Abstracts of the German, Austrian, and Swiss Society for Hematology and Oncology. October 18-22, 2013. Vienna, Austria.

Abstracts Onkologie 2013;36(suppl 7):1–289

Metastasiertes Lungenkarzinom V4

Metastatic lung cancer Pirker R. Univ.Klinik für Innere Med. I – AKH Wien, Wien, Austria

Systemic chemotherapy in metastatic lung cancer depends on tumor histology, performance status and age of patients, organ functions, side effects and other parameters. Chemotherapy decreases cancer-related symptoms and increases survival. Patients with small cell lung cancer receive firstline chemotherapy, preferentially with a platinum plus etoposide, and second-line chemotherapy with topotecan or other protocols. Patients with advanced non-small cell lung cancer (NSCLC) receive palliative chemotherapy with a platinum-based doublet containing a third generation cytotoxic drug. Maintenance therapy with pemetrexed or erlotinib has also been established. Second-line chemotherapy at the time of progression consists of pemetrexed, docetaxel, erlotinib or other protocols. Novel therapeutic strategies have focused on the molecular mechanisms involved in tumor growth and treatment response. While customized chemotherapy remains experimental, targeted therapies have already entered clinical practice. Targeted therapies focused on the inhibition of angiogenesis or growth factor receptor signaling. Current research attempts to define driver mutations and to assess them as potential therapeutic targets. Bevacizumab added to first-line chemotherapy improved clinical outcome compared to chemotherapy alone in selected patients with advanced non-squamous NSCLC and has been approved in combination with first-line chemotherapy for these patients. Blockade of the epidermal growth factor receptor (EGFR) by monoclonal antibodies or tyrosine kinase inhibitors has been studied in phase III trials. Cetuximab added to first-line chemotherapy increased survival compared to chemotherapy alone in patients with advanced NSCLC and high EGFR expression in their tumors. An ongoing SWOG study attempts to validate high EGFR expression and EGFR FISH positivity as predictive biomarkers. Necitumumab is currently evaluated in phase III trials. Erlotinib and gefitinib have shown efficacy in patients previously treated with chemotherapy. In patients who harbor EGFR-activating mutations in their tumors, gefitinib, erlotinib and afatinib increased progression-free survival and improved quality of life compared to first-line chemotherapy. Both gefitinib and erlotinib have been approved for the firstline therapy in these patients. Crizotinib has shown efficacy in patients with ALK-positive tumors and is currently approved for second-line treatment in patients with ALK-positive tumors. Disclosure: Robert Pirker: Financing of Scientific Research: AstraZeneca, Boehringer Ingelheim, Eli Lilly, Merck Serono, Pfizer, Roche.

Expertenseminar

CLL assoziierter Immundefekt V5

CLL associated immunodeficiency Dürig J. Universität Duisburg-Essen, Klinik für Hämatologie, Essen, Germany

The clinical course of chronic lymphocytic leukemia (CLL) is characterized by progressive immunodeficiency and infection remains the commonest cause of death. The immunodeficiency mainly manifests

© 2013 S. Karger GmbH, Freiburg Fax +49 761 4 52 07 14 [email protected] www.karger.com

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as hypogammaglobulinemia but also involves alterations in the T cell compartment, which is often characterized by the expansion of senescent functionally incompetent T effector memory cells. Immune dysregulation appears to be caused by bi-directional cell contact and secretion of cytokines, which both propagate the survival and proliferation of the malignant cell clone and suppress immunity. Cytotoxic treatment of CLL generally makes the immunodeficiency worse, and this is particularly true for the novel highly effective immunochemotherapy protocols employing combinations of fludarabine and monoclonal antibodies such as rituximab and alemtuzumab. More profound immunosuppression observed with the use of modern therapy has led to more frequent infections with or reactivations of viral diseases such as Herpes simplex, varicella zoster, CMV, Hepatitis B and JC virus. Intravenous immunoglobulin is clinically effective but due to its high costs can only be used in defined patient subgroups. Prophylactic antibiotics and antiviral drugs can be useful while vaccination is usually ineffective. Progress in this field is limited by the low number of prospective interventional studies and most data come from observational retrospective studies. In this session we will summarize the available data and discuss management options in a number of clinical cases presenting with infectious complications typically encountered in the context of CLL. Disclosure: Jan Dürig: Advisory Role: Janssen; Financing of Scientific Research: Janssen, Celgene, Binding Site; Expert Testimony: Celgene, Binding Site; Other Financial Relationships: Mundipharma, Janssen, Celgene.

Fortbildung

Gerinnung I – Praktische Hämostaseologie für Hämatologen und internistische Onkologen V11

Thrombocytopenia and anticoagulation Matzdorff A. Caritasklinikum St. Theresia, Hämatologie und Onkologie, Saarbrücken, Germany

Cancer is common. Cancer patients have a high intrinsic risk of both thromboembolism and bleeding. In addition they may be thrombocytopenic from chemotherapy. With a median age of 69 comorbidities requiring anticoagulation such as atrial fibrillation, venous or arterial thromboembolism, myocardial infarction, or stroke are highly prevalent in this population. Immune thrombocytopenia (ITP) is a rare. Half of the ITP patients are older than 50 and as in cancer many have comorbidities in need of anticoagulation. In addition ITP is considered a prothrombotic disorder. Anticoagulation poses a clinical challenge in thrombocytopenic patients because it is usually contraindicated with thrombocytopenia < 50.000/µl. Thrombocytopenic patients requiring anticoagulation have been excluded from all pivotal studies on newer therapeutic agents and clinical data are scarce. Guidelines do not provide guidance in this particular situation. This presentation summarizes experiences from case reports and small series and suggests an approach to the thrombocytopenic patient requiring anticoagulation. Disclosure: Axel Matzdorff: Financing of Scientific Research: AMGEN, GlaxoSmithKline, Leo Pharma.

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Expertenseminar

Fortbildung AML update V19

Transplantation in AML Braess J. Krankenhaus Barmherzige Brüder, Klinik für Onkologie und Hämatologie, Regensburg, Germany

Allogeneic stem cell transplantation (allo-SCT) is unequivocally the most potent antileukemic therapy that is currently available for acute myeloid leukemia (AML). This holds true for patients in advanced disease stages beyond first complete remission (CR-1) but also for certain subgroups in CR-1. The antileukemic effect of allo-SCT depends both on the cytotoxic effect of the conditioning regimen as well as the immunological graft-versus-leukemia (GvL) effect. Therefore different approaches such as myeloablative conditioning (MAC) versus reduced-intensity conditioning (RIC) or even minimally intensive conditioning (mini-transplant) make differential use of these two antileukemic modalities. Whereas no strict chronological age limit exists most centers will offer allo-SCT to biologically fit patients with no major comorbidities up to the age of around (65 to) 70 years if the AML biology or stage necessitates this approach for a potential cure. A clear cut example for the need of allo-SCT in CR-1 are AML with complex cytogenetic aberrations. In this group of patients – even in young patients undergoing intensive induction and chemotherapy-based post-remission therapy – the event-free survival (EFS) at two years is negligible with all patients either being primary refractory, dying during aplasia or relapsing within the first 12 months. In contrast prospective but non-randomized evidence demonstrates that in appropiate patients RIC allo-SCT with the FLAMSA regimen can achieve > 60% EFS at two years with a substantial percentage of cured patients. In AML with intermediate risk or relapse the situation is less clear-cut since the advantages of a higher antileukemic efficacy of allo-SCT are counteracted by increases in non-relapse mortality (NRM). However important advances have been made by a German Intergroup study that has demonstrated the significant reduction of NRM by the modification of the conditioning regimen in a randomized comparison. Even more importantly this group has now initiated the first randomized controlled trial for the comparison of early vs delayed allo-SCT in CR-1 in patients with intermediate risk of relapse which has the potential to answer this highly relevant clinical question. Even in patients with no HLA compatible donor who nevertheless have a clear indication for allo-SCT alternative donor transplantation can often be performed by using cord-blood transplantation or especially haploidentical transplantation. Disclosure: No conflict of interest disclosed.

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Ethische Konflikte und Beratung am Lebensende V22

What are the problems with clinical ethics consultation?

dations and guidelines, or by providing general information or further education in ethical issues. Although the aim to improve ethically reflected clinical decision making is very much welcome in medicine and the general society, formal ethics consultation and its organizational structures remain under constant debate. Its critics state that ethical expertise and judgement should remain a medical core competence and therefore performed by physicians themselves, or criticize its educational or methodological background, amongst others. This congress lecture will highlight the intended aims and proven benefits of formal ethics consultation, as well as the corresponding refutations and the current state of discussion. We will further present final results of a two-step research project that accompanied the implementation process of a clinical ethics committee (CEC) at our University Hospital that 1) investigated expectations, desires or objections against the implementation of a CEC by quantitative methods, and 2) evaluated the first ethical case conferences, focusing on the individual perspectives of all involved actors and decisional / clinical outcome parameters after this intervention, by using qualitative methods. Disclosure: No conflict of interest disclosed.

Wissenschaftliches Symposium

Patientenorientierung und Versorgungsstrukturen V26

Patient expertise and care management – situation appraisal in medical oncology office practices Baumann W., Zamora P., Riese C. Wissenschaftliches Institut der Niedergelassenen Hämatologen und Onkologen – WINHO, Köln, Germany

The ongoing change of approaches in medical oncology implicates an increasing involvement of patients in the management of the treatment process. Success depends more and more on the appropriate cooperation with the patient. Patients need to understand the complex care processes and they have to bring enough resources to meet the self management requirements. This raises the question of the necessary support to enable as many patients to develop an active and independent therapeutic partnership with the oncologist. For the oncology practices, the management tasks increase. Based on surveys in oncology practices, data will be presented to display patients' characteristics and their situation in outpatient cancer care. This involves questions about the continuity in the chain of care and the communication challenges in the doctor-patient relationship. Information is provided on how patients with oral cancer therapy assess their situation and evaluate the supply of care and support. The presentation discusses some of the challenges that a suitable supply management in outpatient oncology care has to face. It is an important issue to what extend the shift of responsibility to the patient can be achieved with more self-determination and not more strain for the patient as a result. Disclosure: No conflict of interest disclosed.

Alt-Epping B., Scherer A., Marx G., Jansky M., Nauck F.

Clinical ethics consultation and its organisational structures have experienced a tremendous surge in Germany during the past 15 years, responding to the increasing demands for ethical expertise in increasingly complex clinical constellations, for instance in the field of intensive care or cancer therapy. Clinical ethics consultation intends to promote ethically reflected decision making by providing concrete consultative advice, by moderating interdisciplinary case conferences, by authoring recommen-

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Onkologie 2013;36(suppl 7):1–289

Abstracts


University Medical Center Göttingen, Dept. of Palliative Medicine, Göttingen, Germany

The stressed oncologist in the clinical encounter – results of the WIN ON-study Pfaff H.1, Ernstmann N.1, Groß S.E.1, Gloede T.D.1, Ansmann L.1, Nitzsche A.1, Jung J.1, Wirtz M.2, Baumann W.3, Osburg S.3, Neumann M.4 Universität zu Köln, Institut für Medizinsoziologie, Versorgungsforschung und Rehabilitationswissenschaft (IMVR), Köln, Germany, 2Pädagogische Hochschule Freiburg, Abteilung für Forschungsmethoden, Freiburg, Germany, 3Wissenschaftliches Institut der niedergelassenen Hämatologen und Onkologen GmbH (WINHO), Köln, Germany, 4Universität Witten/ Herdecke, Fakultät für Gesundheit, Department für Humanmedizin, Witten/ Herdecke, Germany 1

Background: Good communication with patients is an essential skill of oncologists. However, practicing oncologists are exposed to various job stresses, which might affect their communication skills. The aim of the present study is to examine the association between private practice hematologists' and oncologists' (PPOs) job stresses and their perceived communication skills. Methods: This study is part of the WIN ON (Working Conditions in Oncology) project. WIN ON is an interdisciplinary prospective study on the consequences of PPOs' working conditions on their health and as a result on patient care. All members of the Professional Organization of Office-Based Hematologists and Oncologists (BNHO) and additional PPOs (n = 578) were invited to a mail survey. Out of 556 eligible oncologists, N = 157 sent back the completed first questionnaire (response rate 28%). Besides, PPOs who were also willing to enroll patients (N = 47), were asked to fill out a second questionnaire after the initial clinical interview with each enrolled patient (N = 149). Data are analyzed descriptively. Results: More than 50% of the PPOs are between 50 and 59 years old and 80% of the PPOs have over ten years professional experience. Most of the practicing oncologists assess themselves as self-confident when talking to patients. However, 70% of the PPOs feel less confident when assessing the psychological health of their patients. The average workload is high with a median working time of 60 hours per week and a median number of patient consultations of 35 per day. About 46% of the PPOs report moderate symptoms of emotional exhaustion (indication for burnout) and 15% report explicit symptoms. Despite the high workload, 95% of the PPOs, who filled out the second questionnaire, report that they managed to address most or all issues in the initial clinical interview which they intended to address. Moreover, almost 25% of the PPOs report that interruptions during patient consultations were disturbing. However, those disturbances did not affect PPOs' distress after the consultation. Conclusion: In general, PPOs are exposed to high workload and often show burnout-symptoms, but they seem not to transfer their stress on patient interviews. We aim to gain deeper insights into this topic by combining physician and patient data from the WIN ON-study and analyzing the data in multivariate regression models. Disclosure: No conflict of interest disclosed. V28

Psychosocial distress in cancer patients during the course of diagnosis and treatment Weide R. Praxisklinik für Hämatologie und Onkologie, Koblenz, Germany

Introduction: Psychosocial distress in cancer patients is different between patients and changes during the course of the disease. From the perspective of the oncology team central questions need to be addressed: 1. Which are symptoms of psychosocial distress? 2. How can psychosocial distress be measured in routine care? 3. Is distress disease-specific?

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4. 5. 6. 7.

How does distress change during the course of disease? Which persons are considered helpful from the patient´s perspective? How distressed are main care givers? How can the oncology team help to reduce distress?

Purpose: This evidence-based overview aims to give practice-relevant answers to the questions above. Results: 1. Psychosocial distress often shows through psychological and physical symptoms like sadness, depression, sleep disorders, aggressiveness, pain or fatigue. 2. Short and standardised inventories like the NCCN´s distress thermometer and problem list have shown to be very helpful and robust in routine care. 3. Disease-specific distress is highest in patients who just learned about their cancer diagnosis, patients receiving best supportive care only and breast cancer patients receiving adjuvant antihormonal therapy. 4. During the course of the disease distress is highest just after telling the diagnosis of cancer, before telling the result of a cancer-related body imaging procedure or a laboratory test, after breaking bad news and just before a follow up examination. 5. Most helpful persons concerning distress reduction from the patient´s point of view are the oncologist, partner, friends, children, the general practitioner and other relatives. 6. Main care givers are at least as distressed as patients, often even more! 7. Most important factors concerning help from the oncology team are empathy, competence in communication, continuity of care between patients and oncologists, incorporation of main care givers and if necessary other disciplines in the psychosocial care of patients. Conclusion: Oncologists and the oncology team play a central role in detection, treatment and reduction of psychosocial distress in cancer patients during the course of their disease. Disclosure: No conflict of interest disclosed.

Expertenseminar Prostatakarzinom V30

Prostate cancer – discussing treatment options in 2013 Bokemeyer C. Universitätsklinikum Hamburg-Eppendorf, Hamburg, Germany

Prostate cancer is the most frequent tumor diagnosed in men. Typically the disease remains silent and localised for many years in most patients. Screening approaches for prostate cancer based on PSA are controversially discussed and cannot be generally recommended for all men. Treatment of localised disease is based on the expected prognosis of the cancer as well as on patient age and comorbidities and ranges from watchful waiting to active surveillance to localised radiotherapy as well as prostatectomy. Hormone ablative therapy is the preferred approach for patients with metastatic symptomatic prostate cancer. Chemotherapy with docetaxel is typically used when castration refractory disease occurs. However, in recent years several new treatment options have become available including new ways of manipulating the endogenous testosterone levels such as enzalutamide and abiraterone, 2nd line chemotherapy with carbazitaxel after failure of docetaxel as well as radium 223 for patients with mainly bone disease. In addition, immunotherapy for prostate cancer is also under active investigation. Finally, due to the prevalence of bone metastases active treatment with bisphosphonates or denosumab are additional important strategies for many patients. Choosing the right treatment and the right treatment sequence is continuously evolving based on new data over the last years and will be the major part of the discussion in the Expert Seminar. Disclosure: Carsten Bokemeyer: Employment or Leadership Position: Uniklinik Hamburg Eppendorf; Advisory Role: Sanofi Aventis, Jansen-Cilag; Financing of Scientific Research: Sanofi Aventis, Jansen Cilag.

Abstracts


V27

Freier Vortrag

V32

Elevated serum free light chains do not predict event-free and overall survival in two independent cohorts of elderly patients with aggressive B-cell lymphomas

V31

Outcome and prognostic factors in patients with M antle Cell Lymphoma relapsing after autologous stem cell transplantation: A retrospective study of the EBMT 1

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Dietrich S. , Finel H. , Boumendil A. , Avivi I. , Volin L. , Cornelissen J.5, Walewski J.6, Schmid C.7, de Witte T.8, Schouten H.C.9, Kaufmann M.10, Sebban C.11, Trneny M.12, Kobbe G.13, Guardiola P.14, Schetelig J.15, Pfreundschuh M.16, Diez-Martin J.L.17, Bordessoule D.18, Robinson S.19, Dreger P.20 Universitätsklinik Heidelberg, Medizin V, Heidelberg, Germany, 2Working Party, European Society for Blood and Marrow Transplantation, Paris, France, 3 Rambam Medical Center and Bruce Rappaport Faculty of Medicine, Haifa, Israel, 4Helsinki University Central Hospital, Helsinki, Finland, 5Department of Hematology, Erasmus MC- Daniel den Hoed Oncology Clinic, Rotterdam, Netherlands, 6Lymphoid Malignancies, Maria Sklodowska-Curie Memorial Institute and Oncology Centre, Warszawa, Poland, 7Medizinische Klinik, Klinikum, Augsburg, Germany, 8Radboud University Nijmegen Medical Center, Nijmegen, Germany, 9Department of Hematology, University Medical Center, Maastricht, Netherlands, 10Robert-Bosch-Krankenhaus, Stuttgart, Germany, 11Onco-Hematology, Centre Léon Bérard, Lyon, Germany, 12Institute of Hematology and Blood Transfusion, Praque, Czech Republic, 13Department of Hematology, University of Düsseldorf, Düsseldorf, Germany, 14CHU Angers, Université Angers, Anger, France, 15Internal Medicine I, Technical University, Dresden, Germany, 16Innere Medizin I, Universitätsklinikum des Saarlandes, Homburg, Germany, 17Department of Hematology, Gregorio Marañon General University Hospital, Madrid, Spain, 18CHU Limoges, Service d'Hématologie Clinique et Thérapie Cellulaire, Limoges, Germany, 19BMT Unit, Bristol Children's Hospital, Bristol, Germany, 20Universitätsklinik, Heidelberg, Germany 1

Background: It is little known about outcome of MCL recurrence after autoSCT. We therefore conducted a retrospective analysis of patients with MCL who failed autoSCT using the EBMT database. ELIGIBLIBILITY CRITERIA were: age > 18 years, MCL relapse after autoSCT between 2000 and 2010. Results: Median age at autoSCT of 360 included patients was 57 years (range: 37 to 77), 67% had undergone autoSCT as part of 1st-line therapy; 67% and 50% had documented exposure to rituximab (RTX) and highdose ara-C (HA) before autoSCT; and 8% had refractory disease at autoSCT. Only 21 relapses (6%) occurred beyond 5 years after autoSCT.With a median observation time of 40 months, median OS after relapse of the whole study group was 19 months. By univariate analysis, a long (>12mo) interval between autoSCT and relapse (p 1 E-05 and minor when ≤1 E-05; a quantitative discordance was defined as two positive results with a quantitative discrepancy >1 log.

Abstracts

Results: 29 patients were evaluable with at least one method, 15 by both. Here, in 97 FU samples a significant concordance between both MRD methods was shown (r2 = 0.80) (p  100.000/µl after a median of 27 days (range: 11–35), neutrophils > 1000/µl after 24 days (range: 11–42) and hemoglobin after 68 days (range: 42–98). Of note, neutrophil counts decreased in 3 patients before recovery. The spleen sized normalized within days (n = 4/4) as did sCD25 (n = 3/3), which is considered a reliable marker of HCL load. Side effects were arthritis (n = 2) which could be managed with low dose steroids and NSAR, elevated bilirubin (n = 3), wart like lesions and other skin changes (n = 3) and grade 3 hematologic toxicity (neutropenia, n = 3). A heavily pre-treated and refractory patient showed disease recurrence at last follow up and was successfully re-started on vemurafenib. Detailed immunohistological assessments are provided. After vemurafenib treatment p-ERK signaling was almost completely abolished in HCL cells in vivo, followed by apoptosis of HCL cells as shown by Tunnel staining. Conclusion: Targeting of a single mutated oncogene can provide disease control in this leukemia. Further studies with careful assessment of vemurafenib dosing in HCL are urgently needed. Disclosure: No conflict of interest disclosed.

Fortbildung

CML Klinisches Management 2013 V130

Treatment free remission – A spark of hope or realistic option? Hochhaus A. Universitätsklinikum Jena, Abt. Hämatologie/Onkologie, Jena, Germany

The International Randomized Study of Interferon and STI571 (IRIS) demonstrated long-term cytogenetic responses in patients with chronic-phase chronic myeloid leukemia (CML-CP) treated with the tyrosine kinase inhibitor (TKI) imatinib. However, deep molecular responses (MRs), as measured by reductions in BCR-ABL transcript levels below the threshold of major MR, were achieved only by a small proportion of patients. With the advent of the second-generation TKIs nilotinib and dasatinib for the treatment of patients with newly diagnosed CML-CP, the proportion of patients who achieve the deepest levels of MR increased significantly. With these changes, the potential for patient eligibility in TKI cessations studies is becoming a more widely discussed topic and area for research. These developments highlight the need for robust, standardized and workable definitions of deep MRs. Specifically, it is critical that the measurement of MR is standardized in a manner to withstand both intra- and inter-laboratory variability, as well as new methodological developments. Variables to be considered in cessation studies are: Duration of TKI therapy, duration of MR4, MR4.5, time to MR4, MR4.5, use of IFN maintenance, prognostic scores at diagnosis, type of TKI. The prospective multicentre phase III TIGER study (TKI plus IFN initiated by the GERman CML Study Group) is addressing the questions whether IFN in addition to nilotinib may improve the proportion of patients with molecular response and whether a maintenance therapy with pegylated IFN may enhance the stability of molecular response after treatment discontinuation. Details are available at [email protected]. Disclosure: No conflict of interest disclosed.

Abstracts


V126

Fortbildung

Aktuelles zum Keimzelltumor des Mannes

Fortbildung

Einbruch im Aufbruch – onkologische Erkrankungen bei Heranwachsenden und jungen Erwachsenen (AYA)

V132

Bokemeyer C. Universitätsklinikum Hamburg-Eppendorf, Hamburg, Germany

While most male patients with metastatic germ cell cancer can be cured by cisplatin-based combination chemotherapy plus additional resection of residual metastases, patients failing salvage chemotherapy including high-dose chemotherapy or patients with immediate cisplatin-refractory disease have an extremely poor prognosis. Fortunately this situation only occurs in 5–10% of the total patient population. However, since these patients are young and in general in good condition new treatment options for cisplatin-refractory disease are urgently needed. Out of more than 20 chemotherapy agents tested, only gemcitabine, oxaliplatin and paclitaxel as well as daily chronic oral etoposide application have shown clinically relevant efficacy with response rates of around 10–20%. Chemotherapy combinations with doublets of either GemOx or GemTax or TaxOx have been demonstrated feasible and shown responses in up to 20–30% of patients. The most consistent results have been reported with the triple chemotherapy combination of gemcitabine, oxaliplatin and paclitaxel resulting in remission rates of 30–40% and 5–10% longterm survival in this poor prognostic patient population. Targeted agents have also been evaluated, however, no drug has yet proven clear efficacy. In two formal phase-II designs sunitinib has not shown efficacy. A phase-II trial of everolimus is currently ongoing. Future studies based on molecular markers of cisplatin resistance and/or new targets for molecular approaches will hopefully improve the prognosis of these patients. Disclosure: Carsten Bokemeyer: Employment or Leadership Position: Uniklinik Hamburg Eppendorf; Advisory Role: Sanofi Aventis; Expert Testimony: Sanofi Aventis, Pfizer Pharma, Novartis. V133

Late toxicities after curative treatment Fossaa S.D. Oslo University Hospital, Radium Hospital, Oslo, Norway

The 5-year relative survival of testicular cancer is today >95%. The long life expectancy of the testicular cancer survivors (TCSs) together with raising incidence of TC has lead to increased prevalence of TCSs who are at risk of long-term and late adverse effects (AEs) .The most frequent life threatening AEs comprise second cancer (OR: 1.62 [95% CI: 1.51–1.74]) and -after chemotherapy- cardiovascular diseases (CVD; OR: 1.44 [95% CI: 1.06–1.91]) .With the exception of leukemia these late events are typically diagnosed after a latency of >10 years. At that time the survivor no longer is followed-up by cancer specialists. The relation between radiotherapy and second cancer has been recognized for a long time. The etiological role of cisplatin-based chemotherapy for the development of solid second cancer is more unknown. As cisplatin is detectable in TCSs' serum >15 years after their chemotherapy (4), future multi-disciplinary research has to uncover any pathophysiological relation between AEs and the residual cisplatin serum levels. 20–30% of TC survivors suffer from neuropathy, ototoxicity, hypogonadism and sub-infertility (5) leading to impaired psycho-social well-being, not only of the patients but also of his family. Long-term participation in the working force may become another issue of clinical research in TCSs.. After discontinuation of TCSs` oncological follow-up the organization of aftercare of TCSs at risk aiming for prevention, early detection and treatment of late AEs. Treatment of TC has been highly successful, but has also has also emphasized health care professionals` need to be aware about long-term and late toxicity, not only in TCSs ,but in long-term cancer survivors, in general.

V140

Improving outcomes for Teenagers and Young Adults (TYA) with cancer – the European Network for TYA with Cancer (ENTYAC) Stark D.1, Morgan S.2, Lewis I.2 Leeds University, Leeds, United Kingdom, 2Leeds TH NHS Trust, Leeds, United Kingdom 1

The management of TYA with cancer is undergoing a gradual but accelerating revolution across Europe and the world. This is necessary because of: – The unique disease profile in TYA – The challenges within professional structures of care for this group – The clear wishes of many patients and carers to see improvements The key areas of potential improvement are: • Improvement in survival and the quality of survival, where TYA are disadvantaged by existing structures of care • Provision of all the required expertise in dedicated and effective multi-professional teams, sometimes across traditional healthcare boundaries • Provision of an environment for care that meets the specific needs of TYA To help achieve this goal within the EU-FP7 collaborative network ' ENCCA', 'ENTYAC' is working jointly with other work programmes in ENCCA, including bone sarcoma, education, ethics and others, to deliver improvements in 6 key areas: • A European Multidisciplinary Framework for TYA Cancer, to promote collaboration and provide strategic direction • Promoting and developing TYA multi-professional education for the workforce • Improving access to clinical trials for all TYA in the EU • Developing a European TYA research initiative, examining biology, epidemiology and health services • Promoting healthy lifestyles in the TYA population and cancer survivors • Placing patient and support organisations at the heart of what we do Improving outcomes for TYA is proceeding at different speeds in different parts of the world. In some there are established teams, bringing together paediatric and adult specialists from many healthcare professions, reviewing and contributing to the optimal care of all TYA with cancer as part of national health policy. In some there are specialised wards and out-patient services tailored to meet the needs of TYA. In several there are charities and patient groups working alongside professionals to achieve these aims. All those with a stake in improving TYA cancer care can support each other to improve the outcomes of care. Disclosure: No conflict of interest disclosed. V141

The AYA-project Rostock (AYAROSA) Hilgendorf I.1, Kropp P.2, Classen C.-F.3, Krogmann D.3, Reichel K.4, Zettl H.5, Freundt J.1, Nielsen T.3, Kleinke A.M.3, Kundt G.6, Hartmann S.7, Freund M.1 University Rostock, Department of Internal Medicine, Div. of Hematology, Oncology, Palliative Care, Rostock, Germany, 2University of Rostock, Institute of Medical Psychology and Medical Sociology, Rostock, Germany, 3 University Children's Hospital, Rostock, Germany, 4University of Rostock, Oncological Center, Rostock, Germany, 5University of Rostock, Clinical Cancer Registry of the Rostock Region, Rostock, Germany, 6University of Rostock, Institute for Biostatistics and Informatics in Medicine and Ageing Research, Rostock, Germany, 7University of Rostock, Department of Obstetrics and Gynaecology, Rostock, Germany 1

Disclosure: No conflict of interest disclosed.

Background: The diagnosis of a malignant disease always is a severe threat to a person. The treatment of cancer may disturb the vulnerable process of growing-up in adolescents and young adults (AYA), since nearly

Abstracts


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Refractory germ cell tumors – new approaches



Krebs – ein Armutsrisiko? (für Ärzte und Pflegekräfte) V148

Endangers the tumor diagnosis, the economic base of our patients and do this may have an impact on the prognosis of the disease Seifart U. Klinik Sonnenblick, Marburg, Germany

Due to improved treatment options, the prognosis of cancer patients in recent years has significantly improved. For this reason, long-term toxicity in cancer patients in terms of a «long-term survivorship» is becoming increasingly important. In addition to the physical and emotional side effects of disease or tumor therapy especially in younger patients often suffer financial and social cuts. This can be explained by disease or treatment and consequences related power losses, as can worsen the employment situation of the patient as a consequence. This deterioration is either a reduction in working hours with a corresponding reduction in salary, a job loss or retirement due to the tumor or the treatment. The last two situations require a significant reduction of the financial situation because a mean disability pension range up to 661€ in Germany, the unemployment compensation is 840.90 € if there are no children to supply. Studies done by de Boer et al (JAMA 2009) show that cancer patients have a higher risk of unemployment by 37% compared to normal subjects and only about 52% of tumor patients can be sustainable long-term re-integrated back into working life. Apart from the limited quality of life, the question arises if the prognosis of cancer patients is affected through a financial and social decline.

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Studies from Sweden and the UK in patients with malignant lymphomas, leukemias (Kristinsson et JCO 2009Albano et J Natl Cancer Inst 2007, Keegan et al JCO 2009),), breast and lung carcinomas (Coebergh et Eur J Cancer 1998, Sharp et al Lancet 1999, Berrino et al Ann Oncol 2003 Candyce et al JCO 2006) showed that the social and financial situation of cancer patients may be prognostically relevant. Therefore financial and social limitations of cancer patients receive more attention, since they may also have prognostic relevance in view of the tumor in addition to reducing the quality of life. Disclosure: No conflict of interest disclosed. V150

Return to work in the group of health and social service professionals after cancer disease – practical advice and recommendations Barth J. Klinik Nordfriesland, St. Peter-Ording, Germany

Demographically, Europe's population is living longer and getting older, concomitantly increasing the urgency for care services and facilities to serve this aging population. There is already presently a troubling shortage of health care professionals, both patient and geriatric care, as well as other care or social professions. Yet in spite of this, health care professionals retire at an earlier age than nearly all other professions, in 2012, female healthcare professionals retired at an average age of 58,6 years and male at 59,8, with both groups citing work pressure and demands as the cause for early retirement. Cancer is a significant hindrance to a possible return to work, since most cancer diseases occur at a critical age for those considering a return to work. Screening tests in rehab hospitals show a significantly worse subjective work prognosis of healthcare professionals compared to other occupational groups. This is due to the high physical and mental requirements. After a cancer therapy, the combination of physical and mental impairments, exhaustion as well as the fear of confrontation to other cancer patients after return to work, increase the apprehensions that burden a return to work. On the other hand, a premature retirement pension is often combined with a potential poverty risk. Rehabilitation that focuses on a return to work in this context is an outstanding challenge. Since 20% of the patients in our rehab hospital are health care professionals or belong to related sectors, we established a specific program for these patients according to the claims of level 3 of the MBOR-program of the DRV. Screening tests are able to identify patients with a high risk of not returning to work. These patients will be included in a specific ICF-related program that is intended to reflect the particular occupational constellation of health care professionals. Different therapy modules, including workplace simulations, are designed to improve both fine motor and general motor functions related to specific occupational requirements, as well as mental, cognitive and motivation related limitations. Another focus is directed to specific occupational-related coping strategies and specific social work support and advice and also includes preventive skills. The program is able to offer specific occupational related therapies in order to ease a limitation adapted return to work after cancer treatment, if medical conditions make a return to work possible. Disclosure: No conflict of interest disclosed.

Wissenschaftliches Symposium Immunphänotypisierung V152

ICSH guidelines on immunophenotyping in AML and MDS Kern W. MLL Münchner Leukämielabor GmbH, München, Germany

Flow cytometry represents an integral part of the diagnostic work-up of acute myeloid leukemia (AML) and is increasingly used in the diagnosis of myelodysplatic syndromes (MDS). Thus, the definition of disease entities is

Abstracts


all components of meaningful life are in a phase of re-organization and irritability. Besides, risk behavior in this age group is common and may affect the course of treatment. In order to understand the special needs of AYA and to improve their psychosocial care, the project AYAROSA – «AYA with Cancer: Rostock Approach for Pre-emptive Psychosocial Care» – was founded in 2010 at the University of Rostock. AYAROSA is a multidisciplinary team of pediatricians, psychologists, gynecologists, hematologists and oncologists. Results: As a first step, we conducted an epidemiological study and analyzed incidence and reporting of malignant diseases of AYA (15–39 years old patients) to the clinical cancer registry Rostock with regard to diagnoses, gender and distribution of physicians caring for the patients. 1425 cases were collected with a mean follow up time of 33 months. We could demonstrate that the cancer incidence increases from each age cohort to the next, while the distribution pattern of diagnoses changed continuously. In a next step, individual structured interviews were performed with AYA patients, covering the fields of body image, identity, education, work, sexuality, relationship to parents, friendships and social networking. This again was followed by a questionnaire study covering the complete cohort of AYA patients registered in the Rostock cancer registry in 2012. Patients were sent questionnaires covering the subject's quality of life, personality, anxiety and depression, therapy adherence and fertility issues. An additional self-designed questionnaire evaluated patient-doctor relationship. The analysis of data is ongoing. However, first results of the study demonstrate that AYA feel significantly less well informed in comparison to senior controls, and over 60% use the internet to get further information. In addition, 14.1% of AYA considered abandoning all therapy. Conclusions: The treatment and needs of AYA with malignancies show differences compared to older adults, and interdisciplinary concepts to improve information, adherence and care are needed. The work of AYAROSA is based on the idea that active coping and achievement of responsibility for the treatment will help to improve both medical treatment results and quality of life.

Disclosure: Wolfgang Kern: Employment or Leadership Position: MLL Münchner Leukämielabor GmbH, MHP Münchner Hämatologiepraxis. V154

MRD quantification in CLL Böttcher S. Universitätsklinikum Schleswig-Holstein Campus Kiel, II. Medizinische Klinik, Kiel, Germany

With the advent of multiple highly effective treatment options, minimal residual disease (MRD) assessments gained interest as a prognostic marker in chronic lymphocytic leukemia (CLL). This presentation provides a model for the prognostic significance of MRD in this disease. Clinical data corroborating the validity of the model are briefly discussed. A major part of the presentation will be dedicated to technological aspects of the flow cytometric determination of MRD in CLL (MRD flow). Consecutive steps of assay development in the era of multicolor flow cytometry are described. The degree of standardization as well as the sensitivity that can be achieved are discussed. Comparative analyses to ASO primer IGH RQ-PCR are demonstrated. The first standardized assay for MRD flow was developed under the auspices of the European Research Initiative on CLL (ERIC). This initial 4-color assay and its 6-color successor achieved a sensitivity of 10-4 . The initial 4-color assays utilizes 3 different MRD tubes, whereas the 6-color assay comprises 2 tubes. The publication on the 4-color assay contained a detailed description of a gating strategy in order to reduce the inter-lab variability of MRD results. Nevertheless, this assays require additional in-house standardization for maximum reproducibility, as staining procedures and the set-up of the flow cytometers were not fully standardized. More recently, the European research consortium EuroFlow has developed and published technical standards on staining, acquisition, and analysis of flow cytometric data (Kalina et al., Leukemia, 2012). Those standards aim at reproducibility of fluorescence intensity assessments between laboratories and within a laboratory over time. The standardization is regularly confirmed using dedicated quality control rounds. Moreover, the consortium collected several hundreds of extensive immunophenotypic profiles of B-cell non-Hodgkn´s lymphoma cases at diagnosis as as well as immunophenotypes of normal B-cells (van Dongen et al., Leukemia, 2012). Using the technical standardization plus the library of phenotypes that includes more than 100 CLL cases an optimal 8-color combination for MRD flow in CLL was devised. Preliminary data suggest that a sensitivity of 10-5 can be obtained with the novel tube. Of note, the MRD results obtained are highly reproducible between laboratories, making it an ideal end-point for future efficacy assessments in clinical trials.

V156

Standardisation of PNH diagnostics and round robin tests Nebe C.T.1, Hoechsmann B.2, Schrezenmeier H.2 Hämatologie-Labor Mannheim, Mannheim, Germany, 2Institut für Klinische Transfusionsmedizin und Immungenetik Ulm (IKT), Ulm, Germany 1

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired disease most often evolving from bone marrow failure syndromes like aplastic anemia. It is rare but shows a high mortality rate. It is caused by spontaneous mutations in the PIG-A gene, the glycophosphatidyl anchor for a group of cell surface proteins on hematopoietic cells involved in compleent inhibition. It happens on the stem cell level and all progeny of the affected cell shows the defect, i.e. red cells, platelets and leukocyte subpopulations exept of the thymus derived T cells. It confers a certain survival advantage over competing healthy clones and therefore, the PNH clone slowly increases over time with a few exceptions of spontaneous recovery. The clinical symptoms are unspecific and less than 25% of the patients show the name giving PNH. There is no mutational hot spot and therefore no concsensus primer for PCR diagnostics exists. Currently, flow cytometric immunophenotyping is the method of choice. Patients to be tested are either symptomatic with the non immune hemolysis or they are at risk to develop the disease when suffering from bm failure syndromes. In the first case there is a significant clone size i.e. proportion of GPI negative cells, quite often far more than 10%. In the latter you may look in the beginning for small populations far below 1% of the respective hematopoietic lineage. The third situation is monitoring of patients under therapy with a monoclonal antibody against the complement protein C5a (eculizumab). Standardisation and proficiency testing have to cover all three aspects.The PNH testing puts high demands on the team running the test, because you are looking for negative cells and not for positively stained ones as done in regular immunophenotyping. On top of that, you may observe type II and type III cells, where the type II is an intemediate expression between normal and type I, a phenomenon which still awaits scientific explaination. Over the last decade a consensus developed, laid down by national expert groups. According to these guidelines, the testing should be done on at least two lineages (eg. reticulocytes and neutrophils) checking for at minimum two independent GPI anchored proteins on each with immunological gating of the target population to avoid artificial negative events leading to false positive results. The latest strategy incl. FLAER will be presented as well as the results of the robin round tests done so far (INSTAND EQA, UK NEQUAS). Disclosure: C. Thomas Nebe: Advisory Role: ja, PNH-Diagnostik für ALexion; Financing of Scientific Research: für Newsletter Fa. Alexion. Hubert Schrezenmeier: Advisory Role: Fa. Alexion. V158

Functional cell analysis by flow cytometry Sack U. Institut für Klinische Immunologie, Medizinische Fakultät der Universität Leipzig, Leipzig, Germany

Disclosure: Sebastian Böttcher: Financing of Scientific Research: Roche; Expert Testimony: Roche, Celgene.

Flow cytometry can not only be applied for phenotyping and quantification of specific cell populations in peripheral blood and single cell suspensions, but also for functional analysis using targeted immunoassays. These are relevant for the diagnosis of immunodeficiencies and form the basis for personalized treatment. Functions of lymphocytes and phagocytes can easily be investigated by using standardized test systems. Following incubation and stimulation of target cells, read out parameters are commonly detected by fluorescent signals. Stimulation is performed with mitogens, polyclonal activators, or specific antigens. The most common read-out parameters are activation molecules, phosphorylated signal transduction molecules, cytokines, or enzyme activities. Test kits are used to investigate phagocytosis, oxidative burst, migration, cytotoxicity, or degranulation of basophils. Specificity for defined anti-

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accomplished by a combination of clinical, morphologic, immunophenotypic and genetic features. With a variety of published sources being available and in consideration of standard panels or standard procedures for all flow cytometry laboratories not being feasible the aim of the ICSH guideline is to provide both laboratory personnel as well as physicians with an instruction how to apply and use immunophenotyping in patients with recently identified cytopenia or suspected blasts. An international expert panel has been formed with experts recruited from eight countries and three continents. The ICSH guideline will build upon an algorithm detailing the application of immunophenotyping at specified check points in the diagnostic work-up and the way how to derive decisions on further diagnostic and therapeutic steps from available results. The different chapters will comprise data on sample source and sample handling, antigens to be analyzed, principles of data acquisition and data analysis (regarding both diagnostic samples and follow-up samples for minimal residual disease), requirements for the report, essentials on quality control, as well as considerations on personnel. All ICSH guideline contents will rely on published data and will be presented in a focused and comprehensive way. This ICSH guideline thus will serve a valuable source for everyone involved in flow cytometry on AML and MDS.

gens can be added using tetramer technology. One example are modern tests for CMV infections. All these test systems are available as in vitro diagnostic test kits and can be applied within standardized procedures. Based on a correct internal quality control and successful external quality assessment schemes, these tests can be approved for the diagnostic laboratory. Thus, high quality diagnostics of systemic hematological diseases, immune defects, but also allergies and autoimmune diseases can be offered. Disclosure: Ulrich Sack: Financing of Scientific Research: Beckman-Caster, Becton-Dickinson.

Disclosure: Anja Seckinger: Expert Testimony: Novartis Pharma. Dirk Hose: Expert Testimony: Novartis Pharma. V161

A phase II study (PX-171-003-A1) of single-agent carfilzomib in patients with advanced relapsed and refractory multiple myeloma

Myelom – klinisch V160

Bone turnover in multiple myeloma: Impact of bortezomib-based induction therapy, high-dose melphalan, and lenalidomide consolidation on osteoblast and osteoclast function within the GMMG-MM5 trial Seckinger A.1,2, Meißner T.1, Bertsch U.1, Salwender H.3, Dürig J.4, Hänel M.5, Blau I.6, Scheid C.7, Weisel K.8, Klein B.9, Goldschmidt H.1,2, Hose D.1,2 Universitätsklinikum Heidelberg, Medizinische Klinik V, Heidelberg, Germany, 2Nationales Centrum für Tumorerkrankungen, Heidelberg, Germany, 3 Asklepios Klinik Altona, II. Medizinische Abteilung, Hamburg, Germany, 4 Universitätsklinikum Essen, Zentrum für Innere Medizin, Essen, Germany, 5 Klinikum Chemnitz, Klinik für Innere Medizin III, Chemnitz, Germany, 6 Charité – Universitätsmedizin Berlin, Medizinische Klinik mit Schwerpunkt Hämatologie, Onkologie, Berlin, Germany, 7Universitätsklinikum Köln, Klinik I für Innere Medizin, Germany, 8Universität Tübingen, Medizinische Klinik, Tübingen, Germany, 9CHU Montpellier, Institute for Research in Biotherapy, Montpellier, France 1

Introduction: In multiple myeloma, initially, there are increased numbers of osteoclasts showing increased activity, but bone formation by osteoblasts is keeping step. In later stages, parts of the bone remodeling compartments are disrupted by the interaction with myeloma cells leading to increased bone resorption which can no longer be compensated (myeloma bone disease, uncoupling of bone formation and bone resorption). Lenalidomide and bortezomib have been shown to target both, myeloma cells and the microenvironment: lenalidomide inhibits osteoclastogenesis, bortezomib is also able to stimulate osteoblast differentiation leading to increased bone formation. Aim of this study is to evaluate the impact of bortezomib-based induction treatment, high-dose therapy, and lenalidomide consolidation on alterations of bone turnover, i.e. surrogates of osteoblast- (osteocalcin, OC) and osteoclast- (collagen type I fragments, CTX-I) function, and their induction by myeloma cells (DKK1-level). Methods: Serum was collected during routine sampling within the GMMG-MM5 trial (EudraCT 2010-019173-16), and levels of CTX-I, OC, and DKK1 were assessed by ELISA in triplicates using commercially available assays according to the manufacturer's instructions (RnD Systems and Immunodiagnostic Systems). The following time points were assessed: at inclusion (n = 365), after induction therapy with either PAd (n = 88) or VCD (n = 84), stem cell mobilization using CAD (n = 69), high-dose melphalan (n = 92), and 2 months lenalidomide consolidation (n = 92). DKK1 levels were correlated with the expression in CD138-purified myeloma cells (Affymetrix microarrays, n = 365). Results: Prior to treatment, CTX-I levels are increased, those of OC decreased compared to healthy donors (uncoupled bone turnover). DKK1 protein levels are increased and correlate with DKK1-expression in myeloma cells. After induction therapy, osteoclast activity (CTX-I) is decreased below normal values. PAd unlike VCD further decreases osteoblast activity (OC-levels); DKK1-levels are normalized. Subsequent treatment further decreases DKK1-levels below normal values and blocks


Siegel D.1, Martin T.2, Wang M.3, Vij R.4, Jakubowiak A.5, Lonial S.6, Trudel S.7, Kukreti V.7, Bahlis N.8, Alsina M.9, Chanan-Khan A.10, Buadi F.11, Reu F.12, Somlo G.13, Zonder J.14, Stewart A.K.15, Stadtmauer E.16, Harrison B.17, Rajangam K.18, Orlowski R.3, Jagannath S.19 John Theurer Cancer Center, Hackensack, United States, 2University of California, San Francisco, San Francisco, United States, 3MD Anderson Cancer Center, Houston, United States, 4Washington University School of Medicine, St. Louis, United States, 5University of Chicago, Chicago, United States, 6Winship Cancer Institute, Emory University, Atlanta, United States, 7 University of Toronto Princess Margaret Hospital, Toronto, Canada, 8Tom Baker Cancer Centre, University of Calgary, Calgary, Canada, 9H. Lee Moffitt Cancer & Research Center, Tampa, United States, 10Mayo Clinic, Jacksonville, United States, 11Mayo Clinic, Rochester, United States, 12 Taussig Cancer Center-Cleveland Clinic, Cleveland, United States, 13City of Hope National Medical Center, Duarte, United States, 14Karmanos Cancer Institute, Wayne State University, Detroit, United States, 15Mayo Clinic, Scottsdale, United States, 16University of Pennsylvania Abramson Cancer Center, Philadelphia, United States, 17Multiple Myeloma Research Consortium, Norwalk, United States, 18Onyx Pharmaceuticals, South San Francisco, United States, 19Mt. Sinai Medical Center, New York, United States 1

Introduction: Carfilzomib, a selective proteasome inhibitor, is approved in the US based on the single-arm, phase II study PX-171-003-A1 (NCT00511238) assessing single-agent use in patients with relapsed and refractory multiple myeloma (RRMM). Methods: Carfilzomib was administered intravenously over 2–10 minutes twice weekly for 3 weeks of a 28-day cycle at a dose of 20 mg/m2 for Cycle 1 and 27 mg/m2 thereafter. Results: The study population (N = 266; intent to treat population) was heavily pretreated with a median of 5 prior lines of therapy (range, 1-20), including bortezomib (99.6%) and lenalidomide (94%). Overall response rate (ORR, primary endpoint) was 22.9% (95% CI, 18.0-28.5) with a median duration of response (mDOR) of 7.8 months (95% CI, 5.6-9.2). Median overall survival (mOS) was 15.4 months (95% CI, 12.5-19.0). In an exploratory analysis of subgroups defined as refractory to bortezomib (n = 194) and refractory to bortezomib and lenalidomide/thalidomide (n = 169), the ORR was 16.5% and 15.4%, respectively, and the mDOR was 7.8 months for each subgroup. In the safety population (N = 266), the most common adverse events (AEs) of any grade included fatigue (49%), nausea (45%), and dyspnea (34%), and grade 3/4 AEs included thrombocytopenia (29%), anemia (24%), and lymphopenia (20%). The incidence of peripheral neuropathy was 12% overall and 1% for grade 3. Median treatment duration was 3.0 months (range 0.03–16.9) with 18% of patients requiring at least 1 dose reduction. Reason for treatment discontinuation was primarily disease progression (59%) followed by AEs (12%). Five deaths (2%) on study or within 30 days posttreatment were deemed possibly related to carfilzomib. Conclusions: In this heavily pretreated population with advanced RRMM, single-agent carfilzomib provided clinically meaningful, durable responses and was generally tolerable with manageable AEs. The ongoing phase III FOCUS trial (NCT01302392), evaluating single-agent

Abstracts


Freier Vortrag

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osteoclast function. After 2 months lenalidomide consolidation, no normalization of osteoblast activity is found. Conclusion: The main impact on bone turnover by bortezomib-based induction therapy is a reduction of osteoclast activity alongside a decrease in DKK1-levels. During the reported period, no normalization of decreased osteoblast function was observed.

Disclosure: David Siegel: Advisory Role: Millennium and Celgene; Financing of Scientific Research: Millennium and Celgene; Other Financial Relationships: Consultancy for Millennium and Celgene. Sundar Jagannath: Advisory Role: Ortho Biotech, Imedex, Medicom WorldWide, OptumHealth Education, and PER Group.; Financing of Scientific Research: Millennium, Celgene, Onyx Pharmaceuticals, and Merck; Other Financial Relationships: Board of Directors for Ortho Biotech, Imedex, Medicom WorldWide, OptumHealth Education, and PER Group. V162

Lenalidomide, Adriamycin, Dexamethasone (RAD) followed by risk-adjusted transplant is a novel and very effective induction regimen in newly diagnosed multiple myeloma Knop S.1, Langer C.2, Engelhardt M.3, Mügge L.-O.4, Rösler W.5, Reichle A.6, Röllig C.7, Hertenstein B.8, Gramatzki M.9, Bassermann F.10, Ostermann H.11, Sturm I.12, Ringhoffer M.13, Schäfer-Eckart K.14, Falk K.15, Liebert A.16, Einsele H.1, Bargou R.1, Deutsche Studiengruppe Multiples Myelom (DSMM) Universitätsklinikum Würzburg, Zentrum für Innere Medizin, Medizinische Klinik und Poliklinik II, Hämatologie/Onkologie, Würzburg, Germany, 2Universitäsklinik Ulm, Klinik für Innere Medizin III, Ulm, Germany, 3 Universität Klinikum Freiburg, Abteilung für Innere Medizin I, Hämatologie und Onkologie, Freiburg, Germany, 4Klinikum der Friedrich-Schiller-Universität Jena, Klinik für Innere Medizin II, Poliklinik für Hämatologie und Internistische Onkologie, Jena, Germany, 5Universitätsklinikum Erlangen, Medizinische Klinik 5, Hämatologie u. Internistische Onkologie, Erlangen, Germany, 6Uniklinikum Regensburg, Klinik und Poliklinik für Innere Medizin, Regensburg, Germany, 7Universitätsklinikum Carl Gustav Carus an der TU Dresden, Medizinische Klinik und Poliklinik I, Dresden, Germany, 8 Klinikum Bremen-Mitte gGmbH, Klinik für Innere Medizini I, Bremen, Germany, 9Universitätsklinikum Schleswig-Holstein Campus Kiel, Sektion für Stammzell- und Immuntherapie II. Med. Klinik und Poliklinik, Kiel, Germany, 10Klinikum rechts der Isar der TU München, III. Med. Klinik und Poliklinik, Hämatologie und Onkologie, München, Germany, 11Klinikum der Universität München-Großhadern, Hämatologie und Onkologie, München, Germany, 12Charité Campus Virchow Klinikum, Universitätsmedizin Berlin, Medizinische Klinik m.S. Hämatologie, Onkologie u. Tumorimmunologie, Berlin, Germany, 13Städtisches Klinikum Karlsruhe, Medizinische Klinik III Hämatologie/Onkologie, Karlsruhe, Germany, 14Klinikum Nürnberg Nord, 5.Medizinische Klinik/Onkologie/Hämatologie, Nürnberg, Germany, 15 Celgene GmbH, München, Germany, 16ClinAssess GmbH, Leverkusen, Germany 1

III disease and in all except three molecular cytogenetic analysis was performed. Incidences of chromosomal abnormalities were as follows: deletion of (del) 13q, 24.7%; translocation (t) (4;14), 12.4%; t(14;16), 3.4%; and del 17p, 5.6%. Treatment-related mortality with RAD was 0% while 61.8% of pts had treatment-emergent SAEs. Five pts (5.6%) had febrile neutropenia and 7 pts each (8%) had pneumonia and VTE, respectively. All 78 pts with at least stable disease successfully mobilized stem cells. Overall response rate (at least partial response, PR) on an intention-to-treat basis following first SCT was 83%. 12 pts. each (13.5%) achieved centrally confirmed complete response (CR) or stringent (s)CR, respectively and an additional 29 pts (32.6%), very good PR. Compared to post-induction assessment, CR/sCR rate increased from 9 to 27% after SCT. Conclusions: This interim analysis shows RAD to be a well tolerated and effective induction protocol which is currently being compared to bortezomib/len/dex in a phase III trial. Disclosure: Stefan Knop: Financing of Scientific Research: Celgene Deutschland GmbH. Ralf Bargou: Financing of Scientific Research: Celgene Deutschland GmbH; Expert Testimony: Celgene Deutschland GmbH. V163

Safety profile of single-agent carfilzomib in patients with relapsed or refractory multiple myeloma from 4 phase II trials Siegel D.1, Martin T.2, Nooka A.3, Harvey R.D.3, Vij R.4, Niesvizky R.5, Badros A.Z.6, Jagannath S.7, McCulloch L.8, Rajangam K.8, Lonial S.3 John Theurer Cancer Center, Hackensack, United States, 2University of California, San Francisco, San Francisco, United States, 3Winship Cancer Institute, Emory University, Atlanta, United States, 4Washington University School of Medicine, St. Louis, United States, 5Weill Medical College of Cornell University, New York, United States, 6University of Maryland, Baltimore, United States, 7Mt. Sinai Medical Center, New York, United States, 8 Onyx Pharmaceuticals, South San Francisco, United States 1

Background: Induction triplets utilizing at least one of the «novel drugs» and dexamethasone with or without chemotherapy are considered standard of care in newly diagnosed multiple myeloma (MM). Medically fit patients (pts) clearly are candidates for subsequent autologous (auto) stem cell transplant (SCT) while use of allogeneic (allo) SCT remains a matter of debate. As we had previously shown the RAD regimen to be well tolerated and highly effective in relapsed MM, we evaluated this combination in first-line treatment. Methods: The current phase II trial (DSMM XII) was designed to include pts up to 65 years with symptomatic MM. Four 4-week cycles of RAD (lenalidomide 25 mg/day, d1-21; infusional adriamycin 9 mg/m², d1-4; dex 40 mg d1-4 and 17–20; pegfilgrastim 6 mg d 6) preceded stem cell chemo-mobilization. Low molecular-weight heparin for prophylaxis of venous thromboembolic events (VTE) was mandatory. Pts received either tandem auto SCT (melphalan 200 mg/m²) or auto-allo SCT. Allo SCT (treosulfan/fludarabine) was reserved for pts featuring at least one cytogenetic or serologic risk factor (RF) who had a matched sibling or unrelated donor available. Lenalidomide maintenance was administered for 1 year. This is the second pre-planned interim safety and efficacy analysis on 75 pts after their inital SCT. Results: Eighty-nine pts (14 cases progressing) with a median age of 54 (range, 30–65) years are evaluable. 50 pts (56.2%) had ISS stage II/

Introduction: Carfilzomib is a selective proteasome inhibitor recently approved in the US for patients with relapsed and refractory multiple myeloma (RRMM). Results of a cross study analysis of 526 patients in 4 phase II trials including PX-171-003-A0, PX-171-03-A1 (both NCT00511238), PX-171-004 (NCT00530816), and PX-171-005 (NCT00721734) are presented herein. Methods: Carfilzomib was administered intravenously over 2–10 minutes and dosed at 20−27 mg/m2 in a 28-day cycle for all studies except 005 (15−27 mg/m2). Adverse events (AEs) were graded by NCI Common Terminology Criteria for Adverse Events (CTCAE) v3.0. Results: AEs ≥ Grade 3 in ≥15% of patients were thrombocytopenia (23.4%), anemia (22.4%), and lymphopenia (18.1%). Because of AEs, dose reductions and treatment discontinuations were required in 14.6% and 14.8% of patients, respectively. There were 37 deaths on study: 24 due to disease progression, 6 due to other reasons, and 7 due to an AE at least possibly related to carfilzomib (as determined by the investigator) . While hematologic AEs were the most common Grade ≥3 AE, they were transient. Worsening renal function was reported in 13% of patients, of which less than half were transient. Although 73.6% of patients had a past medical history of cardiovascular events, cardiac failure events were reported in 7% of patients, regardless of causality. Dyspnea was the most common AE in the pulmonary analysis (42.2%) and pneumonia was the most common respiratory infection (12.7%). While 71.9% of patients had active peripheral neuropathy at baseline, newly developed peripheral neuropathy was reported infrequently (13.9% overall). Conclusions: These data indicate that single-agent carfilzomib has an acceptable safety profile in heavily pretreated patients with RRMM. Few dose reductions and discontinuations due to AEs were needed. The safety profile of carfilzomib will be further explored in the ongoing phase III FOCUS trial (NCT01302392) evaluating patients with RRMM (sin-

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carfilzomib in RRMM patients versus best supportive care, will provide important information to facilitate regulatory approvals ex-US.

gle-agent carfilzomib versus best supportive care), the results of which will be used to facilitate regulatory approvals ex-US. Disclosure: David Siegel: Advisory Role: Millennium and Celgene; Financing of Scientific Research: Millennium and Celgene; Other Financial Relationships: Consultancy for Millennium and Celgene. Sagar Lonial: Other Financial Relationships: Consultancy for Millennium, Celgene, Novartis, Bristol-Myers Squibb, Onyx Pharmaceuticals, Inc., and Merck V164

Comorbidity (CM) assessment in 820 multiple myeloma (MM) patients (pts): Development of a further improved, weighted MM-risk score defined by host- and diseasespecific risk factors (RFs) Domm A.-S.1, Kleber M.1, Ihorst G.2, Koch B.3, Wäsch R.1, Engelhardt M.1 University of Freiburg Medical Center, Department of Hematology & Oncology, Freiburg, Germany, 2Clinical Trials Unit, University Medical Center, Freiburg, Germany, 3Central Laboratory, Medical Center, University of Freiburg, Freiburg, Germany 1

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Fig. 1. Flow chart of consecutive MM pt assessment. Disclosure: No conflict of interest disclosed. V165

Gain of chromosome 1q21: Pathogenesis, prognostication, and treatment of a time-lapsed multiple myeloma Hose D.1,2, Seckinger A.1,2, Meißner T.1, Hielscher T.3, Rème T.4, Moreaux J.4, Bertsch U.1, Hillengass J.1, Jauch A.5, Goldschmidt H.1,2, Neben K.1 Universitätsklinikum Heidelberg, Medizinische Klinik V, Heidelberg, Germany, 2Nationales Centrum für Tumorerkrankungen, Heidelberg, Germany, 3 Deutsches Krebsforschungszentrum, Abteilung für Biostatistik, Heidelberg, Germany, 4CHU Montpellier, Institute for Research in Biotherapy, Montpellier, France, 5Universität Heidelberg, Institut für Humangenetik, Heidelberg, Germany 1

Introduction: Gain of the chromosomal region 1q21 is a cenerentolesquely investigated frequent genetic alteration in multiple myeloma, poorly understood in terms of pathogenic, prognostic and clinical relevance. Methods: We analyzed 1246 CD138-purified plasma cell samples by iFISH and gene expression profiling (n = 902) alongside clinical parameters, including patients at either early stage (n = 139), or undergoing highdose therapy and autologous stem cell transplantation (n = 1068). Results: i) Patients with 1q21-gain show faster progression from asymptomatic to therapy-requiring myeloma. ii) Symptomatic patients with 1q21-gain reach a faster and deeper remission after induction treatment and autologous stem cell transplantation, but subsequently relapse faster with adverse event-free and overall survival. iii) 1q21-gain is copy-number-dependently associated with proliferation, and iv) shows a characteristic gene expression pattern.

Abstracts


Introduction: CM and a deteriorated functional status have been demonstrated to effect outcome in MM pts. In addition to disease specific and age-related factors, type and severity of CMs play a relevant role in MM pts, influence their tolerance of anti-MM agent treatment, progression-free (PFS) and overall survival (OS; Kleber et al. Clin Lymphoma Myeloma Leuk. 2013). In prior analyses, we have identified an impaired Karnofsky Performance Status [KPS], moderate/severe lung function and renal impairment (eGFR < 30) via multivariate analysis as significant RFs for inferior outcome and combined these variables in a comorbidity score (Freiburg Comorbidity Index [FCI]; (Kleber et al. BCJ 2012)]. Objective of this analysis was to further improve this FCI by adding host-and disease-specific RFs (e.g. cytogenetics), physical function and quality of life. Methods/Results: We are currently assessing 820 consecutive MM pts treated in our department between 1997–2013. (Fig. 1) CMs are assessed as weighted heart-, liver-, gastrointestinal- diseases, disability, frailty, infection, pain, secondary malignancies, amyloidosis, peripheral neuropathy and thrombosis. MM-specific disease parameters, including cytogenetics, are also included therein. Defined RFs are categorized as mild, moderate or severe. Via uni- and multivariate analyses, an improved, weighted FCI of the combined test and validation cohort will be assessable. In addition, this new FCI is being compared with other established comorbidity scores (Kaplan-Feinstein-index and Hematopoietic cell transplantation-specific comorbidity index [HCT-CI]). Conclusions: As CMs in MM are frequent, a weighted CM assessment should allow an improved risk evaluation in MM pts. Our novel FCIscore may eventually also allow more individualized MM treatment allocations and is being assessed in a multicenter approach. Moreover, further prospective analysis will elucidate the value of the FCI in the prediction of treatment-related toxicity, treatment-reduction, -cessation, PFS and OS, thereby allowing to compare safety and feasibility of different therapeutic options in MM.


Posterdiskussion Akute Leukämien I P166

Focussed in vivo RNAi screen to identify pathways that modulate AML chemosensitivity in vivo Lettermann S., Rohde C., Lindtner B., Klosner J., Berdel W.E., Müller-Tidow C., Agelopoulos K. Westfälische Wilhelms-Universität Münster, Molekulare Hämatologie und Onkologie, Münster, Germany

Chemotherapy can induce remission in Acute Myeloid Leukemia (AML). Nonetheless, the majority of patients still relapses. A subset of more chemoresistant leukemia cells might be responsible for this phenomenon. These relapse initiating cells might differ epigenetically from the more chemosensitive leukemia bulk. Since interaction of leukemic cells with the niche might contribute to chemoresistance, we decided to perform an in vivo RNAi screen. We constructed a focussed shRNA library with almost 400 constructs that target 142 genes. The selected genes were known to be associated with stem cell development, stem cell maintainance and leukemia development. The shRNA sequences were cloned with the stem-loop of murine miR-155 to improve processing of the mature shRNA. Primary murine leukemias induced by myc/bcl2 expression were transduced with the library and injected into sublethally irradiated syngeneic mice. Mice (n = 6 in each group) were treated with Cytarabine, Azacytidine or Decitabine or vehicle. DNA was extracted and the shRNA pool was amplified from bone marrow, spleen and liver by PCR. PCR products barcoded and sequenced (n = 64 libraries total). Suppression of HDAC4, Pik3r1, Atr, Ccna2 and HDAC 1 significantly enhanced sensitivity to Cytarabine treatment. Suppression of Stat5a, Ikzf1, Stat4, Myst2 decreased sensitivity of leukemic blasts. Further analyses are underway in vivo and in vitro to analyze the therapeutic potential as well as the mechanism of the synergism. Taken together, these findings establish that in vivo RNAi screening for therapeutic synergism is feasible and that RNAi in vivo screening can identify novel therapeutic targets.

tion of neoplastic cells. Thus, the aim of this study was to define critical pathways explaining the mode of action in two different AML-cell lines. Methods: Based on data from dose-response curves for determining drug concentrations inducing an at least 50% reduction of cell survival, HL60 promyelocytic leukemia cells and KG1 myeloblastic leukemia cells were treated with DAC (0.5 µM for KG1 or 5 µM for HL60) or AZA (2 µM for both KG1 and HL60 cells) for 3 days. RNA was isolated and gene expression was analyzed using Affymetrix human gene 1.0 arrays which cover more than 20000 genes. The Pathvisio software tool was used for comparative quantification of gene-expression patterns from metabolic pathways based on established data banks such as KEGG and Wikipathways. Expression of selected genes was confirmed with QPCR (quantitative real time PCR) and immune blots or enzyme-activity determination for caspases. Results: Drug induced stimulation of FAS and caspase 8 as markers for extrinsic apoptosis was detected in KG1 but not in HL60 cells, where stimulation of caspase 9 further indicates a predominance of the intrinsic apoptotic pathway in this cell line. In addition, the effector caspase 7 which is targeted by both caspase 8 and 9 was upregulated in both cell lines, thus emphasizing the pro-apoptotic effect of DAC and AZA in KG1 and HL60 lines. Expression of autophagosome components (ATGs) was only stimulated in HL60 cells but not in KG1 cells, thus further confirming the difference in pro-apoptotic pathways in these two cell lines. However, it has to be considered that DAPK1, which was stimulated as a consequence of promoter-demethylation in both cell lines may also stimulate FAS expression at the protein level. Conclusions: As stimulation of autophagy is known to induce a differentiation-promoting effect, this could explain the drug-induced upregulation in cell lines and the differentiating effect on dysplastic hematopoiesis with demethylating drugs as observed in clinical practice. Disclosure: No conflict of interest disclosed. P168

Valproic acid can increase apoptosis when FLT3-ITD- positive AML cells are treated with fluvastatin and all-trans-retinoic acid Zirm E., Spies-Weißhart B., Schnetzke U., Hochhaus A.1, Scholl S. Klinik für Innere Medizin II, Universitätsklinikum Jena, Hämatologie und Onkologie, Jena, Germany

Introduction: Epigenetic and apoptosis-regulating mechanisms contribute essentially to the pathogenesis of myelodysplastic syndromes (MDS) and may trigger disease-progression to secondary acute myeloid leukemia (AML). Treatment with the demethylating drugs 5-Azacytidine (AZA) or Decitabine (DAC) appears as a promising strategy for selective elimina-

Introduction: FLT3-ITDs (internal tandem duplications) are associated with an impaired prognosis in AML (acute myeloid leukemia). The pattern of downstream activation by this constitutively activated receptor tyrosine kinase is affected by its localization and depends on the glycosylation status of FLT3-ITD. This process of post-translational maturation can be inhibited by fluvastatin. Furthermore, treatment of AML cells with HDACi (histone deacetylases inhibitor) can influence oncogenic signaling by non-genomic effects, e.g. deceased protein stability. The objective of this study was to investigate whether fluvastatin has an impact on the induction of apoptosis of FLT3-ITD-positive AML cells in the presence of the HDACi VPA (valproic acid) or the differentiation-inducing compound ATRA (all-trans-retinoic acid) and to elucidate a molecular mechanism of VPA-induced alteration of FLT3-ITD signaling. Methods: The murine Ba/F3 leukemia cell line was stably transfected with a FLT3-ITD variant resulting in IL-3-independent growth. Signal transduction after exposing cells to fluvastatin, VPA and/ or ATRA was analysed by Western blotting. Apoptosis, cell cycle analyses and differentiation were detected by flow cytometry. Results: In FLT3-ITD expressing Ba/F3 cells, the combination of VPA and ATRA was not able to induce apoptosis while fluvastatin alone resulted in a slight increase of apoptotic cells compared to the DMSO control. Co-treatment with fluvastatin, VPA and ATRA demonstrates additive effects and is associated with a significant increase of apoptosis in Ba/F3ITD cells. Phosphorylation of the anti-apoptotic protein AKT (Ser473) was strongly decreased in the triple combination while VPA plus ATRA

Abstracts


Disclosure: No conflict of interest disclosed. P167

Demethylating Drugs Promote Expression of Genes Regulating Apoptosis and Autophagy in Acute Myeloid Leukemia (AML) Karlic H.1, Varga F.2, Valent P.3,4, Pfeilstöcker M.5 Ludwig Boltzmann Cluster Oncology, Hanusch Krankenhaus, Wien, Austria, 2Ludwig Boltzmann Institute of Osteology at the Hanusch Hospital of WGKK and AUVA Trauma Centre Meidling and 1st Medical Department, Hanusch Krankenhaus, Wien, Austria, 3Department of Medicine I, Division of Hematology & Hemostaseology, Medical University of Vienna, Wien, Austria, 4Ludwig Boltzmann Cluster Oncology, Medizinische Universität Wien, Wien, Austria, 53rd Medical Department, Hanusch Hospital, Wien, Austria 1

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Conclusions: 1q21-gain hijacks myeloma into a time-lapsed unique biological, clinical, and treatment related entity, that should be assessed in all myeloma patients. Patients with 1q21-gain might benefit from maintenance treatment independent if in (near) complete remission and additional cell cycle targeting treatment.

Disclosure: Elisabeth Zirm: Other Financial Relationships: Reisekosten (Mundipharma). Sebastian Scholl: Financing of Scientific Research: Vortragshonorare (Novartis, Janssen-Cilag); Other Financial Relationships: Reisekosten (Gilead, Novartis, Pfizer). P169

DNMT3B expression impairs leukemic stem cells and delays leukemogenesis in an inducible transgenic mouse model Schulze I.1, Tschanter P.1, Rohde C.1, Bäumer N.1, Linhart H.2, Hebestreit K.3, Klein H.-U.3, Dugas M.3, Berdel W.E.1, Müller-Tidow C.1,4 Universitätsklinikum Münster, MedA, Hämatologie/Onkologie, Münster, Germany, 2Nationales Centrum für Tumorerkrankungen, Heidelberg, Germany, 3Institut für medizinische Informatik, Universität, Münster, Germany, 4 Interdisziplinäres Zentrum für klinische Forschung, Münster, Germany 1

DNA methyltransferases (DNMT) play an important role in regulation of DNA methylation and mutations of DNMT3A are frequently found in AML. We analyzed the effects of DNMT3B overexpression on leukemogenesis and chemoresponse in a doxycycline-inducible transgenic mouse model. To analyze the impact of DNMT3B overexpression on leukemogenesis, we transduced lineage-negative bone marrow cells of wildtype and DNMT3B transgenic mice with MLL-AF9 or cmyc/bcl2 oncogenes and serially transplanted these cells into sublethally irradiated C57BL/6N mice. For verification of a DNA hypermethylation induced by DNMT3B overexpression, we performed Reduced Representation Bisulfite Sequencing (RRBS) on leukemic spleen cells. Chemoresponse in MLL-AF9 leukemic wildtypic or DNMT3B transgenic cells was analyzed by proliferation, apoptosis and colony formation assays after treatment with chemotherapeutics. Expression of the DNMT3B transgene on the mRNA and protein level was strongly induced by the presence of doxycycline. The overexpression itself was not toxic to the cells, as shown by an even increased number of hematopoietic stem cells in DNMT3B expressing mice. Analysis of methylation levels by RRBS revealed a strong hypermethylation in DNMT3B expressing cells from leukemic mice. Differentially methylated regions (DMR) were mainly located in promoter regions and first introns. In transplantation experiments, mice with and without DNMT3B expression developed leukemia with splenomegaly and a tendency of delayed leukemogenesis in DNMT3B overexpressing mice. Recipients of DNMT3B expressing cmyc/bcl2 and MLL-AF9 leukemic cells were severly impaired in leukemogenesis and died significantly later upon secondary transplantation (p 50%) compared to untreated p73-depleted cancer cells. Analyzing the genome-wide gene expression profiles of 286 AML-patients with different karyotypes showed that the top hits from our screen have a prognostic impact in AML-patients. Kaplan-Meier survival analysis showed that AML-patients with high-level expression of p73 displayed a significantly reduced overall survival compared to p73 low-expressing AML-patients. Together, these data may lead to a more individualized AML therapy, resulting in better treatment outcome. This study was supported by grants from Deutsche Forschungsgemeinschaft and Deutsche Jose Carreras Leukämie-Stiftung. Disclosure: No conflict of interest disclosed. P171

Oncostatin M is an important STAT5 target gene in FLT3ITD signalling Müller T.A.1, Grundler R.2, Istvanffy R.2, Hennighausen L.3, Illert A.L.1, Duyster J.1 Universitätsklinikum Freiburg, I. Medizinische Klinik, Freiburg, Germany, Klinikum rechts der Isar der TU München, III. Medizinische Klinik, München, Germany, 3NIDDK, National Institutes of Health, Bethesda, United States 1 2

Activating mutations of FLT3 are among the most frequent alterations in AML. Two distinct types of mutations have been described: Duplication of the juxtamembrane domain (ITD) and point mutations of the tyrosine kinase domain (TKD). Although both lead to constitutive FLT3 signaling, FLT3-ITD signals through the STAT5 pathway whereas FLT3-TKD and FLT3 WT activate STAT5 to a much lesser degree. In a murine bone marrow transplantation model, FLT3-ITD leads to a myeloproliferative

Abstracts


results in the highest phosphorylation level of AKT. VPA alone was able to increase the levels of phosphorylated STAT5 which is accompanied by a significant stabilization of FLT3. Conclusion: We could demonstrate that co-treatment with VPA, fluvastatin and ATRA increases the susceptibility to induce apoptosis. Our data let us hypothesize that compartmentalization of FLT3-ITD by statins combined with non-genomic effects of VPA might represent a promising strategy to overcome TKI resistance in FLT3-ITD-positive AML.


The histone deacetylase inhibitor valproic acid induces proteasomal degradation of the nuclear corepressor Ski in AML and melanoma cell lines Teichler S.1, Brendel C.1, Illmer T.2, Stiewe T.1, Neubauer A.1 Zentrum Innere Medizin, Klinik für Hämatologie, Onkologie, Immunologie; Universitätsklinikum der Philipps-Universität, Marburg, Germany, 2 Medizinische Klinik und Poliklinik I Universitätsklinikum Carl Gustav Carus Technische Universität, Dresden, Germany 1

Introduction: Acute myeloid leukemia (AML) with deletion of chromosome 7 or 7q (-7/ del7q) has a poor outcome. We found the expression of the nuclear oncogene Ski to be highly up-regulated especially in AML with -7/del7q and identified Ski as an inhibitor of vitamin A induced myeloid differentiation through interaction with N-CoR recruiting histone deacetylases (HDAC) (Ritter et al, Leukemia 2006). HDAC inhibitors promote histone acetylation, induce apoptosis and cell growth arrest in tumor cells (see Kraemer et al, Trends Endocrinol Metab 2001). Recently we demonstrated that treatment of primary AML patient cells with HDAC inhibitors like valproic acid (VPA) induces myeloid differentiation and/or apoptosis in cells expressing high Ski (n = 7) compared to cells without Ski expression (n = 7). In addition, we found a decrease of Ski protein level in high Ski expressing AML cells as well as in melanoma cells after VPA treatment. The aim of our investigation was to study the molecular background of Ski reduction by the HDAC inhibitor VPA. Methods: We treated IFB melanoma cells with VPA and/or the proteasomal inhibitor MG132. Mononuclear cells from blood or bone marrow of AML patients were isolated using ficoll density gradient centrifugation. IFB cells were transfected with siRNA oligonucleotides against Arkadia and treated with VPA. Ski and Arkadia expression was tested using Western blot analysis or quantitative RT PCR. Results: Treatment of IFB cells expressing Ski with VPA and MG132 revealed that the decrease of Ski by VPA depends on proteasomal degradation. The ring finger protein Arkadia is an E3-ligase of Ski (see Nagano et al, J Biol Chem 2007). Therefore we tested whether Arkadia is involved in Ski reduction by VPA. We demonstrate that the expression of Arkadia is induced by VPA on protein and RNA level in IFB cells. Furthermore Arkadia expression is inversely associated with Ski expression in several

Abstracts

AML cell lines and primary cells of AML patients. In addition we found that siRNA mediated knockdown of Arkadia impairs reduction of Ski by VPA treatment in IFB cells. Conclusions: We found that treatment with HDAC inhibitors renders AML cells expressing Ski sensitive to myeloid differentiation. VPA reduces the expression of the oncogene Ski by inducing the E3-ligase Arkadia which abolishes Ski by proteasomal degradation. Therefore our data suggest that high Ski expression could be a molecular marker for VPA therapy. Disclosure: No conflict of interest disclosed. P173

Loss of Proteinase-Activated Receptor 1 (PAR1) enhances leukemic stem cell function Bäumer N.1, Krause A.1, Köhler G.2, Lettermann S.1, Evers G.1, Hascher A.3, Bäumer S.1, Berdel W.E.1, Müller-Tidow C.1, Tickenbrock L.3 Uniklinik Münster, Innere Medizin A, Mol. Hämatologie und Onkologie, Münster, Germany, 2Uniklinik Münster, Gerhard Domagk-Institut für Pathologie, Münster, Germany, 3Hochschule Hamm-Lippstadt, Hamm, Germany 1

External signals influence stem cell fates via surface receptors with subsequent intracellular signal transduction. The thrombin receptor PAR1 plays an important role in hemostasis, thrombosis and vascular biology, as well as in in tumor biology and angiogenesis. Its expression and function on hematopoietic stem cells is unknown. Here, we analyzed expression and function of PAR1 in primary hematopoietic cells and their leukemic counterparts. Microarray and real-time RT-PCR revealed that PAR1 mRNA was prominently expressed in the hematopoietic stem cell fraction with lower expression in differentiated granulocytes. We then tested PAR1 levels in microarray analysis and real-time RT-PCR and in tissue arrays of AML patient samples in comparison to CD34+ cells. Indeed, we detected much lower levels of PAR1 mRNA and protein in AML samples than in the controls. We took advantage of a Par1-knockout mouse model to further analyse the function of Par1 in hematopoiesis and in leukemogenesis. Loss of Par1 in this genetic mouse model did not affect blood cell counts or colony formation of bone marrow cells. Also, repopulation capacity after bone marrow transplantation in limiting dilutions was unchanged upon Par1-deficiency. We induced myeloid leukemia both in wild type and Par1-/- bone marrow cells by retroviral introduction of the MLL-AF9 oncogene. Leukemia was initiated in both genotypes with comparable latency, penetrance, and phenotype. However, Par1-deficient MLL-AF9 leukemias showed a significantly higher fraction of leukemic stem cells, since cloning efficiency of Par1-/- leukemias in vitro was significantly increased. Accordingly, the latency of secondary leukemias was significantly shortened in Par1-/- MLL-AF9 cells. To determine the frequency of leukemia initiating cells, we transplanted c-kit+ Par1+/+ vs Par1-/MLL-AF9 secondary leukemic blasts in limiting dilutions. Remarkably, the number of leukemia initiating cells was 4-fold higher in Par1-deficient leukemias. In conclusion, PAR1 is highly expressed on hematopoietic stem cells but its loss does not impair steady state hematopoiesis. In contrast, PAR1 expression is very low on the majority of AML patients´ blasts and the loss of PAR1 enhances leukemia stem cell functions by a cell intrinsic mechanism. Disclosure: No conflict of interest disclosed.


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neoplasm while FLT3-TKD induces a lymphoid disease. In the present study we have analyzed the expression of STAT5 target genes in mice transplanted with FLT3-ITD. One of the most unregulated genes was Oncostatin M, a member of the IL-6 family of cytokines, which is known to be regulated by STAT5. In order to prove the importance of Oncostatin M in FLT3-ITD signaling, we used a murine conditional STAT5 knockout system where we transplanted FLT3 mutants and analyzed for expression of Oncostatin M by Realtime-PCR. In addition, Oncostatin M expression was analyzed in sorted bone marrow progenitor cells. Finally we asked the question, whether Oncostatin M overexpression is sufficient to shift the phenotype of an FLT3-TKD induced lymphoid disease to a myeloid phenotype in mice. We found Oncostatin M highly expressed in the bone marrow of mice that were transplanted with FLT3-ITD, whereas STAT5 deletion strongly decreases its upregulation. Interestingly, when we analyzed bone marrow progenitor populations, we could detect Oncostatin overexpression exclusively in FLT3-ITD+ myeloid progenitor cells, but not in lymphoid progenitor or FLT3-TKD+ cells. We were further interested whether Oncostatin M is sufficient to induce a myeloid phenotype in the absence of STAT5 activation. Indeed, in a murine transplantation model we found that Oncostatin M coexpression together with FLT3-TKD leads to a shift in the hematologic phenotype to a myeloid proliferative disease marked by Gr-1 and CD11b surface expression of bone marrow cells. We thus conclude that Oncostatin M expression can substitute for STAT5 signalling in FLT3-TKD positive disease, and is an important target gene in FLT3-ITD positive myeloid leukemia.

P174

P175

Ben Batalla I.1,2, Schultze A.1,2, Wroblewski M.1,2, Erdmann R.1,2, Heuser M.3, Riecken K.4, Binder M.1, Cubas-Cordova M.1,2, Janning M.1,2, Wellbrock J.1, Fehse B.4, Hagel C.5, Krauter J.3, Ganser A.3, Lorens J.B.6, Fiedler W.1, Carmeliet P.7,8, Pantel K.2, Bokemeyer C.1, Loges S.1,2

Vasold J., Wagner M., Drolle H., Sironi S., Fiegl M.

University Medical Center Hamburg-Eppendorf, Department of Hematology and Oncology, Hubertus Wald Tumorzentrum, Hamburg, Germany, 2 University Medical Center Hamburg-Eppendorf, Department of Tumor Biology, Hamburg, Germany, 3Hannover Medical School, Department of Hematology, Hemostasis, Oncology, and Stem Cell Transplantation, Hannover, Germany, 4University Medical Center Hamburg-Eppendorf, Research Department Cell and Gene Therapy, Clinic for Stem Cell Transplantation, Hamburg, Germany, 5University Medical Center Hamburg-Eppendorf, Institute of Neuropathology, Hamburg, Germany, 6University of Bergen, Department of Biomedicine and Centre for Cancer Biomarkers, Bergen, Norway, 7Versalius Research Center, VIB, Laboratory of Angiogenesis and Neurovascular Link, Leuven, Belgium, 8KU Leuven, Laboratory of Angio genesis and Neurovascular Link, Leuven, Belgium 1

Introduction: We discovered that Axl, the receptor for Growth Arrest-specific protein 6 (Gas6), represents a novel prognostic marker and potential therapeutic target in AML (Blood (ASH Annual Meeting Abstracts), Nov 2011; 118: 940). Here, we validated Axl as a new therapeutic target in AML and investigated its involvement in chemoresistance. Methods: Gas6 levels were measured by ELISA and immunohistochemistry. Axl expression was detected by flow cytometry. Co-cultures of (murine) BM stroma cells (primary, OP9, S17) with Mv4-11 and OCI-AML5 cell lines were performed. Results: We found (i) higher expression of Axl in AML BM compared to healthy BM donors (66.20 ± 10.87 vs. 0.65 ± 0.10%; n = 8/6; p  60 protocol. The patient achieved a complete remission with a duration of CR with a follow up beyond 12 years. All other patients were negative for the interrogated mutations of spliceosomal genes. Conclusion: Since the only mutation in 192 sequence analyses of splicing gene mutational hotspots in 48 APL patients was one K700E mutation it can be concluded that spliceosomal gene mutations in APL are rare and do not play an important role in its molecular pathogenesis. However, the recent available deep sequencing may reveal so far unknown mutation in splicosomal genes. Disclosure: Johann-Christoph Jann: No conflict of interest disclosed. Daniel Nowak: Advisory Role: Celegen Advisory Board.

Introduction: The introduction of ATRA and arsenic trioxide in the therapeutic management of acute promyelocytic leukemia (APL) has led to long term survival in about 75% of de novo APL patients. Approaches to further improve patient outcome by stratifying patients at time of initial diagnosis according to their individual risk and to adjust therapy accordingly have to date been based on clinical features only, such as white blood cell count and platelet count. Molecular markers have not been established for risk stratification as yet. Data on the prognostic implication of Wilms' Tumor 1 (WT1) gene expression in acute myeloid leukemia are controversial. WT1 is known to be highly expressed in APL but information on its impact on prognosis is missing. Methods: WT1 expression levels were retrospectively evaluated by quantitative real-time PCR in bone marrow cells of 86 APL patients after informed consent. All patients were treated in the German AMLCG study. The treatment consisted of simultaneous ATRA and double induction (TAD/HAM), one cycle of consolidation chemotherapy (TAD) and 3 years monthly maintenance chemotherapy. WT1 expression groups were defined as follows: Patients with WT1 expression lower than the 25th or higher than the 75th percentile (WT1low/high) were compared to patients with WT1 expression in between the 25th and 75th percentile (WT1int). Time to complete remission (CR), relapse free survival (RFS) and overall survival (OS) were calculated using the Kaplan-Meier method and a logrank test was used to compare differences between the groups (p A (p.Gly183Ser), in the paired box protein encoding gene, PAX5, was found to segregate with disease in two unrelated kindreds with autosomal dominant pre-B cell acute lymphoblastic leukemia (ALL). Leukemic cells from both families exhibited 9p deletion, with loss-of-heterozygosity and retention of the mutant PAX5 allele at 9p13. Two additional sporadic ALL cases with 9p loss demonstrated PAX5 Gly183 substitution in the leukemic cells. RNAseq analysis of two patients with B-ALL carrying the Gly183Ser mutation as well as further functional and gene expression analyses of the PAX5 germline variants demonstrated reduced transcriptional activity, indicating that the mutation acts as a hypomorphic variant. Interestingly, while sporadic B-ALL cases carrying one of the more deleterious PAX5 mutations (e.g. PAX5 Pro80Arg) generally retain one PAX5 wildtype allele, we found frequent loss-of-heterozygosity (LOH) in B-ALL samples with the PAX5 Gly183Ser mutant. These data extend the role of PAX5 alterations in the pathogenesis of pre-B ALL, and implicate PAX5 in a novel syndrome of germline susceptibility to pre-B cell neoplasia.



Fig. 2. Bone marrow biopsy (Giemsa staining).

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Abstracts


1

CLL

P199

The TNF-family member BAFF is released by NK cells which enables evasion of Chronic Lymphoid Leukemia cells from direct and Rituximab-induced NK cell reactivity Wild J.1, Schmiedel B.1, Maurer A.2, Raab S.1, Kanz L.1, Salih H.R.1 Eberhard Karls Universität, Med. Klinik II, Hämatologie und Onkologie, Tübingen, Germany, 2Eberhard Karls Universität, Interfakultäres Institut für Biochemie, Tübingen, Germany 1

NK cells are cytotoxic lymphocytes that play a major role in anti-tumor immunity. Their ability to mediate antibody-dependent cellular cytotoxicity (ADCC) largely contributes to the therapeutic efficacy of monoclonal antibodies like Rituximab, which meanwhile is an essential component of most treatment strategies for B cell malignancies. In Chronic Lymphoid Leukemia (CLL), the ability of NK cells to target the malignant cells has been reported to be compromised, but the underlying mechanisms are largely unclear (Jaglowski et al., Blood 2010). The TNF family member B cell activating factor (BAFF) regulates the development, differentiation, proliferation and survival of B cells. Here we report that primary NK cells release BAFF in soluble form depending on activation state as revealed by PCR and ELISA. In tetrazolium salt-based assays, NK cell-derived BAFF in turn was found to enhance the metabolic activity of primary B-CLL cells which express high levels of BAFF receptors on their cell surface. The effects of BAFF on CLL cell activity could be blocked by the anti-BAFF antibody Belimumab, which is approved for treatment of systemic lupus erythematosus. Notably, BAFF also facilitated a pronounced resistance of primary B-CLL cells to direct NK cell lysis and also Rituximab-induced NK ADCC as revealed by Europium cytotoxicity assays. Our data on the involvement of NK cell-derived BAFF in metabolic activity and resistance of B-CLL cells to NK cell reactivity provides an explanation for the clinical observation that lymphoblastic leukemia cells are more resistant to NK cell reactivity as compared to myeloid leukemias (Pende et al., Blood 2005) and for the reportedly compromised efficacy of Rituximab to induce NK reactivity in CLL. In addition, our findings point to a potential benefit of Belimumab for immunotherapy of B cell malignancies. Disclosure: No conflict of interest disclosed. P200

Systematic identification of drug sensitivity in chronic lymphocytic leukemia Sellner L.1,2, Oles M.3, Slabicki M.1, Merkel O.1, Blume C.1, Hüllein J.1, Stolz T.1, Sill M.4, Dietrich S.2, Rossi D.5, Zirlik K.6, Seiffert M.7, von Kalle C.1, Ho A.D.2, Glimm H.1, Huber W.3, Zenz T.1,2 National Center for Tumor Diseases and German Cancer Research Center, Department of Translational Oncology, Heidelberg, Germany, 2Heidelberg University Medical Center, Department for Medicine V, Heidelberg, Germany, 3European Molecular Biology Laboratory, Genome Biology Unit, Heidelberg, Germany, 4German Cancer Research Center, Division of Biostatistics, Heidelberg, Germany, 5Amedeo Avogadro University of Eastern Piedmont, Department of Translational Medicine, Novara, Italy, 6Freiburg University Medical Center, Department of Hematology/Oncology, Freiburg, Germany, 7German Cancer Research Center, Division of Molecular Genetics, Heidelberg, Germany 1

Introduction: Different genetic aberrations contribute to heterogeneous clinical course in chronic lymphocytic leukemia (CLL). Roles of specific mutations for the response to drugs have been demonstrated for CLL and other entities. Here we systematically study heterogeneity of treatment response and genetic lesions in CLL using a drug screen platform on primary tumor cells. The identification of pathway dependencies in func-

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tional subgroups can be exploited to develop genetically tailored targeted therapeutic strategies. Methods: Ex vivo screening of primary CLL cells was performed using a drug library containing 67 compounds at different concentrations after careful dose finding. The drug library included inhibitors targeting critical pathways in CLL and other cancers as well as compounds with known activity against CLL cells (e.g. Abt-263, CAL-101, Dasatinib, Fludarabine, Ibrutinib). Cell viability was assessed by quantification of ATP (CellTiter Glo®) 48 and 72 hours after drug application. Genetic characterization was performed by targeted sequencing (BRAF, MYD88, NOTCH1, SF3B1, TP53), whole exome sequencing and low coverage whole genome sequencing. Results: We report on results from 97 CLL samples screened ex vivo. Inhibitors targeting related pathways showed similar response profiles across patients, suggesting that prediction of drug effects can be based on functional and genetic properties of tumors (e.g. drugs targeting the B-cell receptor pathway: CAL-101, Dasatinib, Ibrutinib, R406). In addition, CLL patients can be grouped according to their overall response profile. Clinical relevance of the platform was demonstrated by results on p53-dependent compounds: Nutlin-3 (10µM) and Fludarabine (1 µM) induced cell death in cells with wild-type (n = 68) but not with mutated p53 (n = 14) (mean ± SD [% viability of the untreated control]: 53 ± 18 vs 81 ± 17, p 60 years of age; history of thrombosis; platelet count >1000 × 109/L) with ET who were receiving cytoreductive therapy at the time of study registration were enrolled. The choice of cytoreductive therapy was determined prior to enrolment; patients were managed according to local practice. Data were collected every 6 months for 5 years using an electronic data capture system. Here we present results from a data-cut taken in September 2011 (2.5 years since the last patient was enrolled). Results: Approximately 70% of the patients were continuing in the study at this data-cut. A total of 3643 patients, 61.3% females and 38.7% males, across a wide range of ages (< 40 years, 6.9%; 40–59 years, 25.5%; ≥60 years, 67.6%) were enrolled. The majority of patients (80.6%) had received prior treatment with a cytoreductive therapy at enrolment. The two main monotherapy cytoreductive treatments prescribed were hydroxycarbamide (65.1%) and anagrelide (22.2%). Other treatments included interferon, busulphan, pipobroman, 32P and hydroxycarbamide/anagrelide in combination. At enrolment, a greater proportion of patients receiving anagrelide were < 60 years of age (59.3%), compared with those receiving hydroxycarbamide (19.3%). There was considerable variety in the rates of treatment selection between countries both at registration (hydroxycarbamide 38–80%; anagrelide 10–51%; other 4–50%) and at the time of the data-cut (hydroxycarbamide 33–75%; anagrelide 9–50%; other 6–50%). Conclusions: EXELS provides real-world evidence about the patterns of cytoreductive treatment used for high-risk patients with ET across Europe. Hydroxycarbamide was the most frequent treatment of choice in nearly all of the participating countries across Europe; however, patient age strongly influenced the choice between hydroxycarbamide and anagrelide therapy. The treatment pattern for ET observed in EXELS is in accordance with expert recommendations in Europe. Disclosure: Martin Griesshammer: No conflict of interest disclosed. Gunnar Birgegård: Financing of Scientific Research: Shire.

V406

Characterisation of different regimens for introducing second-line Anagrelide: Results from a multicentre study of 177 patients in France Keddad K.1, Rey J.2, Viallard J.-F.3, Smith J.4, Wilde P.4, Kiladjian J.-J.5 Shire ISP France, Boulogne-Billancourt, France, 2Paoli-Calmettes, Onco-Hematology, Marseille, France, 3Hôpital Haut-Lévêque, Service de Médecine Interne, Bordeaux, France, 4Shire Pharmaceuticals Ltd, Basingstoke, United Kingdom, 5Hôpital Saint Louis, Centre d'Investigations Cliniques, Paris, France 1

Introduction: Anagrelide (ANA) is indicated in the EU for at-risk patients (pts) with essential thrombocythaemia (ET) in whom prior therapy (PT) is not sufficiently effective or well tolerated. This study aimed to identify the switch modalities used when introducing ANA and determine their influence on 6-month (mo) outcomes (efficacy, tolerability and maintenance). Methods: This observational study (NCT01192347) was conducted in 44 clinical centres across France. High-risk pts (aged >60 years; history of thrombosis; platelet count >1000 × 109/L) with ET were enrolled within 1 mo of switching to ANA; data were collected and recorded from pt records after 6-mos follow-up. Results: 177 pts were enrolled: 62% female, 76% aged >60 years, median baseline platelet count 553 × 109/L. Intolerance to therapy (65%) and inefficacy (41%) were the most frequent reasons for treatment switch (factors not mutually exclusive). The Summary of Product Characteristics (SPC)-recommended ANA starting dose (1mg/day) was used most frequently (53%); a notable proportion of pts (41%) started on 0.5 mg/ day, and starting doses ranged from 0.3 to 1.5 mg/day. The median ANA dose at study end was 1.5 mg/day. The method of ANA introduction was consistent with the SPC in 76% of pts. Almost all pts switched to ANA from hydroxycarbamide (93%). Most pts discontinued PT before ANA was introduced (66%; Group A). 22% discontinued PT after introduction of ANA (Group B; 17% within the first mo [Subgroup B1] and 5% in the subsequent 5 mos [Subgroup B2]). 9% had not discontinued PT by the end of follow-up (Group C) and 5 pts (3%) were determined to have no PT. At the end of follow-up, 85% of pts were still continuing on ANA, Groups: A (82%), B1 (93%), B2 (100%), C (81%). 71% of pts achieved platelet responses, Groups: A (67%), B1 (83%), B2 (100%), C (56%); 42% full response (20% blasts in bone marrow and/or peripheral blood), ii) chloroma or iii) lymph node infiltration by lymphoblasts (lymphoid BP). Thirteen pts were FIP1L1-PDGFRA (FP) positive. Five pts were positive for X-PDGFRB fusions genes (ETV6, n = 2; DTD1, n = 1;unknown, n = 2). All pts were male (median age 46 years, range 37–66). Eosinophilia >1.5 × 109/l was present in 12 of 13 (92%) FP pts and 2 of 5 (40%) pts with X-PDGFRB fusion genes. Without knowledge of the fusion gene, 9 pts received intensive chemotherapy. Despite signs of remission, e.g. clearance of blasts, eosinophilia persisted in all pts. The underlying fusion genes (FP, n = 6; X-PDGFRB, n = 3) were subsequently identified and imatinib was initiated in 7 of 9 pts. At diagnosis, FP was identified in 8 pts and imatinib was initiated directly. Overall, CHR was achieved in all 15 imatinib-treated pts after a median of one month (mo, 0.1–15). CMR was detected in all 12 FP pts after a median of 5.4 mo (2.9–32.0). One patient died due to a cerebral hemorrhage while in CMR. The remaining 11 pts are in sustained CMR for a median of 65 mo (7–103). The 3 pts with X-PDGFRB fusions are in CHR for a median of 56 mo (24–56). Three pts (FP, n = 1; ETV6-PDGFRB, n = 2) received an allogeneic stem cell transplantation (SCT) within 9 mo after diagnosis. Two pts relapsed within 3 mo; the third patient remained ETV6-PDGFRB positive while in CHR. Following relapse, the FP positive patient was treated with imatinib and is in sustained CMR up to 34 weeks after SCT. The ETV6-PDGFRB positive patient relapsed 4 weeks after SCT with leptomeningeal involvement. In conclusion, our data highlight several new aspects for the management of MLN-eo: i) mimicking de novo AML, blast phase of MLN-eo with an imatinib-sensitive PDGFR fusion gene is frequently not identified, ii) in absence of CBF fusion genes, pts with

Abstracts


92 patients (pts) (50 male, 42 female; median age at diagnosis and transplant = 45 and 51 years) with primary (n = 60), post-polycythemic (n = 13) or post-thrombocythemic (n = 19) MF underwent allo-HSCT after myeloablative conditioning containing fractionated total body irradiation (TBI) (n = 45), chemotherapy regimen (n = 29) or reduced intensity conditioning (n = 18). Donors were HLA-identical (n = 30) or mismatched (n = 3) siblings and matched (n = 41) or mismatched unrelated (n = 18). Transplants consisted of unmanipulated peripheral blood stem cells (n = 84), bone marrow (n = 6) or highly purified CD34+ cells (n = 2). GVHD-prophylaxis was performed with CSA + MTX (n = 51), 28 pts received anti-thymocyte-globulin (ATG) or alemtuzumab (n = 13). Dynamic international prognostic scoring system (DIPSS) and DIPSS plus scores were generated for each pt. prior to HSCT. The median follow-up was 20 months for all pts and 62 months among surviving pts., 1-year TRM was assessed 29%, primary graft-failure occurred in 4 pts (4%) and relapse was observed in 14% at a median 6 months post-transplant. 5-year overall survival (OS) was 63% in primary and 28% in secondary MF (p = 0.008). Using the Dynamic International Scoring System (DIPSS) risk stratification, low risk pts showed significantly superior OS (p = 0.01) compared to high risk pts. Stratification by DIPSS demonstrated 5-year OS of 66%, 48%, 45%, 0% for low, intermediate-1, intermediate-2 and high risk group. The additional inclusion of thrombocytopenia, abnormal karyotype and transfusion need (DIPSS plus) prior to transplantation resulted in a predicted 5-year OS of 75%, 55%, 50% and 24% for low, int-1, int-2 and high risk groups revealing also significantly higher OS for low vs. int-2 (p = 0.047) and high risk (p = 0.002). OS by DIPSS plus was higher for int-1 vs. high risk (0.037). According to DIPSS plus median survival times were not yet reached for low and int-1 and could be assessed 26 and 11 months for int-2 (95% CI: 0–102) and high risk (95% CI: 6–16) groups. Our data point out superior survival for primary MF and that risk stratification is more accurate and discriminative by using DIPSS plus score. Furthermore, DIPSS plus categorization illustrates the beneficial and life prolonging effect of allogeneic transplantation in all risk groups.

eosinophilia and excess of blast should be screened for PDGFR fusion genes iii) our excellent long-term outcome indicates that monotherapy with imatinib should be initiated early as durable remissions can not be achieved through intensive chemotherapy or allogeneic SCT. Disclosure: Georgia Metzgeroth: Financing of Scientific Research: This work was supported by the ´Deutsche José Carreras Leukämie Stiftung e.V.´(H11/03), Germany. Received honoraria and travel support from Novartis Pharma. Andreas Reiter: Financing of Scientific Research: This work was supported by the ´Deutsche José Carreras Leukämie Stiftung e.V.´(R09/29f and H11/03), Germany. Received honoraria and travel support from Novartis Pharma.

Freier Vortrag

tients 40–49 years the respective rates are 27.5%, 31.4%, 13.7%, 16.3% Conclusions: Using CG for estimation of GFR leads to underdosing in 18% of patients. None of the formulae tested appears to substantially improve the accuracy of CG based C dose calculation in this entire cohort of Seminoma stage I patients. CG disproportionally underestimates GFR in lean (BMI 20–25) and middle aged patients (40–49 years). Physicans need to be aware of these limitations. Measurement of GFR is preferred instead of estimation by formulae. Further improvements are necessary for situations when GFR measurement is not available. Disclosure: No conflict of interest disclosed. V411

V410

Accuracy of different methods for the calculation of carboplatin dose in the adjuvant treatment of patients with Seminoma stage I Fehr M.1,2, Klingbiel D.3, Geldart T.4, Mead G.1, Ellis S.4, Nagaraj N.1, Simmonds P.1, Wheater M.1, von Moos R.5, Cathomas R.5 University Hospital Southampton, Southampton, United Kingdom, Kantonsspital St. Gallen, St. Gallen, Switzerland, 3SAKK Koordinations zentrum, Bern, Switzerland, 4Royal Bournemouth Hospital, Bournemouth, United Kingdom, 5Kantonsspital Graubünden, Chur, Switzerland 1 2

Introduction: Single dose carboplatin (C) AUC7 is an adjuvant treatment option in patients with seminoma I. Correct dosing is key as underdosing of >10% is associated with higher relapse rate. Dosing by the Calvert formula requires determination of glomerular filtration rate (GFR). Measuring GFR by radionuclide method or 24h urine collection is the standard in this situation. However, estimating GFR by Cockroft-Gault formula (CG) is popular amongst oncologist but carries an increased risk of underdosing. We explored alternative formulae of calculating C dose focusing on reduction of underdosing. Methods: All patients with Seminoma stage I treated at Southampton University Hospital 1999–2012 were included. Actual C doses (ACD) based on GFR measurement by Tc99 DTPA were compared with estimated C doses based on various formulae (CG, MDRD, Wright, Martin, Jelliffe, Mayo formulae and a flat dosing strategy). Rates for underdosing (< 90%) and overdosing (>125%), mean percentage error (MPE) as a measure of bias and mean absolute percentage error (MAPE) as a measure of accuracy were calculated. Subgroup analyses of patients at highest risk of underdosing by CG were conducted. Results: 426 patients (median 39 years, range 19–60) with complete documentation of GFR by radionuclide measurement, SCr, age, height and weight were included

First-salvage treatment in patients with recurrent or refractory advanced germ cell cancer after cisplatin-based chemotherapy – A database of the German Testicular Cancer Study Group Berger L.A.1, Lorch A.2,3, Hentrich M.4, Kopp H.-G.5, Gauler T.C.6, Beyer J.7, de Wit M.8, Mayer F.5,9, Bokemeyer C.10, Honecker F.10, Kryselova N.10, Oechsle K.10 Universitätsklinik Hamburg-Eppendorf, 2.Medizinische Klinik und Poli klinik, Hamburg, Germany, 2Klinik für Hämatologie, Hämatologie und Immunologie, Universitätsklinik Gießen und Marburg, Germany, 3Urologische Klinik, Bereich Konservative Urologische Onkologie, Universitäts klinik Düsseldorf, Germany, 4Klinikum Harlaching, Hämatologie, Onkologie und Palliativmedizin, München, Germany, 5Abteilung für Hämatologie und Onkologie, Universitätsklinik Tübingen, Germany, 6Klinik für Innere Medizin, Tumorforschung, Universitätsklinik Essen, Germany, 7Abteilung für Innere Medizin, Hämatologie und Onkologie, Vivantes Klinikum am Urban, Berlin, Germany, 8Abteilung für Innere Medizin, Hämatologie und Onkologie, Vivantes Klinikum Neukölln, Berlin, Germany, 9Onkologische Praxis und Tagesklinik Öttle/Mayer, Friedrichshafen, Germany, 10Abteilung für Hämatologie, Onkologie und Stammzelltransplantation mit der Sektion Pneumologie, Universitätsklinik Hamburg-Eppendorf, Germany 1

For patients with BMI 20–25 rates of underdosing with CG, Wright, Martin formulae and flat dosing are 26.6%, 17.5%, 14.7%, 5.6%; for pa-

Introduction: Relapse rate in patients (pts) with advanced germ cell cancer (GCC) after cisplatin-based chemotherapy is still 20–30%. Aims of the study were the evaluation of prognostic factors at first relapse (International Prognostic Factors Study Group IPFSG; JCO 2010) and the impact of conventional- (CD-CX) or high-dose chemotherapy with autologous stem cell support (HD-CX) as first salvage treatment. Methods: This national database included a total of 143 pts from 16 German centres with relapsed or refractory GCC (73% nonseminoma) undergoing 1st salvage treatment. Results: Subgroups according to the IPFSG prognostic categories, were: very low risk in 13/143 (9%), low risk in 36/143 (25%), intermediate risk in 66/143 (47%), high risk in 22/143 (15%), and very high risk in 6/143 pts (4%). IPFSG prognostic categories significantly correlated with OS (p = 0.033) after 1st salvage treatment (for PFS p = 0.068). First salvage treatment was HD-CX in 95 (66%) and CD-CX in 48 pts (34%). Treatment response was CR/PRm- in 58%, PRm+/SD in 30% and PD in 6%. After a median follow-up (mFU) of 19 months (mos) (range, 2–267), 55% of all pts had relapsed and 33% had died resulting in a median progression-free survival (PFS) of 15 mos (95%CI 9–21) and an median overall survival (OS) of 59 mos (95%CI 27–91). Significantly more pts were progressive during CD-CX than during HD-CX (13 vs. 3%, p = 0.015). Persistent carcinoma was found more often in secondarily resected lesions after CD-CX (22/29=76% vs. 23/48=51%; p = 0.033) and second relapse rate was higher with 75 vs. 44% (p  2nd-salvage treatment. For the total cohort, PFS rate after 2 years was 43%, and OS rate after 3 years was 59%. Even among pts in the high-risk and very high-risk group (n = 28, mFU 11 mos) a PFS of 34% at 2 years was observed. Conclusion: IPFSG prognostic categories correlated significantly with observed OS after first salvage treatment in this cohort of pts with refrac-

Abstracts


Table 1.

MPE (SD)

MAPE (SD)

Underdosing 125% ACD, % (n)

CG

2.1 (15)

11 (10)

18.1% (77)

6.81% (29)

Jelliffe

-13 (12)

15 (9.1)

63.1% (269)

0.9% (4)

Wright

0.4 (14)

11 (8.5)

23.5% (100)

4.9% (21)

Martin

7.3 (15)

12 (12)

9.4% (40)

10.3% (44)

MDRD

-7.8 (14)

13 (8.7)

49.1% (209)

2.1% (9)

Mayo

14 (14)

16 (12)

4% (17)

24.6% (105)

Flat Dosing

1.9 (14)

11 (8.4)

18.8% (80)

5.4% (23)

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Urogenitale Tumore

Disclosure: No conflict of interest disclosed. V412

Pathohistological findings in patients with nonseminomatous germ cell tumours (NSGCT) who undergo postchemotherapy retroperitoneal lymph node dissection (PC-RPLND) for small tumours Pfister D.1, Albers P.2, Busch J.3, Dieckmann K.-P.4, Honecker F.5, Krege S.6, Winter C.7, Schmelz H.8, Schrader M.9, Heidenreich A.1, Deutsche Hodentumorgruppe RWTH Aachen, Urologie, Aachen, Germany, 2Universität Düsseldorf, Düsseldorf, Germany, 3Charite, Urologie, Berlin, Germany, 4Albertinen- Krankenhaus, Klinik für Urologie, Hamburg, Germany, 5Universität Hamburg Eppendorf, Onkologie, Hamburg, Germany, 6Maria-Hilf Kranken haus, Urologie, Krefeld, Germany, 7St. Josefshospital, Urologie, Krefeld, Germany, 8Bundeswehrkrankenhaus Koblenz, Urologie, Koblenz, Ger many, 9Universität Ulm, Urologie, Ulm, Germany 1

Introduction: The current guidelines recommend the resection of all visible residual tumours in NSGCT after a cisplatin based chemotherapy. There are controversial data concerning the necessity of PC-RPLND in patients with residual tumours less than one centimetre in diameter. The aim of our study was to evaluate the pathological findings in a modern series with regard of the residual tumour size. Material and Methods: A retrospective analysis of the patient´s charts was performed including patients who underwent PC-RPLND between 1989 and 2010. Of 408 patients 330 had a NSGCT, 78 patients had a pure seminoma or a primary extragonadal germ cell cancer and were excluded from analysis. The tumour size at the time of surgery was available in 261 patients in the remaining 69 pateints no preopretive data with regard to the tumour size were recorded in the radiology reports. Due to the location of the residual tumour a median laparotomy, a thoracoabdominal approach was used. In one center a laparoscopic approach was preferred. Results: Mean tumour diameter was 4.7 (0 to 32) cm. The patients were stratified in three groups: group 1 n = 28 (RT  1  1.5 cm). The histological specimens contained teratoma in 21.4%, 39.1%, 44.5% respectively, viable cancer in 10.7%, 17.4% and 22% respectively, and fibrosis/necrosis in 64.3%, 52.2% and 37.8% respectively in the three groups respectively. Conclusion: The finding of both teratoma and viable cancer decreases with decreasing sizes of the residual tumour. Nevertheless lesions < = than 1 cm still harbour a significant pathohistology in one third of the patients. As a consequence PC-RPLND must not be omitted even in small RT. Disclosure: No conflict of interest disclosed. V413

Frequency of computed tomography examinations in the follow-up care of testicular cancer patients – an evaluation of patterns of care in Germany Rusner C.1, Dieckmann K.-P.2, Friedel H.3, Stang A.1 Martin-Luther-Universität Halle-Wittenberg, Medizinische Fakultät, Institut für Klinische Epidemiologe, Halle, Germany, 2Albertinen-Krankenhaus, Klinik für Urologie, Hamburg, Germany, 3Universität Duisburg-Essen, Institut für Prävention und Gesundheitsförderung, Essen, Germany 1

Introduction: Exposure to radiation resulting from diagnostic imaging procedures probably increases late cancer risk. Patterns of care regarding the application of computed tomography (CT) imaging in testicular cancer patients were investigated in a population-based patient sample.

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Methods: The database of a large German health insurance company comprising of 850,000 insured men was searched for cases of testicular cancer arising in the years 2005 and 2006. The number of CT scans applied during a 3 year period of follow-up was noted for each individual patient and the resulting cumulative radiation dose was estimated. The number of CT scans actually observed was compared to guideline recommendations. Results: A total of 177 patients [mean age: 36.2; Standard error (SE): 9.5] were identified. Within the 3 years observation period, patients received a mean number of 4.4 CT scans (SE: 0.4) while a number of 6.2 would have been expected according to contemporary guidelines. Patients were exposed to an estimated total median diagnostic radiation dose of 30 mSv (Interquartile range: 10–54 mSv). Conclusion: In contrast to our expectation, there is a considerable gap between recommendation and actual performance regarding the number of CT scans applied to testicular cancer patients. Reasons for this deviation from guideline are elusive. Unfamiliarity of clinicians with guidelines in follow-up care of testicular cancer patients as well as poor acceptance of the high numbers of CT scans scheduled may have contributed to create this particular pattern of care. Disclosure: No conflict of interest disclosed. V414

Adjunctive surgeries in postchemotherapy retroperitoneal lymph node dissection in, PC-RPLND, patients with non seminomatous germ cell cancer, NSGCC after cisplatin based chemotherapy Pfister D.1, Albers P.2, Busch J.3, Dieckmann K.-P.4, Honecker F.5, Krege S.6, Winter C.7, Schmelz H.8, Schrader M.9, Heidenreich A.1, Deutsche Hodentumorgruppe RWTH Aachen, Urologie, Aachen, Germany, 2Universität Düsseldorf, Düsseldorf, Germany, 3Charite, Urologie, Berlin, Germany, 4Albertinen- Krankenhaus, Klinik für Urologie, Hamburg, Germany, 5Universität Hamburg Eppendorf, Onkologie, Hamburg, Germany, 6Maria-Hilf Kranken haus, Urologie, Krefeld, Germany, 7St. Josefshospital, Urologie, Krefeld, Germany, 8Bundeswehrkrankenhaus Koblenz, Urologie, Koblenz, Ger many, 9Universität Ulm, Urologie, Ulm, Germany 1

Introduction: PC-RPLND is an integrated part of the mulitmodal treatment of advanced NSGCC. By this viable cancer or teratoma is resected in about 50% of the cases with curative intention. The oncologic outcome is the first aim and in advanced disease a radical resection of residual tumor can be difficult with the need of resecting adjunctive structures. The aim of the trial is to analyze the frequency of adjunctive surgeries in PC-RPLND. Material and Methods: In 463 patients with NSGCC and a performed PC-RPLND the charts had been evaluated retrospectively for adjunctive surgeries. Results: In 84 patients (19.3%) at least one adjunctive surgery was necessary. Nephrectomy was performed most frequent (n = 24, 28.6%). A resection or reconstruction of major vessels performed in n = 31 (36.9%) (V. cava n = 21, aorta n = 5, V. porta n = 1). In 25 patients a resection or partial resection of the gastrointestinal part was necessary (liver n = 13, duodenum n = 2, ileum n = 6, stomach, pancreas, colon, appendix each in one). An ureteral neoimplantation was needed in 4 patients. Resection of smaller vessels was done in addidional 29 patients. Mean operation time was 248 (70–731) min. In a mean follow-up of 47.9 (0–192) months 17 patients developed recurrent disease and 3 patients are dead of disease. Conclusion: Adjunctive surgeries are necessary in about 20% of the cases. This number decreased compared to older series. To achieve maximum oncologic control and functional outcome patients should be treated in high volume centers. Disclosure: No conflict of interest disclosed.

Abstracts


tory or relapsed GCC. First salvage treatment with CD- or HD-CX resulted in overall 5 year-OS rates of about 50% across all prognostic categories. Even pts with high and very high risk achieved 2yPFS rates of 34%.

V415

Combined treatment with pazopanib and vinflunine in patients with advanced urothelial carcinoma refractory after first-line therapy Gerullis H., Eimer C., Georgas E., Barski D., Otto T. Lukaskrankenhaus Neuss, Neuss, Germany

Introduction: The role of pazopanib in the second-line setting of refractory metastatic transitional cell carcinoma of the urothelium has not been defined clearly. We conducted this phase I/II trial to assess the safety, tolerability, and efficacy of combining pazopanib and vinflunine in patients with metastatic transitional cell carcinoma of the urothelium after failure of first-line platinum-containing therapy. Methods: From May 2011 to December 2011, five patients were enrolled in this trial. Pazopanib was the investigated compound; four levels were planned (200, 400, 600, and 800 mg/day). Vinflunine was dosed at 280 mg/m for the first dose and 320 mg/m every 3 weeks thereafter. After the definition of a tolerated dose for the combined therapy, a subsequent phase II study was planned. Results: At the starting dose for pazopanib of 200 mg/day, dose-limiting toxicities were observed in two of five patients. One patient experienced grade 4 febrile neutropenia, which led to treatment discontinuation. A second patient showed grade 3 hepatobiliary disorder with an increase in γ-glutamyltransferase. The study was interrupted at dose level 1 for safety reasons. The initially planned phase II study was therefore not carried out. Conclusions: This phase I study showed that combined therapy of daily pazopanib (200 mg) and vinflunine (280/320 mg/m) every 3 weeks is poorly tolerated in patients with refractory advanced urothelial cancer. Disclosure: No conflict of interest disclosed.

Fortbildung

Prostatakarzinom

Fortbildung

ZNS-Beteiligung bei malignen Tumoren V425

Management of malignant glioma: Are the old standards still valid? Weller M. University Hospital Zurich, Department of Neurology, Zurich, Switzerland

Anaplastic astrocytomas, oligoastrocytomas and oligodendrogliomas (WHO grade III) as well as glioblastomas (WHO grade IV) are collectively referred to as malignant gliomas. Resection as safely feasible followed by involved-field radiotherapy have remained standard of care for these tumors for decades, but are now changing into three different directions. First, in patients with anaplastic gliomas, alkylating agent chemotherapy was as effective as radiotherapy in the NOA-04 trial, moreover, specifically patients with oligodendroglial tumors with 1p/19q codeletion may benefit from initial combined modality treatment with radiotherapy and alkylating agent chemotherapy regarding overall survival as demonstrated by the EORTC 26951 and RTOG 9402 trials. Second, in elderly patients with glioblastoma, the NOA-08 and Nordic trials suggested that O6-methylguanine DNA methyltransferase (MGMT) promoter methylation might be used as predictive biomarker to select between temozolomide alone and radiotherapy alone as the initial treatment. Third, the addition of the vascular endothelial growth factor (VEGF) antibody, bevacizumab, to radiotherapy and temozolomide in newly diagnosed glioblastoma patients prolonged progression-free, although not overall, survival in the AVAGlio and RTOG 0825 trials, provoking an as yet open discussion on the value of the endpoint of progression-free survival in a cancer that is often rapidly fatal and associated with major burdens on quality of life. Disclosure: Michael Weller: Advisory Role: Antisense Pharma, Magforce, Merck Serono, Roche; Financing of Scientific Research: Antisense Pharma, Merck Serono, Roche; Expert Testimony: Antisense Pharma, Bayer, Merck Serono, Roche.

V420

Omlin A.G. Kantonsspital St.Gallen, Medizinische Onkologie, St.Gallen, Switzerland

In the last three years, five novel treatments have shown to improve survival in metastatic castration-resistant prostate cancer (CRPC). These novel treatments have distinct mechanism of action: immunotherapy (Sipuleucel-T), CYP-17 inhibition (abiraterone), androgen receptor (AR) blockade enzalutamide, tubulin-binding chemotherapy (cabazitaxel) and radioisotope (radium-223). Historically docetaxel was for a number of years the only treatment with a proven survival benefit for patients with CRPC. Somewhat artificially three treatment spaces for drug development in CRPC have emerged: predocetaxel, docetaxel combinations and post-docetaxel. Of the novel drugs currently only abiraterone has been approved in Europe for chemotherapy-naïve patients. However, for patients treated with docetaxel three treatment options are available outside of clinical trials (abiraterone, cabazitaxel, enzalutamide). Prospective data on how to best use the novel agents sequentially are not available and clinicians face the difficult task to choose between treatment options for individual patients to maximize patient benefit. Treatment evaluation in patients with CRPC is challenging due to the predominance of bone metastatic disease, the lack of validated surrogate markers for survival. In this talk the pre-clinical and clinical data available with regards to sequencing of the novel treatments for CRPC and methods of monitoring disease status will be discussed. Disclosure: No conflict of interest disclosed.

Abstracts

Fortbildung

Web-basierte Informationssysteme in der Onkologie und Hämatologie (eHealth): Was gibt es? Was brauchen wir? V429

ONCO T Net: An interactive software for the management of patients with oncohematological diseases Mitterer M.1, Spizzo G.1, Fong D.1, Zabernigg G.2, Oncotyrol World Direct Krankenhaus 'Franz Tappeiner' Meran, Meran, Italy, 2Krankenhaus Kufstein, Kufstein, Austria 1

Introduction: OncoTNet is a web based expert system for diagnosis, treatment and management of patients with oncohematological diseases based on international validated and accepted guidelines.The reason to develop an expertsystem in oncology are various: an increasing number of patients in oncologic centres, an increasing number of medical disciplines involved in treatment, an increasing number of patients treated within studies, an increasing amount of data to process patients have to be treated according to standardized treatment protocols due to an Increasing number of cancer centres with a certification or accreditation a complete or gapless documentation is essential for legal reasons. The software OncoTNet supports the entire process in oncological institutions (hospital, resident physicians etc.) and provides a decision support for therapies and protocols for cancer patients. Furthermore it helps to organise and document clinical trials for oncological diseases.


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Sequential use of novel therapeutics in advanced prostate cancer following docetaxel chemotherapy


SMATOS Waladkhani A.R., Mahlberg R., Clemens M. Klinikum Mutterhaus der Borromäerinnen, Innere Medizin I, Trier, Germany

In haematology and oncology the best quality of treatment can be assured to patients by integration of the national guidelines in the routine. In this case patients will receive exact the same diagnostic procedures and treatments in the whole country. The best way for this integration is the availability of the guidelines in the form of clinical pathways and its integration via software. For the fulfillment of all these requirements SMATOS (Subject Management and Treatment Organisation System) has been developed. SMATOS allows not only the integration of national guidelines in the form of clinical pathways but also the collection and processing of all patient health and treatment data. It supports physicians and health managers in all aspects of their daily work processes. It supports health care professionals to meet the right decisions, documentation of the decisions, providing them with the timetable of the chosen treatments and procedures. Further the timetable of the so planned treatments and procedures can be handed out to the patient. In addition patient outcome reports can be generated. While SMATOS is a multilingual system it allows the generation of the same health report in different languages without need of translation. Further SMATOS allows searching the number of patients with a defined diagnosis and/or stage in a defined period of time. SMATOS has at least the following features: automatic report generation (particularly a list of all planned measures and treatments), integration and acquisition of external findings, recording of all incoming data, the availability of accurate data mining, easy Integration of clinical pathways (guidelines, study protocols), combination of electronic data collection with paper-based data acquisition, automatic way of treatment planning and generating of performance data, providing data for internal quality assurance and external benchmarking requirements. Disclosure: Ali Reza Waladkhani: Employment or Leadership Position: Geschäftsführer. Michael Clemens: No conflict of interest disclosed.

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V431

CANKADO: Support to increase adherence during oral self-medication Schinköthe T. LMU, Brustzentrum am Klinikum der LMU München, München, Germany

Oral self-medication represents an increasing challenge in long-term treatment of cancer patients. Taking loyalty, adverse effects and other eventual occurring events can usually detect only in large intervals. Countermeasures are often only belatedly possible. CANKADO has been designed to support cancer patients during oral self-medication. It's intended to increase communication between stakeholders based on a multi-channel-communication concept. Adherence is supported via a gamification strategy. But before implementing the software, other topics like legal framework, user analysis and sustainability were in front. Legal framework for eHealth is quite complex in Germany. To support treatments regulations and laws like Medical Device Act (MPG, incl. EU directive 93/42/EEC), Therapeutic Products Advertising Law (HWG) must be observed. In addition to that privacy policy and data security are important to note. Gamification must take the future § 299a StGB into account («Corruption and bribery in health care»). To get a better picture of users, hundreds of patients and healthcare professionals were interviewed using a survey with more then 30 items. The survey was intended to analyse currents technical equipment, new media user behaviour and user wishes. Over all around three-quarters of all respondents support the idea of an online-support system like CANKADO. Surprisingly, a high amount of older patients frequently use the internet for health related topics. The idea of CANKADO is to operate a support system, which is independent, non-commercial and free of charge. To combine these aims with a long-term perspective we have founded a non-profit organization as carrier. Disclosure: Timo Schinköthe: Expert Testimony: Sponsoring der Firma GSK, München.

Freier Vortrag

Kolorektales Karzinom II V434

Molecular dissection of effector mechanisms of RAS- induced resistance to monoclonal anti-EGFR antibodies* Kasper S.1, Breitenbücher F.1, Markowetz J.1, Reis H.2, Ting S.2, Worm K.2, Meiler J.1, Schmid K.W.2, Trarbach T.1, Schuler M.1 Universitätsklinik Essen, Westdeutsches Tumorzentrum, Innere Klinik (Tumorforschung), Essen, Germany, 2Universitätsklinik Essen, Westdeutsches Tumorzentrum, Institut für Pathologie und Neuropathologie, Essen, Germany 1

Introduction: RAS mutations are negative predictors of clinical efficacy of anti-EGFR antibodies in patients with colorectal cancer (CRC). Oncogenic RAS activates the MAPK- and PI3K/AKT pathways, which are considered the main effectors of resistance. However, the relative impact of these pathways and their downstream effectors in RAS-mutant CRC is less defined. To this end we conducted functional studies using transgenic cancer models in vitro and in vivo. In addition, we analyzed pathway activation in primary tumor samples from metastatic CRC patients. Methods: Anti-EGFR-antibody sensitive cancer cell lines were retrovirally transduced to stably express (i) oncogenic RAS, (ii) conditionally active mutant RAF-1, or (iii) conditionally active mutant AKT. The impact of oncogene expression on cell proliferation, induction of apoptosis, clonal survival and tumor growth in NOD/SCID mice was analyzed in relation to treatment with cetuximab or panitumumab. Cell lines with endogenous KRAS or PI3KCA mutations were used as additional controls. Formalin-fixed, paraffin-embedded (FFPE) tumor samples from 47 patients with

Abstracts


Methods: The software was developed with the newest Microsoft technologies. Based on user feedback within the pilot project (4 medical doctors in two hospitals), the graphical user interface was developed in order to create a user friendly and intuitive software. OncoT Net is built on knowledge data bases, which are based on international standards (ATC WHO drug code for drugmanagement; TNM and Ann Arbor staging systems, ICD O and WHO histology code for tumour definition, ICD 10 codes for definition of concomittant diseases, ICD9 for documentation of surgery, the LOINC database for the connection with different Lab system and clinical exams) . All these systems guarantee a scientific validated workflow. Furthermore guidelines of International societies are incorporated electronically in OncoTNet, so that the system acts also as a decision support system. Results: The execution of clinical recommendations within a given workflow can be facilitated and the process of «tumorboards» can be standardized. The guidelines represent the current knowledge and guarantee therefore a high quality for individualized cancer therapy (personalized cancer medicine). Another important aspect of Onco TNet is the recognition and identification of mistreatment and reduction of side effects through reporting. For example, chemotherapy dosage is calculated for the individual patient, dependent on continuously updated lab values and clinical exams.

KRAS-wildtype in DNA from archival tumor, but KRAS-mutant in DNA from fresh plasma was identified and may represent subjects whose KRAS mutational status had changed during prior therapy. Correlative subgroup analyses demonstrated that regorafenib mediated a trend for clinical benefit vs. placebo in both KRAS wildtype and mutant subgroups identified by plasma BEAMing (OS: KRAS WT, HR: 0.67, 95% CI: 0.41–1.08; KRAS mutant, HR: 0.81, 95% CI: 0.61–1.09; interaction p = 0.561). Similar results were noted for PIK3CA WT/mutant subgroups (OS: WT, HR: 0.75, 95% CI: 0.57–0.99; mutant, HR: 0.84, 95% CI: 0.47–1.50; interaction p = 0.723). BRAF was not analysed due to the small number of BRAF-mutant samples. Conclusions: The mutational analysis of DNA isolated from fresh plasma is feasible and robust using the BEAMing platform and may better represent the mutational status of the tumor(s) that a mCRC patient harbors at the time of enrollment than does the mutational analysis of archival primary tumor tissue. Regorafenib was associated with clinical benefit (vs. placebo) in all mutational subgroups evaluated. Clinical trial information: NCT01103323.

Supported by the Wilhelm Sander-Stiftung (2005.136.2, M.S.) and a Wiedenfeld Research Fellowship.

Disclosure: Meinolf Karthaus: Other Financial Relationships: Bayer (Travel Grant). Axel Grothey: Advisory Role: Bayer, Bristol-Myers, Genentech, ImClone, Onyx, Roche, Sanofi.

Disclosure: Stefan Kasper: Advisory Role: Merck Serono, Amgen; Financing of Scientific Research: Merck Serono, Amgen; Other Financial Relationships: Merck Serono, Amgen. Martin Schuler: No conflict of interest disclosed.

V436

*

V435

Mutational analysis of biomarker samples from the CORRECT study: Correlating mutation status with clinical response to regorafenib Karthaus M.1, Jeffers M.2, van Cutsem E.3, Sobrero A.F.4, Siena S.5, Falcone A.6, Ychou M.7, Humblet Y.8, Bouche O.9, Mineur L.10, Barone C.11, Adenis A.12, Tebernero J.13, Yoshino T.14, Lenz H.-J.15, Goldberg R.M.16, Wagner A.17, Grothey A.18 Städtisches Klinikum München-Neuperlach, Klinik für Hämatologie und Onkologie, München, Germany, 2Bayer HealthCare Pharmaceuticals, Montville, United States, 3Leuven Cancer Institute, Leuven, Belgium, 4Ospedale San Martino, Genova, Italy, 5Ospedale Niguarda Ca'Granada, Milano, Italy, 6Dipartimento di Oncologia, Universitá di Pisa, Pisa, Italy, 7CRLC Val d'Aurelle, Montpellier, France, 8Saint-Luc University Hospital, Brussels, Belgium, 9University Hospital Robert Debre, Reims, France, 10Institut Sainte-Catherine, Avignon, France, 11Catholic University of Sacred Heart, Roma, Italy, 12Centre Oscar Lambret, Lille, France, 13Vall d'Hebron University Hospital, Barcelona, Spain, 14National Cancer Center Hospital East, Kashiwa, Japan, 15University of Southern California Norris Comprehensive Cancer Center, Los Angeles, United States, 16Ohio State University School of Medicine, Columbus, United States, 17Bayer HealthCare, Berlin, Germany, 18Mayo Clinic, Rochester, United States 1

Background: In the CORRECT Ph3 trial, regorafenib demonstrated significant improvement in OS and PFS vs. placebo in subjects with metastatic colorectal cancer (mCRC) who had progressed on standard therapies. An exploratory biomarker substudy was conducted on samples collected from subjects enrolled in CORRECT. Methods: DNA was isolated from archival tumor tissue and fresh baseline plasma samples that were available from 239 (31%) and 503 (66%) subjects enrolled in CORRECT, respectively. Mutations in KRAS, PIK3CA and BRAF were evaluated via BEAMing technology. Results: Mutations were readily detected in DNA isolated from both tumor and plasma samples: KRAS: 59/69%; PIK3CA: 12/17% and BRAF: 1.5/3.4%. The frequency of KRAS mutation detected in tumor samples via BEAMing (59%) was identical to the frequency determined from pre-existing «historical» KRAS mutation data that was available from 96% of the subjects enrolled in the study. Concordance among the mutations detected via BEAMing in tumor vs. plasma was 76% (KRAS), 88% (PIK3CA), and 97% (BRAF). A subset of CRC which was found to be

Abstracts

Time course of regorafenib-associated adverse events in the phase III CORRECT study Hofheinz R.1, Grothey A.2, Sobrero A.F.3, Siena S.4, Falcone A.5, Ychou M.6, Humblet Y.7, Bouche O.8, Mineur L.9, Barone C.10, Adenis A.11, Tabernero J.12, Yoshino T.13, Lenz H.-J.14, Goldberg R.M.15, Wagner A.16, Cihon F.17, van Cutsem E.18 Universitätsmedizin Mannheim, III. Medizinische Klinik, Mannheim, Germany, 2Mayo Clinic, Rochester, United States, 3Azienda Ospedaliera Universitaria San Martino, Genova, Italy, 4Ospedale Niguarda Ca'Granada, Milano, Italy, 5Universitá di Pisa, Pisa, Italy, 6CRLC Val d'Aurelle, Montpellier, France, 7Centre du Cancer de l'Universite Catholique de Louvain, Brussels, Belgium, 8University Hospital Robert Debre, Reims, France, 9Institut Sainte-Catherine, Avignon, France, 10Catholic University of Sacred Heart, Roma, Italy, 11Centre Oscar Lambret, Lille, France, 12Vall d'Hebron University Hospital, Barcelona, Spain, 13National Cancer Center Hospital East, Kashiwa, Japan, 14University of Southern California Norris Comprehensive Cancer Center, Los Angeles, United States, 15Ohio State University School of Medicine, Columbus, United States, 16Bayer HealthCare, Berlin, Germany, 17Bayer HealthCare Pharmaceuticals, Montville, United States, 18Leuven Cancer Institute, Leuven, Belgium 1

Background: Regorafenib (REG) is an oral multikinase inhibitor that has recently demonstrated significant overall survival benefit vs placebo in the randomized phase III CORRECT study. We examined the time course of adverse events (AEs) in the CORRECT study. Methods: Regorafenib (REG) is an oral multikinase inhibitor that has recently demonstrated significant overall survival benefit vs placebo in the randomized phase III CORRECT study. We examined the time course of adverse events (AEs) in the CORRECT study. Results: The safety population comprised 753 patients (pts): REG n = 500; placebo n = 253. The mean treatment duration was 12.1 ± 9.7 weeks in the REG group and 7.8 ± 5.2 weeks in the placebo group. Treatment-emergent AEs occurred at any grade in 99.6% of REG pts and 96.8% of placebo pts, at grade 1/2 in 21.6% and 47.8%, respectively, at grade 3 in 56.0% and 26.5%, respectively, and at grade 4/5 in 22.0% and 22.5%, respectively. AEs occurring in ≥10% more REG than placebo pts were fatigue, hand-foot skin reaction (HFSR), anorexia, diarrhea, weight loss, voice changes, hypertension, rash/desquamation, oral mucositis, fever, hyperbilirubinemia, low platelet count. The most frequent AEs deemed to be regorafenib related were HFSR, fatigue, diarrhea, hypertension, and rash/desquamation. The frequency of these AEs over time is shown in the Table.


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metastatic CRC were immunohistochemically analyzed for activation of the MAPK and PI3K/AKT pathways in relation to KRAS status. Results: Transgenic RAS, RAF-1 and AKT protected anti-EGFR-sensitive CRC cells against anti-proliferative and pro-apoptotic effects of the antibodies in vitro and in vivo. Transgenic expression of mutant RAS co-activated the MAPK- and the PI3K/AKT pathway in immunoblot analyses. This was in line with immunohistochemical analyses of primary tumour samples, which revealed a strong correlation of MAPK and PI3K/ AKT pathway activation and mutant KRAS (regression coefficient 0.849, p 90%). Conclusion: HER-2 amplification and HER-3 overexpression ist detectable in a significant proportion of hepatic metastases of colorectal cancer. These results suggest that innovative new targeted treatment agents might be possible opportunities for the therapy of patients with HER-2/HER-3 positive metastatic colorectal cancer and should be further assessed within prospective clinical trials.

V438

Impact of a tailpiece cysteine deletion on biochemical and functional properties of an epidermal growth factor receptor-directed IgA2m(1) antibody Brunke C.1, Lohse S.1, Derer S.1, Peipp M.1, Boross P.2, Kellner C.1, Glorius P.1, Beyer T.3, Dechant M.3, Royle L.4, Leusen J.H.W.2, Valerius T.1 Christian-Albrecht-University, II. Department of Internal Medicine, Division of Stem Cell Transplantation and Immunotherapy, Kiel, Germany, 2University Medical Center Utrecht, Department of Immunology, Laboratory for Immunotherapy, Utrecht, Netherlands, 3Christian-Albrecht-University, Department of Internal Medicine IV, Nephrology and Hypertension, Kiel, Germany, 4Ludger Ltd., Oxford, United Kingdom 1

Introduction: Most clinically applied and investigated antibodies are of the human IgG isotype. IgG antibodies activate NK cells, complement and myeloid leukocytes for tumor cell lyses. There is increasing evidence that myeloid cells are critically involved in tumor rejections in vivo. Together with myeloid cells, antibodies of human IgA isotype are key players of the mucosal immune system and in the host defence against bacteria or fungi. However, little is known about the immunotherapeutic potential of IgA antibodies. IgA antibodies carry a C-terminal extension of 18 amino acids, the so-called tailpiece, with a cysteine at position 471. Although this amino acid is important for the formation of dimeric IgA antibodies, its relevance for the function of monomeric IgA has not been demonstrated. In view of a therapeutic monomeric IgA, it would be advantageous to develop an antibody which does not contain reactive side chains. Methods: By site-directed mutagenesis we generated a d471-mutated IgA2m(1) antibody directed against the epidermal growth factor receptor (EGFR). Next, we analysed its biochemical and functional properties using lectin and immune blots as well as 51chromium release cytotoxicity assays, respectively. Results: The mutated IgA2m(1) antibody displayed a reduced presence of aggregated dimers and the formation of stable dimeric antibodies, if the human Joining (J)-chain was co-transfected, was inhibited. Remarkably, the mutated IgA2m(1) antibody demonstrated an altered glycosylation and a higher efficiency in recruiting granulocytes for activating antibody-dependent cell-mediated cytotoxicity (ADCC). Both wild type and mutant IgA2m(1) antibodies were similarly efficient in the recruitment of monocytes/macrophages for ADCC. Conclusions: The presented data show that the deletion of the C-terminal cysteine may further improve an IgA2m(1) antibody towards a therapeutic antibody. Additionally, the results indicate that the glycosylation of IgA2 antibodies does not affect Fc-mediated effector mechanisms. Thus, this report provides further insight into the immunotherapeutic potential of recombinant IgA antibodies. Disclosure: No conflict of interest disclosed.

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Abstracts



V439

A systematic review of the cost-effectiveness of mono clonal antibodies for metastatic colorectal cancer Frank M.1, Lange A.1, Prenzler A.1, Kirstein M.2, Vogel A.2, Graf von der Schulenburg J.-M.1 Center for Health Economics Research Hannover (CHERH), Hannover, Germany, 2Medizinische Hochschule Hannover / Klinik für Gastroentero logie, Hepatologie und Endokrinologie, Hannover, Germany 1

Introduction: Metastatic colorectal cancer (mCRC) imposes a substantial burden on patients and society. In recent years, advances in the treatment of mCRC have resulted from the introduction of monoclonal antibodies (MoAbs). However, the application of these MoAbs considerably increases treatment costs. The objective of this article is to review the economic evidence of MoAB treatment in mCRC. Methods: A systematic literature review was conducted and cost-effectiveness- (CE) as well as cost-utility-studies were identified. Medline, Embase, SciSearch, Cochrane, and 9 other databases were searched from 2000 through February 2013 for full-text publications. The quality of the studies was assessed via a validated assessment tool. Results: A total of 843 publications were screened. Fifteen studies involving the MoAbs bevacizumab, cetuximab and panitumumab met all inclusion criteria. Four studies analyzed the CE of first line treatment with bevacizumab and nine the CE of cetuximab in subsequent treatment lines. Two studies dealt with the CE of panitumumab. The analysis of sequential regimes and the direct comparison of two MoABs were analyzed by only one study each. The quality of the included studies was high with the exception of one study. Conclusion: The treatment with bevacizumab, cetuximab and panitumumab is mainly considered to be not cost-effective in patients with mCRC. However, testing for KRAS mutation prior to the treatment with cetuximab or panitumumab is found to be cost-effective compared to no testing. Future research should focus on the CE of first-line treatment with cetuximab or panitumumab and studies on upcoming agents like regorafenib and aflibercept. Disclosure: No conflict of interest disclosed.

University of Ulm, Institute of Clinical Transfusion Medicine and Immunogenetics, Ulm, Germany, 15University of Ulm, Zentrales Knochenmarkspenderregister Deutschland (ZKRD) Ulm, Ulm, Germany 14

Allogeneic haematopoietic stem cell transplantation (HSCT) offers the chance of cure for patients with non-transformed follicular lymphoma (FL) but is associated with the risk of non-relapse mortality (NRM). Aim of this study was to identify subgroups of FL patients which benefit from HSCT. The Minimum Essential Data (MED)-A data of 146 patients who received HSCT for FL from 1998–2008 were extracted from the DRST data base and completed/verified by direct contact with DRST data managers and reference pathologists. 110 patients had chemosensitive and 33 refractory disease (RD) at time of transplant. Estimated 1-, 2-, 5-year overall survival (OS) was 68%, 60% and 52%, respectively. Day 100 NRM was 16%. Even 40% of patients with RD survived longterm. After transplant 8 patients had progressive disease and 15 relapsed including only 1 of 51 patients surviving >5 years. Univariate statistical analysis revealed limited chronic GvHD, donor age < 40 years, and TBI-based conditioning in chemotherapy-resistant patients to correlate with favourable OS. Multivariate analysis identified chemosensitive disease in general and TBI-based conditioning in chemoresistant patients as independent beneficial prognostic factors. HSCT in FL is associated with an acceptable NRM and offers a substantial chance of cure even to patients being chemotherapy-resistant at time of transplant. * This study was based on data provided by the German Registry for Stem Cell Transplantation (DRST) and was supported by the «Deutsche Krebshilfe e.V.», Deutsche Jose-Carreras Leukämie Stiftung e.V., DKMS e.V and Alfred and Angelika Gutermuth-Stiftung. Disclosure: No conflict of interest disclosed. V441

Outcome of hematopoietic stem cell transplantation in adolescents and young adults with inborn errors of hematopoiesis Albert M.1, Hartrampf S.1, Hauck F.1, Notheis G.2, Engel N.3, Schmid I.1, Tischer J.3 Klinikum der LMU, Pädiatrische Hämatologie/Onkologie, München, Germany, 2Klinikum der LMU, Pädiatrische Immunologie/Infektiologie, München, Germany, 3Klinikum der LMU, Internistische Hämatologie/Onkologie, München, Germany

Freier Vortrag

Allogene Transplantation II

University of Tuebingen, Department of Radiation Oncology, Tuebingen, Germany, 2University of Tuebingen, Department of Internal Medicine II, Tuebingen, Germany, 3University of Essen, Department of Bone Marrow Transplantation, Essen, Germany, 4University of Leipzig, Department of Hematology and Oncology, Leipzig, Germany, 5University of Heidelberg, Department of Medicine V, Heidelberg, Germany, 6University of Freiburg, Department of Hematology and Oncology, Freiburg, Germany, 7University of Ulm, Department of Internal Medicine III, Ulm, Germany, 8University of Munich, Department of Internal Medicine III, Munich, Germany, 9University of Duesseldorf, Department of Hematology, Oncology and Clinical Immunology, Duesseldorf, Germany, 10University of Hamburg, Clinic for Stem Cell Transplantation, Hamburg, Germany, 11University of Regensburg, Department of Hematology and Oncology, Regensburg, Germany, 12University of Dresden, Department of Internal Medicine I, Dresden, Germany, 13 University of Wuerzburg, Data Processing Center, Wuerzburg, Germany,

Introduction: Adolescents and young adults (AYAs) have a significantly worse outcome compared to children with the same type of leukemia due to an increased risk for transplant related mortality (Burke et al, BBMT 2013) even though their outcome has generally improved over the last two decades (Majhail et al, BBMT 2012). This may be one of the reasons why AYAs with inborn errors of hematopoiesis are less frequently referred for allogeneic hematopoietic stem cell transplantation (HSCT) compared to their pediatric counterparts even though they share a dismal natural outcome of their disease without HSCT. Methods: We retrospectively analyzed the outcome of a cohort of 18 consecutive AYAs (15–30 years of age) with inborn errors of hematopoiesis transplanted from MSD (n = 7) or MUD (n = 11) between 2006 and 2012 and compared them to 53 consecutive children (103 cp/ml) or EBV-related symptoms. PTLD or EBV-related symptoms were seen in patients with high vl (>104 cp/ml). Clinically relevant EBV-reactivation occurred only in the first 2 years after HSCT. Other potential risk factors (donor type, in vivo T-cell depletion, GVHD) are currently under investigation. Conclusions: EBV-reactivation is common after allogeneic HSCT at any time. A real-time PCR-based screening for all patients is feasible. There is no evidence for clinical relevance of positive EBV-vl in patients without

P467

Transient secondary graftfailure after allogeneic stem cell transplantation in T-PLL patients having received alemtuzumab induction therapy Szuszies C.J., Wulf G.G., Hasenkamp J., Trümper L. Georg-August-Universität, Hämatologie und Onkologie, Göttingen, Germany

T-cell prolymphocytic leukemia (T-PLL) is a rare, aggressive malignancy which is resistant to conventional chemotherapy treatment. Anti-CD52 humanized monoclonal antibody (alemtuzumab) following by peripheral allogeneic stem cell ransplantation (PBSCT) have emerged as pillars of treatment in curative intent. However precise timing of alemtuzumab and allogeneic PBSCT remains to be defined. Here we document a series of three patients with T-PLL, who received alemtuzumab for remission induction and allogeneic SCT after toxicity-reduced conditioning for consolidate therapy. While all patients engrafted, we observed secondary graft failure in all three patients without evidence for disease recurrence. Between January 2009 and August 2010, three chemotherapy-naïve T-PLL patients underwent PBSC after reduced intensity conditioning (RIC) and alemtuzumab induction therapy. In comparison 6 chronic lymphocytic leukemia (CLL) patients with initial or emerging mutations in the p53 gene product and different previous treatment regimens are demonstrated, even treated with alemtuzumab, RIC and PBSCT. The three consecutive T-PLL patients suffered a loss of donor-chimera in month 3–10 after PBSCT without evidence for disease recurrence. Graft function was reconstituted to 100% in all patients via DLI application, even thought associated with significant graft versus host disease (GvHD). This necessitates a forced immunosuppression, in one case with opportunistic infection and fatal outcome. In comparison the CLL-patients, induced with alemtuzumab and conditioned with RIC, showed a uncomplicatetd course without secondary graft failure. One of these patients relapsed and 5 achieved a complete remission. RIC following alemtuzumab induction is a feasible option in T-PLL treatment with a curative intent. Secondary graft failure seems to be specific to T-PLL, even thought the cumulative dose of alemtuzumab and the treatment free interval prior to PBSCT might play an important role and should be defined in prospective studies. Table 1. Characteristics and patient history

patient

gender

SCT-age (y)

dose of alemtuzumab (mg)

post alemtuzumab interval until SCT (d)

response to alemtuzumab

type uf donor

regimen of conditioning

response to SCT

T-PLL-1

f

40

1483

161

CR

mMURD (HLA-A mM)

FC+ATG

SGF/DLI/CR

T-PLL-2

m

60

523

52

CR

mMRD(HLA-A mM)

FBC-12+ATG

SGF/DLI/CR

T-PLL-3

m

50

733

56

CR

MRD

FC

SGF/DLI/CR

f/m

median (range)

mean (SD)

mean (SD, range)

CLL-patients

1/5

57,5 (50-61)

1013 (302)

85 (62; 48-222

2 PR / 4 CR

3 MRD / 3 MUD

1 FC / 5 FC+ ATG

5 CR / 1 relapse

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Abstracts


EBV-Reactivation after allogeneic HSCT – A single center screening-experience

P469

Dynamics of lymphocyte subpopulations in patients with acute GvHD or allograft rejection during therapy with extracorporeal photopheresis Schmitt A.1, Lorenz K.1, Rommel K.2, Mani J.1, Nan J.1, Hilgendorf I.2, Freund M.2, Schmitt M.1 University Clinic, Heidelberg, Germany, 2University Clinic, Rostock, Germany 1


No impact of platelet-derived growth factor (PDGF) autoantibodies in patients with severe chronic graftversus-host disease (GvHD) after allogeneic stem cell transplantation Spies-Weißhart B.1, Schilling K.1, Böhmer F.2, Hochhaus A.1, Sayer H.G.1, Scholl S.1 Klinik für Innere Medizin II, Universitätsklinikum Jena, Hämatologie und Intern. Onkologie, Jena, Germany, 2Institute of Molecular Cell Biology, Center for Molecular Biomedicine, Universitätsklinikum, Jena, Germany 1

Introduction: The existence of platelet-derived growth factor (PDGF) receptor autoantibodies in systemic sclerosis is conflicting and such antibodies were also detected in patients with chronic graft-versus-host disease (GvHD) after allogeneic peripheral blood stem cell transplantation (PBSCT). We therefore aimed to screen for PDGF receptor autoantibodies in patients with chronic GvHD. Patients and Methods: We evaluated the existence of PDGF receptor autoantibodies in 39 patients while 17 patients presented with a limited and eight patients with an extensive chronic GvHD, respectively. Furthermore, 14 out of 39 patients had no chronic GvHD. Results: We detected at least low levels of PDGF receptor autoantibodies in nearly all (35 of 39) patients after allogeneic PBSCT. Interestingly, only one of six patients with high levels of PDGF receptor autoantibodies presented with an extensive chronic GvHD while the remaining six patients had no clinical signs of chronic GvHD. Thus, there was no correlation between the quantitative detection of antibodies directed against the PDGF receptor and the presence or severity of chronic GvHD. Conclusion: PDGF receptor autoantibodies could easily be detected in patient sera. Nevertheless, we did not observe any correlation between the presence of PDGF receptor autoantibodies and the severity of chronic GvHD in patients who underwent allogeneic PBSCT. Disclosure: Bärbel Spies-Weißhart: No conflict of interest disclosed. Sebastian Scholl: Financing of Scientific Research: Vortragshonorare (Novartis, Janssen-Cilag); Other Financial Relationships: Reisekosten (Gilead, Novartis, Pfizer).

Abstracts


Microangiopathic damage in patients with extracorporeal photopheresis as therapy of graft-versus-host disease Gerlach B.-K., Dohm A., Hasenkamp J., Maas J.-H., Wulf G. Universitätsmedizin Göttingen, Abteilung Hämatologie und Onkologie, Göttingen, Germany

Introduction: Transplantion-associated thrombotic microangiopathy (taTMA) is a potentially fatal complication following allogeneic haematopoietic stem cell transplantation. Methods: We retrospectively analyzed the incidence and clinical course in 39 patients treated with extracorporeal photopheresis (ECP) as therapy of steroid-refractory or -dependent acute or chronic GvHD. The data were compared to those of a control group consisting of 41 patients who did not develop relevant acute or chronic GvHD. Results: The incidence of taTMA according to EBMT Consensus Criteria was only 1 of 39 (ECP group) and 0 of 41 (control group). Minor microangiopathic damage detected by the measurement of schistocytes (≥3‰), low platelet count and elevation of LDH activity above baseline frequently appeared in both groups particularly in the first year after stem cell transplantation. However, in patients with GvHD (ECP group) the


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Fig. 1. Graft function dynamics.

Introduction: Life-threatening complications like acute graft-versushost disease (aGvHD) after allogeneic stem cell transplantation and allograft rejection after solid organ transplantation are associated with poor prognosis. Recently, extracorporeal photopheresis (ECP) has been used at several centers world-wide for the efficient second-line treatment of aGvHD and graft rejection, so far immunological data supporting the efficacy are limited. Methods: Here we present both clinical and immunological analyses of a patient with aGvHD of the gut grade IV and a second patient with BOS grade III after lung transplantation. UVAR XTS device (Therakos, Exton, PA) was used to perform the ECP therapy. In both patients an intensive ECP regimen with two ECP treatments weekly up to 12 weeks was started. Results: Clinically a response of the aGvHD of the gut was observed already two weeks after onset of intensive ECP therapy with a clear reduction of stool volume and a normalization after six weeks. Also a stabilization of the allograft rejection was noted with no further decline of lung function and even more an improvement from a vital capacity (VC) of 5,100 ml at the begin of ECP therapy to a VC of 5,360 ml and a forced expiratory volume (FEV1) of 2,120 ml to a FEV1 of 2,410 ml 15 weeks later. In parallel with clinical responses the frequency of CD4+CD25hiFoxP3+ regulatory T cells as well as the frequency of CD8+CCR7+CD45RA-/+ central memory/naïve T cells increased, while CD8+CCR7-CD45RA-/+ effector memory T(EM/EMRA) cells decreased. Conclusion: ECP treatment was well tolerated and no severe infectious disease complications occurred during the therapy. Clinically a response of the aGvHD and a stabilization of the allograft rejection were observed. Immunologically, the elevated frequency of effector T cells was reduced and the frequency of regulatory T cells, T(CM) and naïve T cells increased. ECP seems to have a complex impact on the immune system and is therefore an immunologically interesting and clinically promising therapeutic option for patients suffering from diseases mediated by the immune system.


Detection of viral DNA in exanthemas after allogeneic Stem Cell Transplantation (aSCT) – relevance for the diagnosis and the clinical course of acute GvHD Weber T., Schmidberger A., Wildgrube R., Müller L.P. Universitätsklinikum Halle (Saale), Innere Klinik IV – Onkologie / Hämatologie / Hämostaseologie –, Halle (Saale), Germany

Introduction: Acute GvHD (aGvHD) initially often presents with isolated exanthema. Clinically, it is difficult to distinguish exanthema due to aGvHD from exanthema due to viral reactivation. We hypothesized that the detection of viral DNA in aGvHD-like exanthema influences the decision-process regarding presumed aGvHD as well as results from a specific pathophysiology of the exanthema. In both cases detection of viral DNA in aGvHD-like exanthema may be related to the transplantation related mortality and outcome after aSCT. Methods: We retrospectively analyzed 33 patients who had undergone skin biopsies including histological examination and PCR for viral DNA due to exanthema. Initial and deferred clinical diagnosis, treatment and outcome of aGvHD as well as incidence of subsequent GvHD episodes were evaluated in relation to the presence of viral DNA. Results: All patients received standard cyclosporine-based immunosuppression in combination with mycophenolic acid or methotrexate. In 13 of the 33 patients (39%) viral DNA was detected in skin biopsies (Ebstein Barr virus in 1, Cytomegaly virus in 4, Parvovirus B19 in 7 patients, all 3 viruses in 1 patient). Clinical diagnosis of exanthema was aGvHD in 8 (62%) patients with and in 18 (90%) patients without viral DNA (p = 0.08). In all of these patients aGvHD treatment was initiated. Four (30%) patients with viral DNA were diagnosed with viral exanthema and 1 (8%) with drug eruption. In these patients aGvHD treatment was not initiated. No relevant differences for maximum skin involvement of aGvHD, overall aGvHD grade as well as incidence of subsequent aGvHD and chronic GvHD were seen for patients with and without detection of viral DNA. Interestingly, 7 patients (54%) with and 2 (10%) without viral DNA developed progression of the underlying disease (p = 0.013). In the group with viral DNA detection, 4 patients with and 3 patients without aGvHD had disease progression. Treatment related death occurred in 3 (23%) patients with and 8 patients (40%) without viral DNA (p = 0.46). Conclusion: Detection of viral DNA in aGvHD-like exanthema tended to be associated with a reduced incidence of clinical aGvHD diagnosis and aGvHD treatment. In contrast to other studies, detection of viral DNA was associated with an increased rate of progression of the underlying hematological disease. This seemed not related to aGvHD diagnosis and immunosuppressive treatment. More detailed analysis will be presented at the meeting. Disclosure: No conflict of interest disclosed.

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DLI provides evidence that differences between GvHD and GvL effect may be rather quantitative and independent of the malignant transformation of the target cell Humpe A.1, Günther A.1, Schub N.1, Kneba M.2, Gramatzki M.1 II. Medizinische Klinik und Poliklinik, Sektion für Stammzell- und Immuntherapie, Universität Kiel, Kiel, Germany, 2II. Medizinische Klinik und Poliklinik, Kiel, Germany 1

Introduction: The therapeutic potential of allogeneic stem cell transplantation (allo-SCT) in patients with malignant diseases depends apart from conditioning therapy on the anti-tumor effect by the immune system of the donor. However, this immune system may exert severe and deadly graft-versus-host-disease (GvHD). Here, we report immune reactions after allo-SCT suggesting that GvHD and graft versus leukemia/lymphoma (GvL) effect are related more quantitatively allowing the extinction of remaining host immune cells regardless whether malignant or not. Clinical Findings: A patient wit relapsed T-PLL was treated with allo-SCT and received cells from a matched unrelated donor (MUD). After being in complete cytological and molecular remission she relapsed molecularly on day +351. While alemtuzumab was used for bridging, on day +383 a first donor lymphocyte infusion (DLI) was applied. Despite a second (on day +421) and third (on day +453) DLI application, a steady increase in the minimal residual disease (MRD) level above 0.02% was noted. The 4th DLI application on day +490 could not be given because the patient had developed acute GvHD of the skin. At the same time MRD was no longer detectable by PCR with a detection limit below 1 × 10-5. GvHD resolved upon treatment and the patient is in continuous complete remission for almost 5 years. Likewise, a quantitative effect in the balance between donor and host immune cells was observed when a patient with blood group 0 Rh pos received an AB0 major incompatible graft from an A Rh pos sibling donor. Frequently, in those situations a delayed recovery of the erythropoiesis is observed due to persisting isohemagglutinin producing donor cells. Here, even after complete tapering of the immunosuppression anti-A was produced and therapeutic approaches including rituximab had no effect. DLIs were started with a dose of 1 × 10E+05 CD3+ cells/kg on day +195. After the third application of DLI the anti-A disappeared and the blood group switched. Conclusion: In these informative situations DLI documented their efficacy in correcting a disbalanced immune system after allo-SCT. While in the MRD situation remaining tumor cells were destroyed in the ABO incompatible scenario host immune cells producing isohemagglutinins were eradicated. This leads to the conclusion that donor immunosurveillance is independent of whether the phenotype of the donor immune cells is malignant or not. Disclosure: Andreas Humpe: Financing of Scientific Research: als Experte im Advisory Board für Mozobil bei Sanofi Aventis. Martin Gramatzki: No conflict of interest disclosed. P473

Excretion of Ascaris lumbricoides following reduced-intensity allogeneic stem cell transplantation and consecutive treatment with mebendazole Luber V.1, Abele-Horn M.2, Einsele H.1, Grigoleit G.U.1, Mielke S.1 Hospital of the Julius-Maximilian University Würzburg, Department of Medicine II, Würzburg, Germany, 2Hospital of the Julius-Maximilian University Würzburg, Institute for Hygiene and Microbiology, Würzburg, Germany 1

A 51-year old German male was diagnosed in 8/2011 with multiple myeloma, received primary induction therapy with PAD and consecutive autologous and allogeneic tandem transplantation. The patient received fludarabin and treosulfan as conditioning regimen for allogeneic stem cell transplantation from his HLA-matched sibling and post transplant immunosuppression with sirolimus and mycophenolate mofetil (MMF). Twelve days after transplantation the patient recovered with stable neutrophil counts and was discharged from hospital afterwards. On day +35

Abstracts


incidence was significantly higher. Classifying the severity of damage into grades 0 through 2 we found that patients with a maximum of grade 2 experienced a significantly shorter overall survival. We also addressed the question whether microangiopathic damage regressed simultaneously to improvement of cGvHD manifestations. Both the reduction of steroids and the amelioration of GvHD symptoms were measured during the course of ECP and we detected significant improvement. However, we found cases of patients developing distinct microangiopathic damage closely after initiation of ECP. Additionally, grade 2 damage occurred in several patients during the initial courses of ECP. Conclusions: While beneficial for patients with cGvHD, ECP may trigger or augment microangiopathic damage in some patients.


Plerixafor significantly increases the hematopoietic stem cell yield in documented poor mobilizers in the autologous and allogeneic setting Haen S.P., Schober-Melms I., Bethge W.A., Möhle R., Schumm M., Kanz L., Vogel W. Medizinische Universitätsklinik Tübingen, Abteilung II für Onkologie, Hämatologie, Immunologie, Rheumatologie und Pulmologie, Tübingen, Germany

Introduction: Insufficient mobilization of hematopoietic stem cells (HSC) after stimulation with G-CSF occurs in up to 20% of cases and represents a serious challenge for both autologous and allogeneic hematopoietic cell transplantation (HCT). The chemokine receptor CXCR-4 plays a pivotal role in the homing of HSC to the bone marrow. Upon CXCR-4 blockade, HSC are released into the peripheral blood. Plerixafor is a clinically available CXCR-4 antagonist that improves HSC yield in poor mobilizers. Methods: We retrospectively analyzed data from leukaphereses performed from 2009 to 2013 and identified 12 individuals (8 men and 4 women, median age 53 years, range 43–70) who received Plerixafor due to inadequate HSC mobilization. We compared data from 30 leukapheresis sessions before and 24 sessions after Plerixafor application. Four individuals were family stem cell donors. Two donors received Plerixafor despite >20/µl CD34+ cells in peripheral blood to enable sufficient collection for in vitro manipulation. Eight patients collected HSC for autologeous HCT (8 poor mobilizers of 277 patients, 2.9%). Autologeous HSC donors received a second cycle of mobilization chemotherapy followed by Filgrastim and Plerixafor application. After interdisciplinary discussion, family donors received Plerixafor in addition to lenograstim before next leukapheresis. Results: Plerixafor application overcame poor mobilization in 2/2 allogeneic HSC donors (100%) and in 5/8 (63%) cases in the autologous setting. The compound also led to sufficient collection in the other 2 family donors (overall response 9/12, 75%). In poor mobilizers (n = 10), CD34+ cell counts in peripheral blood were 8.79/µl (range 0.5–19/µl) before and 19.9/µl (range 0.1–69/µl) after Plerixafor application (p = 0.01). These values were mirrored by the CD34+ cell counts per kilogram bodyweight of the recipient in the leukapheresis products (0.61 × 106/kg, range 0.08– 2.22 × 106/kg vs. 2.37 × 106/kg, range 0.17–6.35 × 106/kg; p = 0.002). White blood cell, T cell and NK cell counts did not differ between products from futile and Plerixafor-facilitated leukapheresis (p > 0.2).

Abstracts

Conclusions: Plerixafor enables improved stem cell mobilization into the peripheral blood in documented poor mobilizers in both the allogeneic and autologous setting and hence permits otherwise impossible HCT. Plerixafor application can also provide sufficient CD34+ cell counts for in vitro manipulation of products like CD34+ selection for HSC boosts. Disclosure: No conflict of interest disclosed. P475

Identification of polyomavirus VP1 specific T cell epitopes Mani J., Wang L., Malcherek G., Schmitt A., Ho A.D., Schmitt M. University of Heidelberg, Heidelberg, Germany

Loss of immune competency due to immuno-suppression of patients after transplantation might result in the reactivation of BK and JC polyoma viruses. In the absence of effective immunity, reactivation of BK and JC polyoma virus may lead to severe complications related to urogenital system and the central nervous system (CNS) named hemorrhagic cystitis (HC) and lethal progressive multifocal leukoencephalopathy (PML). There is an urgent need for curative treatments like adoptive transfer of virus-specific T cells used to fight cytomegalovirus (CMV) infections. Thus, identification of novel, preferentially immunodominant T cell epitopes are of crucial importance. First, in a mixed lymphocyte peptide culture (MLPC), we evaluated T cell response in healthy donors towards HLA-A2 restricted immuno-dominant peptides JCVp36 and p100. By ELISPOT and by tetramer staining we detected specific T cell responses which were almost undetectable in the fresh peripheral blood samples. Tetramer specific CD8+ T cells were successfully expanded and maintained for up to 5 weeks. Most tetramer-specific CD8 positive T cells expressed the T cell activation marker CD137 on the cell surface. Moreover, functional characterization of expanded cells was also performed. In an approach to identify novel T cell epitopes derived from BK and JC polyomavirus in an HLA independent manner, we used a set of overlapping peptides spanning whole VP1 protein. Using matrix pools and/or individual peptide stimulation we detected a number of novel T cell specificities. We are now in the process to verify processing of novel T cell epitopes by stimulation with the entire, recombinant VP1 protein. Restriction analysis is performed using K562 cell lines as target cells transfected with various MHC class I molecules. Several novel BKV and JCV protein derived CD8+ T cell epitopes could be detected which will hitherto broaden our therapeutical armamentarium. Our long term aims includes enrichment and isolation of immuno-dominant T cell specificities by MHC-StreptamerR and usage in an adoptive T cell transfer in the clinical setting. Disclosure: No conflict of interest disclosed. P476

Influence of steroid exposure on CMV specific T cells Link C.1, Rücker-Braun E.1, Tuve S.1, Schmitz M.2, Wehner R.2, Tunger A.2, Sockel K.1, Parmentier S.1, Middeke M.1, Bold Ä.1, Schetelig J.1, Odendahl M.3, Tonn T.3, Bornhäuser M.1, Heidenreich F.1 University Hospital Dresden, Hematology/Oncology, Dresden, Germany, Technical University, CRTD, Institute of Immunology, Dresden, Germany, 3 Blood Donation Center (DRK), Dresden, Germany 1 2

Introduction: CMV-infections are a serious complication in patients after allogeneic stem cell transplantation (SCT). Immunosuppressive therapy and impaired T cell reconstitution result in a high risk for viral infections. Monitoring of CMV-virus-load by PCR and preemptive therapy are important tools in order to prevent CMV disease. CMV specific cytotoxic T cells (CMV-CTLs) are needed to control CMV-infections. More recent-


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the patient visited the outpatient transplant center for a routine check-up and informed the treating physician about an unusual observation regarding his stool. He had observed a worm-like object and brought it with him for further investigation. The suspected parasite was further investigated in the department of microbiology and identified as «Ascaris lumbricoides». A stool analysis for worm eggs remained negative and there was no eosinophilia in the peripheral blood. The patient was commenced on a three-day treatment with mebendazole 100 mg twice daily which was well-tolerated. No serious interactions with the concomitant post transplant medication or negative effects on the hematopoesis were observed. Meanwhile the patient has shown up for his one year check-up post transplant. The myeloma is still in complete remission. This is to our knowledge the first report on excretion of Ascaris lumbricoides in the context of allogeneic stem cell transplantation. Another report described a single case during the conditioning period of autologous transplantation (BMT 1994, 13:491–493). The case is remarkable with view to the fact that the parasite has supposedly survived the conditioning regimen consequently requiring prophylactic treatment in the post allogeneic setting. Parasitosis with Acaris lumbricoides has a world-wide prevalence of more than a billon and is extremely rare in Northern Europe. Most likely the patient got infected during a trip to Egypt years before multiple myeloma was diagnosed.


Posterdiskussion Lymphome I P477

A novel anti-CD37 antibody shows superior antibody dependent cellular cytotoxic activity (ADCC) compared to Rituximab against patient-derived lymphoma cells Wu H.S.1,2, Lunter A.-K.1, Spillner E.2, Schmitz N.1, Heider K.-H.3, Zeis M.1 Asklepios Klinik St. Georg, Department of Hematolgoy, Oncology and Stem Cell Transplantation, Hamburg, Germany, 2Universität Hamburg, Department of Chemistry, Institute of Biochemistry, Hamburg, Germany, 3 Boehringer Ingelheim RCV, Wien, Austria 1

Background: Immunochemotherapy combining Rituximab (R) with CHOP or a CHOP-like regimen has significantly improved treatment results in aggressive B-cell lymphoma. Recently, it has been shown that a novel Fc-engineered monoclonal antibody to CD37 (mAb 37.1) (Boehringer Ingelheim) has superior ADCC and pro-apoptotic activity compared to R against the B-cell lymphoma cell line Ramos. In vitro analyses of mAb 37.1 against other cell lines or patient-derived primary lymphoma cells have not yet been reported. Material and Methods: We in vitro compared antitumoral activities of R and mAb 37.1 utilizing the following assays: CD107a degranulation assay and Europium assay for investigation of ADCC; complement-dependent cytotoxicity (CDC) and apoptosis was measured by Annexin V assay. Freshly separated NK-cells from normal donors or lymphoma patients with follicular lymphoma (FL), mantle cell lymphoma (MCL), or diffuse large B cell lymphoma (DLBCL) were used as effector cells and tested against various lymphoma cell lines and primary malignant cells from the respective lymphoma patients. Results: CD107 degranulation assays using healthy donor NK cells as effector cells and Karpas 422 cell line as target cells showed that the anti-CD37 monoclonal antibody mAb 37.1 resulted in two times higher killing efficiency as compared to R (9.58 vs. 21.16%, p = 0.01, n = 8). Similar results were obtained using other cell lines. Furthermore, in the presence of

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mAb 37.1, ADCC against primary malignant cells from patients with MCL, FL and DLBCL was significantly higher than compared to R. Conclusions: The Fc-engineered anti-CD37 antibody mAb 37.1 is a promising novel therapeutic antibody against various lymphomas of B cell origin. Phase I clinical trials are currently ongoing in order to define the clinical activity of the antibody. Disclosure: Huei Wu: Expert Testimony: Yes. Matthias Zeis: Expert Testimony: Yes. P478

Exosome mediated Wnt-Signaling augments side population in diffuse large B-cell lymphomas Koch R.1, Demant M.1, Güntsch A.1, Cicholas A.1, Aung T.1, Venkataramani V.1, Diering N.1, Wenzel D.2, Chapuy B.3, Trümper L.1, Wulf G.1 Universitätsmedizin, Göttingen, Germany, 2Max Planck Institut für biophysikalische Chemie, Göttingen, Germany, 3Dana-Farber Cancer Institute, Boston, United States 1

Introduction: Patients with aggressive B-cell lymphoma are treated in curative intention. However, some patients experience fatal relapse, originating from refractory lymphoma cells with the capacity for clonogenic regrowth. We here addressed repopulation capacity of lymphoma cell subpopulations and the mechanisms regulating the populational composition in the growing tumor. Material and Methods: We identified side population (SP) cells in diffuse large B-cell lymphoma cell lines and patient samples with the DNA-binding dye Hoechst33342, analyzed clonogenicity in vitro and in vivo and screened for differentially expressed genes and DNA-methylation patterns. A GFP-containing lentiviral vector construct was used to keep track of side population cells cultured among mixed cultures of SP and nonSP cells. Manipulation of canonical wnt-signaling was performed by lentiviral sh-RNA constructs as well as pharmacological tankyrase-inhibition by XAV-939. In vitro data were supported by in vivo experiments using a chorioallantoic membrane-assay. Results: Colony assays and suspension cultures of sorted SP and nonSP cells revealed restriction of clonogenic potential to the SP cell population as well as resurgence of nonSP cells from purified SP cell progenitors, while mixed culture assays using a GFP-vector construct tracing the SP vs. nonSP-population revealed homeostasis between the two populations, showing both SP and nonSP cells contributing to either cell compartment. SP cells show enhanced canonical wnt-signaling and increased exosomal secretion of wnt3a. Suppression of canonical wnt-signaling resulted in reduced clonogenicity. Exosome stimulation of DLBCL cell lines resulted in increased clonogenicity, stabilization of beta catenin and enhanced TOP/FOP activity. Conclusion: Here we show that tumor cells reversibly switch between states of autonomous and non-autonomous clonogenicity, and that such transitions are regulated by exosome-mediated wnt signaling. Disclosure: No conflict of interest disclosed. P479

SFRP4 inhibits proliferation of DLBCL by antagonizing wnt-signaling Koch R.1, Cicholas A.1, Aung T.1, Demant M.1, Venkataramani V.1, Chapuy B.2, Trümper L.1, Wulf G.1 Universitätsmedizin, Göttingen, Germany, 2Dana-Farber Cancer Institute, Boston, United States 1

Introduction: Wnt signaling enhances proliferation in a variety of malignancies including diffuse large B-cell lymphomas. We found differential activity of wnt agonists and the antagonist SFRP4 to drive intratumoral subpopulations and regulate their clonogenic potential. We here focused on the impact of SFRP4 regulating the proliferation of diffuse large B-cell lymphomas.

Abstracts


ly, it has become possible to monitor T cell response by flow cytometric measurement of CMV-CTLs with CMV specific multimers composed of the patients HLA Class I molecule bound to CMV pp65 epitopes. Methods: Here, we present the case of a patient with CMV-reactivation following SCT for AML. This patient as well as his stem cell donor had been tested sero-positive for CMV prior to SCT and levels of CMV-CTLs were closely monitored within a study protocol following SCT. Results: High levels of CMV-CTLs appeared to control CMV, as seen by a non-detectable virus load in standard PCR testing. About three months following transplantation this patient was diagnosed with intestinal GVHD requiring high-dose glucocorticoid treatment. Following steroid exposure, levels of CMV-CTLs dropped and shortly thereafter rising CMV-copy numbers were observed which was accompanied by clinical signs of a CMV enteritis. With the administration of antiviral treatment the CMV specific virus load decreased. However, levels of CMV-CTLs remained low, presumably as a result of ongoing steroid exposure. Conclusion: The close correlation between the drop in CMV-CTL counts and CMV activation highlights the potential of this method to monitor and understand immune responses to CMV following SCT. Of note, early presence of high frequencies of CMV-CTLs did not guarantee CMV-control. Further investigations to clarify the potential of CMV-CTL measurements and to understand the effect of steroid exposure at the functional level are warranted. In future, this tool could provide a chance to select patients at high risk of CMV reactivation who could profit from individualized monitoring and treatment.


A kinome-focused pan-omics approach to chemoresistance in a transgenic mouse model of aggressive B-cell lymphomas Kase J.1, Herrmann A.1, Lenze D.2, Hummel M.2, Lisec J.1, Willmitzer L.3, Dittmar G.4, Leser U.5, Dörken B.1, Schmitt C.A.1

Hematology, Oncology and Tumor Immunology, Charité – Universitäts medizin Berlin/ Molekulares Krebsforschungszentrum, Berlin, Germany, 2 Institute of Pathology, Department of Molecular Biology, Charité – Universitätsmedizin Berlin, Berlin, Germany, 3Max Planck Institute of Molecular Plant Physiology, Postdam/Golm, Germany, 4Max-Delbrück-Center for Molecular Medicine, Berlin, Germany, 5Humboldt Universität zu Berlin, Department of Knowledge Management in Bioinformatics, Berlin, Germany 1

Introduction: Chemoresistance is the key determinant of poor outcome in lymphoma therapy. Unveiling the underlying molecular mechanisms is critical to overcome drug insensitivity and may direct the development of novel therapies. Since patient samples are rarely available as matched pairs at diagnosis and at a resistant state, and cannot be further drug-challenged or subjected to functional validation experiments, we considered transgenic mouse models of cancer as valuable tools for the molecular dissection of treatment responsiveness. We utilize here transcriptomics, proteomics and metabolomics in a «pan-omics» approach to decipher mechanisms of treatment resistance in a Myc-driven lymphoma mouse model with previously documented cross-species predictability for human diffuse large B-cell lymphomas. Methods: 79 immunocompetent recipient mice were transplanted with primary Eµ-myc transgenic mouse B-cell lymphomas, and exposed to cyclophosphamide (CTX) upon tumor manifestation. Affymetrix array-based transcriptomics, mass spectrometry-based proteomics and metabolomics as well as kinome arrays were applied, and the data subjected to bioinformatics processing. Results: After treatment of lymphoma-bearing mice, lasting remissions (reflecting cure) were observed in about half of them. Repetitive treatments of mice harboring relapse lymphomas resulted in progressively shortened remission times and finally led to full-blown resistance, thereby recapitulating clinical courses of patients with drug-insensitive aggressive lymphomas. RNA-, protein- and metabolite-analyzing omics technologies were applied to compare curable vs. relapse-prone and resistant lymphomas, all with or without an additional short-term exposure to CTX to acutely challenge drug-specific response programs. We focused on resistance-related alterations of the druggable kinome, identified numerous kinases with sig-

Abstracts

nificant changes of their activities, and will present functional validation strategies in the Eµ-myc lymphoma model at the meeting. Conclusions: Eµ-myc lymphoma-bearing mice treated in a pre-clinical trial fashion were established as a versatile model of clinical chemoresistance. Going beyond a transcriptome-restricted investigation (which did not identify resistance-conferring alterations), our pan-omics strategy proved instrumental to dissect underlying mechanisms that will be further exploited as targets on their own for novel lesion-based therapies in future cancer precision medicine. Disclosure: No conflict of interest disclosed. P481

SCFFbxo25 and PKC∂ direct the degradation of Hax-1 to regulate apoptosis and suppress B-cell lymphomagenesis Baumann U.1, Fernández-Sáiz V.1, Lemeer S.2, Engel K.1, Nikolova V.1, Rudelius M.3, Targosz B.-S.1, Dietze K.4, Knorn A.-M.1, Hoster E.5, Dreyling M.5, Leitges M.6, Lenz G.4, Miething C.7, Peschel C.1, Keller U.1, Küster B.2, Bassermann F.1 Klinikum rechts der Isar, Technische Universität München, III. Medizinische Klinik, München, Germany, 2Lehrstuhl für Proteomik und Bioanalytik, Technische Universität München, Freising, Germany, 3Institut für Pathologie, Klinikum rechts der Isar, Technische Universität München, München, Germany, 4Molekulares Krebsforschungszentrum, Charité-Universitätsmedizin Berlin, Berlin, Germany, 5Klinikum der Universität München, III. Medizinische Klinik – Campus Großhadern, München, Germany, 6University of Oslo, Biotechnology Centre of Oslo, Oslo, Norway, 7Memorial Sloan-Kettering Cancer Center, Cancer Biology and Genetics Program, New York, United States 1

HS-1-associated protein X-1 (Hax-1) has been identified as a potent anti-apoptotic factor involved in different aspects of hematopoiesis. Hax1 is implicated in B-cell development and survival, while inactivating Hax-1 mutations have been identified as causal aberrations in severe congenital neutropenia (SCN), a human autosomal recessive trait, thus indicating a further role in myeloid progenitor cell survival. In line with its pro-survival function, high expression levels of Hax-1 have been detected in different malignancies, particular in B-cell lineage dependent lymphomas. The mechanism of how Hax-1 abundance is regulated during the apoptotic response and whether Hax-1 can promote lymphoma development has remained unclear. We show that Hax-1 is targeted for ubiquitin-proteasomal degradation in response to apoptotic stimuli by the SCFFbxo25 E3 ubiquitin ligase (Skp1 Cullin-1 F-box complex defined by the F-box protein Fbxo25). This process is initiated by the pro-apoptotic kinase PKCδ, which phosphorylates Fbxo25 in the nucleus, and Hax-1 within a PKCδ-specific phospho-degron at mitochondria. As a consequence, Fbxo25 undergoes mitochondrial translocation to mediate proteasomal degradation of Hax-1. RNAi based experiments reveal increased survival when silencing Fbxo25 in Hax-1 WT MEFs, while no effect could be observed in Hax-1 KO cells. Moreover, mitochondrial translocation of Fbxo25 and Hax-1 degradation is absent in PKCδ KO MEFs, and associates with the loss of phosphorylation of both proteins in these cells. Significantly, we identify monoallelic loss of Fbxo25 and somatic Hax-1 phospho-degron mutations in patient samples of mantle cell lymphoma, which associate with increased Hax-1 expression. Fbxo25 re-expression in Fbxo25-deficient MCL cell lines promotes cell death, while the Fbxo25 shuttling-mutant protects cells from apoptosis. Additionally, using the spontaneus Eµ-Myc lymphoma model, we demonstrate accelerated lymphoma development and decreased survival of mice transplanted with fetal liver cells treated to either overexpress Hax-1 or inactivate Fbxo25. We thus identify the PKCδ-Fbxo25-Hax-1 axis as a novel apoptosis regulatory hub, and suggest that monoallelic deletions of Fbxo25 and phospho-degron mutations of Hax-1 contribute to B-cell lymphomagenesis through stabilization of Hax-1. Disclosure: No conflict of interest disclosed.


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Material and Methods: We identified side population (SP) cells in diffuse large B-cell lymphoma cell lines and patient samples with the DNA-binding dye Hoechst33342 and screened for differentially expressed genes and DNA-methylation patterns. Candidate genes were validated by RTQ-PCR and methylation dependent immunoprecipitation. Beta Catenin-dependent wnt-signaling was manipulated using recombinant wnt3a and SFRP4 proteins and we used lentiviral shRNA to knock down SFRP4 and monitor cell proliferation in vitro in a colony forming assay and in vivo using a chorioallantoic membrane (CAM) assay. Results: We found SFRP4 to be significantly suppressed in side population cells of DLBCL cell lines due to promotor methylation. Proliferation of side population cells is addicted to active beta-catenin dependent wnt-signaling. SFRP4 is effective to antagonize wnt3a induced beta-catenin augmentation and it significantly suppresses proliferation of DLBCL cell lines by reducing the tumors content of side population cells. A knock down of SFRP4 leads to a strong augmentation of beta-catenin and increased lymphoma-proliferation in vitro and in vivo. Treating cell lines with 5-Azacytidine restored expression of SFRP4, induced decline of beta-catenin and suppressed cell proliferation. Conclusion: SFRP4 is highly effective to suppress proliferation of DLBCL cell lines by antagonizing beta-catenin dependent wnt-signaling. Methylation dependent suppression of SFRP4 can be restored by 5-Azacytidine in DLBCL cell lines.

Nonpegylated liposomal doxorubicin is an effective and safe alternative to conventional doxorubicin in the treatment of diffuse large cell B cell lymphoma: A single centre retrospective analysis Rohlfing S., Schöning T., Ho A.D., Witzens-Harig M. Universitätsklinikum Heidelberg, Hämatologie / Onkologie, Heidelberg, Germany

Introduction: Doxorubicin is a major component of R-CHOP, the standard regime in the treatment of diffuse large cell B cell lymphoma (DLBCL), however, cardiac toxicity limits his use. Nonpegylated liposomal doxorubicin (NPLD) might reduce the cardiac toxicity and has been used in the therapy of metastatic breast cancer. The aim of this retrospective study was to investigate the efficacy and safety of NPLD in patients with DLBCL. Methods: All patients with DLBCL, who received NPLD instead of conventional doxorubicin as part of the R-CHOP regime in our department from August 2005 to February 2013, were included. Results: 21 patients could be identified. 19 patients received NPLD as firstline and 2 patients as salvage therapy. The median age was 71 (24–85) years, reasons for NPLD were: presence or history of cardiac diseases in 16, cardiac problems during conventional CHOP in 3 and not known in 1 cases. 1 patient developed a hand-foot-syndrome (HFS) under pegylated liposomal doxorubicin and switched therefore to NPLD. In summary, 103 cycles of CHOP containing NPLD were applied, median dose per cycle was 95 mg. Median cycles per patient was 6 (2–8) and median cumulative dose 485 mg NLPD. The cardiac function did not change clinically or in echocardiography in 13 patients (62%). 2 patients developed an ischemic heart disease without impairment of the left ventricular ejection fraction (LVEF), in 3 patients the LVEF improved and in 3 patients it declined. 4 patients developed fever and shivering after administration of NPLD. No HFS was seen. A grade III/IV hematotoxicity, only due to neutropenia, was documented in 9 (46%) patients. In 3 cases therapy was interrupted due to adverse events: 1x severe pneumonia, 1x deteriorating performance status, 1x ischemic heart attack without impairment of the heart function. With a median follow up of 19 months, only 2 patients have relapsed, one of them received NPLD as salvage. Altogether, 4 patients died. 2 patients died of bleeding under anticoagulation but were both in complete remission, 1 patient died due to infection. The estimated 3years progression-free and overall survivals were 71 and 75%, respectively. Conclusion: NPLD is well tolerated and is an effective alternative to conventional doxorubicin in the treatment of DLBCL. The validation of the results from this retrospective study remains to be shown in a prospective setting. Disclosure: No conflict of interest disclosed. P484

High expression of CXCR4 and CXCR7 is associated with an advanced stage in aggressive lymphoma Deutsch A.1, Pichler M.2, Beham-Schmid C.3, Schaider H.4, Neumeister P.1 Innere Medizin, Hämatologie, Graz, Austria, 2Innere Medizin, Onkologie, Graz, Austria, 3Pathologie, Graz, Austria, 4Dermatologie, Graz, Austria 1

The two chemokine receptors CXCR4 and CXCR7 were identified to be implicated in multiple key processes in tumour cells, including proliferation, survival, migration, invasion and metastasis in more than 20 different types of cancer, providing evidence for the importance of this chemokine signalling pathway in cancer. Because of the limited knowledge on CXCR4 and CXCR7 expression in aggressive lymphomas, we performed a comprehensive study on CXCR4 and CXCR7 expression on diffuse large B cell lymphomas (DLBCL), transformed follicular lymphomas (tFL) and germinal centre B cells (GCB) on mRNA. Comparing expression levels of both chemokine receptors between aggressive lymphomas and their non neoplastic control – GCBs – 22 of 52 (42%) DLBCL- and 16 of 27 (59%) tFL-specimens exhibited an at least two fold over expression of CXCR4, whereas an at least two fold

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over expression of CXCR7 was observed in three of 52 (6%) DLBCL- and in three of 27 (11%) tFL-specimens. Taking all lymphoma samples together and comparing expression levels of both chemokine receptors of aggressive lymphoma patients with clinical stage 1 to lymphoma with an advanced stage (stage 2–4), a significant higher of CXCR4 (five fold, p = 0.017)- and a trend for a higher CXCR7 (3.5 fold, p = 0.087) expression were detected in advanced stage lymphoma specimens . Additionally, a significant positive correlation of CXCR4 expression and bone marrow infiltration was detected (15 of 71, Spearman rho=0.550 and p 5 years. Univariate analysis of prognostic variables showed significance for ECOG (p = 0,000) and CIRS-G (p = 0,002) for OS, Cox-regression analysis showed significance for ECOG (p = 0,016). No significance was shown for disease stage or LDH-levels. The ORR in patients beyond 1stline therapy (median age 64, ECOG-status ≥2 in 17%) was 66% with a median PFS of 8 month and OS of 24 month. Median cumulative dose was 540 mg/m2 in median 4 bouts. Toxicity in the first-line cohort was moderate, mainly grade 1 & 2. Three patients showed grade 3 leukopenia. Other side effects primarily were: inappetence, weight-loss, fever. Conclusion: Bendamustine is active in aggressive NHL of the elderly. Toxicity was well manageable. The GCB-subtype of DLBCL might predict a better outcome. A yet undefined subgroup shows sustained remission, here histologic re-assessment is in progress. Performance and comorbidity assessment is of prognostic value with a greater impact on outcome compared to classic prognostic parameters. Currently, the BRENDA trial prospectively investigates the role of bendamustine in aggressive NHL. Disclosure: Jakob Hammersen: No conflict of interest disclosed. Paul La Rosée: Financing of Scientific Research: Reisekosten und Vortragshonorar von Mundipharma, Hersteller von Bendamustin. P488

Bortezomib-CHOP in 4 HIV-positive patients with plasmablastic lymphomas Mueller M.1, Berger M.1, de Wit M.2, Arastéh K.1 Vivantes Auguste-Viktoria Klinikum, Department of Infectious Diseases and Gastroenterology, Berlin, Germany, 2Vivantes Klinikum Neukölln, Department of Hematology and Oncology, Berlin, Germany 1

Introduction: Plasmablastic lymphoma (PBL) has its highest incidence in HIV-positive patients with a very aggressive clinical course. Suggested regimens are HD-MTX-based chemotherapy while standard CHOP is not regarded as adequate therapy (NCCN guidelines 2012). In HIV-negative patients with DLBCL Bortezomib was added to R-CHOP with acceptable toxicity (Furman, Cancer 2010). This is a single institution experience with Bortezomib-CHOP in HIV-positive patients with aggressive B-NHL. Method: Retrospective analysis of 4 HIV-patients with B-CHOP regarding efficacy and toxicity (WHO toxicity-grading 0-4). Between 12/2010 and 8/2012 4 patients with PBL (patient 3 was reclassified by reference pathology as DLBC with Hodgkin markers, CD20-, CD30+, «unclassifiable») were treated with CHOP-21+Bortezomib 1,3 mg/m2 intravenously day 1+4 for 6 cycles. All patients received HAART during chemotherapy and GCSF.

Introduction: CHOP-based regimens are standard treatment for aggressive NHL, for which elderly patients frequently do not qualify. The use

Abstracts


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P486

Results: Patient 1 (41y) had at baseline a CD4 count of 42/µl, Ann-Arbor stage III with an IPI of 3. He completed 6 cycles of B-CHOP without any delay or dose reduction. In 5/2013 he was in a complete remission (CR) for 26 months. Patient 2 (52y) had CD4-cells of 150/µl, Ann-Arbor stage I (anal) and an IPI of 0. He received 5 cycles of 100% B-CHOP without any delay and stopped therapy because of wasting and fatigue. Since 24 months he is in a CR. Patient 3 (44y) had a history of PML and hepatitis b. He started with 200 CD4 cells, Ann-Arbor stage IV and an IPI of 0. After 6 cycles 100% B-CHOP (no delay) he had a 1,5 months CR before early relapse and death occured. Patient 4 (45y) had 194 CD4-cells. Anorectal PBL (Ann-Arbor stage I, IPI 0) were treated with 6 cycles of 100% B-CHOP (no delay). He is in CR since 10 months.

cohort, in R-CHOP-group and in the R-CHOP and radiation group (with 95% CI in bracket) was 94.2% (89.0–100%) and 95.5% (91.0–100%), 93.4% (86.0–100%) and 92.6% (84.6–99.7%), 85.3% (54.4–100%) and no registered death, respectively. For overall survival, P log rank and hazard ratio were not significant in any BMI category of any group. Conclusion: This study shows an excellent prognosis of patients with DLBCL treated with R-CHOP in the first-line situation. There was no significant association between BMI and treatment response. In the subgroup of patients who were treated with R-CHOP without consolidating radiation, EFS was significantly inferior in overweight patients. However, overall survival in general was excellent and was not impaired in overweight patients. Disclosure: No conflict of interest disclosed.

Table 1. Toxicity data

P1

P2

P3

P4

Leucopenia

4

4

4

3

Thrombopenia

3

3

4

0

Hepatopathy

0

4

1

0

Infection

2

0

2

0

Neuropathy

2

2

1

1

Diarrhea

0

3

2

0

Conclusion: -Bortezomib-CHOP might serve as an effective treatment option in HIV-positive patients with PBL (3 complete remissions, 1 early relapse / death). -Main toxicity was leuco- and thrombopenia grade 3–4. All patients suffered from sensory neuropathy grade 1–2, which was partial reversible after months. 1 patient stopped treatment after 5 cycles because of wasting and fatigue. Disclosure: No conflict of interest disclosed. P489

Impact of body mass index on long term survival of patients with diffuse large B-cell lymphoma after primary treatment with R-CHOP Nguyen T.T.1, Schöning T.2, Ho A.1, Witzens-Harig M.1 1 2

Universität Heidelberg, Medizinische Klinik V, Heidelberg, Germany, Universität Heidelberg, Apotheke des Klinikums, Heidelberg, Germany

Introduction: In diffuse large B-cell lymphoma (DLBCL), the International Prognostic Index (IPI) still remains the most important prognostic factor, however other components, among them the body mass index (BMI), may have an impact on the individual patient's prognosis. In this study, we compared the long term survival of overweight to healthy weight patients with DLBCL who were treated with R-CHOP in our institution. Patients and Methods: We conducted a retrospective data analysis of 190 patients with pathologically confirmed diagnosis DLBCL who were treated in the first-line situation with Rituximab and CHOP at the University of Heidelberg Hospital between 2002 and 2011. 65 patients received additional consolidating radiation therapy. 90 patients were healthy weight (BMI under 25 kg/m2) and 100 patients were overweight (BMI over 25 kg/m2). Results: No significant association between BMI and an achievement of CR was showed by binary logistic regression. Estimated event-free survival for 5 years of healthy weight patient and overweight patient in the whole cohort, in R-CHOP-group and in the R-CHOP and radiation group (with 95% CI in bracket) was 88.4% (80.4–96.4) and 74.4% (64.4–86.7), 89.1% (80.7–97.5) and 83.4% (81.4–85.4), 86.5% (67.5–100) and 93.9% (85.7–100), respectively. With the exception of the R-CHOP without radiation subgroup with a p log rank of .028, HR 2.8 (95% CI 1.1–7.3, p .031), p log rank and HR were not significant altered in any other BMI category of any group. Estimated overall survival for 5 years in the whole

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P490

Case report: Telomere dynamics in a patient with diffuse large cell lymphoma arising from follicular lymphoma Hummel S.1, Wilop S.1, Braunschweig T.2, Fahrenkamp D.3, Müller-Newen G.3, Kaufmann J.1, Crysandt M.1, Ziegler P.1, Jost E.1, Brümmendorf T.H.1, Beier F.1 Klinik für Innere Medizin IV, Universitätsklinikum Aachen, Hämatologie und Onkologie, Aachen, Germany, 2Institut für Pathologie, Universitäts klinikum Aachen, Aachen, Germany, 3Institut für Zellbiologie, Universitätsklinikum Aachen, Aachen, Germany 1

Introduction: Follicular B-cell lymphoma (FL) is characterized by an indolent course, but secondary transformation to a more aggressive form such as the diffuse large B-cell lymphoma (DLBCL) is a known phenomenon. Telomere length (TL) reflects the replicative history of eukaryotic cells and dysfunctional telomeres have been found to play an important role in the development of chromosomal instability and malignant transformation. The mechanisms propagating secondary transformation are not yet understood. We were interested to study whether shortened telomeres in FL propagate disease progression. Here, we present the case of a 63 year old patient with FL and concomitant DLBCL. Patient and Methods: The patient initially presented with significant abdominal and mediastinal lymphadenopathy. DLBCL was diagnosed by mediastinoscopic lymphnode extraction; FL was detected in the bone marrow biopsy showing a strictly localized pattern. Clinical work-up included detailed histopathological and flow-cytometry analysis of both lymphomas. TL analysis of the peripheral blood was carried out using flow-FISH and compared to age-matched controls; tissue sections of the unaffected bone marrow, the FL and the DLBCL were analyzed using confocal Q-FISH. Results: Flow-cytometry analysis revealed similar surface marker patterns supporting the notion of DLBCL arising from FL. Proliferation index in DLBCL was increased to 70% compared to 20% in the FL. TL of the peripheral blood lymphocytes (5.8 kb), but not of the granulocytes (7.5 kb) were found to be significantly shorter compared to the healthy age-matched controls situated in the lowest 10% percentile. Telomere length in the healthy bone marrow (6.6 kb) was similar to the FL (6.7 kb). TL of DLBCL was found to be slightly increased (7.0 kb). Conclusion: Peripheral blood lymphocytes of our patient were significantly shortened in line with recent data showing a predisposition to lymphoma development in individuals with short telomeres. Interestingly, we observed only slightly increased TL in the DLBCL compared to the FL suggesting e.g. increased telomerase activity in the presence of high proliferation rates. In summary, we present a detailed analysis of telomere biology in clonal and non-clonal hematopoietic compartments of a patient with FL and concomitant DLBCL. The role of telomeres as a prognostic biomarker for secondary transformation needs to be further validated in prospective studies. Disclosure: No conflict of interest disclosed.

Abstracts


WHO grade

Assessment of complete response (CR) rate as a surrogate endpoint for overall survival (OS) in relapsed or refractory (r/r) aggressive (a) NHL Clemens M.1, Pettengell R.2, Theocharous P.3, Derigs H.G.4 Klinikum Mutterhaus der Borromäerinnen, Trier, Germany, 2St. Geroge´s Hospital University London, London, United Kingdom, 3CTI Life Sciences, London, United Kingdom, 4Klinikum Frankfurt Höchst GmbH, Hämatologie, Onkologie, Palliativmedizin, Frankfurt, Germany 1

Introduction: OS is the standard measurement of efficacy and patient benefit in oncology clinical studies, but often requires a larger sample size than is practical in clinical settings such as r/r aNHL. If objective surrogate endpoints could predict OS in the context of smaller and shorter studies, the oncology community could potentially bring novel therapies to patients sooner. We used a correlation-based approach to investigate whether CR is an appropriate surrogate for OS in r/r aNHL. Methods: Data on CR and OS in r/r aNHL patients were analyzed from phase I/II trials of pixantrone and PIX301, a randomized phase III trial of pixantrone versus physician's choice of other monotherapies (n = 140); primary objective was demonstration of PIX superiority for CR/CRu using IWG 1999 criteria. Multivariate proportional hazards modeling was used to assess CR rate as a surrogate for OS in PIX301. A metaanalysis of published data from r/r aNHL patients in clinical trials (n = 1263) was also performed, using linear regression and correlation analyses to assess CR/CRu rate as a surrogate for OS. Results: In the pixantrone phase I/II trials, 24-month survival was 77% in patients with a CR vs 30% in patients without. Patients who achieved a CR had a 79% higher chance of OS than those who did not (HR 0.21, 95% CI 0.10–0.43). In pixantrone-treated patients in PIX301, median OS over 24 months' follow-up was not reached for CR responders (24% of the pixantrone ITT population) vs 6.8 months for nonresponders, and 24-month survival was 63% vs 20%, respectively (HR 0.26, 95% CI 0.11–0.61). Under multivariate proportional analysis with treatment and CR as covariates, CR remained a significant predictor of survival (HR 0.35, 95% CI 0.18–0.68). The meta-analysis also demonstrated a strong and statistically significant correlation between CR and 3-year OS (correlation coefficient 0.81, p 9 months). The second case is a 68 years old man with subtotal HCL bone marrow infiltration resulting in °IV neutropenia, transfusion-dependant anemia and °III thrombocytopenia. In view of a concomitant life-threatening invasive pulmonary aspergillosis and a one week history of STEMI requiring dual antiplatelet therapy with ASS and clopidogrel the patient received firstline treatment with vemurafenib (960 mg/d) to avoid prolonged pancytopenia and immunosuppression commonly observed after 2-CdA therapy. After 80 days of therapy the patient had achieved an excellent remission with only residual HCL infiltration in the bone marrow. Subsequently, vemurafenib was stopped and remission duration is currently > 5 months. Conclusion: Vemurafenib is highly active in advanced HCL, induces rapidly durable remissions without hematotoxicity and therefore represents an attractive novel treatment option in particular for patients with refractory disease.


Grey Zone Lymphoma in an elderly patient – stable disease with Rituximab and cyclophosphomide, vincristine, liposomal doxorubicin and prednisone (R-COMP) Piribauer M., Tomka M., Weiss H., Siebert F. Krankenhaus Barmherzige Brüder, St. Veit/Glan, Austria

Introduction: Grey zone lymphomas are rare lymphomas in adults. Their features overlap with those of diffuse large B-cell-lymphoma (DLBCL) and Burkitt lymphoma (BL). Treatment is most often according to regimes like in chronic lymphatic leukemia in younger patients. We are reporting on a case of grey zone lymphoma in an elderly patient where a less aggressive therapy has been chosen. Case Report: A 75 year old patient was admitted to a nearby hospital with increased dyspnea. His medical history showed a chronic obstructive pulmonary disease, a malignant low grade cutaneous T cell lymphoma, which was diagnoses in 2004 and treated with PUVA, and moderate alcohol drinking. On admission, a computertomography (CT) of the chest was performed, showing a pleural effusion on the right side. Abdomen medical ultrasonics showed in addition on steatosis a big central mass in the liver. Magnetic resonance investigation (MRI) was performed, where esophageal cancer was suspected with infiltration of the pericardium, the right pleura, the right and left atrium, the right costophrenic angle, and the V. cava inferior as well as multiple lymphnodes. Gastroscopic findings showed no pathologies in the esophagus but an exophytic mass in the stomach. Biopsies were taken which showed a malignant lymphoma with CD 20 positive cells. There was no evidence of CD 3, CD4, CD5, CD 7 or CD 8. There was no reaction with CD 30, CD 34, bcl 2, GCET1, MUM1, S100, CK and CKAE1/3. There was no evidence for chromosomal ruptures in MYC (8q24). Grey zone lym-

Abstracts

phoma with features of DLBCL and BL, Ann Arbor sage IV, international prognostic factor (IPI) high risk, was diagnosed. As treatment option for grey zone lymphoma, an aggressive therapy regimen as for chronic lymphatic leukemia is recommended, which is available in specialized hematooncological centres. Our patient rejected to be transferred. Therefore, we decided to treat with R-COMP which is usually chosen in treatment of DLBCL, especially. All in all, six therapy cycles were administered, with a dose reduction to 80% starting from cycle 2 because of severe neutropenia despite application of G-CSF. Follow-up 7 months after end of treatment showed stable disease. Discussion: Less is known about therapy of grey zone lymphomas. Immune-chemotherapy with R-COMP is an effective therapy in aggressive lymphomas as well in grey zone lymphomas in elderly patients with comorbitity as we could demonstrate. Disclosure: No conflict of interest disclosed. P505

Complete Remission of a Subcutaneous Panniculitis-like T Cell Lymphoma with Facial Involvement after SMILE Chemotherapy Kreißelmeier K.-P., Riewerts F., Kopp H.-G., Kanz L., Weisel K.C., Sökler M., Vogel W., Möhle R., Bethge W., Müller M.R. Universitätsklinikum Tübingen, Abteilung für Onkologie, Hämatologie und Immunologie, Tübingen, Germany

Background: Subcutaneous panniculitis-like T cell lymphoma (SPTCL) is an aggressive T cell lymphoma which preferentially occurs in the subcutaneous tissue. It accounts for less than 1% of Non-Hodgkin´s Lymphomas in adults and can present at any age. The disease is frequently accompanied by a hemophagocytic syndrome which correlates with a worse prognosis. Approximately 60% of SPTCL are primarily refractory to CHOP-like chemotherapy regimen constituting a challenge in the managment of these patients. Clinical case: A 19 year old male patient with a progressive facial swelling and increasing tricytopenia first presented at our department in December 2012. His white blood count was 2,070/µl with 870 neutrophils/ µL. Hemoglobin was 13.2 g/dL, while the platelet count was 102,000/µL. Bone marrow biopsy showed no evidence for neoplastic infiltration or hemophagocytosis. Ferritin and Il2-Receptor measurements in the peripheral blood on the other hand were significantly elevated, highly suggestive of hemophagocytosis syndrome. An MRI of the head exhibited subcutaneous infiltration of the entire face. A PET-CT scan revealed additional manifestations in the neck, trunk, diaphragm and all four extremities. Pathological evaluation of a biopsy taken from one of the facial manifestations eventually led to the diagnosis of SPTCL of the a/b-subtype. Chemotherapy according to the CHOEP-regimen (cyclophospamide, doxorubicin, vincristin, etoposide and prednisolon) was immediately initiated. After an initial minor response the facial swelling was increasing again. The patient was then switched to the SMILE-regimen (dexamethasone, methotrexate, ifosfamide, L-asparginase, etoposide). After the first cycle the patient exhibited a significant response of his facial infiltration, and after two cycles a complete remission could be documented, and the patient is presently undergoing allogeneic transplantation. Treatment was well tolerated and no significant complications occurred. Remarkably, the patient developed significant hypertriglyceridemia during the second cycle of SMILE with peak levels of 3,500 mg/dL and no apparent clinical symptoms. The hypertriglyceridemia spontaneously resolved by day 25. Conclusions: SMILE is a highly effective regimen for the treatment of SPTCL which are primarily refractory to CHOP-based regimens. Lipid profiles should routinely be determined after the application of SMILE since clinically significant hypertriglyceridemia can occur. Disclosure: No conflict of interest disclosed.


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lymphoma (HL) after failure of autologous blood stem cell transplantation (ASCT) or for patients non-responsive to at least two prior multi-agent chemotherapy regimens and ineligible for ASCT. However, for eligible younger patients with relapsed HL and chemosensitive disease, high-dose therapy and ASCT are considered standard treatment. To date, it is unknown if pretreatment with BV affects the individual patient's ability to mobilize blood stem cells. In addition, it has not been reported if stem cells successfully mobilized after BV retain their full hematopoietic capacities. Methods: In February 2012, a 28-year-old female patient was diagnosed with a gray zone lymphoma (mediastinal bulky disease, stage II AE, aaIPI 0) and was treated with six cycles of R-CHOP-21 and involved field radiation. In December 2012, a biopsy of a newly enlarged supraclavicular lymph node revealed the histology of a nodular sclerosing CD30+ HL. Given the early relapse and, thus, a suspected adverse prognosis, we treated the patient with two cycles of a salvage therapy consisting of BV (1.2 mg/kg BW) in combination with DHAP (dexamethasone, high-dose cytarabine, cisplatin). Cisplatin was dose-reduced to 75% because of concern of potential cumulative neurotoxicity. Results: After the two cycles of BV-DHAP a partial remission was achieved. Neither neurotoxicity nor any other unexpected adverse events occurred during or after treatment. After the second cycle, 7.73 × 106 CD34+/kg BW peripheral blood stem cells (PBSC) could be successfully collected. Subsequently, the patient received high-dose chemotherapy (BEAM) followed by ASCT (6.44 × 106 CD34+/kg BW). The chemotherapy was well tolerated with no concomitant complications except for one day with fever of unknown origin and Clostridia-associated diarrhea. Regular hematological recovery was reached at day 10 after ASCT, and the patient could be discharged 3 days later. Conclusion: As a proof of principle, we here report for the first time that the collection of hematopoietic blood stem cells is feasible after salvage therapy with BV-DHAP. In addition, we demonstrate that PBSC collected after BV-DHAP retain their full hematopoietic capacities. Our observations might be a starting point for further investigations on the role of BV-DHAP in the pre-transplant setting in CD30+ lymphomas.

P506

A case report about the treatment of advanced cutaneous T-cell lymphoma of a 61 years old woman Schulz K.1, Gläser D.1, Große-Thie C.1, Schäd-Trcka S.2, Brückner F.1, Freitag S.1, Kraft F.1, Fritsch A.1, Scheffler J.1, Diwoky S.1, Junghanss C.1 Universität Rostock, Klinik III, Hämatologie/Onkologie/Palliativmedizin, Rostock, Germany, 2Universität Rostock, Klinik für Dermatologie, Rostock, Germany 1

Introduction: We report about a 61 years old woman with mycosis fungoides diagnosed in 2011. Methods: The clinical data and diagnostics of this rare clinical pattern will be presented. Results: In 01/08 the patient reported about symptoms of itching and exanthema. An ambulant treatment with antihistamic drugs and corticoids did not lead to an improvement. In 01/11 patient complained about first erosions. In 07/11 the clinic of dermatology at the University of Rostock histopathologically diagnosed a T-cell lymphoma subtype mycosis fungoides. The initial stage of disease according to ISCL/EORTC 2007 was IIB, with tumor about one centimeter, no infiltrated lymph nodels, no peripheral sezary-cells, and no metastases. As first line treatment a local PUVA therapy in combination with Interferon was started. Interferon was discontinued due to liver toxicity. Following, the patient was treated from 10/11 to 07/12 three times with local radiotherapy at skin at chin, axilla, forearm, abdomen and ear. However, the overall condition of the patient impaired. Therefore, a second line therapy with Bexarotene was added in 12/11. In October of 2012 the patient showed multiple cutaneous tumors. A systemic PUVA therapy was administered at this stage. A CT scan at 10/12 showed a progression of disease with infiltration of subcutis in the right labia majoris and infiltration of inguinal lymph nodes. An interdisciplinary tumor board decision led to a systemic chemotherapy. Nevertheless, in our ambulance the patient showed impressive and painful cutaneous tumors, lesions as well as lymph nodes over one centimeter inguinal on both sides. So at that time the stage of disease according to ISCL/EORTC 2007 has changed to IVA, with a lot of tumors about one centimeter, infiltrated lymph nodes and no peripheral sezary-cells and metastases. A second line chemotherapy with gemicitabine was initated according to the publication of Marchi et al in Cancer 2005 (1) and Zinzani et al in Annals of Oncology 2010 (2). Gemicitabine was given on day 1, 8, 15 in a 28 day cycles. Marchi et al describes overall response rate of 75% in 32 Patients for a total of 6 cycles. Zinzani et al described an overall response rate of 51% in 39 patients. With our chemotherapy the cutaneous lesions and general condition improved. At the moment, the patient is within the 7th cycle with doe reduction of gemcitabine due to a grade 3 neutropenia.

A 29-year old male was diagnosed with an anaplastic lymphoma kinase (ALK)-positive anaplastic large cell lymphoma and subsequently treated with six cycles of CHOP-21 achieving only a partial remission. Salvage therapy with DHAP and Dexa-BEAM did not lead to a reduction of disease activity. Brentuximab, a CD 30 antibody-drug conjugate, was given at a dose of 150 mg resulting in a good response, documented by PET MRI and resolving of B symptoms. Allogeneic transplantation from a matched unrelated donor was successfully performed after a second cycle of brentuximab. A subsequent metabolic CR was documented by PET MRI. Six month after HCT he presented again with B symptoms and lymphadenopathy and relapse was confirmed by PET MRI and histology. In parallel, we detected a decreasing donor chimerism. Given the good response to brentuximab prior to allogeneic transplantation, we restarted brentuximab at a dose of 150 mg every 21 days. Additionally, DLI was given in escalating doses on the day after brentuximab administration to allow for a combined effect. Six month after initiation of brentuximab therapy and a total of five DLIs, a second metabolic CR with a 100% donor chimerism could be achieved. This was accompanied with concomitant signs of cGvHD of the skin. As of today being 13 month after relapse after HCT and seven month after last brentuximab infusion the patient is in ongoing CR with a good quality of life being able to continue his engineering studies. Conclusion: We suggest that the combination of brentuximab and DLI is an attractive option in patients with relapsed CD30+ lymphoid malignancies after allogeneic HCT.

Fig. 2. Chimaerism.


Successful combined anti-CD30 and donor lymphocyte treatment in a patient with refractory ALK-positive anaplastic large cell lymphoma after allogeneic transplantation Wetzko K.1, Middeke J.M.1, Thiede C.1, Ordemann R.1, Beuthien-Baumann B.2, Zietz C.3, Schetelig J.1, Ehninger G.1, Bornhäuser M.1, Platzbecker U.1

Fig. 1. PET. Disclosure: No conflict of interest disclosed.

Universitätsklinikum Carl Gustav Carus, Medizinische Klinik I, Dresden, Germany, 2Universitätsklinikum Carl Gustav Carus, Klinik und Poliklinik für Nuklearmedizin, Dresden, Germany, 3Universitätsklinikum Carl Gustav Carus, Institut für Pathologie, Dresden, Germany 1

144


Abstracts


We here report on the successful use of brentuximab in combination with DLI in a patient with anaplastic large cell lymphoma relapsing after allogeneic stem cell transplantation.

Myelom – präklinisch P508

Using patient-derived B cell immune repertoires as a platform for the selection of antibodies targeting myeloma (neo)epitopes Braig F.1, Hofmann F.1, Viol F.1, Pritzlaff N.1, Thiele B.1, Voigt M.1, Haag F.1, Konthur Z.2, Ayuk F.1, Bokemeyer C.1, Binder M.1 1 2

Universitätsklinikum Hamburg-Eppendorf, Hamburg, Germany, Max Planck Institut für molekulare Genetik, Berlin, Germany

Introduction: Monclonal therapeutic antibodies may be a novel promising approach in the treatment of multiple myeloma. A subset of myeloma patients develops oligoclonal potentially myeloma-directed antibodies after allogeneic stem cell transplant, which seem to confer a better prognosis. We therefore aimed at exploiting such effective humoral anti-myeloma immune responses for the generation of fully human myeloma-directed therapeutic antibodies. Methods: Epitopes targeted by post-transplant antibodies were identified by random peptide phage display screenings. We generated rat anti-epitope antibodies for further evaluation of antigenic targets by a reverse immunization strategy with the phage displayed peptide. To generate antibody libraries from human post-transplant B cell immunoglobulin repertoires, immunoglobulin rearrangements were cloned into a bacteriophage expression vector and expressed as Fab libraries on the phage surface. Myeloma cell binding phage clones were enriched by whole cell biopanning. Results: To explore the epitope landscape targeted by post-transplant antibodies in patients with multiple myeloma, we screened epitope-mimicking random peptide displaying phage on the immunoglobulin repertoires of a cohort of 11 patients after allogeneic transplant. One to three specific epitope-mimicking peptide motifs could be enriched per patient. Time course analyses suggested that epitopes were only transiently targeted and disappeared months to years after transplant. Most patients' serum antibodies recognized a range of epitope-mimics some of which were previously selected on completely unrelated cases. The epitopes identified by this screening appeared to be highly myeloma-specific as they were neither recognized by a broad range of healthy donor derived antibodies nor by antibodies from patients with unrelated diseases after allogeneic transplant. As proof-of-principle one epitope sequence targeted by antibodies from multiple patients could be traced back to its parental antigen using a reverse immunization strategy. Post-transplant B cell immunoglobulin repertoires from patients with highly epitope-reactive antibodies were subsequently expressed as Fab libraries on the surface of bacteriophage. Mining of such Fab repertoires on whole myeloma cells yielded clones reactive with myeloma cell surface structures. Conclusions: Our approach might provide a platform for generating monoclonal therapeutic antibodies against (neo)epitopes in multiple myeloma. Disclosure: No conflict of interest disclosed. P509

Preclinical evaluation of UNC1062, a novel small molecule mer inhibitor for the treatment of multiple myeloma Christoph S.1, Maag S.1, Deryckere D.2, Graham D.K.2, Frye S.3,4, Earp H.S.4,5, Liu J.3, Yang C.3, Zhang W.3, Wang X.3, Beelen D.W.1, Elmaagacli A.1 Universitätsklinikum Essen, Klinik für Knochenmarktransplantation, Essen, Germany, 2University of Colorado Anschutz Medical Campus, Department of Pediatrics, Aurora, United States, 3University of North Carolina at Chapel Hill, Center for Integrative Chemical Biology and Drug Discovery, Division of Chemical Biology and Medicinal Chemistry, Eshelman School of Pharmacy, Chapel Hill, United States, 4University of North Carolina at Chapel Hill, Lineberger Comprehensive Cancer Center, Department of 1

Abstracts

Medicine, School of Medicine, Chapel Hill, United States, 5University of North Carolina at Chapel Hill, Department of Pharmacology, Chapel Hill, United States

Introduction: Multiple myeloma (MM) is an incurable hematopoietic malignancy, although many novel therapeutic agents have been explored. The receptor tyrosine kinase Mer belongs to the TAM (Tyro-3, Axl and Mer) receptor family and is involved in the progression of several human malignancies. Inhibition of Mer expression has been shown to reduce pro-survival signaling, increase chemosensitivity, and delay tumorprogression in vivo suggesting that Mer tyrosine kinase inhibitors are excellent candidates for targeted therapies. Mer receptor tyrosine kinase is also ectopically expressed in MM cell lines. We have previously described the small molecule Mer inhibitor UNC1062, a substituted pyrazolopyrimidine. Here we demonstrate the biochemical and biological effects of treatment with UNC1062 in MM cells (Mer-positive RPMI 8226 and Mer-negative OPM2). Methods: Western blot analysis was used to determine inhibition of Mer activation and downstream signaling by UNC1062 in MM cells. UNC1062-mediated anti-leukemia activity was determined in short(MTT) and long-term (colony-formation) assays. As indicator for apoptosis PARP cleavage and caspase-3 cleavage were detected by western blot analysis. Results: UNC1062 is a novel small molecule Mer tyrosine kinase inhibitor and potently inhibits Mer tyrosine kinase activity in vitro (Mer IC50= 1.1 nM, Morrison Ki = 0.33 nM). In cell-based assays, treatment with UNC1062 inhibited accumulation of phospho-Mer and downstream signaling through AKT in RPMI 8226 cells. UNC1062 also reduced proliferation and/or survival in RPMI 8226 cells (IC50 = 1.2 ± 0.16 µM, n = 3). UNC1062-treated Mer-positive MM cells exhibited increased levels of cleaved caspase-3 and cleaved PARP confirming the induction of apoptosis in response to treatment with UNC1062. Treatment with UNC1062 (100 nM) resulted in a statistically significant, dose-dependent decrease in colony-formation in methylcellulose compared to control cultures in Mer-positive RPMI 8226 cells (418 ± 23 vs. 291 ± 37 colonies, p = 0.04, n = 3). No significant reduction of colony formation was observed in Mer-negative OPM2 cells. Conclusion: UNC1062 inhibits activation of Mer, mediates antineoplastic activity against MM cells in culture, and decreases colony-formation in methylcellulose. Taken together, these data support further development of Mer tyrosine kinase inhibitors as novel and effective MM therapy. Disclosure: No conflict of interest disclosed. P510

«Re-educating» M2 macrophages with Lenalidomide Bruns H.1, Gehlen H.1, Büttner M.2, Busch M.1, Maurberger A.1, Amann K.2, Mackensen A.1, Gerbitz A.1 Uniklinik Erlangen, Medizinische Klinik 5, Hämatologie/Onkologie, Erlangen, Germany, 2Uniklinik Erlangen, Pathologie, Erlangen, Germany 1

Introduction: Multiple myeloma (MM) is a clonal B-cell neoplasia characterized by the accumulation of malignant plasma cells within the bone marrow. MM cells are known to be dependent on support by the stroma of their bone marrow niche. In MM macrophages are an abundant component of the stromal cell compartment, but their role in MM progression remains unclear. Lenalidomide, an IMiD® drug used for the treatment of MM is believed to target the stromal support, but its precise mechanism of action is still unclear to date. Methods: Macrophages in the bone marrow of MM patients, were characterized by immunohistochemistry in situ. In addition, to investigate the role of distinct macrophage subsets in tumorigenesis in MM, we generated macrophages from PBMC either by GM-CSF to create the M1 phenotype, or by M-CSF to obtain the M2 phenotype, coincubated them with different concentrations of Lenalidomide and analyzed the expression of myeloma cell growth factors and the macrophage phenotype by FACS, ELISA and qPCR.


145


Posterdiskussion


Effects of various kinase inhibitors and other targeted drugs on growth and survival of multiple myeloma cells Blatt K.1, Herrmann H.1,2, Peter B.1,2, Berger D.1, Stefanzl G.1, Valent P.1,2 Medical University of Vienna, Department of Internal Medicine I, Vienna, Austria, 2Ludwig Boltzmann Cluster Oncology, Medical University of Vienna, Vienna, Austria 1

Introduction: Multiple Myeloma (MM) is a plasma cell malignancy characterized by monoclonal paraproteinemia, bone marrow plasmocytosis, and osteopathy. Several different effective treatment strategies have been developed for these patients, but MM is still a clinical challenge and many patients relapse after therapy. Therefore, considerable effort is made to identify new effective agents for the treatment of MM. Methods: We examined the effects of 15 kinase blockers and other targeted drugs on growth and survival of five MM cell lines (MM1.S, NCI-H929, OPM-2, RPMI8226, U266). Apoptosis was measured by microscopy, Annexin/PI-staining and Caspase-3-staining. The most effective drugs were further evaluated on apoptosis and proliferation of primary neoplastic cells obtained from patients with MM. We also examined the effects of various drug combinations on proliferation of MM cells. Results: Of all drugs tested, the PI3-kinase blocker NVP-BEZ235, the pan-Bcl-2 inhibitor obatoclax, the Hsp90-inhibitor 17AAG, and the PLK1 blocker BI2536 showed major growth-inhibitory and apoptosis-inducing effects in all cell lines tested. All four compounds were found to inhibit the in vitro proliferation of MM cells at pharmacologically meaningful concentrations (IC50 < 1 µM). Growth-inhibitory effects of all effective agents were accompanied by signs of apoptosis. Major cooperative effects on proliferation of MM cell lines were obtained with the following combinations of targeted drugs: 17AAG+NVP-BEZ235 (MM1.S, OPM2, RPMI8226, U266), 17AAG+BI2536 (MM1.S, OPM2, RPMI8226, U266), 17AAG+obatoclax (MM1.S, NCI-H929, OPM2, RPMI8226), BI2536+NVP-BEZ235 (MM1.S, NCI-H929, OPM2, RPMI8226), BI2536+obatoclax (MM1.S, OPM2, RPMI8226), and NVP-BEZ235+obatoclax (MM1.S, RPMI8226). Conclusions: Several different kinase blockers and other targeted drugs inhibit proliferation and induce apoptosis in MM cells. Most potent effects are obtained by applying drug combinations. Whether these effects also occur in vivo in MM patient remains to be investigated in clinical trials. Disclosure: No conflict of interest disclosed.

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P512

Targeting the protective microenvironment in multiple myeloma (MM): An analysis of CXCR4/SDF-1 inhibition combined with established anti-MM-substances Simon A., Waldschmidt J., Wider D., Follo M., Kleber M., Lorenz J., Wäsch R., Engelhardt M. University of Freiburg Medical Center, Department of Hematology and Oncology, Freiburg, Germany

Introduction: The interaction of malignant plasma cells (PCs) with the bone marrow (BM) microenvironment is crucial for MM pathogenesis. The SDF-1/CXCR4 axis plays a key role in this interaction. SDF-1 induces homing of MM cells to the BM. Binding of SDF-1 to CXCR4 promotes PC survival and facilitates environment-mediated drug resistance, which ultimately renders anti-MM-substances ineffective. Methods: Cytotoxic effects of specific anti-MM agents (bortezomib [B], vorinostat [V], pomalidomide [P]) were tested with (w) and without (w/o) M2-10B4 stroma support and w and w/o the specific CXCR4 inhibitor AMD3100. Experiments were performed w MM cell lines (MMCLs) and MM-pt BM samples using flow cytometry. CXCR4 downstream pathways were investigated by Western blot. The effect of AMD3100 and of the SDF-1 inhibitor NoxA12 in combination w anti-MM-agents is being analysed regarding synergistic cytotoxicity and drug resistance via flow cytometry and image cytometry. Results: Cytotoxic effects on MMCLs determined cytotoxic doses of B, V and P. Cocultivation w stroma reduced apoptosis and induced tumor protective effects. AMD3100 restored sensitivity to drug treatment w/o inducing cytotoxicity itself and inhibited migration of MM cells in vitro (Udi, Engelhardt. BJH 2013). FACS, image cytometry and confocal microscopy verified high CXCR4 expression in >80% of U266 cells; whereby different CXCR4-antibodies (12G5, 44717, 4G10) revealed highest expression levels for 12G5 and 44717, which was decreased by AMD3100, but not w use of 4G10 at various concentrations (20–200µl/ ml). M2-10B4 also showed CXCR4 expression, suggesting that CXCR4-inhibition will likewise influence MM- and stroma cells. A protocol for image cytometry was established that allows imaging of single vital cells and intra- and extracellular-CXCR4. The analysis of CXCR4 in U266 revealed that AMD3100 alone does not alter surface CXCR4, nor does it affect CXCR4/pCXCR4 via Western blot. SDF-1 treatment reduced CXCR4 surface expression by receptor internalization, which was blocked by AMD3100. Conclusions: Our findings stress the central role of SDF-1 and its receptor CXCR4 in MM progression and environment-mediated drug resistance. Targeting these molecules should provide new therapeutic strategies. In ongoing experiments we aim to elucidate the effect of AMD3100 and other CXCR4 inhibitors in order to generate a better understanding of how CXCR4-axis inhibition disrupts the MM-BM interaction. Disclosure: No conflict of interest disclosed. P513

Identification of new cell surface structures by phage display for the development of antibody-based therapeutic approaches in multiple myeloma Klausz K.1, Glorius P.1, Kellner C.1, Peipp M.1, Günther A.1, Burger R.1, Gramatzki M.1 Sektion f. Stammzell- & Immuntherapie, 2Med. Klinik, Uniklinikum Schleswig-Holstein und Christian-Albrechts-Universität Kiel, Kiel, Germany 1

Introduction: Nowadays, antibody-based therapy is clinical standard for the treatment of some hematological and solid tumors. However, for multiple myeloma (MM), a malignant plasma cell disorder, currently no antibody is approved. Even though daratumumab and other antibodies have lately shown encouraging results in clinical trials, there is still a need for MM-specific target structures to enforce tumor specificity and cytotoxic

Abstracts


Results: We demonstrate that infiltrating macrophages in the bone marrow of MM patients display an anti-inflammatory M2-phenotype characterized by the expression of surface marker CD68 and CD163. Furthermore, we reveal that in vitro generated M2 macrophages, in contrast to M1 macrophages express important growth factors such as RANKL, BAFF and APRIL, which indicates that the M2 phenotype promotes tumor growth and bone destruction in MM. Incubation of M2 macrophages with Lenalidomide results in downregulation of the M2 markers CD163 and CD206, whereas the M1 markers CD86 and CD40 were upregulated. Since Lenalidomide is considered «immunomodulatory» we tested generated macrophages for their capacity as antigen presenting cells. In contrast to untreated M2 macrophages treatment with Lenalidomid rendered M2 macrophages to stimulate T-cell responses against several antigens. Conclusion: We show here that in MM, similar to solid tumors, M2 macrophages are the predominant phenotype in the bone marrow. M2 macrophages express several factors known to be important in MM disease pathogenesis and progression. Lenalidomide treatment reverts the M2 phenotype to an more proinflammatory M1 phenotype thereby potentially acting in neoplastic growth in an indirect fashion. Furthermore, changes in macrophage phenotype are associated with improved antigen presentation.


The deubiquitylase USP24 is a determinant of the mitotic spindle assembly checkpoint and is frequently deleted in multiple myeloma Targosz B.-S.1, Lemeer S.2, Reiter C.1, Braun M.3, Baumann U.1, Slotta-Huspenina J.4, Rudelius M.4, Schröder S.1, Perner S.3, Kuster B.2,5, Peschel C.1, Bassermann F.1 Department of Medicine III, Klinikum rechts der Isar, Technische Universität, München, Germany, 2Department of Proteomics and Bioanalytics, Technische Universität München, Freising, Germany, 3Department of Prostate Cancer Research, Institute of Pathology, University Hospital of Bonn, Bonn, Germany, 4Department of Pathology, Klinikum rechts der Isar, Technische Universität, München, Germany, 5Center for Integrated Protein Science Munich (CIPSM), Freising, Germany 1

Multiple Myeloma (MM) is characterized by high levels of genome instability and high clinical response rates to proteasome inhibitors, such as Bortezomib or Carfilzomib. The Ubiquitin Proteasome System (UPS) is an important guardian of the genome by virtue of its involvement in cell cycle checkpoint control and DNA damage response, and marks the target structure of proteasome inhibitors. Its implication in the mitotic spindle assembly checkpoint (SAC) is of high interest, as it marks the last genome surveillance mechanism prior to cell division. Prolonged SAC activation eventually leads to apoptosis, known as mitotic catastrophe (MC). The fate of UPS targets at checkpoints is regulated by opposing ubiquitylation or deubiquitylation activities of ubiquitin ligases and deubiquitylases (DUBs). The identity of altered UPS activies in MM has however remained largely elusive. By analyzing CGH array data sets from different MM cohorts with regard to significantly altered chromosomal loci of orphan DUBs, we found USP24 (located at 1p32.3) as a promising candidate deleted in MM. Indeed, FISH analyses identified deletions of USP24 in 33% within a cohort of 55 MM samples. Moreover, by combining novel techniques of protein extraction from bone marrow biopsies with reverse phase protein array

Abstracts

technology, we detected loss of USP24 protein expression in 30% within a cohort of 31 samples. Functional proteomics and cell biology based assays revealed a role for USP24 in the establishment and maintenance of the SAC and regulation of mitotic cell fate decision. Specifically, USP24 is a cell cycle regulated protein with peak expression in mitosis. RNAi based knock down of USP24 resulted in significantly decreased sensitivity to SAC activation as evidenced by mitotic slippage and decreased MC upon prolonged SAC activation. Likewise, forced expression of USP24 arrested cells in mitosis and potently induced apoptosis. Next, using quantitative SILAC-based mass-spectrometry analyses, we identified the pro-apoptic HECT-domain E3-ligase WWP2 as a novel relevant substrate of USP24, which is implicated in PTEN and TGFβ signaling. Further investigations on the USP24-WWP2 axis concerning novel functions in SAC establishment are ongoing. Together, we identify a previously uncharacterized DUB as a new component of the SAC and demonstrate its deletion and loss of expression in MM, thereby providing a framework of how defective SAC signaling may promote the pathogenesis of multiple myeloma. Disclosure: No conflict of interest disclosed. P515

Novel screening tools identify CD317/HM1.24 as a potent target structure for immunotoxin-based treatment of multiple myeloma Klausz K., Staudinger M., Glorius P., Kellner C., Günther A., Burger R., Gramatzki M., Peipp M. Christian-Albrechts-Universität zu Kiel, Sektion für Stammzell- und Immun therapie, Kiel, Germany

Introduction: Antibody-based targeted immunotherapy represents a promising approach for the treatment of multiple myeloma (MM). Especially antibodies against target antigens with a high internalization capacity are promising candidates for the development of immunotoxins and antibody drug conjugates. The identification of optimal combinations of target antigen, antibody and cytotoxic payload is challenging. To identify antibodies and target antigens with high potential for the development of immunotoxins for targeting multiple myeloma, a novel screening platform was established. Methods: Novel fusion proteins consisting of a truncated Pseudomonas exotoxin A (ETA') and either a kappa light chain (α-kappa-ETA') or an Fc-specific (α-Fc-ETA') domain antibody were generated. These fusion proteins allowed the screening of monoclonal antibodies for their ability to deliver a cytotoxic payload by non-covalent binding to the antibodies. The most potent antibody identified with the screening tool was converted to a single-chain Pseudomonas exotoxin A-based immunotoxin (HM1.24-ETA'). The anti-myeloma activity of HM1.24-ETA' was evaluated in vitro and in vivo. Results: In combination with monoclonal antibodies α-kappa-ETA' and α-Fc-ETA' formed non-covalently linked antibody-toxin complexes and allowed the evaluation a panel of antibodies against MM-associated antigens for their ability to mediate antigen-dependent, ETA'-induced cytotoxicity against human myeloma cell lines. While CD38, CD319 (CS1), IL-6R and CD138 antibodies only ineffectively delivered ETA', a CD317 (HM1.24) antibody inhibited the proliferation of all tested MM cell lines at low nanomolar ETA' and antibody concentrations. The subsequently designed single-chain immunotoxin HM1.24-ETA' inhibited proliferation of MM cell lines at low concentrations (80%) in freshly isolated tumor cells from seven MM patients, and was not cytotoxic against antigen-positive non-malignant cells. Furthermore, establishment of INA-6 plasma cell tumors in SCID mice was significantly inhibited by HM1.24-ETA' (p 90%. 6/11 compounds with anti-myeloma effects were identified (% apoptosis, IC50).

Introduction: Aberrant signaling via RAS/Raf/MAPK has been implicated in multiple myeloma (MM) pathogenesis. Because we have previ-

148


Abstracts


Conclusion: The novel screening tool identified CD317/HM1.24 as a promising target for efficient ETA'-based immunotherapy for the treatment of MM.

Compound N°

1

2

6

7

8

11

Bortezomib

OPM-2

6,8

35,8

2,4

6,2

3,5

40,4

11,8

NCI

5,5

68,1

5,9

1,6

5

14,7

5,5

U-266

5,8

31,6

5,9

0,6

4,3

4,4

5,3

Fibroblasts/PC3

>100

>100

>100

>100

>100

>100

>100

HRT

4

>100

7,9

4,1

9,6

>100

>100

HUVEC

3,4

26,6

2,4

0,9

3,9

3,9

3,4

MDA

3,3

7,4

4,1

1,6

6,5

9,6

7,4

PBMC 1

>100

>100

>100

4,3

>100

>100

>100

PBMC 2

>100

>100

>100

5,7

>100

>100

>100

IC50 was < 10 [nM] for 4/6 effective substances, suggesting activity at least similar to bortezomib. Compounds 1, 2, 6, 8 showed relatively uniform activity in all three cell lines, compound 7 and 11 were most active in U-266 cells. Fibroblasts and PC3 are resistant to all compounds, whereas- HUVECs and MDA were sensitive. Moreover, compounds 1, 2, 6, 7, 8 and 11 showed also potent activity against purified myeloma cells from two patients. Compounds 1, 2, 6, 8, 11 and bortezomib didn´t show any toxic effect on PBMC's up to a concentration of 100 [nM]. Conclusions: We identified new compounds with potential anti-myeloma effects and putative antiangiogenic activities in vitro. Currently, a model is being established to study the antiangiogenic and anti-myeloma effect of these new compounds in vivo. Acknowledgements: Support for this study was provided by EU Research Project FP7 (OPTATIO) and the Innsbruck Medical University. Disclosure: No conflict of interest disclosed. P519

cells, survival rate 28%). Combined knockdown of both Ral isoforms did not significantly enhance cell death compared to single isoform knockdown. Conclusion: RalA may confer tumor cell survival in a subgroup of MM cells with oncogenic Ras. Like Ras, Ral is currently not pharmacologically druggable. Therefore, characterization of Ral signaling effectors is warranted to exploit this pathway for potential therapeutic intervention. Disclosure: No conflict of interest disclosed.

Posterdiskussion Myelom – klinisch P520

Bendamustine and prednisone in combination with Bortezomib (BPV) in the treatment of patients with relapsed or refractory multiple myeloma and light chain induced renal failure Pönisch W.1, Moll B.1, Heyn S.1, Bourgeois M.1, Schmalfeld M.2, Edelmann T.3, Becker C.4, Hoffmann F.-A.4, Schwarzer A.5, Kreibich U.6, Egert M.7, Stiegler R.8, Andrea M.1, Krahl R.1, Remane Y.9, Bachmann A.9, Lindner T.9, Fricke S.1, Al-Ali H.1, Niederwieser D.1 Universitätsklinik Leipzig, Hämatologie/Onkologie, Leipzig, Germany, 2Hämatologisch-Onkologische Gemeinschaftspraxis, Halle/Saale, Germany, 3 Hämatologisch-Onkologische Gemeinschaftspraxis, Schkeuditz, Germany, 4Hämatologisch-Onkologische Gemeinschaftspraxis, Leipzig, Germany, 5 Hämatologisch-Onkologische Gemeinschaftspraxis, Leipzig-Dösen, Germany, 6Heinrich-Braun-Krankenhaus, Zwickau, Germany, 7Hämatologisch-Onkologische Praxis, Werdau, Germany, 8Hämatologisch-Onkologische Gemeinschaftspraxis, Rötha, Germany, 9Universitätsklinik Leipzig, Leipzig, Germany 1

Introduction: Oncogenic Ras is found in 30–40 percent of multiple myeloma (MM) cases and marks the transition from MGUS to MM in these cells. Ral counts among the potential Ras effector proteins which may relay illegitimate cell survival signaling. Because no pharmacological Ral inhibitors are available, we used shRNA-mediated Ral knockdown to analyze the impact of Ral for MM cell survival. Methods: Ral protein expression was analyzed in primary MM samples (n = 21) by immunohistochemical staining of bone marrow trephines and in MM cell lines (n = 10) by Western blotting. ShRNA expression vectors against RalA and RalB isoforms were transiently transfected in MM cell lines (n = 6) by electroporation. Cells were co-transfected with EGFP expression vector and strongly transfected cells were selected by cell sorting. ShRNA specificity for Ral isoforms was validated by depletion of over-expressed HA-tagged RalA and RalB protein, respectively. Cell survival was measured with flow cytometry using annexin V-APC / propidium iodide staining and calculated relative to control cells transfected with empty pSUPER vector. Results: RalA and RalB proteins were heterogeneously expressed in all MM cell lines and primary MM samples tested. However, Ral expression levels did not correlate with the presence of mutant Ras in these cells. Knockdown of RalA impaired cell survival in 4 of 6 tested cell lines and preferentially in Ras mutant cells (most effectively in MM.1S cells, survival rate 44%). In contrast, depletion of RalB only led to minor apoptotic effects and relevantly affected survival in 1 of 6 tested cell lines (INA-6

Introduction: Serious renal failure represents a main complication of Multiple Myeloma (MM). An estimated 25% to 50% of patients are affected during the course of their disease. These patients are at increased risk for infections and have a significantly worse prognosis. Small phase I/II studies suggest that treatment with chemotherapy and/or new substances results in recovery of renal function in more than 25%. The window of opportunity for reversal of renal impairment is rather small making an immediate and highly active treatment strategy mandatory. Bortezomib and bendamustine have turned out to be quickly acting and effective drugs in the treatment of MM. Methods: Between March 2005 and March 2013, 36 patients (median age 64; range 32–81 years) with relapsed/refractory MM and light chain induced renal failure (eGFR 70 are currently uncertain. Therefore, we retrospectively analyzed outcome of ASCT for elderly pts. with newly diagnosed MM at our institution between 01/07 & 09/12. Methods: 202 elderly pts. with newly diagnosed MM underwent ASCT (60– 64 years: 83 pts.; 65–69 years: 93 pts.; >70 years: 26 pts.). Pts. received an average of 3 cycles induction therapy before stem cell mobilization. The proportion of patients receiving novel agents during induction therapy was not significantly different among the groups (60–64: 78%.; 65–69: 62%.; >70: 54%). Tandem ASCT was performed in 43 cases (60–64: 29 pts.; 65–69: 12 pts.; >70: 2 pts.). After ASCT, 94 pts. received maintenance therapy either with interferon alpha or a novel agent (60–64: 40 pts.; 65–69: 43 pts.; >70: 11 pts.). To identify safety & feasibility of ASCT among the three groups, we compared data about hospitalization, reconstitution & 100 day mortality. PFS & OS were analyzed using Kaplan-Meier curves as well as Cox regression analysis, as risk factors ISS, cytogenetics, application of a novel agent as well as remission before and after ASCT were taken into account. Results: Regarding data about hospitalization, reconstitution & 100 day mortality we found no difference between the three groups. The rate of (near)complete remission/(n)CR before & after ASCT was equally distributed among the groups (%before/after: 60–64:19/37; 65–69:16/32; >70:19/30). Pts. receiving novel agents as part of induction therapy achieved significantly higher (n)CR rates than pts. treated with conventional therapy. There was no significant difference for PFS & OS between the groups (median PFS: 60–64:27 mo; 65–69:23 mo; >70:23 mo; median OS: not reached). Cox regression analysis showed no impact of age on PFS & OS. ISS stage & cytogenetics as well as remission before & after ASCT had the highest impact on both, PFS & OS. Maintenance therapy with a novel agent was associated with longer PFS in uni- & multivariate analysis. Conclusion: ASCT is feasible for selected pts. >65 & >70 years & outcome dose not differ from patients between 60 & 65 years. Novel agents for induction & maintenance improve remission & survival among older pts. eligible for ASCT. Cytogenetic analysis should be performed routinely for prognostic assessment in elderly pts.

response rate (ORR). Preclinical studies have demonstrated that vorinostat (V), a histone deacetylase inhibitor, is synergistic with B and Dox. The aim of this phase I/II study was to determine the tolerability and activity of the VBDD combination in RRMM. Methods: Pts received escalated V-doses at 100 (dose level [DL] 0), 200 (DL+1) and 300 mg (DL+2) on days (d, (provided by MSD) 1–4, 9–12, 17–20, combined with B 1.3 mg/m2 d1, 8, 15 (provided by Janssen), D 40 mg d1, 8, 15 and Dox 9 mg/m2 d1+8. The primary objective was the MTD (3+3 dose escalation design). Secondary objectives are safety, responses, PFS, OS and correlative endpoints include prognostic MM-parameters, organ function, QoL-, comorbidity-assessments and translational studies (e.g. HDAC-activity in PBMNCs (Fig. 1). After completion of 6 cycles, pts could continue maintenance with B. Results: To date, 17/30 pts have been enrolled (median age 63 years [55– 75] 53% men). Median prior therapy lines were 3 (1–8): B, thalidomide or lenalidomide were given in 88% and each 24%, respectively; 94% had undergone prior SCTs. Cytogenetic abnormalities included del(17p) (2), t(4;14) (2), gain(1q) (2), t(11;14) (4) and hyperdiploidy (7). No DLTs have been observed; with each 3 pts safely proceeding to DL+2. 6 SAEs occurred in 4/17 pts (24%): sepsis (1) and herpes zoster reactivation (1) were suspected to be related to all VBDD-drugs. No causal relationship to study drugs was suspected for pneumonia (2), 1 syncope and 1 death due to PD with persisting plasma cell leukemia (PCL). The ORR (>PR) was 65%. At a median follow-up of 5 months (range 1–18), there have been only 2 pts with PD (refractory MM + PCL). Conclusions: VBDD is a well tolerated and effective regimen in heavily pretreated RRMM pts. There have been no observed DLTs, the MTD of V was set at 300mg and all reported SAEs are in line with the known safety profile of the investigated drugs. Our alternative V-schedule induced excellent tolerability and may enhance the obvious clinical benefit, warranting completion of this study.

Disclosure: No conflict of interest disclosed. Fig. 1.

Vorinostat (V) in combination with Bortezomib (B), Doxorubicin (D) and Dexamethasone (D) (VBDD) in patients (pts) with refractory or relapsed multiple myeloma (RRMM): An interim phase I/II analysis 1

1

1

2

1

Kleber M. , Wider D. , Groß B. , Keller K. , Reinhardt H. , Jakobs D.1, Möller M.1, Burbeck M.1, Hieke S.3, Claus R.1, Pantic M.1, Pfeifer D.1, Grichina O.4, May A.M.5, Werner M.5, Jung M.2, Wäsch R.1, Engelhardt M.1

Disclosure: Martina Kleber: Expert Testimony: MSD, Janssen-Cilag. Monika Engelhardt: Expert Testimony: MSD, Janssen-Cilag P528

VMP in 1st-Line Treatment of Non-Transplant Patients with Multiple Myeloma in Routine Practice: First Follow- Up Data from the Tumour Registry Lymphatic Neoplasms Knauf W.1, Abenhardt W.2, Harich H.-D.3, Schlag R.4, Grugel R.5, Marschner N.6, on behalf of the TLN Study Group Onkologische Gemeinschaftspraxis, Frankfurt a. M., Germany, 2Münchner Onkologische Praxis, München, Germany, 3Onkol. Schwerpunktpraxis Dr. Harich/Dr. Kasper, Hof, Germany, 4Gemeinschaftspraxis Dres. Rudolf Schlag & Björn Schöttker, Würzburg, Germany, 5iOMEDICO, Freiburg i. Br., Germany, 6Praxis für interdisziplinäre Onkologie & Hämatologie, Freiburg i. Br., Germany

University of Freiburg, Hematology and Oncology, Freiburg, Germany, Institute of Pharmaceutical Sciences, Albert-Ludwigs-University, Freiburg, Germany, 3Institute of Medical Biometry and Medical Informatics, University Medical Center, Freiburg, Germany, 4Clinical Trials Unit, University Medical Center, Freiburg, Germany, 5Institute of Pathology, University Medical Center, Albert-Ludwigs-University, Freiburg, Germany

1

Introduction: The combination of Bortezomib (B), Doxorubicin (Dox) and Dexamethason (D;BDD) is well tolerated and induces a high overall

Introduction: Therapeutic options for patients (pts) with multiple myeloma (MM) have increased in recent years. While clinical trials determine

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efficacy of treatments in highly selected pts, clinical registries collect data on pts with various risk factors and comorbidities. This allows analysis of the effectiveness of treatments in real life pts treated in routine practice. Methods: The open, prospective, longitudinal, multicentre study Tumour Registry Lymphatic Neoplasms (TLN) documents the current treatment of lymphatic neoplasms by German office-based haematologists. A broad set of data regarding various pts' characteristics, all systemic treatments and outcome parameter (progression-free and overall survival) are recorded. Pts are followed for five years. Since May 2009 111 centres have recruited 2,897 pts, of which 510 pts have MM. Non-transplant pts recruited at the start of 1st-line therapy (n = 302) were included into this analysis. Results: Pts are median 73 years old, 49% are male, 26% have International Staging System (ISS) stage III MM (66% Durie & Salmon stage III) and 18% have renal insufficiency. After a median follow-up of 23 months, 32% of pts have received 2nd-line therapy, 22% have died and 11% are lost to follow-up. Two-year overall survival is 75%. The most frequently used 1st-line regimens are VMP (39%), VD (17%), V(mono) and MPT (9% each). Pts treated with VMP were median 74 years old at the start of therapy with 46% aged ≥ 75 years, 26% had ISS stage III disease (73% Durie & Salmon stage III), 19% had renal insufficiency and 76% had at least one documented comorbidity. At the time of analysis, 92% of pts had completed 1st-line treatment with VMP, 30% had received 2nd-line therapy, 17% of pts had died. Objective response rate as assessed by physician in routine practice was 84%. VMP was applied over 23 weeks (median) with a median number of 4 cycles (range 1 to 10). Two-year overall survival is 84%. Conclusion: A variety of 1st-line regimen is used in routine treatment of pts with MM who do not undergo stem cell transplantation, with nearly 40% being treated with VMP. Although pts in routine practice commonly present with comorbidities, objective response data is comparable to pts from the VISTA trial. After a median follow-up of almost two years the majority of pts in the registry have not yet received 2nd-line therapy.

isotype-matched FLC. As shown in Figure 1A and 1B, in pts with IgGκ-MM we observed a significant increase of median IgG-κ-HLC serum levels and HLC-IgG-ratio values in CR/PR vs. SD/PD/newly diagnosed (ED) MM pts (Fig. 1A) and ISS I or II vs. III status (Fig. 1B). Of note, with abnormal HLC-results, pts with MGUS were reclassified as SMM, suggesting that the assay may also assist pt allocation to truely MGUS, SMM or symptomatic MM. Pts with WM-M-spike are very difficult to quantify, but revealed clearly abnormal IgMκ/λ ratio of 231 (normal range: 1.0–2.4; Fig. 1C). Conclusions: The Hevylite-test is of clinical value to observe the progress, particularly in MM pts in remission. Especially in WM pts, the Hevylite-test can be a very helpful additional technique to the M-spike to quantify the amount of monoclonal Ig clone.

Fig. 1a/b. RC_ISSq

D: dexamethasone; M: melphalan; P: prednisone; T: thalidomide; V: bortezomib Disclosure: No conflict of interest disclosed. P529

Hevylite assay (HLC) – an additional indispensable technique to the M-spike in MGUS, Multiple Myeloma (MM) and Waldenstroms macroglobulinamia (WM) patients (pts) Gaiser F.1,2, Kleber M.1, Ihorst G.3, Greil C.1, Becherer U.2, Koch B.2, Bissé E.2, Engelhardt M.1 University Medical Center, Department of Hematology and Oncology, Freiburg, Germany, 2University Medical Center, Central Laboratory, Freiburg, Germany, 3University Medical Center, Clinical Trials Unit, Freiburg, Germany

Fig. 1c. CZE.

Indroduction: A novel polyclonal immunoassay specific for the different light chain types of intact immunoglobulins (Ig; HLC) and the serum free light chain (FLC) enable measurement of changes in the production of clone specific Ig and of the non-involved polyclonal Ig of the same isotype. Methods: We investigated the new HLC compared to the capillary zone electrophoresis (CZE) and FLC. Serum FLC and HLC (IgGκ/IgGλ, IgAκ/ IgAλ, IgMκ/IgMλ) measurements were performed using polyclonal antisera assays (Freelite™ and Hevylite™, Binding Site, Birmingham, UK). In addition, HLC-ratios were calculated and the International Staging System (ISS) and EBMT response criteria status of each pt was recorded. Results: We assessed 146 consecutive MM pts with abnormal M-spike via CZE: 57 IgGκ-MM, 24 IgGλ-MM, 8 IgAκ-MM, 7 IgAλ-MM, 5 SMM, 5 LC-MM, 28 MGUS and 12 IgM WM. The serum concentrations of IgG, IgA and IgM HLC in all pts revealed a high correlation to the

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Abstracts



Heavy/light chain (HLC) and free light chain (FLC) analysis allow sensitive monitoring of multiple myeloma patients and aid detection of potential clonal changes Teleanu V.1, Kull M.1, Kuchenbauer F.1, Liebisch P.2, Graf M.1, Steinbach G.3, Zho S.3, Nold S.4, Döhner H.1, Young P.5, Langer C.1 Universitätsklinikum Ulm, Klinik für Innere Medizin III, Ulm, Germany, Onkologie Moers, Moers, Germany, 3Universitätsklinikum Ulm, Zentrale Einrichtung klinische Chemie, Ulm, Germany, 4The Binding Site Group Ltd, Schwetzingen, Germany, 5The Binding Site Group Ltd, Birmingham, United Kingdom 1 2

Background: Serum free light chains (FLC) assessments have improved detection of subtle plasma cell changes. Nephelometric assays have been developed that measure heavy/light chain (HLC; Hevylite) and FLC

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(Freelite®). Here, we present three case studies showing that HLC/FLC analysis may aid detection of clonal changes at relapse in patients (pts) with multiple myeloma (MM). Methods: Comparison of changes in HLC ratios (IgA κ/λ normal range: 0.80–2.04; IgG κ/λ normal range: 0.98–2.75) and FLC ratios (normal range: 0.26–1.65) during monitoring identified potential clonal events in three MM pts. Clonality at presentation and maximum response was confirmed by immunofixation. Results: Patient 1 was an oligosecretory IgAκ MM patient who presented with 3 g/L IgAκ, an abnormal HLC IgAk/l ratio (142), 3102 mg/L FLCκ and abnormal FLCk/l ratio (3942). Initially, FLC and HLC provided similar clinical information. At third relapse (after day 1000) the HLC ratio normalised and IFE could not detect monoclonal IgAκ. By contrast, FLC ratios progressively increased (119% increase). Patient 2 had an IgGλ MM presenting with 48 g/L IgGλ, an abnormal HLC IgGκ/λ ratio (0.004), 4380 mg/L FLCλ and an abnormal FLCκ/λ ratio (0.001). Following high dose melphalan and autologous stem cell transplantation (Mel200) the patient achieved a complete response (CR). At this time the HLC ratio normalised. At day 523 the patient's FLCλ levels increased to 147 mg/L and the FLC ratio became significantly abnormal (0.05), indicating relapse. By contrast, at this time the HLC ratio (1.6) and IFE remained normal. Patient 3 was an oligosecretory IgGκ MM patient who presented with 5 g/L IgGκ, an abnormal HLC IgGk/l ratio (7.1), 37,710 mg/L FLCκ and abnormal FLCk/l ratio (3712). After Mel200 the patient achieved a CR. At this time the HLC ratio had also normalised. A potential clonal event occurred at first relapse when there was a progressive increase in FLCκ expression (8 mg/L at day 263, 157 mg/L at day 317 and 474 mg/L at day 410) and FLC ratio (1.24 at day 263, 23.5 at day 317 and 601.0 at day 410). By contrast during this period the HLC ratio and IFE remained normal. Conclusions: Measuring HLC and FLC ratios in MM patients may offer a simple method of detecting clonal changes at relapse.

P531

Auto-SCT – A promising attempt in treatment of severe paraproteinemic polyneuropathies: Another small case series Strifler S.1, Wessig C.2, Linke R.P.3, Einsele H.1, Knop S.1 Universitätsklinikum Würzburg, Medizinische Klinik II, Würzburg, Germany, 2Universitätsklinikum Würzburg, Department of Neurology, Würzburg, Germany, 3Ludwig-Maximilians-Universität, München, Germany 1

We present a small case series of severe paraproteinemic polyneuropathies (PNs) of different subtypes, refractory to common immunosuppression but successfully treated with HD-Mel and ASCT. #1 A 45-year old man suffering from progressive hypesthesia and muscular weakness of the upper extremities since 2011 initially was diagnosed with chronic-inflammatory demyelinating polyneuropathy (CIDP) with detection of anti-GM1-/GD1b antibodies and, subsequently, of λ-Bence Jones paraproteinemia. Despite immunosuppression, symptoms aggravated, leading to an almost complete loss of walking ability and severe impairment of upper extremities' motor activity within 14 months. In Aug. and Oct. 2012 he was treated with tandem HD-Mel and ASCT. Today the patient has already regained ability to walk, upper extremities showing significant motor activity improvement. #2 Presenting with a 2-year history of progressive tetraparesis of proximal predominance and concomitant MGUS IgG κ, a 49-year old man finally was diagnosed with sporadic late-onset nemaline myopathy due to κ-LCDD. In mid-2010 he received HD-Mel with ASCT leading to stable disease. After 6 month we initiated treatment with bortezomib/dexamethasone (VD, 4x) followed by a 2nd HD-Mel and ASCT due to decline of muscular strength. Significant reduction of symptoms lasted for another year, but finally strength, including respiratory muscles, deteriorated. In view of prior success we started treatment with VD plus cyclophosphamide to reduce paraprotein, leading to hematologic CR regarding paraprotein and to significant reduction of symptoms. #3 A 57-year old patient initially was diagnosed with multifocal motor neuropathy (MMN) in 1994. With common immunosuppression, symptoms gained momentum until 2010. Then MGUS IgM l just as circulating anti-MAG-antibodies occurred for the first time. He received HD-Mel and ASCT in Sept. 2010 achieving nCR regarding MGUS but resulting in slowly progressive neuropathy. As initially occurred, anti GM 1-antibodies could be detected again, diagnosis had to be revised to MMN. Hence the patient until now is receiving IVIG with lasting stabilization. Regarding previous small case series we suggest that treatment with HDMel and ASCT reducing monoclonal protein provides a noteworthy therapeutic option in refractory cases of PN. Disclosure: No conflict of interest disclosed. P532

Therapy of relapsed/ refractory multiple myeloma with a combination of Lenalidomide plus Dexamethasone: First interim analysis of the non-interventional study «Revlimid» Knauf W.1, Aldaoud A.2, Groschek M.3, Teichmann B.4, Harde J.4, Hasskarl J.4 Onkologische Gemeinschaftspraxis, Frankfurt a. M., Germany, 2Dr. Aldaoud – Dr. Schwarzer Forschungsgesellschaft mbH, Leipzig, Germany, 3 Onkologische Gemeinschaftspraxis, Würselen, Germany, 4iOMEDICO AG, Freiburg, Germany 1


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Introduction: Lenalidomide (Len) has demonstrated significant activity in advanced multiple myeloma (MM). Available data suggest improved efficacy and safety compared to thalidomide. Len in combination with

Abstracts


Fig. 1. Patients 1–3.

Clinical Trial registration: NCT00911105 Disclosure: Wolfgang Knauf: Advisory Role: Celgene; Financing of Scientific Research: Celgene. Jens Hasskarl: No conflict of interest disclosed. P533

Pretransplantation ferritin as a prognostic marker in multiple myeloma Strasser-Weippl K., Ludwig H.

Sixty-five patients (47%) underwent single, 50 (36.5%) double and 22 (16%) three cycles of transplantation. The number of transplantation cycles was not correlated with outcome (P = 0.85), neither were paraprotein type (P = 0.79) or the type of induction treatment P = 0.68). In univariable analysis, age, albumin and ferritin levels were significant prognosticators of OS when used as continuous variables. In addition, patients with ECOG performance status >1 and those with poor cytogenetics had a worse prognosis. Due to a high number of missings these two variables were not included in further analyses. In multivariable analysis a stepwise backward regression revealed only serum albumin (P = 0.011) and ferritin (P = 0.002) to be independent prognosticators for OS, and ferritin only (P = 0.024) for PFS (figure 1).

Fig.1.

Conclusion: In conclusion, our data show a significant prognostic role of pretransplantation ferritin levels, both for PFS and for OS in myeloma patients subsequently subjected to ASCT. Disclosure: No conflict of interest disclosed. P534

Significant improvement in survival post ASCT in myeloma patients after year 2006 Strasser-Weippl K., Ludwig H. Wilhelminenspital, Zentrum für Onkologie und Hämatologie, Wien, Austria

Introduction: Ferritin has been implicated as a prognostic marker in patients with hematologic malignancies undergoing stem cell transplantation (ASCT). It was also found to be associated with overall survival (OS) in newly diagnosed myeloma patients. This prompted us to evaluate the prognostic relevance of pretransplantation ferritin in myeloma patients with long follow-up. Patients and Methods: A cohort of 153 consecutive myeloma patients undergoing ASCT at our unit between May, 1994 and February, 2010, were included. Induction therapy consisted of 4 cycles of VAD until year 2008 and VTD, VTDC or VCD thereafter. We collected pretransplantation parameters when induction treatment was completed: age, gender, paraprotein type, ß2M, haemoglobin, creatinine level, serum calcium, LDH, albumin, ECOG performance status and serum ferritin, cytogenetics (where available), bone marrow infiltration at baseline, the number of transplantation cycles and type of induction treatment. Results: Pretransplantation ferritin and other laboratory parameters were available in 137 patients. Median follow-up of our cohort was 51.9 months and median survival was 83.9 months. At the time of analysis, 66 patients (48.2%) had deceased. Median age was 56 years, (range 27–72). After completion of induction therapy – at time of evaluation of laboratory parameters – only a small proportion of patients had unfavorable laboratory parameters (ß2M >3.5 mg/L: 29.9%; albumin ≤3.5 g/dL 6.6%; LDH >250IU/L: 5.8%).

Introduction: The concept of high dose therapy followed by autologous stem cell transplantation (ASCT) has not significantly been changed since its introduction in the late 1980ies. More recently, myeloma care has been improved by the introduction of novel drugs, better supportive care, and provision of therapy predominantly in specialized centres. We aimed to evaluate the outcome of patients transplanted sequentially between 1994 and 2012 in a single center. Patients were dichotomized into groups before widespread use of bortezomib and lenalidomide (before 2007) and a cohort treated thereafter. Patients and Methods: 153 patients, median age: 56.5 years, (range. 27.5–71.9 years), 43% females, ISS stage I/II/III: 48%/24%/28% were enrolled. Induction therapy consisted of 4 cycles of VAD until year 2008 and VTD, VTDC or VCD thereafter. Conditioning therapy consisted of melphalan 200 mg/m2, or for patients randomized into a group with triple ASCT, of 100mg/m2. Results were analyzed by treatment period: before end of 2006 (A), and after 2006 (B). Results: In the entire cohort progression-free survival (PFS) from ASCT was 26.1 and overall survival (OS) was 76.9 months (med. F/U 54.6 mo). Prognostic parameters such as age, B2M, LDH and Hb, did not change significantly or showed a slight worsening. PFS from end of ASCT was significantly longer after year 2006 (33.4 vs. 22.8 months, P  100U/L) were on the rise. Workup for malignant lymphoma did not reveal any further abnormality and the patient was prepared for a staging splenectomy. Marked drop of serum LDH on the road to splenectomy however suggested a reactive nature of his illness and the splenectomy was cancelled. A serum IgM Titer for leishmaniasis turned out to be highly positive. Repeat bone marrow biopsy did not show parasites on bone marrow cytology again, however a PCR analysis* of bone marrow DNA was positive for leishmania species with sequence homology >99% for leishmania donovani and leishmania chagasi. The patient was placed on i.v. liposomal amphotericin resulting in prompt devervescence and recovery. Spleen size returned to normal within 3 weeks. The patient reported travel to Sri Lanka 20 years ago, to Egypt 10 years ago and to Calabria (Italy) several months ago. He never exhibited any sign of cutaneous leishmaniasis.

Abstracts



Scleroderma renal crisis complicating stem cell mobili zation in a young patient with severe systemic sclerosis Sockel K.1, Ordemann R.1, Kroschinsky F.1, Parmentier S.P.2, Kleymann A.2, Fantana J.2, Ehninger G.1, Aringer M.2, Bornhäuser M.1 1 2

Universitätsklinikum Dresden, Medizinische Klinik 1, Dresden, Germany, Universitätsklinikum Dresden, Medizinische Klinik 3, Dresden, Germany

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Conclusion: Visceral leishmaniasis should be definitively ruled out by bone marrow PCR in patients with fever and splenomegaly, especially in immunocompromised patients. This applies also to patients who deny recent travel to an endemic aera. *provided by Institut für Tropenmedizin University Tübingen Disclosure: No conflict of interest disclosed.

Splenic marginal zone lymphoma was the putative clinical diagnosis and upon appropriate immunization splenectomy was performed. Subsequently, the patient recovered completely but histopathological evaluation did not show any evidence of malignancy. Two months later the patient presented with the same initial symptoms. A repeat bone marrow aspiration showed numerous macrophages filled with inclusion bodies of Leishmania infantum.

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Whole body pain and pancytopenia in a young patient Lang F.1, Webersinke G.2, Erdel M.2, Gruber-Rossipal C.3, Wiesinger K.4, Rechberger E.1, Petzer A.1, Tschurtschenthaler G.1,5 Hospital Barmherzige Schwestern Linz, Internal Medicine: Medical Oncology, Hematology and Gastroenterology, Linz, Austria, 2Hospital Barmherzige Schwestern Linz, Laboratory for Molecular Biology and Tumorcytogenetics, Linz, Austria, 3Hospital Barmherzige Schwestern Linz, Institute of Pathology, Linz, Austria, 4Hospital Barmherzige Schwestern Linz, Central Laboratory, Linz, Austria, 5Hospital Barmherzige Schwestern Linz, Laboratory for Hematology and Immuncytology, Linz, Austria 1

Introduction: Pancytopenia has different reasons and sometimes it is difficult to find the correct diagnosis. We report on a 23 y/o male patient with admission due to pancytopenia and whole body pain 12 months after operation of a medulloblastoma (cerebellum) and adjuvant chemoradiotherapy. Methods: Giemsa staining was performed in peripheral blood and bone marrow and flow cytometry (FACS Calibur) was used to differentiate the blast cells . Conventional chromosomal analysis was done by Giemsa-banding; for FISH Vysis® probes were used. Results: Pancytopenia was caused by an extensive bone marrow infiltration with blasts positive for TdT and CD 56 (NCAM). These blasts were not of hematological origine and could be defined as blasts of a medulloblastoma. Deletion 1p and monosomy 4 were detected in the blast cell population. LDH was elevated to 637U/l. Clinical examination, further laboratory tests and radiology investigations did not show any abnormalities. Conclusion: Pancytopenia was caused by the first recurrence of medulloblastoma limited to bone marrow infiltration. The patient was treated with chemotherapy (EORTC-protocol followed by high dose chemotherapy with autologous stem cell transplantation). Disclosure: No conflict of interest disclosed.

Results from PCR and ELISA confirmed the diagnosis. With the knowledge of VL, silver staining of the former spleen specimens was performed and pathologic re-examination showed sporadic Leishmania infantum inclusion bodies. Liposomal amphotericin b was administered to the patient and after seven days of treatment blood parameters and clinical symptoms normalized steadily. Conclusions: VL is rare and therefore is often not included routinely in the differential diagnosis. Nevertheless, this case emphasizes the importance of an accurate travel history and awareness of VL in patients presenting with the triad of unknown fever, hepatosplenomegaly, and pancytopenia. Disclosure: No conflict of interest disclosed.

Visceral leishmaniasis mimicking malignant lymphoma Evers G.1, Pohlen M.1, Berdel W.E.1, Titze U.2, Köhler G.2, Weckesser M.3, Anthoni C.4, Mesters R.M.1 Universitätsklinikum Münster, Medizinische Klinik A, Münster, Germany, Universitätsklinikum Münster, Gerhard-Domagk-Institut für Pathologie, Münster, Germany, 3Universitätsklinikum Münster, Klinik und Poliklinik für Nuklearmedizin, Münster, Germany, 4Universitätsklinikum Münster, Klinik für Allgemein- und Viszeralchirurgie, Münster, Germany 1 2

Introduction: Visceral leishmaniasis (VL) is a systemic protozoan disease caused by different leishmania species and occurs in multiple endemic areas including the coastal region of the mediterranean sea. Symptoms of VL can be very similar to those of malignant lymphoma. Methods: We report on a patient with VL in whom clinical presentation initially mimicked splenic marginal zone lymphoma. Results: A 57-year old male engineer was admitted to our hospital with fever, night sweats, pancytopenia and hepatosplenomegaly. Past medical history and physical examination revealed besides palpable hepatosplenomegaly nothing remarkable. Travel history revealed frequent stays on the island of Mallorca, Spain, in recent years. Bone marrow aspiration showed normal hematopoiesis with no signs of leishmaniasis (also in retrospect), and PET-CT revealed an enlarged spleen (length 21 cm) with increased metabolic activity.


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Two patients diagnosed with coexisting hematologic and oncologic BRAF V600E mutated malignancies. Which entity is really carrying the BRAF (V600E) mutation? Schlick K., Troch M., Placher-Sorko G., Faber V., Neureiter D., Greil R., Hopfinger G. Paracelsus Medizinische Privatuniversität, Salzburg, Austria

Introduction: During the past years extra- and intracellular signaling pathways in cancer cells have been intensively studied and the development of pathway inhibitors has become of major interest in both hematologic and oncologic malignancies. Recent studies are focusing on gene activating mutation. One of the most common mutations is the BRAFv600e mutation, which is characterized by replacement of valin by glutamine acid – at amino-acid 600. This particularly mutation was found to be targetable by vemurafenib, which causes apoptosis in tumor cells and has a very strong effect against metastatic melanoma and hairy cell leukemia. Methods and Results: At our institution two patients with solid tumors were referred for diagnosis and treatment. One patient suffered from lung cancer and molecular workup of lung cancer specimen revealed the presence of mutation for B-RAF v600e. The bone marrow biopsy done due to

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Fig. 1. Bone marrow with intracellular protozoans (arrow).

pancytopenia led to the diagnosis of hairy cell leukemia and the neoplastic lymphocytes carried mutation of B-RAF v600e. The other patient was diagnosed with metastatic uveal melanoma, also carrying the BRAF v600e mutation in the liver lesion and with coexistent chronic lymphatic leukemia. Under the assumption that solid tumor specimen of the lung and liver probably were contaminated with clonal lymphocytes, we additionally performed immunochemistry staining on NSCLC (Patient 1) and liver metastasis of uveal melanoma (Patient 2) specimen. Interestingly, in both cases immunohistochemistry and histological work up revealed infiltration with lymphocytic cells of hairy cell origin (Patient 1) and chronic lymphocytic leukemia cells (Patient 2), respectively. Conclusion: In view of these findings, we were not clearly able to state that NSCLC or uveal melanoma cells carried the BRAF mutation, which would have provided a basis for treatment with vemurafenib. These two distinct cases demonstrate the major importance of ruling out potential contamination with other malignant cells, potentially harboring the BRAF mutation and to know the limitations of molecular techniques and therefore the danger of misinterpreting pathological results, as treatment strategies change significantly. It is essential to keep in mind that in both cases the more aggressive underlying malignancy might not have been targeted by a BRAF inhibitor due to lack of mutation. Disclosure: No conflict of interest disclosed.

discordances were 16 (6%); minor discordances were 24 (9.1%); quantitative discordances were 11 (4.1%). In 2 ALL clonal evolution hampered straightforward MRD assessment. In one case IGH RQ-PCR underestimated MRD while a second RQ-PCR marker (TCRD) overlapped NGS. In a second case NGS did not detect the tumor diagnostic clone due to loss of the complete IGHV at relapse whereas the preceeding IGHDJ was preserved and detected by RQ-PCR. Conclusions: NGS is effective for MRD monitoring in ALL, MCL and MM showing good concordance with RQ-PCR, but disease-specific pitfalls have to be considered for both methods. Prospective comparative analysis of unselected cases is required to verify the clinical impact of NGS-based MRD assessment. Disclosure: No conflict of interest disclosed. V577

High resolution genomic analysis of serial multiple myeloma samples for the detection of newly acquired alterations and monitoring of clonal evolution Kull M.1, Kuchenbauer F.1, Bullinger L.1, Teleanu V.1, Liebisch P.2, Rücker F.1, Knop S.3, Einsele H.3, Bargou R.3, Doehner H.1, Langer C.1 Universitätsklinik Ulm, Klinik für Innere Medizin III, Ulm, Germany, 2Onkologie, Moers, Germany, 3Universitätsklinik Würzburg, Medizinische Klinik II, Würzburg, Germany 1

Myelom – klinische Forschung V576

Minimal Residual Disease (MRD) detection by next- generation sequencing and real-time quantitative PCR: A methodical comparison in ALL, MCL and MM Brüggemann M.1, Ladetto M.2, Monitillo L.2, Ferrero S.2, Pepin F.3, Drandi D.2, Palumbo A.2, Boccadero M.2, Carlton V.3, Trautmann H.1, Kneba M.1, Faham M.3, Ritgen M.1, Pott C.1 University Hospital Schleswig-Holstein, Second Department of Medicine, Kiel, Germany, 2University of Torino, Division of Hematology, Turin, Italy, 3 Sequenta Inc, San Francisco, United States 1

Introduction: Real-Time Quantitative (RQ)-PCR-based MRD detection using allel specific primers has some limitations, including marker identification failure or false negatives due to clonal evolution or somatic hypermutations. To verify if immunoglobulin heavy chain variable region (IGH)-based next-generation sequencing (NGS) might overcome these limitations we comparatively analysed both methods in a well defined set of patients. Methods: 378 samples (62 DG, 316 FU) were collected from 55 pts (15 ALL, 30 MCL, 10 MM). IGH-RQ-PCR was carried out as previously described by us. NGS was performed at the Sequenta facilities. Using universal primer sets, we amplified IGH variable, diversity and joining gene segments. Products were sequenced with a high degree of coverage (14 reads per each IGH molecule). Tumor-specific clonotypes were identified for each patient and quantitated by an internal reference DNA. Comparability of results was assessed by bivariate correlations (software R 2.15.1 package irr). Discordances were classified as follows: a positive/negative discordance was defined as major when the positive result was >1 E-05 and minor when ≤1 E-05; a quantitative discordance was defined as the presence of two positive results with a quantitative discrepancy >1 log. Results: 51 pts (93%) were evaluable with at least one tool (RQ-PCR 45, NGS 49), 43 (78%) with both methods. Sequences identified with both tools were identical in 41 cases and unrelated in two. Overall, 330 samples (87.3%) were evaluated with at least one tool (RQ-PCR 279, NGS 316) and 265 (70%) with both. In terms of MRD output, concordance was significant (p 2007), age and disease stage by D&S, the latter two were of statistical significance (p 30 days (p  6 months in response to first-line anti-angiogenic therapy was associated with an mOS of 46.8 months compared to 12.1 months for all others suggesting first line PFS > 6 months as an independent prognostic factor. Conclusion: Current data indicate that OS in patients with mRCC improved with the use of sequential treatment of targeted agents. The optimal sequences of drugs for individual patients and potential combinations have to be determined in the future. Disclosure: Thomas Bauernhofer: Advisory Role: Roche; Financing of Scientific Research: Roche, GSK; Other Financial Relationships: Roche. V680

The renaissance of immune modulating therapies in renal cell carcinoma Grünwald V. Medizinische Hochschule Hannover, Klinik für Hämatologie, Hämostase ologie, Onkologie und Stammzelltransplantation, Hannover, Germany

We recently abandoned cytokine-based therapies after more than 2 decades of treatment. Inhibitors of the VEGF, VEGFR or mTOR represent contemporary therapeutic options in renal cell carcinoma (RCC). However, cytokines remain an option in combination with bevacizumab or as intravenous (i.v.) interleukin 2 (IL2) in individual patients. IL2-based regimens are toxic and are limited to specialized centers only. The promise of immune therapy associated long-term remission is challenged by current long-term follow-up

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of VEGFR inhibitors. Overall, the high toxicity of i.v. IL2 and scarce benefit in selected patients has limited the clinical applicability of this approach. Instead, current studies explore the novel concept of targeted immune therapy. Randomized phase III trials test the role of IMA901 – a peptide vaccine – or inhibitors of programmed death (PD)-1 in RCC. In contrast to current therapies, these agents introduce putative biomarkers to the field, which could help to select patients for different therapies. HLA type A02 is a prerequisite for IMA901, whereas expression of PD-1 or its ligand is thought to be predictive for clinical outcome of PD-1 inhibitors. Expectations are high for these novel therapeutic options, because for the first time RCC therapy comes along with a putative biomarker, which could define a rational for selection of therapies in the near future. Disclosure: Viktor Grünwald: Advisory Role: Bayer, Novartis, GSK, Roche, Pfizer, Astellas; Financing of Scientific Research: Bayer, Novartis, GSK, Roche, Pfizer, Astellas.

Freier Vortrag Immun-Therapie V682

A new PD1-CD28 chimeric receptor overcomes PD-1- mediated immunosuppression in adoptive T cell therapy Kobold S., Grassmann S., Peters P., Zeng Y., Schmollinger J.C., Endres S. Ludwig-Maximilians-Universität, Abteilung für Klinische Pharmakologie, München, Germany

Introduction: Although tumor-specific cytotoxic T cells are capable of killing tumor cells, treatment with adoptive T cell transfer does not lead to sufficient tumor regression without adjuvant therapy. Tumor-promoted T cell exhaustion and anergy have been proposed to contribute to this lack of efficacy. We and others have previously shown that programmed death receptor-1 (PD-1) upregulation is a hallmark of tumor infiltrating, adoptively transferred T cells. PD-1 and its ligand (PD-L1) constitute a major immunosuppressive axis driven by tumor cells. Disruption of this axis may hit an Achilles heal of tumor immune escape. Methods: A PD1-CD28 chimeric receptor was cloned into the retroviral vector pMP71 and expressed in primary murine T cells specific for the model antigen ovalbumin (OT-1 cells). Functionality was addressed in vitro using ELISA and flow cytometry. In vivo, ovalbumin and PD-L1 overexpressing Panc02 cells (syngeneic pancreatic cancer cell line) were inoculated subcutaneously in immunocompetent female C57Bl/6 mice. Mice (n = 6 per group) were treated twice i.v. with PD1-CD28 chimeric receptor-transduced T cells or control T cells. Results: In vitro, PD-1-CD28 chimeric receptor-transduced primary T cells released 130-fold more interleukin-2 (IL-2) and 300-fold more interferon-γ than untransduced or control-transduced T cells when stimulated with CD3 and PD-L1, demonstrating the functionality of the chimeric receptor (p = 0.0014). In co-culture experiments with the Panc02 tumor cells, effective co-stimulation through PD1-CD28 was only seen in the presence of the TCR-recognized antigen ovalbumine and PD-L1. Upon blockade of MHC or PD-1, co-stimulation through the receptor was abrogated. Culture of transduced T cells in the presence of CD3 and PD-L1 increased cell numbers 4-fold and significantly increased viability of cells compared to untransduced or control-transduced T cells (p < 0.0001). In vivo, treatment of mice with an established (OVA and PD-L1 expressing) Panc02 subcutaneous tumor (mean tumor size at treatment onset 26 mm2) with PD1-CD28-transduced OT-1 slowed tumor growth compared to treatment with control-transduced OT-1 cells (p < 0.001). This demonstrates the functionality of the chimeric receptor in an immunocompetent organism. Conclusions: Adoptive T cells therapy with PD-1-CD28 chimeric receptor-transduced T cells is a promising approach to overcome PD-1-PD-L1mediated tumor-induced anergy and immunosuppression. Disclosure: No conflict of interest disclosed.

Abstracts



The Dual-Dual-Targeting (DDT) Concept – activating NK cell receptors as trigger molecules for bifunctional antibody-derivatives to enhance anti-lymphoma NK cell responses Kellner C.1, Repp R.1, van de Winkel J.G.J.2, Parren P.W.H.I.2, Valerius T.1, Humpe A.1, Gramatzki M.1, Peipp M.1 Christian-Albrechts-Universität zu Kiel, Sektion für Stammzell- und Immuntherapie, Kiel, Germany, 2Genmab, Utrecht, Netherlands 1

Introduction: NK cells play an important role in cancer immunosurveillance. Their cytotoxic abilities are governed by integrating signals mediated through stimulatory and inhibitory receptors. Activating NK cell receptors such as NKp30 and NKG2D scan host cells for the expression of self-molecules which become upregulated upon malignant transformation. For potent NK cell activation the cell surface expression levels of such self-molecules are critical, and tumor cells escape NK cell recognition by down-modulating the danger ligands. To mimick the induced-self phenotype required for efficient tumor recognition, recombinant bifunctional immunoligands engaging NKG2D or NKp30 and binding to CD20 were generated. Methods: The NKG2D-specific ligand ULBP2 or the NKp30-specific ligand B7-H6 were genetically fused to a CD20-scFv. The molecules were expressed in mammalian cells and purified to homogeneity. Both molecules bound to CD20+ lymphoma cells and simultaneously reacted with the corresponding NK cell receptors, as demonstrated in flow cytometric analysis. The cytotoxic properties and ability to trigger cytokine production was investigated. Results: Lymphoma cells coated with ULBP2:CD20 and B7-H6:CD20 efficiently triggered NK cell cytotoxicity and production of immunostimulatory cytokines (Interferon-γ, TNF-α). B7-H6:CD20 and ULBP2:CD20 induced NK cell-mediated killing of primary tumor cells in a strictly CD20-dependent manner at nanomolar concentrations. When B7H6:CD20 was applied in combination with ULBP2:CD20, strong synergistic effects on NK cell cytotoxicity were observed. When combined with intact antibodies, the recombinant immunoligands synergistically enhanced ADCC by NK cells, which is regarded as an important mechanism of action of therapeutic antibodies. For example, ULBP2:CD20 boosted ADCC by rituximab and the CD38 antibody daratumumab. Conclusions: Stimulatory receptors such as NKG2D and NKp30 deserve further evaluation as activating trigger molecules for antibody-based cancer immunotherapy. With their abilities to trigger NK cell cytotoxicity and to boost ADCC by therapeutic antibodies, recombinant immunoligands directed against tumor-associated antigens and engaging NKG2D or NKp30 may represent attractive biologic molecules to recruit NK cells against tumors. A Dual-Dual Targeting Concept (DDT-Concept) addressing two target antigens and two effector molecules may allow high specific NK-cell activation at the tumor site.

peptides, #1 and #3, induced specific T cell responses in patients with NPM1mut (33% vs 44%). NPM1mut AML patients showed a significantly higher frequency of CTL responses against peptide #3 compared to healthy volunteers. The functional role of NPM1mut for the improved clinical outcome is still under evaluation. Immune responses to NPM1mut may contribute to the favorable prognosis of this AML subtype. Here, we performed survival analysis of 25 NPM1mut patients, analyzed by ELISpot comparing cases with or without specific T cell responses. Our data suggest a better overall survival (OS) of patients with specific CTL responses against peptide #1 or #3 (p = 0.004). In a differential analysis, immune responses seem to differ in dependence of epitopes (p = 0.026), although this finding has to be interpreted with caution given the low number of patients. Due to its exquisite specificity in leukemia, NPM1mut might constitute an ideal target structure for individualized immunotherapeutic approaches. Analysis with material from larger controlled clinical trials with a high number of patients with NPM1mut AML has to be performed. Nevertheless, these data suggest that immune responses might contribute to the clinical outcome by lysis of residual leukemic cells after chemotherapy. Therefore, immunotherapeutic approaches present a promising strategy for NPM1mut patients for maintenance treatment or with persistent minimal residual disease (MRD). In an AML patient with NPM1mut and molecular relapse, we demonstrated polyspecific CTL responses against several known LAAs but also NPM1#3 after preemptive donor lymphocyte infusion (DLI). Importantly, the immune responses against LAAs were associated with MRD negativity. Such persistent responses against NPM1mut epitopes provide a rationale for the development of preemptive maintenance strategies in AML patients with NPM1mut. Taken together, NPM1mut might constitute an interesting target structure for individualized immunotherapeutic approaches in NPM1mut AML patients. We hypothesize that immune responses against mutated NPM1 may contribute to the favorable prognosis. Disclosure: No conflict of interest disclosed. V685

Rescue of impaired NK cell activity in Hodgkin lymphoma with bispecific antibodies in vitro and in patients Pogge von Strandmann E.1, Reiners K.S.1, Kessler J.1, Sauer M.1, Rothe A.1, Hansen H.P.1, Reusch U.2, Hucke C.2, Koehl U.3, Duerkop H.4, Engert A.1 Klinik I für Innere Medizin, Köln, Germany, 2Affimed Therapeutics AG, Heidelberg, Germany, 3Cellular Therapy Centre, Hannover, Germany, 4 Pathodiagnostik, Berlin, Germany 1

Immune responses directed against epitopes derived from the mutated region of NPM1 by NPM1mut-specific CD8(+) cytotoxic T cells (CTLs) might be involved in the rejection of NPM1mut leukemic blasts. Recently, we described specific T cell responses of CD4+ and CD8+ cells against epitopes derived of from NPM1 mutated regions. Two NPM1mut-derived

Introduction: Natural killer (NK) cells represent a key component of the innate immune system against cancer. Here, we tested for functional NK cell defects in Hodgkin lymphoma (HL) patients and suggest an improvement of NK function by therapeutic means. Methods: We screened the sera of healthy donors (n = 50) and HL patients (n>290) using ELISA to detect serum factors known to restrict NK cell function. The expression pattern of activating NK-receptors was analyzed using 4-colour flow cytometry of peripheral blood lymphocytes. The samples were obtained from patients (mean age: 38) before (n = 40), during (n = 39) and on completion/after radio/chemotherapy (n = 17). NK cells from healthy donors and patients were isolated and analyzed for their killing activity in cytotoxicity assays. Finally, the impact of a novel therapeutic bispecific antibody (CD30 × CD16A, Affimed Therapeutics AG, Heidelberg) on NK cell phenotype and function was analyzed in vitro and in CD30 × CD16A treated patients. Results: We demonstrate that peripheral NK cells (pNK) from HL patients fail to eliminate HL cell lines in killing assays. Impaired NK cell function correlated with elevated serum levels of soluble ligands for the NK cell receptors NKp30 (BAG6/BAT3) and NKG2D (MICA), factors known to constrict NK cell function. In vitro, NK cell cytotoxicity could be restored by an NKG2D/NKp30-independent antibody construct (CD30 × CD16A). This bispecific protein targets CD16A on effector cells and binds simultaneously to the surface receptor CD30, which is

Abstracts



Immune responses against the mutated region of cytoplasmatic nucleophosmin 1 (NPM1) might contribute to the favorable clinical outcome of AML patients with NPM1 mutations (NPM1mut) Hofmann S.1, Schneider V.1, Schmitt M.2, Götz M.1, Döhner K.1, Wiesneth M.3, Döhner H.1, Greiner J.1 Universität Ulm, Innere Medizin III, Ulm, Germany, 2Universität Heidelberg, Innere Medizin V, Heidelberg, Germany, 3Universität Ulm, Institut für Transfusionsmedizin, Ulm, Germany 1

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Ratios of IgG1 binding affinities to FcγRIIa and FcγRIIIb determine the efficacy of PMN-mediated ADCC – implications for Fc-engineered antibodies Derer S.1, Glorius P.1, Lohse S.1, Schlaeth M.1, Muchhal U.2, Desjarlais J.2, Valerius T.1, Peipp M.1 Division of Stem Cell Transplantation and Immunotherapy, UKSH Kiel, Kiel, Germany, 2Xencor, Inc., Monrovia, United States 1

Introduction: Antibody-dependent cell-mediated cytotoxicity (ADCC) has been suggested to be an essential effector mechanism for the in vivo activity of tumor-targeting therapeutic monoclonal antibodies (mAbs). Thus, enhancing the affinity of human IgG1 mAbs to NK cell-expressed FcγRIIIa by glyco- or protein-engineering of their Fc moiety has been demonstrated to improve NK cell-mediated ADCC and represents a promising strategy to improve antibody therapy. However, human polymorphonuclear (PMN) cells express the homologous FcγRIIIb isoform, which does not trigger ADCC. The aim of the present study was to analyze a panel of distinct IgG1 mAbs, displaying different affinities for FcγRIIa and FcγRIII, with respect to PMN recruitment for tumor cell destruction. Methods: Affinities of analyzed mAbs to distinct FcγR were determined by surface plasmon resonance technology. Induction of ADCC was assessed by 51Cr release experiments in the absence or presence of FcγRIII- or FcγRII-blocking agents to unravel the contribution of both receptors in these assays. Besides ADCC, antibody-dependent cell-mediated phagocytosis (ADCP) was analyzed by flow cytometry. Results: Non-fucosylated or protein-engineered IgG1 variants with optimized FcγRIII binding capacities demonstrated the expected benefit in triggering NK cell- mediated ADCC. However, these antibodies did not mediate ADCC by PMN, while ADCP mediated by PMN was preserved. Furthermore, ADCC was restored by blockade of FcγRIIIb on PMN. Additionally, eosinophils as well as PMN from paroxysmal nocturnal hemoglobinuria (PNH) patients – expressing no or low levels of FcγRIIIb – mediated effective ADCC with FcγRIII-optimized mAbs. Additional experiments with Fc variants displaying enhanced FcγRIIa binding or with double FcγRIIa/FcγRIII-optimized constructs demonstrated enhanced PMN-mediated ADCC compared to control mAbs. Statistical analyses revealed that FcγRIIa/FcγRIIIb affinity ratios correlated with the extent of human PMN-mediated ADCC. Conclusions: In the case of PMN, improvement of IgG1 mAbs affinities for FcγRIII leads to impairment of FcγRIIa-dependent cytotoxicity against tumor target cells due to enhanced FcγRIIIb ligation. However, low PMN-mediated ADCC triggered by FcγRIII-optimized mAbs might be balanced by a stronger FcγRIIIb-dependent phagocytic activity. Furthermore, protein-engineering strategies can be utilized to further improve IgG1 antibodies' affinities for FcγRIIa and therefore PMN-mediated ADCC. Disclosure: No conflict of interest disclosed.

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V687

The role of the E3 ligase Cbl-b in murine dendritic cells Wallner S.1,2, Lutz-Nicoladoni C.1,2, Tripp C.H.3, Gastl G.1,2, Baier G.4, Penninger J.M.5, Stoitzner P.3, Wolf D.6 Internal Medicine V, Haematology and Oncology, Medical University Innsbruck, Innsbruck, Austria, 2Tyrolean Cancer Research Institute, Innsbruck, Austria, 3Department of Dermatology and Venereology, Medical University Innsbruck, Innsbruck, Austria, 4Department for Medical Genetics, Molecular and Clinical Pharmacology, Medical University Innsbruck, Innsbruck, Austria, 5IMBA, Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna, Austria, 6Department of Haematology/ Oncology, University Hospital Bonn, Bonn, Germany 1

Dendritic cells (DCs) are potent antigen-presenting cells with a promising potential in cancer immunotherapy. Cbl proteins are E3 ubiquitin ligases and have been implicated in regulating the functional activity of various immune cells. As an example, c-Cbl negatively affects DC activation. We here describe that another member of the Cbl-protein family (i.e. Cbl-b) is highly expressed in murine bone-marrow-derived DCs (BMDCs). Differentiation of cblb-/- bone marrow mononuclear cells into classical BMDCs is unaltered, except enhanced induction of DEC-205 (CD205) expression. When tested in mixed-lymphocyte reaction (MLR), cblb-/- BMDCs exhibit increased allo-stimulatory capacity in vitro. BMDCs were next in vitro stimulated by various toll like receptor (TLR)-agonists (LPS, Poly(I:C), CpG) and exposed to FITC-labeled dextran. Upon TLR-stimulation, cblb-/BMDCs produce higher levels of proinflammatory cytokines (IL-1α, IL-6 and TNF-α) and exhibit a slightly higher level of FITC-dextran uptake. To further characterize the functional significance of cblb-/- BMDCs we tested them in antigen-specific T cell responses against ovalbumin (OVA) protein and peptides, activating either CD8+ OT-I or CD4+ OT-II transgenic T cells. However, cblb-/- BMDCs are equally effective in inducing antigen-specific T cell responses when compared to wildtype BMDCs both in vitro and in vivo. The migratory capacity into lymph nodes during inflammation was similarly not affected by the absence of Cbl-b. In line with these observations, cblb-/- peptide-pulsed BMDCs are equally effective vaccines against OVA-expressing B16 tumors in vivo when compared to wildtype BMDCs. We conclude that in contrast to c-Cbl, Cbl-b plays only a limited role in the induction of Ag-specific T cell responses by murine BMDCs in vitro and in vivo. Disclosure: No conflict of interest disclosed.

Wissenschaftliches Symposium CLL Gene, Mikromillieu und Signale V688

Molecular risk prediction of CLL Sellner L., Hüllein J., Zenz T. NCT/DKFZ/Universitätsklinik, Heidelberg, Germany

The identification of genetic alterations in chronic lymphocytic leukemia (CLL) has transformed our understanding of the disease. Different genetic lesions explain -in part- the clinical heterogeneity of CLL. Somatic hypermutation of immunoglobulin genes (IGHV) divides CLL into two major prognostic subgroups. In addition, recurrent gene mutations complement risk evaluation. Besides mutations in well studied genes such as TP53 and ATM, novel genetic alterations were identified by next generation sequencing approaches (e.g. BRAF, MYD88, NOTCH1, SF3B1). The integration of genetic information into improved treatment strategies and clinical care remains largely unresolved.Within genetic markers, only deletions or mutations of TP53 influence the choice of treatment and early allogenic stem cell transplantation.Mutations in ATM are linked to poorer outcome but extensive analysis is missing, mainly due to the complexity of mutation analysis of this relatively large gene without well-defined hot-spot mutations. Recently detected mutations in NOTCH1 and SF3B1 are associated

Abstracts


overexpressed on malignant HL cells. AFM13 belongs to a new group of antibody formats that has two binding sites for each antigen as it is a homodimer consisting of two polypeptides pairing head-to-tail with each other. Interestingly, we observed that NK cells from patients treated with this construct were generally activated and revealed an enhanced expression of the activation marker CD69. They, moreover, displayed restored cytotoxicity against HL target cells. Conclusions: These data suggest that suppression of NK cell activity contributes to immune evasion in HL and can be antagonized therapeutically. We were able to show that the impaired NK cell function in HL patients can be restored: NK cells from HL patients were activated upon treatment with a construct targeting CD16 and CD30 (ex vivo and in vivo), resulting in an improved recognition and killing of an HL derived target cell line (Reiners et al., Mol. Ther. 2013).

Disclosure: Leopold Sellner: No conflict of interest disclosed. Thorsten Zenz: Advisory Role: GSK, Roche; Honoraria: Boehringer Ingelheim; Expert Testimony: Roche Diagnostics. V690

Role of B cell receptor signaling in CLL pathogenesis Jumaa H. Max-Planck-Institut für Immunbiologie, Freiburg, Germany

Introduction: B cell chronic lymphocytic leukemia (CLL) is one of the commonest leukemia in western world and it is widely accepted that B cell antigen receptor (BCR) signaling is important for the development and clinical course of this disease. Since many CLL-derived BCRs from different patients share structural similarities, researchers are greatly trying to identify endogenous antigens that might drive the leukemia development. Blocking such antigens is thought to be a suitable therapeutic approach for CLL. In a recent study, we were able to identify a unique ability of CLL-derived BCRs to induce signaling in cell-autonomous manner suggesting that external antigens are not required for the activation of CLL B cells. Thus, approaches to block external antigens are unlikely to block the activation of B-CLL cells and it appears more important to study CLL-derived BCR expression and function in more detail. The BCR is expressed in different isotypes during B cell development and activation. The majority of B-CLL cells resemble mature B cells and express BCR isotypes IgM and IgD at the same time, but the signaling capacity and the relevance for CLL pathogenesis is unclear. Moreover, it is unclear why some B-CLL cases express the IgG-BCR isotype, which is usually expressed on memory B cells. To investigate the physiological relevance of the differential expression of BCR isotypes, we started to compare the signaling capacities of IgM and IgD BCRs in defined experimental systems. Methods: We expressed IgM and IgD variants of a model BCR in a well-defined cellular reconstitution system and examined receptor function in response to different antigen forms and concentrations. To address potential differences between IgM and IgD in CLL pathogenesis, we tested BCRs derived from TCL1-transgenic mice, which represent an animal model for CLL, and BCRs from human CLL samples. Moreover, we tested CLL development in TCL1-trangenic mice lacking IgM or IgD. Results: Our data using the model BCR suggest that IgD has a higher activation threshold than IgM and requires, therefore, increased amounts of antigen to induce signaling. CLL-derived BCRs induced autonomous signaling only when expressed as IgM but not when expressed as IgD. TCL1-transgenic mice developed CLL-like disease in association with IgM expression but not in association with IgD. Conclusion: BCR isotype is important for signaling and for the clinical course of disease in human CLL patients. Disclosure: No conflict of interest disclosed.

Freier Vortrag

Nichtmaligne Hämatologie V695

Age-associated DNA methylation patterns reveal premature aging in patients with aplastic anemia and dyskeratosis congenita which correlate with telomere shortening Beier F.1, Weidner C.I.2, Lin Q.3, Ziegler P.1, Zenke M.3, Brümmendorf T.H.1, Wagner W.2 Klinik für Innere Medizin IV, Universitätsklinikum Aachen, Hämatologie und Onkologie, Aachen, Germany, 2Helmholtz-Institut für biomedizinische Forschung, Stem Cell Biology and Cellular Engineering, Aachen, Germany, 3Institut für Zellbiologie, Uniklinik, Aachen, Germany 1

Introduction: Telomere length (TL) reflects the replicative potential of a cell and is a useful parameter to assess chronological age of an organism. Aplastic anemia (AA) and dyskeratosis congenita (DKC) are bone marrow failure syndroms (BMFS) characterized by shortened telomeres compared to age-matched healthy individuals. Human aging is associated with DNA methylation (DNAm) changes at specific sites in the genome. In this study, we analyzed if these epigenetic modifications can be used to track donor age and if they reflect premature aging in patients with AA and DKC, too. Methods: Based on a large set of publically available DNAm profiles we identified an Epigenetic-Aging-Signature. The DNAm level of three corresponding CpG sites was then analyzed by pyrosequencing after bisulfite conversion and the results were used for linear regression models for age predictions. This method was applied with peripheral blood leukocytes of a training and a validation set. Furthermore, telomere length analysis by flow-FISH of the peripheral blood leukocytes of 104 healthy donors was used to generate age-adapted reference values. Pyrosequencing and telomere length were carried out on 16 AA and 6 DKC patients Results: Our analysis revealed that DNAm levels at three AR-CpGs – located in the genes ITGA2B, ASPA and PDE4C – were best suited for age predictions. The mean absolute deviation from chronological age in the validation set of 69 donors was less than 5 years. Age-adapted telomere length analysis revealed significant telomere attrition in AA (–1.1 kb) and DKC patients (–3.7 kb, all below the 1% percentile). In analogy, the epigenetic changes revealed premature aging in AA patients – particularly in those which revealed extensive telomere attrition. Conclusion: Our results indicate that patients with AA and DKC undergo massive premature aging on the epigenetic level comparable to the observed telomere shortening in these diseases. Determination of this newly defined Epigenetic-Aging-Signature in combination with telomere length measurement may be used to further refine diagnosis of AA and DKC in the clinical setting. Disclosure: No conflict of interest disclosed. V696

Severe combined deficiency of innate and acquired immunity caused by an IKBKB mutation Pannicke U.1, Baumann B.2, Fuchs S.3, Henneke P.3, Rensing-Ehl A.3, Rizzi M.3, Janda A.3, Borte S.4,5, Schrezenmeier H.1,6, Wirth T.2, Ehl S.3, Schroeder M.L.7, Schwarz K.1,3,6 Institute for Transfusion Medicine, University of Ulm, Ulm, Germany, Institute of Physiological Chemistry, University of Ulm, Ulm, Germany, 3 Centre of Chronic Immunodeficiency (CCI), University of Freiburg, Freiburg, Germany, 4Division of Clinical Immunology and Transfusion Medicine, Department of Laboratory Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden, 5Translational Centre for Regenerative Medicine (TRM), University of Leipzig, Leipzig, Germany, 6Institute for Clincal Transfusion Medicine and Immunogenetics, German Red Cross Blood Service Baden-Wuerttemberg – Hessen, Ulm, Germany, 7Department of Pediatrics & Child Health, University of Manitoba, Winnipeg, Canada 1 2

We analyzed the molecular basis of severe combined immunodeficiency (SCID) in four patients from four independent families of a small pop-

Abstracts


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with poorer outcome as well as particular IgHV usage and (SF3B1) risk of high grade transformation (NOTCH1). Currently available data are not sufficient to guide treatment choice of CLL with these mutations.Different genetic aberrations are accompanied with certain pathway dependencies that may be exploited for targeted treatment approaches. For example unmutated CLL shows stronger on B-cell-receptor (BCR) signaling and may have increased susceptibilities towards inhibition of downstream targets such as Btk or PI3K. In addition BRAF mutated hairy cell leukemia and multiple myeloma were treated successfully with specific BRAF inhibition using vemurafenib and may be an option of BRAF mutated CLL.Despite the euphoria for BCR pathway inhibition and targeted treatment approaches, the development of resistance mechanisms is foreseeable. Targeting of multiple patient-specific vulnerabilities may be the future of CLL management. Such multi-parameter-based treatment approach may lead to «precision medicine» for patients with CLL.


Missense mutation N487S and P488S of the erythropoietin receptor are not associated with constitutively elevated hemoglobin concentration in blood donors Brönnimann C.1, Gassner C.1, Frey B.M.2 Blutspende Zürich, MOC, Schlieren, Switzerland, 2Blutspende Zürich, Schlieren, Switzerland 1

Introduction: Various conditions cause erythrocytosis (EC) that may be discovered by chance at blood donation. Primary EC (pEC) may be triggered by acquired mutations of JAK2, MPL or EPOR. Alternatively, deficient oxygen sensing/delivery conditions such as mutations of VHL, PHD2 or HIF2a gene as well as hemoglobinopathies with increased oxygen affinity may be considered. However, secondary EC (sEC) are more prevalent and their underlying condition (pulmonary or cardiac affections, paraneoplasia or high altitude related) may easily be diagnosed by physical examination. In contrast, following exclusion of inherited conditions, pEC may need molecular investigation to exclude clonal disorders. Deletional and insertional mutations of the inhibitory domain of the erythropoietin receptor (ID-EPOR, exon 7 and 8) are known to cause increased sensitivity to endogenous erythropoietin (EPO) leading to pEC. Methods: 12 patients with pEC (P1), requiring regular phlebotomy and found negative for JAK2, VHL, BCR-ABL mutations, having excluded abnormal EPO concentrations, hemoglobinopathies and sEC, were assessed for mutations of the ID-EPOR by sequencing. Similarly, 24 healthy platelet donors (P2) with high-normal hemoglobin (157–182 g/l) were also assessed for mutations of ID-EPOR. Finaly, 315 (171 m, 144 f) healthy blood donors (P3) with random distribution of hemoglobin concentrations (Hb) were assessed for N487S/A1460G and P488S/C1462T of exon 8 of EPOR, which were found in P2. Results: P1 revealed only wild type sequence of exon 7 and 8 of EPOR. In P2 one donor carried N487S/A1460G and another donor carried P488S/ C1462T. Both individuals were active platelet donors and had Hb values of 176 g/l. In P3, 8 carriers of N487S (4 m,3 f) and 3 carriers of P488S (2 m,1 f) were found. Median Hb (mHb) of mutation carriers was in males 152.5 g/l (144–180 g/l) and in females 138 g/l (135–145 g/l). There was no significant difference in mHb between carrier and non-carrier of EPOR mutations. Conclusions: Although mutations of ID-EPOR are known to cause pEC, our findings suggest, that the missense mutations N487S and P488S of ID-EPOR do not compromise its function. These mutations may repre-

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sent EPOR polymorphisms that do not sufficiently explain pEC. However, these mutations may represent a first hit in the development of clonal disease, since they affect the binding site of Grb2[Le Couedic,1996] and may therefore disturb the intracellular ERK/MAPK signal pathway. Disclosure: No conflict of interest disclosed. V698

Data from German centers in the global PNH Patient Registry – general and subgroup stratified analysis of PNH symptoms Höchsmann B.1, Leichtle R.1, Röth A.2, Panse J.3, Borchmann P.4, Dengler J.5, Aulitzky W.E.6, Port M.7, Platzbecker U.8, Klausmann M.9, Steinmetz T.10, Haferlach T.11, Becker M.12, Schmidt B.13, Schrezenmeier H.1 Institut für klinische Transfusionsmedizin und Immunogenetik Ulm, DRK Blutspendedienst Baden-Württemberg-Hessen und Universität Ulm, Ulm, Germany, 2Universitätsklinik Essen, Abteilung Hämatologie, Essen, Germany, 3Universitätsklinikum Aachen, Klinik für Onkologie, Hämatologie und Stammzelltransplantation, Aachen, Germany, 4Universitätsklinikum Köln, Klinik I für Innere Medizin, Köln, Germany, 5Medizinische Universitätsklinik Heidelberg, Abteilung Innere Medizin V, Heidelberg, Germany, 6Robert Bosch Krankenhaus Stuttgart, Hämatologie und Onkologie, Stuttgart, Germany, 7Medizinische Hochschule Hannover, Abteilung Hämatologie und Onkologie, Hannover, Germany, 8Universitätsklinik Dresden, Abteilung Hämatologie und Onkologie, Dresden, Germany, 9Gemeinschaftspraxis Drs Klausmann und Dr Welslau, Aschaffenburg, Germany, 10Gemeinschaftspraxis Hämatologie und Onkologie, Köln, Germany, 11MLL Münchner Leukämielabor GmbH, München, Germany, 12Onkologische Praxis Minden/Porta, Porta Westfalica, Germany, 13Onkologische Praxis Pasing, München-Pasing, Germany 1

Introduction: PNH is a rare life-threatening, acquired clonal disorder, resulting in a complement mediated intravasal haemolysis. An International PNH Patient registry was started to enhance the understanding of this orphan disease. Recent data confirm a strong correlation between clinical symptoms as well as haemolytic activity and prognosis. Methods: After ethical committee approval and informed consent the pseudomized patient data of the global PNH Registry (n = 2224 pts) and the German subgroup (n = 262 pts) were analysed for physician reported clinical symptoms and laboratory values. In addition quality of life questionnaires (global: 1073 pts, Germany: 87 pts) were analysed with respect to patient reported symptoms. Results: First number represents data of the German subgroup, the number in brackets the global PNH Registry. At enrolment mean age was 43.5 ± 17.9 (43.9 ± 17.6) yrs, mean GPI-deficient granulocyte population was 62.3 ± 32.8 (55.3 ± 37.6) % and 55.3 (46.5)% of pts had classic PNH and 45.4 (46.9)% of pts were male. The most common symptom was fatigue, followed by shortness of breath and haemoglobinuria. There are relevant differences between physician and patient reported symptoms in the German subgroup and global. The table below shows the data of the German subgroup stratified for GPI-deficient granulocyte clone, associated bone marrow disorder and age, including the patient reported symptoms in brackets. Conclusions: Remarkably, a substantial subgroup of pts with small PNH clones reported typical PNH-related symptoms. In particular, fatigue was often and consistently reported in all subgroups. Among all subgroups symptoms are reported more frequently by pts than by physicians. In symptomatic PNH a sufficient complement inhibition was shown to normalize life expectancy (Kelly et al Blood 2011). This emphasizes the need for an optimized collection of symptoms, including patient reported symptoms, to improve treatment decisions and therapeutic success. Disclosure: Britta Höchsmann: Advisory Role: Advisory Board Alexion; Financing of Scientific Research: Vortragshonorare Alexion, Novartis, Genzyme; Expert Testimony: Alexion, Genzyme. Hubert Schrezenmeier: Advisory Role: Advisory Board Alexion; Financing of Scientific Research: Vortragshonorare Alexion; Expert Testimony: Alexion, Genzyme

Abstracts


ulation of homogenous ethnicity. The patients presented within the first months of life with failure to thrive, oral thrush and invasive bacterial and viral infections. All patients showed normal numbers of B and T cells and suffered from agammaglobulinemia. Three patients had markedly reduced numbers of NK cells. B and T cells were of naϊve phenotype. T cells showed reduced responses to anti-CD3/CD28 treatment. Reactivity of NK cells against K562 target cells was extenuated. Assuming an autosomal recessively inherited monogenetic defect, we performed homozygosity mapping. We identified a region of interest around the centromere of chromosome 8. We detected a homozygous insertion of one nucleotide in the open reading frame of IKBKB in all patients predicting a frameshift with premature termination of the protein. Western Blot analysis for the encoded IKKβ protein did not show any residual protein in fibroblasts and PBMC suggesting a null mutation. IKKα and NEMO expression was reduced. Functional testing of NF-κB pathways was performed in fibroblasts. Phosphorylation of IkBa following stimulation with TNFα was considerably decreased. The binding of NF-κB to DNA after stimulation with TNFα or PMA was markedly reduced. Moreover, TLR4 and TLR5 triggered expression of IL-6 was decreased whereas IL-6 production following stimulation with TNFα or IL-1β was normal. These data indicate that IKKβ is not necessary for human B and T cell development but it is indispensable for lymphocyte activation.

Table 1. Lab Values and Symptoms of the German subgroup

Lab Values and Physician (Patient) reported Symptoms at Enrolment in the German PNH Patient Registry

Haemoglobin (g/l) mean ± SD

LDH (U/l) mean ± SD

Abdominal Haemoglobinuria Pain

Fatigue

Shortness of breath

Erectile dysfunction (male)

Any thrombotic event

All pts (n = 262), questionnaire pts (n = 87)

102.1 ± 21.6

772.7 ± 746.3

31.3% (42.5%)

49.2% (57.5%)

68.3% (87.4%)

45% (72.4%)

18.5% (31.3%)

21.8%

GPI-def. 50% (n = 154; questionnaire = 49))

99.1 ± 19.1

1005.9 ± 828.2

37.7% (51.0)

58.4% (63.3%)

68.2% (83.7%)

44.8% (65.3%)

19.5% (35.0%)

22.7%

Aplastic Anemia (n = 98 questionnaire =35)

99.5 ± 18.7

530.1 ± 519.8

29.6% (42.9%)

38.8% (54.3%)

72.4% (91.4%)

43.9% (71.4%)

16.7% (35.3%)

15.3%

Classic PNH (n = 145 questionnaire =48)

104.4 ± 23.3

940.6 ± 841.1

35.9% (43.8%)

59.3% (58.3%)

67.6% (83.3%)

46.9% (75.0%)

20.3% (30.8%)

26.2%

50 years (n = 71 questionnaire = 21)

100.4 ± 17.7

693.0 ± 697.0

22.5% (38.1%)

43.7% (42.9%)

76.1% (85.7%)

56.3% (81.0%)

19.4% (25.0%)

31.0%

The prevalence of erythroblasts in peripheral blood smears of patients with multiple sclerosis treated with natalizumab Robier C.1, Amouzadeh-Ghadikolai O.2, Bregant C.3, Diez J.3, Melinz K.3, Theiler G.4, Neubauer M.1 Laborverbund der Barmherzigen Brüder Graz-Eggenberg, Graz, Austria, Krankenhaus der Barmherzigen Brüder Graz-Eggenberg, Abteilung für Psychiatrie, Graz, Austria, 3Krankenhaus der Barmherzigen Brüder Graz-Eggenberg, Abteilung für Neurologie, Graz, Austria, 4Medizinische Universität Graz, Univ. Klinik für Hämatologie, Graz, Austria 1 2

Introduction: The presence of erythroblasts in the peripheral blood is, excepted from newborn infants or in pregnancy, usually associated with severe disorders, such as haematopoietic or solid neoplasms or various bone marrow diseases. It has been reported that the alpha-4-integrin antagonist natalizumab, which is used for the treatment of relapsing multiple sclerosis (MS), may occasionally cause erythroblastaemia. In this study we determined the prevalence of erythroblastaemia in blood smears of natalizumab-treated patients. Materials and Methods: 14 adult subjects with MS on long-term natalizumab treatment (Tysabri, Biogen Idec, Austria, 300 mg intravenously once every month) and 19 control subjects with MS on chronic interferon beta treatment were examined. In the natalizumab group, blood was drawn immediately preceding the next administration. One blood smear was made from each specimen, stained with May-Grünwald-Giemsa, and was entirely examined in blinded fashion by an experienced analyst for the occurrence of erythroblasts. The results were expressed as erythroblasts per slide. Results: Except for one patient, all sample blood smears of the natalizumab-treated group contained erythroblasts (n = 13, 93%). The median count was 4 erythroblasts per slide (range 0–20). The maturation stages of the erythroblasts were distributed as follows: orthochromatic, 47.5%; polychromatic, 48.75%; basophilic, 3.75%). In the blood smears of the interferon-treated subjects there were no nucleated red blood cells apparent. The differences in erythroblastaemia between the natalizumab- and the control group determined by Fisher's Exact test were highly statistically significant (p 30% with different molecular biology methods. Based on conformational analysis of the protein , 1st generation TKI´s are not expected to bind to the ATP domain in the presence of the T790M mutation. In this paper, we report 1/165 tested pts with a primary T790M mutation. Methods: 165 consecutive patients (s. abstract Halbfass et al.) were molecularly studied for EGFR mutations in exons 18–21. EGFR mutation analysis was performed by Sanger Sequencing or COBAS technology after microdissection of tumor tissue to ascertain a high percentage of tumor tissue. Of 165 patients, 33 had a EGFR mutation and only 1 had a primary T790M. The patient was worked up with appropriate staging and imaging methods. Immunohistochemistry included TTF1 and p63. Remission was measured by RECIST 1.1 criteria. Results: Of 165 patients, there was only 1 patient with a smoking history of 0.25 packyear carrying an activating EGFR exon 21 L858R as well as a resistance exon 20 T790M mutation conveying resistance prior to treatment with TKI. ALK, KRAS, BRAF and p53 were not mutated. Based on the data of Rosell et al., the patient received erlotinib at a dose of 150 mg/ die p.o. After 4 weeks, SD disease was observed in the presence of grade III cutaneous and grade I gastrointestinal side effects. Therefore, erlotinib was dose reduced to 100 mg / die p.o.. After another 4 weeks, the patient was rapidly progressing. Erlotinib was discontinued and the patient was put on a second line trial with an HSP70 inhibitor. No rebiopsy was undertaken at the time of progression.

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Conclusion: The biologic relevance of primary T790M EGFR mutation in the presence of an activating mutation is unknown because it seems to be a rare event. If detected with conventional methods, it most likely confers, like in acquired resistance, primary resistance to 1st generation TKI and might be associated with inferior prognosis. Therefore, T790M mutation should be tested for at diagnosis. Also, treating patients harboring the primary T790M mutation with 2nd generation TKI binding to the ATP domain should be considered even in the presence of T790M. Disclosure: Frank Griesinger: Advisory Role: Roche, Boehringer Ingelheim; Financing of Scientific Research: Vortragstätigkeit Roche und Boehringer Ingelheim Markus Tiemann: Advisory Role: Roche, Boehringer Ingelheim; Financing of Scientific Research: Roche und Boehringer Ingelheim Vortragstätigkeit. P832

ALK Rearrangement in combination with a kras mutation in a smoker with adencarcinoma of the lung: First case report Griesinger F.1, Conradi I.1, Halbfass V.1, Hallas C.2, Falk M.2, Tiemann M.2 Pius-Hospital Oldenburg, Hematology and Oncology, Oldenburg, Germany, 2Hematopathology, Hamburg, Germany 1

Introduction: Molecular characterization of non small cell lung cancer has evolved in recent years with the identification of at least 3 drugable targets, EGFR activating mutations,ALK rearrangements and recently ROS1 translocations. A 4th molecular alteration, KRAS mutations, is frequently observed in lung adenocarcinomas, Overall these muationas are generally mutually exclusive. Especially ALK rearrangements normally occur in younger patients that are never or light smokers and do not carry a KRAS mutation. Cases with concomitant EGFR and ALK and EGFR and KRAS have been observed, however the combination of ALK rearrangement with a KRAS mutation has not been observed as of yet. Material and Methods: Since 2009, all patients with non squamous non small cell lung cancer have been tested for the presence of ALK, KRAS, BRAF, EGFR and p53 (Abstract Halbfass et al.). Testing methods are described in the Abstract of Halbfass et al. Briefly, ALK rearrangement was either detected with RT PCR or in most cases with ALK-FISH. EGFR and KRAS mutations were detected by Sanger-Sequencing, or by hybridization based COBAS test. c Histological and immunohistological analysis was carried out by standardized procedures using the Bondmax (Menarini) . Results: One 51 yo female caucasian patient who had smoked 50 packyears until recently, presented with a poorly differentiated adenocarcinoma of the lung, c2T3c2N3M1b (OSS, BRA). Molecular diagnosis was routinely carried out while the CNS and bone metastases were irradiated. After start of chemotherapy with paclitaxel, carboplatin and bevacizumab, the molecular results became available. These indicated that an ALK rearrangement had occurred in at least 50% of the cells, as analyzed by FISH. Also, the tumor cells carried a KRAS Codon 12 c.34G>T mutation. This is, to the knowledge of the authors, the first case of a concomitant ALK mutation in combination with a KRAS mutation. Conclusion: This case demonstrates that testing for ALK should not be limited to never or light smokers with non squamous cell carcinoma, but instead as already consented for EGFR testing, all patients with non squamous cell carcinoma should be included. Furthermore, sequential test algorithms (only test for EGFR and ALK if KRAS is WT) should be reconsidered. Only combined testing for all drugable targets leads to identification of all patients that potentially benefit from molecular stratified therapies. Disclosure: No conflict of interest disclosed.

Abstracts


Results: A female caucasian 62 y.o. never smoker was diagnosed with TTF1+ adenocarcinoma G3 of the upper lobe of the lung, c2T3 (extension to mediastinal pleura) c2N2 (LN 2R and 4R) c2M0, UICC7 IIIA4 (Robinson). Molecular analysis after microdissection revealed WT for ALK, KRAS and BRAF, but an activating EGFR mutation Exon19 (p.Leu747_ Ala750delinsPro), as well as a TP53 mutation in exon 8 (p.Val272Met (c.814G > A) (Sanger Sequencing). Induction therapy was started with erlotinib 150 mg/die p.o. days -12 to -2 in order to prove responsiveness of the tumour to TKI. On day 0 partial response was achieved. Therapy was continued with 3 cycles of Docetaxel 75 mg/m2 d1 and Cisplatin 50 mg/m2 d 1 and 2 qd22 with intercalated Erlotinib 150 mg/die p.o on days 4–19. Almost complete radiologic remission was achieved after 2 cycles. The patient underwent R0 resection (upper lobe resection and radical lymphadenectomy) 4 weeks after day 1 of the 3rd cycle of chemotherapy, pathologic examination revealed T0N0 (mic+) with only one insula of tumor cells in an N2 lymph node, demonstrating regression grade III in the primary tumor and Grade IIB in the lymph nodes, according to the Junker classification. Molecular analysis of residual tumor cell insula revealed the same EGFR and p53 mutations as the primary tumour. The patient underwent postoperative radiotherapy of the mediastinum. No additional therapy, including TKI was administered postoperatively. Conclusion: Intercalated TKI treatment might be a promising treatment choice in patients with EGFR mutated locally advanced NSCLC. A phase II trial is currently being planned to expand knowledge in this challenging field.

Striking, aggressive synchronous non-small cell lung cancer (NSCLC) in a multiple myeloma (MM) patient (pt) and circulating tumor cell (CTC) detection Rawluk J.1, Pantic M.1, Kleber M.1, Wider D.1, Follo M.1, Passlick B.2, Jakobs D.1, Burbeck M.1, Möller M.1, Herget G.3, Strohm P.3, Knop S.4, Einsele H.4, Martini C.5, May A.6, Werner M.6, Jakob A.7, Wäsch R.1, Engelhardt M.1 University of Freiburg Medical Center, Department of Hematology and Oncology, Freiburg, Germany, 2University of Freiburg Medical Center, Department of Thoracic Surgery, Freiburg, Germany, 3University of Freiburg Medical Center, Department of Orthopaedics and Trauma Surgery, Freiburg, Germany, 4University of Würzburg, Department of Hematology and Oncology, Würzburg, Germany, 5University of Freiburg Medical Center, Department of Radiation Oncology, Freiburg, Germany, 6University of Freiburg Medical Center, Institute of Pathology, Freiburg, Germany, 7 Medical Center Offenburg, Department of Hematology and Oncology, Offenburg, Germany 1

Introduction: Different neoplasms (DN), apart from the MM itself, synchronously detected or after 1.-line- and maintenance-treatment have recently been reported, questioning whether specific risk factors for MM exist. Moreover, 2. tumors have gained more attention, since MM pts live longer and randomized data show associations between newer drugs and excess risk of DNs. Large population-based databases offer powerful sample sizes, but bear the limitation of focusing on primary tumors, therefore well-documented registry analyses are of value. Methods & Results: We report on a 62-year old pt, who was diagnosed with (w) IgM lambda (l) MM w diffuse osteolyses in conventional and CT-radiographs. Via histopathology Waldenstrom´s macroglobulinaemia was excluded. The hemoglobin at initial diagnosis (ID) was 13 g/dl, l-SFLC 32.5 g/l, BM infiltration 15% w del13q14 and ISS I. Due to symptomatic MM, he was included in the DSMM XII trial which applies 4 cycles of RAD (Knop S, Blood 2009) followed by SCT. Neither the initial conventional lung-x-ray nor bone-CT-scans described thoracic abnormalities. After ASCT, the pt reported about haemoptysis; extensive diagnostics revealed central squamous cell NSCLC. Retrospectively, this NSCLC was already detectable w ID of the MM, suspecting that both neoplasms had occurred simultaneously. He underwent R0-resection, the tumor stage was pT2pN2cM0,L2,G2. Since biopsy of the osteolytic lesions was unfeasible, a bilateral BM-re-evaluation was done, that revealed no epithelial tumor cells. Re-staging diagnostics determined no metastases, albeit CTCs were detected by image cytometry and immunofluorescence. CTCs were enriched using CD326 (EpCAM)-microbeads and magnet-assisted cell sorting: w the former, 59 EpCAM+ cells were captured from 1.2e5 cells (0.05%), w the latter, 8 EpCAM+/CD45- cells from 800 cells detected in the EpCAM+ fraction (1%). These CTCs were substantially higher than in 5 other NSCLC pts (range 0-4). VGPR of the MM was obtained after the 1. ASCT, but due to the NSCLC, no 2. ASCT or maintenance were performed. Instead, the pt received mediastinal irradiation, but refrained from adjuvant CTx. 6 months after NSCLC-resection, he showed liver metastases. Despite palliative multi-agent CTx, he died of progressing NSCLC 5 months later and 20 months after ID of his NSCLC. Conclusions: CTCs in this pt preceded detection of aggressive NSCLC-metastases by conventional imaging and may prompt adjuvant CTx application in the future. Disclosure: No conflict of interest disclosed.

Abstracts

Posterdiskussion Sonstige Onkologie P834

Expression of GLI-1 in tumor surrounding stroma in patients with skin cancer Geissler C.1, Grossschmidt P.1, Feldmann R.1, Steiner A.1, Ulrich W.1, Geissler K.1,2 Krankenhaus Hietzing, Wien, Austria, 2Ludwig Boltzmann Cluster of Translational Oncology, Wien, Austria 1

Introduction: The Hedgehog (HH) signaling pathway is a cell program that is active during early development of multicellular organisms and is required for the formation of basic structures in the growing embryo. Activation of HH signaling by mutations of pathway components has been demonstrated in certain malignancies such as basal cell cancer, medulloblastoma and rhabdomyosarcoma (Geissler K and Zach O. Ann Hematol 2012). There is also evidence that aberrant HH signaling in the surrounding stromal cells may contribute to malignant growth in cancers of the pancreas, the prostate and the ovary. The role of HH signaling of stromal cells in skin tumors is unknown. Methods: GLI-1 expression of tumor surrounding stroma was investigated by immunohistochemical staining of paraffin-embedded tumor tissue from 48 patients with skin cancer (17 melanomas, 13 squamous cell carcinomas, 18 basal cell carcinomas). Staininig intensity was scored as no (0), faint (+1), moderate (+2) or strong (+3). Morphological, immunological, and clinical data were obtained from the hospitals database and patient records. Results: Immunohistochemical staining of normal skin showed faint expression of GLI-1 in all layers of the epidermis, as well as faint staining of sweat and sebaceous glands. Strong GLI-1 expression of stromal cells was found in 12/17 (71%) melanomas, 0/13 (0%) squamous cell carcinomas and 3/18 (17%) basal cell carcinomas. GLI-1 was abundantly present in the cytosol and partly in the nucleus of stromal cells showing macrophage morphology. Conclusions: Our results indicate that strong GLI-1 expressing stromal cells can be found in the majority of melanoma patients. Although the morphology of these cells is compatible with macrophages the exact nature of these cells and the functional consequence of this finding remains to be determined. Our findings however suggest that Hedgehog pathway inhibitors may be interesting not only to impact HH signaling in tumor cells of basaliomas but also to target the tumor stroma in melanomas. Disclosure: No conflict of interest disclosed. P835

Case report: palliative management of a V600E-positive, metastasized melanoma Diwoky S., Szibor-Kriesen U., Große-Thie C., Junghanß C. Zentrum für Innere Medizin, Universitätsklinikum Rostock, Klinik für Hämatologie, Onkologie und Palliativmedizin, Rostock, Germany

Background: The melanoma is a malignant neoplasm which saw a steady increase in its incidence figures over the last decades while the viable therapy options, especially for patients in a metastasized state, remained limited to few regimes with often poor response rates. This stagnation was overcome a few years ago with the implementation of new therapy concepts utilizing the new substances Ipilimumab and Vemurafenib. These developments had a great impact on melanoma treatment and have consequentially found their way into the recent guidelines. However, as the following case report illustrates, an effective treatment requires a compliant and adherent patient. Case Report: The Patient, a 53-year old female, was admitted to our palliative care unit several times from March 2012 until her death in April 2012. In the time between initial diagnosis of a melanoma already in metastatic spread in 2009 and the first admission to our department, the patient deliberately refrained from any form of treatment except for some herbal


255


P833


Proliferation and microvascular density predict survival in patients with brain metastases of non-small cell lung cancer Berghoff A.S.1, Ilhan-Mutlu A.2, Wöhrer A.1, Hackl M.3, Widhalm G.4, Hainfellner J.A.1, Dieckmann K.5, Zielinski C.C.2, Birner P.6, Preusser M.2 Institute of Neurology, Medical University of Vienna, Vienna, Austria, Department of Medicine I, Medical University of Vienna, Vienna, Austria, 3 Austrian National Cancer Registry, Vienna, Austria, 4Department of Neurosurgery, Medical University of Vienna, Vienna, Austria, 5Department of Radiotherapy, Medical University of Vienna, Vienna, Austria, 6Institute of Clinical Pathology, Medical University of Vienna, Vienna, Austria 1 2

Background: Non-small cell lung cancer (NSCLC) is the most frequent cause of brain metastases (BM). Survival upon diagnosis of BM is variable and established prognostic scores do not include tissue-based parameters. We studied the prognostic value of proliferation, angiogenesis and hypoxia in NSCLC BM. Methods: All patients treated with neuro-surgery as first line therapy for newly diagnosed NSCLC BM between 01/1990 and 02/2011, were identified from the Neuro-Biobank, Medical University of Vienna. We determined microvascular density (MVD), Ki67 tumor cell proliferation index and hypoxia-inducible factor 1 alpha (HIF1a) score using immunohistochemistry and standard protocols. Clinical and demographic data were retrieved by chart review. We performed statistical correlations including survival analyses using log-rank test and Cox-regression. Results: BM specimens of 210 patients were available for analysis (133 male (63.3%), 77 female (36.7%), median age at diagnosis of BM 56 (range 31–78). 38/210 (18.1%) patients had squamous and 172/210 (81.9%) non-squamous histology of the primary tumour. Median overall survival (OS) from first diagnosis of BM was 8.0 months (range 0.0–158.0). Diagnosis specific graded prognostic assessment (DS-GPA) groups showed statistically significant correlation with survival (class 1: 19 months; class 2: 8 months; class 3: 5 months; class 4: 1 month; p  2 (~Karnofsky-Index < 50). Only 10 (44%) of 23 patients who entered our geriatric department fulfilt the including criteria of the NHL-study for elderly (9 pts > 80 years, 4 pts ECOG=3, 4 pts NYHA III/IV, 4 pts with dementia). Summary: Even the specially for elderly patients (≠ geriatric pts) planned studies exclude frequently the main part of patients of geriatric departments. Disclosure: No conflict of interest disclosed. P860

Achieving interdisciplinary cooperation through a decentralized sequential virtual tumor board Gleißner J.1, Ilg J.2, Schaefer R.M.3 DGU-Die Gesundheitsunion, Urologische Praxis, Wuppertal, Germany, DOCXCELLENCE GmbH, Berlin, Germany, 3Urologische Praxis, BonnBad Godesberg, Germany 1 2

The oncologists' agreement requires joint patient-oriented case consultations taking place regularly i.e. in the course of tumor conferences for all

Abstracts


P857

tumor cases dealt with by the cooperation network. For patients receiving interdisciplinary treatment, case consultations have to be taken up prior to primary and recurrent therapy or changes in the treatment regime, respectively. This demands time and effort from participating urologists as well as clinicians. In order to reduce their harmonization load, we introduce the sequential virtual case consultation in the UroCloud model: For any new case set up for discussion in the UroCloud, a mail notification asking for proposals, comments and amendments regarding the hermeneutic approach is sent out to all participating representatives from the different disciplines. On the basis of these inputs, indications and treatment decisions are elaborated and again published online for feedback. Once published on the virtual platform, case data can be accessed and processed via the internet at any time and therefore highly resource efficiently. To summarize, the UroCloud provides a • cross-sectoral • up-to-date • structural tool allowing for • source control and, via evaluating participation, may contribute to • quality management. We will be giving account of our experiences. Disclosure: No conflict of interest disclosed.

ticed pointing to an association of the CXCR4 expression with hypoxia and tumor proliferation. With respect to normal tissue samples, lymphatic organs were strongly CXCR4 positive, too. All antagonists investigated prevented the binding of the natural ligand SDF-1 (stromal derived factor 1; CXCL12) to the CXCR4. Here, the results showed that even small structural changes lead to significant differences in receptor affinity. Conclusion: UMB-2 may prove to be of great value in the assessment of CXCR4 expression in different human tumor entities and of the mechanisms underlying cancer progression. It may also help to find new strategies in cancer therapy. Disclosure: No conflict of interest disclosed. P862

Opioid receptor activation triggering downregulation of cAMP sensitizes leukemia cells for doxorubicin treatment in vitro and in vivo Friesen C.1,2, Roscher M.1,2, Fichtner I.3, Alt A.2, Hilger R.A.4, Debatin K.-M.5, Miltner E.1,2 Zentrum für Biomedizinische Forschung, Universität, Ulm, Germany, Institut für Rechtsmedizin, Universität, Ulm, Germany, 3Max-Delbrück- Zentrum für Molekulare Medizin, Berlin, Germany, 4West Deutsches Krebszentrum, Universität Essen, Innere Medizin, Essen, Germany, 5 Klinik für Kinder- und Jugendmedizin, Universität, Ulm, Germany 1 2

Tumorbiologie P861

Over-expression of the chemokine receptor CXCR4 in neoplastic tissues: A new target in cancer diagnostics and therapy Reimann C.1, Schulz U.1, Lupp A.1, Baum R.P.2, Wester H.-J.3, Schulz S.1 Universitätsklinikum Jena, Institut für Pharmakologie und Toxikologie, Jena, Germany, 2Zentralklinik Bad Berka, Klinik für Molekulare Radiotherapie, Bad Berka, Germany, 3Technische Universität München (TUM): Klinikum rechts der Isar, Nuklearmedizinische Klinik und Poliklinik, München, Germany 1

Introduction: The CXCR4 is a plasma membrane chemokine receptor which is involved e.g. in organogenesis, hematopoiesis and inflammation. Concerning neoplastic tissues, in the literature an over-expression of CXCR4 is described for many tumors, with a frequency of about 70%. Additionally, an involvement of the CXCR4 in tumor proliferation and metastasis as well as a relation to hypoxia and cancer stem cells is discussed. However, in the current literature, most immunohistochemical attempts to localize the receptor have yielded mainly a nuclear and cytoplasmic staining. Therefore, one aim of the present study was the reevaluation of the CXCR4 expression in a panel of different tumor entities using the novel monoclonal rabbit anti-CXCR4 antibody UMB-2, yielding a predominance of plasma membrane staining. Additionally, the endogenous expression of the receptor in different cancer cell lines was investigated. Furthermore, several cyclic pentapeptide CXCR4 antagonists were tested using HEK-293 cells stably transfected with the human CXCR4. Methods: The CXCR4 expression was assessed by means of immunohistochemistry in a panel of formalin-fixed and paraffin-embedded human normal and neoplastic tissue samples. In parallel, in serial sections, with antibodies against HIF-1α and Ki67 the hypoxic conditions as well as the proliferation within the tumor were investigated. The endogenous receptor expression in different cell lines as well as the effects of the different antagonists were evaluated using immunocytochemistry or Western blot analyses. Results: The CXCR4 was found to be expressed in the majority of the 20 tumor entities investigated, especially in small cell lung cancer, bladder cancer and ovarian carcinoma. Often a specific staining pattern was no-

Abstracts

Introduction: Opioid receptor stimulation activates inhibitory Gi-proteins which in turn block adenylyl cyclase activity reducing cyclic AMP (cAMP). cAMP regulates a number of cellular processes and can modulate cell death induction. The opioid D,L-methadone induces apoptotic cell death and breaks chemo- and radioresistance in leukemia cells. However, the mechanism how opioids trigger apoptosis and activate caspases is not understood. In this study, we analyzed the role of the opioid receptor signalling pathway in killing and sensitizing leukemia cells for doxorubicin treatment in vitro and in vivo. Methods: ALL cell lines and xenograft-derived ALL cells were treated with therapeutic concentrations of D,L-methadone alone or in addition to therapeutic concentrations of doxorubicin. Cell death and apoptosis was measured by flow cytometry and activation of apoptosis pathways was detected by Western Blot analyses. Opioid receptor expression was analyzed using flow cytometry. In vivo, patient-derived ALL-cells were transplanted in NSG-mice. At different time points after D,L-methadone (methaddict) and/or doxorubicin treatment tumor volume of xenografted tumors and body weight of mice were measured. Results: We found that opioid receptor activation using the opioid D,L-methadone induces apoptosis, activates caspases and sensitizes leukemia cells for doxorubicin treatment via downregulation of cAMP. Blocking opioid-receptor signaling, enhancing cAMP levels, strongly reduced D,L-methadone-induced apoptosis, caspase activation and doxorubicin-sensitivity. In addition, cell death induction in leukemia cells by activation of opioid receptors using D,L-methadone depends on critical levels of opioid receptor expression on the cell surface. Doxorubicin increased opioid receptor expression and the opioid D,L-methadone increased doxorubicin uptake and decreased doxorubicin efflux in leukemia cells, suggesting that D,L-methadone as well as doxorubicin mutually increase their cytotoxic potential. Furthermore, we found that opioid receptor activation using D,L-methadone alone or in addition to doxorubicin inhibits tumor growth significantly in vivo. Conclusions: Our results demonstrate that opioid receptor activation via triggering downregulation of cAMP induces apoptosis, activates caspases and sensitizes leukemia cells for doxorubicin treatment in vitro and in vivo. Opioid receptor activation seems to be a promising strategy to improve current anticancer therapies. Disclosure: No conflict of interest disclosed.


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Posterdiskussion

Capture of tissue-factor-bearing microparticles with using annexin-V bound magnetic beads Gamperl H., Theophil F., Stenzel I., Quecke T., Ungefroren H., Gieseler F. Medizinische Klinik I, Experimentelle Onkologie, Lübeck, Germany

A number of recent articles indicate that MPs are important regulators of cellular interactions under physiological and pathological conditions. They have been found in all body fluids and seem to play a regulative role in blood clotting, they enhance cell adhesiveness and increase cell aggregation. In addition, they mediate cell-to-cell communication by transferring cell surface receptors, mRNA, and miRNA from the cell of origin to target cells. MPs have also been found to be active in several diseases such as inflammation, sepsis and cancer where they may contribute to metastasis. They can be released by epithelial cells, thrombocytes and cancer cells and are composed of vesicles formed by double layer membrane and characteristically express tissue factor at the surface. Once exposed, the tissue factor instantly associates with FVII. This TF/FVIIa-complex is as well capable to initiate the extrinsic pathway of coagulation, as to activate PARs by its protease activity. There is an ongoing discussion if the composition of the emerging MP-complexes is decisive for their specific way of action. Thus both the quantitative detection and the possibility of investigating the composition of the microparticles are of high scientific interest. Here, we present a fast and specific method to isolate microparticles from body fluids, such as malignant effusion and plasma, using a sequence of centrifugation and capture by annexin-V magnetic MicroBeads. We proved the specificity and sensitivity by detecting the MPs in a modern flow-cytometer as defined by their size and the expression of tissue factor before and after annexin-V MicroBead capture. In addition, we show that the captured MPs are still functionally active by using a commercially available MP-activity assay. The captures MPs can then be used for further characterization. Disclosure: No conflict of interest disclosed. P864

Antitumor potential of the endocannabinoid reuptake inhibitor OMDM-2 Ammar R.M.1, Ulrich-Merzenich G.1, El-Azab M.2, Moustafa Y.2 Medical Clinic III, UKB of the Rheinische Friedrich-Wilhelms-University of Bonn, Bonn, Germany, 2Faculty of Pharmacy, Suez Canal University, Ismailia, Egypt 1

Introduction: Endocannabinoids are endogenous compounds known to mediate psychotropic effects by stimulating the cannabinoid receptor type1 (CB-1). Endocannabinoid signalling has been shown to be enhanced in several cancer tissues and malignant cells to inhibit their proliferation. Here, we investigated the effect of the endocannabinoid reuptake inhibitor, OMDM-2, on the growth of Ehrlich solid tumor induced in mice. Methods: Three different sets of experiments were carried out: (I) Mice (n = 70) treated either with (1) OMDM-2 (5 mg/kg, i.p.), (2) R-Methanandamide (R-Met) (0.5 mg/kg, i.p.), a direct cannaboid agonist, (3) NIDA 41,020 (0.7 mg/kg, i.p.), CB-1 receptor blocker, (4) R-Met + NIDA 41020, (5) OMDM-2 + NIDA 41020, or (6) Carboplatin (5 mg/kg, i.p.) were evaluated for tumor volume, mean survival time and increase in the life span (%). (II) All groups (n = 70) were assessed for the angiostatic activity using Evan´s blue method (% of control). (III) Time course measurements of tumor weights, serum transforming growth factor-β1 (TGF-β1), and the intra-tumoral receptor (CD-105) were observed on days 7, 14, and 21 post-inoculation (n = 147, 21/ group). The hematological parameters were investigated on day 14. Results: Both, OMDM-2 and R-Met significantly impeded tumor weights and volumes (p 10 × 107 (n = 17; Group C), in 3 patients the cell dose was not available. Twenty patients received escalating doses of CD3+. Results: Twenty-six (37%) patients are alive after DLI with a median OS of 16 (range, 0.6–210) mos. OS at 2 yrs for Group A, B and C were 52, 56 and 33%. Initial CD3+ cell dose >10 × 107/kg was significantly associated with an increased risk of overall mortality (p = 0.038). Forty-five (63%) patients responded to DLI, 21 of them relapsed after initial response, and 20 did not respond. Three patients showed stable disease, in 2 response evaluation is pending. Higher CD3+ doses did not result in lower relapse rates or better response. Sixteen (23%) patients developed acute or chronic GVHD after DLI. The risk for GVHD was significantly higher in patients receiving higher doses of CD3+ (Group B and C) compared to lower doses (Group A; p = 0.033). Five patients died due to severe GVHD.

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GvHD vs. GvL: Epitope specific modulation of CD4+ T-cells by anti-human CD4 antibodies simultaneously facilitates the GvL effect and the long-term suppression of GvHD Hilger N.1,2, Schmidt F.1, Svanidze E.1,2, Emmrich F.1,2,3, Fricke S.1,3,4 Fraunhofer Institute for Cell Therapy and Immunology, Immunology, Leipzig, Germany, 2Institute for Clinical Immunology, University of Leipzig, Leipzig, Germany, 3Translational Centre for Regenerative Medicine, University of Leipzig, Leipzig, Germany, 4Department of Hematology and Oncology, University of Leipzig, Leipzig, Germany 1

Introduction: The major complication after hematopoietic stem cell transplantation (HSCT) is Graft-versus-Host-Disease (GvHD). Common immunosuppressive therapies lead to a general immunosuppression and could increase the risk for tumor relapses and infectious diseases. The influence of CD4+ T-cells responsible for development of acute GvHD and for Graft-versus-Leukemia effect (GvL) can be modulated by administration of anti-human CD4 antibodies. These new therapeutic strategies should not impair the GvL effect. Methods: A full MHC class mismatch transplantation model was developed by using transgeneic C57Bl/6 mice (huCD4+, muCD4-, HLADR3+, triple transgeneic mice, TTG) as donors and Balb/c mice as recipients. Furthermore, murine P815-Balb/c leukemia mice were developed by co-transplantation of P815 leukemic cells to study the GvL effect. Survival, GvHD score, leukocyte subset recovery and chimerism were analyzed for 60 days. Distribution of donor TTG cells in recipient mice was confirmed by flow cytometry and histology. Results: The survival rate of recipients receiving an anti-human CD4 antibody incubated graft of 2 × 107 BM + 2 × 107 splenocytes with co-transplantation of 5 × 103 P815 cells was significantly higher (70%) compared to untreated control mice (0%, P = 0.001, n = 10). Co-transplantation of 2.5 × 103 P815 cells with antibody treatment showed also a higher survival rate of 80% whereas co-transplantation of 1 × 106 P815 cells lead to death of all recipients within 10 days after transplantation. Stable engraftment of TTG donor cells (H-2Kd, huCD4, HLA-DR) and a decrease of murine CD4 after transplantation indicated a full TTG-donor hematopoiesis. Furthermore, flow cytometric analyses showed P815GFP cells in liver (day 4), bone marrow and spleen (day 6) after transplantation of 1 × 106 P815GFP in sublethally irradiated mice (3Gy). 5 × 103 P815GFP cells could not be detected within 6 days after application in 3Gy or non irradiated control mice. However, 5 × 103 P815GFP cells transplanted into lethally irradiated mice (8Gy) could be detected in liver 14 days after transplantation by flow cytometry. Additionally, liver and spleen of recipient mice showed an infiltration of P815 cells with tissue destruction. Conclusion: We could show that an epitope-specific ex vivo modulation of an allogeneic hematopoietic stem cell graft by anti-human CD4 antibodies simultaneously facilitates the GvL effect of the graft and the long-term suppression of the GvHD in this model. Disclosure: No conflict of interest disclosed. V931

HLA-DP mismatch-stimulated alloreactive CD4+ T cells eliminate leukemia blasts in NSG mice Beshay J., Bloetz A., Eichinger Y., Frey M., Hartwig U., Herr W., Thomas S.

Conclusion: Our results show that an initial CD3+ cell dose/kg >10 × 107 is associated with increased risks of mortality after DLI, without reducing

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In allogeneic hematopoietic stem-cell transplantation (HSCT) donor-derived T cells mediate beneficial graft-versus-leukemia (GvL) effects but also graft-versus-host disease (GvHD). To reduce the risk of GvHD, recip-

Abstracts


Universitätsmedizin Mainz, III. Medizinische Klinik und Poliklinik, Hämatologie, internistische Onkologie, Pneumologie, Mainz, Germany

Fig. 1.


Mesenchymal stem cells can modulate the function of CMV-specific CD8+ T cells Jin N., Malcherek G., Wuchter P., Diehlmann A., Schmitt A., Ho A.D., Schmitt M. Universitätsklinikum Heidelberg, Medizinische Klinik V, Heidelberg, Germany

Background: Mesenchymal stem cells (MSCs) have a potential to differentiate into different cell types such as adipocytes, chondrocytes and muscle cells. Moreover MSCs constitute key players of the immune system. Therefore MSCs have been considered for cellular therapies for patients with grafts-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (HSCT). As reactivation of cytomegalovirus (CMV) constitutes a frequent problem after HSCT, we therefore studied how MSCs affect T-cell responses specific to CMV. Methods and Results: Bone-marrow derived MSCs from two different media (human platelet lysate (PL) and fetal calf serum (FCS)) were examined by co-culture with purified subpopulations of immune cells. We first investigated the effects of MSCs on Staphylococcal Enterotoxin B (SEB)- and alloantigen-induced lymphocyte proliferation as well as IFN-gamma secretion by flow cytometry and enzyme-linked immunospot (ELISPOT) assays. We demonstrated that PL-cultured MSCs have similar immunomodulatory capacities when compared to their FCS-cultured counterparts. The immunomodulatory effects of MSCs on CMV-specific CD8+ T cells were studied in mixed lymphocyte-peptide culture (MLPC). Briefly, CMV-specific T cells were expanded by stimulating MACS®-purified CD8+ T cells from buffy coats of HLA-A2/CMV seropositive healthy volunteers with HLA-A*0201-restricted CMVpp65 peptide. MSCs were co-cultured with MLPC for 7 days. Interestingly, when PL- and FCS-cultured MSCs were co-cultured, the proliferation of

Abstracts

CMV-specific CD8+ T cells was inhibited, as well as the frequencies of CD8+CMVtetramer+ T cells and CD137+CMVtetramer+ T cells were reduced. Moreover in ELISPOT assays, MSCs suppressed the number of CMV-specific IFN-gamma- and granzyme B-producing cells. Conclusion: Transfusion of MSCs might constitute a promising therapeutic option for patients with steroid-refractory GVHD. In this work, the immunosuppressive potential in terms of inhibition of proliferation, immunophenotype and function of T cells was paradigmatically demonstrated for antigen-specific CD8+ T cells. Disclosure: No conflict of interest disclosed. V933

Adoptive transfer of T-cell receptor engineered human T cells specifically reduces viral titers in HLA-transgenic NSG mice infected with a humanized cytomegalovirus Klobuch S.1, Lemmermann N.2, Podlech J.2, Theobald M.1, Reddehase M.J.2, Herr W.1, Thomas S.1 Universitätsmedizin der Johannes Gutenberg-Universität Mainz, III. Med. Klinik und Poliklinik, Mainz, Germany, 2Universitätsmedizin der Johannes Gutenberg-Universität Mainz, Institut für Virologie, Mainz, Germany 1

Reactivation of latent human cytomegalovirus (hCMV) infection is a frequent complication in patients after allogeneic hematopoietic stem cell transplantation (HSCT). Preclinical research in murine models as well as clinical phase I/II trials have shown that the adoptive transfer of virus-specific CD8+ T cells is a therapeutic option for preventing CMV disease. However, the feasibility of CMV-specific immunotherapy is currently limited in clinical routine due to technical restrictions. It has also limitations, if the donor is CMV-seronegative or carries only low numbers of CMV-specific memory T cells. In this situation, grafting non-reactive T cells by virus-antigen specific T-cell receptors (TCR) may be an alternative means to transfer CMV-specific T-cell function into HSCT recipients. Nevertheless, improvement of clinical protocols is needed before CMV-specific cell therapy can be implemented in general clinical practice. Here we describe a novel preclinical mouse model that will help to evaluate new hCMV-immunotherapy options. Due to the strict species specificity of CMV, we constructed a recombinant murine CMV (mCMV) coding for the HLA-A2 restricted pp65495-503 (NLV) peptide epitope of hCMV. This peptide is expressed with flanking amino acids within the mCMV-IE2-protein in the context of experimental infection with mCMV-IE2/NLV. The NLV peptide was found to be presented by infected murine fibroblasts from NOD/SCID/IL-2Rγc-/--HLA-A2-transgenic (NSG-A2) mice as shown by T cell specific recognition in ELISPOT and cytotoxicity assays. Interestingly, this presentation proved to be susceptible to the inhibitory function of mCMV immune evasion proteins. NSG-A2 mice were then used to evaluate antiviral control by hCMV-NLV-specific TCR (TCRNLV)-engineered human T cells. After infection with mCMV-IE2-NLV, the NSG-A2 mice showed high viral titers in liver, spleen, lungs, and salivary glands as well as a dramatic virus-induced morbidity and mortality. In contrast, after intravenous injection of TCRNLV-transduced human CD4+/CD8+ T cells, viral infection was reduced in all tested organs, and mice showed improved survival. These findings suggest that the NLV peptide is processed and presented by stromal and parenchymal cells of the NSG-A2 mice and promise validity of the model for preclinical evaluation of adoptive transfer protocols, for instance cell therapy with TCR-engineered T cells alone or in combination with therapeutic vaccination. Disclosure: No conflict of interest disclosed.


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ient and donor are usually matched for HLA-A/B/C as well as DRB1 and DQB1. The HLA class II locus DPB1 is still not taken into account for donor selection and clinical data have suggested that distinct HLA-DPB1 mismatches are permissive and do not adversely influence the results of HSCT. In recent studies the potential of CD4+ T cells as GvL effectors came more and more into focus. Because CD4+ T cells recognize peptides presented by HLA-class-II molecules that are not expressed on non-hematopoietic cells under non-inflammatory conditions, CD4+ T cells might have a more favorable safety profile than CD8+ T cells. We have developed a reliable protocol for the in vitro generation of HLA-DP mismatch-stimulated, AML-reactive CD4+ T cells that could be used for adoptive transfer experiments in AML-engrafted NOD/SCID/2Rgcnull (NSG) mice. Pure naive CD4+ T cells (CD45RA+) were stimulated against single allogeneic HLA-DP alleles, which were previously transfected into autologous dendritic cells by mRNA electroporation. After expansion, HLA-DP reactive CD4+ T cells showed specific IFNg, IL-4 and TNFa secretion as well as cytolysis upon incubation with AML blasts or patient-derived EBV-B lines carrying the mismatched HLA-DP alleles which have been used for T-cell stimulation. To analyze CD4+ T cells in vivo, we engrafted NSG mice with primary AML blasts up to 1–3% infiltration in bone marrow and adoptively transferred 1 × 107 HLA-DP reactive CD4+ T cells together with human Fc-IL-7 and IL-2. One and four weeks later, AML-engrafted NSG mice treated with HLA-DP mismatch-reactive CD4+ T cells showed complete regression of AML blasts in bone marrow, spleen and peripheral blood, not seen with irrelevant control T cells. Homing and persistence of T cells was confirmed as they could still be detected in these organs one and four weeks after transfer. HLA-DP reactive CD4+ T cells can be reliably generated from naive precursors by in vitro stimulation with HLA-DP allele-transfected dendritic cells. As these T cells are capable of strongly reducing leukemia burden in AML-engrafted NSG mice, we suggest to further investigate CD4+ T cells as potent effectors in adoptive immunotherapy of leukemias.

V934

Early B cell reconstitution after allogeneic hematopoietic stem cell transplantation is associated with a distinctive innate-like immune response Mensen A.1, Demski S.1, Ochs C.1, Hemmati P.2, Vuong G.L.2, Penack O.2, Westermann J.2, Dörken B.2, Scheibenbogen C.1, Arnold R.2, Na I.-K.2,3 Institute for Medical Immunology, Charité CVK, Berlin, Germany, 2Depart ment of Hematology, Oncology and Tumorimmunology, Charité CVK, Berlin, Germany, 3Experimental and Clinical Research Center (ECRC), Berlin, Germany 1

B cell dysfunction following delayed immune reconstitution substantially contributes to an increased risk for life-threatening infections after allogeneic hematopoietic stem cell transplantation (alloHSCT). In this study, we phenotypically and functionally investigated B cell subset reconstitution kinetics and kappa-deleting recombination excision circles (KREC), which are non-replicative episomal plasmids generated during bone marrow B cell development, in adult patients diagnosed with acute leukemia after alloHSCT. Additionally, specific B cell antibody responses were analysed by Elispot assay after ex vivo stimulation including CpG. Early onset of bone marrow neogenesis was seen in 52% of patients, characterized by a strong increase of transitional B cells between day 60 and 90 post alloHSCT. KREC quantification by RT-PCR revealed a highly positive and significant correlation between absolute KREC copy numbers and transitional B cells (Spearman: r = 1.00, p = 0.003) indicating ongoing B cell neogenesis in patients with an early recovering B cell reconstitution. Within the next months, patients with early B cell reconstitution also exhibited significantly stronger increases of naïve and IgM memory B cells compared to delayed B cell reconstituting patients, whereas switched memory B cells were lacking in all patients. Interestingly, early reconstituting B cells were paralleled by higher levels of IgM producing B cells against capsular pneumococcal polysaccharide after ex vivo stimulation at day 180 post alloHSCT, whereas polyclonal IgG and Tetanus-specific IgG producing B cells were significantly diminished in all patients. From our data we conclude, that early onset of B cell reconstitution in alloHSCT patients is characterized by an increase of newly regenerated transitional B cells within three months after transplantation. Hereby, KREC quantification is a suitable marker for monitoring of B cell-bone marrow output. Furthermore, IgM B cell responses against Streptococcus pneumonia resulted most likely from the significantly higher levels of reconstituted IgM memory B cells, since naïve B cells were shown not to generate antibody-secreting cells upon CpG stimulation (Capolunghi F et al. 2008) and switched memory B cells were lacking. These specific IgM-producing B cells, distinctive in early B cell-reconstituting patients, represent an innate-like immune response after alloHSCT at a time when conventional adaptive immune response is still developing. Disclosure: No conflict of interest disclosed.

Freier Vortrag

Neoplastische Stammzellen V935

Campath-1 (CD52): A novel target in Neoplastic Stem Cells (NSC) in 5q- patients with MDS or AML Blatt K.1, Herrmann H.1,2, Willmann M.3, Cerny-Reiterer S.1,2, Sadovnik I.1, Herndlhofer S.1, Streubel B.4, Rabitsch W.5, Sperr W.1,2, Rülicke T.6, Valent P.1,2 Medical University of Vienna, Department of Internal Medicine I, Vienna, Austria, 2Ludwig Boltzmann Cluster Oncology, Medical University of Vienna, Vienna, Austria, 3University of Veterinary Medicine Vienna, Department for Companion Animals and Horses, Clinic for Internal Medicine and Infectious Diseases, Vienna, Austria, 4Medical University of Vienna, Department of Obstetrics and Gynecology, Vienna, Austria, 5Medical University of Vienna, Department of Internal Medicine I, Bone Marrow Transplantation Unit, Vienna, Austria, 6University of Veterinary Medicine Vienna, Institute of Laboratory Animal Science, Vienna, Austria 1

Introduction: The humanized anti-CD52 antibody alemtuzumab (MabCampath) induces major clinical responses in a group of patients with myelodysplastic syndromes (MDS). So far, the mechanism underlying this drug effect remains unknown. In the present study, we asked whether CD34+/CD38− neoplastic stem cells (NSC) express CD52 in patients with MDS or acute myeloid leukemia (AML). Methods: A total of 91 patients, including 29 with MDS (males: n = 15; females: n = 14; median age: 70 years) and 62 with AML (males: n = 27; females: n = 35; median age: 62 years) were examined. In a subset of patients, qPCR and FISH analysis were performed on highly purified NSC. We also examined the effects of alemtuzumab on survival of NSC and CD34+/CD38+ progenitor cells in patients with MDS and AML. Normal bone marrow (BM) cells served as control. In a subset of patients with AML, engraftment of NSC in NOD-SCID-IL-2Rgamma-/- (NSG) mice was determined after preincubation of cells with control medium or alemtuzumab (500 µg/ml for 1 hour). Results: CD52 was expressed on NSC in 7/10 patients with MDS and isolated 5q-. In most other patients with MDS, CD52 was weakly expressed or not detectable on NSC. In AML, NSC displayed CD52 in 23/62 patients, namely 4 with complex karyotype including 5q-, one with 5qand t(1;17;X), 2 with inv(3), 2 with t(8;21), one with inv(16), one with isolated del(13q), 3 with trisomy 8, one with monosomy 7, and 8 with normal karyotype. In highly enriched NSC of AML and MDS patients, qPCR confirmed expression of CD52 mRNA, and clonality of NSC was confirmed by FISH. Consecutive studies revealed that CD52-expression correlates with EVI1 mRNA-expression-levels in NSC, but not with CD300a mRNA expression. The CD52 antibody alemtuzumab was found to induce complement-dependent lysis of CD34+/CD38−/CD52+ NSC in all patients examined, but did not induce lysis in CD52− normal stem cells. Finally, alemtuzumab inhibited AML-formation in NSG mice in 3/3 patients tested (66%; 43%; 46%). Conclusions: Our data show that the target-antigen CD52 is expressed abundantly in NSC/LSC in a group of patients with MDS and AML, including 5q- patients. These observations may have clinical implications and may explain clinical effects seen with alemtuzumab in these patients.

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Evaluation of the IL-2R alpha chain (CD25) as novel marker of leukemic stem cells in Ph+ CML Sadovnik I.1, Herrmann H.1,2, Cerny-Reiterer S.1,2, Warsch W.3, Blatt K.1, Hoelbl A.3, Peter B.1,2, Stefanzl G.1, Hoermann G.4, Herndlhofer S.1, Streubel B.5, Sperr W.R.1, Mannhalter C.4, Sexl V.3, Valent P.1,2 Department of Medicine I, Medical University of Vienna, Vienna, Austria, Ludwig Boltzmann Cluster Oncology, Vienna, Austria, 3University of Veterinary Medicine, Vienna, Austria, 4Department of Laboratory Medicine, Medical University of Vienna, Vienna, Austria, 5Institute of Gynecology & Obstetrics, Medical University of Vienna, Vienna, Austria 1 2

Chronic myeloid leukemia is a stem cell neoplasm characterized by the Philadelphia (Ph) chromosome and the related oncoprotein, BCR/ABL. Despite the availability of novel BCR/ABL tyrosine kinase inhibitors (TKI) many patients relapse, which may be due to resistance of leukemic stem cells (LSC) against TKI. So far, little is known about specific markers and targets expressed on LSC in CML. We found that CD34+/Lin-/CD38- stem cells in CML express the IL-2 receptor alpha chain (CD25) in 31/33 patients examined (95%). CML LSC were also found to express IL-1RAP, DPPIV (CD26), Siglec-3 (CD33), KIT (CD117) and the IL-3RA (CD123). Highly purified CD34+/CD38-/ CD25+/CD26+ LSC were found to express BCR/ABL by qPCR and FISH. These LSC were also found to engraft irradiated NOD-SCID-IL-2Rg-/-(NSG) mice with BCR/ABL+ granulomonocytic cells, whereas CD34+/CD38-/ CD26- cells from the same patient failed to express BCR/ABL and engrafted NSG mice with normal BCR/ABL-negative multilineage hematopoiesis. To define underlying signalling cascades contributing to the enhanced CD25 expression on CML LSC we used primary murine hematopoietic cells derived from wild type mice that were co-infected with a BCR/ABL p210 retrovirus in combination with a retrovirus encoding for either, STAT5A or STAT5B. Cell were kept in medium supplemented with IL-3, IL-6 and SCF. The enforced expression of both STAT5 isoforms in this model significantly enhanced CD25 expression establishing a clear correlation between STAT5 activity and CD25 expression in CML cells. However, apparently, STAT5-signalling in CML LSC is regulated by both, BCR/ABL as well as cytokines present in the microenvironment. Correspondingly, BCR/ABL alone was unable to induce a significant expression of CD25 in leukemic cells in mice. Moreover, imatinib did not inhibit expression of CD25 in human CML LSC. However, the more potent BCR/ABL blocker nilotinib and the multikinase inhibitor ponatinib were found to suppress expression of CD25 in CML LSC. Both drugs were also found to inhibit expression of pSTAT5 and CD25 in the CML cell line KU812. In summary our data show that CD25 is a novel robust marker of CML LSC and may be useful for detection and isolation of LSC in BCR/ABL+ CML.

based on surface antigen expression. In recent years, gene expression profiles of minuscule numbers of different hematopoietic stem and progenitor cell fractions have been analyzed, yielding profound insights into hematopoiesis and its diseases. These studies revealed that reducing confounding cell populations is crucial to obtain signal-to-noise ratios that permit reliable conclusions. DNA cytosine methylation is a critical gene regulatory feature. Targeted disruption of methyltransferases results in impaired hematopoiesis, and epigenetic modifiers are frequently mutated in hematologic malignancies. Technical limitations restricted analysis of methylation to pooled human peripheral blood. The analysis of methylation in earliest human HSC from healthy individuals, and changes during their stepwise in vivo differentiation have not yet been reported. Methods: We combined prospective high-speed multiparameter FACS with isoschizomer-based profiling of methylation. The nano-HELP assay, a refined version of the HELP assay (Figueroa et al. Cancer Cell 2010a, 2010b), has been designed to study genomic DNA from low cell numbers (1000–30,000 cells). We profiled methylation and mRNA expression genome-wide from sorted human long-term hematopoietic stem cells, short-term hematopoietic stem cells, common myeloid progenitors and myeloerythroid progenitors from single healthy human individuals, i.e. without pooling the analytes prior to analysis. Results: Contrasting the common perception that somatic stem cells are, with the exception of erythroid progenitors (Shearstone et al. Science 2011), stably methylated we demonstrate distinct dynamic changes in methylation during in vivo differentiation of human HSC. Methylation changes at developmentally critical transitions include loci associated with genes that have been implicated in hematopoiesis and leukemogenesis. A stem cell differentiation-associated methylation signature correlates with clinical features of AML patients. Conclusion: Profiling of methylation from little analytes is feasible with the nano-HELP assay. We here delineate for the first time DNA cytosine methylation profiles of precisely defined human long-term HSC and their progeny, and implicate methylation changes occuring during normal HSC differentiation with the pathogenesis of AML. MC/BB, AV/US contributed equally. Disclosure: No conflict of interest disclosed. V938

Nme2 protein over-expression contributes to Bcr/Abl dependent transformation Tschiedel S., Bach E., Jilo A., Cross M., Niederwieser D. Universität, Leipzig, Germany

Introduction: Human hematopoietic stem and progenitor cells can be prospectively isolated using rigorous lineage depletion/ enrichment,

Introduction: We have previously identified Nme2 to be a tumor antigen in CML. Marked Nme2 protein over-expression is a universal and specific feature of Bcr/Abl+ cells in CP CML at diagnosis, being absent from Bcr/Abl- lymphoid cells and PBMC from normal donors or AML patients. Nme2 up-regulation in Bcr/Abl+ cells is post-transcriptional and can be reversed by TKI treatment. In this analysis we investigated in more detail the connection between Nme2 protein over-expression and Bcr/Abl, and the potential involvement of Nme2 in Bcr/Abl-mediated transformation. Methods: Ba/F3 cells stably transfected with TKI-sensitive or resistant forms of Bcr/Abl were cultured in RPMI medium plus 10% FCS in the presence or absence of 1% WEHI conditioned medium as a source of IL-3. RNA interference (RNAi) for Nme2 was performed using a CMV promotor-based vector (pcDNA 6.2 + EmGFP, Invitrogen) to express an engineered Nme2 miRNA. Cells (4 × 106) were transiently transfected with 10µg of Nme2 miR or miR-control plasmid by electroporation. Productively transfected cells were enriched by FACS sorting (GFP) 24h later. Interleukin-3 conditioned medium was added 24h before transfection where indicated. Sorted cells were analyzed by 3H proliferation assay and by western blotting of whole cell extracts. Messenger RNA levels of nme-2 were determined by qRT-PCR and were normalized to rplp0. Where indicated, Bcr/Abl BA/F3 cells were cultured with 0–10 µM imatinib or nilotinib and Bcr/Abl activity was determined by western blotting for pSTAT5 and pCrkL. Results: The expression of either non-mutated or TKI resistant mutants of Bcr/abl in Ba/F3 cells induces Nme2 protein over-expression and overrides

Abstracts



DNA cytosine methylation during in vivo commitment of human hematopoietic stem cells from healthy individuals, and implications for acute myeloid leukemia Christopeit M.1, Bartholdy B.1, Will B.1, Mo Y.2, Barreyro L.1, Yu Y.2, Bhagat T.D.2, Okoye-Okafor U.-C.1, Todorova T.I.1, Greally J.M.3, Levine R.4, Melnick A.5, Verma A.2,6,7, Steidl U.1,6,7 Albert Einstein College of Medicine, Department of Cell Biology, Bronx, United States, 2Albert Einstein College of Medicine, Department of Molecular and Developmental Biology, Bronx, United States, 3Albert Einstein College of Medicine, Department of Genetics, Bronx, United States, 4Memorial-Sloan Kettering Cancer Center, Human Oncology and Pathogenesis Program and Leukemia Service, Department of Medicine, Manhattan, United States, 5Weill Cornell Medical College, Division of Hematology/ Oncology, Department of Medicine, Manhattan, United States, 6Albert Einstein College of Medicine, Department of Medicine (Oncology), Bronx, United States, 7Albert Einstein College of Medicine, Albert Einstein Cancer Center, Bronx, United States 1

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the IL-3 dependence of these cells. Treatment with the TKI inhibitors imatinib or nilotinib reverses Nme2 over-expression and restores IL-3 dependence to Ba/F3 cells carrying sensitive, but not those carrying resistant forms of Bcr/Abl. Transient expression of an engineered Nme2 miR reduced the level of Nme2 mRNA and protein 24 hr post-transfection in productively transfected cells. This down regulation of Nme2 was associated with a significant decrease in proliferation, which could be rescued by the addition of IL-3. Conclusion: The Bcr/Abl-induced growth factor independence of Ba/ F3 cells requires the post-transcriptional induction of Nme2, suggesting a functional involvement of the tumor antigen Nme2 in Bcr/Abl driven leukemogenesis. Disclosure: No conflict of interest disclosed. V939

Expression of phosphorylated STAT5 in the putative stem cell population of JAK2 V617F transformed myeloprolife rative neoplasms Hadzijusufovic E.1,2,3, Keller A.1, Schur F.3, Cerny-Reiterer S.1,3, Sadovnik I.3, Pecnard E.4, Gouilleux F.4, Valent P.1,3 Ludwig Boltzmann Cluster Oncology, Vienna, Austria, 2University of Veterinary Medicine Vienna, Vienna, Austria, 3Medical University of Vienna, Vienna, Austria, 4Université François Rabelais, Tours, France 1

Recent data suggest that the signal transducer and activator of transcription 5 (STAT5) is a major regulator of growth and survival of neoplastic cells in diverse myeloid malignancies. In essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF), the JAK2 V617F mutant is often detectable, and considered to lead to activation of STAT5 in the cytoplasm of neoplastic cells. We examined the expression and function of STAT5 in primary neoplastic cells obtained from patients with ET, PV, and PMF, and in the JAK2 V617F+ leukemic cell lines HEL and SET2. As assessed by immunocytochemistry, primary neoplastic JAK2 V617F+ cells and HEL cells expressed phosphorylated (p) STAT5 in their cytoplasm as well as in their nuclei. Western blot analysis confirmed that both the cytoplasmic as well as the nuclear extracts of HEL cells contain pSTAT5. We next examined pSTAT5 expression in CD34+/CD38- stem cells in 21 patients with JAK2 V617F+ myeloproliferative neoplasms (MPN) by flow cytometry. In all patients tested, the CD34+/CD38- stem cells were found to express pSTAT5, whereas CD34+/CD38- stem cells in the normal/reactive bone marrow did not express pSTAT5. We next applied JAK-targeting drugs (AZD1480, INCB018424, TG101348) and STAT5-targeting drugs (piceatannol and pimozide) on MPN cells. As assessed by 3H-thymidine uptake, all drugs were found to inhibit the proliferation of HEL and SET2 cells. Moreover, all drugs were found to suppress proliferation in primary neoplastic cells obtained from MPN patients. In summary, our data show that neoplastic cells in JAK2 V617F+ neoplasms express pSTAT5 in their cytoplasm and in nuclei, and that targeting of STAT5 may be a novel interesting approach to control the expansion of neoplastic cells in MPN. Since pSTAT5 is also expressed in stem cell-enriched (CD34+/CD38-) fractions of neoplastic cells, STAT5 may serve as a promising new target in JAK2-mutated MPN. Disclosure: No conflict of interest disclosed. V940

Bone marrow multipotent mesenchymal stroma cells support leukemic cell proliferation and reduce chemotherapy sensitivity in vitro Schelker R.C., Müller G., Gottfried E., Seilbeck A., Hart C., Andreesen R., Grassinger J.

vating them at the perivascular region. The HSC niche supports physiological and aberrant hematopoiesis in a similar manner. In our study we therefore sought to investigate the role of MSC in leukemogenesis. Methods: MSC from patients with acute leukemia (AL) and donors without BM disorder (BD) were obtained from BM aspirates using an plastic adherence method. Phenotypic and functional criteria of MSC were tested by in vitro differentiation cultures and flow cytometry. To compare expansion capacity, liquid culture assays were performed from both donor groups. To test the supportive capacity of MSC, co-cultures with leukemic cells (LC) were initiated and compared to those on mouse fibroblasts (MS5) and MSC derived osteoblasts (OB). Additionally, sensitivity of LC to cytarabin was tested in this co-culture system. Ultrastructural intercellular communication of MSC and LC was investigated by electron microscopy. Finally, transplantation assays with MSC, LC and HSC in NSG mice were performed and analyzed by FACS and immunohistology. Results: MSC were successfully harvested from BM aspirates and could be differentiated into osteoblasts, chondrocytes and adipocytes. MSC showed a typical surface marker profile, however, expression of homing receptors was different to HSC or LC. MSC from patients with AL showed significant decreased expansion capacity compared to MSC from BD. Co-culture of LC on MSC showed a higher proliferation capacity than culture on MS5. Additionally, cytarabin treatment had less effect on LC cultured on MSC than LC cultured on fibroblasts or without feeder cells (82% vs. 55% vs. 49% survival, respectively). Interestingly, electron microscopy showed no evidence for junctional bridging between LC and MSC. Moreover, BM MSC exhibited the same morphology as common fibroblasts. Transplantation experiments demonstrated that HSC and LC had a significant higher homing efficiency into the BM than MSC, the latter being mainly trapped within the lung. Conclusions: MSC from patients with AL show distinct differences to MSC from BD. MSC support growth and reduce chemotherapy sensitivity of LC in vitro. Co-transplantation experiments of MSC and LC into humanized NSG mice are conducted at the moment and will reveal if this is also the case in vivo. Disclosure: No conflict of interest disclosed.

Plenarsitzung

Presidential Symposium / Klonale Heterogenität V941

Darwinian principles and consequences for cancer treatment Maley C.C. UCSF Helen Diller Family Comprehensive Center, Department of Surgery, San Francisco, United States

Neoplasms evolve through selection on both genetic and epigenetic heterogeneity within the neoplastic cell population. Furthermore, microenvironmental heterogeneity likely results in spatial heterogeneity of therapeutic intensity as well as selection for different sub-clones that have reproductive or survival advantages in the different microenvironments. Heterogeneity within neoplasms poses a challenge for both prognosis and cancer treatment. We are developing universal biomarkers that are generally applicable across cancers, based on measuring the evolutionary process itself, rather than the stochastic products of that process. In particular, we are focusing on measures of diversity as predictors of progression and survival. We are also working on evolutionary methods to treat and manage cancers. Disclosure: No conflict of interest disclosed.

Introduction: Multipotent mesenchymal stroma cells (MSC) are a key factor within the bone marrow (BM) niche, maintaining hematopoietic stem cells (HSC) in a quiescent state within the endosteal niche and acti-

288


Abstracts


Uniklinikum Regensburg, Hämatologie/Onkologie, Regensburg, Germany

Author Index

Abdyli L. P224 Abel U. P232 Abele-Horn M. P473 Abendroth A. P855 Abenhardt W. P528 Abramczyk M. P855 Abrey L. V350 Achenbach M. V32 Ackerl M. P838 Adam P. P536 Adam T. V394 Adam Z. P803, V644 Adenis A. P239, P240, V435, V436 Agelopoulos K. P166, P187 Agrawal M. V360 Ahrens T. V604 Aigner M. P888 Aigner R. P535 Aisner J. V117 Akmut F. P176, P178, P190 Aksnes A.K. V73 Aktas B. P538 Al Batran S. V388 Alakel N. V48 Al-Ali H. P520, V444, V652 Al-Batran S.-E. P249, V448 Albers C. V724, V764 Albers P. P268, V412, V414 Albert M. V441 Albus K. V603 Aldaoud A. P523, P532 Alpermann T. V455 Alsina M. V161 Alt A. P231, P862 Alt-Epping B. V22 Altmann B. V32 Amann A. V606 Amann G. P814 Amann K. P510, V669 Ammar R.M. P864 Amouzadeh-Ghadikolai O. V699 Anderson K.C. V715 Andrea M. P520 Andreesen R. P238, P807, V363, V940 Andritsch E. V107, V401 Andrulis M. V127, V714, V768 Angerer-Shpilenya M. V402 Angermeier S. V46 Ansmann L. V27, V759 Anthoni C. P570 Antonini E. V715 Apostolou P. P229, P879 Apperley J. P858 Arastéh K. P488 Aringer M. P567 Arning M. V449 Arnold D. P843, P882 Arnold R. V113, V934 Artmann S. V595 Asslaber D. P205, V650, V747, V748 Attner B. V49 Auer M. V659



Aul C. P859, V772 Aulitzky T. V67 Aulitzky W. V46, V72, V698, V926 Aulwurm S. V71 Aumann K. V752 Aung T. P478, P479, V77, V668 Austein T. P196, P258 Avivi I. V31 Awada A. V357, V356 Awsa S. P184, P185, P186 Ayuk F. P508 Azeh I. P241 Azémar M. P843, P882 Azizi A. V352 Baatout D. V649 Baccarani M. V362 Bach E. V938 Bach V. V700 Bacher U. V455, V744 Bachmann A. P520 Bachtiary B. V451 Backs M. V607 Badalamenti G. V609, V611 Bader O. V394 Badros A.Z. V163 Baerlocher G. V359, V361, V720 Baeumer P. P277 Bahlis N. V161 Bahlo J. V743 Bähring F. P869 Baier G. V687 Bajna E. P227 Baker J. V713 Bakhtair S. P464 Baklayan A. P563 Baldus C.D. V72, V613, V633 Baldus S. V768 Balic M. P541, V600 Ball C.R. P232 Bamberg M. V71 Bamezai S. V657 Bangard C. V395 Banja E. P226 Baran N. P181 Bareiss P.M. V905 Baretton G. P787 Bargou R. P517, P519, V162, V577, V717, V718 Barlow S. P276 Barone C. P239, P240, V435, V436 Barreyro L. V937 Barski D. V415 Bartels M. V615 Bartels S. P825 Barth J. V123, V150 Barth T. V702 Barthel S. P517 Bartholdy B. V937 Barthuber C. V771 Bartsch P. P565 Bartsch R. P552, P554, V353

Bärtschi D. V387 Bartscht T. P871 Bartz-Schmidt K.U. P463 Bäsecke J. P496 Bashari H.M. V715 Bassermann F. P481, P514, V162 Bastholt L. V621 Bauer S. P809, P811, V607, V610, V611, V612 Bäuerlein C.A. V731 Bauernhofer T. V679 Baum R.P. P861, V605 Baumann B. V696 Baumann U. P481, P514, V711 Baumann W. V26, V27, V755, V756, V757, V758, V759 Bäumer N. P169, P173 Bäumer S. P173 Baumgarten A. P495 Baumgartner S. P219 Baurmann H. V408, V409 Bawendi M.G. V766 Becherer U. P529 Bechstein W. P244 Beck J.F. V617 Becker C. P520, V120, V394, V672 Becker E. P781 Becker H.J. P203 Becker H. V724 Becker J.C. V738 Becker M. V698 Beckhove P. V597 Bedke J. P251 Bednařík O. V644 Beeker A. P253 Beelen D. P458, P509, P775, P776, P885, V115, V407, V440, V442, V445, V625 Begemann S. P197 Beham-Schmid C. P219, P484 Behre G. V652 Behringer D. V603 Behringer J. V355 Beier F. P490, V112, V695 Beilhack A. P517, V713, V731 Beissbarth T. P245, V437, V595 Bekeredjian-Ding I. P273, V392, V394 Belleville E. P546 Bellos F. P497 Ben Batalla I. P174, V622 Bendas G. P233 Bender K. P464 Bendszus M. P277 Benett K.L. V719 Benkißer M. P207 Benner A. V768 Benz J. V603 Beraldi A. P562, P563 Berardi Vilei S. P261 Berberich-Siebelt F. V731 Berdel W. P166, P169, P173, P187, P188, P502, P570, P822, V72, V115, V654, V655, V750



Author Index

Bock C. V912 Böckenförde S. P843 Boeckenhoff A. P254 Boege F. V771 Boehm J. V125 Boesch M. V904 Boettcher S. P211 Bohlander S. P790, V613, V655 Bohlmann M.K. P544 Böhm V. V615 Böhme M.U. P197 Böhmer F. P468 Bojic A. V675 Bokemeyer C. P174, P508, P816, P843, P887, V30, V132, V411, V622 Bold Ä. P476 Böll B. V330 Bolt T.A. V448 Bolz M. P804 Bondong A. P210 Boquoi A. V771 Borchert K. P194, P236, P280, P804 Borchmann P. V698 Bordessoule D. V31 Bordihn K. V352 Börger L. P494 Borges jr.U. V755 Borner M. V387 Bornhäuser M. P476, P507, P567, P787, V48, V69, V72, V408, V440, V674 Boross P. V438 Borte M. V711 Borte S. V696 Boruga C. P195 Bos M. P253, V116, V119, V120, V603 Bosmann M. P805 Botezatu L. V642, V653 Böttcher S. P210, V154 Böttger I. P798 Botzenhardt U. V702 Bouche O. P240, V435, V436 Boumendil A. V31 Bourgeois M. P520 Bourquin C. V641 Bouzani M. P876 Braess J. V19, V655, V754 Brähler E. P551 Braig F. P508 Brančíková D. V644 Brandl C. V618 Brandt J. P503 Brandts C. V72, V749 Brauchli P. V387 Braulke F. P496, P792, P793, V767 Braun C. P566 Braun F. V602 Braun H. P791 Braun M. P514, V594, V733 Braun-Dullaeus R. P565 Braunschweig T. P490 Brede C. V713 Bregant C. V699 Brehmer B. P253 Breitenbücher F. V434 Brendel C. P172 Brendel S. V454 Brettner S. V116, V603

Breuer F. V450 Brian T. P464 Bricks J. P206, V594 Briest F. P877 Brinker R. V592 Brodowicz T. P814, V296 Brönnimann C. V697 Brose M.S. V621 Brossart P. P233, P234, P250, P273, P280/1, V42, V67, V722 Brückner F. P506 Brüderlein S. V713 Brügel M. V608 Brüggemann M. V122, V307, V576, V613, V615 Brugger W. V42 Bruland O. V73 Brümmendorf T.H. P215, P490, V112, V654, V695, V767 Brummer T. V651 Brunke C. V438 Brunner A. P500 Bruns H. P510, P888, V669, V901 Buadi F. V161 Bubnoff N.V. V723, V724 Buchenroth M. V116, V603 Buchet O. P239 Buchheidt D. P269, V393 Buchner M. P207 Büchner T. P183, V655 Buchroithner J. V352 Buchtheidt D. V394 Buck A. P483, V899 Budach V. V347 Budach W. P816 Buder R. V446 Buettner R. P250 Bug G. V457, V767 Bugl S. V589 Bühring H.-J. P251, P252, P880, V71 Bullinger L. V67, V577, V630, V702 Bunjes D. V440 Burbeck M. P527, P833 Burchardt C.A. V43 Burger M. V602, V604, V651 Burger R. P513, P515, P516, V716 Burgkart R. V453 Burgstaller S. P220, V404 Busch J. P268, V412, V414 Busch M. P510 Busch R. P211 Buschmann D. P798 Busemann C. P501 Buske C. V656, V657 Buss E. P181, P277 Busse A. P881 Buttkereit U. P885 Büttner M. P510, V669 Büttner R. V116, V119, V120, V603 Buwitt-Beckmann U. P786 Buxhofer-Ausch V. V404 Büyüktas D. V598 Caca K. V45 Capper D. V714 Carlton V. V576 Carmeliet P. P174, V622


291


Berenguer J. V114 Bergemann C. P869 Berger D. P511 Berger J. P206, V594 Berger K. P857, V710 Berger L.A. V411 Berger M. P488 Berger T. P228 Berghoff A. P554, P836, V353 Bergmann F. P232 Bergmann L. P256, P257, V37 Berkessel A. P201 Berlin C. P202, V706, V753 Bernard I. V446 Bernhard H. V389, V597 Bernhardt M. P544 Bertinetti-Lapatki C. V125 Bertram M. V118 Bertsch U. V160, V165, V578 Bertz H. P269 Beschorner R. P566 Beshay J. V931 Besses C.R. V405 Bethge W. P461, P462, P463, P474, P505, P566, V71, V440 Bettelheim P. P789 Betticher D. V387 Beuster G. V899 Beutel G. V38, V383 Beuthien-Baumann B. P507, P820 Beyer J. V411 Beyer T. V438 Bhagat T.D. V937 Bhaumik J. V766 Biehl L.M. V392 Biehl M. V731 Binder C. V595, V598 Binder L. P245 Binder M. P174, P508, V744 Binot E. V603 Birgegård G. V405 Birner P. P836 Bischoff J. V356, V357 Bisht S. P233, P234, P250 Bissé E. P529 Bittenbring J.T. V32 Bjelic-Radisic V. P541 Blackstein M. V609, V611 Blank C. V339, V736 Blatt K. P219, P511, V616, V705, V719, V935, V936 Blau I. V160, V578 Blay J.-Y. V609, V611 Bleckmann A. P245, V595, V598 Blei D. V902 Bloch W. P543 Bloehdorn J. V718 Bloetz A. V931 Bloomfield C.D. V652 Blum H. P790, V613, V655 Blum S. V772 Blume C. P200 Blumenstock G. P190 Bob R. P498 Boccadero M. V576 Bochtler T. P522, V69 Bochum S. P853

292

Dannenmann A. V400 Däßler K.-U. V358 Dauth H. V901 De la Fouchardiere C. V621 De Santis M. P253 De Wit M. P488, V411 De Witte T. V31 De Zwart P. P816 Debatin K.-M. P862 Dechant M. V438 Dechêne A. P855 Decker T. P546, V388, V389 Deligiannis I. P193, P195 Demant A. P502 Demant M. P478, P479 Demetri G. V609, V611 Demirtas D. V745 Demski S. V934 Dengler J. P218, V359, V361, V698 Dengler M.A. V926 Denzinger S. P277 Derer S. V438, V686 Derigs H.G. P491, P492 Deryckere D. P509 Desjarlais J. V686 Desuki A. P873 Deutsch A. P484 Dickerhoff R. V726 Dickinson A.M. P858 Dieckmann K. P836, P838, P839 Dieckmann K.-P. P268, V412, V413, V414 Diehlmann A. V932 Diemar C. P278 Dienst A. P782, V443 Dierbach H. V733 Diering N. P478 Dierks C. V651, V752, V764 Diet F. V603 Dieter S.M. P232 Dieterich S. P280 Dietrich D. V387 Dietrich S. P200, P210, V31, V110, V127, V663, V768 Dietz A. V349 Dietz C. V361 Dietze K. P481, V664, V665 Dietze L. V603 Dietzfelbinger R. V667 Diez J. V699 Diez-Martin J.L. V31, V114 Dill D. P193 Dirnhofer S. V664 Ditschkowski M. P458, V407, V442, V445, V625 Dittmar G. P480 Dittrich C. P545, V117 Diwoky S. P194, P506, P835 Doehn C. V37 Doehner H. V577 Doelken G. V394 Doerfel D. P525 Dohm A. P470, P494 Döhner H. P530, V67, V68, V630, V656, V657, V684, V702, V718 Döhner K. V67, V68, V630, V656, V657, V684, V702 Dombrowski-Lütcke M. P549


Domm A.-S. V164 Dörfel D. P566 Dörken B. P480, V113, V126, V664, V665, V729, V899, V925, V934 Dorn C. P810, P817 Dörr J.R. V899, V925 Drandi D. V576 Drath M. P170 Drebinger K. V400 Dreger P. P210, V31, V110, V440, V663, V768 Dressler C. P554 Drewes C. P850 Dreyling M. P481, P493, V35, V440, V666, V667 Drolle H. P175 Drost A. P777 Dubash T. P232 Dubsky P. V353 Ductus C. V356, V357 Duda D.G. V766 Duerken M. P269 Duerkop H. V685 Dufour A. V655 Dugas M. P169, V654 Dühren- von Minden M. V125 Dührsen U. P213, P801, P878, V591, V642, V648, V653, V662, V763, V898 Dunst J. V766 Dürig J. P213, V5, V160, V284, V578, V591, V648, V662 Dürk H.A. P459 Dutreix C. V719 Duyster J. P171, P785, P837, P866, V608, V658, V659, V704, V709, V723, V724, V733, V764 Earp H.S. P509 Eber S. V726 Eberhardt J. P228, P870 Eberhardt W. P826, P830, V119 Echchannaoui H. P873 Eckart M.J. P206, V594 Eckert F. P816 Eckl C. V764 Eckstein N. P274 Eckstein V. P181 Edeling M. P790 Edelmann T. P520 Edinger M. P858 Egerer G. P526 Egert M. P520 Egger M. P535 Eggert A. V763 Egle A. V123, V404, V650, V748 Ehl S. V696 Ehninger G. P507, P567, P787, P820, V48, V69, V72, V359, V408, V642, V653, V674, V749 Eich H.T. V327 Eichelbrönner I. V928 Eichhorn C. V672 Eichhorst B. P211, V743 Eichinger Y. V931 Eidt S. P230 Eigendorff E. P487 Eimer C. V415

Author Index


Casali P. V609, V611 Castagnetti F. V362 Castoldi R. V641 Cathomas R. P261, V410 Catusse J. V602 Cerny-Reiterer S. P219, V616, V935, V936, V939 Cervantes F. V362 Cevik N. V456 Chakupurakal G. P212 Chanan-Khan A. V161 Chang D.L.T. P272 Chapuy B. P478, P479, V668 Chatterjee M. P517, P519, V717 Chatziioannou M. P229, P879 Chen S. V624, V733, V924 Chen Y. V766 Chinot O. V350 Choidas A. V591 Chopra M. V713, V731 Christ C. V672 Christopeit M. V937 Christoph S. P509, V442 Chung J. V621 Cicholas A. P478, P479 Cierpinski A. P243 Cihon F. P239, P240, V436 Claasen J. V592, V645 Classen C.F. P550, V141 Claus R. P527, V604, V656 Clemens M. P491, P492, V430 Clement J.H. P228, P869, P870 Cleveland J. V900 Cloughesy T. V350 Coleman R.E. V74, V76 Coll R. V405 Conrad H. V597 Conradi I. P829, P830, P831, P832, V121 Conradi L.-C. P245, V437, V598 Corbacioglu A. V68 Cornelissen J. V31 Cornely O. P270, V392, V393, V394, V395, V397 Cortes J. V356, V357 Crabb S. P261 Cramer P. V743 Crevenna R. P838 Crispatzu G. V660 Cross A. V76 Cross M. V444, V652, V938 Cross N.C.P. V409 Crysandt M. P490 Csef H. V400 Cubas-Cordova M. P174, V622 Cui J. V766 Culmann H. V120 Cygon F. P815 D'Abramo F. V673 Däbritz H. V126, V899 Daecke S.N. V722 Daheim M. V924 Dahl A. V749 Dahlhaus M. V720 Daisne J.-F. V386 Dandachi N. P541, V600 Danielzik T. P885

Author Index

Fasshauer M. V711 Fassunke J. V603 Fätkenheuer G. V397 Faul C. P461, P462, P566 Federmann B. P783 Fehm T. V905 Fehr M. V410 Fehse B. P174, V622 Feiten S. P208, P845, P846, P848, P854, V760 Feldhaus V. V592 Feldmann G. P233, P234, P250 Feldmann R. P834 Felthaus J. V751, V927 Felzmann T. V352 Fend F. P536, V905 Fernandez-Cuesta L. V120 Fernández-Sáiz V. P481 Ferrero S. V576 Feuring-Buske M. V656 Fey S. V452, V454, V457 Fichtner I. P862, V348 Fiedler A. P230 Fiedler W. P174, V68 Fiegl M. P175 Fietz T. P842 Fillatreau S. V732 Findeis J. V731 Finel H. V31 Fink A. P211, V743 Fink G. P785 Finke J. P495, V440, V733 Fischer A. V905 Fischer D. P869 Fischer J. P780, P782 Fischer K. P211, V743 Fischer T. P271, P465, P552, P565, P819, V399 Fischer von Weikerstahl L. P249, V388 Fitzal F. V353 Flamme H. V646 Flechl B. P838, P839 Flechtner H.-H. P552 Fleckenstein J. V34, V35 Fletcher J. P809, V607 Flieser-Hartl M. P558 Fließer M. P884 Födermayr M. V353 Föhring D. P801 Follo M. P512, P833, V604, V751, V927 Follows G. V127 Folprecht G. P244 Fong D. P246, V429, V906 Forde A. V752 Förstermann K. V657 Fossaa S.D. V133 Frank M. P824, V439 Frank O. P217, P218 Franke B. V397 Franke G.-N. V444, V652 Franzem M. V41, V391 Freitag H. P877 Freitag S. P194, P506 Frenzel L.P. P203, V592, V645, V649 Freudenberg M. P882 Freund M. P194, P236, P469, P550, V141, V397 Freundt J. V141

Frey B.M. V697 Frey M. V931 Friccius-Quecke H. P887 Frick M. V665 Fricke H.-J. P487 Fricke H. V454 Fricke R. V116 Fricke S. P520, V734, V930 Frickhofen N. V349 Fridrik A. P242 Fridrik M. P220, P242, P545 Friedel H. V413 Friedrich M. V713 Friesen C. P231, P862 Friesenhahn V. P208, P845, P846, P848, P854, V760 Fritsch A. P506 Fritsch K. P495 Fröbel J. P791 Frommer J. P552 Fromm-Haidenberger S. V451 Früh M. V387 Frye S. P509 Fuchs S. V696 Fuhr U. V119 Fuhrmann V. V675 Fukao T. V733 Fukumura D. V766 Füller M. P822, V750 Füllgrabe M. V122 Funovics P. P814 Füreder T. P247, P554 Fusi A. P881 Gabriel C. P789 Gabrysiak T. P217 Gaidzik V.I. V67, V68, V630 Gaiger A. P838 Gaiser F. P529 Galetke W. V116, V120 Galle P.R. P249 Gamerith G. V606 Gampenrieder S.P. P545 Gamperl H. P863 Ganghammer S. P205 Ganser A. P174, P255, V38, V67, V68, V457 Ganster C. P792, V767 Ganten T.M. V446 Garcia-Marquez M. P230 Garcia-Vargas J. P260, P259, V76 Gardizi M. V116, V119, V120, V603 Gary R. P888 Gasparovic A.C. V600 Gassner C. V697 Gastl G. P220, P246, P518, V404, V606, V687, V906 Gattermann N. V770 Gauler T. V349, V411 Gaupmann R. V705 Gebhard C. V769 Geginat G. P271 Gehlen H. P510 Gehrisch S. P820 Geissler C. P834 Geissler K. P184, P185, P186, P191, P192, P216, P226, P227, P796, P806, P834, P841, V721


293


Einsele H. P459, P473, P517, P519, P531, P833, P876, P884, V162, V359, V361, V363, V400, V577, V713, V717, V718, V731, V928 Eisele L. P213, V648 Eisenwort G. V616 Eisterer W. P242 Eiwen K. V68 Ekkehart L. V391 El-Azab M. P864 Elias J. V394 Elisei R. V621 Ellerbroek P. V114 Ellis S. V410 Elmaagacli A. P458, P509, P775, P776, V115, V407, V442, V445, V625, V703 Emmrich F. V734, V930 Endres S. V641, V682 Engel K. P481 Engel N. V441 Engelhardt M. P180, P512, P521, P527, P529, P785, P833, P851, V162, V164, V579, V580, V709, V718, V751, V761, V762, V927 Engel-Riedel W. P826 Engert A. V397, V685 Englund M. V49 Erben P. P223, V360, V361 Erdel M. P569 Erdenetseg R. P226 Erdmann R. P174, V622 Erdmann-Reusch B. P556 Eric B. P226, P227 Erlbeck M. P562 Ernst J. V617 Ernst T. P870, V617 Ernstmann N. V27, V759 Escudier B. P254 Essmann F. V905 Eucker J. P548 Evers G. P173, P570 Ewerth D. P180, V761, V762 Exner A.-K. P553 Fabarius A. P177, P214, V359, V360, V361, V409, V452 Faber V. P571 Fabri M. V669 Faham M. V122, V576 Fahlke J. V449 Fahrenkamp D. P490 Fahrenwaldt C. V901 Falcone A. P239, P240, V435, V436 Faldum A. P183 Falge C. V359, V361 Falk C. V110 Falk K. V162 Falk M. P823, P829, P830, P831, P832, V121 Falkenhorst J. P809 Fallahi M. V900 Faller H. V400 Fan F. V715 Fang F. V73, P259 Fantana J. P567 Farkas C. V401 Fasan A. V614 Fasching P.A. P546

294

Gottfried E. V940 Gottwald E. V765 Götz M. V684, V702 Götze A. P196 Götze K. P793, V67, V453, V457, V767, V772 Gouilleux F. V939 Grabellus F. V612 Grabowski P. P877 Graf A. P790, V613, V655 Graf M. P530 Graf von der Schulenburg J.-M. P824, V439 Graham D.K. P509 Gramatzki M. P472, P513, P515, P516, P786, P883, V162, V683, V701, V716 Grantz T. P558 Grapton D. V642 Grassinger J. V940 Grassmann S. V641, V682 Gratwohl A. V363 Grau M. V664, V665 Graulich T. P868 Greally J.M. V937 Greif P. P790, V613, V655 Greil C. P529 Greil R. P205, P220, P242, P545, P571, V123, V404, V650, V747, V748 Greiner J. V684, V702 Greinix H. P858, V929 Greve G. P828 Grichina O. P527 Griesinger F. P823, P827, P829, P830, P831, P832, V121 Griesshammer M. V86, V405 Grigoleit G.U. P459, P473, V400 Grimm M.-O. P253 Grimmer D. P556 Grohé C. P826 Gromke T. V407, V442, V445, V625 Groschek M. P217, P532, V450 Groß B. P527, P785, P851, V709 Groß S.E. V27, V759 Gross S.H. V719 Grosse-Hovest L. P880, V71 Große-Thie C. P194, P236, P280, P506, P804, P835 Grössinger E.M. V650 Grossmann V. V455 Grossschmidt P. P834 Groten A. V443 Groth C. V115 Grothe W. P784 Grothey A. P239, P240, V435, V436 Grub K. P464 Gruber C. V659 Gruber-Rossipal C. P569 Gruenberger T. P244 Gruenwald V. V349 Grugel R. P528 Gruhn B. V617 Gründer A. P225 Grundler R. P171, V659 Grundmann S. V733 Gruner N. V48 Grunewald S. P809, P811, V607 Grunt T.W. V903 Grunwald U. P501


Grünwald V. P255, V38, V610, V680 Grützmann R. P235 Grzywna S. V897 Gschwend J. P253 Guardiola P. V31 Guderian G. V37 Gugliotta L. V405 Guilhot J. V362 Gülüc S. P195, P193, P799 Gunsilius E. P518 Günther A. P472, P513, P515, P516, P786, V716 Guntinas-Lichius O. V349 Güntsch A. P478 Gutekunst M. V926 Gütz S. P826 Haag F. P508, V744 Haap M. P566, P817 Haarmann J. P233, P234, P250 Haas R. V443, V642, V770, V771, V772 Haase D. P788, P792, P793, V456, V457, V767, V772 Haase G. V394 Haaß W. P214 Habbel P. P548 Habild G. P812 Hackanson B. V604 Hackl C. P789 Hackl M. P836 Hadji P. P546, V596 Hadlich T. P225 Hadzijusufovic E. V719, V939 Haen S.P. P461, P462, P463, P474 Haese A. V74, V76 Häfer R. V617 Haferlach C. P459, P497, V359, V409, V455, V614 Haferlach T. P170, P497, V342, V409, V455, V457, V614, V698 Hagel C. P174 Hagmann H. V624 Hähling D. P804 Hahn M. P210, V581 Hahn-Ast C. P273, P280/1, V394, V396 Hahnfeld S. P487 Haidenberger A. V451 Haider R. P789 Hailfinger S. V665 Hainfellner J.A. P836, P839 Hainzl S. V650 Hake R. P230 Halbe L. P560 Halbfass V. P823, P827, P829, P830, P831, P832, V121 Halbsgut N. P788 Hallas C. P823, P829, P830, P831, P832, V121 Hallek M. P201, P203, P204, P211, P212, P230, V35, V70, V397, V592, V593, V645, V646, V647, V649, V661, V743, V754 Hamilton G. P192, P226, P227 Hammersen J. P487 Hamprecht A. V394 Han H.-S. V766 Handgretinger R. P461 Händschke K. V763, V898

Author Index


Geissler N. P226, P227 Geist T. V120 Geldart T. V410 Gelderblom H. V609, V611 Geller K. V449 Gembicki M. P544 Genuardi E. V122 Georgas E. V415 Gerbitz A. P510, P888, V669, V901 Gerhardt A. V899, V925 Gerigk U. V116, V603 Gerken G. P855, V446 Gerl A. P779 Gerlach B.-K. P470 Gerlach C. P560 Germing U. P798, P791, P793, V68, V88, V443, V457, V642, V767, V768, V770, V771, V772 Gerullis H. V415 Gessner A. V111 Geue K. P551 Gey C. P869 Ghadimi B.M. P245, V437 Giagounidis A. P793, V72, V457, V767, V772 Giebel B. V591 Giehl M. P214 Giers G. P780, P782 Gies U. V47 Gieseler F. P863, P871 Giesinger J. V403 Gil A. V363 Gillessen S. P261 Gillisen M. V766 Giordano F.A. V302 Giselbrecht S. V765 Gisslinger B. V404 Gisslinger H. P216, V404, V721 Gläser D. P194, P236, P280, P506, P804 Glass B. V34, V35 Glauche I. V364, V728 Gleißner J. P860, V78 Gleixner K. V705 Glimm H. P200, P232, V127 Gloede T.D. V27, V759 Glorius P. P513, P515, V438, V686 Gnant M. P242, P554, V353 Göbel M. V591 Goebel A. V596 Goebell P.J. V37, V40, V41 Goel S. V117 Göhler T. V446 Göhring G. P790, V68 Gökbuget N. V613, V615 Gold R. V711 Goldberg R.M. P239, P240, V435, V436 Goldschmidt H. P526, P536, V160, V165, V578, V581, V714, V715 Göllner S. P822, V750, V763 Golpon H. P824 Gopalakrishna P. V117 Gorantla S.P. V723, V724 Görgens A. V591 Gosenca D. P223, V409 Gössel C. P487 Göthert J.R. P213, P878, V648, V763, V898 Göttel R. P263

Author Index

Heim D. V359 Heim M.U. P781 Heimann S. P270, V397 Heimpel H. V359 Heine A. V42, V722 Heinemann V. V450, V388 Heining C. V714 Heinrich B. P248 Heinrich M.C. P176, P178 Heintges T. V388 Heinz W. P269, P270 Heinze K. V713 Heinze T. P870 Heinzelmann F. V440 Heinzer H. V74, V76 Heiß C. P526 Heiss M.M. P887 Heitmann J. V589, V746 Hekmat K. V116, V120, V603 Held G. V32, V33, V34, V35, V67 Held S.A.E. V722 Helle S.I. P260 Hellwig S. P561 Hemmati P. V113, V934 Hemmerling J. V735 Hengeöz Ö. P193 Henke A. V647 Henke R.-P. P829, P830, P831, V121 Henkel J. V641 Henneke P. V696 Hennig J. P779 Hennighausen L. P171 Henrikkson R. V350 Henschler R. P205, V710 Hense J. P538 Hensel M. V124, V711 Henssler A. V47 Hentrich M. P779, V289, V411 Herbrik M. V612 Herbst R. P787, P802 Herget G. P833 Herling M. P204, V647, V660 Hermann A. V675 Hermann E. P885 Hermes K. V757, V758 Herndlhofer S. P219, V935, V936 Herold M. V660 Herold S. V408, V749 Herold T. V655 Herr W. V735, V931, V933 Herrmann A. P480 Herrmann E. V37 Herrmann H. P511, V616, V935, V936 Herrmann R. V387 Herschbach P. V587 Hertenstein B. V162 Herth I. P210, V124 Herzberg C. P493 Herzig J.K. V67 Hess G. P493, P560 Hess V. V387 Hesse E. V622 Heßling J. P852 Heuchel C. V674 Heudobler D. V112, V769 Heukamp L. P250, V116, V119, V120, V603, V661

Heuser M. P174, V68 Heussel C.P. P269 Heußner P. P557, P562, P563, V398, V672 Heymanns J. P208, P845, P846, P848, P854, V760 Heyn S. P520, V652 Hiddemann W. P557, P562, P563, V655, V656, V666, V667, V672, V754 Hieke S. P527, V579 Hielscher J. V388 Hielscher T. V165 Hiemer S.K. P818 Hijazi Y. V618 Hilbe W. V606 Hildebrandt B. V770 Hildebrandt G.C. V923 Hildebrandt M. P858 Hildenbrand B. P882 Hilgarth M. V745 Hilgendorf I. P194, P280, P469, P550, P804, V141 Hilger N. V734, V930 Hilger R.A. P862 Hillbrand M. V451 Hillen U. V445 Hillenbrand T. P813 Hillengass J. P526, V165 Hilton M. V350 Hinke A. V449 Hinterseer E. V650 Hirabayashi S. V769 Ho A. P181, P200, P210, P277, P475, P482, P485, P486, P489, P503, P522, P526, P774, V110, V124, V127, V581, V663, V714, V765, V768, V932 Hoang V.T. P181, P774 Hochhaus A. P168, P280/1, P228, P468, P487, P869, P870, V130, V359, V360, V361, V363, V409, V449, V617, V728, V893 Höchsmann B. V698 Höck K. V580 Höckendorf U. V453 Hoechsmann B. V156 Hoelbl A. V936 Hoelzer D. V613, V615 Hoermann G. V936 Hoerr I. V42 Hofbauer L.C. V596 Hofbauer S.W. P205 Hofer S. V53, V351 Höffkes H.G. V450 Hoffmann C.M. P232 Hoffmann F.-A. P520 Hoffmann I. P181, P774 Hoffmann J. V348 Hoffmann M. V447 Hoffmann O. P538 Hoffmann T. V389 Hoffmann V.S. V362 Hofheinz R. V386, V436, V448 Höfler G. P868 Hofmann B. P802 Hofmann C. V717 Hofmann F. P508 Hofmann M. P789, V71 Hofmann S. V684, V702


295


Hänel M. P802, V34, V160, V361, V363, V578 Hanfstein B. P183, V359, V360, V361, V363 Hanisch U.-K. V640 Hanke M. P774 Hann von Weyhern C. P816 Hannig C. P564 Hansen H.P. V647, V685 Häntschel M. P566, V42 Hanus L. P565 Harbeck N. P279, P849, P850 Harde J. P532, P797 Harich H.-D. P528 Harichandan A. P251, P252 Harms-Effenberger K. V622 Harrison B. V161 Harrison C. V405 Hart C. V940 Hartmann H. P842, V712 Hartmann J.T. P244, P816,V301, V449 Hartmann K. V661 Hartmann M. V398 Hartmann S. V141 Hartmann T.N. P205, V747, V748, V650 Hartrampf S. V441 Hartwig U. V931 Harvey R.D. V163 Hascher A. P173 Hasenburg A. V709 Hasenkamp J. P467, P470 Hasford J. V359, V361, V363 Hass H.G. P547, V599 Hasskarl J. P532, P797 Hassler M.R. P839 Hastka J. P794 Hatem E.N. P802 Haubitz I. V440 Hauck F. V441 Hauer V. P216 Haus U. P538 Hauser A. P858 Häusler C. P220 Haustermanns K. V386 Hauswirth A. V616, V675 Haxel B. V349 He Y. V356, V357 Hebestreit K. P169 Hecht A. P183 Hecht J. V613 Hecker E. V116 Heckl U. V586 Hedberg C. V592 Heeb A. P875 Heesch S. V613 Hefner J. V400 Hegenbart U. P210, P522, V110, V663 Hegewisch-Becker S. P249 Hehlmann R. V359, V360, V361, V362, V363, V728 Heidel F. P465, P552, P781, P819 Heidenreich A. P262, P264, P265, P266, P267, P268, V39, V75, V78, V402, V412, V414 Heidenreich F. P476 Heider K.-H. P477 Heiderich C. V408 Heil G. P193, P195, P799 Heilmeier B. V754

296

Ilg J. P860 Ilhan-Mutlu A. P836 Illert A.L. P171, V658, V723, V724, V764 Illig D. P558, P844 Illing B. P189, P190 Illmer T. P172, V72 Ioannou E. P229, P879 Isermann B. P465 Istvanffy R. P171, V764 Ivanyi P. V38 Iype J. V733 Jacholkowski A. V744 Jackisch C. P543 Jagannath S. V161, V163 Jäger D. P244, P254, V715 Jäger E. P216, P796, P806, V721 Jäger U. P192, P216, P796, V616, V662, V721, V745 Jain M. V117 Jain R.K. V766 Jäkel N. V652 Jakob A. P833, V37 Jakob C. P787 Jakobs D. P527, P833 Jakubowiak A. V161 James R. P212 Jamous A. V902 Janda A. V696 Jänicke M. V354, V355, V358, V391 Janíková A. P803 Jann J.-C. P182, V452, V457 Janni W. P546 Janning M. P174, V622 Jansky M. V22 Janssen J. V37 Janssens J. V386 Janz M. V664, V665 Jarzab B.J. V621 Jauch A. P181, V165 Jawhar M. P223 Jeffers M. V435 Jehn C. V907 Jentsch-Ullrich K. P552 Jentzsch M. V444, V652 Jeromin S. V455 Jilch R. P184, P185, P186 Jilg S. V453 Jilo A. V938 Jin N. V932 Jitschin R. P206, V594, V669 Joensuu H. V609, V611 Joerger M. V708 Johannessen D.C. P260 Johansson P. V662 Jones B. V117 Jordan K. P278, P818 Jordán Garrote A.-L. V731 Jost E. P490, V112 Jost P. V453, V608, V660 Josten K. P254 Josupeit R. V597 Jumaa H. V651, V690 Jung A. V388 Jung G. P880, V71 Jung J. V27, V759 Jung M. P527, P851


Junghanß C. P194, P236, P280, P506, P550, P804, P835 Jürgens K.-U. V47 Kaeferstein A. P273 Kaemmerer D. V605 Kaergel E. V665 Kafka A. P248 Kahl C. V388 Kähnert H. P553 Kaiser F. P844 Kaiser G. P855 Kaiser L. P882 Kaiser S. P851, V709 Kakadia P. V655 Kalanovic D. P256, P257, P493 Kalhs P. V929 Kambartel K. V116, V603 Kaminsky B. V120 Kamoun W.S. V766 Kampa-Schittenhelm K. P176, P178, P179, P189, P190 Kämpfe D. P856 Kancha R.K. P837, P866, V704 Kang Y.-K. V609, V611 Kanz L. P176, P178, P179, P189, P190, P199, P202, P215, P251, P252, P461, P462, P463, P474, P505, P525, P536, P566, P777, P783, P800, P810, P817, P865, P880, V42, V71, V359, V361, V589, V643, V706, V746, V753, V897, V905 Kanzler S. P249, V389 Kapelle M. P273 Kapp M. V400 Kappeler C. V611 Kapp-Schwoerer S. V68 Karamustafa S. V110 Karcher A. V391 Karlic H. P167 Karthaus M. P240, P779, V394, V435 Kasal A. P246, V906 Kase J. P480 Kashkar H. V645 Kasper B. P813, V610 Kasper S. P538, P855, V390, V434 Katay I. V116 Kaufmann J. P490 Kaufmann K.B. P225 Kaufmann M. V31 Kaufmann P.A. V356, V357 Kaufmann R. P228 Kaufmann T. V116, V603 Kavina A. P841 Kayser G. V602 Kebschull M. P250 Keddad K. V406 Kegel T. P818 Keil F. P220, V36 Keilani M. P838 Keilholz U. P881, V347, V348, V349 Kekec-Dadas M. V442, V445, V703 Kekulé A. V394 Keller A. V939 Keller K. P527 Keller P. V394 Keller U. P481, V899, V900 Keller-Matschke K. P568

Author Index


Hofmann W.-K. P177, P182, P183, P214, P223, P269, P787, P794, P795, V88, V360, V361, V363, V393, V409, V452, V454, V457, V613, V772, V894 Hohberger A. P464 Hoheisel M. P827 Hohenberger P. P813, V609, V610, V611 Höhler T. V389, V390 Hoiczyk M. V612 Holderried T.A. V42 Höllein A. V900 Hollenbach C. V581 Hollenbeck A. V898 Holler E. V111, V112, V440 Hölscher A.H. P230 Holtick U. P212, V661 Holzer P. V314 Holzner B. V403 Homann N. V448 Homayounfar K. P245, V437 Honecker F. P268, V290, V411, V412, V414 Hönes J. V642, V653 Hopfinger G. P571, V123 Hopfner K.P. V655 Höpker K. V624 Hoppe S. P552 Horger M. P525, P816, V42 Hörmann G. P219 Horn C. V744 Horn M. V728 Horn P. P878, P885 Hörsch D. V623 Horst H.-A. V67 Horstmann K. V124 Horwitz M. P198 Hose D. P526, V160, V165, V578 Hossfeld D.K. V359 Hoster E. P481, P562, P563, V754 Hu B. P207 Huang Y. V766 Huber E. V112 Huber W. P200 Huberle C. V453 Hubmann R. V745 Hucke C. V685 Huellein J. V714 Hufnagl C. P545 Hug M.J. P851, V709 Hügli B. V720 Hüllein J. P200, V688 Hülsemann M. P203 Humblet Y. P239, P240, V435, V436 Hummel M. P480, V664, V665 Hummel S. P490, V112 Humpe A. P472, P786, P883, V683, V701 Hundemer M. V127 Hundsamer A. V36 Hünerlitürkoglu A.-N. V116 Hurtz H.-J. V41 Huscher D. V711 Hutter G. V666, V667 Hütter G. V114, V452 Hüttmann A. V72, V591 Hutzschenreuther U. P217 Idzko M. V733 Ihorst G. P529, V164, V579, V580

Author Index

Klinghammer K. V348 Klingner K. P821, V601, V751 Klobuch S. V735, V933 Klosner J. P166, P187 Kloß S. V37 Kluba T. P816 Kluge A. P830 Klvačová L. V644 Knauf W. P528, P532 Kneba M. P210, P472, V122, V359, V361, V576, V615 Knipp H. V390 Knipping S. V349 Knöbl P. V675 Knoedler M. V349 Knop S. P531, P833, V162, V577, V718 Knorn A.-M. P481 Ko Y.D. V116, V603 Kobbe G. V31, V440, V443, V772 Kobold S. V641, V682 Koch B. P521, P529, V164, V579, V580 Koch M.S. P188 Koch M. P232 Koch R. P478, P479, V668 Kochanek M. V754 Köchel C. V928 Kocher T. V748 Koeberle D. V387 Koehl U. V685 Koehler A. P272 Koehler M. P552, V399 Koenig F. P260 Koenig J. P249 Koeppen S. V377 Koestler J. V111 Koestler W.J. P814 Köhl U. P858 Köhler A. P842, V354 Köhler A.-C. P827 Köhler G. P173, P502, P570 Kohlhofer U. V905 Kohlmann A. V457 Köhne C.-H. P244, V67 Kohrt H.E. V730 Kolb H.-J. V359, V453 Koldehoff M. P458, P775, P776, V115, V407, V442, V445, V625, V703 Kollar A. V387 Koller E. V36 Kollmeier J. P826 Kolluri S.B. V723 Kominowski A. P788 Konantz M. V897 Kondakci M. V443 König J. V453 König K. V116, V603 Königsrainer A. P816 Konowski P. P870 Konrad F. V116, V603 Konschak R. V347 Konstandin N. V613, V655 Konthur Z. P508 Kopfmann S. V118 Kopp B. V718 Kopp H.-G. P505, P800, P810, P817, V411, V589, V746 Kopp J. V729

Köppler H. P208, P845, P846, P848, P854, V760 Korbenfeldt E. P254 Korfee S. P556 Korger M. P220 Kornek G. P247 Körner S. P880 Koschmieder S. V654 Koslowsky T. P230 Kossak-Roth U. V363 Köstler W. V675 Koukalová R. P803 Kourtidou E. P229, P879 Kovacs G. P224 Kowalewski D.J. P202, P215, V706, V753 Kqiku X. P224 Kraft F. P506 Kraft K. P236 Krahl R. P520, V444, V652 Král Z. V644 Krallmann S. P201 Kralovics R. V404 Kramer M. V69, V72, V674 Krämer A. V69, V72 Krammer P.H. V597 Krammer-Steiner B. P561 Kratzat S. V899 Kratzsch T. V640 Kraus S. P517, V713, V731 Krause A. P173 Krause S. P253, V72, V359, V361, V394 Krauter J. P174, V68 Krauth M.-T. V455 Krebs S. P790, V613, V655 Kreft A. P805 Krege S. P268, V412, V414 Kreibich U. P520 Kreil S. P794 Kreißelmeier K.-P. P505 Krejčí M. V644 Krekeler G. P256, P257, P493 Kremer S. P886 Kremers S. V363 Krenn P.W. P205 Kreutmair S. V764 Kreutzer C. V597 Kreuzeder J. P543, P546 Kreuzer K.-A. P201, P211, V70, V593, V646, V754, V772 Krieg V. V116 Krieger O. P220 Kristen A. P522 Kriz J. V327 Kroeger N. V440 Krogel C. P819 Krogmann D. P550, V141 Krohn A. V604 Krohn R. V758 Krohs J. P180, V927 Krönke J. V68 Krönke M. V647 Kropf-Staub S. V104 Kropp K.N. P800 Kropp P. P550, V141 Kroschinsky F. P567 Krug U. P188, P502, V72, V115, V654 Krüger M. P201


297


Kellner C. P513, P515, P883, V438, V683, V701 Kelly G. V660 Kemmler G. V403, V700 Kempa S. V899 Kempf B. V34, V35 Kenner H. P261 Kerkmann M. P275 Kerle I. P837, V608 Kern J. P518 Kern P. P538 Kern W. P497, V152, V455, V614 Kerschbaum H.H. V650 Kessler J. V647, V685 Kestler H. V657 Ketzer J. V607 Khandanpour C. V642, V653 Khurshid S. V624 Kiani A. V388 Kiecke C. V77 Kiehl M. V394 Kielbasa S. V125 Kier A. P841 Kieschke L. P258 Kiewe P. P787, P852 Kiladjian J.-J. V405, V406 Killing F. P856 Kimmich C. P522 Kimmig R. P538 Kindler M. V37, V450 Kindler T. V67, V68 Kingreen D. P852 Kintzelé L. V649 Kirch W. V711 Kirkovits T. P849 Kirstein M. V439 Kissel S. V752 Klade C. V404 Klaile Y. V654 Klameth L. P226, P227 Klappacher J. V656 Klapper W. P196, P516 Klatt E. P781 Klaumünzer M. P177, P182 Klausmann M. V698 Klausz K. P513, P515 Kleber M. P512, P521, P527, P529, P785, P833, P851, V164, V579, V580 Klebl F. P238 Kleboth K. P208, P845, P846, P848, P854, V760 Klech J. P247 Klein B. V160, V578 Klein C. V452, V641 Klein H.-U. P169 Klein M. P822, V389, V750 Kleine M. V603 Kleiner H. P214 Klein-Hitpass L. V662 Kleinke A.M. P550, V141 Klemm F. V595, V598 Kleymann A. P567 Kliche S. P465 Klier J. V78 Klingbiel D. P261, V410 Klingeberg C. V658, V764 Klinger M. V618

298

Lathan B. P217 Lauseker M. V359, V361, V363 Lauten M. V393 Le Cesne A. V609, V611 Le Coutre P. P218, V113 Leahy M. V609, V611 Lebenatus A. V615 Lechner D. V404 Lechner K. V721 Lechtenberg L. P274 Lee B.H. P538 Lee K. P865 Lee S. V899, V925 Leffler H. P234 Leha A. V595 Lehmann J. P258 Lehne F. V906 Lehners N. P486, V714 Lehnert H. P871 Lehnhardt D. V601 Leibbrand B. P553 Leibold J. P880 Leichtle R. V698 Leimer L. V46 Leitges M. P481 Leithäuser M. P280 Leitner G. V929 Leitzke S. P212 Lemeer S. P481, P514 Lemmermann N. V933 Lengerke C. V643, V897, V905, V910 Lengfelder E. P183, V57 Lenz G. P481, V59, V664, V665 Lenz H.-J. P239, P240, V435, V436 Lenz P. V664, V665 Lenze D. P480, V664 Leonhardt F. V602 Leonhartsberger N. P254 Lerchenmüller C. V388 Leser U. P480, V925 Letsch A. P881, V457, V772 Lettermann S. P166, P173, P187 Leusen J.H.W. V438 Leutner C. P273 Levine R. V937 Lewens F. P877 Lewis I. V140 Liebert A. V162 Liebert T. P870 Liebisch P. P530, V577, V718 Liersch T. P244, P245, V437, V598 Lietz C. P886 Lin Q. V695 Lindauer M. V44 Lindemann M. P885 Linden G. V390 Lindner T. P520 Lindörfer D. V362 Lindtner B. P166 Linhart H. P169 Link C. P476 Link H. P275 Linke R.P. P531 Linkesch W. P219, P220, P224, P460, V36, V109 Lipka D.B. P465 Lis A. P872


Lisec J. P480, V899 Lithman T. V49 Liu J. P509 Loeffler M. V728 Löffler J. P876, P884 Loges S. P174, V622 Lohmann G. P204 Löhr A. P821 Lohse S. V438, V686 Lonial S. V161, V163 Lopez C.D. P189, P190 Lorch A. V411 Lordick F. P244, P887, V108 Lorens J.B. P174 Lorenz J. P512 Lorenz K. P469 Lorenzen D. P882 Lorenzen S. P245 Losem C. P218, P797 Lotichius P. P209 Lotze C. P556 Lowdell M.W. P858 Lowe S. P198 Lowenberg B. V653 Loyd A.J. V74 Lübbert M. P828, V68, V457, V631, V772 Luber V. P459, P473 Lubrich B. V709 Lück A. V40 Ludwig H. P533, P534 Ludwig W.-D. V335 Luedders D.W. P544 Luft T. V110, V663, V768 Lüftner D. P543, P546, V907 Luley K. V448 Lunter A.-K. P477 Lupp A. P861, P875, V605 Lustig D. P276 Lutz C. V911 Lützkendorf J. V902 Lutz-Nicoladoni C. V687 Lux M.P. P546 Ma J. P254 Maag S. P509 Maas J.-H. P470 Maas S. P888 Mack T.S. P465 Mackensen A. P206, P510, P888, V594, V669, V901 Madle H. V664, V665 Mahlberg R. V430 Mähr B. P554 Maier J. V608 Mainka D. V603 Maintz C. V116 Maki R.G. V609, V611 Makino E. V643 Maksimovic O. V42 Malcherek G. P475, V932 Malenke E. P810 Malessa C. P228 Maley C.C. V941 Malfertheiner P. P565, V446 Mandoli A. V656 Mani J. P469, P475 Maniera C. P776

Author Index


Krüger S. V116, V603 Krüger W. P501 Kruggel L. V358 Kruis W. V116 Kruse C. V643 Kryselova N. V411 Ksienzyk B. V613 Kuball J. V114 Kube U. V37 Kuchenbauer F. P530, V577, V718 Kuchenmeister A. P851 Kufer P. V618 Küffer S. V77 Kügelgen B. V761, V762 Kuhfahl J. V908 Kuhlmann M. P502 Kuhn F. V351 Kuhn M. V749 Kuhn R. P847 Kuhn S.A. V640 Kührer I. P247 Kukreti V. V161 Kull M. P530, V577, V718 Kullmann F. P249, V388 Kumar K. V117 Kumar Das S. P868 Kummer W. P868 Kündgen A. V67, V770 Kundt G. V141 Küng M. V387 Künstlinger H. V603 Kuntz R. V44 Kunz C. V454 Kunzmann V. P547, V72, V599, V908 Küpferling S. V907 Küppers R. V662 Kurbacher C. V908 Kuriakose R. P778 Kurreck A. P548 Kurschat P. V661 Kurts C. V722 Kurtzer R. P197 Kuss I. V609, V611 Küster B. P481, P514 Kvainickas A. P180, V761 Kwon M. V114 Kyriazopoulou E. V44 Kzienzyk B. V655 La Guarde M. P541 La Rosée P. P487, V394 Ladetto M. V122, V576 Ladinek V. V401 Lamm W. P814 Lamparter A. V67 Lampe H. P236 Landgren O. V580 Lang A. P220, P242, P545 Lang F. P569 Lang H. P244 Lange A. P824, V439 Lange T. V444, V608, V652 Langer C. P530, V162, V577, V713, V718 Langerbeins P. P212, V743 Lanning R.M. V766 Laryionava K. P557, V398, V672 Laskowski U. V116

Author Index

Menck K. V595, V668 Menge F. P813 Mensen A. V934 Merkel O. P200, V747 Merkelbach-Bruhse S. V116, V603 Mertens P. P565 Merz B. V643 Merz H. P564, P487 Merz M. P526 Mesters R.M. P502, P570 Metz M. V767 Metzgeroth G. P794, V409, V452 Meyer A. P852 Meyer B. P501 Meyer R.G. P464, P466, V735 Meyerhuber P. V597 Michalka J. P803 Michel L.C. V642 Middeke M. P476, P507, V69 Middel P. V437 Mielke S. P459, P473, V400 Miething C. P198, P481, V658 Mikesch J.-H. P502 Mikolajewska A. P274 Milanovic M. V899, V925 Milkovic L. V600 Miltner E. P231, P862 Minck C. P812 Mineur L. P239, P240, V435, V436 Minichsdorfer C. P554 Minkov M. P808 Mischak-Weissinger E. P858 Mitterbauer M. V929 Mitterbauer-Hohendanner G. V616 Mitterer M. P220, V429 Mlineritsch B. P545 Mo Y. V937 Modest D.P. V388 Moehler M. P249, V388 Möhle R. P474, P505, P777, P783 Mohr B. V69 Mohring S. V668 Moi S. P888 Moll B. P520 Möller M. P527, P833 Möller P. V713 Möllmann M. P213, V591, V648 Molnar I. V621 Monahan J.E. P538 Monitillo L. V122, V576 Moosmann A. P888 Morche M. P196, P258 Moreau P. V579 Moreaux J. V165 Morgan S. V140 Morgner A. P802 Moroy T. V642 Moser P. P246 Mossmann P. V387 Mossner M. P182, V454, V452, V457 Möstl M. V36 Mottok A. P517, P519, V713, V731 Mougiakakos D. P206, V594, V669 Mousset S. V637 Moustafa Y. P864 Mrózek A. P852 Muchhal U. V686

Müdder K. V452 Mueller M. P488 Mügge L.-O. V162 Mühlenberg T. P809, P811, V607 Mühlfeld C. P868 Mulabecirovic A. V36 Mulaw M. V656, V657 Müllauer L. P219 Müller A. V440 Müller A.M. V730 Müller C. V33, V440 Müller E. P517 Müller F. V397 Müller G. V940 Müller L. V40, V41 Müller L.P. P471, P524, P784, V902 Müller M. P218, V728 Müller M.C. V313, V361, V363, V359, V360 Müller M.R. P505, P810, P817, P865, V42, V589, V746 Müller N. P794, V452 Müller S. V388, V901 Müller T.A. P171 Müller V. V622 Müller-Hagen S. V358 Müller-Leisse J. P876 Müller-Newen G. P490, V112 Müller-Schmah C. P785 Müller-Thomas C. V453, V767 Müller-Tidow C. P166, P169, P173, P187, P188, P502, P822, V72, V115, V654, V657, V750, V763 Mullighan C. P198 Munder M. P873 Munn L.L. V766 Murawski N. V33, V34, V35 Muth M. P543, P546 Na I.-K. V934 Nachbaur D. V67, V68 Nachtkamp K. V443 Nagaraj N. V410 Nagel F. P875 Nagel I. V615 Nägele V. V618 Nagelmeier I. V437 Nan J. P469 Nauck F. V22 Navarrete M. V125 Nebe C.T. V156 Neben K. P503, P526, V165, V714 Necke M. V898 Neesen J. P792 Nerger K. V902 Nerl C. V359, V363 Nerreter T. V928 Nestorova V. V666 Neubauer A. P170, P172, P555, V72, V359, V361 Neubauer M. P221, P535, V699 Neuhaus P. V449 Neukirchen J. V770, V772 Neumann C. P548 Neumann M. V27, V759 Neumann M. V613 Neumann S. V394


299


Manjara I. P799 Manley P. V719 Mann A. P875 Mannhalter C. P191, P219, V616, V773, V936 Manns M.P. V447 Mantoan B. V122 Manzl C. P500 Marciniak-Czochra A. P791 Marcucci G. V652 Mardan P. V643 Mardi D. P196, P258 Mardiak J. P254 Marino-Enriquez A. P809 Märklin M. P865, V589, V746 Markowetz J. V434 Marks R. P495 Markus P. P855, V390 Marosi C. P838, P839, V352 Marschner N. P528, P842, V40, V41, V118, V354, V355, V358, V391, V450, V712, V890 Martens J.H.A. V656 Martens U. P853, V44 Marth C. V908 Martin J.D. V766 Martin T. V161, V163 Martini C. P833 Martowicz A. P246 Marx A. P177, V452 Marx G. V22 März W.J. P568 Maschmeyer G. P537, V349 Mason W. V350 Massenkeil G. V113 Mattenheimer K. V731 Matter K. V387 Mattonet C. V120, V603 Matzdorff A. V11 Maurberger A. P510, V901 Maurer A. P199 Maurer C. V743 Maurer H. V752 May A. P527, P833 May A.M. P527 May T. P878 Mayer F. V411, V448 Mayer J. P803, V644 Mayer K. P273, P280/1, V396 Mayer P. P204 Mazier A. P777 Mazzoleni G. P246, V906 McCulloch L. V163 Mead G. V410 Mechl Z. P803, V644 Medyouf H. V452 Meggendorfer M. V455 Mehnert K. P556 Meiler J. P855, V390, V434 Meincke M. P217, P218 Meintker L. P270 Meissner J. P503 Meißner T. V160, V165, V578 Melchardt T. V123, V404 Melinz K. V699 Melnick A. V937 Memmer M.-L. P486

Oberndorfer S. V352 Obiditsch M. V675 Obländer J. V452, V454, V457 Obrist P. P246 O'Bryan-Tear G. P259, P260, V76 Ochs C. V934 Ochsenreither S. P881 Ockert D. P244 Odendahl M. P476 Oduncu F. P779, P808 Oechsle K. V411 Oefner P. V111 Oelschlägel U. V69 Oettle H. V449, V450, V908 Oexle H. P220 Offit K. P198 Öhler L. P248, V721 Okoye-Okafor U.-C. V937 Okun J. V110 Oles M. P200 Olivo M. V356, V357 Olschewski H. P224 Olsson H. V49

300

Omlin A.G. V420 Oostendorp R. V764 Opalka B. P213, P878, V653, V898 Opatz S. V655 Opeker K. V709 Opferman J. V715 Oppel F. P232 Oppliger Leibundgut E. V720 Orchard K.H. P778 Ordemann R. P507, P567 Orlowski R. V161 Ortner P. P275 Osburg S. V27, V757, V759 Oskay-Özcelik G. V907 Ossenkoppele G. V362 Ostřížková L. V644 Ostermann H. P276, P857, V162, V710 Ostermann J. P276 O'Sullivan J.M. P260 Oswald E. P821, V601 Oswald F. V656 Otremba B. P209, V354 Ott G. V32 Otte M. P249 Otte P. V709 Ottillinger B. P272 Ottinger H. P885, V115, V440, V442, V445 Ottmann O. P218, V615 Otto T. V415 Overkamp F. V37, V40 Pachmann K. P539, P540, P874 Pachmann U. P539, P540, P874 Pacini F. V621 Paczulla A. V897, V905 Padera T. V766 Padur W. P856 Pahernik S. P253 Pahl H. P225, V724, V761 Pallasch C.P. P203, V592, V645 Palmer M.R. P538 Palumbo A. V576 Pannicke U. V696 Panse J. V116, V120, V603, V698 Pantel K. P174, V622 Pantic M. P527, P833 Papachristou I. V119 Papasotiriou I. P229, P879 Papendorf F. V447 Parker C. P259, P260, V73, V74, V76 Parmentier S. P476, P567, P820, V72 Parren P.W.H.I. V683 Parsons S.L. P887 Paschka P. V67, V68, V630 Paschke R. V621 Pascolo S. V42 Pasemann S. V669, V901 Passlick B. P833 Paul A. P855, V390 Paulduro A. P561 Pauligk C. V448 Pearce K.F. P858 Pechloff K. V660 Pecnard E. V939 Peiffer L. V593 Peil-Grun A. P555 Peine A.-C. P849


Peipp M. P513, P515, P883, V438, V683, V686, V701, V716 Pellagatti A. V768 Penack O. V934 Penninger J.M. V687 Penzel R. V714 Pepin F. V576 Perez E.A. V356, V357 Perner F. P465 Perner S. P514, V905 Peschel C. P481, P514, V453, V608, V658, V659, V660, V900 Peter B. P511, V705, V719, V936 Peter S. V392 Peters M. P538 Peters P. V682 Peters U. P852 Petersen I. P487 Peterson L. P276, V392 Petersson I.F. V49 Petrides P.E. P222 Petru E. P545 Petrylak D. V117 Pettengell R. P491, P492 Petzer A. P220, P569 Peyn A. V47 Peyrade F. V127 Pezzutto A. P852, V729 Pfaff H. V27, V759 Pfeifer D. P527, V651 Pfeifer H. V615 Pfeifer M. V664, V665 Pfeilstöcker M. P167, V89, V772 Pfirrmann M. V359, V361, V362, V363 Pfister D. P262, P264, P265, P266, P267, P268, V39, V75, V78, V412, V414 Pfreundschuh M. V31, V32, V33, V34, V35, V359, V361, V363 Pfründer D. P209 Pham A. V720 Pham M. P277 Pichler B. V905 Pichler M. P484 Pichler U. P205 Pickert A. V44 Pilarsky C. P235 Piñón J.D. V650 Pinon Hofbauer J. V747, V748 Piper C. P262, P264, P265, P266, P267, V39, V75, V78 Pircher A. P500, V606 Pircher M. P545 Piribauer M. P504 Piringer G. P242 Pirker R. V4 Pittrow D. V711 Pizon M. P539, P540, P874 Placher-Sorko G. P571 Plass C. V631, V656, V657 Plath T. V348 Platzbecker U. P507, P787, P792, P793, V69, V91,V332, V408, V457, V642, V698, V767, V772 Platzek I. P820 Pletsch N. V359 Ploenes T. V604 Pober M. P220

Author Index


Neumeister P. P460, P484 Neureiter D. P571 Nguyen T.T. P489 Nguyen-Tat M. V446 Niederfellner G. P252, V641 Niedergethmann M. V449 Niederle N. V389 Niedermann G. V601 Niederwieser D. P520, V440, V444, V608, V652, V938 Nielsen T. P550, V141 Niemeyer C. V769 Niemeyer E. V766 Niesvizky R. V163 Nietert M. P245 Nietsch J. P275 Nijhuis M. V114 Nikolova V. P481 Nilsson S. V73, V74, V76 Nilsson U. P234 Nimmrich I. P826 Nishida T. V609, V611 Nishikawa R. V350 Nitschke-Gérard C. P568 Nitzsche A. V27, V759 Nocera D.G. V766 Noesslinger T. V36 Nogai H. V664, V665 Nogová L. V116, V119, V120, V603 Nold S. P530 Nolte F. P183, P787, P793, P794, P795, V452, V454, V457, V767 Nooka A. V163 Nordlinger B. V386 Noreen D. V49 Noriega J. P780 Notheis G. V441 Nowak D. P177, P182, P183, V452, V454, V457 Nowak V. P183, V452 Nowosielski M. V352 Nuebling T. V71 Nutting C. V621

Author Index

Rajangam K. V161, V163 Rammensee H.-G. P202, P215, P463, V42, V706, V753 Randerath W.J. V116, V603 Ranjan S. P465 Rao A. V589 Rasp K.-H. P176, P190 Rath P. V393 Rauh J. V389, V712 Rautenberg B. V709 Rawat V. V656, V657 Rawluk J. P833 Řehák Z. P803 Rebmann V. P878 Rechberger E. P569 Reddehase M.J. V933 Regitz E. V32 Rehli M. V769 Reichardt P. V609, V610, V611 Reichel K. P550, V141 Reichert D. P218 Reichle A. P238, P807, P867, V162, V923 Reijden B. V653 Reimann C. P861 Reimann M. V899 Reimer D. V904 Reimer P. V390 Reinel H. V386 Reiners K.S. V647, V685 Reinhardt H.C. V624, V924 Reinhardt H. P527, P851, V709 Reinhardt M. P830 Reinold S. V606 Reinwald M. P269, V393 Reis H. P538, V434 Reiser M. V116, V603, V711 Reiter A. P223, V409 Reiter C. P514 Reiter M. V745 Reitter S. P224 Remane Y. P520 Rème T. V165 Rensing-Ehl A. V696 Rentsen E. P227 Repp R. V683 Ressler S. P545 Rethwisch V. V349 Retz M. P253 Reu F. V161 Reusch U. V685 Reuss-Borst M. P847 Reuter C. P255, V38 Rey J. V406 Reyher-Klein S. P812 Rhode C. V657 Riabinska A. V924 Richly H. P538 Richman D. V114 Richter D. P551 Richter S. P820 Rickmann M. P255 Ridwelski K. V449 Riecken K. P174, V622 Riedel S. V713 Riedl E. V452 Rieger C. P276 Rieger H. P276

Rieger M. P210, P485, V124 Riese C. V26, V755, V756 Riess H. V449 Riethdorf S. V622 Riewerts F. P505 Ringhoffer M. V67, V162 Rinke J. V617 Rinnerthaler G. P545 Rissiek A. V744 Ritgen M. P210, V576 Ritter P. V671 Rittig S.M. V42 Ritz M. V731 Rix U. V719 Rixecker T. V33 Rizzi M. V696 Robak O. V675 Roberge S. V766 Robier C. P221, P535, V699 Robinson S. V31 Rochau U. P220 Rody A. P544 Roedder P. P850 Roeder I. V364, V728, V749 Roehl H. V452 Roers A. V732 Roesel J. V719 Rohde C. P166, P169, P187, P822, V750 Rohde H. V392, V394 Rohlfing S. P482, V663 Roigas J. P256, P257 Rojewski M. V702 Roller A. V455 Röllig C. V69, V72, V162, V674, V749 Rommel K. P469 Roscher M. P862 Rosenfeldt M.T. V126 Rosenhahn A. P774 Rosenwald A. P517, P519, V917 Rosien B. P871 Rösler W. P888, V162 Ross R.S. V442 Rossi D. P200 Rössler K. P839, V352 Rössler L. P248 Rößler S. V48 Rost S. P213, V648 Roth A. V387 Roth N. P463 Röth A. P801, V698 Rothe A. V685 Rothfuß O. V643, V905 Rothmann F. P537 Rox J. P780, P782 Royle L. V438 Rübe C. V34, V35 Rubin B. V607 Ruch M. V709 Rücker F. V577 Rücker-Braun E. P476 Rückert F. P235 Ruckser R. V352 Rudas M. V353 Rudelius M. P481, P514, V724 Rudolph N. P523 Rudolph R. V642 Ruhnke M. P274, V394


301


Pobiruchin M. P853 Pöchlauer S. P796 Podar K. V715 Podlech J. V933 Podleska L. V612 Pogge von Strandmann E. V647, V685 Pohl S. V769 Pohlen M. P570 Poljak A. V589, V746 Poll-Wolbeck S.J. P201, V593, V646 Polzer H. V655 Ponath E. V745 Pönisch W. P520, P523, V652 Popescu R. V387 Popper H. V606 Porres D. P262, P264, P265, P266, P267, V39, V75, V78, V402 Port M. V698 Pöschel V. V33 Possinger K. P548 Postina P. V393 Potenberg J. P549, P812 Pott C. V122, V576 Pöttgen C. V612 Potzner M. P826 Pour L. V644 Prayer D. P839 Prenzel R. P823, P827, P830 Prenzler A. P824, V439 Preusser M. P836, P839, V353 Price T. V386 Prinz C. P204 Pritzlaff N. P508 Proetel U. V359, V361, V363 Pröll J. P789 Promberger R. V353 Prosch H. V739 Proschmann R. V364 Protivánková M. V644 Prüfer S. P805 Publicover A. P778 Pukrop T. V595, V598 Pullwitt J. V47 Purfürst B. V899 Putz E. V928 Qianli J. V750 Quack H. P245 Quecke T. P863 Quidde J. P843 Quintanilla-Fend L. V656 Quintanilla-Martinez L. V905 Raab H.-R. P244 Raab M.S. P526, V714 Raab S. P199, P800 Rabenhorst A. V661 Rabitsch W. V675, V929, V935 Rachlis E. V443 Rachner T.D. V596 Rachow T. P280/1, V396 Raderer M. V281, V288 Radsak M.P. P805, V732 Radujkovic A. V768 Raff T. V613 Raftopoulos H. V117 Raguse J.D. V348

302

Scheffler M. V116, V119, V120, V603 Scheibelhofer J. P191 Scheibenbogen C. V934 Scheid C. P212, V160, V578 Scheidereit C. V665 Scheithauer W. P247, V388 Schelker R.C. V940 Schellongowski P. V675 Schemionek M. P215, V654 Schepler H. P193 Scherer A. V22 Scherer E. P785 Scherer F. V125 Scherer R. V447 Scherer S. P821 Scherwitz P. P230 Schetelig J. P476, P507, V31, V48, V69, V72, V408, V749 Scheu F. P524 Scheulen M.E. P538 Schieferdecker A. V744 Schiel X. P779 Schilbach K. P252 Schild H. P805, V732 Schildhaus H.-U. V116, V119, V603 Schildmann E. V670 Schildmann J. V337, V670, V671, V673 Schiller J. P212, V70 Schilling K. P468 Schiltz D. P279 Schima W. P248 Schimanski C. P249, V389 Schinköthe T. P279, P849, P850, V431 Schirmacher P. V714 Schirrmacher-Memmel S. P253 Schittenhelm M.M. P176, P178, P179, P189, P190 Schlaak M. V661 Schlaeth M. V686 Schlag R. P528 Schlee C. V613 Schlegel M.-A. P883 Schlegel R. P538 Schlegelberger B. P790, V68 Schlenk F. P869 Schlenk R. V67, V68, V457, V630, V772 Schlesinger A. V116, V603 Schlesinger M. P233 Schletter L.M. P784 Schlichting A. P840 Schlick K. P571 Schlimok G. V754 Schlögl E. P220, V404 Schlösser H. P230, V661 Schlumberger M. V621 Schlüter D. P271 Schmalfeld M. P520 Schmalfeldt B. V908 Schmelz H. P268, V412, V414 Schmick A.N. V598 Schmid C. V31, V333, V754 Schmid I. V441 Schmid K.W. P538, P855, V119, V434 Schmid P. P779 Schmidberger A. P471 Schmidt B. V453, V698 Schmidt C.A. P501


Schmidt E. P502, P822, V750 Schmidt F. V734, V930 Schmidt G. V602 Schmidt K. V110 Schmidt M. P232 Schmidt P. V386 Schmidt R. P231 Schmidt S. P220 Schmidt V. V709 Schmidt-Graeff A. V125 Schmidts A. V761, V762 Schmiedel B. P199 Schmiedl N. V717 Schmitt A. P469, P475, P778, V932 Schmitt C.A. P480, V126, V899, V925 Schmitt M. P469, P475, P778, V581, V684, V932 Schmitt T. P464 Schmitz J. V37 Schmitz M. P476 Schmitz N. P477, V33, V34, V35 Schmitz S. P797, P798, V116, V120, V389, V603 Schmoll H.-J. P243, P249, P278, P524, P784, P815, P818, V386, V902 Schmoll J. P196 Schmollinger J.C. V641, V682 Schmölzer J. V650 Schnabl S. V745 Schnallinger M. P220 Schnattinger T. V657 Schneeweiss A. P546 Schneider F. V710 Schneider J. P204, V902 Schneider N. V658 Schneider P. P212 Schneider S. V655 Schneider V. V684, V702 Schneiderat S. P815 Schneider-Kappus W. P218 Schnell R. V116, V120, V603 Schnerch D. V751, V761, V927 Schnetzke U. P168, P280/1, V396 Schnittger S. P497, V359, V361, V455, V614 Schnitzler P. V581 Schnoeder T.M. P465 Schober-Melms I. P474 Schobert R. P204 Schoeffmann S. V900 Schöffski P. V609, V611 Scholl S. P168, P280/1, P468, P487, V396 Schollenberger L. P249 Scholtysik R. V662 Scholz C.W. P852 Scholz M. V388 Schönefeldt C. P787 Schönfelder H. V78 Schöning T. P482, P489 Schönland S. P522, V663 Schopohl D. P857 Schott E. V446 Schouten H.C. V31 Schrader A.J. V73 Schrader K. P198 Schrader M. P268, V412, V414 Schramm W. P853

Author Index


Ruland J. V660 Rülicke T. V935 Rummel M. V43, V123 Rupp C. P191 Rusch M. V592 Rüschoff J. V437 Rusner C. V413 Rüssel J. P243, P278, P524, P815, P818 Ruthardt M. P177 Rutkowski P. V609, V611 Rycak L. P170 Sack U. V158 Sadovnik I. V616, V935, V936, V939 Saffrich R. V765 Sahin U. V889 Šálek D. P803 Salazar P. V117 Saletti P. V387 Salih H. P179, P199, P202, P215, P800, P865, P880, V71, V706, V753 Salinas-Riester G. P792 Salitzky O. P179 Sallaberger E. V36 Salloch S. V671 Salwender H. V160, V578 Sam I. V700 Samonigg H. V401 Sand C. P258 Sandlund J. P198 Sandner R. V355 Saran F. V350 Sartor O. V73, V74, V76 Sattler M. V715 Sauer A. P217, P218, P798 Sauer M. V647, V685 Sauer S. P277, P485, P526 Sauer T. P188 Sauerland C. P183, P188, V655 Saußele S. V359, V360, V361, V362, V363 Sawall S. V622 Sax C. P838, P839 Sayer H.G. P468 Schabath R. P497 Schacher S. V387 Schäd-Trcka S. P506 Schaefer R.M. P860 Schaefer U. P859 Schäfer B. V388 Schäfer N. V351 Schäfer V. V617, V731 Schäfer-Eckart K. V72, V162 Schaffer S. P888 Schafhausen P. V363, V392, V394 Schaich M. V69, V72, V749 Schaider H. P484 Schairer R. V905 Schakols K. V444 Schalk E. P271, P565, P819, V394 Schambye H. P234 Schanz J. P792, P793, V456, V767 Schardt C. P241 Schaub F. P783 Schauwecker J. V453 Scheding S. P858 Scheede C. P847 Scheffler J. P506

Author Index

Seggewiß-Bernhardt R. V928 Sehouli J. V908 Seidel C. P255, V38 Seifart U. P555, V148 Seifarth W. P177, P214, V393 Seifert H. V392, V397 Seifert M. V662 Seiffert M. P200 Seilbeck A. V940 Seipelt G. V388 Seitz V. V665 Selak E. V712 Sellinger S. P841 Sellner L. P200, V688 Semmlinger A. P884 Sender A. P551 Sengpiel J. V642 Serke M. P826, V116, V120, V603 Serve H. V72, V749 Settmacher U. P228 Severin K. P798, V116, V603 Sexl V. V936 Shah S. P198 Shan M. P260 Shehata M. V745 Sherman S.I. V621 Shimabukuro-Vornhagen A. P230, V395, V661 Shirneshan K. P792, P793, V457, V767 Shizuru J.A. V730 Shlyakhto V. V361 Shong Y.K. V621 Siebert F. P504 Siebert R. V615 Siebert U. P220 Siedentop H. V621 Siegel D. V161, V163 Siegel F. P222 Siegmund B. P877 Siena S. P239, P240, V435, V436, V621 Sifft E. V747 Sill H. V616 Sill M. P200 Silling G. V115, V394 Simmonds P. V410 Simon A. P512 Simons R. V356 Simonsson B. V362 Sinn M. V449 Sironi S. P175 Sitter S. P485 Sivasubramaniyan K. P251, P252 Siveke J.T. P249 Sjövall K. V49 Sklenarova H. V398 Skottky S. P538 Slabicki M. P200 Sliwa T. P184, P185, P186, P191, P192, P220 Slotta-Huspenina J. P514 Šmardová L. P803 Smit J.W.A. V621 Smith J. V405, V406 Sobrero A. P239, P240, V435, V436 Sockel K. P476, P567 Sohlbach K. P555 Sökler M. P505, V363

Somlo G. V161 Sommer M. P487, V610, V735 Sopper S. V904 Sörensen A. V45 Sos M.L. V119 Spadaro S. V709 Späth D. V67, V68 Specht E. V605 Specht K. V608 Sperr W. P192, P219, V616, V675, V705, V772, V935, V936 Spiekermann K. P183, V359, V361, V363, V655, V754 Spiess B. P269, V393 Spies-Weißhart B. P168, P468 Spillner E. P477 Spinner S. V660 Spizzo G. P246, V429, V906 Sporrer D. V111 Sprenger T. P245, V437 Spring L. V355 Sprissler C. V752 Sproßmann-Günther G. P549 Sprüssel A. V763 Stadler R. V661 Stadtherr P. P210 Stadtmauer E. V161 Staebler A. V905 Staehler M. V37 Staib P. V394 Stam A. V114 Stang A. V413 Stange T. V749 Stangel M. V711 Stark D. V140 Stassen M. P805 Stauber R. V446 Stauch K. P254 Stauch M. V388 Staudacher K. V74, V76 Stauder R. V700, V772 Staudinger M. P515, P516, P883, V701, V716 Staudinger T. V675 Steckel N. P458, P775, P776, V115, V407, V442, V445, V625, V703 Stefanzl G. P219, P511, V705, V719, V936 Steffen J. V641 Stegelmann F. P218, V359, V361 Steger G.G. V353 Steger U. P244 Stegmayr J. P234 Steguweit H. V661 Stehle M. P214 Steidl U. V937 Stein A. P843 Stein H. P498, V47 Stein M. V363 Stein P. P805, V732 Stein T. P841 Steinbach G. P530 Steinbrunn T. P517, P519, V717 Steinemann D. P790 Steiner A. P834 Steiner G. V37 Steiner N. P518 Steiner T. P256, P257, V37


303


Schraven B. P465 Schreiber A. V359, V363 Schrezenmeier H. V156, V440, V696, V698, V702 Schröder S. P514 Schröder T. V642 Schroeder M. V349, P859 Schroeder M.L. V696 Schroeder T. P270, V443, V770 Schruckmayer G. V700 Schub A. V618 Schub N. P472, P786, V701 Schubert J. V361, V363 Schubert S. V392 Schuetz F. P543 Schuler M. P538, P809, P811, P820, P855, V119, V390, V434, V607, V612 Schuler U. V674 Schüler J. P821, V601, V651, V751, V762 Schulte J.H. V763 Schulte K. V772 Schulte W. V603 Schultens A. P196 Schultheis B. P269 Schultze A. P174, V622 Schulz A. V645 Schulz C.-O. P548 Schulz C. P271, P565 Schulz H. V116, V603 Schulz K. P506, V394 Schulz S. P861, P875, V605 Schulz U. P238, P861 Schulze C. V744 Schulze E. V908 Schulze I. P169, P188, P822, V750 Schulze M. V450 Schulze-Bergkamen H. V715 Schulze-Osthoff K. V643, V905 Schumacher B. P204 Schumacher G. V374 Schumacher M. V579 Schumann C. P795, P826 Schumm M. P461, P474 Schur F. V939 Schur S. P814 Schuster C. P787 Schuster H. P202, V706 Schütte U. P233, P234, P250 Schwaab J. P223, V409 Schwartz S. V394, V613 Schwarz K. V696 Schwarzenbach H. V622 Schwarzer A. P520, P523 Schwarzer G. P523 Schweiger B. V581 Schweitzer N. V447 Schwella N. V45 Schwind S. V444, V652 Schwinger U. P218 Schwinn S. V713, V731 Scriba D. P823, P827, P830 Sebban C. V31 Sebesta M. V353 Seckinger A. V160, V165, V578 Seeber A. P246, V906 Seeger J. V645 Seeger S. P886

304

Szturz P. P803, V644 Szuszies C.J. P467 Tabatabai G. V620 Tabernero J. P239, P240, V436 Taipaleenmaeki H. V622 Tanev I. P565 Targosz B.-S. P481, P514 Tari S. P562 Taromi S. V602 Tauscher J. P222 Tawadros S. V647 Tebbe S. P217 Tebernero J. V435 Teichgräber U. P487 Teichler S. P172 Teichmann B. P532, P797 Teleanu V. P530, V577, V718 Terracciano L. V906 Terwey T.H. V113 Tesch H. P217, P272, P543, P546, V354, V355, V358, V365 Tessen H.-W. P263, P825, P840, V118, V450 Tewes M. P538 Thaler J. P220, P242, P545, V404 Thalhammer T. P226, P227 Theiler G. P221, P535, V699 Theobald M. P464, P466, P560, P873, V735, V933 Theocharous P. P491, P492 Theophil F. P863 Theurich S. P203, P212, P230, V661 Thiede C. P459, P507, V72, V364, V408, V653, V749 Thiel M. P872 Thiel V. V656 Thiele B. P508 Thilo N. V47 Thiruchittampalam D. P501 Thissen A. P262, P265, P266, P267, V39, V75, V78 Thissen A.K. P264 Thoennissen G.B. P502 Thoennissen N.H. P502 Thol F. V67, V68 Thomalla J. P208, P845, P846, P848, P854, V760 Thomas M. P826 Thomas R. V116, V119, V603 Thomas S. V735, V931, V933 Thomay K. P790 Thome M. V665 Thomssen H. V47 Thudium J. V754 Thuss-Patience P. P249 Tickenbrock L. P173 Tiegs G. V744 Tiemann M. P196, P823, P829, P830, P831, P832, V121 Timms A. P198 Tinchon C. V36 Ting S. P538, V390, V434 Tinhofer I. V347 Tischer J. V441 Titgemeyer J. P217, P218 Titze U. P570 Todorova T.I. V937


Tolosa E. V744 Toloudi M. P229, P879 Tometten M. P855, V390 Tomka M. P504 Tonn J.-C. V305 Tonn T. P476 Tonon G. V715 Töpelt K. V116, V120, V603 Topolar D. V647 Topp M.S. V618 Tran L. V395 Trarbach T. P244, P855, V390, V434 Trautmann H. V122, V576, V615 Trenschel R. V115, V407, V442, V445, V625 Trepel M. V744 Tripp C.H. V687 Trittler R. P851 Trneny M. V31 Troch M. P571 Trost B. P786 Trudel S. V161 Trummer A. P255, V38 Trümper L. P467, P478, P479, P494, P496, V77, V668, V767 Trumpp A. V452 Tschanter P. P169, P188 Tscherkes A. P818 Tschiedel S. V938 Tschmelitsch J. P242 Tschurtschenthaler G. P569, V662 Tunger A. P476 Tuve S. P476 Twelves C. V356, V357 Tzankov A. V664, V665 Übelhart R. V651 Uckert W. V597 Ullmann A. V394 Ulmer H. P545 Ulrich W. P184, P185, P186, P834 Ulrich-Merzenich G. P864 Ulshoefer T. P272 Ulsperger E. P841 Ungefroren H. P863, P871 Unger A. V591 Unteregger M.J. V36 Untergasser G. P518 Unverzagt S. V902 Vajen B. P790 Vajkoczy P. V640 Valdix A.-R. P840 Valent P. P167, P192, P219, P220, P511, V616, V705, V719, V935, V936, V939 Valerius T. V438, V683, V686 Valk P. V653 Vallet S. V715 Van Cutsem E. P239, P240, V386, V435, V436 Van de Winkel J.G.J. V683 Van der Burgt M. V125 Van der Kuip H. V926 Van Erps T. V402 Van Gool R. V74 Van Roye C. P208, P845, P846, P848, P854, V760

Author Index


Steinle A. P800 Steinmetz T. P798, V116, V390, V603, V698, V712 Stelljes M. V115 Stemmler H.J. V754 Stenzel I. P863 Stenzl A. P251, V42 Stern S. P218 Sternfeld T. P499, P558, P808, P844 Stettin M. P221 Steuber T. V74, V76 Steudel N. V450 Steurer M. P500 Stevanovic S. P202, P215, V706, V753 Stewart A.K. V161 Stickel J. P202, P215, P817, V706, V746, V753 Stickeler E. V709 Stiegler R. P520 Stiehl T. P791 Stiewe T. P170, P172 Stift J. P247 Stilgenbauer S. P211 Still H. P460 Stintzing S. V388 Stippel D.L. P230 Stöbel-Richter Y. P551 Stoehlmacher-Williams J. V349 Stöhlmacher J. P244 Stoitzner P. V687 Stolz T. P200 Stölzel F. V48, V69 Storek B. V664 Straka C. V718 Stranzl N. P247 Strasser A. V660 Strasser U. P500 Strasser-Weippl K. P533, P534 Strauß A. P259 Strauss U.P. P254 Strecker K. V404 Streubel B. V935, V936 Strifler S. P531 Ströbel P. V77 Strohm P. P833 Strölin P. V74, V76 Stromberger C. V347 Stropiep U. P823 Strupp C. V770 Stübler J. P248 Stühmer T. P517, P519, V717 Stunnenberg H.G. V656 Sturm I. V162 Stutz M. V720 Styczen H. V437 Südhoff T. P244 Suessner S. P789 Superti-Furga G. V719 Suppan C. P541, V600 Surmann B. P188 Sustmann C. V641 Sutanto J.H. P873 Suttorp M. V364 Svanidze E. V734, V930 Symons J. V114 Szibor-Kriesen U. P559, P835 Sztankay M. V403

Author Index

Wagner W. P791, V695 Waizenegger J. V622 Wajant H. V731 Waladkhani A.R. V430 Walczuk E. P806 Walder A. P220 Waldmann H. V592 Waldschmidt D. V450 Waldschmidt J. P512 Walenda T. P791 Walewski J. V31 Waller C. V359, V361 Wallner M. P205 Wallner S. V687, V722 Wallwiener D. P546, V905 Walpinski I. P238 Walser M. V451 Walter M. P179, P189 Walther J. V451 Wanders J. V356, V357 Wang H. V643, V905 Wang L. P475 Wang M. V161 Wang X. P509 Warsch W. V936 Wäsch R. P180, P512, P521, P527, P785, P833, V164, V579, V580, V751, V761, V762, V927 Wäscher S. V671 Wattad M. V68 Weber M. P560, P805, V732 Weber S. P213, V648, V763, V898 Weber T. P471, V394 Weber W. V602 Webersinke G. P569 Weckesser M. P570 Wedding U. P487, V101 Wedeken K. P827 Wehler T. P560 Wehmeyer J. P797 Wehner R. P476 Wehrle J. P225, V604 Weichert W. P232 Weide R. P208, P218, P845, P846, P848, P854, V28, V760 Weidner C.I. V695 Weigel A. P274 Weikert S. V37 Weilbacher A. V926 Weimer K.J. V42 Weinzer M. P841 Weis J. V586 Weisel K. P505, P525, P536, P777, V160, V578 Weiß C. P826 Weiss H. P504 Weiss L. V123, V650 Weiss N. V48 Weißer A. P804 Weissinger F. P249 Weitschies W. V707 Weitz J. P232, P235, P244 Wellbrock J. P174 Weller M. V425 Welslau M. P218, V756, V908 Welt A. P538 Weltermann A. P545, V353

Wendtner C.-M. P203, P211, V369, V592, V645, V649, V743 Wennhold K. V661 Wensing A. V114 Wenz F. V302 Wenzel D. P478 Wenzel F. P780, P782 Wenzel S.-S. V664, V665 Wermke M. P787 Werner D. V448 Werner J. P232 Werner K. P235 Werner M. P527, P833 Wesselmann J.S. P211 Wessels L. P868 Wessig C. P531 Wessling J. P502 Weßling M. P273 Wester H.-J. P861 Westermann A. P211 Westermann J. V729, V934 Wetzko K. P507 Wheater M. V410 Wick W. V350 Wickenhauser C. V608 Wider D. P512, P527, P833, V751, V761, V762 Widhalm G. P836, P838 Wiehr S. V905 Wierecky J. V712 Wiesinger K. P535, P569, P789 Wiesneth M. V684, V702 Wiest R. P238 Wiesweg M. P538, P855, V119 Wijdenes J. V716 Wild J. P199, P865 Wilde P. V406 Wildgrube R. P471 Wilhelm S. P263, P825 Wilke H. P249 Will B. V937 Will S. V393 Willborn K.-C. P823, P827, P830 Willenbacher E. P500, V404 Willenbacher W. P500, P518 Willmann M. V935 Willmitzer L. P480, V899 Wilop S. P490 Wimberger P. P887 Windhager R. P814 Winkler E. P557, V337, V398, V672 Winkler J. P888 Winter C. P268, V412, V414 Winter G. V719 Winterberg T. P561 Winterhalder R. V387 Wintner L. V403 Wirth T. V696 Wirths S. P810, V589, V746 Wirtz M. V27, V759 Wirtz R.M. V605, V889 Wisplinghoff H. P270, V395, V397 Wittke C. P194 Witzens-Harig M. P277, P482, P485, P486, P489, P503, V34, V35, V124, V663 Witzke V. V622 Wlodarski M. V769 Woermann B.J. V655


305


Varga F. P167 Vasold J. P175 Väth M. V731 Veelken H. V125, V651 Vegi N.M. V656 Vehling-Kaiser U. V389, P272, P499, P558, P808, P844 Vehreschild J. P270, V393, V395, V397 Vehreschild M. P269, P270, V392, V394, V395, V397 Velikova G. V356 Venkataramani V. P478, P479, V77, V668 Verhling-Kaiser U. V388 Verma A. V937 Vesely M. P184, P185, P186 Viala A. P801 Viallard J.-F. V406 Viardot A. V34, V35 Vietzke R. P555 Vij R. V161, V163 Vijay J. P198 Viol F. P508 Vogel A. P239, V439, V447 Vogel C. V447 Vogel D. V668 Vogel W. P461, P462, P474, P505, P566, P817, V392 Vogelhuber M. P238, P807 Vogelzang N. P259, P260, V74, V76 Vogl U.M. P248 Vogt J. V116 Vogt M. V40, V41, V712 Voigt K. P870 Voigt M. P508 Volin L. V31 Vollmann J. V671, V673 Von Bergwelt-Baildon M. P203, P212, P230 V395, V661 Von Bonin M. P787, V72 Von Bubnoff N. P837, V608 Von Campe G. V352 Von Deimling A. V714 Von Kalle C. P200, P232, V127 Von Lilienfeld-Toal M. P273, P280/1, V392, V638 Von Mehren M. V609, V611 Von Moos R. V387, V410 Von Pawel J. P826 Von Tempelhoff G.-F. V908 Von Verschuer U. P253, V41, V118, V389, V642 Von Wichert G. V389 Vormittag L. P248 Vosberg S. P790, V613, V655 Vucinic V. V444, V652 Vuong G. V113, V934 Waanders E. P198 Wachsmuth-Melm M. P466 Waeg G. V600 Wagner A. P239, P240, V110, V435, V436 Wagner B. P878 Wagner E.M. P466 Wagner K. P228, P870 Wagner M.C. P465 Wagner M. P175 Wagner T. V389

306

Yang C. P509 Ychou M. P239, P240, V435, V436 Yelle L. V356, V357 Yildirim S. P783 Yomade O. P280/1, V396 Yonekawa Y. V351 Yoshino T. P239, P240, V435, V436 Young P. P530 Yu C. V704 Yu Y. V126, V899, V937 Zabernigg A. P242, V403 Zabernigg G. V429 Zabieglinski T. P547, V599 Zaborsky N. V748 Zach O. V353 Zago M. P525, P536 Zahriychuk O. V404 Zaiss M. P253, P797 Zamora P. V26, V756 Zander T. V116, V603 Zasada C. V899 Zeimet A.G. V904 Zeis M. P477 Zeiser R. V602, V733 Zellmeier E. V655 Zellmer L. P561 Zemanova M. P254 Zeng Y. V682 Zenger M. V455 Zenke M. V695 Zens B. V452 Zenz T. P200, P210, V127, V688, V714 Zepeda-Moreno A. P181


Zesewitz M.-L. V648 Zettl H. P550, V141 Zey A. P211 Zeynalova S. V33, V34, V35 Zhang L. V702 Zhang W. P509 Zho S. P530 Zhou F. V657 Ziad I. P274 Ziegler P. P490, V112, V695 Zielinski C. P554, P836, V353 Ziepert M. V32, V34, V35 Zierler S. V650 Zietz C. P507 Zimmermann A. V757, V758 Zimmermann K. V925 Zimmermann Y. V666, V667 Zimmer-Roth I. P841 Zimon D. P539, P540, P874 Zink M. V45 Zinke-Cerwenka W. P460 Zipperer E. V770 Zirlik K. P200, P207, V651 Zirm E. P168 Zloklikovits S. V401 Zober A. P495 Zoellner A.-K. V666, V667 Zoerer M. V404 Zonder J. V161 Zopf A. P789 Zou J. V76 Zugmaier G. V618 Zülke C. V449 Zwick C. V33, V34, V35

Author Index


Wohlfarth P. V675 Wohlrab A. P542 Wöhrer A. P836, P839 Woike M. P256, P257 Wolf A. V618, V664, V665 Wolf A.M. V606 Wolf D. P273, V404, V606, V687, V722, V904 Wolf J. V116, V119, V120, V603 Wolff D. P464, V735 Wolff H.A. P245, V437 Wölfler A. P460 Wöll E. P220, V375 Wollert-Wulf B. V664, V665 Wolleschak D. P465, P819 Wollina K. P556 Wömpner C. V119 Worbs D. V589, V746 Worel N. P858, V929 Worm K. P538, V434 Wotschadlo J. P870 Wroblewski M. P174, V622 Wu H.S. P477 Wuchter P. P485, P774, V765, V932 Wulf G. P467, P470, P478, P479, P494, V77, V668 Wulf-Goldenberg A. V348 Würstlein R. P279, P849, P850 Wuttig K. P781 Xu J. V609, V611 Yajnanarayana S.P. V722 Yaktapour N. V651 Yalcin A. V604

Imprint

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The 2013 austrian-swiss-german traveling fellowship tour.

Abstracts from the 3rd International PPRI Conference, October 12-13, 2015, Vienna, Austria.

Abstracts of the 41st Annual Meeting of the Austrian Diabetes Society. November 21-23, 2013. Salzburg, Austria.

2014 Austrian-Swiss-German Fellowship.

Abstracts of the 16th ESOT Congress, 8-11 September 2013, Vienna, Austria.

Abstracts of the 48th Annual Meeting of the German-Speaking Mycological Society (DMykG) together with the Austrian Society of Medical Mycology (ŌGMM), 4-6 September 2014, Salzburg, Austria.

Abstracts from 18th International Meeting of the European Society of Gynaecological Oncology (ESGO), 19-22 October 2013, Liverpool, UK.

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The history of pediatric hematology and oncology in Austria.

Abstracts of the 21st International Congress of the European Association for Endoscopic Surgery (EAES), 19-22 June 2013, Vienna, Austria.

Antimicrobial prescribing practices by Swiss, German and Austrian equine practitioners.

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Abstracts of the Annual Meeting of the Austrian Society of Cardiology, May 28-31, 2014, Salzburg, Austria.

IX Congress of the European Society for Urological Oncology and Endocrinology (ESUOE). Trento, Italy, October 29-31, 1992. Abstracts.

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Photopatch testing: the 5-year experience of the German, Austrian, and Swiss Photopatch Test Group.

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Abstracts of the XXXI Annual Scientific Meeting of the British Blood Transfusion Society. October 16-18, 2013. Birmingham, United Kingdom.

[Supplements in age-related macular degeneration: recommendations by the German Ophthalmological Society, the German Retina Society and the German Professional Association of Ophthalmologists--October 2014].

Abstracts of the Irish Society for Rheumatology, Autumn Meeting 2013, 19-20 September, 2013, Co.Meath, Ireland.