFP þ fraction amongst the most mature single positive thymocytes (7%). In contrast, the majority of thymic dendritic cells (55%) was EYFP þ suggesting that they are predominantly derived from Cebpa-expressing thymic progenitors. Although myeloid colony-forming potential was generally low in thymic progenitors, it was almost exclusively found in EYFP þ cells. In conclusion, these results establish Cebpa expression as a common marker for myeloid lineage specification in multipotent haematopoietic as well as early thymic progenitor cells.

P27 A single nucleotide polymorphism in the intelectin 1 gene (ITLN1) is associated with acute intestinal graft-vs.-host-disease H. Hauser, O. Krieger, H. Kasparu, J. König, D. Lutz, O. Zach 1st Medical Department, Hospital Elisabethinen, Linz, Austria

Current concepts suggest a major role for innate immunity in the initiation of graft-vs.-host-disease (GvH-D) as well as in Crohn’s disease (CD). Recently, the non-coding C=T polymorphism rs2274910 in intron 3 of the intelectin 1 (ITLN1) gene (human lactoferrin receptor) has been associated with CD. Thus, we tested whether this polymorphism of the ITLN1 gene is also associated with gut GvH-D. We retrospectively typed this polymorphism by TaqMan PCR from peripheral blood from AML patients and their donors. Results from a pilot study with 19 patients were confirmed in a second cohort with 40 patients. In total, 50.8% of patients had a CC genotype. In the pilot study, 2 out of 12 patients with a CC genotype versus five out of seven patients with a T allele had acute intestinal GvH-D (p ¼ 0.045). In the second cohort acute intestinal GvH-D was found in 3=18 patients (16.7%) with a CC genotype and 12=22 patients (54.5%) with a T-allele (p ¼ 0.014). In a combined analysis (n ¼ 59) we found acute intestinal GvH-D in 58.6% of patients with a T-allele versus 16.7% of patients with a CC genotype (p < 0.001). No difference was seen with regard to skin or liver GvH-D. We did not see any association between donor genotypes and GvH-D. The strong association (RR 3.52; 95% CI 1.5–8.2) between this SNP and the incidence of acute intestinal GvH-D is in accordance with reports about SNPs in other genes associated with both acute GvH-D and CD.

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P28 Human MSC expansion in an animal serum-free system: a GMP-compliant protocol for safe and genomically stable MSCs K. Schallmoser1,2, E. Rohde1,2, A. Reinisch1,3, A. C. Obenauf4, C. Bartmann1,3, G. Lanzer2, W. Linkesch3, D. Strunk1,3 1

Stem Cell Research Unit Graz, Medical University of Graz, Graz, Austria 2 Department of Blood Group Serology and Transfusion Medicine, Medical University of Graz, Graz, Austria 3 Department of Hematology and Stem Cell Transplantation, Medical University of Graz, Graz, Austria 4 Institute of Human Genetics, Medical University of Graz, Graz, Austria

Human multipotent mesenchymal stromal cells (MSCs) are currently tested in clinical trials for immunomodulatory and regenerative therapy. Genomic instability in fetal bovine serum (FBS)-driven MSC cultures and tumour formation by MSCs in animals has raised safety concerns. We and others have recently established MSC propagation with pooled human platelet lysate (pHPL) substituting FBS. This study was performed to determine safety of MSCs expanded under humanized conditions Unmanipulated bone marrow aspirates (n ¼ 4) were seeded in A-MEM supplemented with pHPL without antibiotics for clinical scale propagation. MSC quality and function were assessed according to defined release criteria. Array-comparative genomic hybridization (aCGH) was performed using a whole genome oligonucleotide microarray platform. PHPL is highly efficient in stimulating MSC expansion resulting in 780  150 million MSCs or up to four application doses after one passage. Flow cytometry revealed  95% viability, >95% CD73=90=105 reactivity and <2% haematopoietic contamination. We could show adipo=osteo=chondrogenic differentiation potential, endotoxin levels <0.025 EU=mL and negative bacterial= fungal=mycoplasma testing. Array-CGH analysis revealed balanced profiles for all propagated MSC products. Several copy number variations (CNVs; 7 kb C1.8 Mb; n ¼ 33) were also observed previously in normal individuals not associated with phenotype changes. Five detected CNVs ( >60 kb) were not documented in the database of genomic variants. Despite high proliferation rate MSCs propagated in a pHPL-driven system are genomically stable in aCGH. These data extend earlier results showing that MSCs expanded under humanized conditions did not form tumours in experimental animals in vivo, indicating superior safety of the rapidly available MSC transplants compared to currently used FBS-expanded MSCs.

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P29 Successful unrelated haematopoetic stem cell transplantation in two HLA alloimmunized patients U. Posch1, W. Zinke-Cerwenka2, S. Ulrich1, K. Schallmoser1, C. Drexler1, G. Lanzer1 1

Department of Blood Group Serology and Transfusion Medicine, Medical University of Graz, Graz, Austria 2 Division of Hematology and Oncology, Department of Internal Medicine, Medical University of Graz, Graz, Austria

HLA alloimmunization is a significant risk to allogeneic stem cell recipients: it causes platelet refractoriness and may even contribute to graft rejection. We report on two patients with secondary AML transformed from MDS, scheduled for non-myeloablative allogeneic stem cell transplantation (SCT). Second induction chemotherapy was complicated by refractoriness to platelet transfusions. Patient sera were tested for HLA and platelet specific antibodies by complement-dependent cytotoxicity (CDC) and ELISA using commercial test kits (Biotest, GTI). Both the patients were negative for platelet specific antibodies but positive for HLA class I antibodies (patient I: male, polytransfused, anti HLA A2 and B22; patient II: female, anti HLA A1, son positive for HLA A1). Further on all platelet products for these patients consequently had to be HLA compatible, selected either according to the HLA type of single donors or by platelet crossmatch. Whereas in patient II antibodies had already disappeared prior to transplantation, anti HLA A2 of patient I could be identified till day þ130. Stem cell grafts were HLA matched 12=12 and 11=12 (A*2901 vs. A*2902), which assured us that antibodies were not directed against donor antigens. Both patients achieved complete remission on day þ28 with 100% donor chimerism. Respectively, 6 and 11 HLA compatible platelet units were transfused after SCT until discharge and no bleeding events were recorded. Allogeneic SCT is feasible in alloimmunized patients provided that antibodies are not directed against donor antigens and optimal HLA compatible platelet donors are selected to reduce the risk for refractory bleeding events.

P30 Storage of cryopreserved haematopoietic progenitor cells for 5–10 years Konrad Rosskopf, E. Rohde, A. Bahadir, M.-L. Stubenrauch, L. Gerhard Department of Blood Group Serology and Transfusion Medicine, Medical University of Graz, Graz, Austria

It is unclear how long cryopreserved haematopoietic progenitor cells (HPC) keep their functionality. memo Suppl 2/09

Depending on cryopreservation solutions, methods of freezing and storage conditions 5–10 years of storage could be achieved. We examined our freezing strategy of 10% DMSO, computer-controlled freezing and storage in liquid nitrogen by measuring HPC older than 5 years from patients after their death (study group) and compared it with quality controls of autologous HPC which were thawed 6–50 days after cryopreservation (control group). A total of 29 Cryopreserved HPC (n ¼ 17, study group; n ¼ 12, control group) were thawed at 37  C. Nucleated cell counts (NC) were analyzed by automated cell counting. Viable CD34þ and CD45þ cells (7-AAD-) were assessed by FACS. Cell viability was determined by trypan blue exclusion. Clonogeneic capacity ((CFU-GM þ BFUE)=CD34þ) was performed using a commercially available stem cell culture kit. The median (range) duration of cryopreservation was 7.5 years (5.8–10.8) in the study group. In 15 out of 16 cases clonogeneic capacity was detectable. One additional culture was contaminated. The Viability (%) of Study and Control Group was 72 (12–96) and 90 (74–95); CD34þ=7-AAD-(%) was 97 (0– 100) and 98 (74–100) resp. Clonogeneic capacity (%) was 6 (0–52) and 9 (5–64). Long-term storage of HPC apheresates with freezing strategy of 10% DMSO, computer-controlled freezing and storage in liquid nitrogen leads to acceptable viability of CD34þ HPC compared to short-term storage. Functional capacity is retained in 94% (15=16) after a storage time up to ten years in vitro.

P31 Atherogenesis as a risk factor accompanying cellular therapeutic angiogenesis? E. Rohde1,2, K. Schallmoser1,2, A. Reinisch1,3, N. Hofmann1,3, T. Pfeifer4, E. Fröhlich5, G. Rechberger6, G. Lanzer2, D. Kratky4, W. Linkesch3, D. Strunk1,3 1

Stem Cell Research Unit Graz, Medical University of Graz, Graz, Austria 2 Department of Blood Group Serology and Transfusion Medicine, Medical University of Graz, Graz, Austria 3 Division of Hematology and Oncology, Department of Internal Medicine, Medical University of Graz, Graz, Austria 4 Institute of Molecular Biology and Biochemistry, Medical University of Graz, Graz, Austria 5 Center for Medical Research, Medical University of Graz, Graz, Austria 6 Cener for Molecular Biosciences, Karl-Franzens-University, Graz, Austria

Current clinical trials for therapeutic angiogenesis use blood- or marrow-derived transplants containing endothelial progenitor cells (EPCs), monocytes and mesenchymal stromal cells (MSCs) to support vascular regeneration. Safety concerns regarding atherosclerosis aggravation have emerged since all three cell types can also

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Abstracts

contribute to atheroma formation. We examined foam cell formation potential of these cells after lipid exposure in vitro as a surrogate marker for potential pro-atherogenic side effects. Foam cell development was tested with human EPCs, monocytes and MSCs either immediately exposed to lipoproteins or subjected to a three day pro-angiogenic induction prior to lipid loading. Intracellular lipid accumulation was detected by Nile Red staining with fluorescence laser-scanning microscopy. After 3D mapping, the number of lipid droplets per cell was determined using ImageJ software. Morphometric observations were confirmed by flow cytometry and cholesterol measurement. Characteristic foam cells developed from monocytes with and without pro-angiogenic induction. However, increased lipid accumulation was found after pro-angiogenic culture. EPCs and MSCs did not accumulate lipids or form lipid droplets under culture conditions mimicking those applied in clinical trial protocols. The foam cell potential of monocytes in vitro was strikingly evident, especially after prior pro-angiogenic culture. These data raise serious concerns that cellular therapy with monocyte-containing haematopoietic cell preparations may be counterproductive or even aggravate atheroma formation in patients with cardiovascular disease if underlying pathologic conditions are not appropriately reverted. We therefore argue that the role of transplanted cells in the various aspects of vascular homeostasis and regeneration must be re-examined prior to further clinical trials.

P32 Allogeneic haematopoietic stem cell transplantation for refractory Hodgkin's disease with modified Campath-BEAM conditioning S. Eder, W. Rabitsch, P. Kalhs, A. Schulenburg, M. Mitterbauer, C. Zielinski, H. Greinix Department of Internal Medicine I, Bone Marrow Transplantation, Medical University of Vienna, Vienna, Austria

Introduction. Refractory Hodgkin’s disease has a poor prognosis and myeloablative conditioning for allogeneic haematopoietic cell transplantation (allo-HCT) in these patients has been associated with high transplantrelated mortality (TRM). Meanwhile, reduced-intensity conditioning is available for patients with high risk of TRM including elderly patients, ones with comorbidities and patients after autologous HCT. Usually, Campathbased conditioning protocols investigate a total of 100 mg of Campath-1H and resulting immunodeficiency with severe infections has been of concern. Therefore, we administered at our centre a modified Campath-BEAM protocol with dose reduction of Campath-1H. Patients and methods. Eleven patients (8 male, 3 female) with a median age of 35 (range 24–44) years with refractory Hodgkin’s disease were referred to our centre for allo-HCT after a median of 4 (range 3–6) previous

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chemotherapy regimens. In addition, 7 patients had received a previous autologous HCT. Duration of Hodgkin’s disease was a median of 43 (range 13–100) months until allo-HCT. Reduced-intensity conditioning for allo-HCT consisted of chemotherapy according to the BEAM regimen given in the usual dose with BCNU (carmustin) at 300 mg=m2 on day-6, etoposide at 200 mg=m2 on days-5 until -2, cytarabine at 200 mg=m2 on days-5 until -2 and melphalan at 140 mg=m2 on day-1. Campath-1H (alemtuzumab) was given in a dose of 10 mg on days-5 to -1. Graft-versus-host disease (GvHD) prophylaxis consisted of cyclosporine A (CSA) and all patients received peripheral blood stem cells from a HLA-identical related (n ¼ 7) or unrelated (n ¼ 4) donor. Results. Ten of 11 patients achieved rapid complete haematologic engraftment whereas one rejected the allogeneic graft and died of Hodgkin’s disease 17 months after allo-HCT. Three of ten eligible patients experienced acute GvHD. One patient died of multi-organ-failure on day 146 after allo-HCT. Around 3 months after allo-HCT in five patients complete donor chimerism in multiple cell lineages and in another five patients mixed chimerism in either the CD3 þ subset (n ¼ 4) or unseparated peripheral blood cells (n ¼ 1) was observed. Best response after alloHCT was complete remission (CR) in six of ten evaluable patients (60%), and partial remission (PR) in three patients (30%) including one very good partial remission. Relapse= progression of Hodgkin’s disease was seen in 4 of 11 patients (36%). One of these achieved another CR after donor lymphocyte infusions (DLI). Currently, he has been alive for 49 months after allo-HCT. Three patients experienced chronic GvHD including one after DLI. As of February 15, 2009 seven of 11 patients (64%) are alive after a median of 20 (range 4–53) months after allo-HCT with six in continuous complete remission and one in very good PR. Conclusions. Allogeneic HCT with reduced-intensity conditioning according to the modified Campath-BEAM protocol is an efficient and well-tolerated therapeutic option for patients with refractory Hodgkin’s disease. Administration of DLI should be considered for residual disease to improve long-term treatment outcome in these patients.

5. Solid Tumours A16 Analysis of common germline polymorphisms to predict metastasis in node-positive breast cancer G. Knechtel1, A. Gerger1, W. Renner2, T. Langsenlehner3, G. Hofmann1, J. Szkandera1, H. Samonigg1, P. Krippl4, U. Langsenlehner5 memo Suppl 2/09

Abstracts

1

Department of Internal Medicine, Division of Oncology, Medical University Graz, Graz, Austria 2 Clinical Institute of Medical and Laboratory Diagnostics, Medical University Graz, Graz, Austria 3 Clinic of Therapeutic Radiology and Oncology, Medical University Graz, Graz, Austria 4 Department of Internal Medicine, Regional Hospital of Fuerstenfeld, Fuerstenfeld, Austria 5 Internal Outpatient Department, Steiermaerkische Gebietskrankenkasse, Graz, Austria

Women with breast cancer which initially involves local lymph nodes have a higher risk for local recurrence or developing metastases. Recent data suggest that germline polymorphism is a significant, previously unrecognized factor in breast cancer progression and metastasis. We assessed the influence of common germline genetic polymorphisms in disease-free survival amongst women diagnosed with lymph-node-positive breast cancer. DNA samples from 216 women with histological confirmed breast cancer were analyzed for a comprehensive panel of 16 selected polymorphisms in a variety of pathways. The association of polymorphisms and disease-free survival was analyzed using univariate and multivariate Cox-regression analysis. The rare allele of the transforming growth factor-beta 1 (TGFB1) 29T > C polymorphism was significantly associated with poor disease-free survival (p ¼ 0.026, risk ratio of recurrence (RR) ¼ 1.361, 95% confidence interval (CI) ¼ 1.037– 1.786). After statistically considering known prognostic factors (age at diagnosis, tumour size, clinical stage, histological grade, oestrogen receptor status, progesterone receptor status and treatment modalities) the association remained significant (p ¼ 0.005, RR ¼ 1.503, 95% CI ¼ 1.134–1.993). No association was found for MTHFR 677C > T, VEGF 936C > T, CCND1 870G> A, FASLG 844C > T, FAS 1377G> A, FAS 670A> G, GNB3 825C > T, ITGA2 807C > T, ITGA2 1648G> A, ITGB3 176T > C, MMP1 -1607 1G=2G, MMP3 5A=6A, PTGS2 8473T > C, IL10 592C > A and SULT1A1 638G> A polymorphisms and disease-free survival. We suggest that the TGFB1 29T > C polymorphism could modify the risk for metastasis in women with lymph-node-positive breast cancer.

P33 Colorectal cancer survival and its association to a common hereditary polymorphism in the gene of death receptor 4 (TNFRSF10A) G. Hofmann1, T. Langsenlehner2, F. Fuerst3, A. Gerger1, U. Langsenlehner4, G. Knechtel1, J. Szkandera1, P. Krippl5, W. Renner6 1

Division of Oncology, Department of Internal Medicine, Medical University of Graz, Graz, Austria 2 Department of Therapeutic Radiology and Oncology, Medical University of Graz, Graz, Austria 3 Division of Rheumatology, Department of Internal Medicine, Medical University of Graz, Graz, Austria memo Suppl 2/09

4

Internal Outpatient Department, Steiermärkische Gebietskrankenkasse, Graz, Austria 5 Department of Internal Medicine, Regional Hospital Fürstenfeld, Austria 6 Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Graz, Graz, Austria

Background. Cell surface death receptors, such as death receptor 4 (DR 4, TNFRSF10A, TRAILR-1), are well established key players in the apoptotic cascade. A single nucleotide polymorphism within the extracellular domain of the DR 4 gene, Glu228Ala (683A> C), has been associated with modulation of the risk for a variety of different cancers and tumour progression. Aim of the present study was to evaluate the role of this polymorphism for survival of colorectal cancer patients. Methods. A retrospective analysis including 433 patients with histologically confirmed colorectal cancer was performed. Genotypes were analyzed by a fluorogenic exonculease assay (TaqManTM). A Cox regression model including DR 4 genotypes, age at diagnosis, tumour grading, primary tumour size, number of lymph nodes examined, number of metastatic lymph nodes, tumour stage (AJCC) and application of fluorouracil-based adjuvant chemotherapy was used to estimate the effect of the DR 4 genotype on survival. Results. DR 4 Glu228Ala genotype frequencies were 63.2% (AA), 33.5% (AC) and 3.3% (CC). Twenty eight patients were excluded from the Cox-regression analysis because of missing values. Of the remaining 408 patients, 69 (18%) died during a follow-up of maximum 10 years. Mean follow-up time was 58  34 months (median 55 months). There was no correlation between the different genetic variants of DR 4 and survival in this study population detectable (relative risk 0.97, 95% confidence interval 0.638–1.51; p ¼ 0.902). Conclusion. We conclude that the DR 4 Glu228Ala polymorphism is not correlated with survival in colorectal cancer patients.

P34 The FASL -844T>C polymorphism is not associated with metastasis-free survival in patients with colorectal cancer G. Hofmann1, T. Langsenlehner2, F. Fuerst3, A. Gerger1, U. Langsenlehner4, H. Samonigg1, H. Clar5, P. Krippl6, W. Renner7 1

Division of Oncology, Department of Internal Medicine, Medical University of Graz, Graz, Austria 2 Department of Therapeutic Radiology and Oncology, Medical University of Graz, Graz, Austria 3 Division of Rheumatology, Department of Internal Medicine, Medical University of Graz, Graz, Austria 4 Internal Outpatient Department, Steiermärkische Gebietskrankenkasse, Graz, Austria 5 Department of Orthopaedic Surgery, Medical University of Graz, Graz, Austria

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Abstracts

6

Department of Internal Medicine, Regional Hospital Fürstenfeld, Austria 7 Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Graz, Graz, Austria

Background. Apoptosis is an important biological process for maintaining homeostasis and abnormal regulation of apoptosis is likely to contribute to tumourigenesis and development of metastases. FAS-ligand (FASL) can trigger the cell death cascade via cross-linking with FAS (TNFRSF 6) and hence is a key player for apoptosis. A single nucleotide polymorphism in the promoter-region of the FASL gene, -844T> C, modulates the signalling cascade. Aim of the present study was to evaluate the role of this polymorphism in the risk of developing metastases for patients with colorectal cancer. Methods. We performed a retrospective analysis including 436 patients with histologically confirmed colorectal cancer. A Cox-regression model including FASL -844 genotypes, age at diagnosis, tumour grade, tumour size, Stage (AJCC), number of lymphnodes evaluated, number of pathologic lymph nodes and admistration of adjuvant 5-FU containing chemotherapy was used to estimate the effect of the FASL genotype on the risk of developing metastases. Results. FASL -844 genotype frequencies were 40.9% (TT), 45.9% (TC) and 13.2% (CC). Fifty-five patients were excluded from the Cox-regression analysis because of missing values. Of the remaining 381 patients, 149 (39.1%) developed metastases during a median followup time of 41 months (range 0–198). In this study population there was no correlation detectable between the FASL -844T > C polymorphism and development of metastases (relative risk per allele 1.11; 95% confidence interval 0.88–1.39; p ¼ 0.364). Conclusion. The FASL -844T> C polymorphism is not associated with an increased risk for developing metastases in patients with colorectal cancer.

P35 Vasohibin induces remodelling of the vascular network G. Untergasser, J. Kern, M. Steurer, G. Gastl, E. Gunsilius Tumor Biology and Angiogenesis Laboratory, Division of Hematology and Oncology, Medical University of Innsbruck, Innsbruck, Austria

The murine homologue of human vasohibin (mVASH1), a putative anti-angiogenic protein, was investigated for its inhibitory effects on in vitro and in vivo angiogenesis. Cell growth and migration were analyzed in murine fibroblasts, smooth muscle cells and endothelial cells. Moreover, angiogenic sprouting was studied in human umbilical vein endothelial cells (HUVEC) in the spheroid sprouting assay. In vivo

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effects on blood vessel formation were investigated in the chorioallantoic membrane (CAM) assay and in the C57BL=6 melanoma model. Purified mVASH1 protein induced apoptosis and inhibited cell growth of murine fibroblasts, but not of vascular aortic smooth muscle cells (AoSMC) or endothelial cells. Adenoviral overexpression of mVASH1 inhibited capillary sprouting of HUVEC in the spheroid assay. Administration of recombinant murine VASH1 inhibited growth of large vessels in the CAM assay and promoted the formation of a dense, fine vascular network. Murine VASH1-overexpressing B16F10 melanomas displayed a reduction in large vessels and a significant increase in CD31-positive microvessels. Moreover, tumours showed more mature microvessels that stained positive for the mural cell markers alphasmooth muscle cell actin (ASMA) and proteoglycan (NG2). Our data imply that murine VASH1 causes angiogenic remodelling by inhibiting large vessel growth and supporting the formation of a vascular bed consisting predominantly of more mature microvessels.

P36 Association of Interleukin-10 gene variation with breast cancer prognosis A. Gerger1, W. Renner2, T. Langsenlehner3, G. Hofmann1, G. Knechtel1, J. Szkandera1, H. Samonigg1, P. Krippl4, U. Langsenlehner5 1

Department of Internal Medicine, Division of Oncology, Medical University Graz, Graz, Austria 2 Clinical Institute of Medical and Laboratory Diagnostics, Medical University Graz, Graz, Austria 3 Clinic of Therapeutic Radiology and Oncology, Medical University Graz, Graz, Austria 4 Department of Internal Medicine, Regional Hospital of Fuerstenfeld, Fuerstenfeld, Austria 5 Internal Outpatient Department, Steiermaerkische Gebietskrankenkasse, Graz, Austria

Genetic polymorphisms are responsible for interindividual variation and diversity and have been recently considered as the main genetic elements involved in the development and progression of cancer. We examined associations between common germline genetic variants in 7 genes involved in folate metabolism, cell proliferation and apoptosis, prostaglandin synthesis, detoxification of compounds and inflammation, and disease free survival amongst women diagnosed with invasive breast cancer. DNA from up to 432 women was genotyped for 8 polymorphisms. The genotypes of each polymorphism were tested for association with disease-free survival using univariate and multivariate Cox-regression analysis. The model was adjusted for known breast cancer prognostic factors. The rare allele of the IL-10 592C > A polymorphism was significantly associated with poor disease-free survival (p ¼ 0.018, risk ratio of recurrence (RR) ¼ 1.45, 95% confidence interval (CI) ¼ 1.06–1.98), which was not attenuated after memo Suppl 2/09

Abstracts

adjusting for age at diagnosis, tumour size, lymph node status, clinical stage, histological grade, oestrogen receptor status, progesterone receptor status and treatment modalities (p ¼ 0.019, RR ¼ 1.48, 95% CI ¼ 1.066–2.044). No association was found for MTHFR 677C> T, TGFB1 29T> C, FASLG 844C> T, FAS 1377G> A, FAS 670A > G, PTGS2 8473T > C and SULT1A1 638G> A polymorphisms and disease-free survival. Our data suggest that the rare allele of IL-10 592C > A is an independent prognostic marker in breast cancer for disease-free survival.

but not PFS in ovarian cancer, whereas IL-23 expression did not appear to be independently associated with either OS or PFS. In summary, in contrast to the recently proposed opposing roles of IL-12 and IL-23 for cancer growth in rodents, our data from a large patient cohort rather suggest that high intratumoural expression levels of IL-12 mRNA in advanced and IL-23 mRNA in early phase disease is associated with a superior outcome.

P38 P37 The role of IL-12 and IL-23 in ovarian cancer

Etablierung von FISH und Real Time PCR zum Nachweis von TMPRSS2 Rearrangements bei Prostatakarzinomen

A. M. Wolf1, H. Rumpold2, D. Reimer3, G. Gastl1, C. Marth3, A. Zeimet3, D. Wolf1

M. Schaschinger1, R. Marschon1, F. Stoiber2, H.-C. Bösmüller3, W. Loidl2, G. Webersinke1

1

1

Interleukin-12 (IL-12) and IL-23 are members of a small family of cytokines, which share a common p40 subunit that is either covalently linked to the p35 subunit giving rise to IL-12 or to the p19 subunit forming IL-23. Recent data from genetic animal models suggest that in contrast to the central role of IL-12 for tumour-immunosurveillance, its close relative IL-23 promotes cancer incidence and growth-supporting inflammatory responses involved in cancer development (i.e. MMP-9 induction, increased angiogenesis but reduced CD8 T-cell infiltration) [Langowski JL, Nature 2006]. Thus, it was the aim of our current project to evaluate the clinical significance of these findings by quantification of mRNA expression level of the IL-12 specific subunit p35 and the IL-23 specific subunit p19 in a large and well-documented cohort of ovarian cancer patients (n ¼ 115) as well as in respective control samples from healthy ovary tissue specimens (n ¼ 20). Both the cytokines were expressed at higher levels in ovarian cancer specimens as compared to healthy control samples (highest expression levels in serous carcinoma). Surprisingly, there was a significant positive correlation of IL-12 and IL-23 expression in both, cancerous and healthy ovarian tissue. High IL-12 expression (the median was chosen as cut-off value for all analyses) was associated with a significant better overall survival (OS) in advanced stage disease (FIGO III and IV) but not in early stage (FIGO I and II) disease. Vice versa, high IL-23 expression was correlated with a superior OS solely in early stage disease (FIGO I and II). IL-23 expression had no impact on progression-free survival (PFS), whereas in parallel to OS, high IL-12 expression was significantly associated with superior PFS in advanced phase disease. Finally, multivariate analysis revealed that high IL-12 expression represents an independent predictor for OS

Einleitung. Chromosomale Rearrangements spielen in der Genese von Leuka¨mien, Lymphomen und Sarkomen eine wesentliche Rolle. Rezente Studien zeigten erstmals auch beim Prostatakarzinom einen hohen Prozentsatz an Fusionen des androgen-regulierten TMPRSS2 Gens (Transmembran-Protease Serin 2) mit diversen Mitgliedern der ETS-Transkriptionsfaktor Familie (ERG, ETV1 und ETV4). Diese Erkenntnis ist ein erster Hinweis darauf, dass derartige Rearrangements auch eine entscheidende Rolle in der Entwicklung von epithelialen Tumoren spielen ko¨nnten. Ziel dieser Arbeit war, Methoden zum Nachweis der Rearrangements zu etablieren. ¨ r ERG, ETV1 und ETV4 Methoden. FISH-Sonden fu wurden durch Fluoreszenzmarkierung von BAC-Klonen (Bacterial Artificial Chromosomes) hergestellt und an Paraffinschnitten von Prostatakarzinomen (Gleason 5–10) getestet. Ein Real-Time PCR Verfahren zum Nachweis von TMPRSS2-ERG-Rearrangements wurde an einer Zelllinie mit Fusion (VCaP) etabliert und ko¨nnte in weiterer Folge zum Nachweis von Tumorzellen im Urin dienen. Ergebnisse. An 15 bisher mittels FISH getesteten Pra¨paraten konnten zwei TMPRSS2-ERG-Rearrangements nachgewiesen und am Gewebe auch mittels PCR verifiziert werden. Durch Verdu¨nnung von VCaP-Zellen in Urin konnte die derzeitige PCR-Nachweisgrenze bei 20 Tumorzellen pro ml Urin ermittelt werden. Zusammenfassung. Zum Nachweis von TMPRSS2Rearrangements beim Prostatakarzinom konnten FISHMethoden sowie ein PCR-Verfahren fu¨r Urinproben erfolgreich etabliert werden. Speziell die nicht invasive PCR-Methode ko¨nnte die Diagnostik von Prostatakarzinomen erga¨nzen, jedoch sind weitere Untersuchungen notwendig, um die Praxistauglichkeit der Tests zu verifizieren.

Internal Medicine V, Hematology and Oncology, Medical University of Innsbruck, Innsbruck, Austria 2 KH der Barmherzigen Schwestern, Linz, Austria 3 Department of Gynecology and Obstetrics, Medical University of Innsbruck, Innsbruck, Austria

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Department of Molecular Biology, Hospital Barmherzige Schwestern, Linz, Austria 2 Department of Urology, Hospital Barmherzige Schwestern, Linz, Austria 3 Department of Pathology, Hospital Barmherzige Schwestern, Linz, Austria

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P39 Sensitivität der Bestimmung von KRAS-Mutationen: Vergleich zwischen DHPLC, Sequenzierung und Real-Time PCR J. Sunzenauer1, M. Bernkopf1, H.-C. Bösmüller2, G. Webersinke1, W. Kranewitter1

P40 Partnerschaft und Krebs – erleben Krebspatienten und deren Partner in ihrer Beziehung durch die Erkrankung Veränderungen – kann ein sekundärer bzw. tertiärer Krankheitsgewinn festgestellt werden?

1

Department of Molecular Biology, Hospital Barmherzige Schwestern, Linz, Austria 2 Department of Pathology, Hospital Barmherzige Schwestern, Linz, Austria

Einleitung. Moderne Antiko¨rpertherapien des Colorectalkarzinoms (Cetuximab bzw. Panitumumab) hemmen die Funktion des EGFR, wodurch die nachgeschaltete RAS=RAF=MAPK-Signalkaskade inaktiviert wird. Ca. 30– 50 % der Colorectalkarzinome weisen jedoch Mutationen im KRAS-Gen auf, die zu einer konstitutiven Aktivierung dieser Signalkaskade und damit zu einer Unwirksamkeit der Antiko¨rpertherapie fu¨hren. Dabei sind im KRAS-Gen nahezu ausschließlich die Codons 12, 13 und 61 von Mutationen betroffen. Zur Detektion dieser Methoden werden verschiedene kommerzielle Testkits angeboten (vor allem mutations-spezifische Real-Time PCR), die jedoch insbesondere Mutationen im Codon 61 nicht erfassen. Um auch die seltener auftretenden Mutationen zu erfassen, die nicht von kommerziellen Testkits abgedeckt werden, haben wir zusa¨tzlich zur Sequenzierung eine DHPLC-Methode entwickelt, die ein sensitives Screening auf Mutationen erlaubt. Methoden. Zur Bestimmung der Nachweisgrenze fu¨r die Mutationen G12A, G12 V und G13D wurden jeweils 3 unabha¨ngige Verdu¨nnungsreihen von Zellkulturen, die die jeweilige Mutation im KRAS-Gen tragen, in KRASWildtyp Zellen hergestellt. Die daraus pra¨parierte DNA wurde mit DHPLC, Sequenzierung und einem kommerziell erha¨ltlichen KRAS-Real-Time-PCR Testkit (Therascreen, Roche) analysiert. ¨ r die Kombination aus DHPLC und Ergebnisse. Fu Sequenzierung wurde die Nachweisgrenze mit bis zu 5% mutierten Zellen (2,5 % mutierter DNA-Anteil) bestimmt und wird mit den Daten der Real-Time PCR verglichen. Mit dieser Methode wurde bei u¨ber 300 untersuchten Colorectalkarzinomen ca. 6 % der detektierten Mutationen in Bereichen gefunden, die mit kommerziellen Testkits nicht abgedeckt werden (vor allem Codon 61: Q61 K, Q61 L, Q61H). Zusammenfassung. Mit der entwickelten Methode ist ein sensitiver Nachweis von KRAS-Mutationen mo¨glich. Zusa¨tzlich zu den ha¨ufigen sind damit auch selten auftretende Mutationen nachweisbar, die von kommerziellen Testkits nicht erfasst werden.

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A. Kier, S. Kral, K. Geissler Hospital Hietzing, Vienna, Austria

Partnerschaft und Krebs – erleben Krebspatienten und deren Partner in ihrer Beziehung durch die Erkrankung Vera¨nderungen – kann ein sekunda¨rer bzw. tertia¨rer Krankheitsgewinn festgestellt werden? Hintergrund. Die vorliegende Untersuchung hatte zum Ziel, die Vera¨nderungen in der Partnerschaft wa¨hrend einer Krebserkrankung zu beleuchten und nach einem mo¨glichen sekunda¨ren bzw. tertia¨ren Krankheitsgewinn zu suchen. Methodik. Es nahmen 32 Patienten unserer Abteilung und deren Partner teil. Dafu¨r wurde der bereits standardisierte Fragebogen BSSS (Die Berliner Social Support Skalen) von Schwarzer und Schulz (2000) herangezogen und teilweise adaptiert. Ergebnisse. 27 von 32 Patienten (das sind 84 %) erleben eine Vera¨nderung der Partnerbeziehung in posi¨ tzung tiver Richtung. Die vom Partner erhaltene Unterstu nimmt stark zu, was im Sinne des sekunda¨ren Krankheitsgewinns als Vorteil fu¨r den Erkrankten gesehen wird. 69 % der Patienten gaben bei Fragen nach einer Vera¨nderung der Rollenaufteilung innerhalb der Partnerschaft an, ¨bernehmen. 72 % dass ihre Partner vermehrt Aufgaben u der Partner zeigen eine Tendenz in Richtung vermehrte ¨llung. 37 % der Patienten mit malignen Aufgabenerfu Erkrankungen versuchen ihre Partner vor zusa¨tzlichen ¨tzen. Interessanterweise schu ¨tzen Belastungen zu schu Patienten mit einem soliden Tumor ihren Partner weniger (65 %) als Patienten mit einer ha¨matologischen Systemerkrankung (35 %) (p < 0,04). 24 von 32 (76 %) aller ¨tPatienten bzw. 63 % der Partner erleben mehr Unterstu zung durch das soziale Netzwerk. Der Anteil der Patienten mit Wunsch nach Autonomie (54 %) war a¨hnlich dem Anteil der Patienten mit Wunsch nach vermehrter Unterstu¨tzung (46 %). 28 von 32 Patienten erleben durch die ¨ nderung ihrer Ansicht u ¨ber den Sinn Erkrankung eine A des Lebens. Schlussfolgerung. Da die Mehrzahl der Patienten eine positive Vera¨nderung der Partnerbeziehung und eine ¨ bernahme von Aufgaben durch den Partner vermehrte U angibt konnte die Theorie des sekunda¨ren Krankheitsgewinns besta¨tigt werden. Die Theorie des tertia¨ren Krankheitsgewinns konnte allerdings nicht besta¨tigt werden. Unsere Beobachtung, dass ha¨matologische Patienten ¨ tzen als onkoloeher dazu neigen ihren Partner zu schu gische Patienten sollte Ausgangspunkt fu¨r weitere Untersuchungen sein.

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Abstracts

P41 Effects of various targeted drugs on growth of breast and colon cancer cell lines: results from a pilot screen of the LB-CO S. Laffer1, T. Grunt1,2, H. Herrmann1, B. Marian3, C. Zielinski1,2, P. Valent1,4 1

Ludwig Boltzmann Cluster Oncology, Vienna, Austria Department of Internal Medicine I, Division of Clinical Oncology, Medical University of Vienna, Vienna, Austria 3 Department of Internal Medicine I, Institute of Cancer Research, Medical University of Vienna, Vienna, Austria 4 Department of Internal Medicine I, Division of Hematology and Hemostaseology, Medical University of Vienna, Vienna, Austria 2

Solid tumours are one of the leading causes of morbidity and mortality in industrialized countries. Despite improved diagnostics and increasing efforts to develop new therapies, the prognosis in most advanced tumours remains poor. During the past few years, an increasing number of novel targeted anticancer drugs have been developed. However, little is known about the relative potency of these agents in various solid tumours. We examined the effects of 12 novel targeted drugs on growth of four breast cancer cell lines (MDA-MB-231, MDA-MB-453, T-47D and MCF7), A431 cells and four colon cancer cell lines (Colo201, HT-29, SW480 and HCT116) in an MTT assay (48 hours, 37 C). Drugs included the Mcl-1=Bcl-2= Bcl-xL-inhibitor obatoclax (GeminX), Aurora kinase-inhibitor VX-680, HDAC-inhibitor vorinostat, Hsp90-inhibitor 17-AAG, EGFR=ERB family kinase-inhibitors erlotinib, gefitinib and lapatinib, and multikinase-inhibitors imatinib, nilotinib, dasatinib, sorafenib and sunitinib (ChemieTek). Of all drugs, obatoclax was found to induce a reproducible growth arrest in most cells. Major effects of obatoclax were seen in MDA-MB-231 (IC50: 0.1–1 mM), HT-29 (IC50: 0.1– 1.0 mM) and SW480 cells (IC50: 0.1–1 mM). Some growthinhibitory effects were also seen with 17AAG in MDA-MB453 and HT-29 cells (IC50: 1.0 mM), and dasatinib in A431 (IC50: 1–5 mM) and Colo201 cells (IC50: 5–10 mM). The other inhibitors including major blockers of EGFR=ERB and KIT, showed no effects on neoplastic cell growth under the conditions applied. Together, certain targeted drugs including the BH3-mimetic obatoclax, affect in vitro growth of breast and colon cancer cell lines. Whether these agents also exhibit anti-neoplastic effects in tumour patients remains unknown.

memo Suppl 2/09

P42 Immunological effects after application of Cetuximab in combination with chemotherapy in NSCLC patients – correlation with CD4 þ CD25 þ regulatory T cells number and functional activity A. Pircher1, G. Gamerith1, D. Wolf1, M. Wolf1, A. Gächter2, G. Gastl1, W. Hilbe1 1

Department of Internal Medicine V (Hematology and Oncology), Innsbruck Medical University, Innsbruck, Austria 2 Department of Internal Medicine I, Innsbruck Medical University, Innsbruck, Austria

Background. The combination of chemotherapy and anti-EGFR monoclonal antibodies has proven clinical efficacy in NSCLC. However, the mechanism involved remains elusive so far. Direct effects on tumour cells as well as immuno-modulatory effects might both be involved. CD4 þ CD25 high regulatory T-cells (Treg) play a key role for maintenance of tolerance in rodents and men. Recent reports emphasized the important role of increased Treg levels in pateints with epithelial cancer for tumour immune escape. The aim of the current study is to determine the impact of neoadjuvant chemo-immunotherapy on Treg number and proliferative potential. Patients and methods. Twenty patients suffering from NSCLC (stage IB–IIIA) were included in an ongoing neoadjuvant clinical study protocol (INN06 study) and received up to two cycles of chemo-immunotherapy (Cetuximab þ Docetaxel þ Cisplatin) prior to surgery. Treg measurement was performed in a fixed schedule, which included assessment prior to initiation of therapy and prior to every cycle thereafter as well as at the end of neoadjuvant therapy. Relative Treg quantification was performed by FACS (co-staining of CD4, CD25 and FoxP3). Absolute quantification of Treg was done by Trucount staining. In parallel, the proliferative capacity of Treg was assessed by proliferation assays of magnetically isolated CD4 þ CD25 þ and the respective control CD4 þ CD25- T-cell populations from peripheral blood. Results. Preliminary data from serially analyzed 20 patients revealed consistently higher peripheral blood Treg levels during therapy as compared to baseline values. Interestingly, at the end of neoadjuvant treatment, Treg levels dropped back to basal levels. Ex vivo cultures confirmed previous results showing that in contrast to CD4 þ CD25- cells, CD4 þ CD25 þ are anergic towards T-cell receptor stimulation. Conclusion. The observed increase of Treg throughout chemo-immunotherapy suggests that due to chemotherapy-induced homeostatic expansion, Treg compartment restoration is more efficient as compared to CD4 þ CD25- control T-cells. Future studies will clarify whether the observed increase of Treg in PB can be correlated with an inferior clincial response to therapy via inhibition of mAb-induced anti-cancer effects (i.e. ADCC).

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P44

P43 ERCC1, MAPK1 and TUBB3 gene expression and its correlation with multiple immunohistochemical and molecular parametres in non-small cell lung cancers (NSCLCs) G. Gamerith1, A. Pircher1, S. Skvortsov2, M. Dlaska1, H. Rumpold1, A. Gächter1, H. Zwierzina1, G. Gastl1, W. Hilbe1 1

Departments of Internal Medicine I and V, Medical University Innsbruck, Innsbruck, Austria 2 Department of Radiotherapy – Radiooncology, Medical University Innsbruck, Innsbruck, Austria

Rationale. Conventional chemotherapy has reached a therapeutic plateau. Recently, by inclusion of new targeting strategies outcome was slightly improved. Biomarkers predicitive for efficacy or toxicity may identify specific subgroups and enable a more individualized therapeutic strategy to further improve survival. For the present study NSCLC patients were included in the ongoing neoadjuvant INN06 study, being treated with a triple combination of cisplatin, docetaxel and cetuximab. To better understand response or resistance, three pivotal genes ERCC1, MAPK1 and TUBB3 were analyzed by real-time reverse-transcription polymerase chain reaction (rt RT-PCR). Patients and methods. Fifty-four tumour probes of chemonaive NSCLC patients, 8 probes of patients treated with the triple therapy (‘treated’)) and, as a control, 22 tumour-free probes were analyzed with rt-RTPCR. Subsequently, these results were tested for their prognostic relevance and correlated with a panel of immunological and molecular markers (ERB-B2, ERB-B3, EGFR, pEGFR, EGFRvIII, hnRNP A2=B1, hnRNP B1, S100 A2, p53, bcl-2, ki-67, CD31 (MVD), FOX P3 and K-RAS mutations). Results. When comparing the three different cohorts only MAPK1 gene expression was significantly elevated in chemonaive and downregulated in pretreated tumour probes. None of these genes proved to be of prognostic relevance at the present state of data accrual. Testing the correlations with the mentioned panel of markers a strong correlation of K-RAS activating mutations and the three genes was seen. MAPK1 expression showed a negative correlation with ERB-B2, ERB-B3 and bcl-2. FOX-P3 correlated with an elevated TUBB3 expression. Conclusion. The three pivotal genes involved in chemoresistance are differentially expressed in chemonaive and treated patients. One might speculate that there exists some form of crosstalk between ERCC1, MAPK1, TUBB3 and K-RAS or the EGFR signalling cascade.

Analysis of the association between single nucleotide polymorphisms and haplotypes in the VEGF gene and prostate cancer risk T. Langsenlehner1, G. Hofmann2, A. Gerger2, U. Langsenlehner3, G. Tauber1, R. Partl1, K. Kapp1 1

Clinic of Therapeutic Radiology and Oncology, Medical University of Graz, Graz, Austria 2 Division of Oncology, Department of Internal Medicine, Medical University of Graz, Graz, Austria 3 Internal Outpatient Department, Steiermaerkische Gebietskrankenkasse, Graz, Graz, Austira

Purpose. Vascular endothelial growth factor (VEGF) plays a key role in the regulation of angiogenesis and has been related to cancer development and progression. Aim of the present study was to evaluate the role of VEGF single nucleotide polymorphisms (SNPs) and haplotypes in prostate cancer risk. Experimental design. We performed a case–control study including 702 prostate cancer patients and 702 male age-matched healthy control subjects. Seven VEGF candidate polymorphisms with a minor allele frequency of at least 0.10 and location in the promoter region, coding region or untranslated region of the VEGF gene were selected and genotypes were determined by 50 -nuclease (TaqMan) assays. Furthermore, VEGF plasma levels and genotypes were analyzed in a group of 64 healthy men. Results. Haplotype analysis showed two separate blocks of high-linkage disequilibrium, formed by five polymorphisms upstream of the coding sequence (promoter and 50 untranslated region) and two polymorphisms downstream of the coding sequence. None of the single polymorphisms or haplotypes was significantly associated with the presence or stage of prostate cancer. In a multivariate regression analysis including age, VEGF genotypes, and haplotypes as covariates and VEGF plasma level as dependent variable, none of the VEGF polymorphism or haplotypes was a significant predictor of VEGF plasma levels. Conclusions. The present data suggest that polymorphisms or haplotypes in the VEGF gene do not modify the risk of prostate cancer.

P45 Auto immune haemolytic anaemia and thrombocytopenia, a paraneoplastic phenomenon in patients with solid tumours J. Puthenparambil1, K. Lechner1, G. Kornek2 1

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Division of Hematology and Hemostaseology, Department of Medicine I, Medical University of Vienna, Vienna, Austria memo Suppl 2/09

Abstracts

2

Division of Oncology, Department of Medicine I, Medical University of Vienna, Vienna, Austria

Various paraneoplastic syndroms are known in cancers. Autoimmune haemolytic anaemia (AIHA) and autoimmune thrombocytopenia (AITP) are typical paraneoplastic phenomena in non-Hodgkin lymphomas, but little is known on the association of this disorders with solid tumours. We screened the literature for cases of an association of AIHA and AITP with solid tumours and classified the association as probable (typical haematological findings temporal association with tumour and remission of AIHA or AITP after surgery) or possible. (Typical haematological findings but no response to surgery or chemotherapy) We identified 52 cases of an association of AIHA and 51 cases of an association of AITP with a solid tumour. We found 9 cases of a probable association of AIHA with a tumour (amongst them 6 cases of renal cell cancer). In addition we found 2 possible associations. There were no cases of a probable and 4 cases of a possible association between a solid tumour and AITP. We suggest that AIHA has to be added to the list of paraneoplastic syndroms in renal cell cancer.

6. Cases

eine Druckdolenz im re. Oberbauch keine weiteren Auffa¨lligkeiten. Laborchemisch signifikant war eine ma¨ßige Erho¨hung der LDH und der Leberfunktionsparameter sowie eine grenzwertig erho¨hte NSE. Computertomographisch zeigten sich nebst einem ausgepra¨gten infrarenalen Aortenaneurysma multiple Leber- und Lungenherde. ¨ brigen fanden sich keine weiteren Manifestationen Im U der Erkrankung. Endoskopisch unauffa¨llige Befunde. Eine Biopsie der Leber ergab die Diagnose einer LangerhansZell-Histiozytosis X. Noch vor Therapiebeginn kam es zu einer Spontanruptur des Aortenaneurysmas, welches mittels Stent saniert werden konnte. Im Anschluß wurden dem Patienten gema¨ß den allgemeinen Richtlinien eine strikte Nikotinkarenz sowie eine Cortisontherapie mit 1 mg=Kg=KG empfohlen. Eine engmaschige radiologische Observanz wird zwischenzeitlich fortgefu¨hrt. Die hepatale Manifestation der Histiozytosis X gilt als extrem rar und ist demnach in der Literatur nur selten beschrieben.

P47 Complete remission after a single cycle of azacytidine in a case of relapsed acute myeloid leukaemia C. Valentiny, M. Steurer, F. Räbiger, K. Moser, W. Willenbacher, R. Stauder

P46 Pulmonale Histiozytosis X mit hepataler Beteiligung S. Sprung1, E. Szabo1, D. Gartner2, G. Weixler3, P. Bratusch-Marrain1 1

Department of Internal Medicine=Oncology, Landesklinikum Waldviertel Horn, Austria 2 Department of Radiology, Landesklinikum Waldviertel Horn, Austria 3 Department of Pathology, Landesklinikum Waldviertel Horn, Austria

Pulmonale Histiozytosis X mit hepataler Beteiligung Langerhans Zell Histiozytosis X (LCH) ist eine klinisch sich vielfa¨ltig pra¨sentierende Erkrankung gekennzeichnet durch eine pathologische Ansammlung und Proliferation von Entzu¨ndungszellen. LCH ist meist eine pa¨diatrische Knochenerkrankung, beim Erwachsenen pra¨sentiert sie sich typischerweise als nikotin-assozierte Lungenmanifestation. Wir pra¨sentieren hier den Fall eines 61 ja¨hrigen Mannes bei welchem im Rahmen einer extramuralen Abkla¨rung eines infrarenalen Aortenaneurysmas multiple Leberherde sonographisch entdeckt wurden. Anamnestisch bestanden seit kurzer Zeit Oberbauchschmerzen; eine Dyspnoe oder Gewichtsverlust konnte nicht erhoben werden. Weiters bekannt ist ein chronischer Nikotinabusus. Im Status fanden sich bis auf ein Systolikum sowie memo Suppl 2/09

Department of Internal Medicine V (Haematology and Oncology), Medical University Innsbruck, Innsbruck, Austria

The prognosis of elderly patients with AML remains poor. Preliminary results suggest that azanukleosides are active in AML. We report the case of 75-year-old female patient presenting with a late relapse of an AML-M2 achieving a complete remission after a single cycle of Azactidine (VidazaTM). The diagnosis of a de novo AMLM2 with a normal karyotype was established in August 1993. After induction chemotherapy with one cycle MICE (mitoxantrone, cytarabine, etoposide) she achieved a complete remission (CR). Due to pulmonary and hepatic infiltrates, highly suspicious for aspergillosis, only a mitigated consolidation therapy consisting of three cycles of low-dose cytarabine (LDAra-C) could be applied. The CR lasted until October 2008 when the patient presented with pancytopenia and a severe bone marrow infiltration with M2-blasts displaying a normal karyotype. As the patient refused a classical induction for AML as well as LDAra-C, the patient received Azacitidine (VidazaTM) 75 mg=m2, intravenously for 7 days. This therapy was well tolerated; however, she refused to continue. Two months after Azacitidine application she presented with an unexpected normalization of peripheral blood counts; bone marrow cytology revealed a disappearance of blasts ( < 5%) by morphological and flow cytometric analysis, thus fulfilling the criteria of a CR. In parallel the geriatric assessment demonstrated a pronounced improvement in perfor-

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Abstracts

mance status WHO-Scale (improved from 2 to 0), Karnofsky-Index (70!90) as well as in self-perceived quality of life (EORTC QLQ-C30). The favourable and surprising response in this case suggests to analyse the effectiveness and the duration of Azacitidine therapy in AML in prospective clinical trials.

P48 Rhabdomyolysis as a complication of trabectedin (Yondelis) treatment in metastatic leiomyosarcoma: a case report M. Pichler, H. Samonigg, T. Bauernhofer Division of Oncology, Department of Internal Medicine, Medical University of Graz, Graz, Austria

Trabectedin (Yondelis+) has been recently established as treatment option for soft tissue sarcoma in adults after the failure of standard therapy. Many studies concerning the activity of this drug in solid tumours are ongoing. Commonly reported toxicities of trabectedin were neutropenia and hepatic toxicity. We present a case of trabectedin-induced severe rhabdomyolysis in a female patient receiving trabectedin as third line chemotherapy for metastatic leiyomyosarcoma. Ten days after the fifth cycle of trabectedin the patient presented with three-day history of generalized muscle pain, weakness and fatigue. Laboratory results showed elevation of creatine kinase (CK) 3936U=l, lactate dehydorgenase (LDH) 1538 U=l and myoglobin > 3000 ng=ml. There was no history for seizures, trauma, extreme exercise or co-medication with statins as a potential secondary cause of rhabomyolysis. To avoid acute renal failure intravenous fluid and electrolyte rerplacement was initiated. On the following days the level of muscle enzymes returned towards normal level. However, therapy with trabectedin was discontinued to avoid reoccurrence of rhapdomyolysis. Although the mechanism by which trabectedin causes rhabdomyolysis is currently unknown, levels of creatine kinase (CK) should be monitored closely in patients receiving this agent. In case of elevated CK levels treatment should be stopped until return of CK levels to normal range. Physicians should be aware of this rare side effect to avoid potential severe toxicity in patients treated with trabectedin.

P49 Treating aggressive fibromatosis with chemotherapy followed by target therapy with imatinib – a case report and a review of literature G. Knechtel1, H. Stöger1, A. Beham2, H. Samonigg1

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1

Division of Oncology, Department of Internal Medicine, Medical University of Graz, Graz, Austria 2 Institute of Pathology, Medical University of Graz, Graz, Austria

Desmoid tumours also known as aggressive fibromatosis are benign tumours which grow slowly but locally aggressive and do not metastasize but can be potentially life threatening when infiltrating vital structures. The therapy strategy contains surgery, radiation and systemic therapy with non-steroidal anti-inflammatory drugs, antioestrogen compounds and cytotoxic chemotherapy. We report on a 40-year-old male patient with advanced fibromatosis of the neck who has been treated with seven cycles of polychemotherapy followed by target therapy with imatinib. He received 40 mg=m2 adriablastin at the first day and 1500 mg=m2 ifosfamide and 200 mg=m2 dacarbacine daily for 4 days. After chemotherapy the therapy continued with imatinib 400 mg orally per day. Tumour response was evaluated with magnetic resonance imaging every two cycles. The tumour decreased significantly after the first cycle of chemotherapy already and tumour-related symptoms declined. The response continued after switching to target therapy with imatinib which is ongoing currently. The best treatment combination for this rare tumour remains under discussion. Doxorubicine and dacarbacine are frequently used agents. We included ifosfamid in our regimen which is standard in the treatment of soft tissue tumours. The tyrosin kinase inhibitor imatinib seems to offer new possibilities and is currently investigated in randomized trials. We conclude that combination chemotherapy including doxorubicin, ifosfamid and dacarbacine in the treatment of aggressive fibromatosis should be considered for patients who suffer from unresectable, advanced disease and clinical symptoms which require a rapid response to therapy.

P50 Prolonged response of trabectedin in patient with refractory metastatic liposarcoma after failure of prior chemotherapy: a case report D. Voskova, R. Greul, G. Hochreiner, G. Wahl, J. Preining, M. Fridrik Department of Internal Medicine 3, Oncology, General Hospital Linz, Linz, Austria

Trabectedin (ET743, YONDELIS+ Pharma Mar, Madrid, Spain) is a new marine-derived compound that binds the DNA minor groove and has an original mechanism of action involving proteins of the nucleotide excision repair machinery. Several Phase II Studies has shown response in 5–10% of patients with soft tissue sarcoma failing prior chemotherapy and disease stabilization in 30–40%. We report on one patient with metastatic liposarcoma with 12 months response on the trabectedin after failure of primary chemotherapy. In December 2006, a memo Suppl 2/09

Abstracts

71-year-old man was presented with an abdominal pain. The computer tomography showed a large mass in retroperitoneal space. On Januray 5, 2007, wide resection was performed. The histology report was myxoid liposarcoma. In December 2007, the retroperitoneal lesion recurred and metastases to the abdomen and to the lung were identified. The patient received the chemotherapy with doxorubicin. After the four cycles a disease progression was reported. Because of severe paraneoplastic polyneuropathy the chemotherapy with paclitaxel could not be given. In October 2007 the chemotherapy with trabectedin as a 24-hour continuous infusion of 1.5 mg=m2 every 3 weeks was introduced. The partial response was performed after three cycles and the best response after eight cycles of therapy with more than 50% reduction of size of all metastases. In October 2008 progression of disease was detected. As side effects transient elevation of liver enzymes (grade 3), fatigue and mild haematological toxicity with grade 1 neutropenia and grade 1 anaemia were observed.

P51 Gender differences in functional capacities in elderly cancer patients C. Valentiny1, G. Kemmler2, K. Moser1, R. Hofer1, R. Stauder1 1

Department of Internal Medicine V (Hematology and Oncology), Medical University, Innsbruck, Austria 2 Department of Biological Psychiatry, Medical University, Innsbruck, Austria

Geriatric assessment is essential in decision making in elderly cancer patients. Aim of this study was to investigate the impact of gender in geriatric assessment (GA) in tumour patients. GA was applied in 78 tumour patients of age 60 þ , treated at the ward of the Department of Internal Medicine V (Haematology and Oncology), Medical University, Innsbruck, Austria, to assess the domains functional capacities, comorbidities, quality of life, cognition and social support. In this cohort women were older (median 75 yrs) than men (72 yrs) (p < 0.03). A trend towards a gender effect was observed in iADL (Instrumental activities of daily living) as women displayed better functional activities than men: iADL (median 8 vs. 6) p ¼ 0.063; this effect even increased in age adjusted analysis (p ¼ 0.05). In contrast the Timed up and go test was performed faster by men than by women (14.22 ($) vs. 10.35 seconds (#); p ¼ 0.048); however, this difference was less pronounced in age adjusted models (p ¼ 0.0889). Interestingly social support was somewhat lower in women and was not age-dependent (F-Sozu 4.23 vs. 4.53, p ¼ 0.094). In WHO-Scale, Karnofsky Index (KI), Activities of Daily Living (ADL), Mini Mental State Examination (MMSE), Geriatric Depression Scale (GDS-30), Cumulative illness rating scale for geriatricians (CIRS-G), Charlson Comorbidity Index, VES-13, PPT and FACT-G memo Suppl 2/09

no gender effects were observed. These results emphasize the fact that gender differences should be considered and recognized in the comprehensive evaluation of elderly tumour patients.

P52 Rituximab for the treatment of acquired von Willebrand's syndrome as complication of chronic graft-versus-host-disease after allogeneic stem cell transplantation Z. Kuzmina, M. Mitterbauer, P. Kalhs, I. Pabinger, P. Quehenberger, H. Greinix Department of Internal Medicine I, Bone Marrow Transplantation and Division of Hemostaseology, Department of Medical and Clinical Laboratory Diagnostics, Medical University of Vienna, Vienna, Austria

Acquired von Willebrand’s syndrome is a rare cause of serious bleeding. Here, we report a case occurring in the context of chronic graft-versus-host disease (GVHD) after allogeneic haematopoietic cell transplantation (HCT). A 40-year-old Caucasian male was diagnosed with peripheral T-cell lymphoma (TCL) in stage IVB in November 2003. He underwent multidrug chemotherapy achieving a partial response and received an autologous HCT after BEAM conditioning in October 2005. After relapse he underwent in January 2006 allogeneic HCT in second complete remission (CR) from a HLA-identical sibling blood stem cell donor after reduced-intensity conditioning with fludarabine and cyclophosphamide. GVHD prophylaxis consisted of cyclosporine A and mycophenolate mofetil. Starting on day 130 after HCT severe chronic GVHD with involvement of skin, gastrointestinal tract, eyes and liver was observed. Under immunosuppressive therapy with steroids, cyclosporine A and extracorporeal photopheresis chronic GVHD activity decreased to mild involvement of eyes. In September 2007 the patient was hospitalized with diffuse gastrointestinal bleeding, a drop in haemoglobin levels, drop in Factor VIII (FVIII) to 5%, von Willebrand Factor (vWF) Antigen of 4% and Ristocetin Cofactor < 10% consistent with acquired von Willebrand’s syndrome. After initial response to cyclophosphamide with normalization of FVIII and vWF other gastrointestinal bleeding episodes occurred and decreased levels of FVIII to 16–34% were observed. Therefore, in July 2008 four doses of Rituximab at 375 mg=m2 were administered every other week. FVIII was 14% before treatment and increased to 92% after the first dose of Rituximab. Then, it remained normal and the patient had no more bleeding episodes without further therapy. His coagulation parametres remained normal with FVIII of 117%, aPTT of 34.9 sec, vWF Antigen of 137% and Ristocetin Cofactor of 164%. Currently, no signs of chronic GVHD are present. Thus, Rituximab induced complete resolution of acquired von Willebrand’s syndrome after allogeneic HCT.

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