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Jahrestagung der ­Deutschen, Österreichischen und ­Schweizerischen ­Gesellschaften für Hämatologie und ­Medizinische Onkologie, Leipzig, 14.–18. Oktober 2016: Abstracts

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Oncol Res Treat 2016;39(suppl 3):1–348


Plenarsitzung – Eröffnungsveranstaltung

V14 – Nuclear waste disposal – what do we pass to our children?

Stumpf T.1

1Institut für Ressourcenökologie / HZDR, Dresden, Germany

The commission of the German Parliament which deals with the law for the site selection will finish their work until this year. With the final report a new chapter in the field of searching and building a nuclear waste repository in Germany will be opened.

Which kind of criteria are of importance for the site selection process?

Where are the potential sites in Germany located?

Are there any alternatives to a disposal in deep geological formations?

All these questions will be discussed within the talk “Nuclear waste disposal – what do we pass to our children?”. Furthermore, the scientific background of the formation, conditioning and disposal of high radioactive waste will be illustrated.

Disclosure: No conflict of interest disclosed.

Fortbildung – Management der chronischen myeloischen Leukämie

V15 – Therapy goals for first-line treatment in CML and implications for the doctor

Saußele S.1

1Medizinische Fakultät Mannheim der Universität Heidelberg, III. Med. Klinik, Mannheim, Germany

In CML, three tyrosine kinase inhibitors (TKI, dasatinib, imatinib, and nilotinib) are registered and recommended by expert groups for first-line treatment in CML. In order to choose optimal treatment for the individual patient with newly diagnosed CML different aspects have to be considered, e.g. impact of the TKI on overall survival (OS), progression-free survival (PFS), molecular response, side effects, and quality of life (QoL) as well as pre-existing comorbidities.

First line studies comparing 2nd generation TKIs vs. imatinib (ENESTnd and DASISION study) demonstrated significant higher rates for all molecular remission levels like MMR (BCR-ABL (IS) level < 0.1%), MR4 (BCR-ABL (IS) < 0.01%), and MR4.5 (BCR-ABL (IS) < 0.0032%) and lower rates of PFS in patients randomized to the nilotinib and dasatinib arms compared with the imatinib arms; however, both studies failed to show a significant difference in OS. This could be due to the fact that patients often switch to a second- or third-line therapy, leading to a more difficult assessment of a benefit in OS. In addition, it was shown that comorbidities at diagnosis have major influence on the outcome and hence CML unrelated-deaths influence OS. Another hypothesis could be that the lower rate of PFS with the more potent TKIs cpuld be antagonized by a negative effect of these drugs in regard of adverse events; e.g. pulmonary toxicity with dasatinib and vascular toxicity with nilotinib.

On the other hand, for the majority of patients, CML is now a chronic condition maintained by regular TKI therapy, so tolerability of the TKIs and their impact on Qol are of highest interest. In addition, the possibility to probably stop treatment (treatment-free remission (TFR)) moved in the focus since the publication of the STIM trial in 2010. If this option will become general recommendation in a well-defined patient population, the next step will be to think about how to increase patient numbers fulfilling these definitions. In this context, many factors have to be considered, e.g. optimizing first-line therapy, switching patients in MMR to other drugs to gain deeper MR. With the initial use of 2nd generation TKIs faster and deeper MR is achievable. The specific impact on TFR should be validated and current 1st line treatment strategies should be critically assessed. Further, the addition of other drugs, e.g. IFN or stem cell active drugs, may also increase the proportion of candidates for cessation attempts.

Disclosure: Susanne Saußele: Advisory Role: Novartis, BMS, Pfizer, Ariad; Financing of Scientific Research: Novartis, BMS, Pfizer, Ariad; Expert Testimony: Novartis, BMS

Fortbildung – Gynäkologische Tumoren

V25 – Immunotherapeutic approaches in gynecological cancers

Letsch A.1

1Med. Klinik m.S. Hämatologie, Onkologie und Tumorimmunologie, Charité Campus Benjamin Franklin, Berlin, Germany

The immune system is able to control cancer through various and dynamic interactions with cancer cells. Long-term remissions can be achieved by sustained immune responses against recurring cancer cells. The identification of immune checkpoint inhibitors, seems to be one of the most promising approaches of immunotherapy, currently known. The correlation between T cells in the tumor microenvironment and improved progression-free and overall survival in ovarian cancer, is only one factor indicating that immunotherapy could hold promise in gynecological cancers.

The use of immune checkpoint inhibitors directed against programmed death receptors PD-1 and PD1-ligand and combinatorial approaches covering immunotherapeutics and conventional treatments will be discusses with focus on gynecological cancers. Furthermore the need for specific consideration of immune-related adverse effects, specific response criteria and potential biomarkers will be addressed.

Although the clinical experience with novel immunotherapeutics remains limited in gynecologic cancer so far, early trials and clinical activity at least in specific subgroups of patients strongly supports the need for further investigation of immunotherapy approaches and their a potential role in the future treatment of gynecological cancers.

Disclosure: Anne Letsch: Expert Testimony: Celgene, Novartis

Fortbildung – Palliativmedizin

V27 – Palliative Care for patients in clinical trials

Simon S.T.1

1Uniklinik Köln, Zentrum für Palliativmedizin, Köln, Germany

Palliative Care is part of high quality cancer care for patients with incurable cancer. National and international evidence-based guidelines state that palliative care should be integrated early after the diagnosis of incurable cancer. Palliative care should be offered regardless of whether cancer-specific and disease modifying therapies are implemented. Clinical trials evaluate new and innovative approaches in order to optimize clinical care. Clinical trials are essential to improve both cancer care and palliative care. Patients in clinical trials should not be excluded from palliative care and have the same right to receive high quality cancer care including palliative care. Clinicans and researcher in oncology need to ensure that patients with incurable cancer in clinical trials have access to palliative care. Palliative care teams need to offer palliative care regardless of whether the patient take part in a clinical trial or not.

Disclosure: Steffen Simon: Expert Testimony: Teva GmbH (Finanzierung einer IIT clinical trial); Otsuka GmbH (Finanzierung retrospektive Studie); Immaterial Conflict of Interests: Mitglie der Dt. Gesellschaft für Palliativmedizin und der Dt. Gesellschaft für Innere Medizin

V28 – When is it time to discontinue cancer treatment?

Jahn-Kuch D.1

1Medizinische Universität Graz, Klin. Abteilung für Onkologie/Universitäre Palliativmedizinische Einrichtung ; UKIM, Graz, Austria

The launch of new tumorspecific agents has significantly increased the life expectancy of patients suffering from advanced cancer during the past decades. Therefore a lot of studies show the increasing use of antineoplastic agents even in end-stage cancer patients with the intention to prolong life and to reduce cancer related symptoms regardless to the very small benefit in third-, forth or later therapy lines. Research data regarding the positive effects on patient survival or even more important quality of life of aggressive cancer treatment at the end of life are still scarce. The available publications demonstrate that up to a fifth of all patients with extremely advanced cancer are undergoing chemotherapy in the last month of life without apparant signs of benefit. It has been clearly demonstrated that antineoplastic therapy administered to patients at end stage disease produces a lot of adverse effects, leads to an increase in emergency department visits , increases days spent in hospital towards end of life, prevents patients from engaging in meaningful life review and preparing for death and is associated with a considerably underuse of hospice service. Studies document that even in patients with good performance status and advanced disease tumorspecific therapy can have a negative impact on quality of life. Patients of young age or with short history of metastatic disease are most likely to receive chemotherapy near the end of life. One of the main reasons for the overuse of tumorspecific therapy in this peculiar setting could be the overestimation of survival by medical oncologists. The prediction of a cancer patient’s life expectancy by physicians is accurate in only about 30-40% of cases. The decision to continue or to end antitumor-therapy is usually based on patient´s performance status, stage of disease, sensitivity of the tumor to antineoplastic therapies, blood tests, organ function and present symptoms. Nevertheless it is equally important for the process of decision making on further tumorspecific therapy to clearly address the limitations of antitumor therapy at end-stage cancer in time, to evaluate and be responsive to the patients wishes and to openly discuss end-of-life issues with the patient. This might help to avoid the application of useless treatments at the end of life.

Disclosure: No conflict of interest disclosed.

Freier Vortrag – Management hämatologischer Erkrankungen älterer Patienten

V29 – Hematopoietic Stem Cell Transplantation (HSCT) as compared to Consolidation Chemotherapy (CT) increases leukemia free survival in patients between 60 and 75 years with Acute Myelogenous Leukemia (AML) irrespective of the genetic risk: Report from the AML 2004 of the East German Study Group (OSHO)

Niederwieser D.1, Al-Ali H.K.1, Krahl R.1, Kahl C.2, Wolf H.-H.3, Kreibich U.4, Vucinic V.1, Hähling D.5, Hegenbart U.6, Krämer A.6, Hirt C.7, Peter N.8, Opitz B.9, Florschütz A.10, Reifenrath K.11, Schulze A.12, Zojer N.13, Scholl S.14, Jakob C.15, Junghanss C.16, Pönisch W.1, Heyn S.1, Sayer H.G.12, Hochhaus A.14, Heinicke T.17, Fischer T.17, Dreger P.6, Maschmeyer G.18

1University Hospital of Leipzig, Division of Hematology and Oncology, Leipzig, Germany, 2Hospital Magdeburg, Department of Hematology and Oncology, Magdeburg, Germany, 3University of Halle, Department of Hematology and Oncology, Halle, Germany, 4Heinrich-Braun Hospital Zwickau, Department of Hematology, Oncology and Palliative Care, Zwickau, Germany, 5Oncology Practice, Schwerin, Germany, 6University Hospital of Heidelberg, Department of Hematology, Oncology and Rheumatology, Heidelberg, Germany, 7University Hospital Greifswald, Clinic of Internal Medicine, Greifswald, Germany, 8Carl-Thiem-Hospital, Divison of Hematology and Oncology, Cottbus, Germany, 9St. Elisabeth and St. Barbara Hospital Halle, Medical Clinic II, Halle, Germany, 10Städtisches Klinikum Dessau, Department of Hematology and Oncology, Dessau, Germany, 11Hospital Zittau, Zittau, Germany, 12Helios Hospital Erfurt, Hematology, Oncology and Hemostaseology, Erfurt, Germany, 13Wilhelminenspital Wien, Centre of Hematology and Oncology, Wien, Austria, 14University Hospital Jena, Divison of Hematology and Oncology, Jena, Germany, 15Imperial College of London, London, United Kingdom, 16University Hospital Rostock, Clinic of Hematology, Oncology and Palliative Care, Rostock, Germany, 17University Hospital Magdeburg, Clinic of Hematology and Oncology, Magdeburg, Germany, 18Hospital Ernst von Bergmann, Department of Hematology, Oncology and Palliative Care, Potsdam, Germany

HSCT has been reported to be an option for elderly patients with AML. In this analysis the outcome was compared in regard to CT or HSCT and analyzed according to genetic risk groups defined by the ELN.

By May 2015, 789 patients (60-75 a) of the AML 2004 study were identified as eligible. After induction with cytarabine (AraC; 1 g/m² i.v. bid d1, 3, 5, 7)/mitoxantrone (Mito; 10 mg/m² d1-3), 492 (62%) patients entered complete remission (CR) 1 with ranges from 79% for good risk (GR), 65% for intermediate (IM)-I, 59% for IM-II to 59% for high risk (HR) patients. A total of 355 patients were eligible either for CT with AraC (0.5 g/m2 i.v. bid d1, 3, 5)/Mito (10 mg/m² d1-2) (n = 205) or for matched HSCT (n = 119), if a donor was available. Most of the patients with HCT had unrelated (74.8%) donors and received low dose TBI (80.6%) as conditioning regimen.

Median age was 68 and 66 a in the CT and HCT groups, respectively (p < 0.0005). There were no statistical significant differences regarding gender, AML type, NPM1 and FLT3 mutation status, but IM-II and HR cytogenetics were less frequent in the CT than in the HCT arm (p < 0.002). In addition, the interval from CR to CT was significantly shorter (43 d) than from CR to HCT (65 d; p < 0.0005].

Patients receiving matched HCT had superior LFS at 9 a than those receiving CT (25 ± 5% vs. 14 ± 3%, respectively; p < 0.001). As expected, relapse incidence (RI) was lower after HCT (42 ± 5%) than after CT (78 ± 3%; p < 0.0001) and non-relapse mortality (NRM) was higher in HCT (34 ± 5%) than in CT patients (8 ± 2%; p < 0.0001). Analyses of risk categories identified differences in LFS at 9 a between matched HCT and CT for IM-I (28 ± 8% vs. 16 ± 4%; p = 0.007), for IM-II (30 ± 10% vs. 4 ± 4%; p = 0.08) and for HR (12 ± 7% vs. 0 ± 0%; p < .05). The differences in LFS were due to decreased RI in matched HCT vs. CT for IM-I (35 ± 7% vs. 74 ± 4%; p < .0001), for IM-II (38 ± 10% vs. 96 ± 5%; p < .0001) and for HR (59 ± 11% vs. 96 ± 7%; p < .004). These effects outweigh the higher NRM in HCT vs CT for IM-I (37 ± 9% vs. 10 ± 3%; p = 0.0005), for IM-II (33 ± 10% vs. 0%; p = 0.003) and for HR (29 ± 10% vs. 4 ± 4%; p = 0.11). Independent prognostic factors for LFS were genetic risk group and HCT (p < 0.0005), for OS genetic risk group (p < 0.01), for RI were genetic risk group and CT (p < 0.0005) and for NRM HCT (p < 0.005).

HCT from related/unrelated donors improves LFS in elderly patients with AML. This improvement is noted in all genetic risk groups from IM-I to HR.

Disclosure: No conflict of interest disclosed.

V30 – Outcome of patients = 65 years of age suffering myelofibrosis and treated with RIC allogeneic hematopoietic stem cell transplantation

Christopeit M.1, Alchalby H.1, Zabelina T.1, Zeck G.1, Ayuk F.A.1, Wolschke C.1, Kröger N.1

1Universitätsklinikum Eppendorf, Klinik für Stammzelltransplantation, Hamburg, Germany

Introduction: Median age of patients with myelofibrosis is 65 years at diagnosis. Frequently, patients of higher age are considered not fit for allogeneic transplantation. We hypothesized that RIC plus allogeneic transplantation is feasible for this population of patients and assessed outcomes of 53 patients =65 years with myelofibrosis.

Patients and methods: Patients’ (22 female, 42%) median age at transplantation was 68 (65-75) years. Primary Myelofibrosis (PMF) had been diagnosed in 37 patients (70%), the disease was of post-ET/-PV origin in 16 patients (30%). At diagnosis, 24 patients showed normal karyotype (45%). Bone marrow fibrosis scored 1° in 3 patients (6%), 2° in 7 patients (13%), and 3° in 34 patients (64%). DIPSS was intermediate-1 in 3 patients (6%), intermediate-2 in 21 patients (40%), and high in 22 patients (42%). CMV IgG pairing between donor/ patient was negative/ negative in 17 patients (32%), positive/ positive in 26 patients (49%), positive/ negative in 4 patients (8%), and negative/ positive in 6 patients (11%). Intravenous Busulfan (10 doses of 0.8 mg/kg BW) and Fludarabin (6 doses of 30 mg/m²), combined with Anti-Lymphocyte Globulin at 30-90 mg/kg, was used for conditioning in 33 patients (62%), 17 patients (32%) received an intensified RIC scheme consisting of sequential FLAMSA-chemotherapy and Busulfan/ Fludarabine RIC, combined with 30-90 mg/kg Anti-Lymphocyte Globulin. The median of transplanted PBSC was 6.3×106 (1.5-16.1×106)/kgBW. A matched related donor was present for 7 patients (13%), matched unrelated for 27 patients (51%), 18 patients (34%) received their transplant from a mismatched unrelated donor, 1 patient from a haploidentical donor. Immunosuppression mainly (85%) consisted of cyclosporine and mycophenolic acid.

Results: Leucocyte engraftment occurred in 49 patients (92%), 4 patients (8%) experienced primary graft failure. Median time to leucocyte engraftment was 13 (7-34) days, median time to platelet engraftment 20.5 (5-293) days. Acute GVHD grades 2-4 was present in 22 patients (42%), chronic GVHD in 19 patients (36%). Cumulative incidence (CI) of relapse was 21 ± 6% after 3 years. CI of non-relapse mortality (NRM) was 28 ± 6% after 1 year. OS at 3 years was 46 ± 8%.

Conclusion: Allogeneic stem cell transplantation of patients with myelofibrosis is feasible even at and above 65 years of age. Further research will have to focus on further reducing NRM in this elderly population.

Disclosure: No conflict of interest disclosed.

V31 – Prevalence and dynamics of leukemia-associated mutations in elderly individuals without hematologic disorders

Ernst T.1, Rinke J.1, Müller V.1, Waldau A.1, Midic D.1, Pester F.2, Landschulze J.2, Rudolph L.3, Hochhaus A.1

1Klinik für Innere Medizin II, Universitätsklinikum Jena, Abteilung Hämatologie und Internistische Onkologie, Jena, Germany, 2HausArztZentrum, Kahla, Germany, 3Leibniz Institute for Age Research, Fritz Lipmann Institute e.V. (FLI), Jena, Germany

Introduction: Clonal hematopoiesis is frequently observed in elderly people and may represent a premalignant condition. Evidence suggests that genetic alterations found in patients with myeloid malignancies may also be causative for induction of clonal hematopoiesis in healthy elderly. Given these circumstances, discrimination between age-associated and leukemia-associated mutations is of major clinical importance.

Methods: In order to investigate the prevalence and dynamics of genetic alterations among elderly individuals without hematologic/oncologic disorders, we genotyped a cohort of 50 elderly individuals (29 women; median age 84 years; range 80-90 years) for a panel of 30 commonly mutated leukemia-associated genes by targeted deep next-generation sequencing (NGS). Buccal cells were analyzed to confirm the somatic origin of mutations.

Results: A total of 16 somatic mutations in leukemia-associated genes were identified in 13 of 50 (26%) elderly individuals. One subject presented with two, another subject with three different mutations. Ten of 16 mutations (63%) affected epigenetic modifier genes (DNMT3A, n = 8; TET2, n = 1; IDH2, n = 1). Four somatic mutations affected genes involved in the RNA splicing machinery (SRSF2, n = 2; SF3B1, n = 1; U2AF1, n = 1). Mutations in TP53 and NRAS were identified in two individuals. All but one mutation were missense mutations with cytosine to thymine transitions being the most common base pair change (n = 7). Mutations occurred at low variant allele frequencies (VAF) with a median of 11.7% (range: 1.0% to 30.7%) indicating that mutations were presented in only a subset of blood cells. Mutation kinetics remained virtually stable over a follow-up observation of two years as measured by targeted NGS (median VAF 13.1%, range 1.0% to 35.3%).

Conclusions: These findings indicate that the appearance of low-level clones characterized by mutations in epigenetic regulator genes and the RNA splicing machinery is a common age-associated phenomenon which may represent a premalignant condition in the development of hematologic cancers and may also predispose to other aging associated disorders. The question why these subclones are selected in the elderly but apparently enter a stage of clonal size stability without further selection in healthy elderly in contrast to patients with myeloid disorders is of particular interest and needs further investigation.

Disclosure: No conflict of interest disclosed.

V32 – The distinct genetic and epigenetic landscape of elderly AML: Data of the SAL registry

Baldus C.D.1,2, Silva P.1, Neumann M.1,2, Vosberg S.3,4,5, Schlee C.1, Isaakidis K.1, Schroeder M.P.1, Ortiz Tanchez J.1, Fransecky L.R.1, Hartung T.1, Türkmen S.6, Graf A.3, Krebs S.3, Blum H.3, Thiede C.7,8, Ehninger G.7,8, Serve H.9,10, Berdel W.11, Greif P.A.4,5,12, Röllig C.7,8, Studienallianz Leukämie (SAL)

1Charite, Campus Benjamin Franklin, Hematology, Oncology, Berlin, Germany, 2DKTK, Berlin, Germany, 3Gene Center LMU München, München, Germany, 4DKTK, München, Germany, 5Medizinische Klinik III, Klinikum der Universität München, München, Germany, 6Labor Berlin Charité Vivantes GmbH, Berlin, Germany, 7Medizinische Klinik und Poliklinik I, Universitätsklinikum Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany, 8DKTK, Dresden, Germany, 9Medizinische Klinik II, Universitätsklinikum Frankfurt am Main, Frankfurt, Germany, 10DKTK, Frankfurt, Germany, 11Medizinische Klinik, Universitätsklinikum, Münster, Germany, 12German Cancer Research Center (DKFZ), Heidelberg, Germany

Despite advances in the characterization of molecular alterations in younger AML patients, comprehensive studies in elderly AML are lacking to uncover its distinct molecular alterations. In this project, we investigated genetic and epigenetic modifications to unravel the specific molecular background of this unfavourable disease.

To characterize elderly AML, we studied bone marrow samples of 93 AML patients enrolled on the Studienallianz Leukämie (SAL) registry. We selected a cohort of patients 65 to 90 years of age (median 72 years). To capture a broad spectrum of alterations we performed target enrichment of 555 candidate genes and sequenced them on an Illumina HiSeq 1500. We further investigated the DNA methylation profiles assessed them with an Infinium® HumanMethylation450 BeadChip on 79 available DNA samples (from the 93 samples).

Overall, 814 molecular alterations were detected in 281 of 555 genes with a median of 7 genes mutated per patient (range, 1 to 23). Particularly high mutation frequencies were detected in DNMT3A 33%, TET2 24%, SRSF2 23%, ASXL1 21%, RUNX1 18%, IDH1 17%, NPM1 15%, IDH2 and BCOR 10%. We observed a high frequency of mutations in epigenetic regulators, which affected 85% (79/93) of patients and also found frequent alterations in splicing factors (SRSF2, U2AF1, SF3B1, ZRSR2,DDX5) affecting 38% (35/93) of patients. Notably, 17% of elderly AML patients had mutations in the DNA repair genes (TP53, NBN, ATM, FANCA, FANCC), which were associated with an inferior median overall survival (OS) of only 4 months, compared to 16 months for patients without alterations in these DNA repair genes (p < 0.001). Interestingly, mutations in DNA repair proteins predicted poor OS independently of the TP53 mutational status (p = 0.007). The unsupervised analyses of DNA methylation revealed a profile of hypermethylation specific to AML samples with IDH1/2 mutations. Samples defined by a triple mutation pattern (DNMT3A, NPM1, FLT3) presented with a markedly distinct profile, largely characterized by hypomethylation of specific regions.

In conclusion, elderly AML harbours a high frequency of molecular alterations in epigenetic regulators, spliceosome components and DNA repair factors, the latter being associated with poor prognosis. This molecular categorization of elderly AML underscored a distinct biology and the need for specific molecularly driven therapeutic approaches.

Disclosure: No conflict of interest disclosed.

V33 – Evaluation of the international prognostic index for chronic lymphocytic leukemia (CLL-IPI) in elderly patients with comorbidities: Analysis of the CLL11 study population

Bahlo J.1, Goede V.1,2, Kutsch N.1, Fischer K.1, Fink A.-M.1, Stilgenbauer S.3, Bergmann M.4, Eichhorst B.1, Hallek M.1,5

1Uniklinik Köln, Deutsche CLL Studiengruppe, Klinik I für Innere Medizin, Centrum für Integrierte Onkologie Köln-Bonn, Köln, Germany, 2St. Marien Hospital, Klinik für Altersmedizin, Köln, Germany, 3Uniklinik Ulm, Klinik für Innere Medizin III, Ulm, Germany, 4Klinikum München-Schwabing, Klinik für Hämatologie, Onkologie, Immunologie, Palliativmedizin, Infektiologie & Tropenmedizin, München, Germany, 5Exzellenzcluster Cellular Stress Responses in Aging-Associated Diseases (CECAD), Köln, Germany

Introduction: CLL-IPI is a validated tool for prognostication of overall survival (OS) in chronic lymphocytic leukemia using age, stage, ß2-microglobulin, 17p deletion/TP53 mutation, and IGHV mutational status as weighted factors to stratify patients for low, intermediate, high, or very high risk of death. To date, validation studies of CLL-IPI have been restricted to patient populations with only moderately advanced age and little comorbidity. We here evaluated CLL-IPI in a large sample of elderly patients with comorbidities who represent the majority of patients with CLL outside of clinical trials.

Methods: CLL-IPI was analyzed in the CLL11 study which enrolled 781 patients with previously untreated CLL and increased comorbidity for treatment with obinutuzumab (GA101) plus chlorambucil (G-Clb), rituximab plus chlorambucil (R-Clb), or chlorambucil alone (Clb). Patients with all five CLL-IPI factors available were stratified into CLL-IPI risk groups. Kaplan-Meier methodology was used to estimate OS for low, intermediate, high, and very high risk. Log-rank test and Cox regression were used to compare OS among CLL-IPI risk groups.

Results: Among 781 patients enrolled in the CLL11 study, 691 patients were evaluable in this analysis while 90 subjects had to be excluded due to missing information for ß2-microglobulin, 17p deletion/TP53 mutation, or IGHV mutational status. Of the 691 subjects, 299 were treated with G-Clb, 294 with R-Clb, and 98 with Clb. Median age, cumulative illness rating scale (CIRS), and ECOG performance status were 74 years, 8 and 1, respectively. Median observation time was 41.8 months. Stratification according to CLL-IPI was as follows: 62 (9%) low risk, 206 (30%) intermediate risk, 361 (52%) high risk, 62 (9%) very high risk. In a pooled analysis of all 691 evaluable patients, OS was significantly different across CLL-IPI risk groups (p < 0.001, see figure), with statistically satisfying values regarding both discrimination and calibration.

Conclusions: This is the first validation study of CLL-IPI in elderly patients with previously untreated CLL and comorbidities. Results suggest good performance of the CLL-IPI in this patient population.


Disclosure: Jasmin Bahlo: Financing of Scientific Research: RocheMichael Hallek: Advisory Role: Roche; Financing of Scientific Research: Roche

V34 – International, randomized phase 3 study results: Ibrutinib versus Chlorambucil in patients 65 years and older with treatment-naïve CLL (RESONATE-2™)

Burger J.1,2, Tedeschi A.3, Barr P.M.4, Robak T.5, Owen C.6, Ghia P.7, Bairey O.8, Hillmen P.9, Bartlett N.L.10, Li J.11, Simpson D.12, Grosicki S.13, Devereux S.14, Mccarthy H.15, Coutre S.16, Quach H.17, Gaidano G.18, Maslyak Z.19, Stevens D.A.20, Janssens A.21, Offner F.22, Mayer J.23, O’Dwyer M.24, Hellmann A.25, Schuh A.26, Siddiqi T.27, Polliack A.28, Tam C.S.29, Suri D.30, Cheng M.30, Clow F.30, Styles L.30, James D.F.30, Kipps T.J.31, for the RESONATE-2 Investigators

1The University of Texas MD Anderson Cancer Center, Houston, United States, 2Albert-Ludwigs University, School of Medicine, Freiburg, Germany, 3Azienda Ospedaliera Niguarda Cà Granda, Milano, Italy, 4Wilmot Cancer Institute, University of Rochester, Rochester, United States, 5Medical University of Lodz and Copernicus Memorial Hospital, Lodz, Poland, 6Tom Baker Cancer Centre, Calgary, Canada, 7Università Vita-Salute San Raffaele and IRCCS Istituto Scientifico San Raffaele, Milano, Italy, 8Rabin Medical Center, Beilinson Hospital and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel, 9The Leeds Teaching Hospitals, St. James Institute of Oncology, Leeds, United Kingdom, 10Washington University School of Medicine, St. Louis, United States, 11Jiangsu Province Hospital, Nanjing, China, 12North Shore Hospital, Auckland, New Zealand, 13Department of Cancer Prevention, School of Public Health, Medical University of Silesia, Katowice, Poland, 14Kings College Hospital, London, United Kingdom, 15Royal Bournemouth Hospital, Bournemouth, United Kingdom, 16Stanford University School of Medicine, Stanford, United States, 17St. Vincent’s Hospital, University of Melbourne, Melbourne, Australia, 18Department of Translational Medicine, Amedeo Avogadro University of Eastern Piedmont, Novara, Italy, 19Institute of Blood Pathology and Transfusion Medicine of NAMS of Ukraine, Lviv, Ukraine, 20Norton Cancer Institute, Louisville, United States, 21University Hospital Leuven, Leuven, Belgium, 22University Hospital Gent, Gent, Belgium, 23Fakultni Nemocnice Brno, Brno, Czech Republic, 24University College Hospital Galway, Galway, Ireland, 25Department of Hematology, University Clinical Center of Medical University of Gdansk, Gdansk, Poland, 26University of Oxford, Oxford, United Kingdom, 27City of Hope National Medical Center, Duarte, United States, 28Hadassah University Hospital, Hebrew University Medical School, Jerusalem, Israel, 29Peter MacCallum Cancer Centre and St. Vincent’s Hospital, Melbourne, Australia, 30Pharmacyclics LLC, an AbbVie Company, Sunnyvale, United States, 31University of California San Diego, Moores Cancer Center, San Diego, United States

Introduction: Chronic lymphocytic leukemia (CLL) primarily affects older patients (pts) with medical comorbidities. Alkylating agents, such as chlorambucil (clb), are commonly used in these pts, but novel therapies are needed. Ibrutinib (ibr), a first-in-class, once-daily inhibitor of Bruton’s tyrosine kinase, is approved in the EU for pts with CLL after =1 prior therapy and for pts with del17p CLL and/or TP53 mutation unsuitable for chemoimmunotherapy. Ibr is also indicated by the US FDA for the treatment of pts with CLL and for CLL with deletion 17p. Ibr has shown high activity in treatment-naïve (TN) pts age =65 y. This randomized open-label phase 3 trial evaluated efficacy and safety of single-agent ibr vs clb in TN older pts with CLL.

Methods: Pts age =65 y with TN CLL were randomized 1:1 to receive 420 mg ibr daily until progression or clb 0.5 mg/kg (max 0.8 mg/kg) on d1 and 15 of a 28-d cycle (=12 cycles). Primary endpoint was independent review committee (IRC)-assessed PFS per iwCLL 2008 criteria, with 2012 clarification for treatment-related lymphocytosis. Secondary endpoints were OS, ORR, rates of hematologic improvements, and safety.

Results: 269 pts enrolled; median age 73 y. Baseline characteristics were balanced between arms. For clb-treated pts, 40% completed 12 cycles of therapy (mean dose 0.6 mg/kg). At median follow-up (18.4 mo), ibr significantly prolonged IRC-assessed PFS vs clb (median not reached vs 18.9 mo, P < 0.0001). Investigator-assessed 18-month PFS was 93.9% vs 44.8% for ibr vs clb (P < 0.0001). Ibr also significantly prolonged OS vs clb (P = 0.0010). Three deaths occurred on the ibr arm vs 17 deaths on the clb arm. IRC-assessed ORR was 86.0% with ibr vs 35.3% with clb; investigator-assessed ORR was 90.4% vs 35.3%. Rates of sustained hematologic improvement were significantly higher for ibr vs clb (84% vs 45%, P < 0.0001, anemia; 77% vs 43%, P = 0.0054, thrombocytopenia). Median duration of treatment was 17.4 mo (ibr) and 7.1 mo (clb). The most frequent (=20% of pts) adverse events with ibr were diarrhea, fatigue, cough, and nausea and for clb were nausea, fatigue, neutropenia, anemia, and vomiting. Major hemorrhage occurred in 4% with ibr over median follow-up of 1.5 years and in 2% with clb. At study closure, 87% of ibr pts continue to receive ibr.

Conclusions: Single-agent ibr was superior to clb for PFS, OS, ORR, and hematologic improvements in TN older CLL pts, with an 84% reduction in risk of death and an acceptable safety profile.

Disclosure: Jan Burger: Advisory Role: Janssen, Boehringer Ingelheim, Portola; Expert Testimony: Pharmacyclics, Gilead; Other Financial Relationships: Travel, Accommodations, Expenses: Roche, JanssenThomas Kipps: Advisory Role: AbbVie, Genentech, Gilead; Expert Testimony: AbbVie, Genentech, Pharmacyclics

Fortbildung – Kopf-Hals-Tumoren

V35 – Squamous cell carcinoma of head and neck: surgical and adjuvant treatment concepts in 2016

Dietz A.1

1HNO-Universitätsklinik, Leipzig, Germany

In this update presentation, the current major considerations for the primary surgical treatment of squamous cell carcinoma of the head and neck region are presented and discussed. The reader will be introduced in less detail of surgica techniques than in the operational conceptual background of currently recommended treatment concepts. The Europe 5 - year survival rate of squamous cell carcinoma of the head and neck region (HNSCC) is currently at 42%. Especially in the last 3 years, various guidelines have been established based on limited evidence for standardization of therapeutic concepts in HNSCC. If functional operability is possible In Europe It is found predominantly that primary surgical approaches are preferred. Postoperative adjuvant therapy is standardized due to clear indications based on defined risk situations. Indications are related to assessment of surgical margins, cervical lymph node metastases and extracapsular tumor growth. Ablative surgical procedures are competitive with so-called organ preservation programs which are currently addressed in clinical trials since many open questions regarding late functional outcome are still under discussion.

Disclosure: Andreas Dietz: Advisory Role: Merck Serono, Astra Zeneka, MSD Merck, BSM,

Freier Vortrag – Infektionsmanagement

V42 – Invasive mucormycosis in patients with hematological diseases identified in the global FungiScope™ registry

Seidel D.1, Duran Graeff L.1, Vehreschild M.J.G.T.1, Liss B.1, Köhler P.1, Müller F.1, Wisplinghoff H.2, Vehreschild J.1, Cornely O.1

1Uniklinik Köln, Klinik I für Innere Medizin, Cologne, Germany, 2Uniklinik Köln, Institut für Mikrobiologie, Cologne, Germany

Background: Invasive fungal diseases (IFD) are a frequent complication in hematological patients. Hematological malignancies and their treatment are main risk factors for IFD. Less frequent IFD, such as invasive mucormycoses (IM), are increasing worldwide and are associated with mortality up to 100%. Efficient diagnostic and treatment approaches for IM are not known or validated yet. Thus, it is urgent to better understand clinical presentation and management of IM in hematological patients to eventually improve patient outcome.

Methods: Clinical data on IM were collected in FungiScope™, an international web-based retrospective registry. Cases with cultural, histological or molecular evidence of IM are enrolled. Data collected include demographics, underlying conditions and their treatment, clinical signs and symptoms, sites of infection, diagnostic tests, antifungal treatment and outcome. Clinical isolates are collected for centralized identification, molecular analyses and exchange between collaborators.

Results: To date, 158 cases of IM with an underlying hematologic disease (HD) were captured in the FungiScope database. AML, ALL, Non-Hodgkin’s Lymphoma and CLL were the most frequent malignancies (46.8%, 19%, 7%, and 5.7%, respectively). Treatment of HD was chemotherapy in 75%, for 70% of these it was the primary course. Prolonged neutropenia (>10 days) was reported in 51% of cases. The majority of patients received antifungal agents (79%; median 42 days, range 1-733 days) and 37% underwent surgical debridement. Antifungals known to be active against Mucorales (amphotericin B, isavuconazole, itraconazole, posaconazole) were administered in 86% of cases, in 23% of those amphotericin B was used as a single agent. Median followup time was 46 days (range 0-1394 days). Favourable response (complete or partial response or stable disease) resulted in 42% of cases. Overall mortality was 65% and death due to IM was reported for 78% of cases. For patients who were not treated for IM, mortality exceeded 90%. Depending on the underlying disease overall mortality ranged from 61% for ALL and AML to 100% for CLL.

Conclusion: The FungiScope registry is a feasible approach to collect data on a relevant amount of rare cases of IM. IM remains a life threatening disease in hematological patients. Despite aggressive antifungal treatment strategies outcome remains poor. More effective treatment strategies are urgently needed to improve patient outcome.

Disclosure: Danila Seidel: No conflict of interest disclosed.Oliver Cornely: Advisory Role: Amplyx, Anacor, Astellas, Basilea, Cidara, Da Volterra, Daiichi Sankyo, F2G, Genentech/Roche, Gilead, Matinas, MedPace, Merck/MSD, Merck Serono, Pfizer, Sanofi Pasteur, Scynexis, Seres, Summit, Vical, Vifor; Financing of Scientific Research: Astellas, Basilea, Gilead, Merck/MSD, and Pfizer; Expert Testimony: 3M, Actelion, Astellas, AstraZeneca, Basilea, Bayer, Celgene, Cubist/Optimer, Duke University (NIH UM1AI104681), Genzyme, Gilead, GSK, Leeds University, Merck/MSD, Miltenyi, NanoMR, Pfizer, Quintiles, Scynexis, Viropharma; Other Financial Relationships: supported by the German Federal Ministry of Research and Education

V43 – Parainfluenza in hematologic patients – impact of prolonged viral shedding and nosocomial infection

Lehners N.1, Puthenparambil J.1, Schiller M.1, Ho A.D.1, Schnitzler P.2, Egerer G.1

1Universitätsklinikum Heidelberg, Abteilung für Hämatologie, Heidelberg, Germany, 2Universitätsklinikum Heidelberg, Zentrum für Infektiologie, Heidelberg, Germany

Introduction: Parainfluenza virus (PIV) is often cause of self-limiting upper respiratory tract infection (URTI), but can result in severe and even life-threatening lower respiratory tract infection (LRTI) in the immunocompromised host. In contrast to seasonal influenza, PIV is transmitted all year round. Here, we describe outcome and risk factors of PIV infection in hematologic patients and report on prolonged viral shedding in this particular population.

Methods: Clinical characteristics and outcome of hematologic patients with documented PIV infection treated at our institution from July 2013 to June 2015 were retrospectively evaluated. In patients with available consecutive tests for PIV, duration of viral shedding was assessed.

Results: 60 patients were identified, 47 with PIV 1/3, 13 with PIV 2/4. Median age was 59 years [range 26-76], 38 patients were male. 41 patients had received a stem cell transplantation (20 allogeneic, 17 autologous, 4 both). Nosocomial infection, defined as diagnosis of PIV infection =1 week after hospital admission, was apparent in 23 patients. In regard to outcome, 36 patients had URTI only, 24 (40%) developed a LRTI. Of the latter, 3 patients (13%) were transferred to the ICU and subsequently died. Neither type of PIV nor underlying hematologic disease had a significant impact on outcome. Leukopenia as well as nosocomial infection were significantly associated with LRTI (p = 0.02 and p = 0.01, resp.), a trend was seen for status post allogeneic transplantation (p = 0.06). Duration of viral shedding could be evaluated in 23 patients. Median duration of viral shedding was 14 days, but shedding of up to 79 days was observed. A significant association between LRTI and prolonged shedding for >20 days was observed (p = 0.01).

Conclusion: PIV infection can cause significant morbidity in hematologic patients. Nosocomial infection was frequently present and was associated with a higher rate of LRTI in our patient population. Prolonged viral shedding was observed which might facilitate nosocomial spread of PIV and should be taken into account in infection control management.

Disclosure: No conflict of interest disclosed.

V44 – Prophylactic exchange of central venous catheters (CVC) for prevention of CVC-related bloodstream infections (CRBSI) in patients with haematological malignancies: indirect evidence from pooled data of the SECRECY registry and the COAT study

Schalk E.1, Biehl L.M.2,3, Färber J.4, Huth A.2, Panse J.5, Krämer C.5, Hentrich M.6, Engelhardt M.7, Schäfer-Eckart K.8, Kofla G.9, Kiehl M.10, Wendtner C.-M.11, Karthaus M.12, Cornely O.A.2,13,14, Fischer T.1, Vehreschild M.J.G.T.2,3

1Otto-von-Guericke University Magdeburg, Medical Centre, Department of Haematology and Oncology, Magdeburg, Germany, 2University Hospital of Cologne, Department I of Internal Medicine, Cologne, Germany, 3German Centre for Infection Research (DZIF), Site Bonn/Cologne, Cologne, Germany, 4Otto-von-Guericke University Magdeburg, Medical Centre, Department of Medical Microbiology, Infection Control and Prevention, Magdeburg, Germany, 5RWTH Aachen University Hospital, Department of Haematology, Oncology, Haemostaseology, and Stem Cell Transplantation, Aachen, Germany, 6Red Cross Hospital Munich, Department of Medicine III, Munich, Germany, 7Medical Centre – University of Freiburg, Department of Medicine I, Haematology, Oncology and Stem Cell Transplantation, Freiburg, Germany, 8Klinikum Nuernberg, Medical Clinic V, Haematology and Oncology, Nuernberg, Germany, 9Charité – University Medicine Berlin, Department of Medicine, Division of Oncology/Haematology, Berlin, Germany, 10Clinical Centre Frankfurt/Oder, Medical Clinic I, Haematology and Medical Oncology, Frankfurt/Oder, Germany, 11Klinikum Schwabing, Department of Haematology, Oncology, Immunology, Palliative Care, Infectious Diseases and Tropical Medicine, Munich, Germany, 12Klinikum Neuperlach, Department of Haematology and Oncology, Munich, Germany, 13University of Cologne, Centre for Integrated Oncology CIO Köln/Bonn, Cologne, Germany, 14University of Cologne, Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), Cologne, Germany

Introduction: CVC exchange in cancer pts. is not general recommended to prevent CRBSI, but it is sometimes still practice and the optimal time point is unclear. However, this recommendation is based on studies that were underpowered for haematological pts. Therefore, we aimed to verify this question in a larger cohort of pts. with haematological malignancies.

Methods: Prospective, multicentre analysis of CRBSI due to short-term CVC (=1 day in situ) in pts. with haematological malignancies. Pooled data of the SECRECY registry (DRKS00006551) and the COAT study (NCT01544686) were used. For CRBSI definition the 2012 AGIHO/DGHO criteria were used; only definite and probable CRBSI were considered. Length of catheterization (LOC) was used to determine a cut-off for CRBSI risk using area under the receiver operating characteristic curve (AUC); AUC< 0.50 was considered to be of none predictive value. Also, CRBSI risk was calculated comparing LOC for quartiles 1 to 4 (Q1 to Q4).

Results: Altogether, 1083 CVC (median age = 59 years [range 18-86], 59.5% men) with 18,327 CVC days (median LOC = 17 days [range 1-60]) were analysed in 857 pts. in 10 centres. Underlying diseases were acute leukaemia (46.8%), multiple myeloma (26.4%) or lymphoma (19.0%). Most of the CVC were inserted in the jugular vein (n = 699, 64.5%). The CRBSI rate was 11.6% (n = 126). The CRBSI incidence and the cumulative incidence were 6.9/1000 CVC days and 30.3%, respectively. The CRBSI onset was in median on day 13 (range 2-40). The predominant pathogenic agents were coagulase-negative staphylococci (n = 95, 75.4%). The LOC quartiles were day 1-12 for Q1, day 13-17 for Q2, day 18-23 for Q3 and day 24-60 for Q4, respectively. The CRBSI risk was higher for LOC Q2 vs. Q1 (hazard ratio [HR]=3.62 [95%CI 1.94-6.74]; p < 0.001), for Q3 vs. Q2 (HR = 4.92 [95%CI 2.57-9.42]; p < 0.001) and for Q4 vs. Q3 (HR = 2.36 [95%CI 1.23-4.52]; p = 0.01). For the LOC cut-off for CRBSI prediction an AUC of 0.45 (95%CI 0.40-0.49) was calculated. There was no higher CRBSI risk for LOC more than the median LOC (Q3-Q4 vs. Q1-Q2) (relative risk [RR]=1.02 [95%CI 0.73-1.43]; p = 0.89), but for LOC more than median CRBSI onset (equivalent to Q2-Q4 vs. Q1) (RR = 1.89 [95%CI 1.25-2.86]); p = 0.003).

Conclusions: The CRBSI risk is increasing with LOC in pts. with haematological malignancies. However, there is no LOC cut-off for prediction CRBSI. Therefore, within the first 2 weeks of LOC no recommendation can be given for CVC exchange to prevent CRBSI.

Disclosure: Enrico Schalk: No conflict of interest disclosed.Maria Vehreschild: Advisory Role: MJGT has been a consultant to Astellas Pharma, Berlin Chemie, Merck/MSD and DaVolterra.; Financing of Scientific Research: MJGTV has served at the speakers’ bureau of Pfizer, Merck/MSD, Gilead Sciences and Astellas Pharma.; Expert Testimony: MJGT received research funding from 3M, DaVolterra, Gilead Sciences and Astellas Pharma.

V45 – Epidemiology and outcomes of infections during neutropenia after 166 allogeneic stem cell transplantations in one year at one center

Samek M.1, Wolschke C.1, Langebrake C.1, Adjallé R.1, Ayuk F.A.1, Kröger N.1, Christopeit M.1

1Universitätsklinikum Eppendorf, Klinik für Stammzelltransplantation, Hamburg, Germany

Introduction: During allogeneic stem cell transplantation (allo-SCT), infections significantly contribute to morbidity and mortality. We performed a prospective analysis of allo-SCT in 2015 in order to assess epidemiology and outcome of infections during the neutropenic phase.

Methods: Data of pts during allo-SCT were recorded in a prospective fashion using a predefined questionnaire.

Results: In 2015, 163pts (61f, 102m) were transplanted during 166 procedures. Median age was 59(18-79)y. Median duration of neutropenia was 14.5(4-43)d. Fever (in neutropenia) occurred in 138(117)pts (83.1/70.5%). Fever occurred before an increase in CRP (n = 34; 20.5%), CRP rose before fever (n = 60; 36.1%), CRP and fever co-occurred (n = 44; 26.5%), CRP rose without apparent fever (n = 17; 10.2%) or there was neither an increase of CRP nor fever (n = 11; 6.6%). Severe sepsis developed in 95 (57.2%), septic shock in 26pts (15.7%). BC were drawn in 142pts (85.5%) on a median of 4(1-38)d. At least 1 positive BC was obtained in 34pts (23.9%), 1/2/3/4 BC were positive in 23/7/2/2. In 7/34pts (21%) the same species grew in more than 1 BC (3xStaph.epi.,2xC.paraps.,Crypt.neof.,Ps.aerug.). 1/2/3/4/5/6/9 CVC were inserted in 121/27/10/4/3/1/1pts. The mean duration for the 1st/2nd CVC was 25.6 ± 8.5/13.3 ± 6.2d. At least 1 tCT was done in 94pts, exactly 1/2/3/4/5/6/7 tCT in 34/35/15/5/1/1/3pts. No tCT was necessary for 72pts (43.4%). Lung infiltrates were seen in 64/94pts (68.1%), 51/64 (79.7%) received 1-5 BL. There, Aspergillus antigen or DNA was positive for 14pts. Once, Rhizopus spp. DNA was detected. Fever (n = 73; 44%), fever and increase in CRP (n = 15;9%), increase in CRP (n = 43; 25.9%), or other (n = 35; 21.1%) were reasons for antibiotic therapy. No empiric antibiotic therapy was necessary for 13pts (7.8%). 1st line therapy (CEF,V/D/L) was prescribed in 129pts (77.7%). Escalation to 2nd line therapy (MERO,V/D/L) occurred for 75/129 (58.1%), to 3rd line therapy for 42/75pts (56%). 1st line therapy sufficed for 36pts (21.7%). 2pts died during neutropenia. 145 stays (87.3%) resulted in discharge. 18/21 deaths were due to infectious causes.

Conclusion: Immediate broad spectrum antimicrobial therapy, CVC exchange and diagnostic efforts result in infection-related mortality below historical comparatives even in a population of heavy pretreatment and fairly advanced age.

Disclosure: Markus Samek: No conflict of interest disclosed.Maximilian Christopeit: Advisory Role: Basilea, MSD; Financing of Scientific Research: Basilea, MSD

V46 – Community acquired respiratory viruses in healthy controls and allo-SCT recipients – a prospective comparative study

Rachow T.1,2, Konowski P.1,2, Kalkreuth J.1, Kurze S.1, Hammersen J.1, Klink A.1, Hilgendorf I.1, Hochhaus A.1, von Lilienfeld-Toal M.1,2

1Universitätsklinikum Jena, Klinik für Innere Medizin II, Abt. Hämatologie und Internistische Onkologie, Jena, Germany, 2Leibniz-Institut für Naturstoff-Forschung und Infektionsbiologie e.V., Hans-Knöll-Institut, Jena, Germany

Introduction: Community acquired respiratory viruses (CRV) are an important cause of morbidity and mortality in immunocompromised patients, especially after allogeneic hematopoietic stem cell transplantation (allo-SCT). There are just few prospective trials focussing on epidemiological and clinical aspects of CRV in hematologic patients.

Methods: Samples of throat gargles were obtained from all participants at 2 or more different time points during the influenza season of each winter regardless of symptoms (for healthy controls 2013/14 and 2014/15 and for patients after allo-SCT 2014/15 and 2015/16). Incidence of CRV and clinical presentation were assessed for both groups. A molecular multiplex PCR assay for 21 respiratory pathogens (FTD® Respiratory Pathogens 21 kit, Fast-track diagnostics Ltd. Malta) was performed for detection of CRV.

Results: In the patient cohort 301 throat gargles of 142 patients and in the control group 464 throat gargles of 232 subjects could be analysed. More throat gargles were tested positive for viruses patients when compared to controls (n = 46/301 [15.2%] vs. n = 22/464 [4.7%], p < 0.001). The three most common viruses in the patient cohort were respiratory syncitical virus (RSV, n = 11), adenovirus (n = 8) and coronaviridae (n = 8) whereas in the control group coronaviridae (n = 8), rhinovirus (n = 5) and influenza A (n = 4) were the most common pathogens. Most persons with presence of a virus exhibited symptoms of respiratory tract infection. However, some participants were asymptomatic despite presence of a virus and the rates of asymptomatic virus carriers significantly differed between groups (14/142 patients [9.8%] in patients and 2/232 [0.8%] subjects in the control group, p < 0.001).

Conclusions: Patients who have undergone allo-SCT are at higher risk for carrying respiratory viruses. The high rate of asymptomatic carriers might be explained by a prolonged clearance of CRV from the respiratory tract in the condition of immunosuppression. The clinician should keep in mind that even asymptomatic patients after allo-SCT might be a potential source of virus infection for other immunocompromised patients.

Disclosure: Tobias Rachow: Financing of Scientific Research: Pfizer; Other Financial Relationships: Reise- und Fortbildungskosten: GILEAD, Baxalta, MedacMarie von Lilienfeld-Toal: Advisory Role: MSD, Celgene, Honorare: MSD, Celgene, Janssen Cilag, Gilead; Expert Testimony: MSD, Pfizer, Deutsche José Carreras Leukämie-Stiftung e.V.; Other Financial Relationships: Reisekosten: Celgene, Janssen Cilag, Gilead, Astellas

V47 – The impact of a LysM-specific NFATc1 knockout on neutrophil antifungal defense and myelopoiesis in NFATc1LysM mice infected with Aspergillus fumigatus

Teschner D.1, Michel C.1, Prüfer S.2, Theobald M.1, Schild H.2, Radsak M.1

1University Medical Center of the Johannes Gutenberg-University, Department of Hematology, Medical Oncology, & Pneumology, Mainz, Germany, 2University Medical Center of the Johannes Gutenberg-University, Institute of Immunology, Mainz, Germany

Introduction: Immunosuppressive medication e.g. by calcineurin inhibitors substantially contribute to the risk for opportunistic fungal infections in patients after allogeneic transplantation (HSCT). The nuclear factor of activated T cells (NFAT) is an important transcription factor downstream of calcineurin especially in T cells. Additionally, NFAT also seems to play a substantial role in innate antifungal immune responses by polymorphonuclear neutrophils (PMN), as well as in regulation of myelopoiesis.

Methods: LysM-specific NFATc1 knockout (NFATc1LysM) mice were generated by crossing LysM-Cre mice with NFATc1flox/flox mice to obtain NFATc1 deficiency solely in myelomonocytic cells. NFATc1LysM mice were firstly infected with Aspergillus fumigatus (A. f.) conidia intratracheally. PMN recruitment to the lungs, several chemokines/cytokines and pulmonary fungal clearance were examined by analyzing bronchoalveolar lavages (BAL) and peripheral blood (PB) using flow cytometry and cytometric bead array and murine lungs by fungal culture assays and histopathologic examination. In addition, survival was studied with neutropenic animals serving as controls. In addition, we investigated myelopoiesis under steady state conditions by quantifying bone marrow derived myeloid progenitor cells from NFATc1LysM mice using flow cytometry compared to PMN in PB.

Results: NFATc1LysM mice infected with A. f. showed unimpaired survival. However, there were no detectable differences in PMN recruitment or fungal clearance, whereas pulmonary inflammation, chemokines/cytokines production and PMN counts in PB seemed to be more pronounced in knockout mice (0.9 inflammation points/lung ± 0.12 (NFATc1LysM) vs. 0.6 ± 0.08 (control), p = 0.04; 1.5 x 103 PMN/µl ± 0.2 vs. 0.9 ± 0.1, p = 0.04). Distribution of bone marrow derived myeloid progenitor cells was clearly impaired in NFATc1LysM mice especially in megakaryocyte-erythroid progenitor cells (1.2 x 105 cells ± 0.2 vs. 2.7 ± 0.6, p = 0.02) whereas PMN blood counts in PB were unchanged.

Conclusion: NFATc1 downregulation in PMN of A. f. infected NFATc1LysM mice leads to enhanced pulmonary inflammation and elevated PMN blood counts compared to controls. Additionally, NFATc1LysM mice display constrained myelopoiesis under steady state conditions without affecting peripheral PMN blood counts compared to wild type controls. Further studies are needed to clarify underlying mechanisms and clinical relevance in HSCT of our findings.

Disclosure: Daniel Teschner: Advisory Role: Pfizer Deutschland GmbH, MSD Sharp & Dohme GmbH; Financing of Scientific Research: Gilead Sciences GmbH, MSD Sharp & Dohme GmbH; Other Financial Relationships: Astellas Pharma GmbH , Gilead Sciences GmbH, Jazz Pharmaceuticals Germany, MSD Sharp & Dohme GmbH (Reisekostenerstattungen)Markus Radsak: Advisory Role: Novartis Pharma GmbH; Financing of Scientific Research: Celgene GmbH, Novartis Pharma GmbH; Expert Testimony: Celgene GmbH; Other Financial Relationships: Astellas Pharma GmbH (Reisekostenerstattungen)

Freier Vortrag – Rehabilitation

V48 – Rehabilitation means no risk for patients with hemato-oncological diseases also and especially after allogeneic blood stem cell transplantation

Kiefer-Trendelenburg T.1

1Klinik am See, Onkologie, Rüdersdorf, Germany

Introduction: Patients with hemato-oncological diseases show a weakened immunsystem, which is especially pronounced after high-dose chemotherapy followed by allogeneic blood stem cell transplantation (HDC/BSCT). To avoid infections patients are recommended to limit contact with other people. In this context, the question arises to what extent the stay in a rehabilitation clinic means a hazard, or whether it may be recommended.

Methods: We report a total of 94 rehabilitation stays (RSs) of 91 patients with hemato-oncological diseases, who have been treated under special hygienic conditions during the period from 01-July-2014 to 31-Dec-2015. As the criterion for serious complication early termination of RS has been selected and risk factors were analyzed.

Results: Eight of the 94 RSs (8.5%) had to be terminated prematurely. The RSs of patients with prior HDC/alloBSZT had to be discontinued in 21.4% (6 of 28). In comparison the drop-out rate of RSs of patients with so-called solid tumours is 2.4% (29 of 1220). The reasons for stopping RSs were cytomegalovirus reactivation in three patients and in three other cases the occurrence of fever immediately after the start of RS (within the first 48 hours). One can assume, that the patients have been infected before starting RS. In one case RS could not be started due to a low number of leukocytes. The RS of the last patient has to be stopped after one week, since the reduced general condition, vomiting and malnutrition could not be improved.

Analyzing possible risk factors for premature termination the platelet count and the period of time between the last treatment and the start of the RS show significant correlation. Age, sex, body mass index, kidney function, CRP and leukocyte count show no significant correlation.

Conclusions: Since all reasons for premature rehabilitation stop were not caused by the RS itself, we conclude that RSs do not mean a risk for patients with hemato-oncological diseases, assuming an individually tailored hygiene program. On the contrary during a RS life-threatening complications can be detected early and appropriate treatment can be initiated.

Disclosure: No conflict of interest disclosed.

V49 – Oscillating pole therapy – The best option to treat urinary incontinence after radical prostatectomy? - Follow-up-data

Heydenreich M.1, Zermann D.-H.1

1Vogtland-Klinik Bad Elster, Urologie/ Uroonkologie, Bad Elster, Germany

Introduction: There is evidence that specialized continence training in combination with an oscillation pole therapy has a major effect on the early recovery of continence after prostatectomy. Aim of this investigation was the question, if the results of a prospective randomized controlled study can be confirmed in rehabilitation daily routine.

Methods: 336 patients (Ø 64.1 years) with urinary incontinence after prostate cancer surgery were evaluated. All patients passed a standard treatment program (continence training, endurance training, moderate strength training). Additionally the intervention and follow-up group participated in a guided training with an oscillating pole (daily, 30 minutes). Urinary incontinence was evaluated using 1-h (ICS) and 24-hour pad-test.

Results: The data of 336 participants could be evaluated. We report the follow-up-data of 168 patients within the framework of normal rehabilitation in comparison to the results of the prospective randomized study published last year.


Conclusion: Results of urinary continence therapy after prostate cancer surgery were significantly improved up by complex approach combining standard continence therapy and the new training using an oscillating pole. After integration of the new therapeutic option in rehabilitation daily routine the primary study results were confirmed. Therefore this new treatment option should be offered to all patients suffering urinary incontinence.

Disclosure: No conflict of interest disclosed.

V50 – Prospective, randomized evaluation of the influence of sports therapy on quality of life after hematopoietic stem cell transplantation

Stüwe S.1, Schumacher H.1, Kropp P.2, Diedrich D.3, Greger N.1, Freitag S.1, Junghanss C.1, Hilgendorf I.1,4

1University Medicine Rostock / Department of Internal Medicine III, Rostock, Germany, 2University Medicine Rostock / Department of Medical Psychology and Medical Sociology, Rostock, Germany, 3University Medicine Rostock / Institute for Biostatistics and Informatics in Medicine and Ageing Research, Rostock, Germany, 4University Hospital Jena / Department of Internal Medicine II, Jena, Germany

Introduction: Hematopoietic stem cell transplantation (SCT) causes severe physical and psychological side effects. However, sports therapy may have positive impact on sentiment. Here we evaluate the influence of classical physiotherapeutic treatment (PT) in comparison to a multimedia sensor-based practice on psychological aspects and quality of life (QOL).

Methods: Patients undergoing SCT were randomized into the control group (n = 23) receiving PT or the experimental group exercising on the Nintendo-Wii® (n = 19). Patients of both groups completed the FACT-BMT, HADS-D and Distress Thermometer at the date of hospital admission (T1) and on day 14 (T2), 28 (T3) and 100 (T4) after SCT.

Results: The level of distress was comparable between both groups. However, at T2 distress increased above the cut-off level of 5 in both groups (Wii-group p = 0.006, PT-group p = 0.276). This was accompanied by an increase of anxiety (p = 0.705) and depression (p = 0.006) in the PT-group, while both parameters decreased in the Wii-group (p = 0.087 and p = 0.220), respectively. Of note, the level of anxiety in the Wii-group stayed below the PT-group at all times.

Concerning QOL, social and emotional well-being (SWB/EWB) scored highest in both groups. In Wii-group EWB increased significantly between T1-T4 (p = 0.015) and ranked above PT-group at all times. Physical well-being (PWB) showed the strongest fluctuation of all domains. It declined significantly between T1-T2 in both groups (PT p = 0.015, Wii p = 0.019), followed by a significant increase between T2-T4 (both groups p = 0,001). However, only in Wii-group results of PWB at T4 ranked significantly above T1 (p = 0.028). Lowest scores were proved for functional well-being (FWB) in both groups at all times. The score of bone marrow transplant scale (BMTS), the second lowest score in both groups, was always higher in Wii-group.

Conclusion: The results indicate less psychological distress and higher QOL after SCT when exercising with Nintendo-Wii®, which therefore should be considered as an alternative of physiotherapy.

Disclosure: No conflict of interest disclosed.

V51 – Reducing the symptoms of chemotherapy-induced peripheral neuropathy with specific exercise interventions

Streckmann F.1,2, Bloch W.2, Lehmann H.3, Faude O.1, Baumann F.T.2

1Universität Basel, Department für Sport, Bewegung und Gesundheit, Basel, Switzerland, 2Deutsche Sporthochschule Köln, Institut für Kreislaufforschung und Sportmedizin, Köln, Germany, 3Universitätsklinikum Köln, Neurologie, Köln, Germany

Chemotherapy-induced peripheral neuropathy (CIPN) is a highly prevalent and clinically relevant side-effect of chemotherapy. Depending on the neurotoxic agent, 60-90% of patients are affected. The motor and sensory symptoms not only severely diminish patients’ quality of life, but represent a decisive limiting factor for medical therapy, consequently affecting the clinical outcome. To date approved and effective treatment options are lacking. Promising results have now been achieved with specific exercise interventions.

In a first randomized, controlled trial (RCT) with lymphoma patients, positive effects on motor as well as sensory symptoms of CIPN could be achieved with an exercise intervention, concomitant to therapy, comprising endurance-, strength- and sensorimotor training (SMT). Patients, who had exercised, showed improved peripheral deep sensitivity, balance control and level of activity, which enhanced their quality of life. Furthermore, 87% of the patients in the intervention group were able to reduce the symptoms of CIPN, while they persisted in the control group (0%). A tendency towards a potentially preventive effect could also be detected. Lacking further references in oncology, we conducted a systematic review, investigating beneficial exercise interventions for patients with peripheral neuropathies independent of the pathophysiology. It revealed that for toxically-induced peripheral neuropathies such as CIPN, exercise interventions involving balance components were essential, while studies conducting endurance- or strength training alone showed no effect on the relevant symptoms. We therefore conducted a further RCT, isolating SMT and comparing it to whole body vibration training (WBV). The two intervention groups were compared to an oncological control group as well as a healthy, age- and gender-matched control group. First analysis showed that both SMT and WBV not only proved feasible for patients with CIPN, but were able to improve reflex activity, peripheral deep sensitivity, balance control and quality of life. We are currently also investigating the preventive effects of SMT and WBV on the incidence of Oxaliplatin- as well as vinca-alkaloid-induced peripheral neuropathy.

We therefore propose that SMT as well as WBV are promising exercise interventions to reduce and possibly even prevent symptoms of CIPN. Consequently, specific exercise therapy should be taken more seriously as a supportive therapy for oncological patients.

Disclosure: No conflict of interest disclosed.

V52 – Return to work following a work-related oncological rehabilitation: The pilot study Perspective Job

Kähnert H.1, Exner A.-K.2, Muckel E.1, Leibbrand B.3

1Institut für Rehabilitationsforschung, Norderney, Bad Salzuflen, Germany, 2Universität Bielefeld, Fakultät für Gesundheitswissenschaften, AG Epidemiologie & International Public Health, Bielefeld, Germany, 3Salzetalklinik, Bad Salzuflen, Germany

Introduction: Returning to work is a crucial factor for cancer patients. Accordingly, work-related orientation in medical rehabilitation (MBOR) gains an increasing importance. However, only little is known about contents and processes of MBOR measures in oncological rehabilitation. The aim of the study was to develop and evaluate a multimodal MBOR module called Perspective Job for cancer patients. Furthermore the supporting needs for occupational integration were examined from the patient’s point of view.

Methods:Perspective Job was developed by a rehabilitation team. The pilot-study had a sequential control group design with an intervention group (IG: n = 120) and a control group (CG: n = 86). Participants were oncological patients with substantial work-related problems. Patients were asked how helpful they estimated the work-related therapies and defined work-related outcomes (self-assessed working capacity (SWC), functional capability in occupational (FCO), return to work (RTW)) over 3 months after discharge. Furthermore telephone based interviews with participants were conducted 6 and 18 weeks after discharge. Chi-square tests and analysis of covariance were applied to examine differences between CG and IG.

Results:Perspective Job consists of occupational therapies and job trainings. The IG emphasized that most therapies were classified as work-related (p < 0.001) than the CG. Three months after discharge, the IG felt better prepared for returning to work (p < 0.05), were considerably less limited in their FCO (p < 0.01) and estimated their SWC more often as re-established (p < 0.05) than the CG. 65% of the IG and 57% of the CG returned to work 3 months after discharge (p = 0.551).

The telephone interviews illustrate: A stepwise occupational reintegration is described as an essential instrument for a successful RTW. Moreover, the RTW can be promoted by good health status, work-related rehabilitation with planned occupational aftercare, support by doctors and good working climate.

Conclusions: The pilot study provides evidence that Perspective Job is a useful intervention to enhance work-related outcomes for cancer patients with substantial work-related problems. Facilitators for RTW were good functional abilities, health status, supports by doctors and positive working atmosphere. Moreover, a work-related rehabilitation and occupational aftercare activities facilitate return to work. Further studies should be carried out to verify these results.

Disclosure: Heike Kähnert: No conflict of interest disclosed.Birgit Leibbrand: Employment or Leadership Position: Vorstandsmitglied im Verein zur Förderung der Rehabilitationsforschung e. V. Norderney

V53 – Sustainability of a urooncological rehabilitation program for prostate cancer patients

Heydenreich M.1, Zermann D.-H.1

1Vogtland-Klinik Bad Elster, Urologie/ Uroonkologie, Bad Elster, Germany

Introduction: Specialized rehabilitation programs allow an early social and occupational reintegration of patients after prostate cancer surgery. Aim of this prospective study was an evaluation of the sustainability and efficacy of a complex training program two years after rehabilitation.

Methods: 150 former patients after prostate surgery were contacted and asked to fill out a questionaire. All former patients had a standardized functionally oriented, specialized 3-week-rehabilitation program completed two years ago.

Results: 47 patients (Ø 67.2 years) completed the questionnaire.

The following results were obtained:

  1. 93,6% of patients had a good to excellent state of health

  2. 40,4% of patients were without stress incontinence

  3. 53,2% of patients performed continence training

  4. number of used pads (Ø 1,15 pads (SD = 1,37)) for security reasons or persistent urinary incontinence

  5. the most common daily life activities were: walking (31,4%), cycling (19,8%), gymnastic (14%) and swimming (9,3%)

  6. 97,9% of patients evaluated the rehabilitation program with good to excellent

  7. 100% of patients evaluated the medical care with good to excellent

  8. 100% of patients evaluated the workshops with good to excellent

  9. 91,5% of patients would visit Vogtland-Klinik Bad Elster for a second time, if necessary

Conclusion: Specialized rehabilitation has a strong sustainability! This applies for all aspects of general health, healthy behavior and also functional deficits. Two years after rehabilitation patients still benefit from rehabilitation after prostatectomy. Therefore rehabilitation should be offered to all prostate cancer patients.

Disclosure: No conflict of interest disclosed.

Freier Vortrag – Chronische lymphatische Leukämie – Biologie

V56 – Genetic loss of NFAT2 leads to CLL transformation

Fuchs A.R.1, Märklin M.1, Heitmann J.S.1, Truckenmüller F.M.1, Saur S.J.1, Ganser M.1, Bugl S.1, Kopp H.-G.1, Kanz L.1, Rao A.2, Wirths S.1, Müller M.R.1

1Universitätsklinikum Tübingen, Medizinische Klinik und Poliklinik, Abteilung für Onkologie, Hämatologie, Rheumatologie, Klinische Immunologie und Pulmonologie, Tübingen, Germany, 2La Jolla Institute of Allergy and Immunology, La Jolla, United States

NFAT2 is a transcription factor which regulates developmental and activation programs in diverse cell types. CLL constitutes a heterogeneous disease with some patients exhibiting an indolent course for many years and others progressing rapidly and requiring early treatment. A defined subgroup of patients shows enhanced responsiveness to stimulation of the B cell receptor (BCR) complex and more aggressive disease. In contrast, another subset of CLL patients with more indolent course is characterized by an anergic B cell phenotype referring to B cell unresponsiveness to IgM ligation. Here, we analyzed the role of NFAT2 in CLL with respect to the maintenance of the anergic phenotype and the development of Richter´s syndrome.

For this purpose, we generated conditional NFAT2fl/fl CD19-Cre knock out mice on the Eµ-TCL1 transgenic background. These mice exhibit NFAT2 deletion limited to the B cell lineage and develop a human-like CLL. To extend our observations to the human disease, we further analyzed primary blood samples from patients with CLL (n = 30) and Richter´s syndrome (n = 5).

Mice with genetic loss of NFAT2 in their B cell compartment showed a significantly more aggressive disease course and a dramatically reduced life expectancy. Spleen and lymph nodes exhibited a transformed disease phenotype in the NFAT2 ko cohort with diffuse distortion of their architecture by large B cells with blastic chromatin. CLL cells with regular NFAT2 expression were unable to mobilize calcium upon IgM ligation and thus exhibited the typical anergic phenotype. Using affymetrix microarrays we defined a genetic signature of anergy in CLL cells consisting of Lck, Pacsin1 and the E3 ligases Cbl-b and Grail, which were highly expressed in CLL cells with normal NFAT2 expression. Furthermore Lck was constitutively activated in anergic CLL cells while it was essentially inactive in NFAT2 ko cells suggesting an important role for Lck in mediating the anergic phenotype. Primary human CLL samples showed a significant overexpression of NFAT2 and Lck, predominantly in its active conformation. Tissue samples from patients with Richter’s Syndrome on the other hand demonstrated a significantly reduced expression of NFAT2 and complete loss of Lck expression.

In summary, our data provide strong evidence that NFAT2 and its target gene Lck are important mediators of anergy induction in CLL and that their loss may lead to disease transformation to aggressive B cell lymphoma (Richter´s syndrome).

Disclosure: No conflict of interest disclosed.

V57 – Loss of NFAT2 leads to an acceleration of clonal evolution in CLL

Müller D.J.1, Märklin M.1, Kanz L.1, Wirths S.1, Müller M.R.1

1Universitätsklinikum Tübingen, Medizinische Klinik und Poliklinik, Abteilung für Onkologie, Hämatologie, Rheumatologie, Klinische Immunologie und Pulmonologie, Tübingen, Germany

Chronic lymphocytic leukemia (CLL) may be defined as a clonal expansion of mature B cells with stereotypic B cell receptors (BCR). Somatic hypermutation of the BCR heavy chains (IGVH) defines subgroups with different prognosis. In up to 10% of cases Richter’s transformation to a high grade lymphoma with a mostly dismal prognosis is observed. The TCL1 transgenic mouse is a well accepted model of human CLL. Upon B cell specific knock out of NFAT2, TCL1 mice develop a disease resembling Richter’s syndrome with a significantly more aggressive disease phenotype. While TCL1 B cells exhibit tonic anergic BCR signaling characteristic of human CLL, loss of NFAT2 leads to readily activated BCRs indicating different BCR usage with altered down-stream signaling.

Here, we analyzed BCR usage in C57BL/6 wildtype, TCL1, and TCL1-NFAT2-/- mice. Splenocyte cDNA of the respective animals at an age of 7 months was amplified by multiplex PCR covering known heavy chain VDJ alleles. The PCR products were subsequently cloned into standard bacterial plasmids and subjected to conventional DNA sequencing. In addition, PCR products were analyzed by next-generation-sequencing (NGS). For data analysis, the IMGT/HightV-QUEST online tool was applied.

A very diverse and polyclonal BCR usage was found in wild type mice with more than 4000 different clones identified. Although TCL1 mice at 7 months of age exhibit all features of CLL, their BCR usage as analyzed by unambiguously identified VDJ recombination was still polyclonal. Only with respect to VH usage, TCL1 mice were found to use a more limited set of V alleles compared to wild type mice. Loss of NFAT2 by conditional knockout on the contrary, leads to an oligoclonal usage of VDJ recombination, to a further limitation of V alleles, and also to the usage of identical VDJ recombination in different mice with high frequency – indicating BCR selection either by antigen or self-signaling in these mice. Further analyses by sequencing of hypervariable regions showed preferential usage of mutated BCRs in TCL1 mice and of unmutated BCRs in NFAT2 ko mice reflecting benign and aggressive disease in humans.

In summary, the loss of NFAT2 signaling in CLL precipitates the selection of unmutated BCRs as well as the preferential usage of certain VDJ recombinations which results in the accelerated development of true oligoclonal disease.

Disclosure: No conflict of interest disclosed.

V58 – Knock-down of the NFAT2 long and intermediate isoforms leads to CLL acceleration

Heitmann J.S.1, Märklin M.1, Truckenmüller F.M.1, Fuchs A.R.1, Kopp H.-G.1, Kanz L.1, Rao A.2, Wirths S.1, Müller M.R.1

1Universitätsklinikum Tübingen, Medizinische Klinik und Poliklinik, Abteilung für Onkologie, Hämatologie, Rheumatologie, Klinische Immunologie und Pulmonologie, Tübingen, Germany, 2La Jolla Institute of Allergy and Immunology, La Jolla, United States

NFAT2 is a highly phosphorylated transcription factor which regulates developmental and activation programs in diverse cell types. We and others have previously described a significant overexpression of NFAT2 in CLL cells as compared to physiological B cells. Three major isoforms of NFAT2 with different regulatory properties have been described (700 aa short isoform, 800 aa intermediate isoform, 900 aa long isoform). Here, we analyzed the role of different NFAT2 transcripts in CLL with respect to disease phenotype and cell proliferation.

We investigated primary samples from CLL patients (n = 30) for their expression profile of different NFAT2 isoforms using RT-PCR. Applying an shRNA approach, we generated stable knock-down cells of the CLL cell line MEC-1 for the long and intermediate isoforms and for the entire NFAT2 gene resulting in the complete ablation of all isoforms. The proliferation properties of the different MEC-1 cell line were subsequently assessed in xenotransplant experiments into NSG mice.

While physiological B cells express comparable levels of the short and intermediate/long isoforms, we could detect a fivefold overexpression of the intermediate/long isoforms in primary CLL samples. To further analyze the differential regulation of the different NFAT2 transcripts on tumor cell proliferation and cell cycle regulation, we injected NSG mice with MEC-1 cells with intact NFAT2 (n = 6), MEC-1 cells with a knock-down of the intermediate and long isoforms (n = 6) and MEC-1 cells with a complete NFAT2 knock-down (n = 6). MEC-1 cells with selective ablation of the intermediate and long NFAT2 isoforms grew significantly faster in NSG mice than MEC-1 cells with intact NFAT2 expression or MEC-1 cells with a complete NFAT2 knock-down. MEC-1 cells selectively lacking the intermediate and long isoforms led to accelerated tumor proliferation upon subcutaneous injection. Cell cycle analysis as assessed by flow cytometry showed a significantly increased number of cells in the G1/S-Phase for the group without expression of the short isoform, while the group with complete NFAT knock-down exhibited a compromised growth pattern as compared to wild-type MEC-1 cells.

In summary, our data demonstrate that genetic loss of the intermediate and long isoforms of NFAT2 leads to CLL acceleration

Disclosure: No conflict of interest disclosed.

V59 – AKT activation in Chronic Lymphocytic Leukemia cells promotes transformation towards aggressive Richter´s syndrome lymphoma

Nickel N.1, Al-Maarri M.2, Pal M.2, Roth A.1, Schäfer S.3, Büttner R.3, Hallek M.1, Wunderlich T.2, Pallasch C.P.4

1Klinik I für Innere Medizin, Universität Köln, CIO, Köln, Germany, 2MPI für Stoffwechselforschung, Köln, Germany, 3Institut für Pathologie, Köln, Germany, 4Klinik I für Innere Medizin, Universität Köln, CIO, CECAD, Köln, Germany

Introduction: Richter´s syndrome (RS) is an aggressive transformation of Chronic Lymphocytic Leukemia (CLL) refractory to current therapies with dismal prognosis. RS arises from CLL cells independent of common DLBCL-mutations. Frequently mutations in p53, CDKN2 or cMyc genes are involved, but a significant proportion displays no specifically acquired driver mutation. Here we aim to elucidate the role of AKT in transformation towards Richter´s syndrome in human patients and functionally address constitutively AKT-activation in vivo. Ultimately, we aim to develop mouse models that can be used to develop novel treatment options towards Richter´s syndrome.

Methods: We have developed a mouse model allowing for Cre-activatable expression of a myristoylated AKT constitutive active allele from the Rosa26 locus (AKT-C). We crossed that mice with Cd19Cre- and Cg1Cre-mediated B cell-specific AKT-C expression in the Eµ:Tcl1 CLL mouse model.

Results: In biopsies revealed from patients with RS that one third of cases showed enhanced AKT activation. Strikingly, Richter´s transformation was recapitulated when AKT was genetically over-activated in Eµ-Tcl-1 mice, where AKT-C expressing cells developed a high-grade lymphoma phenotype leading to significantly decreased survival. AKT-C expression in TCL1 CLL cells furthermore induced morphology changes displaying features of aggressive lymphoma such as large transformed B-cell phenotype and frequent mitotic figures, enhanced proliferation indicated by KI67.

Thus, Eµ:Tcl1 transformed CLL cells act in concert with constitutively active AKT to develop RS. Noteworthy, Cd19Cre;AKT-C double transgenic mice fail to develop leukemia and lymphoma, indicating that CLL development in Eµ:Tcl1 mice is a precondition to transformation. As for the downstream mechanisms of AKT-mediated transformation we identified GSK-3b inhibition and subsequent cMyc and Mcl-1 stabilization. This might confer contribute to resistance of AKT-C transfomed lymphoma cells against genotoxic and targeted drugs. Here we see resistance towards conventional chemotherapeutics as against BTK/PI3K/BCL2-inhibiting compounds in this novel model of Richter´s syndrome.

Conclusions: Collectively, we have generated the first murine RS model providing novel mechanistic insights into the molecular understanding of Richter´s transformation that is amenable to model therapeutic strategies and to address the efficacy of synergistic treatment combinations.

Disclosure: Nadine Nickel: No conflict of interest disclosed.Christian Pallasch: Advisory Role: Gilead Sciences

V60 – The microenvironment alters CLL cell response to NF-kB inhibition

Foerster K.1, Simon-Gabriel C.-P.1, Bleckmann D.1, Benkisser-Petersen M.1, Thornton N.1, Claus R.1, Duyster J.1, Zirlik K.1

1University Medical Center Freiburg, Department of Hematology, Oncology, and Stem cell transplantation, Freiburg, Germany

Introduction: NF-kB transcription factor is constitutively active in CLL and has been suggested as promising therapeutic target. NF-kB inhibition induces apoptosis in CLL cells in vitro. However, whether this effect pertains in vivo remains unclear. Since the microenvironment is crucial for CLL cell viability circumventing apoptosis, we tested whether NF-kB inhibition modulates CLL viability in the presence of microenvironment.

Methods: The specific NF-?B inhibitor Dehydroxymethylepoxyquinomicin (DHMEQ) was used alone or combined with fludarabine, BAFF, APRIL, CXCL12, or CD40 ligand on primary CLL cells in mono- or in co-culture with stromal cells (BMSC). Apoptosis was measured using AnnexinV/PI stainings. Protein expression was analyzed by western blot. NF-kB DNA-binding activity was measured by ELISA and p65 knockdown was performed by siRNA transfection.

Results: NF-kB inhibition using DHMEQ led to apoptosis in monocultured CLL cells (viability 74% vs. 24%, n = 17, p < 0.0001) but surprisingly had no effect on cell viability of cells co-cultured with BMSC (viability 96% vs. 95%, p = 0.9995). In monoculture, apoptosis induction was accompanied by downregulation of the NF-kB target protein TRAF-1, upregulation of the proapoptotic protein Bax, and increased PARP cleavage. Conversely, in co-culture, downregulation of TRAF1 and several NF-kB subunits were observed without concomitant Bax upregulation or PARP cleavage. NF-kB DNA-binding activity of all NF-kB subunits was equally suppressed by DHMEQ treatment in mono- and co-cultured cells. Knock down of the NF-kB subunit p65 solely induced apoptosis in monocultured CLL cells. Adding soluble BAFF to monocultured CLL cells, attenuated DHMEQ efficiency (viability 1% vs. 16%, n = 9). Finally, the combined use of DHMEQ with fludarabine in co-cultured CLL cells led to a higher rate of apoptosis than DHMEQ (viability 57% vs. 37%, p = 0.0202) or fludarabine alone (viability 50% vs. 37%, p = 0.1828).

Conclusion: NF-kB inhibition in primary CLL cells shows great discrepancy between in vitro and in vivo scenarios. While DHMEQ treatment leads to apoptosis in mono-cultured cells, CLL cell viability is not affected in the presence of microenvironment, suggesting that the NF-kB pathway can be bypassed in vivo. Soluble ligands appear to be involved in mediating this protective effect. However, the combination of NF-kB inhibition with standard chemotherapy might represent a fruitful approach and warrants further clinical assessment.

Disclosure: No conflict of interest disclosed.

V61 – Pathway inhibitors for bridging to allogeneic hematopoietic stem cell transplantation (HSCT) for chronic lymphocytic leukemia (CLL)

Hahn M.1, Dietrich S.1, Hain S.-A.2, Hegenbart U.1, Rieger M.3, Scheuer B.4, Ho A.D.1, Dreger P.1

1Universität Heidelberg, Medizinische Klinik V, Heidelberg, Germany, 2St. Marien-Krankenhaus, Medizinische Klinik III, Siegen, Germany, 3Onkologische Schwerpunktpraxis, Darmstadt, Germany, 4Onkologische Praxis, Pirmasens, Germany

Pathway inhibitors (PI), such as ibrutinib and idelalisib, have dramatically increased the treatment options for high-risk CLL. There is only limited information on the feasibility and efficacy of HSCT if used as consolidating treatment after PI salvage for relapsed/refractory high-risk CLL.

Aim: of this single centre retrospective analysis was to investigate the outcome of HSCT after previous PI exposure in patients with CLL.

Results: Between Nov 2013 and Feb 2016, 9 patients (m/f= 6/3; median age 53y (33-61)) underwent HSCT for relapsed/refractory high-risk CLL (RR-CLL) after prior exposure to ibrutinib (6), CC-292 (2), idelalisib (2), and/or venetoclax (1) (multiple exposure included). Indications for HSCT were (hierarchical) RR-CLL with poor-risk cytogenetics (6), previous PI failure (1), and Richter transformation (RT) (2). Before start of conditioning, 8 patients were still responding to the most recent PI with incipient progression under ibrutinib in 2 of them. A single patient with RT in CR was off PI. Patients had been on their most recent PI for 9 (1-15) months. Donors were well-matched unrelated (8) or related (1); conditioning comprised fludarabine/ATG with Treosulfan (6) or TBI 8Gy (3). PI was generally stopped on the day before conditioning. However, a single patient developed a fulminant CLL relapse under fludarabine conditioning within 3 days after ibrutinib discontinuation and was put back on ibrutinib until day -1. With 10 (2-22) months of follow-up, low-grade acute and chronic GVHD each occurred in one patient. Whilst unexpected toxicity was not observed, one patient who had been bridged with ibrutinib dynamically progressed at day +72, and another patient had a relapse of her RT on day +214 resulting in rapid death. The 7 remaining patients live progression-free, 5 of them MRD-negative.

Conclusion: This preliminary data does not raise safety concerns about the use of PI prior to HSCT. Ibrutinib seems to be effective for bridging patients with high-risk CLL to transplant. Although disease control after HSCT does not appear to be affected by pre-transplant PI exposure, the optimum strategy for transition from PI treatment to transplant remains to be defined.

Disclosure: Michael Hahn: No conflict of interest disclosed.Peter Dreger: Advisory Role: Janssen, Gliead; Financing of Scientific Research: Janssen, Gilead

Fortbildung – Indolente Non-Hodgkin-Lymphome

V63 – WHO-Update 2016: What is relevant?

Rosenwald A.1

1Institut für Pathologie, Universität Würzburg, Würzburg, Germany

The 2016 revision of the World Health Organization (WHO) classification of lymphoid neoplasms reflects the progress that has been made in our biological, genetic and clinical understanding of some lymphoma entities over the last years impacting on the development of more targeted therapeutic strategies in the future.

In the arena of indolent Non-Hodgkin lymphomas, the concept of ‘precursor’ or ‘early’ lesions as well as very indolent forms of the disease is emphasized. Monoclonal B-cell lymphocytosis (MBL) is now recognized as a precursor of chronic lymphocytic leukemia (B-CLL). However, ‘low count’ MBL that very rarely progresses to overt B-CLL is now distinguished from ‘high count’ MBL, since the latter has many phenotypic, molecular and genetic features of Rai stage 0 B-CLL. The concept of ‘tissue-based’ MBL (discrete lymph node involvement by SLL cells) with a low rate of progression is introduced.

Precursor lesions of follicular lymphoma (FL) and mantle cell lymphoma (MCL) have been renamed to ‘in situ follicular neoplasia (ISFN)’ and ‘in situ mantle cell neoplasia (ISMCL)’ to reflect the low rate of progression to overt lymphoma. The ‘duodenal type’ of FL is specifically mentioned, since it appears to remain localized for a long period and to be associated with an excellent outcome. The previous term of ‘pediatric FL’ will be changed to ‘pediatric-type FL’ to reflect the finding that these B-cell proliferations that are almost always localized can occasionally occur also in adults.

Finally, novel genetic findings that were made possible by modern next generation sequencing technologies are introduced in the 2016 revision. Almost all cases of hairy cell leukemia carry the BRAF V600E mutations, whereas this mutation appears to be absent in hairy cell leukemia variant which in half of the cases carries a MEK1 mutation instead. Lymphoplasmacytic lymphoma (LPL, Waldenström macroglobulinemea) is associated in about 90% of cases with MYD88 (L265P) mutations. In this context, monoclonal gammopathy of undeterminded significance (MGUS) of IgM-type may be more closely related to LPL than to plasma cell myeloma.

In summary, the 2016 update of the WHO classification provides updated diagnostic lymphoma categories including their precursor lesions, together with the description of more refined biological and clinical correlates.

Disclosure: No conflict of interest disclosed.

V64 – Follicular Lymphoma Grade 3 - To CHOP or not to CHOP?

Scholz C.W.1

1Vivantes Klinikum Am Urban, Hämatologie und Onkologie, Berlin, Germany

Follicular lymphoma (FL) is the most prevalent indolent lymphoma. Depending on the frequency of interspersed centroblasts, FL are divided into grade 1 to 2 (grade 1/2), if less than 16 centroblasts per high power field (HPF) are detected among centrocytes, and grade 3, if more than 15 centroblasts are counted per HPF. Grade 3 is further grouped into grade 3A, if neoplastic follicles are composed of centroblasts and centrocytes, and grade 3B, if only centroblasts are detectable. Noteworthy, CD10 expression and t(14;18)/BCL2 rearrangement, two distinguishing marks of FL, are less frequently found in FL grade 3 as compared to grade 1/2. Remarkably, FL grade 3A frequently contains areas of grade 1/2 in the same specimen, while this rarely is the case for FL grade 3B. This circumstance likely further increases interobserver variability when it comes to diagnosing FL grade 3. Therapy regimen for individuals with FL grade 1/2, which account for approximately 85% of all FL patients, have been investigated in distinct phase II and III trials, challenging the need for anthracyclines in first line treatment. As a consequence, Rituximab Bendamustin has replaced R-CHOP as first line therapy in many centers. In contrast, treatment for patients with FL grade 3 is far less evidence based, as most prospective clinical trials with FL patients either exclude individuals with FL grade 3 or contain only small subsets of patients with FL grade 3A or 3B. FL grade 3B is deemed to be a more aggressive disease and therefore is treated like diffuse large B cell lymphoma with R-CHOP. The same is applicable for cases where a diffuse large B cell lymphoma component is detected concomitantly with FL grade 1/2 or 3. In contrast, the situation is less settled for FL grade 3A, which account for approximately 10% of all FL. Here, evidence resulting from retrospective register analyses and post-hoc evaluations of prospective trials, suggests that patients with FL grade 3A, progress similar to or, in respect to patients with pure FL grade 3A, fare better than patients with FL grade 1/2. While many of the individuals from these analyses received anthracycline-containing regimen like CHOP or R-CHOP as first line treatment, it remains unresolved, whether anthracyclines are truly required for first line therapy of all patients with FL grade 3A, or whether for some of them a regimen like Rituximab Bendamustin could be an alternative similar to the situation in FL grade 1/2.

Disclosure: Christian Scholz: Advisory Role: Gilead, Janssen-Cilag, Novartis, Roche, Sepropharm; Financing of Scientific Research: Gilead, Janssen-Cilag, Novartis, Pfizer, Roche, Sepropharm; Expert Testimony: Mundipharma, Novartis

V65 – Mantle cell lymphoma: has the era of immun-kinom-chemotherapy come?

Hess G.1

1III. Medizinische Klinik, Universitätsmedizin Mainz, Mainz, Germany

Mantle cell lymphoma still has a dismal prognosis, however new treatment approaches have entered the therapeutic arena recently with the potential to ultimately improve prognosis of patients: optimized use of conventional immune-chemotherapy and secondly the introduction of more specific targeted agents.

Among the first group the introduction of cytarabinoside in first line treatment combined with dose intense consolidation treatment combined with antibody treatment has resulted in unprecedented long term responses. In addition early results show that maintenance treatment using rituximab may further improve PFS and potentially OS. For patients not suitable for intensive treatment receiving R-CHOP, R-maintenance already can be considered standard of care, whereas optimal regimen incorporating cytarabinoside need yet to be defined for this group.

As the susceptibility to chemotherapy approaches is in general limited to very few treatment lines, alternative approaches have been investigated early on, proofing the suitability of drugs like bortezomib, lenalidomide and temsirolimus. All of these drugs were able to induce reasonable responses in relapsed disease, however responses to single agent treatment used to be short lived. Ibrutinib now has become the best targeted agent available with responses observed in up to 70% of patients and a median remission duration for more than one year. Although superior to other treatments, still this cannot be considered optimal and attempts have been initiated to improve the use of these agents.

In general 2 approaches have been used: development of so called chemo-free regimen and all-in combinations testing these agents together with chemotherapy and immunotherapy. The first type of combinations aims to achieve optimized response rates and remission duration primarily with treatment until progression und comparison is made against conventional regimen in terms of efficacy and side effects. Trials like the ENRICH (Ibr + R vs. Chemo + R)( will help to understand the value of these approaches. In contrast, approaches combining all available treatment modalities try to achieve deep responses with limited treatment duration. Key trials to investigate these concepts are the TRIANGLE (intensive treatment +/- Ibr) trial and to some extent the SHINE (BR+/- Ibr) trial.

Taken together, improvements have been made using all available therapeutic options, with the optimal use still needs to be defined.

Disclosure: Georg Hess: Advisory Role: Pfizer, Celgene, Roche, Janssen; Expert Testimony: Pfizer, Roche, CTI, Celgene

Fortbildung – CUP-Syndrom

V66 – CUP: Molecular Diagnostics

Weichert W.1

1Technische Universität München, Institut für Pathologie, München, Germany

Carcinoma of unknown primary (CUP) is a challenging disease “entity” not only on the clinical side but specifically when it comes to the selection of diagnostic modalities to apply in this setting. Diagnostically, initial conventional histology aided by immunohistology aims at diminishing the number of cases, which have to be assigned as CUP and helps to identify putative primaries. Although this approach is not successful in all cases, it might still delineate a groups of special CUP cases with likely primary sites. However, the majority of CUP cases remain entirely non-classifiable and usually present as adenocarcinomas with an uncharacteristic immune phenotype. For these cases multimarker expression analyses investigating either mRNA or microRNA profiles have been proposed as an adjunct for diagnosis but these approaches have failed to enter clinical routine testing so far. Recently, massive parallel sequencing (MPS) approaches have enabled us to interrogate comprehensive molecular profiles from human tumors and it has been shown that this technology can be implemented into the routine diagnostic setting. For CUP cases MPS approaches are specifically interesting, since they a) might supply additional information with respect to entity classification and b) might detect molecular alterations that are indicative for the response to specific targeted drugs. Consequently, targeted multigene and broader exomic/genomic sequencing approaches have been used to define the molecular landscape of these tumors and aid in CUP therapy selection. The respective research approaches and available data are discussed.

Disclosure: No conflict of interest disclosed.

V67 – Chemotherapeutic treatment of cancer of unknown primary

Krämer A.1

1Klinische Kooperationseinheit Molekulare Hämatologie/Onkologie, Medizinische Klinik V, Universität Heidelberg und Deutsches Krebsforschungszentrum, Heidelberg, Germany

Cancer of unknown primary (CUP) is a heterogeneous group of histologically confirmed metastatic cancers for which the anatomical site of origin remains occult despite appropriate examination. Approximately 3-5% of all human cancers manifest as CUP, which is the seventh most frequent cancer diagnosis and the fourth most common cancer-related cause of death in patients of both sexes. CUP can be divided into favorable and unfavorable subsets. Patients with unfavorable CUP account for 70-80% of cases and have a poor prognosis with a median survival of 8-11 months. Two opposing hypotheses exist with regard to the biology of CUP, which is still poorly understood. One hypothesis considers CUP to be a separate type of cancer with unique biologic features that account for the absence of a primary tumor and early metastatic disease. Based on this view, many patients with unfavorable CUP uniformely receive platinum-based standard chemotherapy in combination with a taxane or gemcitabine. Alternatively, CUP represents different, site-specific, unrelated groups of malignancies. In that case, gene expression profiling approaches to identify the tissue-of-origin might allow to extend the management of known cancers to subtypes of CUP in order to advance therapies for this disease. Most recently, mutational profiling has been suggested to alleviate the need for tissue-of-origin identification, making CUP an epitome of personalized medicine, with individualized treatment driven by the mutational status rather than the primary site of each patient.

Disclosure: Alwin Krämer: Advisory Role: Roche; Expert Testimony: Merck

Fortbildung – Lebertumoren

V69 – Rational imaging of hepatic lesion: How to do it

Wiggermann P.1

1Universitätsklinikum Regensburg, Institut für Röntgendiagnostik, Regensburg, Germany

The purpose of this lecture is to address capabilities and limitations of different imaging techniques such as ultrasound (US), contrast enhanced US (CEUS), magnetic resonance imaging (MRI), computed tomography (CT) and positron emission tomography-CT (PET-CT) for detection and differentiation of hepatic lesions. Relevant implications from national and international guidelines for different tumor entities regarding hepatic imaging will be discussed and set into a framework for a rational imaging pathway of hepatic lesions in a clinical setting.

Disclosure: Philipp Wiggermann: Expert Testimony: Bayer Vital GmbH - 51368 Leverkusen

Wissenschaftliches Symposium – Neue immuntherapeutische Ansätze nach allogener Stammzelltransplantation

V75 – Gamma-delta T-cells: graft-versus-leucemia effect without graft-versus-host disease?

Kunzmann V.1, Smetak M.2, Schaefer-Eckart K.2, Kimmel B.1, Birkmann J.2, Einsele H.1, Wilhelm M.2

1Medizinische Klinik und Poliklinik II, Universitätsklinikum Würzburg, Hämatologie/Onkologie, Würzburg, Germany, 2Klinikum Nürnberg, Medizinische Klinik 5, Nürnberg, Germany

Introduction: Human gammadelta (?d) TCR-expressing cells constitute 1-5% of total peripheral T cells and have been shown to exert potent antitumor activity in a MHC-independent manner. Recent analysis of various cancer types revealed the presence of intratumoral ?d T cells as the most significant favourable prognostic immune population. Within this pilot study we want to determine the feasibility and safety of an adoptive transfer and in vivo expansion of human haploidentical ?d T lymphocytes.

Methods: Four patients with advanced haematological malignancies (AML, NHL or Multiple Myeloma) who were not eligible for allogeneic transplantation received peripheral blood mononuclear cells from haploidentical family donors. For that, a single unstimulated leukapheresis product was incubated with both the anti-CD4 and anti-CD8 antibodies conjugated to paramagnetic particles. On average, patients received 2.17 × 106/kg (range 0.9-3.48) ?d T cells with < 1% CD4-or CD8-positive T cells remaining in the product. All patients received prior lymphopenia-inducing chemotherapy (fludarabine 20-25 mg/m2 day -6 until day -2 and cyclophosphamide 30-60 mg/kg day -6 and -5) and were treated with 4 mg zoledronate on day 0 and 1.0×106 IU/m2 IL-2 on day +1 until day +6 for the induction of ?d T cellproliferation in vivo.

Results: This treatment resulted in a marked in vivo expansion of donor ?d T cells and, to a lower extent, natural killer cells and double-negative aß T cells (mean 68-fold, eight-fold, and eight-fold, respectively). Proliferation peaked by around day +8 and donor cells persisted up to 28 days. Although refractory to all prior therapies, three out of four patients achieved a complete remission, which lasted for 8 months in a patient with plasma cell leukaemia. None of the patients showed any signs of acute or chronic GVHD.

Conclusions: This pilot study shows that adoptive transfer and in vivo expansion of haploidentical ?d T cells is feasible and suggests a potential role of this donor ?d T cell infusion in the treatment of haematological diseases.

Disclosure: No conflict of interest disclosed.

Wissenschaftliches Symposium – ALL: Molekulare und zelluläre Grundlagen für zielgerichtete Therapien

V77 – Clonal heterogeneity and evolution in acute lymphoblastic leukemia

Brüggemann M.1

1Sektion für Hämatologische Spezialdiagnostik, Klinik für Innere Medizin II, UKSH, Campus Kiel, Kiel, Germany

Recent genomic studies have provided insight into tumor heterogeneity of acute lymphoblastic leukemia (ALL) and the evolutionary trajectory from diagnosis to relapse. In fact, continuing clonal expansion itself might be accompanied by genetic and functional diversification of cancer cells. Their subclonal diversity is inter alia held accountable for primary or secondary resistance to systemic therapy. Clonal heterogeneity and subclonal hierarchies have been substantially studied particularly in childhood BCR-ABL fusion positive (BCR-ABL+) BCP-ALL: linear and branching patterns of clonal evolution have been documented and diverse individual subclones showed leukemia maintaining or initiating potential in xenograft models. Furthermore, founder mutations like the BCR-ABL fusion may originate at distinct stages of hematopoietic development. All these findings translate into a clinically heterogenous disease also regarding treatment response and prognosis. Molecular analyses of other recurrent genetic alterations like intrageneic deletions of the BTG1, IKZF1 and ERG indicate that also these aberrations can arise independently in multiple subclones. Detailed sequence analyses of intrageneic breakpoints strongly suggest illegitimate RAG1/RAG2-mediated recombination as the responsible mechanism for aberrations. Comparison of the clonal architecture of ALL between diagnosis and relapse may help to characterize the prognostic meaning of subclonal events for treatment resistance as resistant mutated subclones may survive therapy, acquire additional mutations and become the relapse founder clone. In childhood all, NT5C2, CREBBP, WHSC1, TP53, USH2A, NRAS and IKZF1 are enriched at relapse indicating their potential importance for drug resistance. E. g. recurrent NT5C2 mutations potentially allow for resistance to nucleoside analogs. These mutations predominantly emerge early during maintenance therapy in which nucleoside analogs assume a predominant role in treatment. In summary, analysis of the dynamics of the subclonal structure of ALL and may help to get insights into the biological meaning of particular aberrations and to optimize therapy accordingly.

Disclosure: No conflict of interest disclosed.

V78 – Personalized medicine for ALL? The pediatric INFORM project

Worst B.C.1,2, van Tilburg C.M.1,2,3, Balasubramanian G.P.1, Fiesel P.2, Pfaff E.1,2, Pajtler K.1, Freitag A.3, Witt R.3, Kulozik A.E.2, von Stackelberg A.4, Meisel R.5, Borkhardt A.5, von Deimling A.1,2, Eggert A.4, Lichter P.1, Capper D.1,2, Witt O.1,2, Pfister S.M.1,2, Jones D.T.W.1

1Deutsches Konsortium für Translationale Krebsforschung (DKTK) und Deutsches Krebsforschungszentrum (DKFZ), Heidelberg, Germany, 2Heidelberg University Hospital, Heidelberg, Germany, 3NCT Trial Center, Heidelberg, Germany, 4Charité – University Hospital, Berlin, Germany, 5Düsseldorf University Hospital, Düsseldorf, Germany

Introduction: Relapses from high-risk tumors, including several solid tumor entities and also hematopoietic malignancies, pose a major clinical challenge in pediatric oncology. The German INFORM registry study (INdividualized therapy FOr Relapsed Malignancies in children) addresses this problem using integrated next-generation sequencing to rapidly identify patient-specific potential therapeutic targets.

Methods: Whole-exome, low-coverage whole-genome and RNA sequencing is complemented with microarray-based expression and DNA methylation profiling. Identified alterations are discussed and prioritized according to biological significance and potential druggability in a weekly molecular tumor board.

Results: To date, 182 tumors have been profiled, with acute lymphoblastic leukemia accounting for 5% of these (n = 9). Turnaround time from tissue arrival to molecular results was 21 days on average. For patients post stem cell transplantation, we established an analysis pipeline to eliminate donor germline variant ‘contamination’ and generate reliable results within the same timeframe. In the total cohort, we detected one or more high-confidence druggable alterations in 118/182 (65%) patients. This ratio was higher for ALL-patients, where we identified druggable alterations in 9/9 (100%). Tyrosine kinases, the PI3K/mTOR pathway, MAPK pathway, and cell-cycle regulators were commonly affected. In one ALL patient with multiple relapses who had already undergone two stem cell transplantations, we identified a very rare INPP5D(SHIP1):ABL1 fusion. Treatment with tyrosine kinase inhibitors (dasatinib, later switched to ponatinib) combined with a multimodal immunotherapeutic treatment strategy lead to the induction of a complete remission for approximately 9 months.

Conclusion: In summary, comprehensive profiling of pediatric tumors is feasible in a clinically meaningful timeframe, and provides valuable diagnostic information and potential therapeutic targets. We now aim to expand this approach to early-phase biomarker driven interventional trials. The relatively low number of ALL patients included so far, compared with the disease prevalence, may partly be explained by the high diversity of alternative diagnostic and therapeutic approaches. We are therefore establishing collaborations, e.g. with the iVAC-ALL peptide vaccination study, to maximize the available biological information and identify the optimal therapy concept for every individual patient.

Disclosure: No conflict of interest disclosed.

V79 – CAR-T cell strategy – hype or hope?

Rössig C.1

1Universitätsklinikum Münster, Klinik für Kinderheilkunde und Jugendmedizin – Pädiatrische Hämatologie und Onkologie -, Münster, Germany

Chimeric antigen receptors (CARs) have recently emerged as a powerful means of redirecting T-cell functions toward B-lineage leukemias. CARs combine the ability of antibodies to recognize leukemia-associated surface antigens with the capacity of (co)stimulatory signal domains to activate T cells. Clinical trials now clearly demonstrate the potency of CAR-expressing T cells to eliminate human cancers. In a large proportion of patients with refractory CD19-expressing leukemias, T cells that express a CD19-specific CAR by genetic modification efficiently induced complete and even molecular remissions. The most significant toxicity of CAR T cell therapy is cytokine release syndrome that can be immediately life-threatening but has become manageable in most cases. The strength of CAR T cell therapy compared to bispecific T cell engagers is the ability of CAR gene-engineered T cells to persist in vivo and establish protective tumor-antigen specific memory responses. Indeed, CAR T cells were detected in the peripheral blood of many treated patients for several years, resulting in durable remissions at least in some patients. Another advantage is effective CNS penetration of CAR T cells and the ability to eliminate leukemic cells from this extramedullary compartment. On the other hand, a major challenge to broad implementation of the strategy in B cell cancers remains the need for individualized manufacturing of the T cell products. A drawback shared with other CD19 targeting strategies is the emergence of CD19-negative resistant clones by either CD19 splice variants or outgrowth of CD19-negative myeloid subclones under the selective pressure of CD19-directed CAR T cell therapy which has caused relapses in around 20% of acute lymphoblastic leukemia patients. The identification of additional CAR target antigens is a critical step also for extending the promise of this immunotherapeutic approach to hematological malignancies not derived from the B cell lineage, such as AML, and to solid cancers. A critical barrier against the use of engineered T cells for the treatment of bulky lymphomas and solid tumors is the tumor microenvironment which prevents both the recruitment and functional activity of therapeutic T cells. To overcome local immunosuppression and remain active in the tumor niche, CAR T cells may have to be administered in combination with agents that block the relevant inhibitory immune pathways.

Disclosure: No conflict of interest disclosed.

Fortbildung – Hirnmetastasen und ZNS-Lymphome

V80 – Targeted-Therapy of brain metastasis of NSCLC, malignant melanoma and breast cancer

Pukrop T.1

1Universitätsklinikum Regensburg, Innere Medizin III, Regensburg, Germany

In the last years, we were pleased to achieve a significant improvement in the targeted treatment of metastatic tumor diseases, in particular in breast cancer, NSCLC and malignant melanoma. However, parallel to this positive development we observed an increase in CNS metastases. In contrast to the treatment of metastases in other organs, the body of evidence of systemic therapy in CNS-metastasis is sparse. One reason for this was the discussion about whether the blood-brain-barrier (BBB) is intact or not within CNS metastases. Today we know that the blood-brain barrier is disrupted and systemic therapy can be effective. For example, many TKI therapies have demonstrated a significant response even with CNS metastases, including meningeal metastasis. Thus, nowadays we have to consider systemic therapy for brain metastases not only in the context of clinical trials, but also in everyday clinical practice. For this reason it is also necessary and important to develop decision algorithms and clinical trials for these critically ill patients.

Disclosure: Tobias Pukrop: Financing of Scientific Research: MSD, Lilly, Mundipharma; Other Financial Relationships: Reisefinanzierungen von Amgen und Mundipharma

V82 – Targeted therapy in CNS lymphoma

Korfel A.1

1Charité – Universitätsmedizin Berlin, Hämatologie, Onkologie und Tumorimmunologie, Berlin, Germany

Introduction: Despite some progress made in management of CNS lymphoma (CNSL) its prognosis remains worse as compared to other lymphoma localization. Moreover, toxicity, including both acute side-effects and delayed CNS toxicity, represents a major problem, particularly in the elderly and after combined radio-/chemotherapy. Thus, there is a need for development of novel drugs targeting key survival pathways in CNSL.

Methods: English language literature has been reviewed for clinical studies with target therapy in CNS lymphoma.

Results: CNSL are mostly aggressive lymphomas. While secondary CNS lymphoma remain to be nearly characterized, primary CNS lymphomas (PCNSL) are usually diffuse-large B-cell lymphomas exhibiting an activated B-cell like immunophenotype with ongoing immunoglobulin gene somatic hypermutation, Candidate targets and pathways identified thus far include the NFkB pathway, the B-cell receptor, the JAK/STAT pathway, IRF4, BCL-6 and PIM kinases as well chemokine pathways, macrophages and T-cells in tumor microeviroment. A phase II study of the mTOR inhibitor temsirolimus in relapsed/refractory PCNSL is the first completed and published trial on a target drug in PCNSL. A relatively high response-rate of 54% was found in the 37 patients treated, however, most responses were short-lived. In ongoing studies, the B-cell receptor inhibitor ibrutinib, the IRF/MUM1inhibitor lenalidomide, the dual PI3K/mTOR inhibitor PQR309 and the check-point inhibitor nivolumab are being tested.

Conclusions: A better definition of therapeutic targets will probably allow the development of new drugs to improve the outcome in CNSL.

Disclosure: No conflict of interest disclosed.

Fortbildung – Thrombophilie

V85 – Perioperative anticoagulation: Is heparin bridging still relevant?

Angelillo-Scherrer A.1

1Universitätsklinik für Hämatologie und hämatologisches Zentrallabor / Inselspital, Bern, Switzerland

Background: More than 6 million Europeans are on long-term oral anticoagulation (OAC) therapy and about 10% of them require annually anticoagulation discontinuation for procedures. The approval of direct oral anticoagulants (DOAC) and novel data on perioperative anticoagulation with heparins render the management of perioperative anticoagulation therapy complex.

Method: Relevant articles were selected in PubMed according to abstract content. To supplement the search, citations in pertinent review articles were examined. Current guidelines were studied.

Results: Observational studies, large meta-analyses and a recent large randomized trial (the BRIDGE trial, NEJM 2015, 373: 823-33) revealed significant perioperative bleedings without reduction of thromboembolic events (TE) when heparin bridging is prescribed. These data point to an unbalanced effect of bridging toward bleeding. However, despite these evidences, bridging remains a common practice. Updated clinical guidelines suggest that heparin bridging is unnecessary for patients anticoagulated with DOACs. Heparin bridging should be considered for patients on long-term therapy with vitamin K-antagonists (VKA) temporarily interrupted in the periprocedural context if these patients are at highest risk of TE (= 10% per year). The decision of heparin bridging in these patients should be carefully weighted in presence of a concomitant high bleeding risk. Conversely, patients at low risk for TE should not be bridged. Interestingly, evidences from the literature suggest that patients with intermediate or unclear risks of TE or bleeding display higher perioperative bleedings than TE rates. Consequently, the individual patient and the procedure/surgery need to be very well considered before the decision to bridge with heparin is made.

Finally, the management of patients under VKA or DOAC therapy with prothrombin complex concentrates and novel antidotes specific for DOACs in the context of an urgent procedure or surgery will be discussed.

This review will be illustrated by cases from the daily clinical practice.

Conclusion: Scientific evidence supports the current guidelines and treatment algorithms for perioperative anticoagulation. Heparin bridging is proposed for a limited group of patients. Larger-scale studies are however still needed to determine the optimal periprocedural management of patients receiving DOAC therapy.

Disclosure: No conflict of interest disclosed.

Freier Vortrag – Zellbiologie

V86 – Influence of the JAK inhibitor Ruxolitinib on the migration process of dendritic cells (DC)

Wolf D.1, Heine A.2, Quast T.3, Kolanus W.3, Trebicka J.4, Brossart P.2, Rudolph J.2

1Universitätsklinikum Bonn, Bonn, Germany, 2Department of Internal Medicine III, Oncology, Hematology and Rheumatology, University Clinic Bonn (UKB), Bonn, Germany, 3Molecular Immunology & Cell Biology, Life and Medical Sciences Institute (LIMES), University of Bonn, Bonn, Germany, 4Department of Medicine I, Gastroenterology and Hepatology, University Clinic Bonn (UKB), Bonn, Germany

Introduction: Ruxolitinib is a potent JAK inhibitor approved for treatment of MPN and with a potential also for acute and chronic GvHD therapy. Ruxolitinib modified various immune cells including DC, that are essential antigen-presenting cells migrating via afferent lymphatics into T cell areas of draining lymph nodes (LN) for T cell priming. This process is guided by chemokine gradients and requires cytoskeletal rearrangement allowing cell movement. The aim of this study was a detailed analysis of the impact of ruxolitinib on DC migration with a particular focus on short-term effects of the drug and the identification of potential target molecules mediating these effects.

Methods: Migration of human monocyte-derived DCs (moDC) or murine bone marrow-derived DCs (bmDCs) was analyzed in Transwell assays or dynamically by time-lapse microscopy within three dimensional collagen gels towards CCL-19 gradients. Morphological changes and signaling events were analyzed by immunofluorescent staining and Western Blot for changes in phosphorylation levels, respectively. siRNAs were used to knockdown talin, JAK1 or JAK2.

Results: 2D migration of ruxolitinib-exposed DC is dose-dependently reduced in vitro. Analysis of the migratory phenotype of moDCs within 3D collagen gels revealed that ruxolitinib-exposed DCs are still able to sense chemokine gradients and form lamellipodia at the leading edge of the cell, whereas uropod retraction is inhibited. siRNA knockdown experiments revealed that this inhibitory effect is JAK1- and JAK2-independent. Moreover, ruxoltinib-mediated inhibition of migration is independent of integrin expression or signalling, as ruxolitinib-exposed DC express unaltered integrin levels and migration of talin 1-deficient DC (which are integrin decoupled) is also efficiently inhibited by ruxolitinib. On a molecular level we could show a reduced phosphorylation of the Rho-associated protein kinase (ROCK) in ruxolitinib-treated moDC upon CCL-19 stimulation. Finally, the observed migration phenotype induced by ruxolitinib could be mimicked by the ROCK inhibitor Y-27632.

Conclusions: RhoA family members control important steps of cell migration, such as protrusion formation and consequently cell polarization. ROCK is a downstream effector of RhoA and stabilizes the actin cytoskeleton and acto-myosin II contraction. The observed reduction of ROCK phosphorylation may reveal an important mechanism of ruxolitinib-induced inhibition of DC migration

Disclosure: Dominik Wolf: Advisory Role: Bexalta, Novartis; Financing of Scientific Research: Bexalta, Novartis; Expert Testimony: Bexalta, NovartisJanna Rudolph: No conflict of interest disclosed.

V87 – APC/CCdh1 controls differentiation and self-renewal in normal and malignant hematopoietic cells

Ewerth D.1, Kreutmair S.1, Wider D.1, Felthaus J.1, Schüler J.2, Schmidts A.1, Duyster J.1, Illert A.-L.1, Engelhardt M.1, Wäsch R.1

1Klinik für Innere Medizin I, Hämatologie, Onkologie und Stammzelltransplantation, Universitätsklinikum Freiburg, Freiburg, Germany, 2Oncotest GmbH, Freiburg, Germany

Introduction: The balance between differentiation and self-renewal in hematopoietic stem and progenitor cells (HSPCs) is crucial and deregulation may lead to malignant transformation. The differentiation is predominantly initiated in G1 phase of the cell cycle when the E3 ligase anaphase-promoting complex or cyclosome (APC/C) is highly active. In association with its coactivator Cdh1 the APC marks proteins for proteasomal degradation. There is growing evidence that Cdh1 is a regulator of differentiation which has already been demonstrated in neurons, muscle cells or osteoblasts.

Methods: Human CD34+ cells were collected from peripheral blood of G-CSF mobilized donors and cultured in presence of different cytokine combinations. The knockdown of Cdh1 was achieved by lentiviral delivery of shRNA into target cells. Transduced CD34+ cells were used for in vitro differentiation in liquid culture or CFU assay. For in vivo experiments purified cells were transplanted into NSG mice. The differentiation of AML cell line HL-60 with or without Cdh1 knockdown was induced by ATRA or PMA.

Results: A detailed analysis of different subsets from both in vitro differentiated CD34+ cells and BM showed high Cdh1 level in CD34+ cells and low expression in myeloid or lymphoid cells. The depletion of Cdh1 (Cdh1-kd) had no effect on proliferation detected by CFSE and by measuring the cell cycle length of single cells. However, Cdh1-kd cells showed a significant maintenance of CD34+ cells under self-renewal conditions and during erythropoiesis with lower frequency of glycophorin A+ cells. In CFU assays, the Cdh1-kd resulted in less primary colony formation, notably CFU-GM and BFU-E, but significantly more secondary colonies. In a xenotransplant mouse model, human Cdh1-depleted CD34+ cells engrafted to a much higher degree in the murine BM 8 and 12 weeks after injection as shown by higher frequencies of human CD45+ cells. Moreover, we also found increased frequencies of human CD19+ B cells after transplantation of CD34+ Cdh1-kd cells. Primary AML blasts showed a significant downregulation of Cdh1 expression compared to normal CD34+ cells. The Cdh1-kd in AML cell line HL-60 led to less differentiated cells and a delay in PMA- and ATRA-induced differentiation.

Conclusions: Loss of the APC/C coactivator Cdh1 supports self-renewal of CD34+ cells in vitro, facilitates engraftment capacity and B cell development of human HSPCs in vivo and contributes to the differentiation block in AML.

Disclosure: No conflict of interest disclosed.

V88 – NAMPT-NAD+-SIRT2-triggered deacetylation of LMO2 protein is indispensable in the early hematopoiesis through TAL1 complex formation

Morishima T.1, Lindner C.1, Bernhard R.1, Zahabi A.1, Dannenmann B.1, Nasri M.1, Samareh B.1, Kanz L.1, Welte K.1, Skokowa J.1

1Universitätsklinikum Tübingen, Tübingen, Germany

Introduction: Nicotinamide phosphoribosyltransferase (NAMPT)- adenine dinucleotide (NAD+)- sirtuins (SIRTs) pathways regulate transcriptions through protein deacetylase function of SIRTs. In this study, we analyzed the roles of this pathway in early hematopoiesis.

Methods: We tested NAMPT- or SIRT-inhibitors in human induced pluripotent stem (iPS) cells hematopoietic differentiation model.

Results: We differentiated iPS cells with the specific inhibitor of NAMPT and found marked reduction of VEGFR2+CD34+ early hematopoietic progenitor cells at day 6. As qRT-PCR results shows SIRT2 mRNA expression is increasing in this stage, we tested SIRT2 specific inhibitor and found significant reduction of these progenitor cells. Furthermore, the emergence of CD43+ hematopoietic progenitor cells and colony forming ability at day 13 were completely suppressed by inhibition of SIRT2. These results indicate that NAMPT-NAD+-SIRT2 signaling is needed for early hematopoiesis. To evaluate downstream targets, we analyzed mRNA and protein expressions of early hematopoietic genes (GATA2, RUNX1, LMO2, and TAL1) on day 6 and found that they were not down-regulated by NAMPT or SIRT2 inhibition. With the idea of post-transcriptional modifications, we quantified acetylation of these proteins in the cells treated or not with SIRT2 inhibitor and found that only LMO2 was deacetylated by SIRT2. Interaction between SIRT2 and LMO2 was confirmed with Duolink and co-immunoprecipitation. LMO2 is known as a component of TAL1 complex, which consist of LMO2, TAL1, LDB1, E47 and GATA1/2 proteins and has transcriptional activity as a complex on hematopoiesis-specific target genes. Interestingly, mRNA expressions of all these target genes were markedly downregulated by inhibition of NAMPT or SIRT2. We made acetylation- (KQ) and deacetylation- (KR) mimic mutants of LMO2 protein at K74 and K78 lysine residues and compared LMO2-LDB1 interaction by co-immunoprecipitation. This experiment revealed that KQ-mutant had impaired interaction in comparison to WT and KR-mutant. These results indicate that deacetylation of LMO2 protein by SIRT2 is required for the interaction between LMO2 and LDB1 proteins.

Conclusions: NAMPT-NAD+-SIRT2 pathway plays an indispensable role in the early hematopoiesis by deacetylation of LMO2 protein which enables the interaction between LMO2 and LDB1 proteins and complete TAL1 complex formation.

Disclosure: No conflict of interest disclosed.

V89 – Unaffected myeloid differentiation of iPS cells derived form a cyclic neutropenia (CyN) patient with ELANE mutation

Zahabi A.1, Morishima T.1, Bernhard R.1, Kanz L.1, Welte K.2, Skokowa J.1

1University Hospital Tuebingen, Oncology, Hematology, Immunology, Rheumatology and Pulmonology, Tübingen, Germany, 2University Children’s Hospital, Tübingen, Germany

Background: Cyclic neutropenia (CyN) is a hematologic disorder in which blood cell counts particularly granulocytic neutrophils, monocytes, platelets, and reticulocyte numbers show cycles at appr. 21 day intervals. The majority of CyN patients (ca 90%) harbor inherited mutations in the ELANE gene. Intriguingly, same ELANE mutations are present in two different bone marrow failure syndromes: congenital as well as in cyclic neutropenias. It is unclear how mutation in the same gene cause congenital (CN) or cyclic neutropenia (CyN). The pathomechanism of cycling hematopoiesis downstream of ELANE mutations is also unclear. Recent studies using inducible pluripotent stem cells (iPSCs) derived from severe congenital neutropenia (CN) patients harbouring ELANE mutations demonstrated markedly diminished granulocytic differentiation of these cells in vitro (Nayak RC, et al. JCI 2015; Hiramoto T, et al. PNAS 2013). Interestingly, correction of ELANE gene mutation in iPSCs from a CN patient using CRISPR/Cas9 technology restored defective granulopoiesis, suggesting the monogenic origin of the ELANE mutation caused congenital neutropenia (Nayak RC, et al. JCI 2015). These data suggest that additional gene mutations or epigenetic defects in combination with mutated ELANE might be responsible for the pathogenesis of cyclic neutropenia.

Aims: In the present study we evaluated the in vitro myeloid differentiation of iPSCs derived from the CyN patient harboring sporadic heterozygous ELANE mutation (c.761C>G p.W241L), in comparison to iPSCs derived from healthy individuals.

Methods: We used embryoid-body (EB)-based protocol of granulocytic differentiation from iPSCs described by N. Lachmann et al. (Stem Cell Reports 2015).

Results: Interestingly, we found that myeloid differentiation of iPSCs derived from CyN patient, was comparable to that of healthy individual iPSCs, as revealed by the analysis of the percentage and absolute numbers of CD45+/CD11b+, CD45+/CD16+ and CD45+/CD15+ cells as well as by the examinations of the cell morphology on cytospin preparations.

Summary/ Conclusion: These data suggest that the in vitro hematopoiesis in CyN is normal as judged by iPSC technology and that in vivo additional humoral or bone marrow niche components might influence myeloid cell cycling in CyN.

Disclosure: No conflict of interest disclosed.

V90 – Differential use of metabolic substrates during erythropoiesis and granulopoiesis

Böhme C.1,2, Billing C.1, Walker M.3, Noack N.1, Pompe T.2, Niederwieser D.1, Whetton A.3, Cross M.1

1Universitätsklinikum Leipzig AöR, Selbständige Abteilung für Hämatologie/int. Onkologie, Leipzig, Germany, 2Universität Leipzig, Institut für Biochemie, Leipzig, Germany, 3University of Manchester, Stem Cell Proteomics Laboratory, Manchester, United Kingdom

Introduction: Erythrocytes and neutrophilic granulocytes are constantly produced in large numbers from a common progenitor in the same region of haematopoietic marrow. We are interested in the possibility that these lineages use different patterns of metabolic activity to fuel the high levels of proliferation required.

Materials and methods: CD34+ progenitors were purified from umbilical cord blood by MACS. Following short term culture, CD34+ CD133+ (multipotent and lympho-myeloid progenitors, MPP / LMPP) and CD34+ CD133low (erythro-myeloid progenitors, EMP) were purified by FACS. The metabolic requirements of colony-formation were assessed in a system developed to allow the exchange of media without disturbing the cells. After 14 days, cells were recovered from the methylcellulose for FACS and PCR analysis. Metabolite exchange with the medium during erythropoiesis and granulopoiesis was assessed in liquid cultures of murine FDCP-Mix cells. Mitochondria purified from the same cells were used in proteomic analysis (iTRAQ).

Results: Even in the absence of detectable glucose, regular exchange of the medium over the first 3 days of culture was sufficient to generate robust colony formation over 14 days, suggesting that at least a subpopulation of progenitor cells is capable of gluconeogenesis. Consistent with this, the expression of both PEPCK 1 and 2 was detected in colonies.

Colony formation from all populations tested required glutamine. However, high glutamine concentrations selectively blocked the development of erythroid colonies from EMPs.

Similarly, FDCP-Mix cells undergoing either granulocytic or erythroid differentiation take up glutamine from the medium, but large amounts of glutamate are released back into the medium only under erythroid conditions. This suggests a block in glutaminolysis specifically in the erythroid lineage. A proteomic analysis of mitochondria from FDCP-Mix cells differentiating along each lineage showed erythroid differentiation to be accompanied down-regulation of Gpt2, an amino transferase responsible for channelling glutamate into the TCA cycle.

Discussion: The novel colony assay procedure has identified previously unknown features of haematopoietic metabolism, consistent with the lineage-specific use of limiting substrates. Metabolic compartmentalisation may help to make maximal use of limiting substrates and/or provide a level of metabolic feedback between the lineages.

Disclosure: No conflict of interest disclosed.

V91 – Immunosuppressive capacity of mesenchymal stromal cells depends on allogeneic donor T-cells and correlates with metabolic activity

Nold P.1, Killer M.1, Henkenius K.1, Fritz L.1, Hackstein H.2, Neubauer A.1, Brendel C.1

1Universitätsklinikum Gießen und Marburg GmbH, Hämatologie/Onkologie/Immunologie, Marburg, Germany, 2Universitätsklinikum Gießen und Marburg GmbH, Zentrum für Transfusionsmedizin und Hämotherapie, Giessen, Germany

Introduction: Mesenchymal stromal cells (MSC) have entered the clinic as Advanced Therapy Medicinal Product (ATMP) and are currently evaluated in a wide range of studies for tissue regeneration or in autoimmune disorders. Various efforts have been made to standardize and optimize expansion and manufacturing processes but until now reliable potency assays reflecting therapeutic effectiveness of MSC are still lacking. As recent findings suggest superior therapeutic efficacy of freshly administered MSC in comparison to frozen cells we sought to determine the immunosuppression capacity of MSC in relation to their metabolic activity.

Methods: Human MSC were obtained from bone fragments of patients and were employed in co-culture with peripheral blood mononuclear cells (PBMC) in an allogeneic T-cell proliferation assay to measure immunosuppressive function. Metabolic activity of MSC was measured real-time as aerobic glycolysis quantified by the extracellular acidification rate (ECAR) and mitochondrial respiration quantified by the oxygen consumption rate (OCR).

Results: We show that MSC induced suppression of T-cell proliferation varied enormously between different healthy donors of PBMC. Moreover, direct contact of PBMC and MSC increased the metabolic activity of MSC in a PBMC donor dependent manner. Enhanced lactate production as a surrogate parameter for glycolysis and increased oxygen consumption was paralleled by higher T-cell suppression capacities of MSC. The cryopreservative dimethylsulfoxide decreased metabolic and immunosuppressive performance of MSC while valproic acid enhanced metabolism and T-cell suppression.

Conclusion: Metabolic and immunosuppressive capacity of MSC are linked closely but are highly dependent on the predisposition of interacting T-cells which enhance MSC metabolism upon direct contact in co-culture. Hence functional fitness and quality of MSC can be determined by measuring metabolic activity and can be enhanced by exposure to valproic acid.

Disclosure: No conflict of interest disclosed.

Freier Vortrag – Infektionen / supportive Therapie

V92 – Detection and characterization of Aspergillus fumigatus azole resistance and cyp51A combinations of mutations in clinical isolates and primary clinical samples of hematological patients – preliminary results comparing two molecular assays

Spiess B.1, Postina P.1, Boch T.1, Miethke T.2, Dietz A.2, Merker N.1, Hofmann W.K.1, Buchheidt D.1

1Universitätsmedizin Mannheim; III. Medizinische Klinik, Mannheim, Germany, 2Universitätsmedizin Mannheim; Institut für Medizinische Mikrobiologie und Hygiene, Mannheim, Germany

Introduction: In hematological patients, there is a rising incidence of invasive aspergillosis (IA) caused by azole resistant Aspergillus fumigatus (A. fumigatus) that reveals a combination of novel mutations. As the diagnosis of IA is rarely based on positive culture yield in this group of patients, molecular detection of resistance mutations directly from clinical samples is crucial.

Methods: In addition to our established azole resistance PCR assays detecting the frequent mutation combinations TR34/L98H/M220I and TR46/Y121F/T289A in the A. fumigatus cyp51A gene, we investigated in parallel the commercially available AsperGenius® real time PCR detection system (PathoNostics BV, Maastricht, The Netherlands) detecting the cyp51A alterations TR34/L98H and Y121F/T289A directly from clinical samples and isolates. We analyzed phenol-chloroform extracted DNA aliquots and compared both PCR methods, not the preceding different DNA extraction protocols.

Results: Our novel Y121F and T289A PCR assays showed a detection threshold of 300 fg genomic A. fumigatus DNA. As positive control for the feasibility of these assays, we detected the TR46/Y121F/T289A mutation combination in two isolates of hematological patients with known cyp51A alterations and in a lung biopsy sample of a patient with AML and proven IA. Results comparing the two test systems are shown in table 1.


Conclusions: In order to detect A. fumigatus azole resistance directly from clinical samples, we broadened the spectrum of our PCR assays. The combination of our results with these of the AsperGenius® assay revealed that our conventional PCR assays are more suitable for the analysis of biopsy samples. Negative results of the AsperGenius® assay in biopsy samples could most likely be due to different PCR conditions and the interference of the PCR reactions caused by high amounts of human DNA. Despite this, we consider non-culture based molecular detection of Aspergillus azole resistance to be of high epidemiological and clinical relevance.

Disclosure: Birgit Spiess: No conflict of interest disclosed.Dieter Buchheidt: Advisory Role: Basilea, Gilead Sciences; Financing of Scientific Research: Astellas, Gilead Sciences, Merk Sharp & Dohme/Merck, Pfizer; Expert Testimony: Gilead Sciences, Pfizer

V93 – Peptide vaccination against cytomegalovirus (CMV) can clear the virus load after allogeneic stem cell transplantation even from a CMV seronegative donor

Schmitt M.1, Schmitt A.1, Wiesneth M.2, Hückelhoven A.1, Wu Z.3, Kuball J.4, Wang L.1, Schauwecker P.2, Hofmann S.5, Götz M.5, Michels B.1, Maccari B.2, Wuchter P.1, Mertens T.3, Schnitzler P.6, Döhner H.5, Ho A.D.1, Bunjes D.W.5, Dreger P.1, Schrezenmeier H.2, Greiner J.5,7

1Medizinische Klinik V, Universitätsklinikum Heidelberg, Heidelberg, Germany, 2Institut für Klinische Transfusionsmedizin und Immungenetik Ulm (IKT), Ulm, Germany, 3Institut für Virologie, Universitätsklinikum Ulm, Ulm, Germany, 4Department of Hematology, University Medical Center, Utrecht, Netherlands, 5Klinik für Innere Medizin III, Universitätsklinikum Ulm, Ulm, Germany, 6Department für Infektiologie, Virologie, Universitätsklinikum Heidelberg, Heidelberg, Germany, 7Medizinische Klinik, Diakonie-Klinikum Stuttgart, Stuttgart, Germany

Introduction: Cytomegalovirus (CMV) reactivation occurs particularly in patients receiving an allogeneic stem cell graft from a seronegative donor. CMV reactivation is associated with a high risk of disease and mortality. The nonamer peptide NLVPMVATV derived from CMV phosphoprotein 65(CMVpp65) is highly immunogenic. Here we report on a clinical phase I peptide vaccination trial with this peptide in a water-in-oil emulsion (Montanide™) plus administration of granulocyte-macrophage colony stimulating factor (GM-CSF) (EudraCT number: 2010-018884-40).

Methods: Four vaccines were administred subcutaneously at a biweekly interval to ten patients. Clinical course and CMVpp65 antigenemia were monitored. CMV-specific CD8+ and gamma-delta T cells were analyzed by multi-color flow cytometry. We established a neutralizing anti-CMV antibody assay and correlated it to clinical parameters.

Results: Peptide vaccination was well tolerated and no drug-related serious adverse events were detected. Seven of nine patients with CMVpp65 antigenemia cleared the CMV after four vaccinations and are still free from viremia until now. Two patients with CMV reactivation showed no clinical response, i.e. persisting CMV antigenemia. One patient received prophylactic vaccination and did not develop viremia.

In four patients an increase in frequency of both CMV specific CD8+ T cells Vdelta2-negative gamma-delta T cells was detected by factor 6 and 7, respectively. In four patients also titers of neutralizing antibodies increased up to tenfold over the time of vaccination. Humoral and cellular immune responses correlated with clearance of the CMV load in the patients.

Conclusion: In patients after allogeneic stem cell transplantation with high risk for CMV reactivation, we demonstrated that administration of CMVpp65 peptide vaccination was safe, well tolerated and clinically encouraging. Further studies with larger patient cohorts are planned. A study with prophylactic CMVpp65 peptide vaccination is ongoing in patients receiving a kidney transplantation.

Disclosure: No conflict of interest disclosed.

V94 – Different localization of Dectin-1 isoforms governs its recognition of ß-glucan and signaling quality

Fischer M.1, Müller J.P.2, Spies-Weisshart B.1, Gräfe C.1, Hochhaus A.1, Scholl S.1, Schnetzke U.1

1Universitätsklinikum Jena, Klinik für Innere Medizin II, Abteilung Hämatologie und Internistische Onkologie, Jena, Germany, 2Universitätsklinikum Jena, Institut für Molekulare Zellbiologie, CMB, Jena, Germany

Introduction: The incidence of invasive fungal infections (IFI) in hematological patients has increased substantially during the last decades. Thus, there is a need for a better understanding of antifungal immunity. Dectin-1, a C-type-lectin-like receptor, is recognized as a major pattern-recognition receptor for fungal ß-glucans and contributes to anti-fungal immunity. The two major human isoforms A and B are expressed on human monocytes as well as on macrophages and dendritic cells. Both proteins share the same carbohydrate recognition domain while isoform A is characterized by an additional stalk region and its N-linked glycosylation site.

Methods: Expression of Dectin-1 isoform A and B was analyzed in monocyte-derived cells. Cell lines (THP-1, HEK293) stably expressing Dectin-1 isoform A or B were generated by retroviral transduction. Subcellular localization of Dectin-1 was analyzed using flow cytometry and confocal laser scanning microscopy (CLSM). Furthermore, binding capacity of ß-glucan was examined by flow cytometry and signaling quality using immunoblotting.

Results: Cell line models demonstrate a ten times higher surface membrane expression of isoform A compared with B, despite comparable mRNA und whole cell protein levels. CLSM imaging validates an intracellular retention of isoform B on protein level. The reduced surface membrane expression of isoform B correlates with a reduced ß-glucan binding capacity and an impairment of Syk and p38 dependent signaling quality. Inhibition of N-linked glycosylation of isoform A by tunicamycin leads to an equalization of surface membrane expression comparable to isoform B.

Conclusions: This work reveals the substantial role of N-linked glycosylation of Dectin-1 and its impaction on surface membrane expression and consequently ß-glucan mediated signal transduction. Different expression profiles of human Dectin-1 isoforms on monocyte-derived cells may be associated with cell-type-dependent effector mechanisms of antifungal immunity.

Disclosure: Mike Fischer: No conflict of interest disclosed.Ulf Schnetzke: Expert Testimony: Firma Gilead

V95 – Selenium for the prevention and management of adverse effects of cancer treatments

Renner P.1, Dennert G.2, Kalisch A.3, Horneber M.3

1Universitätsklinik für Innere Medizin 8, Paracelsus Medizinische Privatuniversität, Klinikum Nürnberg, Schwerpunkt Kardiologie, Nürnberg, Germany, 2Angewandte Sozialwissenschaften, Fachhochschule Dortmund, Sozialmedizin und Public Health mit Schwerpunkt Geschlecht und Diversität, Dortmund, Germany, 3Universitätsklinik für Innere Medizin 5, Paracelsus Medizinische Privatuniversität, Klinikum Nürnberg, Schwerpunkt Onkologie/Hämatologie, Nürnberg, Germany

Introduction: This is an updated version of the original Cochrane review published in 2009. Selenium is an essential trace element and is involved in antioxidant protection and the redox-regulation in humans. It has been claimed for its efficacy in supportive cancer care. Selenium supplements are frequently used by cancer patients during anticancer treatments. The aim of this systematic review was to evaluate the efficacy of selenium for the prevention and management of adverse effects of chemotherapy or radiotherapy and for after-effects of tumor surgery.

Methods: For this update we searched electronic databases, including those of Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE and EMBASE, up to September 2015. We included randomised controlled trials of selenium for the prevention or management of adverse effects of chemotherapy, radiotherapy and of after-effects of tumour surgery. At least two review authors independently determined suitability for inclusion and extracted and assessed study data.

Results: We found four new studies with a total of 253 participants for this update. Together with the three previously identified studies, the update is now based on a total of seven studies with 411 participants suffering from various cancers at different stages. All studies used different preparations and doses of oral selenium to prevent a heterogenous set of side effects or toxicities. Therefore, no meaningful metaanalysis was possible. Conflicting evidence was found for protective effects of selenium on renal toxicity of cisplatin and other chemotherapeutic agents. Conflicting evidence was also found for effects of selenium to prevent oral mucositis during chemotherapy, chemoradiation or radiation therapy. Due to high risk of bias in the reporting studies, only low quality evidence was found for protective effects of selenium on either radiation-induced diarrhoea during pelvic radiotherapy, or the recurrence of erysipelas in lymphedematous skins after mastectomy or on postoperative lymphedema after oral surgery.

Conclusions: In summary, we found no convincing evidence for beneficial effects of selenium for the prevention or management of chemotherapy- or radiotherapy-related adverse effects or for after-effects of tumor surgery in cancer patients. Four additional identified studies for this review did not change our conclusions.

Funding: Ernst und Anita Bauer-Stiftung, Deutsche Krebshilfe

Disclosure: No conflict of interest disclosed.

V96 – Glutamine in the prevention of tumortherapy-induced diarrhea

Leithold C.1, Jahn F.1, Rüssel J.1, Unverzagt S.2, Müller-Tidow C.1, Jordan K.1

1Universitätsklinikum Halle-Wittenberg, Halle, Germany, 2Institut für Medizinische Biometrie, Epidemiologie und Informatik, Halle, Germany

Introduction: Glutamine is a main energy resource for cells with high turnover rates, such as enterocytes or cells of the immune system. There are a lot of animal studies supporting the important role of glutamine in mucosal integrity and gastrointestinal function. Also numerous studies in human showed benefit in improving gastrointestinal function using glutamine as a supplement. Due to the presumed mode of action it is assumable that glutamine might be of benefit in preventing mucosal damage in patients receiving chemo- or radiotherapy. This systematic review assessed the effects of oral or parenteral glutamine in preventing diarrhea due to tumor therapy.

Methods: We conducted a systematic literature search using Medline (via Ovid and PubMed) and Cochrane Library to find randomized controlled trials which studied the efficacy of orally or parenterally administered glutamine for the prevention of diarrhea in patients receiving chemo- or radiotherapy. Duration, incidence and severity of diarrhea were documented as outcome measurements. We conducted subgroup analyses for chemo- and radiotherapy-induced duration of diarrhea and oral or parenteral application of glutamine.

Results: We found 2891 records and identified a total of 14 studies that met the inclusion criteria, eight studies were finally included in meta-analysis. Neither oral (MD: -0.44 d; 95% CI: -1.56 d-0.68 d) nor parenteral administration of glutamine (MD: -0.68 d; 95% CI: -2.72 d-1.36 d) could significantly reduce duration of chemotherapy-induced diarrhea (CID). For the prevention of radiotherapy-induced diarrhea (RID) we found inconsistent study results suggesting that the administration of oral glutamine might even increase the incidence of diarrhea (OR: 13.03; 95% CI: 2.68-63.22).

Conclusions: Glutamine is not effective to prevent duration or severity of CID or RID and has no impact on the incidence of CID or RID. Since study results for RID even described an increase of incidence of diarrhea, there is no point using glutamine as a preventive agent. These results are in contrast with former meta-analysis. The results of our systematic review formed the basis for the recommendation of the S3 guidelines of “Supportive therapy”.

Disclosure: No conflict of interest disclosed.

V97 – Bisphosphonates for patients with bone metastases from prostate cancer. A Cochrane review update including more than 5,000 patients

Macherey S.1, Jahn F.2, Jordan K.2, Skoetz N.1

1Cochrane Haematological Malignancies Group, Klinik I für Innere Medizin, Universitätsklinik Köln, Köln, Germany, 2Klinik IV für Innere Medizin, Universitätsklinik Halle/Saale, Halle/Saale, Germany

Introduction: Patients with advanced prostate cancer frequently present with bone metastases. These patients are usually affected by bone pain, pathological fractures or spinal cord compression and often need interventions like surgery or radiation of the bone (skeletal-related events, SRE). Bisphosphonates are well established in the multimodal therapy of bone pain in patients with bone metastasis from prostate cancer. Only few studies have investigated the effect of bisphosphonates on overall survival (OS). Hence, we performed a meta-analysis aimed at the effect of bisphosphonates on the patients’ general outcome.

Methods: In December 2015, we performed a literature search by screening the electronic databases MEDLINE and the Cochrane library or databases of ongoing clinical trials. Furthermore, we hand searched conference proceedings of the American Society of Clinical Oncology and the references of any identified trial. We defined pain response as primary end point and mortality, SRE, analgesic consumption, disease progression, renal adverse events and osteonecrosis of the jaw (ONJ) as secondary end points for the statistical analysis. The GRADE software was used to evaluate each trial’s quality of evidence. This study was partly funded by the German Cancer Aid (No. 110645).

Results: A total of 21 randomized controlled trials reporting on approximately 5,000 patients could be included. Bisphosphonates probably improved the patients’ pain response (RR: 1.21; 95% CI: 0.99, 1.48; p = 0.05), reduced the number of skeletal-related events (RR: 0.90; 95% CI: 0.81, 0.99; p = 0.03) and the patients’ disease progression (RR: 0.94; 95% CI: 0.89, 0.99; p = 0.03) in comparison to the control group. There is no evidence for a difference between bisphosphonates and placebo considering mortality (RR: 0.98, 95% CI: 0.91, 1.05) or renal adverse events (RR: 1.04, 95% CI 0.76, 1.42). Bisphosphonates increase the rate of ONJ (RR: 6.77, 95% CI: 3.17, 14.46). It is uncertain whether bisphosphonates improve analgesic consumption (RR: 1.19, 95% CI: 0.87, 1.63) in those patients.

Conclusions: Bisphosphonates lead to a significant reduction of skeletal-related events and disease progression in patients with prostate cancer and bone metastases. The use of bisphosphonates showed an improvement in pain response, but is associated with a higher incidence for osteonecrosis of the jaw. There is no evidence for an effect of bisphosphonates on mortality or analgesic consumption.

Disclosure: No conflict of interest disclosed.

Fortbildung – Interprofessionelle Sitzung: Geriatrische Onkologie(für Ärzte und Pflegekräfte)

V100 – Decision-making capacity in elderly cancer patient

Weidmann-Hügle T.1

1Institut für Biomedizinische Ethik und Medizingeschichte, Universität Zürich, Zürich, Switzerland

Patient autonomy is a fundamental and indispensable moral principle in contemporary medicine as well as in medical ethics. It is the basis of much theory and most regulation. The main legal process to promote patient autonomy is through informed consent. Within this process the patient is being comprehensively informed about a pending treatment. Informed consent involves discussion of the nature of a procedure, its possible risks and benefits, and alternative treatments. Ideally the patient then makes a free and non-pressured decision about further treatment, which is consistent with his values and life´s plan. Key to the legal validity of a patient’s consent (or dissent) is her decision-making capacity (in German: “Urteilsfähigkeit” or “Einwilligungsfähigkeit”).

In general terms, the requirement of having decision-making capacity is then met, if a person is capable 1) of understanding and appreciating relevant information, 2) of processing this information, 3) of assessing possible consequences of available treatment options, and, finally, if she is capable of coming to a decision.

Cancer occurs frequently among older adults. Cognitive impairment and depression are also common in this population. Both cognitive impairment and depression, coupled with stress, uncertainty, or side effects from treatment may have an impact on a person’s ability to make decisions. Decisions about cancer treatment can be demanding and require sufficient cognitive capacity, especially given the fact that there is often uncertainty in the outcomes and the decisions rely on weighing probabilities of side effects versus benefits Therefore, knowledge about approaches and instruments for the assessment of a patient’s decision-making capacity is crucial for healthcare professionals in oncology. Furthermore, healthcare professionals must be aware of the various factors which can impact decision-making capacity.

In this presentation I will focus on the theoretical and legal concepts of decision-making capacity and factors influencing this, on assessment approaches, and on how nurses can support elderly cancer patients in the process of decision-making.

Disclosure: No conflict of interest disclosed.

V101 – Geriatric cancer patients- Comprehensive geriatric assessment and patient-reported quality of life

Schmidt H.1, Boese S.1, Lampe K.2, Jung M.1, Jordan K.3, Fiedler E.4, Müller-Werdan U.5, Vordermark D.2

1Martin Luther University Halle-Wittenberg, Institute of Health and Nursing Science, Halle (Saale), Germany, 2University Hospital Halle Saale, Department of Radiation Oncology, Halle (Saale), Germany, 3University Hospital Halle Saale, Department of Hematology and Oncology, Halle (Saale), Germany, 4University Hospital Halle Saale, Department of Dermatology, Halle (Saale), Germany, 5Charite – Universitätsmedizin Berlin, Chair of Geriatrics, and Protestant Geriatric Centre, Berlin, Germany

Introduction: Oncologic therapy of elderly cancer patients is often complicated by comorbidities, reduced functioning and the organization of care at home. For long term care the maintenance of functionality and health related quality of life (HRQOL) is important. Therefore, prior to cancer specific therapy, the identification of relevant risk factors by comprehensive geriatric assessment (CGA) is recommended for elderly cancer patients.

Methods: Aiming to maintain HRQOL we developed an interdisciplinary care program based on comprehensive geriatric assessment (CGA) and patient reported HRQOL comprising tailored supportive measures and telephone based counselling during 6 months aftercare. The intervention was pilot-tested in three centres at the University Hospital Halle Saale to examine feasibility, acceptance and potential benefit. Inclusion criteria: Oncologic patients >70 years with at least one comorbidity and/or one functional impairment with written informed consent. Primary endpoint is HRQOL (EORTC QLQ-C30, ELD14), measured at admission and 6 month-follow-up.

First Results: Out of n = 226 eligible patients n = 100 participated (44%), mean age: 76.3 years (SD = 4.8), 47% female, comorbidities mean: n = 5 (SD = 2.8). Follow-up will be completed by July 2016. Preliminary analyses show large inter-individual differences regarding functional status, reported symptom intensity and supportive needs. Individualized supportive care was triggered by summarized individual results that were presented to the physicians in charge of the respective patients (e.g. malnutrition, reduced HRQOL, reduced physical functioning, high symptom-intensity and depression). Preliminary analyses for the primary endpoint global HRQOL (n = 46) showed clinical relevant improvement of HRQOL (=10 pts.) for 35%, no change for 41% and worsening for 24%. Concurrent with worsening of global HRQOL we found a deterioration of physical function, mobility and fatigue. Further case based analyses comparing professional assessments and self-assessments including HRQOL will be presented in connection with follow-up data.

Conclusion: First results show feasibility and potential usefulness of the combination of CGA and HRQOL to complement standard assessments and to decide on individualized therapeutic measures and after care.

Disclosure: Heike Schmidt: No conflict of interest disclosed.Dirk Vordermark: Advisory Role: Roche, Boehringer, Bristol-Myers Squibb,; Financing of Scientific Research: Merck, Lilly

Freier Vortrag – Mastozytose, myeloproliferative Neoplasien

V103 – Expression, regulation and function of the cell adhesion molecule CD44 in neoplastic mast cells and stem cells in patients with systemic mastocytosis

Mueller N.1, Wicklein D.2, Eisenwort G.1,3, Boehm A.1,4, Herrmann H.1,3, Stefanzl G.1,3, Hoermann G.3,5, Sperr W.R.1,3, Arock M.6, Schumacher U.2, Valent P.1,3

1Medical University of Vienna, Internal Medicine I, Division of Hematology and Hemostaseology, Vienna, Austria, 2University Medical-Center Hamburg-Eppendorf, Institute of Anatomy and Experimental Morphology, University Cancer Center, Hamburg, Germany, 3Medical University of Vienna, Ludwig Boltzmann Cluster Oncology, Vienna, Austria, 4Elisabethinen Hospital, Linz, Austria, 5Medical University of Vienna, Department of Laboratory Medicine, Vienna, Austria, 6Ecole Normale Supérieure de Cachan, LBPA CNRS UMR8113, Paris, France

The Hermes antigen CD44 is a multifunctional adhesion molecule that plays an essential role in the homing and expansion of neoplastic stem- and progenitor cells in various myeloid malignancies. Although mast cells (MC) are known to express CD44, little is known about the regulation and function of this receptor on neoplastic mast cells and stem cells in patients with systemic mastocytosis (SM).

In the current study, we found that CD34+/CD38- stem cells, CD34+/CD38+ progenitor cells, and KIT+/CD34- MC invariably express CD44 in all patients with indolent SM (ISM), SM with associated hematologic non-MC-disease (SM-AHNMD), aggressive SM (ASM), and MC leukemia (MCL). In addition, all human MC lines examined, including HMC-1.1, HMC-1.2, ROSAKIT wt, ROSAKIT D816V and MCPV were found to express cytoplasmic and cell surface CD44. We next examined the regulation of expression of CD44 in neoplastic MC. Incubation with the MEK inhibitor RDEA119 (0.1-5 µM) or the STAT5 blocker pimozide (2.5-10 µM) for 48 hours resulted in a significant, dose-dependent downregulation of surface expression of CD44 in all MC lines examined. By contrast, incubation with the demethylating agents decitabine (0.1-5 µM) or azacitidine (0.1-5 µM) for 96 hours resulted in a dose-dependent and significant upregulation of CD44 expression in all MC lines tested. We were also able to detect soluble CD44 in the sera of patients with ISM, SM-AHNMD, ASM, and MCL as well as in the supernatants of neoplastic MC lines. In order to define a functional role for CD44 expressed on neoplastic MC, we transduced shRNA against CD44 as well as a control shRNA into HMC-1.2 cells, and injected these cells into the skin of severe combined immunodeficient (SCID) mice. In this xenotransplantation model, the shRNA-mediated knockdown of CD44 was found to lead to reduced MC expansion, reduced tumor formation, delayed ulceration, and to a significantly prolonged survival compared to cells transduced with control shRNA. The formation of lung metastases, quantified by human Alu-sequence-specific qPCR, was found to decrease particularly (15-fold) in the CD44 knock-down group compared to control mice.

Together, we show that CD44 is a relevant homing molecule expressed on neoplastic MC and CD34+ neoplastic stem- and progenitor cells in advanced SM. Our data also suggest that CD44 may serve as an interesting new target of therapy in patients with advanced SM.

Disclosure: Niklas Mueller: No conflict of interest disclosed.Peter Valent: Financing of Scientific Research: Novartis; Expert Testimony: Novartis

V104 – Mast cell leukemia: clinical heterogeneity, molecular aberrations and prognostic factors

Jawhar M.1, Schwaab J.1, Meggendorfer M.2, Naumann N.1, Kluger S.1, Horny H.-P.3, Sotlar K.3, Haferlach T.2, Fabarius A.1, Hofmann W.-K.1, Reiter A.1, Metzgeroth G.1

1University Medical Centre, Department of Hematology and Oncology, Mannheim, Germany, 2MLL Munich Leukemia Laboratory, Munich, Germany, 3Ludwig-Maximilians-University, Institute of Pathology, Munich, Germany

Introduction: Mast cell leukemia (MCL) is a rare variant of systemic mastocytosis (SM). Data on disease characteristics, treatment, and prognosis are limited due to small patient numbers.

Methods: Here, we report on 25 MCL patients (median age 67 years, range 45-82; male 64%).

Results: An associated hematologic neoplasm (AHN), e.g. CMML (n = 7), MDS/MPNu (n = 6) or MDS (n = 4) was diagnosed in 17/25 (68%) patients. Primary MCL was found in 15/25 (60%) patients, and secondary MCL evolving from SM-AHN or aggressive SM (after median 20 months; range, 4-71), in 10/25 (40%) patients. Median percentage of MC in BM smears and trephine biopsies were 25% (range 20-95) and 60% (range, 20-60), respectively. MC in peripheral blood (PB) =10% (leukemic MCL) were only detected in 2/25 (8%) patients. Median serum tryptase level was 549 µg/L (range 160-1854). Hematologic C-findings such as hemoglobin < 10 g/dL and/or platelets < 100×109/L were identified in 23/25 (92%) patients. Most important non-hematologic signs of organ dysfunction included elevated alkaline phosphatase in 17/25 (68%) patients and splenomegaly (=450 mL) or even marked splenomegaly (=1200 mL) in 15/15 (100%) and 8/15 (53%) patients, respectively, with available spleen volumetry (n = 15). Mutations in KIT were identified in 22/25 (88%) patients (D816V, n = 18; D816Y, n = 2; D816H, n = 1; F522C, n = 1) with a median KIT D816V allele burden of 42% (range 20-98) in PB. Additional mutations in SRSF2, ASXL1 or RUNX1 (S/A/R gene panel, Jawhar et al., Leukemia 2016) were identified by NGS in 13/21 (62%) patients. Cytoreductive treatment in 22/25 patients included midostaurin (n = 21), cladribine (n = 9), and allogeneic stem cell transplantation (n = 1). Median observation from MCL diagnosis was 13 months (range, 0-86); 18/25 patients (72%) have died. Median overall survival (OS) was 17 months (range, 10-24) with a 2-year OS probability of 24% for all patients. OS of S/A/R mutated patients (n = 13) was significantly inferior as compared to S/A/R non-mutated patients (n = 8, P = 0.007, hazard ratio, HR: 5.8, 95% confidence interval: 1.3-26.1, median OS 13 months vs. not reached).

Conclusion: Leukemic MCL and MCL without C-findings are rare, b) KIT D816V mutations were more frequently detected as previously reported, c) the prognostically relevant mutations in the S/A/R gene panel are present in approximately 60% of patients, and d) median OS is approximately 1.5 years with significantly inferior survival observed in S/A/R mutated patients.

Disclosure: No conflict of interest disclosed.

V105 – Incidence and prognostic impact of cytogenetic aberrations in systemic mastocytosis

Naumann N.1, Jawhar M.1, Schwaab J.1, Metzgeroth G.1, Khaled N.1, Horny H.-P.2, Sotlar K.2, Valent P.3, Haferlach C.4, Göhring G.5, Schlegelberger B.5, Meggendorfer M.4, Cross N.C.P.6,7, Hofmann W.-K.1, Reiter A.1, Fabarius A.1

1Department of Hematology and Oncology, University Medical Centre, Mannheim, Germany, 2Institute of Pathology, Ludwig-Maximilians-University, Munich, Germany, 3Department of Internal Medicine I, Division of Hematology and Ludwig Boltzmann Cluster Oncology, Medical University of Vienna, Vienna, Austria, 4MLL Munich Leukemia Laboratory, Munich, Germany, 5Institute of Human Genetics, Hannover Medical School, Hannover, Germany, 6Faculty of Medicine, University of Southampton, Southampton, United Kingdom, 7Wessex Regional Genetics Laboratory, Salisbury, United Kingdom

Introduction: In chronic myeloid neoplasms, the type, relative frequency and prognostic impact of cytogenetic aberrations are highly heterogeneous. With increasing knowledge on an adverse prognostic impact of specific gene mutations, contemporaneous presence and potential interaction of cytogenetic aberrations and mutations may become prognostically highly relevant.

Methods: We analyzed clinical, cytogenetic and molecular characteristics of 109 (KIT D816V positive n = 102, 94%) patients (pts.) with indolent systemic mastocytosis (ISM, n = 27) and advanced SM (advSM, n = 82) with or without an associated hematologic neoplasms (AHN) [aggressive SM, n = 3; SM-AHN, n = 60; mast cell leukemia (MCL), n = 9; MCL-AHN, n = 10)].

Results: In advSM, an aberrant karyotype (KT) was identified in 16/82 (18%) pts., which was complex (n = 7), associated with a bad prognosis (e.g. monosomy 7, n = 3) or associated with a good/unknown prognosis (e.g. +8 or del(5q), n = 6). In subsequent studies, KT anomalies were grouped and split into good KT (normal KT + good KT) and poor KT (complex KT + bad KT) based on e.g. MDS, AML. The median overall survival (OS) of pts. with advSM and poor KT (n = 10) was 4 months and significantly shorter than the OS of pts. with good KT (n = 72, 38 months) (HR 6.1, 95% confidence interval [2.6-14.2], p < 0.0001). Notably, progression into secondary AML or acute MCL was observed in 8/10 (80%) and 1/10 (10%) pts., median 4 months (range, 0.3-24) and 2 months after diagnosis of advSM and first detection of the cytogenetic aberration, respectively. Mutations in SRSF2/ASXL1/RUNX1 (S/A/Rpos.), which were recently shown to be associated with adverse OS in SM (Jawhar et al., Leukemia 2016), were identified in 5/10 (50%) pts. with poor KT and 31/72 (43%) pts. with good KT, respectively. Significant differences regarding OS were observed between good KT – S/A/Rneg. (n = 29) vs. good KT – S/A/Rpos. (n = 31) vs. poor KT – S/A/Rneg. (n = 5) vs. poor KT – S/A/Rpos. (n = 5, Figure).

Conclusion: In addition to clinical and morphological baseline staging, molecular and cytogenetic analyses should be routinely performed in pts. with advSM because they may indicate overt disease progression/transformation.


Disclosure: No conflict of interest disclosed.

V106 – Next-generation sequencing identifies MPL-TET2 mutated clones in a subset of patients with JAK2V617F-positive myelofibrosis

Schulze S.1, Stengel R.2, Jäkel N.1, Wang S.-Y.1, Hubert K.3, Roskos M.2, Schneider M.2, Franke G.-N.1, Niederwieser D.1, Al-Ali H.K.1

1Universitätsklinik Leipzig AöR, Hämatologie/ internistische Onkologie, Leipzig, Germany, 2Oncoscreen Synlab, Jena, Germany, 3Medizinische Fakultät/ Universität Leipzig, Hämatologie/ internistische Onkologie, Leipzig, Germany

The diagnostic workup via PCR or melting analysis is limited to analyzing exon 14 of JAK2, exon 10 of MPL, and exon 9 of CALR as the phenotype driver mutations (mut.) in JAK2, CALR, and MPL are considered to be mutually exclusive in patients (pts) with myelofibrosis (MF). We compared a next-generation sequencing (NGS)-based approach to standard PCR in detecting driver mut.

Patients and methods: Genomic DNA was isolated from whole-blood samples of 129 pts with MF (68 males; median age 60y). JAK2, MPL, and CALR mut. analysis via PCR were performed as published. Then, NGS was performed to detect mut. in 23 genes known to be involved in myeloid malignancies.

Results: Standard PCR detected JAK2V617F, MPLW515L, and mutated CALR in 81 (63%), 1 (0.8%), and 25 (19.4%) pts respectively. One patient (0.8%) harboured both mutated JAK2 and CALR. NGS identified further mut. in JAK2 [exon 24 at position R1063H (n = 6) and exon 20 at position R893T (n = 1)] in 7 JAK2V617F+ pts and in MPL at positions W515L + E335K, E259K, Y591D (n = 3), and Y591D (n = 2). None of the pts with JAK2R1063H or JAK2R893T mut. carried a TET2 or ASXL1 mut. JAK2-TET2 double-mutant cells were present in 16 (12%) pts. Interestingly, 4 of 5 MPL mut. were found in the JAK2-TET2 mutated group and non in the CALR mutated or triple negative cohorts. Patients with the JAK2-MPL-TET2 mut. were older, had a higher JAK2V617F allele burden, and larger spleens compared to the JAK2V617F+ “only” cohort (table).


Conclusions: With NGS, additional activating mut. such as MPLY591D (a gain of function mut. in exon 12 of MPL) could be detected. Our data provide evidence that driver mutations are not always mutually exclusive as was the case in 4% of the pts and imply that combination of mut. could influence the clinical phenotype. Analysis of larger cohorts will provide accurate estimates of the frequency of concomitant JAK2 and MPL mut. Serial NGS permits the study of the chronology of mut., which might influence the biology of the disease and/or response to therapy.

Disclosure: Susann Schulze: No conflict of interest disclosed.Haifa Al-Ali: Financing of Scientific Research: yes; Expert Testimony: yes

V107 – Cell intrinsic induction of IP-10 in MPN

Schnöder T.M.1, Eberhardt J.2, Nimmagadda S.C.2, Weinert S.3, Wolleschak D.2, Sammt A.2, Fahldieck C.2, Schönborn U.2, Fischer T.2, Heidel F.H.1,4

1 Universitätsklinikum Jena, Klinik für Innere Medizin II, Jena, Germany, 2Otto-von-Guericke Universität Magdeburg, Klinik für Hämatologie und Onkologie, Magdeburg, Germany, 3Otto-von-Guericke Universität Magdeburg, Klinik für Kardiologie und Angiologie, Magdeburg, Germany, 4Otto-von-Guericke Universität Magdeburg, Magdeburg, Germany

Introduction: Myeloproliferative neoplasm (MPN) is a group of diseases which include polycythemia vera (PV), essential thrombocytosis (ET) and primary myelofibrosis (PMF). In advanced phases, especially in post-ET and post-PV myelofibrosis, the clinical hallmark of the disease is a striking inflammatory syndrome. At a molecular level, PV, ET and PMF are characterized by an activating point mutation (V617F) of JAK2. Currently, the cellular and molecular basis of the inflammatory response syndrome is incompletely understood and therapeutic options for MPN patients ineligible for allogeneic stem cell transplantation are limited to symptomatic approaches. Here, we aimed to evaluate the role of JAK2V617F mutation for aberrant cytokine expression in MPN.

Methods: IP-10 mRNA expression was analyzed in the peripheral blood of patients diagnosed with PV, ET or MF after informed consent using quantitative real-time PCR (qPCR). Moreover, BaF3 cells were stably transduced with EpoR and Jak2WT or Jak2V617F, respectively. Cytokine Array was performed to identify Jak2V617F-triggered inflammatory response. Validation of differentially expressed targets was performed using qPCR and immunoblotting.

Results: IP-10 mRNA expression was significantly elevated in JAK2V617F+ MPN patients compared to healthy donors or JAK2V617F- MPN patients. High V617F allele burden correlated with high IP-10 expression (p = 0.0003). To exclude paracrine/autocrine stimulation as a potential mechanism, we used BaF3 cells harboring the Jak2V617F mutation but lacking IP-10 receptor. IP-10 mRNA was also highly expressed in BaF3 Jak2V617F cells and pharmacological treatment with Ruxolitinib (RUX) abrogated IP-10 expression. NF?B signaling was induced in Jak2V617F-positive cells as compared to Jak2WT controls and its activation was reduced after RUX treatment. Using a luciferase promoter assay, NFkB signaling was modulated by the presence or absence of Jak2V617F-mediated signaling, suggesting direct induction of IP-10 by NFkB signaling.

Conclusions: Taken together, we could show that IP-10 expression correlates with JAK2V617F allele burden in MPN patients and is differentially up-regulated in the respective MPN entities. Our data provide first evidence for a link between oncogenic JAK2V617F signaling and cell intrinsic induction of chemokines/cytokines regulated through NFkB signaling. These data encourage further investigation to elucidate the intercellular pathomechanism of chronic inflammation in MPN.

Disclosure: Tina Schnöder: No conflict of interest disclosed.Florian Heidel: Advisory Role: Advisory Board Novartis; Financing of Scientific Research: Vortragshonorar Novartis; Expert Testimony: Research Funding Novartis

V108 – Differential NK-suppressive capacity of the JAK2 inhibitor Pacritinib when compared to the JAK1/2 inhibitor Ruxolitinib

Wolf D.1, Rudolph J.2, Brossart P.2, Schoenberg K.2

1Universitätsklinikum Bonn, Bonn, Germany, 2Department of Internal Medicine III, Oncology, Hematology and Rheumatology, University Clinic Bonn (UKB), Bonn, Germany

Introduction: We previously demonstrated the NK cell suppressive potential of the JAK1/2 inhibitor ruxolitinib, which is also linked to infections (Schönberg et al, Cancer Res 2015). More specific JAK inhibitors may exert a differential immune-modulatory potential, leading to a less pronounced immunosuppression. The JAK2 inhibitor pacritinib is currently evaluated in phase III clinical trials. We here evaluated the NK cell suppressive potential of pacritinib as compared to ruxolitinib.

Methods: NK cells from healthy volunteers were isolated by magnetic beads and purity was regularly controlled. NK cell function (killing, cytokine production), proliferation and receptor expression were evaluated by standard immunological techniques. The doses used were 0.1, 1 and 10 µM of both JAK inhibitors. The relevant in vivo achieved dose of ruxolitinib is 1-2µM, whereas the one of pacritinib is 0.1 µM.

Results: In line with out previous results, ruxolitinib potently inhibits proliferation, activation, killing activity and cytokine production at doses of 1µM. When therapeutically achieved doses of pacritinib are compared to ruxolitinib, its NK suppressive potential is clearly lower, with only a slight but insignificant reduction of killing against K562 target cells and degranulation, whereas production of IFN-g is significantly reduced but clearly less potent when compared to ruxolitinib. More strikingly, 1 µM of ruxolitinib reduced proliferation by approximately 75%, whereas 0.1µM of pacritinib only marginally reduces NK cell proliferation. When comparing induction of apoptosis, ruxolitinib does not induce significant amounts of cell death up to doses of 10µM, whereas pacritinib at a concentration of 10µM almost completely eliminated NK cells. When NK cells are activated by activating NK receptor antibodies (i.e. NKp46-mAb) instead of cytokine stimulation by IL-2, both compounds are ineffective with respect to NK cell suppression, which is again in line with our previous results showing that JAK-inhibitors predominantly inhibit cytokine-mediated NK cell activation.

Conclusions: The more specific JAK2 Inhibitor pacritinib is less potent with respect to NK cell suppression when compared to the JAK1/2 inhibitor ruxolitinib. This may help to understand differential immune-modulatory effects of different types of JAK inhibitors.

Disclosure: Dominik Wolf: Advisory Role: Bexalta, Novartis; Financing of Scientific Research: Bexalta, Novartis; Expert Testimony: Bexalta, NovartisKathrin Schoenberg: No conflict of interest disclosed.

Fortbildung – Immuntherapie in der Hämatologie und Onkologie

V110 – Checkpoint-inhibition in oncology

Wolf D.1

1Universitätsklinikum Bonn, Medizinische Klinik 3; Onkologie, Hämatologie, Immunonkologie und Rheumatologie, Bonn, Germany

It was in 2011, when Hanahan and Weinberg renewed their initial “hallmark of cancer” concept by adding the ability of malignant tumors to escape from an efficient immune cell attack (termed cancer immune-evasion). It was already assumed for decades that tumors are able shape the immune system, thereby actively preventing their elimination. Visionaries such as Paul Ehrlich more than a century ago already envisioned that “magic (e.g. immunological) bullets” may overcome tolerance to cancer. Immune cell activation is fine-tuned by a number of receptor/ligand-pairs, such as immune-activating (CD28, GITR or CD137) and immune-inhibitory (PD1, CTLA-4, LAG3, TIM3) proteins. CTLA-4 and PD-1 on T and PD-L1 on cancer cells represent the most prominent checkpoint-molecules, since both targets can be succesfully blocked in a therapeutic setting by antibodies, leading to their approval in various solid metastatic diseases (melanoma, NSCLC and RCC). In addition, various proof-of-concept Phase 1/2 studies documented very encouraging response and survival rates in other tumors, (e.g. mismatch repair deficient colorectal cancer, Merkel cell carcinoma, ovarian cancer). Most strikingly, these compounds induce long-term survival in a subgroup of immune-susceptible patients. Clinically applicable response predictors are not available so far, even though in some tumors a greater overall survival can be seen in PD-L1 positive tumors. Moreover, it appears that “immunological visibility” based on the genetic instability appears to be associated with improved response rates to checkpoint-blocking agents. This explains superior response rates seen in smoking NSCLC patients carrying a higher mutational load leading to T cell activation based on the generation of mutated neo-antigens. The tolerability of checkpoint-antibodies is very good. In addition to infusion-related reactions, physicians particularly have to check for immune-related side-effects (e.g. pneumonitis, colitis, hypophysitis), as they may need a rapid therapeutic intervention with corticosteroids. In summary, immune-oncology (IO) represents a new therapy pillar in the treatment of solid tumors, which complements the classic treatment modalities of surgery, radiation and chemotherapy, as well as targeted drugs. Combination studies with classical therapeutics and different IO-agents fuel the hope that IO therapies will help to improve outcome of more cancer patients in the near future.

Disclosure: Dominik Wolf: Financing of Scientific Research: BMS, MSD, Roche; Expert Testimony: BMS

V112 – Development of chimeric antigen receptor (CAR) T-cell immunotherapy

Borchmann P.1

1Uniklinik Köln, Klinik I für Innere Medizin, Köln, Germany

Chimeric antigen receptor (CAR) T-cell immunotherapy involves the adoptive transfer of patient-derived T-cells, which are genetically modified to express antigen-targeted receptors. The CAR is designed to recognize the tumor cell and induce a potent T-cell attack and therefore has extra- and intracellular domains. The extracellular domain usually consists of a tumor-antigen specific single-chain variable fragment coupled to a transmembrane linker. The intracellular signaling modules trigger the T-cell functions. One commonly used intracellular effector domain is the CD3? chain from the T-cell receptor; however, co-stimulatory signals are needed to ensure enhanced T-cell function and persistence. Co-stimulatory domains include CD28, 4-1BB, OX40, ICOS. There are many different options to combine different intracellular effector domains in order to improve specific T-cell functions. The CAR encoding gene is delivered into the T-cell using viral vectors and the final cell product is then re-transfused into the patient. CAR T-cell expansion upon antigen-recognition may result in severe cytokine release with relevant clinical symptoms like high fever and low blood pressure. Thus, CAR-T-cell therapy needs highly sophisticated logistics. This presentation will focus on the development of CAR T-cell therapy for malignant lymphomas.

Disclosure: No conflict of interest disclosed.

Fortbildung – Mammakarzinom: (Neo)adjuvante Therapie

V114 – AGO recommendations for the diagnosis and treatment of patients with early breast cancer: update 2016

Janni W.1

1Universität Ulm, Ulm, Germany

For the last 15 years, the Breast Committee of the Arbeitsgemeinschaft Gynäkologische Onkologie (German Gynecological Oncology Group, AGO) has been preparing and updating evidence-based recommendations for the diagnosis and treatment of patients with early and metastatic breast cancer. The AGO Breast Committee consists of gynecological oncologists specialized in breast cancer and interdisciplinary members specialized in pathology, radiological diagnostics, medical oncology, and radiation oncology. The lecture will give an overview of the 2016 update, which has been performed according to a documented rule-fixed algorithm, by thoroughly reviewing and scoring chapter by chapter the recent publications for their scientific validity (Oxford level of evidence (LoE), www.cebm.net) and clinical relevance (AGO grades of recommendation (GR); table 1). I will present a summary of the 2016 update; the full version of the updated slide set is available online as a PDF file in both English and German.

Disclosure: No conflict of interest disclosed.

Fortbildung – ZNS-Tumoren

V117 – The 2016 WHO classification of brain tumors: What’s new?

Riemenschneider M.J.1

1Universitätsklinikum Regensburg, Abteilung für Neuropathologie, Regensburg, Germany

Since its first launch in 1979 the WHO classification of tumors of the (central) nervous system has been continuously developed. In 1993 immunohistochemistry had been added and in 2000 the classification was extended by the description of molecular alterations though diagnoses still remained based on histology and immunohistochemistry alone. The ratio for brain tumor therapies in the meantime, however, has radically changed. Clinicians on a regular basis ask for a number of well-established molecular markers and patients often are primarily treated according to molecular factors rather than to histological diagnoses. It is in this context, that the new 2016 (revised 4th) edition of the classification describes a major paradigmatic change: For the first time, molecular parameters will be defining for certain diagnoses and an integrated histological and molecular diagnosis becomes state-of-the-art for some brain tumor entities. This makes perfect sense for molecular parameters such as combined allelic deletions on chromosome arms 1p/19q that are closely associated with defined histological appearances. Consequently, oligoastrocytoma will be a dying entity that will prospectively only exist in a histological context with molecular diagnostics not yet performed. Also, the IDH mutational status will be part of an integrated diagnosis, stigmatizing a diffusely infiltrating IDH-wildtype astrocytoma with non-glioblastoma histology as a weird tumor with divergent histological and molecular features. It is a major strength of the new classification that such tumors that clinically might behave irregularly are now readily identified and can be treated accordingly. They can also be omitted from clinical studies making groups more homogeneous and results more meaningful. In this context, also new entities like the genetically defined medulloblastomas might open up options for more individualized therapies both inside and outside of clinical studies. While most academically-based brain tumor centers with specialized neuropathology departments should be able to promptly implement the new procedures in their diagnostic work-up, the new classification poses considerable challenges to smaller local medical centers. Though an NOS category is possible in settings where molecular tests cannot be performed, collaborative clinical networks are more than before recommended to secure best possible diagnoses and up-to-date treatment options for all patients affected.

Disclosure: No conflict of interest disclosed.

Fortbildung – Kontroverse Indikationen zur Stammzelltransplantation

V119 – Allogeneic stem cell transplantation as primary treatment of multiple myeloma

Mielke S.1

1Universitätsklinikum Würzburg, Medizinische Klinik und Poliklinik II, Zentrum für allogene Blutstammzelltransplantation, Würzburg, Germany

Standard treatment for fit patients with multiple myeloma (MM) includes induction therapy followed by autologous stem cell transplantation (SCT) providing substantial treatment-free intervals for the majority of patients. Although there is promising evidence of an allogeneic immune system’s principal ability to provide long term immune-surveillance or even cure to patients suffering from MM, there is ongoing discussion whether allogeneic SCT represents a standard of care treatment option for fit and younger patients. With low rates of non-relapse mortality (NRM) in place, which is largely a consequence of the introduction of reduced-intensity conditioning regimens and improved supportive care options, constantly high rates of relapse and possibly impaired long-term quality of life (QoL) aspects drive today’s controversial discussions particularly in light of several new treatment options surfacing. Nevertheless the genetic instability and the clonal evolution associated with the progression of MM leave allogeneic SCT more than ever as a valuable treatment option to achieve long-term disease control. This however, does require an optimal selection of patients most likely benefitting from this approach. Here, the scenario of primary allogeneic stem cell transplantation for patients with high risk disease upfront after induction therapy or closely following autologous SCT as a tandem approach will be discussed in the light of results for allogeneic SCT as a salvage treatment for patients relapsing after autologous SCT. With regard to dramatically increasing health care costs associated with the approval of several new drugs in the field of MM allogeneic SCT may represent indeed a very reasonable treatment option also from an economical point of view particularly when being capable of offering cure to patients. Certainly, these questions have to be answered in prospective clinical trials in the context of new drugs used for induction treatment before and possibly for consolidation after allogeneic SCT.

Disclosure: Stephan Mielke: Financing of Scientific Research: Celgene, Cellex; Other Financial Relationships: Celgene

V122 – Allogeneic hematopoietic stem cell transplantation (HSCT) with an unrelated donor as first-line therapy in patients with severe aplastic anemia (SAA)

Tichelli A.1, Passweg J.1

1Universitätsspital Basel, Hämatologie, Basel, Switzerland

Allogeneic HSCT is standard first-line treatment for patients with SAA younger than 40 years with an eligible matched sibling donor (MSD). Patients not eligible for HSCT should receive immunosuppressive therapy (IST). Candidates for a matched unrelated donor (MUD) are patients having failed at least one course of IST.

The outcome of unrelated HSCT has greatly improved, as the consequence of better supportive care, improved conditioning regimens and superior donor selection by high resolution HLA-typing. Therefore, the paradigm on algorithm of first-line therapy in SAA is questioned. So far, most data on MUD HSCT are of retrospective nature and based on patients who failed first-line IST. Compared to MSD, transplants from MUD lead to more acute and chronic GVHD, but survival is not inferior. The negative predictors for survival are the same in MSD and MUD HSCT: the use of peripheral blood, longer interval from diagnosis to HSCT, older age at transplantation. Excellent results are obtained with a 8/8 or 10/10 MUD, but significantly inferior with < 8/8 MUD transplants. In a recent French cohort study, three risk factors (age >30 years; interval longer >12months; 9/10 mismatch), have been integrated into a risk score. The 4-year survival was 74% for patients with low-risk and 49% for patients with high-risk score. In a retrospective study, 29 children receiving upfront MUD transplant were compared to 87 MSD HSCT and 58 IST. The 2-year overall survival was >90% for all groups, but event-free survival was significantly lower for IST controls. Concern about upfront MUD transplantation in SAA is the delay in processing from diagnosis to HSCT. The median time interval from diagnosis to MUD HSCT was 4.5 months, which is barely longer than the time needed for MSD HSCT. Histocompatibility laboratories are able to estimate the probability to identify a compatible unrelated donor. About 30% of patients have a high probability to find fast a MUD.

In conclusion, time for revision of the current treatment algorithm for first-line therapy of SAA patients has come. An upfront 10/10 MUD HSCT should be considered as first-line treatment for children and young adults, who lack a MSD, provided there is a high chance for fast donor availability. If a suitable MUD is available to donate bone marrow within 3-4 months from diagnosis, upfront MUD transplantation seems reasonable. The risk score proposed by the French group could help in patient selection for an upfront MUD HSCT.

Disclosure: No conflict of interest disclosed.

Fortbildung – Lebensqualitäts-Assessment

V123 – Assessing quality of life in clinical trials (CTs) - what should be paid attention to?

Engelhardt M.1, Ihorst G.2, Waldschmidt J.1, Keller A.1, Deschler B.3, Dold S.1, Zober A.1, Messner C.1, Möller M.1, Wünsch A.4, Wirsching M.5, Antes G.6, Vach W.7, Duyster J.1, Wäsch R.1

1University of Freiburg Medical Center, Hematology & Oncology + Comprehensive Cancer Center Freiburg (CCCF), Freiburg, Germany, 2Clinical Trials Center, University of Freiburg, Freiburg, Germany, 3Department of Internal Medicine II, CCC Mainfranken, University Hospital Würzburg, Würzburg, Germany, 4Technische Universität München, München, Germany, 5Department of Psychosomatic Medicine and Psychotherapy, University Medical Center, Freiburg, Germany, 6Cochrane Center University of Freiburg, Freiburg, Germany, 7Center for Medical Biometry and Medical Informatics, University of Freiburg, Freiburg, Germany

Introduction: Growing budget pains caused by new products and concerns about novel drugs´ financial toxicity have fueled an acrimonious debate over explosive costs. Health technology assessments (HTA) have been included in CTs, providing additional information on Quality of life (QoL) and health care resource utilization. Albeit these QoL assessments are desirable, if not mandatory, doubts persist about their relevance to clinical practice and which tools should be used.

Methods: We assessed all relevant literature via search functions “QoL”, “CTs”, “oncology”, “State-of-the-art”, obtaining ~30.000 references, limited to “last 5 yrs, humans, English, core journals and cancer”. Moreover, of >130 cancer CTs in our hem&onc clinic, all were screened for quality measures; and compared to results obtained within functional comorbidity assessments (CMAs), initially used in MDS/AML and currently in multiple myeloma (MM) patients (pts).

Results: Pt-reported outcomes (PROs) allow to include pt perspectives into CTs. Many PRO survey instruments are however lengthy and few facilitate care management. The analysis of available QoL tools demonstrate, that most cancer CTs use the EORTC QLQ-C30 or shorter SF-12 with most consistently sensitive QoL scales being appetite loss, fatigue, pain, role, social and physical dysfunctions. Core principles for the development of health care quality measures should address

1. pt-centered outcomes,

2. support robust scientific evidence linked to improved health outcomes,

3. include anticipated benefits,

4. balance in time and resources required to acquire the data.

CMA instruments in older pts with MDS/AML have been implemented in wise decision-making; our brief prospective CMA in MM pts includes the IADL, ADL, Timed Up and Go Test, malnutrition, pain, fitness, SF12 and depression. This CMA is performed to assess pts´ fitness, tolerability of multiagent treatment and to predict OS.

Conclusion: QoL indicators remain relevant, their perfection being under extensive debate. Today, treatment decisions need to be driven by efficacy, QoL being affected, SAE risks, pt preference and costs/affordability. Most predictive CMA tools should be determined and consistently included in future CTs. Scientific advice sessions with regulators, HTA experts and drug developers seem vital, discussing premarketing CT designs openly to support effective and safe new treatments.

Disclosure: Monika Engelhardt: Advisory Role: Janssen, Celgene, Novartis, MSD, Amgen; Financing of Scientific Research: Janssen, Celgene, Novartis, MSD, Amgen; Expert Testimony: Janssen, Celgene, Novartis, MSD, AmgenRalph Wäsch: Advisory Role: Celgene, Novartis, MSD, Amgen; Financing of Scientific Research: Celgene, Novartis, MSD, Amgen

V124 – Electronic collection of quality of life data – one option to suit them all?

Holzner B.1, Sztankay M.2, Gamper E.-M.1, Rumpold G.3, Wintner L.M.1

1Medical University of Innsbruck, Department of Psychiatry, Psychotherapy and Psychosomatics, Innsbruck, Austria, 2University of Innsbruck, Institute of Sport Science, Innsbruck, Austria, 3Medical University of Innsbruck, Department of Medical Psychology, Innsbruck, Austria

Patient-reported outcomes (PRO) like quality of life add the patient’s perspective to traditional clinical outcomes and facilitate patient-centred care. The feasibility of electronic PRO collection (ePRO) grows with decreasing costs for technical devices, comprehensive availability of internet services and rising acceptance of computer-based measures and internet-enabled devices, even in the elderly. In Austria, about 40% of persons older than 65 years regularly access the internet and nearly as many cancer patients stated having internet access and its occasional use. Overall, ePRO is well accepted and only a small proportion of patients state a preference for paper-pencil measures (ranging from 10-18%). ePRO offers several benefits, as it simplifies data collection procedures, improves data quality, eases and accelerates data calculation, presentation and storage and saves human resources.

Available software differs considerably concerning features, applicability and adaptability to the particular needs of users respecting the individual clinical workflow. Useful functionalities include PRO monitoring in daily clinical routine, study monitoring, clinical data bases, and patient portals. One major benefit of routinely collected ePRO data is its versatility, enabling research with real word data, health economy and quality assurance analyses.

The Computer-based Health Evaluation System (CHES) is an ePRO software used for various clinical conditions (e.g. oncology, orthopaedics, neurology, psychiatry/psychosomatics, dementia) in multiple institutions worldwide (e.g. medical universities, rehabilitation centres, county hospitals in AUT, CH, GER, CAN, the UK). Within the longstanding collaboration with the EORTC Quality of Life Group and the EBMT, CHES has shown to be a reliable and user-friendly software for daily clinical routine and research.

Literature shows that ePRO in oncology is feasible both technically and contentwise. The success of ePRO applications depends on reliable software as well as a proper implementation strategy considering several aspects (e.g. clear purpose and expectations, throughout inclusion of stakeholders, adaption of software, training and process evaluation). Systems with integrated patient portals offer an additional benefit to patients, providing disease and treatment-related information, education and self-management advice tailored to PRO results to empower active involvement into their own care.

Disclosure: Bernhard Holzner: Honoraria: Bernhard Holzner and Gerhard Rumpold are owner of the intellectual property rights of the software CHES.Lisa M. Wintner: No conflict of interest disclosed.

V126 – Assessment of quality of life in patients with aplastic anemia and/or PNH

Panse J.1

1Universtiätsklinikum Aachen, Onkologie, Hämatologie und Stammzelltransplantation, Aachen, Germany

Acquired Aplastic Anemia (AA) and Paroxysmal Nocturnal Hemoglobinuria (PNH) are so-called ultra-rare diseases. While much is known about patho-physiology and treatment of these interrelated diseases, less is known about pts. psycho-social issues. Quality of life (QoL) evaluation tools used in studies for AA and PNH are rather unspecific and were designed for cancer patients (pts) (e.g. the EORTC QLQ-C30). Given the complexity of AA and PNH, the variation in symptoms and treatment approaches, the young age of the pts, and the fact that marrow failure syndromes are not classified as malignant diseases, it is likely that cancer-specific questionnaires are inappropriate to adequately assess the QoL and illness intrusiveness in these pts. Hence, we initiated the development and validation of an AA/PNH-specific QoL-instrument (QLQ-AA/PNH). The generation of a QLQ-AA/PNH was performed according to EORTC QoL group guidelines: after identification of QoL issues by literature review, a focus group of pts and physicians were interviewed (phase I). After screening of documented interviews, QoL issues were generated and reworded in a preliminary questionnaire (phase II). In phase III the questionnaire with generated items was tested in a representative patient group and psychometrically validated. In addition, patients were asked to complete a questionnaire regarding their supportive care needs, (e. g. information, support by medical staff, psychosocial counseling, patient support groups etc.) and potential iatrogenic problems (e. g. delay in diagnosis, appreciation of QoL problems by health care professionals). 102 patients in more than 25 German and Swiss cities were interviewed. In phase I, interviews of 19 pts and 8 physicians specialized in AA/PNH-treatment resulted in 649 QoL issues; these were condensed to 175, and graded according to their importance by 30 pts and 14 physicians (phase II). 97 issues were rated important. 12 EORTC QLQ-C30 items were not rated important, while several new QoL aspects were brought up. Modifications led to two questionnaires with 77 items regarding general QoL-aspects and 20 items regarding medical care. Pre-testing of the questionnaire including 97 items was done with 52 pts, followed by psychometric validation. The final QLQ-AA/PNH includes 54 questions. It has been translated into English, Italian and French and is currently validated through phase III studies for AA pts and by use of the PNH registry for PNH pts.

Disclosure: No conflict of interest disclosed.

Fortbildung – Eosinophilie, Mastozytose

V127 – Differential diagnosis and therapy of hypereosinophilia

Metzgeroth G.1

1III. Medizinische Klinik, Hämatologie und internistische Onkologie, Universitätsmedizin Mannheim, Mannheim, Germany

Sustained eosinophilia (>0.5×109/l) is observed in a wide range of reactive/non-clonal and neoplastic/clonal disorders. The clinical presentation may be complicated by a life-threatening organ damage, e.g. of heart, lung and/or nervous system. In the majority of cases, eosinophilia is reactive, e.g. due to an autoimmune disorder or hypereosinophilic syndrome (HES), through overproduction of eosinophilopoietic cytokines. In the presence of distinct molecular aberrations, the underlying entities are either classified as ‘myeloid/lymphoid neoplasms associated with eosinophilia and rearrangements of PDGFRA, PDGFRB, FGFR1 or JAK2 (MLN-eo)’ or ‘chronic eosinophilic leukemia, not otherwise specified (CEL-NOS)’, other cases as eosinophilia-associated myeloproliferative neoplasm (MPN-eo). The FIP1L1-PDGFRA fusion gene, which is only identified by FISH or RT-PCR, is the most frequent molecular aberration. Cytogenetic analysis from a bone marrow aspirate is mandatory for the detection of rearrangements of 5q31-33 (PDGFRB), 8p11 (FGFR1) or 9p24 (JAK2) and subsequent identification of fusion partners by FISH/PCR. FIP1L1-PDGFRA negative patients with normal karyotype should be screened for KIT D816V and JAK2 V617F leading to diagnosis of systemic mastocytosis (SM-eo) and MPN-eo, respectively. All those patients are candidates for treatment with tyrosine kinase inhibitors, however, sustained responses are not seen in all entities. Patients with PDGFRA/-B fusion genes achieve durable remissions on imatinib, irrespective of the disease stage (chronic or blast phase). Resistance, e.g. due to T674I or D842V point mutations in PDGFRA, is extremely rare. Responses may also be observed in patients with JAK2 fusion genes on ruxolitinib and to a lesser extent in patients with FGFR1 fusion genes on ponatinib, however, resistance and/or progression are common. Because of an aggressive clinical course, eligible patients should be offered an early allogeneic stem cell transplantation. In CEL-NOS and MPN-eo, hydroxyurea and possibly also imatinib or ruxolitinib remain treatment options, SM-eo should be treated with midostaurin and/or cladribine. In reactive eosinophilia with organ damage, the disease is usually diagnosed as autoimmune disorder or HES. It is most important to find a good balance between response and potential adverse effects of corticosteroids. Careful attention should therefore be paid to the need of steroid-sparing drugs such as MTX, azathioprin or cyclophosphamide.

Disclosure: No conflict of interest disclosed.

V130 – Treatment options in systemic mastocytosis

Reiter A.1, Jawhar M.1, Metzgeroth G.1, Schwaab J.1

1Universitätsmedizin Mannheim, Hämatologie und Onkologie, Mannheim, Germany

Systemic mastocytosis (SM) is a clonal hematologic neoplasm characterized by accumulation of neoplastic mast cells (MC) in various tissues, e.g. bone marrow (BM), visceral organs and skin. The diagnosis is based upon the presence of aggregates of spindle shaped mast cells with abnormal immunophenotype (CD117+/CD25+), elevated serum tryptase levels and presence of the KIT D816V mutation in 80-90% of patients. Indolent SM (ISM) shows little or no evidence of organ damage and a normal life expectancy. Treatment is based on the avoidance of triggers and symptomatic treatment with H1- and H2-inhibitors, cromoglycin acid and steroids. Advanced SM (advSM) comprises several subtypes that are characterized by the overlapping presence of an associated hematologic neoplasm (SM-AHN, predominantly myelodysplastic/myeloproliferative or myeloproliferative neoplasms, SM-MDS/MPN, SM-MPN), C-findings (e.g. cytopenia, hypoalbuminemia, malabsorption, ascites, weight loss) indicating organ damage (aggressive SM, ASM) or >20% mast cells in BM smears (mast cell leukemia, MCL). More than 80% of advSM patients have one or more (>60%) additional mutations in genes such as TET2, SRSF2, ASXL1, RUNX1, CBL, JAK2, RAS and others. Mutations in SRSF2, ASXL1 and/or RUNX1 have a strong negative impact on phenotype, clinical course and prognosis. Organ damage and clinical symptoms frequently require rapid initiation of cytoreductive therapy. For many years, only IFN-alpha and cladribine have been available achieving variable degrees of clinical remissions, which are rarely complete and durable. Currently, the main focus lies on KIT-inhibitors, e.g. midostaurin, and allogeneic stem cell transplantation (ASCT). In a phase-II-study, midostaurin induced significant responses (C-findings, BM infiltration, serum tryptase levels, clinical symptoms) in the majority of patients, independently of the subtype, KIT mutation status or prior therapy. A retrospective study of 57 patients revealed that ASCT was associated with responses and complete remissions in 40 (70%) and 16 (28%) of patients, respectively. Overall survival at 3 years was 57% for all patients, 74% for SM-AHN, 43% for ASM and 17% for MCL, respectively. Future studies should gain a better insight into the impact of the complex molecular profile on treatment outcome and they should evaluate combined treatment strategies including conventional cytoreductive therapy, KIT inhibitors and ASCT.

Disclosure: Andreas Reiter: Advisory Role: Novartis Pharma; Financing of Scientific Research: Novartis PharmaJuliana Schwaab: Financing of Scientific Research: Novartis Pharma

Freier Vortrag – Nichtkleinzelliges Lungenkarzinom

V131 – Summary of EGFR-Biomarker data from the randomized, controlled phase 3 trial SQUIRE on adding Necitumumab to first-line Gemcitabine-Cisplatin (GC) for squamous Non-Small Cell Lung Cancer (sqNSCLC)

Schumann C.1, Reck M.2, Thomas M.3, Mezger J.4, Socinski M.A.5, Hozak R.6, Mi G.6, Depenbrock H.7, Krause T.7, Hirsch F.R.8, Thatcher N.9

1Klinikum Kempten, Pneumologie, Thoraxonkologie, Schlaf- und Beatmungsmedizin, Kempten, Germany, 2LungenClinic Großhansdorf, Department of Thoracic Oncology, Airway Research Center North (ARCN), German Center for Lung Research (DZL), Großhansdorf, Germany, 3Internistische Onkologie der Thoraxtumoren, Thoraxlinik im Universitätsklinikum Heidelberg, Translational Lung Research Center Heidelberg (TLRC-H), Heidelberg, Germany, 4St. VincentiusKliniken, Medizinische Klinik II, Karlsruhe, Germany, 5University of Pittsburgh, Department of Hematology/Oncology, Pittsburgh, United States, 6Eli Lilly and Company, Indianapolis, United States, 7Lilly Deutschland GmbH, Medical – Oncology, Bad Homburg, Germany, 8University of Colorado Cancer Center, Aurora, United States, 9The Christie Hospital, Manchester, United Kingdom

Introduction: In SQUIRE (N = 1093), the addition of the IgG1 EGFR-antibody necitumumab to first-line GC in advanced sqNSCLC significantly improved overall survival (OS); the safety profile was acceptable (Thatcher et al., Lancet Oncol 2015;16:763-774). SQUIRE included mandatory tissue collection from archived tumor. Here, we summarize analyses exploring the relationship of EGFR protein expression and EGFR gene copy number with efficacy outcomes (OS, PFS).

Methods: Tissue samples were centrally assessed for EGFR protein expression by immunohistochemistry (IHC, Dako EGFR PharmDx kit) and for EGFR gene copy number by fluorescence in situ hybridization (FISH). OS and PFS were evaluated by Kaplan-Meier analysis and Cox proportional hazard models, separately for patients with EGFR-protein expressing (EGFR>0) and non-expressing (EGFR = 0) tumors (stratified, pre-specified analysis), and for patients with and without increased EGFR gene copy number (Hirsch et al., J Clin Oncol 2008;26:3351-3357; exploratory, unstratified interaction analysis).

Results: Tissue samples were evaluable for EGFR protein expression in 982 of the 1093 randomized patients (89.8%). The majority of these patients (935/982, 95.2%) had EGFR-expressing tumors, and this subgroup benefitted from the addition of necitumumab similar to the overall population (Table). Benefit was not apparent in the small subgroup of patients with non-EGFR expressing tumors. Samples for EGFR gene copy analysis were available for 557 patients (51.0%). Of these, 208 (37.3%) had an increased EGFR gene copy number (FISH positive tumor). Patients with increased EGFR gene copy number showed more favorable hazard ratios for both OS and PFS (0.70 and 0.71, respectively; vs. 1.02 and 1.04 for non-increased copy number, but with a non-significant treatment-by-marker interaction (Table).

Conclusions: Patients with EGFR expressing tumors benefitted from the addition of necitumumab to first-line GC treatment. EGFR gene copy number gain showed a trend for a more favorable HR, but the interaction was not statistically significant. Further investigations to better understand the roles of these biomarkers are ongoing.


Disclosure: Christian Schumann: No conflict of interest disclosed.Nick Thatcher: Financing of Scientific Research: Eli Lilly and Company

V132 – Distribution of histological subtypes of lung cancer in Germany (1999-2012)

Hermann S.1, Friedrich S.1, Arndt V.1

1Deutsches Krebsforschungszentrum (DKFZ), Epidemiologisches Krebsregister Baden-Württemberg, Heidelberg, Germany

Introduction: Earlier publications from other countries have shown that the proportion of adenocarcinoma lung cancer has increased while squamous cell carcinoma has decreased over the last decades presumably due to changes in cigarette design, composition and smoking habits. Our aim is to evaluate if this shift in histological subgroups has also taken place in Germany.

Methods: The study is based on all primary lung cancer cases (ICD-10 C34) diagnosed in 1999–2012 and reported by six German epidemiological cancer registries (Bremen, Hamburg, Mecklenburg-Vorpommern, Münster, Sachsen, Saarland) after exclusion of death certificate only cases. Histology of lung cancer cases was classified into: squamous cell carcinoma (SCC), adenocarcinoma (ADC), small cell carcinoma (SCLC), large cell carcinoma (LCC), other, and unspecific morphology. Overall, sex-, and age-specific trends in the distribution histological subtypes was assessed.

Results: A total of 87.558 lung cancer cases (males: 63.109, females: 24.449), diagnosed 1999–2012, were included. The overall distribution according to histological subtype was ADC 26.9%, SCC 24.2%, unspecific histology 22.7%, LCC 7.2%, SCLC 3.6%, and other 15.4%. After exclusion of cases with unspecific histology, the proportion of ADC continuously increased significantly between 1999 and 2012 in men (25.6%?38.2%) and in women (37.0%?50.5%) whereas the proportion of SCC significantly decreased from 39.5% to 32.4% in men and from 22.1% to 16.2% in women. This pattern was consistent over all age-groups, least pronounced for patients =75 years of age and most pronounced for age-group 65-74 years of age. In absolute numbers, ADC increased between 1999 and 2012 from 1158 to 2114 cases (+ 134% in women, + 60% in men) whereas the number of all other lung cancer cases with specified histology addition increased by 36% in women and decreased by 10.5% in men.

Conclusion: As in other countries, both the proportion as well as the absolute number of adenocarcinoma lung cancer has increased in Germany between 1999 and 2012. Increases in ADC are considered to reflect a wider use of filter-tripped and low-tar cigarettes.

Acknowledgement: The here used data originates from six cancer registries (Bremen, Hamburg, Mecklenburg-Vorpommern, Münster, Sachsen, Saarland) and was provided via the German Centre for Cancer Registry Data. Our thanks goes to each individual cancer registry for the contribution of the data.

Disclosure: No conflict of interest disclosed.

V133 – P53 non-disruptive mutation is a negative predictive factor in EGFR M+ NSCLC treated with TKI

Griesinger F.1, Netchaeva M.1, Lüers A.1, Prenzel R.2, Scriba D.3, Willborn K.C.4, Stropiep U.5, Hallas C.6, Tiemann M.6, Falk M.6, Neemann N.7, Heukamp L.7, Roeper J.1

1Pius-Hospital Oldenburg, University of Oldenburg, Universitätsklinik Innere Medizin-Onkologie, Oldenburg, Germany, 2Pius-Hospital Oldenburg, Department Pneumology, Oldenburg, Germany, 3Pius-Hospital Oldenburg, Department Thoracic Surgery, Oldenburg, Germany, 4Pius-Hospital Oldenburg, Department Radiotherapy, Oldenburg, Germany, 5Pius-Hospital Oldenburg, Oldenburg, Germany, 6Hematopathology Hamburg, Hamburg, Germany, 7New Oncology, Köln, Germany

Introduction: P53 mutations are common in lung cancer, and have also been described in EGFR M+ patients. The impact of p53 mutations in EGFR M+ patients is controversial, especially if classified as disruptive and non-disruptive according to their functional effect on the p53 protein as proposed by Poeta and colleagues. The aim of the study was therefore to systematically analyze EGFR and p53 mutations within a cohort of patients with lung cancer stage IV (UICC 7), to correlate alterations with clinical characteristics and to investigate a potential impact of p53 mutations on treatment outcome.

Methods: 409 patients from a single center diagnosed with lung cancer stage IV were studied for the presence of EGFR as well as inactivating p53 mutations. Methods for the detection of EGFR mutations included Sanger Sequencing and hybridization based COBAS testing, hybrid cage next generation sequencing. P53 mutations were detected by Sanger Sequencing and either MiSeq or hybrid cage NGS. Clinical characteristics including smoking status were available for more than 95% of the patients.

Results: 409 consecutive patients at the lung cancer center of the Pius-Hospital Oldenburg were studied. The overall EGFR M+ rate was 18% (73/409) in all patients, 73% (53/73) showing common mutations of exon 19 or 21. In 21/73 (29%) patients’ p53 analysis was not successful. P53 disruptive mutations were demonstrated in 25% (13/52) of successfully tested patients, and p53 non-disruptive mutation occurred in 31% (16/52) whereas p53 WT configuration was found in 44% (23/52). Median OS was 28 months in p53 disruptive mutation and 42 month in p53 WT compared to 23 months in p53 non-disruptive mutation (p < 0.018). PFS on 1st line TKI therapy was 14 months in p53 disruptive mutation, 18 months in p53 WT and 7 months in p53 non-disruptive mutation (p < 0.001). Similar results were shown in the EGFR common mutation but not in the uncommon mutation subgroup. The ORR was 49.2% in patients with a disruptive p53 mutation/p53 WT constellation compared to 14.3% in patients with a non-disruptive p53 mutation and 17.5% in patients with an unknown p53 status.

Conclusion: Significant differences in PFS and OS in EGFR M+ patients were observed depending on p53 mutation status. P53 mutational status is only predictive when disruptive and non-disruptive p53 mutations are differentiated. P53 should be tested prospectively in EGFR M+ patients as management of patients on 1st line TKI may be different.

Disclosure: No conflict of interest disclosed.

V134 – A comprehensive analysis of potentially targetable genetic aberrations and clinical findings in 821 patients with squamous-cell NSCLC – a comparison of NGM and TCGA LUSC data

Koleczko S.1, Schäpers C.1, Scheffler M.1, Ihle M.2, Kostenko A.3, Michels S.1, Fischer R.1, Nogova L.1, Serke M.4, Kaminsky B.5, Benedikter J.6, Brümmendorf T.H.7, Ficker J.H.8, Lorenz J.9, Schulte C.10, Schulze-Olden S.11, Brandes V.1, Abdulla D.1, Ueckeroth F.2, Thurat M.1, Merkelbach-Bruse S.2, Büttner R.2, Wolf J.1

1LCGC, Universitätsklinikum Köln, Köln, Germany, 2Institut für Pathologie, Universitätsklinikum Köln, Köln, Germany, 3Klinik I für Innere Medizin – NGM, Universitätsklinikum Köln, Köln, Germany, 4Lungenklinik Hemer, Hemer, Germany, 5Krankenhaus Bethanien, Solingen, Germany, 6Städtisches Klinikum München, München, Germany, 7RTWH Aachen, Aachen, Germany, 8Klinikum Nürnberg, Nürnberg, Germany, 9Klinikum Lüdenscheid, Lüdenscheid, Germany, 10Gemeinschaftspraxis für Hämatologie und Onkologie, Dortmund, Germany, 11Florence-Nightingale-Krankenhaus der Kaiserswerther Diakonie, Düsseldorf, Germany

Introduction: In contrast to the improvements which have been made in the treatment of adenocarcinoma NSCLC, squamous-cell carcinoma NSCLC (SCC) remains a therapeutic challenge. While there are recent advantages with immunotherapy approaches, targeted therapy still lacks of evidence regarding the frequency of driver aberrations in advanced SCC. We set out this study in order to characterize a large-scale set of patients with SCC genetically and clinically and compared the findings to the early-stage The Cancer Genome Atlas (TCGA) LUSC cohort.

Methods: Tumor biopsies of 821 patients were analyzed within the Network Genomic Medicine (NGM) lung cancer using next-generation parallel sequencing (NGS). The panel used consisted of 102 amplicons and 14 genes: KRAS, PIK3CA, BRAF, EGFR, ERBB2, NRAS, DDR2, TP53, ALK, CTNNB1, MET, AKT1, PTEN and MAP2K1. In subsets of patients, fluorescence in-situ hybridization (FISH) was performed for amplification detection of FGFR1 and MET. We queried the TCGA dataset with respect to the panel used and compared the findings. For the NGM patients, therapy and outcome were also collected.

Results: Beside expected frequencies of TP53, DDR2, PTEN and PIK3CA mutations, we detected EGFR mutations in 3.2% and BRAF mutations in 1.8%. Unlike the TCGA dataset, where the frequencies were 2.8% and 3.9%, respectively, the detected mutations in the NGM cohort consisted of activating mutations (i. e., EGFR del19 and L858R, and BRAF V600E). FISH data revealed a presence of MET amplification in 14.2% and of FGFR1 amplification in 20.0%. The association and correlation of these aberrations with clinical findings and prognosis as well as with PD-L1 expression status and mutational load is still being analyzed. HER2 amplification, which occurred in 2.2% of the TCGA cohort, will be analyzed in a subset of patients.

Conclusions: Our data suggest that the presence of a potential targetable aberration might occur in up to 40% of SCC all-comers. Further analyses are warranted in order to characterize SCC patients according to their biomarker profiles for potential treatment recommendations.

Disclosure: Sophia Koleczko: No conflict of interest disclosed.Jürgen Wolf: Advisory Role: ASTRAZENECA, Boehringer Ingelheim, Bristol-Myers Squibb, Clovis, MSD, Novartis, Pfizer, Roche; Financing of Scientific Research: siehe oben; Expert Testimony: MSD, Novartis, Pfizer, Roche; Other Financial Relationships: siehe oben

V135 – CheckMate 017 and 057 studies of nivolumab vs docetaxel in patients with advanced NSCLC: 2-year-update and exploratory cytokine profile analyses

Eberhardt W.E.E.1, Borghaei H.2, Brahmer J.R.3, Horn L.4, Ready N.5, Steins M.6, Felip E.7, Paz-Ares L.G.8, Arrieta O.9, Barlesi F.10, Antonia S.J.11, Fayette J.12, Rizvi N.A.13, Crinò L.14, Reck M.15, Hellmann M.13, Desai K.16, Li A.16, Healey D.16, Spigel D.R.17

1University Hospital – University Duisburg-Essen, West German Tumor Center, Essen, Germany, 2Fox Chase Cancer Center, Philadelphia, United States, 3The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, United States, 4Vanderbilt University Medical Center, Nashville, United States, 5Duke University Medical Center, Durham, United States, 6Thoraxklinik-Heidelberg gGmbh, Heidelberg, Germany, 7Vall d’Hebron University Hospital, Vall d’Hebron Institute of Oncology, Medical Oncology Department, Barcelona, Spain, 8Virgen del Rocio University Hospital, Seville, Spain, 9Instituto Nacional de Cancerologia – INCAN, Mexico City, Mexico, 10Aix-Marseille University, Assistance Publique Hôpitaux de Marseille, Hopital Nord, Marseille, France, 11H. Lee Moffitt Cancer Center, Tampa, United States, 12Centre Léon Bérard, Lyon, France, 13Memorial Sloan Kettering Cancer Center, New York, United States, 14Ospedale di Perugia, Perugia, Italy, 15Lungen Clinic Grosshansdorf, Airway Research Center North (ARCN), Grosshansdorf, Germany, 16Bristol-Myers Squibb, Princeton, United States, 17Sarah Cannon Research Institute, Nashville, United States

Background: Nivolumab, a fully human IgG4 programmed death-1 immune checkpoint inhibitor, is approved in the US and EU for patients with previously treated advanced/metastatic NSCLC, based on results of 2 phase 3 trials: CheckMate 017 (NCT01642004) in squamous (SQ) NSCLC and CheckMate 057 (NCT01673867) in non-SQ (NSQ) NSCLC.

Methods: Patients were randomized 1:1 to receive nivolumab 3 mg/kg Q2W or docetaxel 75 mg/m2 Q3W until progression or discontinuation. OS was the primary objective in both studies. Multivariate analyses of baseline serum cytokines (exploratory) were performed separately in patients with SQ and NSQ NSCLC. Cytoscores derived from evaluable patients in three studies (CheckMate 017, 057, and 063 [a single-arm phase 2 study of nivolumab in SQ NSCLC] were calculated to quantify the effect of identified cytokine sets on 18-month OS. Cytoscores were categorized as high or low based on median cutoffs.

Results: CheckMate 017: median OS was 9.2 vs 6.0 months with nivolumab vs docetaxel (18month OS: 28% vs 13%; HR: 0.62 [0.48, 0.81]; P = 0.0004). CheckMate 057: median OS was 12.2 vs 9.4 months with nivolumab vs docetaxel (18-month OS: 39% vs 23%; HR: 0.72 [0.60, 0.88]; P = 0.0009). Patients with NSQ NSCLC that expressed PD-L1 achieved a greater magnitude of benefit with nivolumab; in patients with SQ NSCLC, PD-L1 expression was neither prognostic nor predictive of benefit. In both trials, treatment-related AEs were less frequent with nivolumab than with docetaxel. Preliminary results showed an association of high vs low SQ- and NSQ-cytoscores with OS (SQ NSCLC: nivolumab, HR = 0.48, P < 0.0001; docetaxel, HR = 0.39, P < 0.0001; NSQ NSCLC: nivolumab, HR = 0.52, P = 0.0001; docetaxel, HR = 0.60, P = 0.0001; figure).

Conclusion: Nivolumab demonstrated improved OS and a favorable safety profile compared with docetaxel in patients with SQ and NSQ NSCLC. Two-year OS and safety results will be presented. Levels of baseline serum cytokines (details to be presented) correlated with OS benefit in patients with advanced SQ and NSQ NSCLC. Prospective validation of these preliminary results is needed.


Disclosure: Wilfried Ernst Erich Eberhardt: Advisory Role: Consulting/advisory role: BMS, Eli Lilly, Astra Zeneca, Pfizer, Roche, Novartis, Boehringer-Ingelheim, Merck, Hexal, Bayer, Astellas, Daichi Sankyo; Financing of Scientific Research: BMS, Eli Lilly, Astra Zeneca, Pfizer, Roche, Novartis, Boehringer-Ingelheim, Merck, Hexal, Bayer, Astellas; Expert Testimony: Eli Lilly; Other Financial Relationships: Speakers Bureau: Roche, Pfizer, Novartis, Merck, Boehringer-IngelheimDavid Spigel: Advisory Role: Consulting/advisory role: Novartis/Genentech; Other Financial Relationships: Travel, accommodations, expenses: Novartis, Genentech, Pfizer; Speakers Bureau: Novartis (uncompensated)

Fortbildung – Interprofessionelle Sitzung: Therapiebegrenzung(für Ärzte und Pflegekräfte)

V138 – The EPAL-project (Ethics policy for advanced care planning and limiting treatment)

Winkler E.1, Mehlis K.1, Jaeger E.2, Laryionava K.1, Hiddemann W.2, Heussner P.2

1Universität Heidelberg, Nationales Centrum für Tumorerkrankungen, Heidelberg, Germany, 2Klinikum der Ludwig-Maximilians Universität, Medizinische Klinik und Poliklinik III, München, Germany

Introduction: Decisions to limit treatment (DLT) are important in order to protect the patient from burdensome treatment at the end of life but constitute one of the most ethically challenging situations in oncology practice. Thus, they are often accompanied by clinical and psychological conflicts for patients and the medical team. The aim of the EPAL-project is to investigate relevant factors influencing End-of-life decision-making and to develop an ethics policy for these decisions. The policy intends to establish a framework for responding to patients’ preferences in an ethically responsible way and supporting the concept of advance care planning.

Methods: This prospective quantitative study recruited 50 cancer patients from 5/2014-6/2015 in the Dept. of Hematology/Oncology at the University Hospital of Munich with treatment limitations. The patients and their respective physician and nurse completed a set of validated instruments on moral distress, patient’s information needs, involvement in DLT and perceived quality of communication.

Results: Nurses state to have moral distress in 71% (n = 30) of the cases. The average level of stress lies under the cut-off point of 5 (mean/SD: 1.8/1.7) and especially occurs in situations, when DLT is surrounded by a challenging communication within the medical team (p = 0.047). Nurses do not know patients’ preferences on treatment limitation in 77% and only 61% (n = 30) of the patients feel sufficiently involved in DLT. Those who show a higher degree of information seeking behavior are more often involved (p = 0.037). Patients perceiving a good communication feel significantly better informed about their diagnosis (p = 0.003), treatment options (p = 0.019) and progress of the disease (p = 0.000) and are more satisfied with the decision-making process (p = 0.010) compared to those perceiving a bad interaction.

Conclusion: Given the fact that good interaction within the medical team and with the patient is strongly related to the nurses’ level of stress and the patients’ level of information, participation and satisfaction concerning DLT, there is a need to ensure good communication and respect for patient autonomy in these crucial situations. Our policy focuses on these aspects and aims to define conditions and standards for early discussions about treatment limitation, shared decision-making and a respectful communication between physicians, nurses, patients and relatives. A post-implementation study will evaluate the policy’s impact.

Disclosure: No conflict of interest disclosed.

V140 – Referral to palliative care without prognostic awareness – how to deal with diaappointment

Schuler U.S.1, Freitag J.1

1Universitätsklinikum Carl Gustav Carus, PalliativCentrum und Medizinische Klinik I, Dresden, Germany

Prognostic understanding, prognostic awareness of patients is frequently lacking even in advanced stages either due to insufficient communication or due to denial. Even the trial of early integration of palliative care (Temel et al. 2011, JCO 29:2319) reported a rate of ~20% of patients regarding their cancer as curable at 12-24 weeks in the intervention group. Lack of prognostic awareness comes in two variants: (1) The complete lack of information about missing curative options and (2) the acceptance of incurability combined with unrealistic expectations about survival duration. PCUs have to deal with both variants, as important decisions arise. Even at admission, it is sometimes necessary to confront patients with issues like do-not-resuscitate orders (DNR). Other issues (e.g. unnecessary medication, artificial nutrition etc.) are left for clarification in the ensuing days. Even if no longer indicated for physical reasons, these may be continued at least for some time for psychosocial reasons, until cessation becomes acceptable for all parties involved. It is of utmost importance to include (wherever permitted by the patient) close relatives in the decision making. Relinquishing artificial nutrition sometimes leads over-ambitious relatives to almost force-feed their relatives. Especially nutrition is an issue, which frequently involves nursing staff in further communication. But for all goals of care, it is important to keep the multidisciplinary team informed and communication with the patient and family transparent and consistent. Representative examples will be given.

Disclosure: No conflict of interest disclosed.

Freier Vortrag – Chronische lymphatische Leukämie – Therapie

V141 – Ibrutinib (I) plus bendamustine and rituximab (BR) in previously treated chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL): a 2-year follow-up of the HELIOS study

Cramer P.1, Fraser G.2, Chanan-Khan A.3, Demirkan F.4, Santucci Silva R.5, Pylypenko H.6, Grosicki S.7, Janssens A.8, Pristupa A.9, Mayer J.10, Dilhuydy M.-S.11, Loscertales J.12, Goy A.13, Avigdor A.14, Rule S.15, Phelps C.16, Mahler M.16, Salman M.16, Howes A.17, Balasubramanian S.16, Hallek M.1

1Uniklinik Köln, Klinik I für Innere Medizin, Köln, Germany, 2McMaster University, Juravinski Cancer Centre, Hamilton, Canada, 3Mayo Clinic, Division of Hematology and Oncology, Jacksonville, United States, 4Dokuz Eylul University, Division of Hematology, Izmir, Turkey, 5IEP SÃO LUCAS, Hemomed Oncologia e Hematologia, São Paulo, Brazil, 6Cherkassy Regional Oncological Center, Division of Hematology, Cherkassy, Ukraine, 7Community of Hospitals, Division of Hematology, Chorzow, Poland, 8Universitaire Ziekenhuizen Leuven, Leuven, Belgium, 9Regional Clinical Hospital, Ryazan, Russian Federation, 10University Hospital Brno, Division of Internal Medicine, Hematology and Oncology, Jihlavska, Czech Republic, 11Hopital Haut Leveque, Bordeaux, France, 12Hospital Universitario La Princesa, IIS-IP, Division of Hematology, Madrid, Spain, 13John Theurer Cancer Center, Hackensack University Medical Center, Hackensack, United States, 14The Chaim Sheba Medical Center, Institute of Hematology, Division of Hematology and Bone-Marrow Transplantation, Tel Hashomer, Israel, 15Derriford Hospital, Division of Hematology, Plymouth, United Kingdom, 16Janssen Research & Development, Raritan, United States, 17Janssen Research & Development, High Wycombe, United Kingdom

Introduction: The phase 3 HELIOS study evaluated I + BR vs placebo (P) + BR in patients (pts) with previously treated CLL/SLL. At first analysis (median follow-up: 17.0 months) progression-free survival (PFS) was significantly improved for I + BR vs P + BR (HR [95% CI]: 0.203 [0.15-0.28], p < 0.0001). Prior studies have shown deepening responses with continued ibrutinib treatment; long-term follow-up assessing durability and depth of response is important.

Methods: 578 pts received BR (= 6 cycles) and were randomized 1:1 to I (420 mg daily) or P (n = 289/arm). Pts with del17p (= 20% of cells) were excluded. Primary end point was PFS. Key secondary end points: overall survival (OS), overall response rate (ORR), and rate of minimum residual disease negative (MRD -ve) response.

Results: Median follow up is 25.4 months. I + BR continues to show improvement in PFS vs P + BR (investigator [INV]-assessed median: not reached [NR] vs 14.2 months; HR [95% CI]: 0.199 [0.15, 0.26], p < 0.0001; 2-yr rate: 74.8% vs 20.9%). Median OS is still NR in either arm (HR [95% CI]: 0.670 [0.44, 1.02], p = 0.0587; 2-yr rate: 86.2% vs 81.5%); 142 pts (49.1%) in the P + BR arm with confirmed PD have crossed over to receive I, as permitted by protocol. The updated INV-assessed ORR is 87.2% for I + BR vs 66.1% for P + BR (p < 0.0001); updated rates of CR/CRi are 33.9% vs 7.2% (rates at first analysis: 21.4% vs 5.9%). Rates of MRD -ve response for the intent-to-treat population are 18.0% (52/289) for I + BR and 4.8% (14/289) for P + BR (p < 0.0001) (rates at first analysis: 12.8% vs 4.8%). Median PFS2 is unreached in both arms, but PFS2 is significantly longer for pts assigned to I + BR vs P + BR, despite crossover (HR [95% CI]: 0.622 [0.42, 0.92], p = 0.0162). Safety is consistent with first analysis.

Conclusions: I + BR continues to demonstrate superiority vs P + BR with significantly longer PFS and higher ORR. Responses continue to deepen with continuous ibrutinib therapy with rates of CR/CRi and MRD -ve response increasing over time. At every MRD level (< 0.01% or =10% CLL+), I + BR showed a more sustained PFS than P + BR. These 2-year follow-up data confirm the important role of ibrutinib in pts with previously treated CLL.

Trial Registration: NCT01611090; EudraCT No. 2012-000600-15; UTN No. U1111-1135-3745

Disclosure: Paula Cramer: Advisory Role: Janssen, Hoffman-LaRoche; Financing of Scientific Research: Speakers Bureau: Janssen, Hoffman-LaRoche; Expert Testimony: Gilead, GSK, Janssen, Hoffman-LaRoche; Other Financial Relationships: Travel grants: Astellas, Gilead, Janssen, Hoffman-LaRoche, MundiPharmaMichael Hallek: Advisory Role: Abbvie; Financing of Scientific Research: Abbvie; Expert Testimony: Abbvie

V142 – In CLL a complex karyotype is an adverse prognostic parameter that is associated with shorter survival independent of TP53 alteration and IGHV mutation status

Haferlach C.1, Jeromin S.1, Kern W.1, Haferlach T.1

1MLL Münchner Leukämielabor, München, Germany

Introduction: First studies demonstrated a prognostic impact of chromosome banding analysis (CBA) in addition to FISH, TP53 mutation and IGHV mutation status (Haferlach et al. GCC 2010, Rigolin et al. Blood 2012, Thompson et al. Cancer 2015). The aim of this study was to evaluate the impact of a complex karyotype in untreated CLL patients.

Patients and methods: 1046 CLL cases were evaluated at diagnosis or prior to first therapy by CBA and FISH with probes for 17p13 (TP53), 13q14 (D13S25, D13S319, DLEU), 11q22 (ATM), the centromere of chromosome 12 and IGH-CCND1. Further, the TP53 and IGHV mutation status was determined in all patients. Median age was 67 years (range: 30-89 years).

Results: Patients were categorized according to the number of chromosome abnormalities (CA) detected per case by CBA. None, 1, 2, 3, 4 and =5 abnormalities were observed in 228 (21.8%), 449 (42.9%), 208 (19.9%), 72 (6.9%), 36 (3.4%), and 53 (5.1%) cases, respectively. In 46 (4.4%) cases a TP53/17p deletion was identified by FISH and in 76 (7.3%) TP53 mutations by sequencing. 39 patients carried both a TP53 deletion and a TP53 mutation. A significant association was observed between the number of CA and TP53 alterations: 0-2 CA: 5.5%, 3 CA: 11.1%, 4 CA: 13.9%, and =5 CA 39.6% (p < 0.001). The number of CA was also associated with an unmutated IGHV status (p < 0.001). OS at 5 years differed significantly according to the number of CA (0-2 CA: 84.8%, 3 CA: 83.7%, 4 CA: 77.7%, =5 CA: 52.6%, p < 0.001). Three different definitions for complex karyotypes (CK) were evaluated: CK3: =3 CA, CK4: =4 CA, and CK5: =5 CA. In patients without a TP53 alteration overall survival (OS) at 5 years was significantly lower in cases harboring a CK than in those without (CK3: 80.3% vs 86.4%, p = 0.03; CK4: 73.6% vs 86.4%, p = 0.004; CK5: 66.4% vs 86.3%, p = 0.001). Also in patients with TP53 alterations a CK was associated with a negative impact on OS (OS at 5 years: CK3: 41.0% vs 59.6%, p = 0.01; CK4: 35.7% vs 59.4%, p = 0.01; CK5: 31.3% vs 59.1%, p = 0.002). In multivariate Cox regression analysis an independent negative impact on OS was identified for TP53 deletion (relative risk (RR): 3.1, p = 0.001), TP53 mutation (RR: 1.7, p = 0.05), number of chromosome abnormalities (RR: 1.1 per CA, p = 0.006) and unmutated IGHV status (RR: 2.0, p < 0.001).

Conclusion: The number of CA as determined by CBA is a negative prognostic parameter in CLL which is independent of the presence of TP53 alterations and the IGHV mutation status.

Disclosure: No conflict of interest disclosed.

V143 – The Pan-PIM kinase inhibitor LGB321 targets apoptotic pathways and microenvironmental interactions in CLL

Decker S.1, Kissel S.1, Aumann K.2, Zenz T.3, Zirlik K.1, Claus R.1, Duyster J.1, Dierks C.1

1Uniklinik Freiburg/Innere Medizin/Hämatologie, Onkologie und Stammzelltransplantation, Sektion Molekulare Hämatologie, Freiburg, Germany, 2Uniklinik Freiburg, Pathologie, Freiburg, Germany, 3NCT Heidelberg, Heidelberg, Germany

In recent years, the emergence of kinase inhibitors like Ibrutinib has drastically altered treatment strategies and improved outcomes in CLL patients, but lack of cure and resistance to therapy still remain serious problems. PIM kinases are involved in various important disease mechanisms in CLL, with PIM1 regulating CXCR4 surface expression impacting its interaction with the microenvironment, and PIM2/3 affecting the apoptotic machinery by regulating BAD. The Pan-PIM kinase inhibitor LGB321 (Novartis) targets all 3 PIM kinases and therefore affects both, CLL apoposis and its interaction with the microenvironment. In the study presented here, we investigated the effect of LGB321 on CLL in vitro and in vivo. LGB321 was highly effective in inducing apoptosis in primary CLL cells, independent of risk factors like 17p deletions or the mutation status. Apoptosis induction correlated with reduced pBAD and BAD levels. Furthermore, LGB321 was also effective in the presence of protective stromal cells and could completely overcome the stroma protective effects. Mechanistically, we found that LGB321 treatment blocked the CXCR4/CXCL12 axis by dephosphorylating the CXCR4 receptor on Ser339, by reducing total CXCR4 protein levels and by blocking the externalization of the CXCR4 receptor. Concordantly, PIM inhibition blocked CXCR4 functions like migration towards a CXCL12 gradient (P < .0001), and reduced homing of LGB321-pretreated primary CLL cells towards the bone marrow (P = 0.0001) of NOG mice invivo. In vivo experiments comfirmed the efficacy of LGB321 in 4 different CLL xenograft studies. Transplantation of primary human CLL cells into NOG mice and treatment with LGB321 for 2 weeks strongly reduced WBC counts, spleen size and spleen infiltration with human CLL cells (P = 0.0295) in all four CLL cases, and blocked BAD as well as CXCR4 phosphorylation also in vivo. Our results demonstrate, that the Pan-PIM kinase inhibitor LGB321 might be an effective treatment option for CLL patients by impairing PIM2/3 mediated CLL-cell survival, and by blocking the PIM1/CXCR4-mediated interaction of CLL cells with their protective microenvironment in vitro and in vivo. Future clinical trials should be performed to validate its efficacy in human CLL.

Disclosure: No conflict of interest disclosed.

V144 – Checkpoint inhibition reverses immunometabolic dysfunctions of monocytes in chronic lymphocytic leukemia

Mougiakakos D.1, Qorraj M.1, Bruns H.1, Böttcher M.1, Saul D.1, Jitschin R.1, Mackensen A.1

1Medizinische Klinik 5, Universitätsklinikum Erlangen-Nürnberg, Erlangen, Germany

Introduction: CLL patients display immune defects at early disease stages. Increasing evidence suggests that immune alterations negatively impact disease course. The intrinsic tumor immune surveillance is severely attenuated. Monocytes are key components of anti-tumor immunity and important mediators of effects triggered by therapeutic antibodies. The myeloid compartment of CLL patients displays substantial alterations. However, the underlying mechanisms remain largely elusive. It is well accepted that the immune cells’ metabolism strongly determines differentiation and (anti-tumor) function. Current reports suggest that a “metabolic interplay” between cancer and T-cells contributes to tumor immune escape. As yet, nothing has been reported on immunometabolic dysfunctions of monocytes (in CLL). Similar to activated effector T-cells monocytes require an increased glycolytic flux for functioning efficiently.

Methods: CLL-monocytes were divided into classical (CD14++CD16-), intermediate (CD14++CD16+), and non-classical (CD14-CD16++) subsets. We assessed the metabolic phenotype of these subsets using gene-expression, FACS-based, and functional analyses. The impact of metabolic interference on the monocytes’ phagocytic function was tested using E. coli and CLL-cells opsonized with a therapeutic anti-CD20 antibody. The role of PD-L1/PD-1 crosstalk in mediating immunometabolic alterations was investigated in co-cultures of CLL-cells and monocytes.

Results: CLL-monocytes exhibit several changes of their metabolic phenotype: glucose uptake, glucose transporters, and expression of key glycolytic molecules are significantly reduced. Increasing the glycolytic rate enhances the monocytes’ efficacy in terms of eliminating primary CLL-cells. PD-1/PD-L1 interaction is involved in the CLL-mediated T-cell suppression (=immune checkpoint). In a recent study, PD-1/PD-L1 pathway activation was shown to elicit metabolic reprogramming in T-cells by inhibiting glycolysis. We observed similar effects when triggering PD-1 signaling on monocytes. As anticipated, disrupting the PD-1/PD-L1 axis reversed the metabolic and phagocytic dysfunctions of (CLL-)monocytes.

Conclusions: Our data suggests an immunometabolic crosstalk between CLL-cells and monocytes that hampers the monocytes’ function. It provides a strong rationale for exploiting PD-1/PD-L1 checkpoint blockade (e.g. in combination with anti-CD20 antibodies) to restore metabolic together with antitumor activity of monocytes.

Disclosure: No conflict of interest disclosed.

V145 – HLA ligandome analysis of primary chronic lymphocytic leukemia (CLL) cells under lenalidomide treatment approves lenalidomide as a suitable adjuvant for T-cell based immunotherapy

Nelde A.1, Kowalewski D.J.1, Backert L.2, Schuster H.1, Kanz L.3, Salih H.R.3,4, Rammensee H.-G.1,5, Stevanovic S.1,5, Stickel J.S.3

1Universität Tübingen, Abteilung für Immunologie, Tübingen, Germany, 2Universität Tübingen, Center für Bioinformatik und Abteilung für angewandte Computerwissenschaften, Tübingen, Germany, 3Universitätsklinik Tübingen, Hämatologie und Onkologie, Tübingen, Germany, 4Clinical Cooperation Unit Translational Immunology, German Cancer Consortium (DKTK), DKFZ partner site Tübingen, Tübingen, Germany, 5German Cancer Consortium (DKTK), DKFZ partner site Tübingen, Tübingen, Germany

Several studies have proven the positive immunomodulatory effect of lenalidomide on T-cell responses in CLL, in particular regarding the repair of the immune synapse between CLL and T cells. Therefore, lenalidomide seems to be a promising adjuvant for T-cell based immunotherapy approaches. We recently conducted a study, which characterized the antigenic landscape of CLL by mass spectrometric analysis of naturally presented HLA ligands and identified a panel of CLL-specific T-cell epitopes (Kowalewski/PNAS 2015). For a rational combination of T-cell based immunotherapy with lenalidomide, it is of great importance to characterize the effects of this immunomodulatory drug on the antigenic landscape of the target cells.

Here we present a mass spectrometry-based study, which longitudinally maps the HLA ligandome of primary CLL cells under in vitro lenalidomide treatment. We quantified HLA surface expression on CLL cells (n = 4) at t0, t24h and t48h after incubation with lenalidomide. With regard to HLA class I expression, no impact of lenalidomide treatment was observed (fold-change 0.92-1.02, t48h), whereas a slight increase of HLA class II molecules after treatment was detectable (fold-change 1.25-1.43, t48h) with absolute molecule counts ranging from 40,000-125,000 class I and 30,000-200,000 class II molecules/cell.

Implementing label-free quantification data, we then quantitatively assessed HLA class I ligand presentation during lenalidomide treatment. We observed a higher plasticity of the HLA ligandome over time compared to treatment with lenalidomide. 2.5 ± 3.0% of HLA class I ligands showed significant modulation (fold change =4, p = 0.01) after 24h of mock treatment, whereas only 0.9 ± 1.2% of the ligands were modulated through lenalidomide treatment. At t48h similar proportions of modulation were observed with 4.0 ± 1.7% of HLA ligands significantly altered in their abundance over time, while lenalidomide treatment only resulted in 0.9 ± 1.2% modulated ligands.

Out of the >6300 different HLA class I ligands we could identify on primary CLL cells (n = 3), we were able to detect 39 CLL-associated epitopes described in our previous study. Importantly, these antigens showed robust presentation under lenalidomide therapy.

Taken together our study shows that lenalidomide has no relevant influence on and rather seems to stabilize the HLA ligandome of primary CLL cells. Therefore, lenalidomide appears to be an ideal adjuvant for T-cell based immunotherapy in CLL patients.

Disclosure: No conflict of interest disclosed.

V146 – Clinical response to ibrutinib is accompanied by normalization of the T-cell environment in CLL-related autoimmune cytopenia

Schliffke S.1, Akyüz N.1, Ford C.1, Mährle T.1, Thenhausen T.2,Krohn-Grimberghe A.2, Knop S.3, Bokemeyer C.1, Binder M.1

1Universitäsklinikum Hamburg-Eppendorf, Hamburg, Germany, 2LYTIQ GmbH, Paderborn, Germany, 3Universitätsklinikum Würzburg, Würzburg, Germany

B-cell receptor-targeted therapy with ibrutinib is of increasing importance for the therapeutic management of chronic lymphocytic leukemia (CLL). However, limited data is available regarding the therapeutic potential of ibrutinib in the treatment of autoimmune cytopenias (AIC), which frequently affect patients with CLL. The pathophysiological mechanisms driving AIC in CLL are insufficiently understood, but the majority of evidence points towards a polyclonal B- and T-cell-mediated response, rather than direct involvement of the malignant clone. Beyond targeting of malignant B-cells, ibrutinib has a broader immune modulating profile through inhibition of Bruton’s tyrosine kinase (BTK) and the interleukin-2-inducible T-cell kinase (ITK) in a variety of immune cells and – in line with this – ibrutinib is currently under investigation for autoimmune disorders. This was the rationale behind our decision to initiate treatment with ibrutinib in three cases of severe CLL-related AIC. In addition to close clinical monitoring, immune profiling from these patients with multicolor-flow cytometry and next generation sequencing (NGS) of the B- and T-cell repertoire was performed. Pre-treatment analysis by flow cytometry suggested exceptionally high levels of effector memory CD8+ T-cells and activated CD8+ T-cells compared to a cohort of 100 CLL patients without AIC. In parallel with rapid clinical improvement, effector memory and activated CD8+ T-cell counts normalized after the initiation of ibrutinib. Circulating B-cells, however, were almost exclusively of CLL phenotype pre- and post-treatment. Deep sequencing of the immunoglobulin heavy chain and the T-cell receptor beta chain locus revealed circulating T-cell immune environments that were heavily biased towards hyperexpanded and – most likely – autoimmunity-related T-cell clones. After initiation of ibrutinib, these T-cell immune environments normalized significantly with a loss of the hyperexpanded clones. In contrast, the B-cell immune environment remained essentially unchanged with dominance of the malignant CLL clone throughout the treatment. We believe that this observation provides additional evidence that CLL-related AIC is driven by abnormal T-cell environments that can be effectively targeted with ibrutinib and that this supports a clinical trial of early treatment with ibrutinib in CLL-related AIC. Furthermore our data point to response kinetics that vary throughout different ibrutinib target structures.

Disclosure: No conflict of interest disclosed.

Fortbildung – Intensivmedizin und Hämatologie/Onkologie

V150 – Hemophagocytic Lymphohistiozytosis (HLH): A Mimic of Sepsis

La Rosée P.1

1Schwarzwald-Baar Klinikum, Villingen-Schwenningen, Germany

Hemophagocytic Lymphohistiocytosis (HLH) is a hyperinflammatory syndrome, which depending on disease severity can mistakenly be diagnosed as SIRS, Sepsis or multiorgan dysfunctional syndrome (MODYS) on an intensive care unit (ICU). It is a cytokine storm syndrome affecting in particular immunosuppressed patients, and patients with malignant or autoimmune diseases. While HLH is better known in pediatrics due to well characterized hereditary defects of the immune synapse, HLH in adult patients only recently comes into focus of hematologists and ICU physicians. Diagnostic criteria developed for pediatric trials (HLH-2004) are cytopenia, organomegly, fever, elevated Ferritin and soluble IL2-receptor, hemophagocytosis, loss of NK-cell function and lowered fibrinogen or/and elevated triglyzerides. When 5/8 criteria are rated positive, the probability of HLH is high. It is recommended to include S-Ferritin into the lab panel for ICU admission. Early recognition and rapid suppression of overt inflammation are critical for prognosis. Hence, suppression of the cytokine storm by polyvalent immunoglobulins, corticosteroids and etoposide in selected patients, and maintenance with cyclosporine A or tacrolimus under certain circumstances are therapeutic strategies with evidence from case series. Recently, targeted agents like Jak1/2-inhibitors, Interferon-gamma antibodies or IL6-antibodies have been used in early phase clinical trials or case reports. It is important to note that definitive treatment of the underlying trigger disease is the only way to permanently control HLH. Close cooperation of ICU-physicians, hematologists, rheumatologists, infectious disease specialists and diagnostic institutions is pivotal for successful treatment. Management of HLH in adults is discussed based on clinical cases derived from the German HLH registry www.hlh-registry.org.

Disclosure: Paul La Rosée: Advisory Role: Novartis Pharma; Financing of scienti-fic Research: Novartis Pharma

V151 – Non-invasive mechanical ventilation for prevention of invasive mechanical ventilation?

Schellongowski P.1, Wohlfarth P.1, Sperr W.R.2, Knöbl P.2, Buchtele N.1, Gelbenegger G.1, Rabitsch W.3, Staudinger T.1, AG für Hämato-Onkologische Intensivmedizin der ÖGIAIN; AK für Intensivmedizin in der Hämatologie und Onkologie der DGHO; Intensive Care in Hematologic and Oncologic Patients (iCHOP)

1Universitätsklinik für Innere Medizin I, Medizinische Universität Wien, Intensivstation 13i2, Wien, Austria, 2Universitätsklinik für Innere Medizin I, Medizinische Universität Wien, Abteilung für Hämatologie und Hämostaseologie, Wien, Austria, 3Universitätsklinik für Innere Medizin I, Medizinische Universität Wien, Knochenmarktransplantation, Wien, Austria

The acute respiratory failure (ARF) is the most frequent reason for intensive care unit (ICU) admissions in cancer patients. It is of strong prognostic impact, especially, if invasive mechanical ventilation becomes necessary. Non-invasive mechanical ventilation (NIMV) is established for the prevention of intubation in case of cardiac pulmonary edema and obstructive pulmonary disease. However, solid data on patients with hypoxic ARF due to pneumonia or sepsis – both most relevant in cancer patients – are lacking.

A small historic trial showed reduced intubation and mortality rates in immunosuppressed patients with hypoxic ARF (n = 52) receiving pressure support NIMV when compared to O2 insufflation. However, as mortality rates of control group patients were very high, these results may not be relevant nowadays anymore (Hilbert, NEJM 2001). Observational studies revealed inhomogeneous results regarding the association of NIMV, intubation and mortality in hematologic patients, and identified risk factors for NIMV failure (=intubation): severity if illness, ARDS, high respiratory rate during NIMV, delay between admission and NIMV, vasopressors or renal replacement therapy, NIMV dependency > 72h, nonrapidly reversible or unknown causes for ARF, or presence of airway involvement by the malignancy.

A meta-analysis showed successful NIMV to be associated with increased survival. However, NIMV failure was common (61%) and associated with increased risk of death (Amado-Rodríguez, Crit Care Med 2016).

A recent trial in immunosuppressed, mainly hematologic patients (n = 374) with hypoxic ARF compared NIMV with O2 insufflation and found no differences of intubation or survival rates (Lemiale, JAMA 2015). These results may have been influenced by the unequal use of high-flow nasal oxygen (HFNO) between the groups, as HFNO was shown to reduce intubation and 90-day mortality rates in non-immunocompromised patients with hypoxic ARF when compared to NIMV or O2 insufflation (Frat, NEJM 2015).

Conclusively, recent data question the benefits of NIMV in hematologic patients with hypoxic ARF. Furthermore, NIMV failure is common and associated with mortality. If NIMV is used in such patients, meticulous screening for risk factors associated with NIMV failure and early evaluation of intubation in case of their occurrence seems advisable. HFNO seems to be of benefit in non-immunocompromised patients with hypoxic ARF. Confirmation of these data in cancer patients is needed.

Disclosure: Peter Schellongowski: Financing of Scientific Research: Fisher & PaykelThomas Staudinger: No conflict of interest disclosed.

Freier Vortrag – Chronische myeloische Leukämie – Erstlinien- und Absetzstudien

V153 – Treatment free remission in CML patients after first line nilotinib therapy

Eigendorff E.1, Saussele S.2, Gattermann N.3, Le Coutre P.4, Illmer T.5, Brümmendorf T.H.6, Giles F.7, Hochhaus A.1

1Klinik für Innere Medizin II, Universitätsklinikum, Jena, Germany, 2III. Medizinische Klinik, Universitätsmedizin, Mannheim, Germany, 3Klinik für Hämatologie, Onkologie und Klinische Immunologie, Universitätsklinikum, Düsseldorf, Germany, 4Medizinische Klinik m.S. Hämatologie und Onkologie, Charité, Berlin, Germany, 5BAG Freiberg-Richter, Jacobasch, Illmer, Wolf, Dresden, Germany, 6Klinik für Onkologie, Hämatologie und Stammzelltransplantation, Universitätsklinikum, Aachen, Germany, 7Division of Hematology/Oncology Northwestern University, Chicago, United States

Introduction: Tyrosine kinase inhibitors (TKI) are the standard of care for patients (pts) with chronic myeloid leukemia in chronic phase (CP-CML). Although TKI therapy may result in a stable deep molecular response (DMR) in a significant proportion of pts, CML management guidelines recommend indefinite continuation of TKI therapy. According to current opinion, pts are eligible for TFR studies after >3 y of TKI therapy including at least one y of stable DMR (MR4.5, BCR-ABL =0.0032% on the International Scale [IS]) and known BCR-ABL transcript type. Within ENESTnd (NCT00471497), these crucial, but not exclusive prerequisites were met in 37.9 vs 21.6% of pts after nilotinib (NIL) 2*300 mg/d vs imatinib 400 mg/d therapy, respectively. In ENEST1st (NCT01061177), 38.6% of pts achieved MR4.5 after 24 mo of NIL 2*300 mg/d therapy.

Methods: The ENESTfreedom (NCT01784068) study prospectively analyzed the stability of TFR in CP-CML pts after 1st line NIL therapy. Eligible pts had typical b2/3a2 BCR-ABL transcripts, =2 y frontline NIL and had achieved MR4.5. On study, pts continued NIL for 1 y; pts meeting predefined response criteria during the consolidation phase were eligible to stop NIL (TFR phase). NIL reinitiation was triggered by loss of major MR (MMR [BCR-ABLIS =0.1%]).

Results: 215 pts (50 pts from 27 sites in Germany and Austria) were enrolled and entered the consolidation phase; 190 of these pts stopped NIL. Median age was 55 y, and median NIL duration prior to TFR was 43 mo (range, 33-89 mo). At wk 48 of the TFR phase, 51.6% (95% CI, 44.2-58.9%) of 190 pts remained in MMR, and 47.4% (95% CI, 40.1-54.7%) were in MR4.5 without reinitiation of treatment. A total of 86 pts lost MMR; 85 (98.8%) and 76 (88.4%) pts regained MMR and MR4.5 after NIL reinitiation, respectively. After NIL discontinuation, 47 pts (24.7%) reported musculoskeletal pain.

Conclusions: Rate of DMR is significantly improved with 1st line NIL vs imatinib therapy. In ENESTfreedom, >50% of pts remained in TFR 48 wk after stopping NIL, a clinically meaningful rate for pts with a relatively short duration of NIL therapy (median 3.6 y). To test the impact of immune response for TFR, the ongoing TIGER trial (NCT01657604) investigates (i) the rate of molecular response after NIL vs NIL+PEG-Interferon (IFN) therapy and (ii) the stability of DMR after NIL vs NIL+PEG-IFN and PEG-IFN maintenance therapy. So far, 530 pts have been recruited from 123 sites in Germany, Switzerland and the Czech Republic.

Disclosure: Ekkehard Eigendorff: Financing of Scientific Research: Novartis, BMS, Pfizer, AriadAndreas Hochhaus: Financing of Scientific Research: Novartis, BMS, Pfizer, Ariad; Expert Testimony: Novartis, BMS, Pfizer, Ariad

V154 – Treatment free remission (TFR) in CML patients after 2nd line nilotinib (NI) therapy

Dengler J.1, Stegelmann F.2, Sauer A.3, Saußele S.4, le Coutre P.5

1Onkologische Schwerpunktpraxis Heilbronn, Heilbronn, Germany, 2Department of Internal Medicine III, University Hospital Ulm, Ulm, Germany, 3Medizinisches Versorgungszentrum für Blut- und Krebserkrankungen, Potsdam, Germany, 4II. Medizinische Klinik, Universitätsmedizin, Medizinische Fakultät Mannheim der Universität Heidelberg, Mannheim, Germany, 5Medizinische Klinik mit Schwerpunkt Onkologie und Hämatologie, Campus Charité Mitte, Charité – Universitätsmedizin Berlin, Berlin, Germany

Introduction: Achievement of a sustained deep molecular response (DMR) is a key prerequisite for successful TFR following frontline tyrosine kinase inhibitor (TKI) therapy; however, limited data are available on TFR following 2nd-line TKI therapy. The single-arm phase 2 study ENESTop explores the discontinuation of NI therapy in pts who achieved sustained MR4.5 (BCR-ABL1 = 0.0032% on international scale IS) after switching from imatinib (IM) to NI. To estimate the proportion of pts who may be eligible to attempt TFR following 2nd-line NI, recently shown 4-y data from ENESTcmr has been considered.

Methods: In ENESTop CP-CML pts were enrolled with previous TKI therapy for = 3y (including IM for > 4 wk followed by NIL for = 2 y) and achieved MR4.5 on NI. On study, pts continued NI for 1y (consolidation phase CONS). After 1y, pts without confirmed loss of MR4.5 were eligible to stop NI. Primary endpoint was the proportion of pts with successful TFR (neither loss of MMR nor confirmed loss of MR4by 48 wk of TFR) after = 48wk follow up.

Results: In ENESTop, 126 of 163 pts (13 pts contributed from Germany) in CONS entered TFR (median duration of TKI prior to TFR, 87.7mo; median duration of NI, 53mo). At data cut-off 57.9% remained in TFR at 48 wk. During TFR, 18 pts had confirmed loss of MR4, 34 lost MMR. Of the 51 pts who re-initiated NI, 98% regained at least MMR by data cut-off, and 94.1% and 92.2% regained MR4 and MR4.5, respectively. Median time to regain MR4/MR4.5 was 12/13.1 wk. No new safety findings were observed on treatment.

Conclusion: ENESTop provides to date the largest prospective TFR data set in pts who achieved a sustained DMR after switching from IM to NI. Transfer from ENESTcmr to ENESTop is limited due to limited follow-up in ENESTcmr study. Yet overall, these results suggest that for pts lacking DMR on frontline IM, switching to 2nd-line NI may provide a route to TFR eligibility after shorter treatment duration.

Disclosure: Jolanta Dengler: Employment or Leadership Position: Selbstständige Ärztin; Financing of Scientific Research: NovartisPhilipp le Coutre: Employment or Leadership Position: Angestellter Arzt; Financing of Scientific Research: Novartis, BMS, Ariad, Pfizer

V155 – Treatment optimization of newly diagnosed BCR-ABL positive patients with chronic myeloid leukemia (CML) in chronic phase with nilotinib vs. nilotinib plus interferon a induction and nilotinib or interferon a maintenance therapy: Interim analysis of the TIGER (CML V) study

Hochhaus A.1, Saussele S.2, Baerlocher G.M.3, Brümmendorf T.H.4, Burchert A.5, Eigendorff E.1, La Rosée P.6, Hasford J.7, Hehlmann R.2, Heim D.8, Krause S.W.9, Le Coutre P.10, Niederwieser D.11, Lange T.12, Schenk T.1, Fabisch C.1, Pfirrmann M.7, Ostdeutsche Studiengruppe Hämatologie und Onkologie (OSHO), Schweizerische Arbeitsgemeinschaft für Klinische Krebsforschung (SAKK), Deutsche CML-Studiengruppe

1Klinik für Innere Medizin II, Universitätsklinikum, Jena, Germany, 2III. Medizinische Klinik, Universitätsmedizin, Mannheim, Germany, 3Universitätsklinik für Hämatologie und Hämatologisches Zentrallabor, Inselspital, Bern, Switzerland, 4Klinik für Onkologie, Hämatologie und Stammzelltransplantation, Universitätsklinikum, Aachen, Germany, 5Klinik für Hämatologie, Onkologie und Immunologie Universitätsklinikum Gießen und Marburg GmbH, Marburg, Germany, 6Klinik für Innere Medizin II, Schwarzwald-Baar Klinikum, Villingen-Schwenningen, Germany, 7Institut für Med. Informationsverarbeitung, Biometrie und Epidemiologie, Ludwig Maximilians-Universität, München, Germany, 8Klinik für Hämatologie, Universitätsspital, Basel, Switzerland, 9Medizinische Klinik 5 - Hämatologie und Internist. Onkologie Universitätsklinikum, Erlangen, Germany, 10Medizinische Klinik m.S. Hämatologie und Onkologie, Charité, Berlin, Germany, 11Abt. Hämatologie/Onkologie, Universitätsklinikum, Leipzig, Germany, 12Klinik für Hämatologie und internistische Onkologie, Asklepios-Klinikum, Weissenfels, Germany

Introduction: The TIGER study (NCT01657604) is a multicenter, randomized phase III trial to evaluate efficacy and tolerability of nilotinib (NIL) 2*300mg/d monotherapy vs NIL 2*300mg/d + pegylated interferon a2b (Peg-IFN) and the option to discontinue therapy after Peg-IFN maintenance. A pilot phase preceded the main phase to evaluate the tolerability of NIL 2*300mg/d combined with PEG-IFN (30-50µg/week) commenced after >6 weeks NIL monotherapy. Here, data from the 1st interim analysis according to the protocol are presented.

Methods: Between 8/2012 and 1/2013, 25 patients (pts) were recruited for the pilot phase. During the main phase of the study, newly diagnosed pts are being randomized between NIL 2*300 mg/d and NIL/PEG-IFN combination as confirmed during the pilot phase. An interim analysis was performed for the initial 200 pts with a minimum of 6 mo therapy. Efficacy and safety data are presented without specification of the randomized treatment arm due to ongoing recruitment.

Results: As of 4/2016, a total of 511 pts (300 male; median age 53, range 18-85 years; 13% EUTOS high risk) were recruited from 123 sites in Germany, Switzerland, and the Czech Republic. The pilot phase of 25 pts confirmed the feasibility of the combination of NIL 2*300mg/d and PEG-IFN 30µg/week. With regard to efficacy, 143/183 pts (73%) reached MMR (BCR-ABL1 transcripts < 0.1% according to the international scale) at 12 mo, 141/158 pts (89%) at 18 mo, and 85/89 pts (96%) at 24 mo. 63 pts concluded the induction phase and reached the maintenance phase of the study. Probabilities of adverse events grade 3-5 after 6 mo. of therapy are 21 and 26% for the two treatment arms. Five pts progressed to accelerated phase or blast crisis; one of them died from blast crisis, 2 received an allogeneic stem cell transplantation. Of 4 death total, 2 were CML related, one resulted from vascular complications, and one from an accident.

Conclusions: Our data demonstrate feasibility of 1st-line treatment with NIL 2*300 mg/d combined with PEG-IFN 30-50 µg/week commenced after >6 weeks of NIL therapy. Molecular response during the first 24 mo compares favorably with data from recent studies and allows access to the maintenance phase (NIL vs PEG-IFN monotherapy) for the majority of patients. Randomization will be continued until the protocol defined recruitment goal (n = 652) will have been reached.

Disclosure: Andreas Hochhaus: Financing of Scientific Research: Novartis, BMS, Pfizer, Ariad; Expert Testimony: Novartis, BMS, Pfizer, AriadMarkus Pfirrmann: No conflict of interest disclosed.

V156 – Expression of the CTLA-4 ligand CD86 (B7.2) on plasmacytoid dendritic cells (pDC) predicts risk of disease recurrence after treatment discontinuation in chronic myeloid leukemia

Burchert A.1, Inselmann S.1, Saußele S.2, Dietz C.T.2, Müller M.C.2, Eigendorff E.3, Brendel C.A.1, Metzelder S.K.1, Brümmendorf T.H.4, Waller C.5, Dengler J.6, Goebeler M.E.7, Herbst R.8, Freunek G.9, Stefan H.10, Illmer T.11, Wang Y.1, Lange T.12, Finkernagel F.13, Hehlmann R.2, Huber M.14, Neubauer A.1, Hochhaus A.3, Guilhot J.15, Mahon F.X.16, Pfirrmann M.17, Schütz C.1, CML-Studiengruppe

1Universitätsklinikum Marburg, Marburg, Germany, 2Universität Heidelberg, Universitätsmedizin Mannheim, Mannheim, Germany, 3Universitätsklinikum Jena, Jena, Germany, 4Universitätsklinik der RWTH Aachen, Aachen, Germany, 5Universitätsklinik Freiburg, Freiburg, Germany, 6Onkologische Praxis Heilbronn, Heilbronn, Germany, 7University Hospital Würzburg, Würzburg, Germany, 8Klinikum Chemnitz, Chemnitz, Germany, 9MVZ Klinikum Straubing GmbH, Straubing, Germany, 10Klinikum Kempten-Oberallgäu GmbH, Kempten, Germany, 11Gemeinschaftspraxis für Hämatologie und Onkologie, Dresden, Germany, 12Asklepios Klinikum Weißenfels, Weißenfels, Germany, 13Zentrum für Tumor- und Immunbiologie, Marburg, Germany, 14Biomedical Research Center (BMFZ), Marburg, Germany, 15Clinical Investigation Center, Poitiers, France, 16Hematology Laboratory, CHU Bordeaux, Bordeaux, France, 17IMBE, LMU München, München, Germany

Introduction: It is unclear, why only a minority of chronic myeloid leukaemia (CML) patients eventually sustains molecular remission after discontinuation of tyrosine kinase inhibitor (TKI) therapy in deep molecular remission. Within the EURO-SKI TKI discontinuation study (EUDRACT 2011-000440-22) we studied expression of the CTLA-4 immune checkpoint ligand and co-stimulatory molecule CD86 (B7.2) on BDCA2+/CD123+ plasmacytoid dendritic cells (pDC) using flow cytometry. We asked, whether CD86 expression on these specialized, interferon producing dendritic cells governs relapse risk after TKI stop.

Methods: Peripheral blood samples of 122 patients were analysed before TKI treatment discontinuation. The expression of CD86 on BDCA2+/CD123+ pDC and PD-1 on CD8+ T-cells, and CD8+ proteinase-3 specific T-cells was studied. Molecular relapse in this study was defined as loss of major moleuclar remission (loss of MMR).

Results: The 1-year molecular relapse-free survival (RFS) was 30.1% (95% confidence interval [CI] 15.6-47.9) for patients with > 95 CD86+pDC / 105 lymphocytes prior to TKI stop versus 70.0% (95% CI 59.3-78.3) for patients with < 95 CD86+pDC (hazard ratio [HR] 3.4, 95%-CI: 1.9-6.0; p < 0.0001). The RFS for patients with < 95 CD86+pDC was significantly better in case of > 8 years TKI exposure before discontinuation (HR 0.3, 95% CI 0.1-0.8; p = 0.0263), whereas there was no apparent benefit from longer TKI pre-treatment with > 95 CD86+pDC. High CD86+pDC counts significantly correlated with leukaemia specific T-cell exhaustion (Spearman correlation: 0.74, 95%-CI: 0.21-0.92; p = 0.0098).

Conclusion: CD86+pDC counts predict relapse risk of CML patients discontinuing a TKI in deep molecular remission. Only patients with low CD86+pDC counts seem to benefit from long TKI treatment duration before TKI cessation. Blocking CTLA-4 and PD-1 using immune checkpoint inhibitors could be a new treatment strategy to improve RFS in CML.

Disclosure: Andreas Burchert: Financing of Scientific Research: BMS, PfizerChristin Schütz: No conflict of interest disclosed.

V157 – Deregulated CD62L expression predicts molecular response to nilotinib therapy in early chronic phase Chronic Myelogenous Leukemia (CML-CP)

Wolf D.1, Mustjoki S.2, Gjertsen B.-T.3, Gastl G.4, Baldauf M.5, Trajanoski Z.5, Giles F.6, Hochhaus A.7, Ernst T.7, Schenk T.7, Janssen J.8, Porkka K.9, Sopper S.4

1Universitätsklinikum Bonn, Bonn, Germany, 2Hematology Research Unit Helsinki, University of Helsinki and Department of Hematology, Helsinki University Hospital Comprehensive Cancer Center, Helsinki, Finland, 3Institute of Medicine, Hematology Section, University of Bergen, Bergen, Norway, 4Department of Hematology and Oncology, Innsbruck Medical University, Innsbruck, Austria, 5Divison for Bioinformatics, Biocenter, Innsbruck Medical University, Innsbruck, Austria, 6NMDTI, Northwestern University, Chicago, United States, 7Klinik für Innere Medizin II, Universitätsklinikum Jena, Jena, Germany, 8Department of Hematology, VU University Medical Center, Amsterdam, Netherlands, 9Hematology Research Unit Helsinki, University of Helsinki and Department of Hematology, Helsinki, Finland

Introduction: Immunological surveillance of minimal residual disease (MRD) in chronic myelogenous leukemia (CML) may relevant for long-term control or cure of the disease. Little is known about immunomodulatory effects of nilotinib in vivo potentially predicting response to therapy.

Methods: A prospective and comprehensive flow cytometry-based immunomonitoring program paralleled the ENEST1st clinical study (NCT01061177), investigating 52 nilotinib-naïve CP-CML patients and after treatment with 300 mg BID nilotinib at months 6 and 12. Molecular data were analyzed in central EUTOS reference laboratories. The data were verified in an independent validation cohort (n = 21) from the CML IV TIGER trial.

Results: T cells of CML patients at diagnosis displayed an aberrant phenotype characterized by very low L-selectin (CD62L) expression levels, which was not due to proportional shifts from CD62Lhigh to CD62Llow or negative T cell subsets. At diagnosis, low numbers of CD62L-expressing CD4+ and CD8+ T cells highly correlated with important baseline disease characteristics, such as higher SOKAL score, increased spleen size and high leukocyte as well as peripheral blood blast counts. Six months after nilotinib-therapy initiation, expression of CD62L returned back to levels seen in healthy individuals. Of note, the amount of reduced CD62L expression on T cells directly correlated with the extent of soluble CD62L (sCD62L) elevation in the plasma of diagnostic CML patients, which returned back to normal levels at months 6 and 12 during CML therapy. In parallel to the increase of sCD62L at diagnosis, the proteolytic activity but not the total amount of TACE (ADAM17, CD156b), the metalloproteinase known to shed CD62L is clearly increased at diagnosis and significantly decreased during nilotinib therapy. High CD62L expression on both CD4+ and CD8+ T cells and vice versa low sCD62L levels at CML diagnosis were linked to superior molecular responses (MMR, MR4 and MR4.5). These findings were corroborated in the independent validation cohort from the german CML IV TIGER trial.

Conclusions: We demonstrate for the first time the prognostic impact of increased CD62L shedding from T cells at CML diagnosis for later molecular response to nilotinib in early CML-CP. Decreased CD62L is most likely mediated by increased TACE-activity. This may impair T cell-mediated leukemia control, as CD62L is important for T cell entry to secondary lymphoid organs.

Disclosure: Dominik Wolf: Advisory Role: Novartis; Financing of Scientific Research: Novartis; Expert Testimony: NovartisSieghart Sopper: Expert Testimony: Novartis

V158 – The predictive value of early molecular response by relative risk in patients (pts) with chronic myeloid leukemia in chronic phase (CML-CP) treated with dasatinib (DAS) or imatinib (IM) from the phase 3 DASISION trial

Stegelmann F.1, Shah N.P.2, Saglio G.3, Hochhaus A.4, Bilmes R.5, Li L.5, Cortes J.E.6

1Universitätsklinikum Ulm, Ulm, Germany, 2UCSF School of Medicine, San Francisco, United States, 3University of Turin, Turin, Italy, 4Universitätsklinikum Jena, Jena, Germany, 5Bristol-Myers Squibb, Princeton, United States, 6University of Texas M.D. Anderson Cancer Center, Houston, United States

Introduction: Improved outcomes were observed in treatment-naive CML-CP pts treated with DAS or IM who achieved the early molecular milestone of BCR-ABL1 =10% at 3 mo in an exploratory landmark analysis of the phase 3 DASISION trial, and BCR-ABL1 =10% at 3 mo was achieved by more pts on DAS (DAS: 84%, IM: 64%). We therefore evaluated the impact of EURO (Hasford) risk score on the achievement of this predictive molecular milestone and investigated other predictive factors of survival.

Methods: In DASISION, newly diagnosed CML-CP pts were randomized to receive DAS 100 mg once daily (n = 259) or IM 400 mg once daily (n = 260). BCR-ABL1 transcript levels were measured on the International Scale. EURO score =780 is low, >780 to =1480 is intermediate, and >1480 is high risk.

Results: Based on EURO score, 33% of pts in both treatment arms were at low risk, 48% and 47% of pts receiving DAS and IM, respectively, were at intermediate risk, and 19% in both arms were at high risk. Across all risk groups, more pts on DAS had BCR-ABL1 =10% at 3 and 6 mo vs IM (3 mo: low 91% vs 73%, intermediate 80% vs 66%, high 83% vs 44%; 6 mo: low 99% vs 89%, intermediate 84% vs 82%, high 84% vs 71%). More pts on DAS vs IM also had BCR-ABL1 =1% at 3 mo (low 56% vs 20%, intermediate 48% vs 12%, high 33% vs 5%) and at 6 mo (low 79% vs 58%, intermediate 66% vs 50%, high 60% vs 29%). Five-y molecular responses and outcomes were higher for pts with BCR-ABL1 =10% vs >10% at 3 mo in all risk groups (Table), as were 5-y response and outcomes for pts with BCR-ABL1 =10% vs >10% at 6 mo in all risk groups. Multivariate analysis of survival at 5 y showed women on DAS were at a lower risk of death than men; there was no association between EURO score or age and survival.

Conclusions: More pts achieved BCR-ABL1 =10% at 3 and 6 mo on DAS vs IM regardless of EURO risk score, with a significantly higher proportion of pts on DAS with BCR-ABL1 =1%. The largest difference in BCR-ABL1 levels between the treatment arms was observed in those at high risk. BCR-ABL1 =10% vs >10% at 3 and 6 mo was predictive of improved long-term outcomes in all risk groups.


Disclosure: Frank Stegelmann: Advisory Role: ARIAD, PFIZER; Honoraria: NON; Financing of Scientific Research: ARIAD, BMS, NOVARTIS, PFIZER; Other Financial Relationships: BMS, NOVARTIS (travel expenses for scientific meetings)Jorge E. Cortes: Advisory Role: Ariad, BMS, Novartis, Pfizer; Expert Testimony: Ariad, BMS, Novartis, Pfizer, Teva

Wissenschaftliches Symposium – Neue Optionen beim Hodgkin-Lymphom

V160 – PET guided therapy of Hodgkin Lymphoma

Borchmann P.1

1Uniklinik Köln, Klinik I für Innere Medizin, Köln, Germany

Treatment with current radio- and chemotherapy regimens cures the majority of patients suffering from Hodgkin lymphoma (HL). Treatment allocation is based on the Ann-Arbor system, which mainly reflects the burden of disease. Within the three risk groups (early, intermediate, and advanced stage HL), the individual HL patients still may have very different risk profiles. Reliable predictive markers at baseline are not available. Therefore, current standard therapies such as ABVD (doxorubicin, bleomycin, vinblastine, and dacarbazine), or BEACOPP (bleomycin, etoposide, doxorubicin, cyclophosphamide, vincristine, procarbazine, prednisone) are administered to all patients with newly diagnosed HL without knowing the individual risk for treatment failure. Consequently, over- and undertreatment may occur and may result in either excessive toxicity or preventable relapse. To overcome these limitations regarding the individual upfront risk assessment, the concept of response-adapted therapy has been developed. According to this concept, treatment intensity is either de-escalated or intensified depending on the observed early response to therapy. This more individualized treatment approach has become possible by the introduction of functional imaging using 18F-fluoro-2-deoxy-D-glucose positron emission tomography (18FDG PET) for response assessment. Determining the metabolic response to therapy seems to generate more prognostic information than the radiological assessment alone.

This concept has been tested in all different stages of HL in large phase III studies. Results have become available in the last few years and will be summarized.

Disclosure: No conflict of interest disclosed.

Fortbildung – Standardisierte Leukämie-Diagnostik

V168 – Next-generation sequencing

Haferlach T.1

1MLL Munich Leukemia Laboratory, München, Germany

The recent ten years demonstrated an outstanding success for molecular approaches in hematology: This is true for research discoveries, to define markers at diagnosis, for prognostication, for follow-up studies including minimal residual disease and also for target detection. This has been possible due to ground breaking developments in techniques such as NGS, instruments, automation and also in software tools, to make data appropriate for science as well as for routine diagnostic purposes.

Several techniques have been introduced to the market, some only for research, some also with FDA or CE market labels, accredited for routine diagnostic purposes. Allover, the turnaround time decreased, the specificity and the sensitivity increased, assays included the investigation of single genes, gene panels, but also whole exome sequencing and whole genome sequencing is now possible. In parallel, costs are going down, making it even possible to implement next generation sequencing not only for highly skilled research institutes but also for routine laboratory investigations. Although the recent technological opportunities with respect to assay development, data read-out and turnaround time is impressive, the most challenging field so far is to interpret the data correctly for any research project but especially for report generation in patients care. Several international available databases in the www have to be addressed in parallel to interpret the respective findings on patients DNA or RNA. In parallel, an in-house database should be developed and curated. As the recent opportunities also lead to findings that could not be clearly interpreted as being damaging (malignant) or benign but maybe so-called a “variant of unknown significance” (VUS), the management of data today is more complex than the technique NGS itself. Several important approaches, also guided by the FDA and the ASH including the task force for precision medicine are working in the field of standardization, accreditation and data management in the upcoming era of NGS driven molecular diagnostics. However, the options are opening a new field not only to understand the pathobiology of hematology but also to foster diagnostic approaches and lead more and more directly to targeted treatment.

Disclosure: Torsten Haferlach: Employment or Leadership Position: MLL Muich Leukemia Laboratory

V169 – Development and application of WHO standards for standardization of molecular monitoring in CML

Cross N.C.P.1

1University of Southampton, Salisbury, United Kingdom

Serial quantification of BCR-ABL1 mRNA levels is an important therapeutic indicator for patients with chronic myeloid leukaemia, but historically there has been substantial variation in results reported by different laboratories. To help improve the comparability of results, an International Scale (IS) for BCR-ABL1 has been developed and implemented by many testing laboratories worldwide, initially by the derivation of laboratory-specific conversion factors (CFs) following sample exchange with a reference laboratory. Whilst the development of CFs was a major step forward and provided an important proof of principle, it is obvious that this approach is not sustainable in the long term. Ideally, any testing laboratory should be able to access reference standards or use a kit that enables them to convert patient results directly to the IS. The development of standards and kits initially required the development of a process by which these tools could be calibrated to the IS. An important milestone in this process was the establishment in 2010 of the 1st World Health Organization International Genetic Reference Panel for quantitation of BCR-ABL1 mRNA. The reference panel comprises four different dilution levels of freeze dried preparations of K562 cells diluted in HL60 cells that were assigned fixed % BCR-ABL1/reference gene values on the IS following an international calibration process. Due to the scale of molecular monitoring, it was not physically possible to manufacture and validate a sufficiently large quantity of reference material to satisfy worldwide demand and thus the principal function of these primary reagents was limited to the calibration of secondary reference reagents. These secondary reference reagents may be manufactured and calibrated by companies, reference laboratories or other agencies and made available to testing laboratories either on a commercial basis or as part of specific national or regional standardization initiatives. Several different kits, systems and secondary reagents are available that enable testing laboratories to derive patient results on the IS. Comparative data is, however, very limited at present and it is not possible to say which of these approaches is best. It is clear, however, that use of an IS kit does not guarantee accurate results and additional quality control measures are required.

Disclosure: Nicholas Cross: Financing of Scientific Research: Novartis; Expert Testimony: Novartis

Freier Vortrag – Versorgungsforschung

V170 – Use of local oncological Standard Operating Procedures (SOPs) and oncological guidelines: A survey of 1600 physicians at 4 oncology sites/centers of excellence

Krebs S.1, Starbatty B.1, Skoetz N.1, Schmidt-Wolf I.2, Brandts C.3, Bischoff M.4, Wolf J.1, Glossmann J.P.1, Centers of excellence working group SOP

1Universitätsklinikum Köln, Centrum für Integrierte Onkologie (CIO) Köln Bonn, Köln, Germany, 2Universitätsklinikum Bonn, Centrum für Integrierte Onkologie (CIO) Köln Bonn, Bonn, Germany, 3Universitätsklinikum Frankfurt, Universitäres Centrum für Tumorerkrankungen (UCT), Frankfurt am Main, Germany, 4Universitätsklinikum Freiburg, Department für Medizinische Biometrie und Medizinische Informatik, Freiburg i. Br., Germany

Introduction: All centers of excellence in Germany are required to develop and provide standards for cancer care and treatment. The center of excellence network, funded by the German Cancer Aid, established a working group for SOPs. Objectives of this group are to analyze SOPs in the centers to standardize content and structure of SOPs, to develop new SOPs for rare cancers and to give impetus to the evidence based national S3-Guidelines. While guidelines of the medical societies are nationally important and build the base for SOPs, the SOPs also implement local specific conditions and circumstances of the center.

Methods: We developed a survey for physicians practicing in the centers of excellence to assess knowledge and usage of SOPs. A pretest of the survey was conducted, involving ten physicians. The final version was approved by the Employee Committees, Data Protection Supervisors and ethics committees. The survey was web-based and anonymous and conducted in the centers Cologne, Bonn, Frankfurt and Freiburg. We assessed baseline characteristics as well as questions related to local web-based SOPs and national S3-guidelines. Only datasets with at least 30% of the survey completed were evaluated.

Results: 1669 doctors were invited. 1175 responsed and 424 responses reached the =30% completion limit. 60% were male, 40% female. 6% are chief physicians, 33% consultant physicians, 18% board certification and 43% assistant physicians.


Conclusion: 64% of physicians are familiar with the SOPs. Rating of the topics importance is similar for SOPs and guidelines. Therapeutic aspects are the most important information. According to frequency of usage, oncological guidelines seem to be used more regulary compared to SOPs.

Disclosure: No conflict of interest disclosed.

V171 – Systemic palliative treatment in patients with metastatic colorectal cancer tested for their (K)RAS mutation status – Real-life data from the clinical Tumour Registry Colorectal Cancer (TKK)

Zahn M.O.1, Schnell R.2, Karcher A.3, Schnitzler M.4, Wetzel N.4, Fleitz A.4, Marschner N.5, for the TKK registry group

1Studienzentrum MVZ Onkologische Kooperation Harz, Goslar, Germany, 2Praxis Internistischer Onkologie und Hämatologie (PIOH), Frechen, Germany, 3Onkologische Schwerpunktpraxis, Heidelberg, Germany, 4iOMEDICO, Freiburg, Germany, 5Praxis für Interdisziplinäre Onkologie & Hämatologie, Freiburg, Germany

Background: RAS has been identified as a predictive biomarker for treatment response and outcome in patients (pts) with metastatic colorectal cancer (mCRC). Pts with RAS mutation (mt) do not benefit from treatment with EGFR-inhibitors. Since 2008 KRAS mutation testing was mandatory before applying the EGFR-inhibitors cetuximab (CET) and panitumumab (PAN). Since 2014 expanded RAS mutation testing is required. Pts with RAS mt can be treated with chemotherapy (CT), with or without the VEGF-inhibitor bevacizumab (BEV). A VEGF- or EGFR-inhibitor in combination with CT or as single agent is recommended for RAS wild-type (wt) pts. Data from recent studies indicate a better survival of pts with RAS wt treated with a regimen with an EGFR-inhibitor in 1st-line and a regimen with a VEGF-inhibitor in 2nd-line.

Methods: Since 2006, the Tumour Registry Colorectal Cancer (TKK) prospectively documents treatment of pts with mCRC by medical oncologists in Germany. Pts receive treatments according to physician’s choice. The TKK collects data on all systemic treatments, biomarker testing, pts and tumour characteristics and outcome. By March 2015, 166 sites recruited 3500 mCRC pts. Data on mutation status, referred to as (K)RAS mutation status, has been collected for KRAS since 2008 (n = 2264) and for RAS since 2014 (n = 226).

Results: The number of pts tested for KRAS increased from 52% in 2008 to 72% in 2013. In 2015 79% of pts have been tested for extended RAS mt status. 66% of pts were tested with KRAS wt until 2013. With expanded RAS mutation testing, this number decreased to 48% of pts tested with RAS wt in 2014–15. In pts with (K)RAS wt the most frequently applied 1st-line regimens from 2010–2013 were FOLFIRI or FOLFOX, with or without BEV or CET. The proportion of pts treated with CET was 33%. In 2014–15 the use of a regimen with CET or PAN increased to 48% with FOLFIRI+CET and FOLFOX+PAN as the two most frequently applied regimens. In 2nd-line treatment of (K)RAS wt pts the use of FOLFIRI+CET decreased from 2010–2015, while treatment with FOLFIRI+BEV increased and is the most frequently applied regimen in 2014/15. In pts with (K)RAS mt the most frequently used regimens were FOLFIRI+BEV, FOLFOX+BEV, FOLFOX and FOLFIRI in 1st- and 2nd-line.

Conclusions: In 2014/15 the majority of pts were tested for RAS mutation status before the start of 1st-line therapy. Upcoming analyses will show how recent study results on treatment with EGFR-VEGF sequence will influence routine treatment.

Disclosure: No conflict of interest disclosed.

V172 – Oncologists´ consultation: Attitudes of migrant patients

Riese C.1, Borges jr. U.1, Mödder M.1, Baumann W.1

1Wissenschaftliches Institut der Niedergelassenen Hämatologen und Onkologen – WINHO, Köln, Germany

Background: Since one in five residents in Germany have a migration background, we focused on the implications of language barriers and cultural differences for the patients’ comprehension of treatment and on their attitudes concerning cancer care. Differences between patients with and without migration experience should be considered.

Methods: The WINHO patient survey is held annually and serves as benchmarking for the participating office-based oncology practices as well as baseline study for specific topics. In 2015, the participants were surveyed in: a) perception of the practice structure and conversation with the doctor, b) communication and attitudes, c) sociodemographic questions. The answers of patients with migration experience were compared to those born in Germany. Group differences were tested for significance via ?²-test.

Results: 9540 patients participated (return rate: 84%; 53.3% female, M = 64.93 years, SD = 13.2). 858 (9%) were not born in Germany. Migrants with a lower command of the German language more often reported the impression that crucial parts of the conversation with the oncologist remained incomprehensible (27.7%) than migrants with good knowledge of German (9.2%) and native German speakers (5.8%), p < .001. The understanding of written information relevant for treatment provided by practices was more often difficult for migrants with less knowledge of German than for those with good knowledge and for native speakers (respectively 10.4%, 4.1% and 2%), p < .001. Regarding expectations of treatment, foreign patients in general have a lower tendency to a) tell doctors about their needs and concerns regarding their treatment (89.3% compared to 94.2% of patients born in Germany), p < .01, b) tell their opinion on the intentions of the doctor (81.4% to 87.3%), p < .01, and c) desire to participate in crucial decisions regarding diagnosis and treatment (85.8% to 91.6%), p < .01.

Conclusions: Survey participants with migration experience, especially those with less language proficiency, stated more difficulties with understanding of both written information and the verbal explanation by the oncologist during a consultation. As foreign patients with different cultural backgrounds are a very heterogeneous group, such differences need to be taken into account in further analyses.

Disclosure: No conflict of interest disclosed.

V173 – Sensorimotor vs. endurance exercise in cancer survivors with chemotherapy-induced peripheral neuropathy

Müller J.1, Wehrle A.2, Kneis S.1, Gollhofer A.3, Bertz H.1

1Medical Center – University of Freiburg, Department Medicine I, Haematology, Oncology and Stem Cell Transplantation, Freiburg, Germany, 2Medical Center – University of Freiburg, Institute for Exercise- und Occupational Medicine, Center for Medicine, Freiburg, Germany, 3Department of Sport and Sport Science, Albert-Ludwigs-University Freiburg, Freiburg, Germany

Introduction: Symptoms of peripheral neuropathy often occur after treatment with neurotoxic chemotherapy, depending on agent and dose. Affected patients suffer from sensory and motor dysfunction resulting in limitations of their daily activities culminating in a necessary change of therapy regime. Up to now, there is no evident therapy to effectively reduce or prevent symptoms of chemotherapy-induced peripheral neuropathy (CIPN). Due to motor dysfunction in terms of balance problems, gait instability and risk of falling exercise seems to be a promising instrument to improve postural control in CIPN patients. Therefore, the aim of this study was to evaluate the relative benefit of sensorimotor exercise, which is known to improve neuromuscular function, versus endurance exercise, which is proven to reduce diabetes-induced neuropathic symptoms.

Methods: 50 cancer patients after active treatment with persisting symptoms of CIPN were randomly allocated either to a sensorimotor exercise group (SG) or an endurance exercise group (EG). Both groups exercised three times per weeks over 12 weeks and underwent pre- and post-assessments: objective clinical tests and subjective symptom questionnaires to determine the level of CIPN, balance tasks to evaluate motor dysfunction and a maximum cardiopulmonary exercise test.

Results: After intervention both groups reduced their subjective sensory symptoms significantly (SG: P = 0.028; EG: P = 0.037), while only SG reported also improved motor function (P = 0.006) measured by EORTC QLQ-CIPN20. Clinical tests revealed no significant changes. With increased difficulty of balance tasks, difference between groups appeared: SG reduced their sway path in semitandem stance significantly (P = 0.031) and improved their one leg stance time on instable surface (P = 001). Furthermore, cardiorespiratory fitness (VO2peak) descriptively increased in both groups, this change, however, did not reach statistical significance

Conclusion: All patients benefited from exercising. However, the study revealed no clear superiority of sensorimotor training in terms of influencing motor dysfunction as hypothesized. Nonetheless patients of SG showed more positive changes than patients of EG. We speculated that the averaged expression of CIPN in our cohort was too weak and could therefore not be influenced by our interventions due to a ceiling effect. Further studies need to differentiate between moderate and severe CIPN to ensure a higher reliability.

Grant: Janssen Cilag

Disclosure: No conflict of interest disclosed.

V174 – Overall survival of 4865 patients with metastatic solid tumours treated in German routine practice – combined results from four prospective, multicentre cohort studies

Marschner N.1, Potthoff K.2, Schnell R.3, Tesch H.4, Zahn M.-O.5, Hegewisch-Becker S.6, Jänicke M.2, iOM-Registry group

1Praxis für interdisziplinäre Onkologie & Hämatologie, Freiburg i.Br., Germany, 2iOMEDICO, Freiburg i.Br., Germany, 3Praxis Internistischer Onkologie und Hämatologie (PIOH), Frechen, Germany, 4Onkologische Gemeinschaftspraxis, Frankfurt a.M., Germany, 5Studienzentrum MVZ Onkologische Kooperation Harz, Goslar, Germany, 6Onkol. Schwerpunktpraxis HH Eppendorf, Hamburg, Germany

Introduction: Published data on overall survival (OS) of patients (pts) with metastatic solid tumours are mostly based on randomized clinical trials conducted at university hospitals. In Germany, cancer care is predominantly delivered by office-based medical oncologists and oncologists based in community hospitals. They mainly treat cancer pts with various comorbidities generally excluded from clinical trials. Prospectively collected data on OS in the routine clinical setting are rare.

Methods: Data source are prospective, multicentre, longitudinal cohort studies recruiting pts with metastatic colorectal (mCRC), breast (mBC), non-small cell lung (mNSCLC) or pancreatic (mPC) cancer. Pts were recruited at the start of first-line treatment by more than 250 study sites in Germany, mainly office-based medical oncologist practices and few community hospitals. Pts receive standard treatments according to physician’s choice. A broad set of data including pts and tumour characteristics, systemic treatments and outcome are collected. OS was estimated with the Kaplan-Meier method. Common clinical trial exclusion criteria were defined as ECOG=2 or presence of renal disease, moderate or severe liver disease, heart failure or brain metastases at the start of first-line treatment (“trial ineligible”).

Results: All pts started their first-line treatment between 2006 and 2015. Table 1 presents pts characteristics and OS. About 70–80% of pts had one or more relevant comorbidities, mainly hypertension. In mBC only 53–65% of pts had comorbidities. The percentage of pts who were trial ineligible ranges from 12% (HR/Her2 positive mBC) to 41% (mNSCLC). About 20% of pts with mCRC and mPC met at least one exclusion criterion of a clinical trial.


Conclusions: OS of pts with metastatic solid tumours in German routine clinical practice is similar to published data from randomized clinical trials although pts are older and most often affected with comorbidities.

Disclosure: No conflict of interest disclosed.

V175 – Recommendations for diagnosis, treatment and follow-up in oncology – an inventory of Standard Operating Procedures (SOPs) of centers of excellence

Glossmann J.-P.1, Starbatty B.2, Bischoff M.3, Follmann M.4, Skoetz N.2, Centers of excellence working group SOP

1Centrum für Integrierte Onkologie (CIO) KölnBonn, Köln, Germany, 2Centrum für Integrierte Onkologie (CIO) KölnBonn, Koordinationsstelle der AG SOP, Köln, Germany, 3Uniklinik Freiburg, Institut für Medizinische Biometrie und Informatik, Freiburg, Germany, 4Leitlinienprogramm Onkologie der AWMF, DKG und DKH, Berlin, Germany

Introduction: Currently 13 centers of excellence exist, all funded by the German Cancer Aid (DKH). It is mandatory for them to provide up-to-date standard operating procedures (SOPs). Usually, SOPs are developed in one center and consist of clinical pathways which reflect recommendations given by current evidence-based guidelines provided by the Guideline Program in Oncology (OL). In addition, latest research results from clinical trials and specific in-house features are integrated to adapt the recommendations on center-specific needs. If questions of interest may not be covered by guidelines, interdisciplinary experts from the center develop a new SOP.

Representatives from all centers, one representative from DKH and one from OL are part of the working group SOP to guarantee excellent communication between organizations and high-quality pathways in all centers in accordance with evidence-based guidelines. Moreover, the working group SOP reduces duplication of effort by developing public available network SOPs.

The objective of this project is to give an overview of all SOPs, harmonized methods and identified supply gaps.

Methods: Since 2012, each year all centers were asked to provide information, which SOPs in their center exist.

As the first survey in 2012 identified differences in the actuality, methods used to develop SOPs, members of the working group developed recommendations how to prepare, update and implement a SOP.

Results: In 2016, at least one SOP in the network exists for almost any kind of solid cancer (89 out of 90 ICD- codes), haematological malignancies and aspects of supportive care. This list of SOPs is available at www.ccc-netzwerk.de and interested centers or physicians may ask via der co-ordinating office (funded by the DKG, No. 111493) for access to these SOPs. The first network SOP thyroid cancer is currently in the final review process and will soon be freely available on this website. Moreover, recommendations how to prepare and update SOPs can be downloaded as a handbook, giving suggestions for harmonized methodology and evidence processing.

One topic identified so far, without any published guidelines and SOPs, is familial cancers. The working group SOP has already started to co-ordinate these SOPs.

Conclusions: SOPs are important for standardized diagnostic and treatment of patients and exist for almost all types of cancer in the network. Harmonized processes might increase the quality of cancer care throughout Germany.

Disclosure: Jan-Peter Glossmann: Employment or Leadership Position: Sprecher AG SOP des Netzwerks onkologischer SpitzenzentrenNicole Skoetz: Employment or Leadership Position: Wissenschaftliche Koordinatorin AG SOP des Netzwerks onkologischer Spitzenzentren

Freier Vortrag – Akute myeloische Leukämie – Therapie 1, Mutationsprofil

V178 – Response-adapted sequential Azacitidine and induction chemotherapy in patients > 60 years with newly diagnosed AML eligible for chemotherapy: Interim analysis of the DRKS00004519 study (RAS-AZIC)

Jäkel N.1, Hubert K.2, Krahl R.1, Hänel M.3, Maschmeyer G.4, Herbst R.3, Jacob C.4, Schulze S.1, Wang S.-Y.1, Cross M.1, Brosteanu O.5, Niederwieser D.1, Al-Ali H.K.1

1Universitätsklinik Leipzig AöR, Hämatologie/ internistische Onkologie, Leipzig, Germany, 2Medizinische Fakultät/ Universität Leipzig, Hämatologie/ internistische Onkologie, Leipzig, Germany, 3Klinik für Innere Medizin III/ Klinikum Chemnitz gGmbH, Hämatologie, Onkologie, Stammzelltransplantation, Chemnitz, Germany, 4Klinikum Ernst von Bergmann gGmbH, Klinik für Hämatologie, Onkologie und Palliativmedizin, Potsdam, Germany, 5Universität Leipzig AöR, Koordinierungszentrum für Klinische Studien Leipzig, Leipzig, Germany

Treatment for AML in patients (pts) >60 years (y) includes intensive chemotherapy (IC) and/or azacitidine (AZA). Yet, AZA and IC need not be mutually exclusive. In this multicenter study of the East German Study Group, first-line treatment with AZA followed by AZA or IC in pts >60 y was evaluated. Data of a planned interim analysis are presented.

Patients and methods: Patients >60y with newly diagnosed AML were included. Upfront AZA (75 mg/m2/day s.c.) for 7 days was followed by AZA or IC (mitoxantrone 10 mg/ m²/day on day (d)1–3 and cytarabine 1g/ m²/BID on d1, 3, 5, 7) based on d15 bone marrow (BM) blasts (< 45 versus =45%) and response on d56 previously identified as early predictors for response to AZA in AML. The primary endpoint was response (OR) [CR, CRi, PR] at d90 according to IWG criteria. Safety, OS, and TRM were secondary endpoints. On the basis of an optimal two-stage design, an expected OR of 61% at the end of the trial was estimated. Thus, if = 19 of the first 40 pts did not achieve the OR, protocol treatment would be considered inferior to standard IC. All pts gave written informed consent.

Results: The median age of the 40 pts was 70y (55% males). de novo AML was present in 63% of pts. Median BM blasts was 39%. Cytogenetics was high- in 29% and intermediate-risk in 71% of 38 pts. FLT3 and NPM1 were mutated in 11% and 17% of 36 pts respectively. All pts received upfront AZA. 37 (92.5%) pts received protocol assigned treatment based on BM blasts on d15 (54% continued with AZA; 46% received IC). Overall, in the 33 (82.5%) pts who continued therapy per protocol until d90, an OR was achieved in 27 (82%) pts [CR/CRi (n = 17/7); PR (n = 3)]. In the intention-to-treat cohort (n = 40), this represents an OR of 67.5% [CR/CRi (60%); PR (7.5%)] with AZA + one IC cycle (n = 28) or two IC cycles (n = 3). TRM on d30 and d90 was 0% and 5% respectively. After a median follow-up of 202 days, OS was 84.5%. OS for responding pts was 95.5%. Upfront AZA was well tolerated. Constipation grade 1+2 was the most frequently reported AE under AZA (45%). Overall, the most frequent grade 3+4 non-hematologic AE was infection [11 times in 31 IC-cycles (35%); 7 times in 60 AZA-cycles (12%)].

Conclusions: Response-based sequential epigenetic therapy and intensive chemotherapy is feasible in elderly pts with AML with a very low treatment-related mortality. The final results of the trial will scrutinize the impact of this approach on survival.

Disclosure: Nadja Jäkel: No conflict of interest disclosed.Haifa Al-Ali: Financing of Scientific Research: yes; Expert Testimony: yes

V179 – Only SETBP1 hotspot mutations in myeloid neoplasm are associated with refractory disease and reduced overall survival- a single center analysis of 442 patients

Winkelmann N.1, Schäfer V.1, Reichert J.1, Waldau A.1, Rinke J.1, Hochhaus A.1, Ernst T.1

1Klinik für Innere Medizin II, Universitätsklinikum Jena, Hämatologie und internistische Onkologie, Jena, Germany

Introduction: SKI-homology domain aberrations of SETBP1 have been characterized as a diagnostic biomarker in myeloid neoplasm and are associated with inferior survival. Since there is limited data on the clinical impact of these aberrations and their stability during disease progression, we aimed to follow up and study affected patients from our institution.

Methods: Bidirectional Sanger sequencing of gDNA from 442 unselected patients with WHO-defined myeloid disease was performed at diagnosis or first encounter (AML; n = 61, sAML; n = 117, MDS; n = 98, MPN; n = 111, MDS/MPN; n = 43, aplastic anemia; n = 12). Secondly, we conducted a follow up analysis on 190 samples from 123 patients at a median of 8 months (range: 1–62 months) from first screening.

Results: Ten patients (2.3%) had aberrations in the highly conserved 11-nucleotide region of codons 868 to 871 (MDS/MPN, n = 6, sAML, n = 3, AML, n = 1). Germline material was available in 5/10 patients and confirmed the somatic origin of mutations. Five patients (1.1%) showed SETBP1 aberrations that were spread within the SETBP1 SKI homology domain (MPN, n = 1, MDS/MPN, n = 1, MDS, n = 2, sAML, n = 1). In three patients, germline material was available and showed the same aberration, indicating a polymorphism. These patients (median age: 64 ys) received demethylating therapy (n = 1) or supportive care (n = 4) and had a median overall survival of 34 months. The 10 patients with hotspot mutations (median age: 65 ys) underwent leukemia induction therapy (n = 1), allogeneic transplantation (n = 4) or palliative care (n = 6) and had a median overall survival of 4.5 months. All 3 transplanted patients had an early relapse after 2, 6 and 14 months, respectively. In 2/3 patients, the clone carrying the SETBP1 mutation was detectable by sanger sequencing at relapse. In univariate analysis of the entire cohort patients harboring SETBP1 hotspot mutations were significantly associated with unfavorable overall survival (4.5 vs. 14 months, p = 0.009). Targeted sequencing on cooperating mutations of patients with and without SETBP1 hotspot mutations is currently ongoing.

Conclusions: Patients with SETBP1 hotspot mutations suffer from aggressive disease with rapid evolution and relapse after allo SCT. Diagnostic screens for SETBP1 hotspot mutations may help identifying this dismal patient group and treat them in multicenter clinical studies. With emerging data on the biologic function of these mutations, a targeted therapy may be available in the future.

Disclosure: No conflict of interest disclosed.

V180 – Individual drug exposure to clofarabine measured by AUC predicts liver toxicity in patients with relapsed or refractory acute myeloid leukemia

Büttner B.1, Middeke J.M.2, Knoth H.1, Oertel R.3, Seeling A.4, Kramer M.2, Sockel K.2, von Bonin M.2,5,6, Stölzel F.2, Alakel N.2, Platzbecker U.2, Röllig C.2, Ehninger G.2, Bornhäuser M.2, Schetelig J.2,7

1Universitätsklinikum Carl Gustav Carus der TU Dresden, Klinikapotheke, Dresden, Germany, 2Universitätsklinikum Carl Gustav Carus der TU Dresden, Medizinische Klinik und Poliklinik I, Dresden, Germany, 3Medizinische Fakultät Carl Gustav Carus der TU Dresden, Institut für Klinische Pharmakologie, Dresden, Germany, 4Friedrich-Schiller-Universität Jena, Institut für Pharmazie, Jena, Germany, 5German Cancer Consortium (DKTK), Heidelberg, Germany, 6German Cancer Research Center (DKFZ), Heidelberg, Germany, 7DKMS, German Bone Marrow Donor Center, Dresden, Germany

Introduction: Clofarabine (clo.) is a nucleoside analogue with activity in acute myeloid leukemia (AML). Liver toxicity, particularly a transient elevation of transaminases is a common side effect of this substance. Especially in the context of the allogeneic HSCT, liver toxicity is a major concern. We studied the correlation of pharmacokinetic parameters with the liver toxicity and antileukemic activity of clo. in patients (pts.) with relapsed or refractory (r/r) AML.

Methods: We analyzed samples of pts. treated within the BRIDGE trial, a Phase II trial with clo. in combination with cytarabine as salvage therapy prior to hematopoietic stem cell transplantation (HSCT) in pts. with r/r AML. The pharmacokinetic parameters area under the curve (AUC) from 0–24 h after end of the infusion and the maximum plasma level (cmax) were analyzed and correlated with daily transaminase and bilirubin levels on days 0–15 after the first clo. infusion, with the maximum levels of transaminases and with response to salvage chemotherapy. Linear mixed models with random intercept and random slope and AUC per day exposure as influential variable were fitted in order to assess the impact of drug exposure on courses of transaminase and bilirubin levels. Multiple regression models were fitted to adjust for age, sex, clo. dose, baseline transaminase and treatment cycle.

Results: The mean transaminase levels rose within 10 days after start of clofarabine therapy with a maximum of more than 3.5 times the upper limit of normal between day 5 and 7. Up to day 15 the levels declined to the baseline values in all pts.

Results of the plasma levels showed large inter-individual differences in the pharmacokinetic parameters. A higher clo. AUC was significantly associated with liver toxicity reflected by a higher transaminase elevation (p = 0.02 for ALAT, adjusted for age, sex, Clo. dose, baseline ASAT and treatment cycle).

No statistical significant correlation could be found between cmax and the liver toxicity parameters. Furthermore, no association between the response to salvage chemotherapy evaluated at day 15 and the AUC or cmax of clo. was observed.

Conclusion: We were able to show that a higher individual clo. exposure is associated with more severe liver toxicity reflected by elevated liver enzymes without having an impact on anti-leukemic efficacy. Therefore, pharmacokinetic analysis might help to calibrate the optimal dosing of clo. for individual pts.

Disclosure: Bozena Büttner: No conflict of interest disclosed.Johannes Schetelig: Expert Testimony: Sanofi (Genzyme)

V181 – Mutational profiling of AML patients with CEBPA-mutations reveals differences between biallelic and monoallelic TAD and bZIP mutant subgroups

Georgi J.-A.1, Taube F.1, Kramer M.1, Herold S.1, Röllig C.1, Eberlein C.1, Stasik S.1, Krämer A.2, Berdel W.E.3, Serve H.4, Platzbecker U.1, Bornhäuser M.1, Ehninger G.1, Schetelig J.1, Thiede C.1, Studienallianz Leukämie (SAL)

1Medizinische Fakultät Carl Gustav Carus der TU Dresden, Medizinische Klinik und Poliklinik 1, Dresden, Germany, 2Deutsches Krebsforschungszentrum, Molekulare Hämatologie/Onkologie, Heidelberg, Germany, 3Universitätsklinikum Münster, Medizinische Klinik A, Münster, Germany, 4J.-W.-Goethe-Universität, Medizinische Klinik II, Frankfurt, Germany

Mutations of the key myeloid transcription factor CCAAT/enhancer binding protein alpha (C/EBPa) are found in 5–10% of patients with acute myeloid leukemia (AML). Two mutational clusters exist, in the aminoterminal transcription activation domains (TAD1 or 2) and in the basic leucine zipper domain (bZIP) located at the c-terminus of the protein. Biallelic mutations (biCEBPA) have been found to be associated with improved outcome and are now included as an independent entity in the WHO-classification. In contrast, monoallelic CEBPA-mutations (moCEBPA) do not appear to provide prognostic information. We analyzed the mutational spectrum of mono- and biallelic CEBPA-mutant AML patients to better understand potential differences in the biology of these groups.

Patients and methods: Patients included were registered clinical protocols of the Study Alliance Leukemia (SAL)(AML96, AML2003 or AML60+, SORAML) or the SAL-register. Screening for CEBPA mutations was done using PCR and capillary electrophoresis. All CEBPA mutant samples were analyzed using next generation sequencing (Trusight Myeloid Panel, Illumina).

Results: 239 patients with AML and CEBPA-mutations were identified. We restricted the analysis of coexisting mutations to 204 patients with normal karyotype (NK) or intermediate-2 mutations according to the ELN-classification. In this group, 104 patients presented with biCEBPA, whereas 100 had moCEBPA (57 bZIP, 43 TAD). Interestingly, these groups showed significantly differing patterns of co-mutations. In patients with biCEBPA, the most common alterations were seen in GATA2 (39%), TET2 (24%), WT1 (22%), and NRAS (15%). A similar distribution was found in bZIP-moCEBPA mutant patients (GATA2 (37%), TET2 (23%), WT1 (11%), and NRAS (14%)). In contrast, patients with isolated mutations in the TAD-domains had only 2% GATA2 mutations, predominant lesions were found in TET2 (42%), NPM1 and DNMT3A (35% each) and FLT3-ITD (26%), a pattern reflecting the distribution seen in the general AML population with NK. An analysis comparing the effect of these alterations on outcome indicated that patients with GATA2 mutations had an improved EFS but no significant difference in the OS and CR rate, whereas TET2-mutations were associated with significantly worse OS and EFS.

Conclusions: These results support functional differences between the mutational subgroups in CEBPA and reveal for the first time clinically relevant differences among the moCEBPA patient subgroup.

Disclosure: Julia-Annabell Georgi: No conflict of interest disclosed.Christian Thiede: Employment or Leadership Position: AgenDix GmbH; Stock Ownership: AgenDix GmbH

V182 – Single cell genotyping and epigenotyping of acute myeloid leukemia

Renz N.1, Niemöller C.1, Riba J.2, Bleul S.1, Metzeler K.H.3, Stosch J.M.1, Pfeifer D.1, Duyster J.1, Lübbert M.1, Zimmermann S.2, Claus R.1, Becker H.1

1Medical Center – University of Freiburg, Department of Medicine I, Freiburg, Germany, 2University of Freiburg, Department of Microsystems Engineering – IMTEK, Freiburg, Germany, 3University of Munich, Department of Medicine II, Munich, Germany

Clonal genetic and epigenetic heterogeneity impacts outcome in acute myeloid leukemia (AML). Deciphering the clonal heterogeneity requires analyses in single cells. Here, we studied gene mutations and DNA methylation in AML bulk samples and single cells.

In bulk samples, mutations were analyzed by targeted next generation-, Sanger- or pyrosequencing and DNA methylation by HumanMethylation450K array (Illumina). Single cells were deposited into microtiter plates by a Single-Cell Printer (SCP). Mutations in single cells were studied by PCR/Sanger sequencing directly on single cell DNA or after whole genome amplification (WGA); WGA success was assessed by a PCR on LINE1 transposons. DNA methylation in single cells was analyzed by methylation sensitive restriction enzyme digest (MSRE-D).

Mutations in KIT (p.N822K; variant allele frequency [VAF] 84%) and TP53 (p.R248Q; VAF 100%) were identified in the AML cell line Kasumi-1. Of the 450,000 DNA methylation sites analyzed in Kasumi-1, we selected 4 loci in DNMT3A with 3%, 70%, 77% and 96%, respectively, methylation level for further analyses. We printed 33 single Kasumi-1 cells and subjected them to WGA. Median DNA yield per cell was 14µg; the LINE1 PCR was positive in all cells. The KIT mutation was confirmed in 30, the TP53 mutation in 25 cells; PCR in the remaining cells failed. All cells with results for both mutation sites harbored both the KIT and TP53 mutation. The DNMT3A site with 3% methylation level in the bulk expectedly was unmethylated in 7 cells analyzed by MSRE-D. Preliminary analysis of both DNMT3A methylation and TP53 within the same cell was successful in 2 cells. We also studied single cells of a patient AML that in bulk sequencing only had the G-allele detectable in the TP53 polymorphism rs1042522 (p.P72R) and had a subclonal monosomy 17 (hosting TP53). Among 23 single cells, median WGA DNA yield was 16µg; the LINE1 PCR was positive in all cells. The TP53 PCR yielded a product in 20 cells. In 5 cells we detected the C-allele which was not found in bulk sequencing, suggesting its subclonal loss due to monosomy 17.

In conclusion, we confirmed the co-occurrence of mutations and verified the DNA methylation status in single Kasumi-1 cells. Single cell genotyping also confirmed subclonal genetic changes in an AML patient. Evaluation of mutation and methylation status in the same cell allows unique insights into clonal architecture; the SCP proved to be an efficient tool for single cell isolation.

Disclosure: No conflict of interest disclosed.

V183 – The Lef1 isoform, lacking the ß-catenin binding domain, is not acting as a dominant negative Lef1 variant, but is a hematopoietic active protein with unique DNA binding properties

Feder K.1, Edmaier K.1, Eshraghi P.1, Vegi N.1, Mulaw M.1, Ihme S.1, Spiekermann K.2, Metzeler K.2, Hiddemann W.2, Döhner K.3, Döhner H.3, Feuring-Buske M.3, Buske C.1

1Institute for Experimental Cancer Research, University Hospital Ulm, Ulm, Germany, 2Department of Internal Medicine III, University Hospital Munich, Grosshadern, Munich, Germany, 3Department of Internal Medicine III, University Hospital Ulm, Ulm, Germany

The Wnt pathway is essential for maintenance, activation and proliferation of normal hematopoietic stem cells. The lymphoid enhancer factor 1 (Lef1) is a member of the Lef/T-cell-factor family of transcription factors that directly interacts with ß-catenin in transcriptional complexes to induce expression of Wnt downstream target genes. Aberrant expression of Lef1 was shown to perturb normal hematopoietic stem cell (HSC) and progenitor function, induces AML in the murine bone marrow transplantation model and is a novel prognostic independent factor in patients with normal karyotype AML (Petropoulos et al. JEM 2008; Metzeler et al., Blood 2012). Furthermore we could show that Lef1 is essential for LT-HSC function (Edmaier et al. Leukemia, 2014). To dissect the role of the Lef1 isoform lacking the ß-catenin binding domain at the N-terminus (Lef1?N), we retrovirally engineered primary murine bone marrow cells to express the Lef1?N in comparison to the full-length Lef1 (Lef1WT) and the empty retroviral YFP control. Loss of binding to ß-catenin was validated by Co-IP for Lef1?N. The mutant did not impact Lef1 activity at the level of the short-term repopulating stem cells (CFU-S) but reduced the CRU frequency substantially (1:597.197(Lef1WT) versus 1:1.675.238 (Lef1?N), respectively, and 1: 1.233.152 for the EGFP control), indicating the necessity of Lef1 to collaborate with ß-catenin at the level of the LT-HSCs. ChIP-Seq in hematopoietic murine cells revealed unique binding sites for Lef1?N compared to Lef1WT with binding of the Lef1?N to the promoter of Wnt5a, which is known to regulate HSC proliferation and repopulation (Povinelli et al., Stem cell 2014).

To further understand the relevance of expression of the different LEF1 isoforms in patients with AML, over 100 normal karyotype AML cases were analysed by qRT-PCR, showing a dominant expression of the long isoform of LEF1 in the leukemic bulk population. Dependence of leukemic growth on binding of LEF1 to ß-catenin was further indicated by a high sensitivity of AML cell lines to the compounds Cercosporin and Calphostin C, which were shown to block LEF1-ß-catenin interaction. Our data clearly indicate that loss of the ß-catenin binding site creates a ‘neomorphic’ isoform with distinct biological and DNA binding properties. Differential expression of LEF1 isoforms in normal versus leukemic stem and progenitor cells might open the way to target leukemic cells by blocking LEF1-ß-catenin crosstalk.

Disclosure: No conflict of interest disclosed.

Posterdiskussion – Akute myeloische Leukämie

P186 – Non-psychoactive cannabidiol exerts proapoptotic activity in acute leukemia cells in vitro and ex vivo

Kampa-Schittenhelm K.1, Häusser L.1, Kanz L.1, Schittenhelm M.1

1Universitätsklinikum Tübingen, Medizinische Klinik II, Tübingen, Germany

We recently demonstrated that (-)-?9-Tetrahydrocannabinol (THC) has potent antileukemic activity in acute myeloid and lymphoblastic leukemia (AML/ALL) in vitro and in vivo. However, clinically effective doses may not be achieved due to compliance and side effect issues – especially with regard to the unwanted psychotropic effects of THC.

Cannabidiol (CBD) is a natural cannabinoid derivative with minimal psychotropic activity. It is part of a combination compound approved as Sativex for the treatment of spasms in multiple sclerosis. We herein provide evidence that CBD is even more potent with regard to the proapoptotic antileukemic activity compared to THC in several acute leukemia cell models in vitro as well as in ex vivo native blasts.

The AML cell lines MOLM13 and MOLM14 and the T-lymphoblastic Jurkat cell line as well as native blasts from patients were comparatively treated with CBD and THC in dose-dilution series and assessed for induction of apoptosis (Annexin V-based flow cytometry assay) and anti-proliferative effects (XTT).

MOLM13/14 and Jurkat cells displayed potent induction of apoptosis upon exposure to CBD with IC50s that were significantly lower compared to THC (5–7 microM vs.10–15 microM). This observation was confirmed treating native leukemia blasts of 10 patients with AML (n = 7) or ALL (n = 3) with CBD and THC ex vivo. Knockout (ko) of the cannabinoid receptors CB1 and CB2 in different leukemia cell models was performed using an shRNA approach. Pathway signaling analyses via the different cannabinoid receptors is ongoing. First data suggests apoptosis induction via both receptors, since CB1 as well as CB2 ko cells demonstrate attenuated apoptosis for both substances compared to the empty vector controls.

Besides a pro-apoptotic effect on leukemia blasts we have recently demonstrated that THC is capable of overriding the differentiation block in vivo resulting in loss of CD34 expression and an increase of CD11c, CD14 and/or CD15 in two patients treated with dronabinol for best supportive care – resulting in a maturing monocytic population of leukemic origin. These findings were reproducible in in vitro cell models. So far, similar effects were not seen for CBD, but shedding light into the mechanisms of differentiation is topic of our ongoing research.

Our findings provide a strong rationale to clinically explore CBD as an agent with remarkable antileukemic efficacy.

Disclosure: No conflict of interest disclosed.

P187 – Proof of clinical activity of Dasatinib in a patient with mutant-KIT D816V positive core binding factor leukemia (CBF AML)

Schittenhelm M.1, Bonzheim I.2, Witte K.1, Soekler M.1, Kanz L.1, Kampa-Schittenhelm K.1

1Universitätsklinikum Tübingen, Medizinische Klinik II, Tübingen, Germany, 2Universitätsklinikum Tübingen, Institut für Pathologie, Tübingen, Germany

Activating KIT D816V mutations are frequently found in CBF AML and are predictive for an unfavorable outcome. Dasatinib is a potent inhibitor of wildtype and mutant-KIT isoforms – including D816V. However, clinical efficacy as monotherapy is in question. In this study, we proof antileukemic efficacy via overriding the differentiation block in a patient with mutant-KIT D816V CBF AML.

Clinical activity of dasatinib was monitored using (1) Western immunoblotting for target-specificity (2) a plasma-inhibitory assay (PIA) using serum of this patient and our well established KIT D816V-dependent Ba/F3 model cell line to demonstrate clinically active concentrations (3) cytomorphology and immunophenotyping to analyze differentiation effects (4) a cell sort to isolate the maturing population and screen for presence of KIT D816V in order to proof leukemia origin.

This 77 year old unfit male patient with relapsing disease (WBC 13 000/microL, blasts 59%) and a high KIT D816V mutation burden was put on dasatinib 70 mg BID. Target-specificity was demonstrated via dephosphorylation (i.e. inactivation) of KIT on day 2 and 4 and PIA revealed clinically active doses of dasatinib (49% apoptotic cells after 48hrs). Intermittent cytoreduction with hydroxyurea was necessary due to leukocytosis, which did not allow to definitely proof proapoptotic efficacy of dasatinib in vivo. Intriguingly however, an increasing monocytic population (up to 80%) was noted – going along with disappearance of morphologic blasts and rise of neutrophils (~20%). Immunophenotyping argued for maturation of the leukemia clone, shifting from a CD14+/CD34+ positive phenotype to CD34 negativity. However, due to suspected pneumonia and questionable disease recurrence, therapy was halted. High doses of dexamethasone quickly improved the symptoms strengthening the differential diagnosis of differentiation syndrome. Cytoreduction with cytarabine and 6-thioguanin resolved leukocytosis and dasatinib was readministered with 100 mg/d. Again, signs of maturation were documented. Release of differentiation block was confirmed ex vivo demonstrating loss of CD34 upon exposure to dasatinib. Ultimately, sanger sequencing of the maturing population provided molecular proof of the leukemic origin with presence of KIT D816V in the suspected CD14+/CD34- population.

To conclude, dasatinib has clinical activity in mutant-KIT CBF AML via effective release of the differentiation block – providing a novel therapeutic approach.

Disclosure: No conflict of interest disclosed.

P188 – Oncometabolite 2-hydroxyglutarate impacts metabolism but not function of T cells

Böttcher M.1, Renner K.2, Mentz K.1, Mackensen A.1, Kreutz M.2, Mougiakakos D.1

1University Hospital Erlangen, Department of Internal Medicine 5, Hematology and Oncology, Erlangen, Germany, 2University Hospital Regensburg, Department of Internal Medicine III, Regensburg, Germany

Elevated levels of the oncometabolite 2-hydroxyglutarate (2HG) have been described in different tumor entities such as glioma, breast cancer, and AML both in serum and in the tumor. It was shown that 2HG can be generated via conversion of a-ketoglutarate by mutated isocitrate dehydrogenases (IDH), a process taking also place with non-mutated IDH but at a much lower level, or in a myc-driven manner via glutaminolysis. However, current research has thus far only focused on 2HG-mediated intrinsic effects on the tumor itself, especially its role as a competitive inhibitor of a-ketoglutarate resulting in epigenetic changes as well as metabolic deregulation. Moreover, results are often converse. Here, we show for the first time the effects of D-2HG (the most common enantiomer in this context) on activated T cells. Upon treatment with D2HG T cell respiration (basal and maximal), as well as respiration-related ATP production were enhanced while glycolysis was decreased. Additionally, glucose uptake, mTOR phosphorylation, as well as the content of a-ketoglutarate and citrate were elevated while lactate production was reduced. On the other hand, no influences on proliferation, survival, oxidative and suppressive capacity, T cell activation and memory formation, as well as mitochondrial biomass and membrane potential could be shown. Taken together, tumor-derived D-2HG affects T cell metabolism by enhancing mitochondrial respiration and decreasing glycolysis. Considering the elevation of tricarboxylic acid cycle intermediates D-2HG might fuel respiration by anaplerosis. However, the underlying mechanism and the possible impact on T cell activation and function remains to be elucidated. Ultimately, long-term effects of D-2HG as found in tumor patients should be explored as well.

Disclosure: No conflict of interest disclosed.

P189 – Assessment of prognosis and minimal residual disease in acute myeloid leukemia based on the use of NPM1 gene mutations in patients of Gomel Region, Belarus

Kozich Z.1, Silin A.1, Martinkov V.1, Tropashko I.1

1Das Republikanische Centrum für Strahlungsmedizin und Humanökologie, Hämatologie, Gomel, Belarus

Introduction: Considering the heterogeneity of acute myeloid leukemia (AML), the intensity of chemotherapy depends on the specific number and combination of morphological and molecular genetic markers. The additional use of specific leukemic markers (such as mutations in NPM1 gene) will increase the effectiveness of the therapy through the timely identification of them in the period of remission as an early sign of recurrence, as well as evaluate the prognosis of the disease.

Methods: The material for the study served as samples of venous blood and bone marrow of 111 patients with AML, who were treated in the period 2009–2014 in hematology department of SI “RRCRM & HE”, Gomel. To identify mutations in NPM1 gene there was used PCR method (PCR) with specific primers followed by electrophoretic detection.

Results: As a result of molecular-genetic analysis it was found that 26 of 111 analyzed patients (23.4%) were carriers of NPM1 mutations. In 10 cases, the mutation was identified together with FLT3-ITD mutation. The highest frequency of mutations was observed for M1 variant (28.6%). In groups of patients with M2, M3 and M4 variants NPM1 mutations were identified in 24.0%, 16.0% and 23.5% respectively.

In patients with M0 variant NPM1 mutations were absent. In the analyzed group of patients in the age of less than 60 years at the moment of diagnosis of AML the presence of NPM1 mutations was statistically significant adverse prognostic factor especially in combination with FLT3-ITD mutation. In addition survival rate in patients with leukocytosis in blood of more than 20 *10^9 /L and the blood Hb less than 90 g/L was significantly lower in relation to patients with normal levels of Hb and indicators of leukocytes of less than 20 *10^9 L.

A mutation in NPM1 gene, preserved after the first course of chemotherapy was associated with a high risk of relapse after three years of follow-up treatment and low overall survival. Although this mutation is connected with preleukemic clone, in 2 patients it was determined throughout remission after chemotherapy. During relapse, the mutation in NPM1 gene was identified in all patients that initially had this mutation.

Conclusion: Determination of mutations in NPM1 gene can supplement the prognostic information of other risk factors that will help to improve the survival of patients with AML as well as to increase the effectiveness of treatment when using NPM1 mutation as a molecular marker of relapse.

Disclosure: No conflict of interest disclosed.

P190 – Differential expression of Pri-miR-320a impacts on outcome in acute myeloid leukemia patients undergoing non-myeloablative allogenic stem cell transplantation

Gaber T.1, Bill M.1, Jentzsch M.1, Schubert K.1, Weidner H.1, Grimm J.1, Schulz J.1, Kloss L.1, Schmalbrock L.1, Bonifacio L.1, Wildenberger K.1, Pönisch W.1, Vucinic V.1, Franke G.-N.1, Lange T.1, Cross M.1, Behre G.1, Niederwieser D.1, Schwind S.1

1Universitätsklinik Leipzig, Hämatologie und internistische Onkologie, Leipzig, Germany

MicroRNA (miR) expression has been shown to be altered in AML & miR-based therapies are entering clinical trials. MiR-320a maps to chromosome 8p21.3 & its high expression has been associated with improved outcomes in some solid cancers. In AML miR-320 inhibits cell proliferation, likely by targeting the transferrin receptor 1. The objective of this study was to investigate whether a differential expression of miR-320a also impacts on outcome in AML pts.

We analyzed 121 AML patients (pts) (median age at diagnosis 63 years [y]; range 37–75 y) who were treated with cytarabine-based chemotherapy & received non-myeloablative allogeneic stem cell transplantation (NMA-SCT, Fludarabine 3×30mg & 2 Gy total body irradiation) as consolidation therapy at our institution. At diagnosis, pts were characterized for presence of FLT3-ITD, CEBPA, DNMT3A, IDH1, IDH2 & NPM1 mutations & EVI1 expression. European LeukemiaNet (ELN) genetic classification was: 26% favorable, 26% intermediate I, 21% intermediate II & 26% adverse. Pretreatment pri-miR-320a expression in bone marrow (BM) was analysed by reverse transcription polymerase chain reaction & normalized to a housekeeping gene (18S). The median normalized gene expression defined high & low pri-miR-320a expressers. Median follow-up was 4.3y for pts alive.

High pri-miR-320a expression associated with younger age (P = 0.02), lower platelet count (P = 0.04) & by trend lower BM blast count (P = 0.08) at diagnosis. High pri-miR-320a expressers more frequently were IDH2 (P = 0.04) & DNMT3A (P = 0.05) mutated & were less likely to be EVI1 positive (P = 0.02). High pri-miR-320a expressers had significantly longer overall survival (OS; P = 0.04; Figure 1a) & event-free survival (EFS; P = 0.02, Figure 1b). In multivariate analysis, pri-miR-320a expression kept its prognostic impact on EFS (Hazard Ratio 0.5 [95% Confidence Interval 0.3–0.9], P = 0.01).


We showed for the first time that pretreatment pri-miR-320a expression levels impact on outcome in AML pts. Increasing miR-320a expression prior to AML treatment, e.g. by miR-replacement therapies, might improve outcomes of AML pts, especially ones with low miR-320 expression.

Disclosure: No conflict of interest disclosed.

P191 – In AML with normal karyotype lacking mutations in NPM1, CEBPA, RUNX1 and MLL partial tandem duplication array CGH and fluorescence in situ hybridization can add prognostic information

Haferlach C.1, Kern W.1, Zieschang K.1, Zenger M.1, Perglerova K.2, Haferlach T.1

1MLL Münchner Leukämielabor GmbH, München, Germany, 2MLL 2, Paríkova, Czech Republic

Introduction: A subset of AML with normal karyotype lacking mutations in NPM1, CEBPA, RUNX1 and a partial tandem duplication within the MLL gene (MLL-PTD) is still poorly characterized. Array CGH and fluorescence in situ hybridization are able to detect abnormalities which are undetectable by chromosome banding analysis either due to a higher resolution, ability to detect copy neutral loss of heterozygosity (CN-LOH) and independence of in vitro proliferation.

Patients and methods: For 1473 AML cases with normal karyotype complete data on mutation status of NPM1, CEBPA, RUNX1 and MLL-PTD was available. Of these 303 cases (21%) did not carry any of these mutations. Out of these 159 cases with de novo AML (median age: 68 years (range: 19–93)) were selected based on the availability of material for array CGH (SurePrint G3 ISCA CGH+SNP Microarray, Agilent) and FISH screening with probes for MLL, RUNX1, CBFB, NUP98, MECOM/EVI1, NPM1, ETV6 and DEK-NUP214 (MetaSystems, Abbott).

Results: In total in 67 of 159 patients (42%) abnormalities were identified by FISH and/or array CGH. In detail, 12 balanced rearrangements were detected by FISH screening involving NUP98 (n = 7), MLL (n = 2) and MECOM, RUNX1 and CBFB (one each). In addition, 27 gains, 42 losses and 41 CN-LOH were observed in 58 (37%) patients. Recurrent gains affected regions 6q23.3q23.3 (n = 2) and 8q24.21q24.21 (n = 2) while recurrent losses were found for the regions 21q22.12q22.12 encompassing the RUNX1 gene (n = 5), 5q31.2q31.2 including i.a. EGR1 and CTNNA1 (n = 3), 2q34q34 including i.a. IKZF2 (n = 2), 7q22.1q22.1 encompassing i.a. CUX1 (n = 2), and Yq11.223q12 (n = 2). Recurrent CN-LOH were observed on chromosomes 11q (n = 10), 2p (n = 5), 4q (n = 4), 21q (n = 3), 1p (n = 2), 17q (n = 2) and 18q (n = 2). Survival analysis was performed for 90 intensively treated patients. Overall survival (OS) and event-free survival (EFS) were significantly shorter in patients with abnormalities detected by FISH and/or array CGH compared to those without (median OS: 19 vs 49 months, p = 0.027; median EFS: 9 vs 21 months, p = 0.016).

Conclusions: 42% of AML with normal karyotype lacking a disease defining molecular mutation harbor balanced rearrangements, copy number gains or losses as well as CN-LOH. The presence of these abnormalities has a negative impact on survival demonstrating that FISH and array CGH can add prognostic information in AML with normal karyotype.

Disclosure: No conflict of interest disclosed.

P192 – Three cases of cyclic neutropenia with acquired CSF3R mutations, one developing AML

Klimiankou M.1, Zeidler C.2, Mellor-Heineke S.2, Reinel E.1, Kandabarau S.1, Uenalan M.3, Kanz L.4, Welte K.5, Skokowa J.1

1University Hospital Tübingen, Department of Hematology, Oncology, Immunology, Rheumatology and Pulmonology, Division of Translational Oncology, Tübingen, Germany, 2Hannover Medical School, Severe Chronic Neutropenia International Registry (SCNIR), Hannover, Germany, 3Hannover Medical School, Department of Experimental Hematology, Hannover, Germany, 4University Hospital Tübingen, Department of Hematology, Oncology, Immunology, Rheumatology and Pulmonology, Tübingen, Germany, 5University Hospital Tübingen, Dept of Pediatrics, Tübingen, Germany

Introduction: We recently reported a 17-year-old female with cyclic neutropenia (CyN) harboring ELANE mutations presenting with cycling hematopoiesis involving neutrophils, platelets and reticulocytes who developed AML (FAB M2) (presented at ASH 2015, Blood 126 (23) abstract 885). At the time of AML diagnosis, all CSF3R-expressing BM leukemic blasts were positive for the p.Gln741X mutation. We tested the patient’s BM MNCs, obtained at the time of leukemia development, for RUNX1 mutations, finding that the RUNX1 mutation p.Asp171Asn was present at an allele frequency of 10%. So far, CSF3R mutations have been reported in congenital neutropenia patients (CN) only but never in patients with CyN. Next, we aimed to determine whether other CyN patients harbor CSF3R mutations.

Methods: We performed deep sequencing of CSF3R critical region in 19 additional CyN patients.

Results: Out of these 19 patients we identified two additional patients harboring acquired CSF3R mutations. One CyN patient aged 15.4 years and her sister inherited the ELANE mutation from their father. Time-course analysis of the CSF3R mutations in this patient showed that at the age of 13 years frequency of the CSF3R p.Gln749X allele in her BM MNCs sample reached 2.6%. After additional 1.5 and 2.4 years, respectively, the mutant allele frequency increased to 9% and 8%. A third CyN patient, a 7 year old girl, harboring spontaneous ELANE mutations revealing typical cycling of neutrophils, platelets, and monocytes has been treated with G-CSF at a dose of 4.5 µg/kg/day. She has been recently detected with 30% of frequency of CSF3R p.Gln739X mutant allele. The latter 2 CyN patients have not developed AML or myelodysplastic syndrome (MDS).

Conclusion: Taken together, these findings suggest that CyN patients with typical CyN-associated ELANE mutations may also acquire CSF3R mutations and are therefore at risk for leukemic development. Long-term data of the Severe Chronic Neutropenia International Registry (SCNIR) have shown that the risk of acquisition of CSF3R mutations and myeloid transformation is very low but not absent in patients with CyN compared to patients with CN. This new knowledge is important for prognostic counseling and long-term management of ELANE-CyN patients.

Disclosure: No conflict of interest disclosed.

P193 – Idiopathic intracranial hypertension resulting from ATRA therapy in acute promyelocytic leukemia: an important adverse effect

Teichmann L.L.1, Thieltges F.2, Brossart P.1, Mayer K.1

1Universitätsklinikum Bonn, Medizinische Klinik III, Bonn, Germany, 2Universitätsklinikum Bonn, Augenklinik, Bonn, Germany

Introduction: All-trans retinoic acid (ATRA) is highly effective in inducing remission in patients with acute promyelocytic leukemia (APL) by causing the differentiation of abnormal promyelocytes to mature neutrophils. Retinoids and vitamin A, however, are strongly associated with the development of idiopathic intracranial hypertension (IIH), a disorder of elevated intracranial pressure with no established pathogenesis.

Case: A 21-year old woman, who received idarubicin plus ATRA as induction therapy for high-risk APL, presented with refractory headache and nausea accompanied by intermittent brief graying-out of vision. Bilateral enlargement of the blind spot was noted in the visual field test. Funduscopic evaluation revealed bilateral swelling of the optic disc with obscuration of major blood vessels and retinal hemorrhages. These findings were suggestive of papilledema due to raised intracranial pressure. The presence of papilledema was confirmed by optical coherence tomography. Magnetic resonance imaging of the brain showed no secondary causes of increased intracranial pressure such as intracranial bleeding or cerebral venous sinus thrombosis. Thus, the patient was diagnosed with IIH. Lumbar puncture opening pressure was not determined due to thrombocytopenia and a prolonged prothrombin time. ATRA was immediately discontinued and the patient was treated with the carbonic anhydrase inhibitor acetazolamide. A rapid clinical improvement was seen and swelling of the optical nerves decreased. After symptoms resolved ATRA was reinitiated at a 50% dose with concomitant prophylactic acetazolamide, which was initially well tolerated. Yet, during the 1st consolidation cycle IIH recurred with sixth nerve palsy of the left eye. Symptoms were successfully treated by discontinuing ATRA and administering topiramate, an anticonvulsant that also blocks carbonic anhydrase, in addition to acetazolamide. Subsequently, the patient received the 2nd and 3rd consolidation cycle including ATRA at reduced dose without any major complications.

Conclusion: The diagnosis of IIH should be kept in mind in patients with APL. Owing to the use of ATRA its incidence is expected to be much higher in this group than in the general population. Female gender and obesity are considered additional risk factors for IIH. Untreated IIH can lead to retinal atrophy and permanent visual loss. After resolution of symptoms reinitiation of ATRA is possible when the patient is closely monitored.

Disclosure: No conflict of interest disclosed.

P194 – The prognostic impact of tetraploidy in patients with Acute Myeloid Leukemia

Mohr B.1, Röllig C.2, Kramer M.2, Oelschlägel U.2, Thiede C.2, Jost E.3, Schetelig J.2, Middeke M.2, Brümmendorf T.H.3, Serve H.4, Berdel W.E.5, Ehninger G.2, Bornhäuser M.2, Stölzel F.2, SAL

1Universitätsklinikum Dresden, Medizinische Klinik und Poliklinik 1, Dresden, Germany, 2Universitätsklinikum Dresden, Dresden, Germany, 3Uniklinik RWTH Aachen, Aachen, Germany, 4Universitätsklinikum Frankfurt, Frankfurt, Germany, 5Universitätsklinikum Münster, Münster, Germany

Introduction: Karyotypes of AML blasts reveal different disturbances of chromosomal integrity such as structural aberrations and numerical changes of whole chromosomes. Most of these recurring aberrations have been integrated in prognostication systems. Gains of whole chromosomes often appear together with structural aberrations and could be later events during disease progression whereby distinct structural aberrations rather function as earlier lesions with a strong impact on prognosis. Patients with trisomy 8 fare well in the presence of translocation t(8;21) whereas patients presenting with a sole trisomy 8 do not have a favorable prognosis (Schaich M 2007). In addition, a sole trisomy 13 was shown to be a marker conferring an adverse prognosis in AML (Herold T 2014) as well as several trisomies resulting in a pure hyperdiploid karyotype revealed an adverse prognosis, too (Stölzel F 2016). A rare but recurring phenomenon in AML is the tetraploid karyotype showing not only the doubling of several chromosomes but rather the doubling of the whole diploid karyotype. Therefore, the aim of this study was to investigate the prognostic impact of tetraploid karyotypes in AML.

Methods: The AML databases of the German SAL group were examined for tetraploid cases. The respective chromosome analyses were performed at diagnosis. The probability for overall survival (OS) was estimated according to the Kaplan-Meier method.

Results: Between 1996 and 2015 n = 17 patients, median age 55 years, fulfilled the criterion of tetraploidy (median OS: 18.7 months, 95%-CI 6.1-Inf). Taking into consideration additional aberrations two groups of patients were defined: (1) pure tetraploidy and =2 additional aberrations (n = 8) with a CR rate of 75% and a median OS of 26.2 months (95%-CI 18.7-Inf). (2) tetraploidy and =3 additional aberrations and at least one additional adverse-risk aberration (n = 9) with a CR rate of 67% and a median OS of 9.6 months (95%-CI 5.5-Inf). By comparison, in our SAL trials AML patients with a normal karyotype have a median OS of 24.9 months (95%-CI 22-27.9) and patients with adverse cytogenetics a median OS of 7.9 months (95%-CI 7.4-8.6).

Conclusions: However, taking into consideration the low patient numbers, tetraploid AML patients without additional adverse risk aberrations have an OS comparable to patients with a normal karyotype. Tetraploid AML patients with additional adverse risk aberrations have an inferior OS comparable to adverse risk patients.

Disclosure: No conflict of interest disclosed.

P195 – Characterization of patients with relapsed or refractory AML in continued follow-up after treatment with vosaroxin/cytarabine vs placebo/cytarabine in the valor trial

Horst H.-A.1, Ravandi F.2, Ritchie E.K.3, Lancet J.E.4, Craig M.D.5, Pigneux A.6, Maertens J.7, Derigs H.-G.8, Heuser M.9, Wei A.10, Hogge D.11, Clark R.12, Ward R.13, Craig A.R.13, Stuart R.K.14

1Klinik fuer Innere Medizin II, University Hospital Schleswig-Holstein, Kiel, Germany, 2University of Texas MD Anderson Cancer Center, Houston, United States, 3Weill Cornell Medical Center, New York, United States, 4Moffitt Cancer Center, Tampa, United States, 5West Virginia University, Morgantown, United States, 6Université de Bordeaux, Bordeaux, France, 7Universitair Ziekenhuis, Leuven, Belgium, 8Klinikum Frankfurt Main, Frankfurt, Germany, 9Hannover Medical School, Hannover, Germany, 10The Alfred Hospital and Monash University, Melbourne, Australia, 11Vancouver General Hospital, Vancouver, Canada, 12Royal Liverpool University Hospital, Liverpool, United Kingdom, 13Sunesis Pharmaceuticals, Inc., South San Francisco, United States, 14Hollings Cancer Center, Medical University of South Carolina, Charleston, United States

Introduction: Patients with relapsed/refractory (R/R) AML have a median overall survival (OS) less than 1 year. In the phase 3 VALOR trial, vosaroxin/cytarabine prolonged median OS in patients with R/R AML by 1.4 mo vs placebo/cytarabine (7.5 vs 6.1 mo; HR = 0.87 [95% CI 0.73–1.02]; p = 0.061). Of 711 enrolled patients, 134 (19%) were alive in follow-up at the primary analysis. Here, we characterize patients in continued follow-up.

Methods: In VALOR, patients with R/R AML were randomized 1:1 to receive cytarabine (1 g/m2 IV over 2 h, d 1–5) plus either vosaroxin (90 mg/m2 IV over 10 min d 1, 4; 70 mg/m2 in subsequent cycles) or placebo. After the primary analysis, ongoing patients were followed for survival.

Results: As of Jan 22, 2016, 83 patients (12%) were alive in follow-up: 46/356 (13%) in the vosaroxin/cytarabine arm and 37/355 (10%) in the placebo/cytarabine arm. Median follow-up in these patients was 40 mo (range 28–60). Patient characteristics are presented (Table); a higher proportion of patients were = 60 years in the vosaroxin/cytarabine arm (50% vs 27% with placebo/cytarabine). Most achieved complete remission (CR) on study (70% with vosaroxin/cytarabine; 51% with placebo/cytarabine); over half maintained CR at database lock (59% with vosaroxin/cytarabine; 49% with placebo/cytarabine). Nearly all had subsequent therapy (93% with vosaroxin/cytarabine; 100% with placebo/cytarabine). Most patients on vosaroxin/cytarabine (85%) and all patients on placebo/cytarabine received posttreatment stem cell transplantation (SCT). Seven patients in the vosaroxin/cytarabine arm did not undergo SCT; all were = 60 years of age. Median follow-up in these 7 patients was 33 mo (range 31–48).


Conclusions: A small proportion of patients with R/R AML continue to be followed for survival in VALOR. Typically, these patients achieved CR followed by SCT; however, some patients = 60 years treated with vosaroxin/cytarabine achieved long-term survival without SCT.

Disclosure: Heinz-August Horst: Advisory Role: Amgen, AbbVie, BMS; Financing of Scientific Research: Amgen, AbbVie; Expert Testimony: Amgen, Boehringer Ingelheim; Other Financial Relationships: MSD, BMS, CelgeneRobert Stuart: Advisory Role: Sunesis; Financing of Scientific Research: Sunesis; Expert Testimony: Sunesis

P196 – Evaluation of tyrosine kinase inhibitor treatment in patients with FLT3-ITD positive acute myeloid leukemia

Fleischmann M.1, Schrenk K.G.1, Schnetzke U.1, Hilgendorf I.1, Hochhaus A.1, Scholl S.1

1Klinik für Innere Medizin II, Universitätsklinikum Jena, Hämatologie/ internistische Onkologie, Jena, Germany

Introduction: Activating mutations of the receptor tyrosine kinase FLT3 (fms-related tyrosine kinase 3) represent the most frequent molecular aberration in acute myeloid leukemia (AML) and predominantly comprise FLT3 internal tandem duplications (FLT3-ITD) that are associated with a worse outcome. Targeted therapy with tyrosine kinase inhibitors (TKI) addressing FLT3-ITD has been introduced into the treatment of relapsed or refractory AML.

Methods: We retrospectively analysed the efficacy of TKI treatment in 15 patients (median age 54 years, range 21–74, 10 female) with relapse of FLT3-ITD positive AML. All patients underwent sorafenib treatment while 3 of 15 patients also received 2nd line therapy with ponatinib. Nine of 15 patients (60%) were characterised by AML relapse after allogeneic stem cell transplantation. Response to TKI treatment was evaluated according to the criteria published by Metzelder et al. (Blood 2009). Furthermore, TKI-related toxicity was analysed.

Results: Hematologic response (HR) was observed in 7 of 15 patients (47%) while only two patients (13%) achieved bone marrow response (BMR) during treatment with sorafenib. In contrast, there was no patient who achieved a complete (CR) or even complete molecular remission (CMR). The median duration of sorafenib therapy was 73 days (11–213). Importantly, in the subgroup of patients with AML relapse after allogeneic stem cell transplantation only 3 of 9 patients (33%) responded to sorafenib treatment (all with HR only). In addition, all patients who received ponatinib as 2nd line treatment had no response. TKI-induced toxicity was moderate and only one patient developed grade 3 toxicity (rash) related to sorafenib.

Conclusions: Treatment of relapsed FLT3-ITD positive AML remains a huge challenge and in most cases treatment with TKIs like sorafenib is not sufficient to control this disease. Especially in patients with AML relapse following allogeneic stem cell transplantation the results of TKI treatment are dissatisfying. Results of prospective clinical trials investigating 2nd generation TKIs (e.g. quizartinib, AC220) or concepts combining epigenetic approaches with FLT3-ITD directed treatment are needed.

Disclosure: Maximilian Fleischmann: No conflict of interest disclosed.Sebastian Scholl: Expert Testimony: Förderprogramm Infektiologie der Firma Gilead

P197 – Patients with acute myeloid leukemia admitted to intensive care units: Scores predicting outcome and post-ICU survival

Pohlen M.1, Braess J.2, Thudium J.3, Schmid C.4, Kochanek M.5, Kreuzer K.-A.5, Görlich D.6, Gerth U.7, Rhode C.8, Müller-Tidow C.8, Stelljes M.1, Büchner T.1, Schlimok G.4, Hallek M.5, Waltenberger J.9, Hiddemann W.3, Berdel W.E.1, Heilmeier B.2, Krug U.10

1Universitätsklinikum Münster, Medizinische Klinik A – Hämatologie und Onkologie, Münster, Germany, 2Krankenhaus der Barmherzigen Brüder, Onkologie und Hämatologie, Regensburg, Germany, 3Universitätsklinikum Großhadern, Hämatologie und Onkologie, München, Germany, 4Klinikum Augsburg, Medizinische Klinik II, Augsburg, Germany, 5Universitätsklinikum Köln, Medizinische Klinik I, Köln, Germany, 6Universitätsklinikum Münster, Institut für Biometrie und Statistik, Münster, Germany, 7Universitätsklinikum Münster, Medizinische Klinik D – Innere Medizin, Nephrologie und Rheumatologie, Münster, Germany, 8Universitätsklinikum Halle-Wittenberg, Medizinische Klinik IV, Halle/Saale, Germany, 9Universitätsklinikum Münster, Department für Kardiologie, Münster, Germany, 10Klinikum Leverkusen, Medizinische Klinik 3, Leverkusen, Germany

Introduction: Despite encouraging survival rates of ICU survivors compared to non-ICU patients, assumed high mortality represents a major reason for the widespread hesitation to refer AML patients for treatment in the ICU. This retrospective, multicenter study aimed to reveal risk predictors for mortality in the ICU as well as survival after ICU discharge in patients with AML requiring treatment in the ICU.

Methods: The AML in ICU score was established in a cohort of 187 adults with AML admitted to the ICU at the University Hospital of Muenster between 11/2004 and 09/2011. Validation was performed on a cohort of 264 patients with AML admitted to the ICU at the University Hospital of Grosshadern in Munich, the University Hospital of Cologne and the Municipal Hospital of Augsburg (all located in Germany) between 01/2004 and 02/2010.

Results: Multivariate analysis of training cohort revealed the following as independent prognostic factors for death in the ICU: arterial oxygen partial pressure below 72 mmHg, active AML and systemic inflammatory response syndrome upon ICU admission, and need for hemodialysis and mechanical ventilation. Based on these variables, we developed an ICU mortality score and validated the score in the validation cohort. Compared with the Simplified Acute Physiology Score (SAPS) II, our score yielded a better prediction of ICU mortality in the receiver operator characteristics (ROC) analysis (AUC = 0.84 vs AUC = 0.73 in the validation cohort).

Factors predicting decreased survival after ICU discharge were as follows: relapse or refractory disease, previous allogeneic stem cell transplantation, time between hospital admission and ICU admission, time spent in ICU, impaired diuresis, Glasgow Coma Scale < 8 and hematocrit of =25% at ICU admission. Based on these factors, an ICU survival score was created and used for risk stratification into three risk groups. This stratification discriminated distinct survival rates after ICU discharge.

Conclusions: Based on data from a large multicenter cohort, we identified and validated relevant risk predictors, which provided a basis for two scores distinguishing between survival differences both in the ICU as well as after ICU discharge. However, while these scores might aid the prognostication of patients with AML treated in the ICU, decisions about initiating or pursuing intensive treatment must not rely solely on the results of these scores. This study should encourage further prospective analyses.

Disclosure: No conflict of interest disclosed.

P198 – Comparison of ciprofloxacin and colistin as prophylactic antibiotics for the treatment of chemotherapy-induced neutropenia in patients with acute myeloid leukaemia: Two parallel patient cohorts treated in a single centre

Pohlen M.1, Marx J.1, Melmann A.2, Becker K.3, Mesters R.M.1, Mikesch J.-H.1, Schliemann C.1, Lenz G.1, Müller-Tidow C.4, Büchner T.1, Krug U.5, Stelljes M.1, Karch H.2, Peters G.3, Gerth U.6, Görlich D.7, Berdel W.E.1

1Universitätsklinikum Münster, Medizinische Klinik A – Hämatologie und Onkologie, Münster, Germany, 2Universitätsklinikum Münster, Institut für Hygiene, Münster, Germany, 3Universitätsklinikum Münster, Institut für Medizinische Mikrobiologie, Münster, Germany, 4Universitätsklinikum Halle-Wittenberg, Medizinische Klinik IV, Halle/Saale, Germany, 5Klinikum Leverkusen, Medizinische Klinik 3, Leverkusen, Germany, 6Universitätsklinikum Münster, Medizinische Klinik D – Innere Medizin, Nephrologie und Rheumatologie, Münster, Germany, 7Universitätsklinikum Münster, Institut für Biometrie und Statistik, Münster, Germany

Introduction: Patients with acute myeloid leukaemia (AML) are at high risk for bacterial infections during chemotherapy-related neutropenia. However, the use of antibiotic prophylaxis in afebrile neutropenic AML patients is controversial.

Methods: This was a retrospective evaluation of 172 AML patients who were randomly allocated to receive antibiotic prophylaxis with colistin or ciprofloxacin during chemotherapy-related neutropenia (expected duration of >7 days). A total of 322 treatment courses with at least one chemotherapy course per stay were analyzed. During a total of 44 courses (14.9%) in 35 patients, antibiotic prophylaxis was not administered. The remaining 138 patients received antibiotic prophylaxis over 252 treatment courses; in detail, 57 patients received ciprofloxacin (in 130 courses), and 72 patients received colistin (in 122 courses).

The differences between groups were analyzed through statistical methods capable of modelling repeated measurements, specifically generalized estimation equations (GEEs) were applied.

Results: The infection rate was reduced from 88.6% to 74.2% through antibiotic prophylaxis (vs. without prophylaxis, p = 0.04). A comparison of both antibiotic drugs revealed a trend towards fewer infections associated with ciprofloxacin prophylaxis (69.2% vs. 79.5% in the colistin group, p = 0.07), as determined by univariate analysis. This result was confirmed through multivariate analysis (odds ratio [OR] 0.475, 95% confidence interval [CI] 0.236 - 0.958; p = 0.041). The prophylactic agents did not differ with regard to the microbiological findings (p = 0.6, n.s.). The risk factors for higher infection rates were the presence of a central venous catheter (p < 0.0001), mucositis grade III/IV (p = 0.0039), and induction/relapse courses (vs. consolidation, p < 0.0001).

Conclusions: Ciprofloxacin prophylaxis appears to be of particular benefit during induction and relapse chemotherapy for AML, but it may be safely replaced by colistin during consolidation cycles of AML therapy. The selection of prophylactic agents should take into account variables like therapy stage (consolidation vs. induction/relapse), the risk of developing mucositis, and the local distribution of resistant pathogens.

Disclosure: No conflict of interest disclosed.

P199 – Pan-mammalian target of rapamycin (mTOR) inhibitor PP242 synergistically enhance ABT-737 induced apoptosis in acute myeloid leukemia (AML) cells

Ahmed F.1, Ilyas A.1

1King Abdulaziz University, Center of Excellence in Genomic Medicine Research, Djidda, Saudi Arabia

Pro-survival members of the BCL-2 family proteins are known to be overexpressed in hematological malignancies, including acute myeloid leukemia (AML). These pro-survival BCL-2 members that include BCL-2, BCL-xL, MCL-1 and BCL-w gives the cancer cells a selective survival advantage and resistance against a broad range of apoptotic stimuli. Therefore, several agents targeting pro-survival BCL-2 proteins have been developed and are being currently tested in the clinical settings. ABT-737 and its orally available derivative ABT-263, and more recently ABT-199 have displayed promising early results in CLL and AML. However, it is unlikely that these inhibitors will be sufficient as single agents. Finding combinations that could potentiate the effects of BCL-2 inhibitors has become imperative.

Here, we screened two PI3K/AKT/mTOR pathways inhibitors for possible synergistic killing of multiple AML cell lines and patient derived primary cells. AML cells were cultured and incubated with dual PI3K/mTOR inhibitor PKI402 or mTORC1&2 inhibitor PP242 and combined with BCL-2 specific inhibitor ABT-737. Cell proliferation assays were performed to assess the cytotoxicity. Chou-Talalay method of multi-drug combination was used to determine combination index (CI) values as a measure for the interaction between two drugs. Flow cytometry was used to study apoptosis, mitochondrial potential and Caspase 3 activity. Intracellular mapping of target proteins was performed by flow cytometry.

PKI-402, demonstrated additive effect in combination with ABT-737 in HL60, TF1 and K562 cells and showed synergistic effect in MV-411 and NB4 cells. PP242, the dual mTORC1/2 inhibitor, demonstrated synergistic inhibition of AML cell proliferation with ABT-737 and induced apoptosis in multiple AML cell lines. The increased induction of apoptosis by PP242 in combination with ABT-737 was associated with increased caspase 3 activity and depolarization of mitochondrial membrane. Phospho protein analysis by flow cytometry revealed that combination of PP242 and ABT-737 significantly down regulated phosphorylation of BCL-2 and ribosomal protein S6 to induce apoptosis. Ex vivo treatment of AML patient derived blast cells revealed enhancement of ABT-737 induced apoptosis by PP242.

In conclusion, the combination of pan-mTOR inhibitor PP242 with ABT-737 represent a potentially promising approach in the treatment of AML and has the potential to overcome the limited efficacy of single agents.

Disclosure: No conflict of interest disclosed.

P200 – Biomodulatory therapy with low-dose azacitidine, pioglitazone and ATRA induces myeloid differentiation and phagocytosis in primary AML blasts

Klobuch S.1, Steinberg T.1, Reichle A.1, Herr W.1,2, Thomas S.1,2

1Universitätsklinikum Regensburg, Klinik und Poliklinik für Innere Medizin III, Regensburg, Germany, 2Regensburger Centrum für Interventionelle Immunologie, Regensburg, Germany

Clinical outcome of elderly patients with chemorefractory acute myeloid leukemia (AML) is very poor and allogeneic hematopoietic stem cell transplantation is rarely feasible. We recently described in a small cohort of 5 chemorefractory elderly AML patients a novel biomodulatory therapy consisting of low-dose 5-azacitidine (AZA) in combination with the PPAR? ligand pioglitazone (PGZ) and all-trans retinoic acid (ATRA). The therapy induced complete remissions in 4/5 patients and was associated with strong myeloid differentiation and a rapid increase of peripheral blood neutrophils. Interestingly, peripheral blood neutrophils expressed the same AML-specific mutations than primary AML blasts, suggesting that the early increase of neutrophils observed during treatment included a considerable fraction of differentiated blasts.

To analyze these clinical observations in AML cultures in vitro, we treated primary AML blasts from different patients with AZA, PGZ and ATRA and subsequently evaluated myeloid cell differentiation by means of morphological changes, the expression of differentiation antigens and phagocytosis capacity. In samples from 4 of 8 patients we detected a 2 to 4-fold increase of cells showing morphological features of myeloid differentiation (i.e. nuclear lobulation), an increase of CD11b surface expression as well as growth control during 14 days of treatment with AZA/PGZ/ATRA. However, ATRA monotherapy was less efficient to induce myeloid differentiation in AML blasts from the same patients, suggesting that combination therapy might act synergistically on leukemic differentiation. Interestingly, differentiated cells that arise during in vitro treatment with AZA/PGZ/ATRA also showed an increase in phagocytosis, as demonstrated by a 2-fold increase in the uptake of GFP transfected E. coli as well as ROS production. Again, ATRA treatment was less efficient to induce phagocytosis in analyzed AML samples.

We conclude that biomodulatory combination therapy with low-dose AZA, pioglitazone and ATRA can be sufficient to drive differentiation of primary AML blasts into functional neutrophils, which may lower the neutropenia associated infection rates in AML patients. Clinical impact on infections rates as well as overall survival of low-dose AZA/PGZ/ATRA treatment will be further investigated in a planned randomized clinical trial on chemorefractory elderly AML patients.

Disclosure: No conflict of interest disclosed.

Posterdiskussion – Chronische myeloische Leukämie

P201 – Major route additional chromosomal aberrations (ACA) precede increase of blasts in chronic myeloid leukemia (CML) independent of therapy. An analysis of CML studies III, IIIA and IV

Voskanyan A.1, Dietz C.T.2, Fabarius A.C.1, Lauseker M.3, Saußele S.1, Kalmanti L.4, Rinaldetti S.1, Haferlach C.5, Pfirrmann M.3, Hasford J.3, Baerlocher G.M.6, Hochhaus A.7, Baccarani M.8, Hehlmann R.1, for the SAKK and the German CML Study Group

1Medizinische Fakultät Mannheim, Universität Heidelberg, III. Medizinische Klinik, Mannheim, Germany, 2IHO Institute for Hematology and Oncology, Mannheim, Germany, 3Ludwig-Maximilians Universität München, Institut für medizinische Informationsverarbeitung, Biometrie und Epidemiologie, München, Germany, 4DKD HELIOS Klinik Wiesbaden, Zentrum für Blutstammzell- und Knochenmarktransplantation, Wiesbaden, Germany, 5MLL Münchner Leukämielabor, München, Germany, 6Inselspital Bern, Universitätsklinik für Hämatologie und Hämatologisches Zentrallabor, Bern, Switzerland, 7Universitätsklinikum Jena, Abteilung Hämatologie/Onkologie, Klinik für Innere Medizin II, Jena, Germany, 8University of Bologna, Department of Hematology and Oncology, Bologna, Italy

Progression of CML to blast crisis is poorly understood. The aim was to determine the possible impact of major route and non-major route ACA in Ph+ cells in the course of disease on progression of CML. Patients (pts.) with primary treatments interferon-alpha (CML studies III and IIIA) and imatinib (CML study IV) were analyzed for correlation of ACA with blast increase (BI). 1287 pts. recruited to CML-studies III/IIIA were evaluable with a 10-year survival of 48% and 61%, respectively, of whom 258 (20%) progressed to blast crisis (BC). 195 pts. displayed ACA, 45 at baseline. 109 pts. showed major route ACA.

1536 pts. recruited to CML-study IV were evaluable with a 10-year survival of 83% of whom 81 (5,3%) progressed to BC. 183 pts. displayed ACA,105 at baseline. 86 pts. showed major route ACA.

In a univariate analysis of CML studies III/IIIA, pts. with ACA had a hazard ratio (HR) for BI ranging between 2.45–3.27 (p < 0.001) for blast counts from 1% to > 30%compared with pts. without ACA. For major route ACA, HRs of 3.5–4.5 were found. For non-major route ACA HRs were 1.7-2.2 (p < 0.001).

In the same model with CML study IV, pts. with ACA had a HR for BI ranging between 4.5-8.9 (p < 0.001) compared with pts. without ACA. For major route ACA, HRs of 13.1-27.8 (p < 0.001) were found. For non-major route ACA HRs were 1.05-1.07 (p = n.s.).

In both studies the cumulative incidences of BI by 5 years after diagnosis of major route ACA was 40% (Fig.1). In CML III/IIIA pts. cumulative incidences of BI by 5 years after diagnosis of non-major route ACA was 25-30%, in CML IV pts. 1- 5% (Fig.2).


ACA, particularly major route ACA preceded an increase of blasts. For pts. with major route ACA the 5-year-probability of a BI was 40% independent of treatment type. The impact of non-major route ACA on BI appeared to be eliminated by imatinib in contrast to IFN treatment.

Disclosure: No conflict of interest disclosed.

P202 – Protein-tyrosine phosphatases modulate therapy response in CML cells

Drube J.1, Albert B.V.1, Pfirrmann M.2, Ernst T.3, Hochhaus A.3, Böhmer F.-D.1

1Universitätsklinikum Jena, Institut für Molekulare Zellbiologie, CMB, Jena, Germany, 2Ludwig-Maximilians-Universität München, Institut für Medizinische Informationsverarbeitung, Biometrie und Epidemiologie (IBE), München, Germany, 3Universitätsklinikum Jena, Klinik für Innere Medizin II, Abteilung Hämatologie und Internistische Onkologie, Jena, Germany

Introduction: Most patients with chronic myelogenous leukemia (CML) can be successfully treated with tyrosine kinase inhibitors (TKIs) targeting the protein tyrosine kinase (PTK) BCR-ABL, but some do not reach molecular remission on the 4log level (MR4) or not even a 3log reduction (MMR). The counteractors of PTK are protein tyrosine phosphatases (PTPs). It is likely, that expression levels of specific PTPs modify the response to BCR-ABL inhibitors and that they have an impact on treatment outcome.

Methods: A qPCR-platform for the expression analysis of 38 PTPs was established, and the PTP expression status in leukocytes of 66 newly diagnosed, untreated CML patients, was analyzed. This data for each individual PTP was correlated with BCR-ABLIS after 9 months of nilotinib treatment (2*300 mg/d) using logistic regression analysis.

Several PTPs, including CD45, PTPRG, and SHP-1 were selected for further functional analysis using cell lines with shRNA-mediated depletion, CRISPR/Cas9 knockout, and stable re-/overexpression. The engineered lines were treated with TKI and the IC50 values were determined.

Additionally we investigated the effect of BCR-ABL on PTP expression in a set of newly created control-, BCR-ABL-, and TKI resistant BCR-ABL-T315I-expressing cells.

Results: Comparing patients with MR4 or better (n = 36) to patients not having a log4 reduction (n = 30) after 9 months of therapy, a higher expression of CD45 significantly increased the probability of MR4 at 9 months (p = 0.007). A significant association was also found for higher PTPRG levels (p = 0.012).

Interestingly, cell culture studies revealed lowering of the nilotinib IC50 in case of the CD45 depletion, indicating increased responsiveness. In contrast, SHP-1 deficient cells were more resistant to nilotinib compared to control cells.

PTPRG (encoding PTP?) was identified as a novel BCR-ABL regulated PTP gene. We observed an increase of PTPRG expression in the BCR-ABL and BCR-ABL-T315I-expressing cells, that decreased to control-levels upon nilotinib-treatment in case of the BCR-ABL cells, but not in the T315I-mutant cell lines. The role of PTPRG levels and the mechanisms of PTP-mediated modulation of TKI responses are currently being investigated.

Conclusions: Several PTPs correlate in expression with the therapy response to nilotinib and appear to causally affect responses in cell line models. Understanding the underlying mechanisms may aid in further optimizing TKI treatment.

Disclosure: No conflict of interest disclosed.

P203 – BCR-ABL independent gene mutations in children and adolescents with chronic myeloid leukemia

Busch M.1, Rinke J.1, Schäfer V.1, Waldau A.1, Ernst J.2, Hochhaus A.1, Gruhn B.2, Ernst T.1

1Abteilung Hämatologie und Internistische Onkologie, Klinik für Innere Medizin II, Universitätsklinikum Jena, Jena, Germany, 2Klinik für Kinder- und Jugendmedizin, Universitätsklinikum Jena, Jena, Germany

Introduction: Chronic myeloid leukemia (CML) in children and adolescents is rare. Many diagnostic standards and therapy options have been adapted from adult CML. We have previously identified BCR-ABL independent gene mutations in 33% of newly diagnosed adult CML patients that may function as important cofactors in the evolution of CML (Schmidt M, Rinke J, et al. Leukemia 2014; 28(12): 2292-2299). Here we sought to investigate whether BCR-ABL independent gene mutations can also be observed in children and adolescents with CML.

Methods: The cohort included 20 children and adolescents (male, n = 14; median age: 14 years, range: 10 months to 27 years) suffering from CML. Patients’ DNA was isolated from peripheral blood leukocytes obtained at diagnosis. Targeted deep next-generation sequencing (NGS) was applied to analyze a panel of 30 commonly mutated genes in myeloid disorders, affecting signal transduction, transcription factors, epigenetic regulation, RNA splicing and the cohesin complex.

Results:BCR-ABL independent gene mutations were detected in 8 of 20 patients (40%). In total, 14 mutations were detected in 7 different genes, namely ASXL1, DNMT3A, EZH2, JAK2, KRAS, STAG2, and UTX. Ten mutations affected epigenetic modifier genes. Mutations included 10 missense mutations, 2 nonsense mutations and 2 deletions. To investigate whether mutations originated in the myeloid lineage or are likely to be germline, purified T-cell DNA of selected patients will be analyzed until the meeting.

Conclusions: Analogous to adult CML, BCR-ABL independent gene mutations can also be frequently found in children and adolescents with CML. This implicates that BCR-ABL independent gene mutations are not age-related events. Thus, such molecular aberrations may play an important role in the evolution and persistence of the disease and may affect therapy in both children and adults with CML. Our findings underline a potential multistep pathogenesis and the important role of epigenetic events in CML biology.

Disclosure: No conflict of interest disclosed.

P204 – The Jak2-inhibitor Pacritinib with wide kinome-profile overcomes cytokine-mediated and mutation-driven resistance in CML cell lines

Hammersen J.1, Haase J.1, Becker C.1, Clement J.1, Hochhaus A.1, La Rosée P.1

1Universitätsklinikum Jena, Klinik für Innere Medizin II, Hämatologie/Onkologie, Jena, Germany

Introduction: Targeting the JAK/STAT-pathway in chronic myelogenous leukaemia (CML) interfers with the ability of BCR-ABL independent survival attributed to pathologic stem- and progenitor cells (SPC). However, this primary resistance of CML-SPC is not driven “one way” via JAK-signaling, but regulated by multiple kinase-signals. Pacritinib (PA) is a new JAK2/FLT3 inhibitor with a wide kinome profile. Aim of this study was to investigate the effects of PA in CML cell lines and primary CML SPCs in combination with Nilotinib (NI).

Material/Methods: Cells: BCR-ABL transfected murine Ba/F3p210 wildtype/TKI-resistant mutants & human M07p210. Incubation +/- cytokines IL3/GM-CSF. Antiproliferative effects tested in MTS assays, signaling by Western blot (WB), synergism analysis by the method of Chou (Combination-Index (CI) >1.1: antagonism; < 0.9: synergism). Methocult-assays (MA) to assess effects on the clonogenic capacity of primary CD34 sorted cells from CML patients.

Results: IL3-exposed Ba/F3p210 lost sensitivity to NI, which served as model for primary cytokine-driven resistance. It was overcome by adding PA: IC50 IL3+NI (mono) > 64nM vs. IC50 IL3+NI+PA[500nM] 4nM. M07p210 reacted analogously. NI&PA showed synergism in both cell lines (CI = 0.7/0.5).

Ba/F3 transfected with BCR-ABL mutants G250E or T315I showed resistance to NI [IC50>64nM], a model for mutation induced secondary resistance. PA alone was active in unmutated and resistance mutation expressing BaF3 cells (IC50 non-mutant p210: 500nM; G250E: 638nM; T315I: 785nM). In Ba/F3 PA was more active in p210 transfected than in parental cells IC50 = 800nM, suggesting partially BCR-ABL-dependent activity. Combination treatment in IL3-depleted media showed an antagonistic effect (CI = 1.7). Cytokine-mediated activation of STAT5 is inhibited by PA [500nM]/PA+NI [500nM+20nM], as detected by WB. Incubation of NI/PA/NI+PA (2000/750/2000+750nM) in MA from 3 patients lead to reduction of CFU-GM colonies of 47 / 72 / 93% with high inter-patient variability.

Discussion: PA shows antiproliferative effects in CML cell lines & primary CML SPCs. It is able to overcome cytokine driven TKI resistance, partially explained by inhibition of STAT5 phosphorylation. Synergism of PA/NI and its activity against NI-resistant BCR-ABL mutants can be explained by interference with cytokine signaling and a wide kinome profile. PA is identified as potential partner in combination therapy of CML to prevent and overcome resistance.

Disclosure: No conflict of interest disclosed.

P205 – Resistance in chronic myeloid leukemia: Therapeutic targeting of escape via CSF2RB

Becker C.1, Poser I.1, Wohlmann A.2, Clement J.1, Friedrich K.-H.2, Hochhaus A.1, La Rosée P.1

1Universitätsklinikum Jena, Hämatologie und Internistische Onkologie, Jena, Germany, 2Universitätsklinikum Jena, Institut für Biochemie II, Jena, Germany

Introduction: Persistence of minimal residual disease constitutes a risk of disease recurrence in patients with chronic myelogenous leukaemia (CML) treated with Abl-inhibitors. Persistent BCR-ABL+ leukemic stem and progenitor cells are promoted by bone marrow stroma cytokines such as interleukin 3 (IL-3) and granulocyte/macrophage-colony stimulating factor (GM-CSF) mediating BCR-ABL-independent survival via CSF2RB. We demonstrate upregulation of CSF2RB in BCR-ABL-transformed cells and primary cell samples. Disruption of the CSF2RB axis by the Janus kinase1/2-inhibitor ruxolitinib overcomes cytokine-mediated resistance in vitro as seen in an indepth molecular and functional analysis.

Methods: Gene expression analysis of CSF2RB was performed in M07p210 cells and patient derived BCR-ABL+ CML cells from bone marrow in response to nilotinib. Functional relevance of CSF2RB-mediated rescue of leukemic cells was investigated by gene knockdown of CSF2RB using siRNA in human M07p210. Viability and colony formation of BCR-ABL positive CML CD34+ progenitor cells in cytokine enriched nilotinib or dasatinib with or without ruxolitinib supplemented media were analyzed in vitro.

Results: Upon TKI treatment of M07p210 cells and CML CD34+ progenitor cells, compensatory upregulation of CSF2RB exert distinct survival signals including cytokine-induced STAT5 activation, despite effective BCR-ABL-inhibition. CSF2RB is regulated on the transcriptional level as demonstrated by quantitative RT-PCR. Knockdown of CSF2RB or combination treatment of M07p210 and treatment naïve CML CD34+ progenitor cells with nilotinib or dasatinib and ruxolitinib overcomes cytokine-mediated TKI-resistance as demonstrated by additive growth inhibition in the presence of GM-CSF. Increased mRNA-expression of CSF2RB is demonstrated in bone marrow harvested at 3 months of nilotinib (n = 8). No correlation with molecular response could be demonstrated in this exploratory cohort.

Conclusion: Upregulation of CSF2RB in response to TKIs is a consistent finding in CML in vitro and ex vivo with high pre-clinical activity of combined ruxolitinib. Exploratory analysis of CSF2RB as biomarker for response fails to demonstrate a robust relationship in individual patients. Further analysis is ongoing.

Disclosure: No conflict of interest disclosed.

P206 – BCR-ABL suppresses canonical BMP-signalling

Knobloch U.1, Elsner M.1, La Rosée P.1, Hochhaus A.1, Clement J.H.1

1Klinik für Innere Medizin II, Universitätsklinikum Jena, Hämatologie und Internistische Onkologie, Jena, Germany

Introduction: Chronic myeloid leukemia (CML) is a myeloproliferative disease of pluripotent hematopoietic stem cells and is characterized by the Philadelphia chromosome and the fusion protein BCR-ABL. Bone morphogenetic proteins (BMP) are essential regulators of differentiation and cell death during embryogenesis as well as in adults.

The aim of this study was to evaluate the presence of key components of the BMP signalling cascade in BCR-ABL-positive cells and their responsiveness for BMP signals.

Methods: The cell lines M07e (acute megakaryoblast leukemia), M07ep210 (stably overexpressing bcr-abl) and K562 (CML, blast crisis) were cultivated with RPMI1640 + 20% FCS under standard conditions; for M07e supplemented with GM-CSF. Incubation experiments were performed with BMP-2, BMP-4, BMP-6 or the BMP inhibitor dorsomorphin. Gene expression was measured by reverse transcriptase PCR and qPCR. Protein expression and activity was estimated by immunoblotting. Cell vitality was monitored by AnnexinV/Propidium iodide staining.

Results: PCR analysis showed that most of the BMPs, their receptors and molecules involved in canonical BMP signalling are expressed in the investigated cell lines on a comparable level except BMP-2 (high expression in M07e) and Activin receptor 2A (ACVR2A) (low expression in M07e). The most dramatic changes were observed for Smad1. Its expression was reduced 5-fold in K562 compared to M07e and was undetectable in M07ep210. Thus, a reduced sensitivity for BMPs acting via the canonical Smad signalling pathway was expected for bcr-abl-expressing cells. Interestingly, the well-known BMP target gene ID1 was highly expressed in bcr-abl-expressing cells, but not in M07e. On the protein level, ID1 was not regulated by BMP-2, BMP-4 or BMP-6. These observations point to alternate activation of ID1 expression and to a more undifferentiated state of the bcr-abl-expressing cells. BMPs are involved in regulation of apoptosis. Nether the application of BMP-2 nor the BMP inhibitor dorsomorphin affected the viability of the bcr-abl-expressing cells. In contrast, BMP-2 enhanced the vitality of M07e slightly, but dorsomorphin reduced vitality more than 6-fold compared to untreated cells.

Conclusion: The canonical BMP signalling via the Smad pathway is suppressed in the presence of BCR-ABL. This may keep the cells in a more undifferentiated state and protect them from pro-apoptotic signals transmitted via receptor-serine/threonine kinases.

Disclosure: No conflict of interest disclosed.

P207 – European survey on the assessment of deep molecular response in chronic phase CML patients after at least two years of therapy with tyrosine kinase inhibitors (TKI) (EUREKA).

Schenk T.1, Lange T.2, Saussele S.3, Pott C.4, Ernst T.1, Cross N.C.P.5, Hochhaus A.1

1Klinik für Innere Medizin II, Universitätsklinikum, Jena, Germany, 2Klinik für Hämatologie und internistische Onkologie, Asklepios-Klinikum, Weissenfels, Germany, 3III. Medizinische Klinik, Universitätsmedizin, Mannheim, Germany, 42. Med. Klinik, Universitätsklinikum Schleswig-Holstein, Campus Kiel, Kiel, Germany, 5Wessex Regional Genetics Laboratory, Salisbury District Hospital, Salisbury, United Kingdom

Introduction: The advent of highly effective therapies has changed the natural history of chronic myeloid leukemia (CML). The European LeukemiaNet (ELN) recommends quantification of BCR-ABL transcripts by real-time quantitative polymerase chain reaction (RQ-PCR) every 3 mo during TKI therapy. Recently, interest has focused on the assessment of deep molecular response (DMR, MR4, MR4.5, MR5) because a proportion of patients (pts) maintain remission after treatment stop. Molecular (mol) data monitored with a sensitive and standardized assay collected systematically do not exist so far outside of a clinical trial setting.

Methods: The purpose of this ongoing registry is to collect data on the standardized assessment of molecular response in the context of real life practice. BCR-ABL transcript levels after >2 yrs of TKI therapy are evaluated for DMR and its impact on pts’ management. Since standardized mol monitoring is a prerequisite for treatment discontinuation, central surveillance of the performance of the participating labs has been introduced.

Results: Between 2014-16, 1768 samples were sent after pts’ informed consent to 9 laboratories in 6 countries (D, I, H, CZ, HR, BIH). Pts’ median age was 59 yrs (range, 19-90), 55.5% of pts were male. Transcript type was b2a2 in 460 (26.0%), b3a2 in 685 (38.7%), b2a2/b3a2 in 47 cases (2.7%). 10 samples showed atypical transcripts (0.6%; e19a2, n = 5; e1a2, n = 3; b2a3, n = 2). In 555 cases (31.4%), no initial transcript type was reported. 1114 (63.0%), 451 (25.5%), and 187 (10.6%) samples were from pts on 1st, 2nd, or 3rd line therapies, respectively. Line of therapy was unknown in 16 cases. Current therapy was imatinib, n = 939 (53.1%), nilotinib, n = 517 (29.2%), dasatinib, n = 180 (10.2%), ponatinib, n = 9 (0.5%), bosutinib, n = 7 (0.4%), other, n = 12 (0.7%), unknown, n = 104 (5.9%). Response levels were: No MMR, n = 102 (5.8%); MMR, n = 283 (16.0%); MR4, n = 411 (23.2%); MR4.5, n = 538 (30.4%); MR5, n = 392 (22.2%). RQ-PCR failed in 42 cases (2.4%). Previous judgement as “complete” mol response was specified in 47 cases.

Conclusions: Improving the monitoring of deeper and sustained mol responses is critical for the optimal management of BCR-ABL+ CML patients and will assist to define parameters for treatment discontinuation. Multicenter DMR assessment is feasible in the context of real life clinical practice. Information on the BCR-ABL transcript type at diagnosis is crucial to accurately follow up pts’ response on or after therapy.

Disclosure: Thomas Schenk: No conflict of interest disclosed.Andreas Hochhaus: Financing of Scientific Research: Novartis, BMS, Pfizer, Ariad; Expert Testimony: Novartis, BMS, Pfizer, Ariad

P208 – High molecular response rates in patients with chronic myeloid leukemia – a retrospective health care analysis of more than 1000 patients

Tesch H.1, Jost P.2, Kisro J.3, Springer G.4

1Centrum für Hämatologie und Onkologie Bethanien, Frankfurt, Germany, 2III. Medizinische Klinik, Klinikum rechts der Isar, München, Germany, 3Lübecker Onkologische Schwerpunktpraxis, Lübeck, Germany, 4Praxis für Onkologie, Hämatologie und Palliativmedizin Stuttgart, Stuttgart, Germany

Introduction: Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder of the hematopoietic stem cells. The aim of this retrospective analysis was to analyze guideline adherence and treatment results in German CML centers under real life condition.

Methods: The quality of molecular monitoring and health care for CML patients was evaluated by a questionnaire according to current CML recommendations. With data of more than 1000 patients from 49 centers (7 clinics, 42 oncology practices) distributed all over Germany, it is representative for the health care situation of CML patients in German oncology centers.

Results: For the initial analysis, data from 1002 patients registered in the period from 08-2013 to 09-2015 were analyzed. Patients characteristics at diagnosis such as age, sex and phase of disease at diagnosis were similar to literature. 24.8% of patients were enrolled in first-line therapy CML studies. While cytogenetic analysis was not performed in 68.1% of patients during the last 12 months, quantitative PCR analysis was regularly performed in 92.5% of patients. Monitoring with quantitative PCR analysis was reported for 59.8% of patients every 3 months until reaching the BCR-ABL value of < 0.1. The majority of patients (60.8%) received first-line therapy with tyrosine kinase inhibitors (TKIs), whereas 26.2% were treated in second-line therapy. Response parameters corresponded to published data of clinical trials. In 82.9% of the patients, hematological response was maintained during therapy. Cytogenetic response was maintained in 63.2% and molecular response in 70.7% of patients. A high proportion of 69.5% of patients had achieved major molecular response (MR3.0) and 58.4% deep molecular response (MR4.0) at the time point of last visit. In an extension of the analysis, further data of 300 patients will be included from the ongoing project.

Conclusion: As the cohort investigated covers about 1/10 of the CML patients in Germany the data from this extensive retrospective chart analysis complements current knowledge about management of CML patients in Germany outside of clinical trials in a real-world setting. Importantly current CML recommendations are being followed in general and therefore demonstrate a high standard in molecular monitoring of CML patients in Germany.

Disclosure: Hans Tesch: Employment or Leadership Position: Niedergelassener Onkologe im Centrum für Hämatologie und Onkologie Bethanien Frankfurt.; Advisory Role: Novartis, BMS; Financing of Scientific Research: Novartis, BMSGregor Springer: Financing of Scientific Research: Novartis; Other Financial Relationships: Novartis, BMS

P209 – Molecular monitoring in assessing minimal residual disease and risk of progression in chronic phase CML: our data at King Fahad Medical City

Alsohaibani L.1, Altahan R.1, Alshehri N.1, Tailor I.K.1, Motabi I.1, Almishari M.1, Tashkandi S.1, Peer Zada A.A.1

1King Fahad Medical City, Riyadh, Saudi Arabia

Background: The World Health Organization and European Leukemia Net classification has set up criteria for the definition of the chronic phase (CP), accelerated phase (AP), and blast crisis (BC) of CML. In CP-CML patients on therapy, the reduced disease burden is an important prognostic indicator and minimal response to therapy or relapse suggests a therapy change. Monitoring minimal residual disease in CP-CML in view of its clinical and therapeutic outcome therefore, becomes essential.

Aim: We sought to assess levels of MRD in CP-CML patients by measuring the levels of BCR-ABL1 p210 fusion gene using an automated real-time RT-PCR test, the Xpert BCR-ABL (p210) Monitor Assay and assess the progression free survival, and the rate of relapse in our patients. We also assessed the rate of TKI response in CP-CML cases. Cytogenetic analysis (FISH) on bone marrow or peripheral blood samples was used to assess cytogenetic remission (or not) in all CP-CML cases.

Results: We describe here 51 CP-CML patients consisting of 29 females (~57%) with a median age of 44 ± 4.9 years (95% CI 26.6- 62.39) and 22 males (43%) with a median age of 36 ± 7.7 years. In patients with cytogenetic remission, BCR-ABL1 p210 level was detectable in 30 (30/51, 58.8%) and undetectable (< LOD) in 16 (16/51, 31.3%). 5 patients showed very high levels of p210 either due to relapse, intolerance, or compliance issues. Of the 30 CP-CML patients, 21 (70%) patients revealed BCR-ABL1/ABL ratio < 0.1% IS (MMR) are in continuous remission while 9 (30%) patients showed >0.1% IS p210 levels and did not achieve MMR. In response to TKI treatment, 48 (94%) CP-CML patients received imatinib as first-line, of whom 35 (72.9%) achieved molecular remission while 13 (27%) revealed treatment failure based on molecular data. Desatinib and nilotinib as second-line were effective in reducing p210 levels (MMR) in patients who failed imatinib treatment. 2 patients received desatinib as first-line and both (100%) achieved molecular remission. 25 (/48, 52%) CP-CML patients showed >5 years of progression free survival with imatinib with respect to p210 levels (continuous MMR).

Conclusion: In summary, depending upon the individual patient co-morbidities, all TKIs (Imatinib, desatinib, Nilotinib) showed good molecular response in CML patients albeit with some degree of intolerance. In CP-CML patients who failed imatinib treatment, desatinib and nilotinib were effective in achieving molecular milestones.

Disclosure: No conflict of interest disclosed.

P210 – Results of the non-interventional TARGET study: Efficacy and safety of nilotinib in CML patients (pts) failing prior therapy in routine healthcare

Dengler J.1, Müller M.C.2, Buß E.3, le Coutre P.4, Stegelmann F.5, Ulshöfer T.6, Sauer A.7, Schardt C.8, Reichert D.9, Schwinger U.10, Grunewald R.11, Waller C.12, Meincke M.13, Rupprecht S.13, Tesch H.11

1Onkologische Schwerpunktpraxis, Heilbronn, Germany, 2Universitätsklinikum Mannheim, Mannheim, Germany, 3Universitätsklinikum Heidelberg, Heidelberg, Germany, 4Charité – Universitätsmedizin Berlin, Berlin, Germany, 5Universitätsklinikum Ulm, Ulm, Germany, 6Schwerpunktpraxis für Hämatologie und Onkologie, Ludwigsburg, Germany, 7Medizinisches Versorgungszentrum für Blut- und Krebserkrankungen, Potsdam, Germany, 8Onkologische Praxis und Tagesklinik, Gelsenkirchen, Germany, 9Gemeinschaftspraxis für Hämatologie und Onkologie, Westerstede, Germany, 10Onkologikum, Stuttgart, Germany, 11Onkologie Bethanien, Frankfurt a.M., Germany, 12Universitätsklinikum Freiburg, Freiburg, Germany, 13Novartis Pharma GmbH, Nürnberg, Germany

Introduction: Nilotinib (NI) as a potent and highly selective BCR-ABL tyrosine kinase inhibitor is indicated for Ph+ CML pts in CP and AP with intolerance to or resistance of prior therapy including imatinib (IM) as well as for de novo Ph+ CML in CP.

Methods: Follow-up analysis of an observational study of NI in 479 pts with Ph+ CML resistant to or intolerant of prior treatment within routine clinical management in 156 centres in Germany (Jan 2008-Mar 2016).

Results: The median age of pts was 65.1 yrs, with 7.9% being older than 80 yrs. 99.2% (0.6%) presented in CP (AP)(phase missing in 1pt). 91% had a good performance status (Karnofsky index = 1). 92.7% of all pts were pretreated with IM (any dose). Further prior drug treatments were chemotherapy (23.4%), IFN (16.3%), dasatinib (19%) and other unspecified drugs (12.5%). Three pts had received SCT in the past. 56.6%/45% (n = 129) were treated with NI mostly due to resistance/intolerance against IM and 26.4%/75.8% (n = 91) against dasatinib, respectively (multiple responses possible). At initial visit, a dose of 800 mg NI/d was prescribed in 50.7% and 600 mg/d in 27.1%. Median duration of NI was 633 days at the data cut-off for this analysis. Remission status at study entry was 60.8% in CHR, 42.2% in MCyR/26.3% in CCyR (missing data in 20.5%), 30.5%/10.6% in MMR/CMR. These responses improved significantly under NI, reaching cumulative incidences of CHR, MMR and CMR of 90.2%, 63% and 35.3%, respectively. Of note, cytogenetic response improved as well but is not conclusive enough due to missing examinations (87.7% after 12 months). Dose reduction or therapy interruption at any time occurred in 29.6% or 17%. 77.7% experienced at least one AE during the observation period which was considered serious in 15.2%. 42.1% of pts with documented final visit (n = 409) prematurely discontinued the study. Hematologic toxicity / non-hematologic toxicity were observed in 12.7%/41.1% of pts. The most frequently reported AEs (AEs =5%) were skin reactions (pruritus 11.7%, rash 9.6%, alopecia 6.7%), fatigue (10.9%), thrombocytopenia (6.9%), anaemia (5.6%), arthralgia (5.4%), dyspnoea (5.2%), nausea (6.9%), and upper abdominal pain (6.1%) as well as headache (9.8%). Coronary disease occurred in 5.7% of pts, whereas peripheral arterial occlusive disease was reported in 4.2%.

Conclusions: This interim analysis supports NI as an efficacious and safe treatment option for CML pts with poor response or intolerance to a previous treatment.

Disclosure: Jolanta Dengler: Employment or Leadership Position: Selbständige Ärztin; Financing of Scientific Research: Vortragshonorare der Firma NovartisHans Tesch: Employment or Leadership Position: Niedergelassener Onkologe im Centrum für Hämatologie und Onkologie Bethanien Frankfurt; Advisory Role: Beratungstätigkeit Novartis; Financing of Scientific Research: Novartis

P211 – Efficacy and safety of Nilotinib in newly diagnosed Ph+ CML patients in chronic phase: Results of the 4th interim analysis of the non-interventional MOMENT II-study

Lathan B.1, Sauer A.2, Tebbe S.3, Ulshöfer T.4, Lange E.5, Schulze M.6, Nusch A.7, Janssen J.8, Losem C.9, Meincke M.10, Rupprecht S.10, Tesch H.11

1Gemeinschaftspraxis für Hämatologie und Onkologie, Dortmund, Germany, 2Medizinisches Versorgungszentrum für Blut- und Krebserkrankungen, Potsdam, Germany, 3Praxis für Hämatologie und Onkologie, Kassel, Germany, 4Schwerpunktpraxis für Hämatologie und Onkologie, Ludwigsburg, Germany, 5Evangelisches Krankenhaus, Hamm, Germany, 6Praxis und Tagesklinik für Hämatologie/Onkologie und Palliativmedizin, Zittau, Germany, 7Onkologische Praxis, Velbert, Germany, 8Gemeinschaftspraxis für Hämatologie und Onkologie, Westerstede, Germany, 9Praxis für Hämatologie und Onkologie, Neuss, Germany, 10Novartis Pharma GmbH, Nürnberg, Germany, 11Onkologie Bethanien, Frankfurt a.M., Germany

Introduction: Nilotinib (NI) is a potent and highly selective BCR-ABL TKI approved for treatment of newly diagnosed Ph+ CML pts in CP based on ENESTnd data showing improved treatment with higher molecular response rates vs. imatinib (IM). NI is also indicated for CP and AP Ph+ CML pts who failed prior therapy including IM.

Methods: 4th interim analysis of a non-interventional study of NI in 362 pts with de novo Ph+ CML in CP within routine clinical management (Aug 2011- Mar 2016; 118 centres in Germany).

Results: The median age was 58 yrs (17-88). 43.9% and 2.5% of the pts were older than 60 and 80 yrs. The median time since diagnosis of CML was 12 days (0-84). 93.1% of the pts had a good performance status (ECOG: =1; missing data in 3.9%). The median observation period was 586 days (4-1064).The median daily dose of NI was 600 mg (150-800 mg) with an initial NI daily dose of 600 mg in ~94% of the cases and a final NI daily dose of 600 mg >87% of the cases. There were 19.1% of pts with at least one therapy interruption and 15.5% of pts with at least one dose reduction. The most common reason for dosage adaption and/or therapy interruption were the occurrence of AEs (40,4%).At last visit 81.6% (of 315 pts with a hematologic examination) had a CHR (7% with missing data), 84.4% (of 32 pts with a cytogenetic examination) had a CCyR (96.9% with PCyR), 63.4% (of 268 pts with a molecular examination) had an MMR (1.5% with missing data). In the subgroup of pts with molecular response (MMR or better, n = 241) the median time to response was 191 days (56-783). A premature treatment discontinuation took place in 18.2% of pts mostly due to AEs/non-hematologic toxicity, in 5 cases (7.6%) because of disease progression, which is mainly characterised by new BCR-ABL mutations. Altogether, 76.8% of pts experienced AEs. Hematologic toxicity was observed in 6,6% of pts (5.3% with thrombocytopenia), non-hematologic toxicities occurred in 37.3% of pts. The most frequently reported AEs were skin reactions (rash 8.8%, pruritus 8.3%, alopecia 5.8%), fatigue (9.1%), headache (5.5%), gastrointestinal symptoms (nausea 7.2%, upper abdominal pain 6.1%). No cardiac / vascular disorders were = 2%. The most frequent biochemical abnormalities were increases in GGT (5.8%) and blood bilirubin (5.3%).

Conclusions: NI is supported as an effective and safe treatment for newly diagnosed Ph+ CML pts in CP by these data from routine clinical management.

Disclosure: Bernd Lathan: No conflict of interest disclosed.Hans Tesch: Employment or Leadership Position: Niedergelassener Onkologe im Centrum für Hämatologie und Onkologie Bethanien Frankfurt; Advisory Role: Beratungstätigkeit Novartis; Financing of Scientific Research: Novartis

P212 – DasPAQT: Treating patients with Chronic Myeloid Leukemia (CML) in Chronic Phase (CP) with Dasatinib: PCR-Monitoring, adherence, quality of life, therapy satisfaction (OMC 2014-I; BMS CA180-565) - an analysis of the first 150 patients

Tesch H.1, Pelz H.2, Janssen J.3, Hansen R.4, Fietz T.5, Anhuf J.6, Haeberle L.7, Belleville E.8, Schardt C.9, Azeh I.9, Steinmetz T.10

1Niedergelassener Onkologe im Centrum für Hämatologie und Onkologie Bethanien Frankfurt, Frankfurt, Germany, 2Ambulantes Therapiezentrum für Hämatologie und Onkologie, Offenburg, Germany, 3Gemeinschaftspraxis für Hämatologie und Onkologie, Westerstede, Germany, 4IDGGQ, Institut für med. Dokumentation GbR, Kaiserslautern, Germany, 5Schwerpunktpraxis für Hämatologie und Internistische Onkologie, Singen, Germany, 6Onkologie Duisburg Nord, Duisburg, Germany, 7Comprehensive Cancer Center Erlangen-EMN, Erlangen, Germany, 8ClinSol GmbH & Co.KG, Würzburg, Germany, 9Onko.Logix GmbH & Co. KG, Gelsenkirchen, Germany, 10Praxis für Hämatologie und Onkologie, Köln, Germany

Introduction: Despite tyrosine kinase inhibitors (TKI) remarkable efficacy in clinical trials, there is a lack of published data on how CML is managed in the real- world clinical practice settings. The non-interventional study (NIS) DasPAQT is designed to document real-world data on Dasatinib treatment of patients with CML in chronic phase in clinical routine. As most CML patients are treated outside of clinical studies focus lies on health care provided in office based physicians.

Methods: Patient population consists of subjects with newly diagnosed Ph+ CP-CML and CML patients in chronic phase resistant or intolerant to prior therapies that are treated with Dasatinib according to the clinical routine. Follow up is documented for a maximum of 24 months. A total of 300 patients from up to 75 sites (hematological and oncological hospitals or practices) will be documented in order to answer the following questions: What is the treatment pattern of Dasatinib in CML patients in a real-world environment, including Dasatinib treatment strategies in first-line chronic CML or in a switch setting? What is the effectiveness and outcome of the Dasatinib treatment and which prognostic clinical and scientific factors determine the specific treatment strategy? What is the patient-reported benefit and the impact of first-line Dasatinib CML treatment on patient quality of life? What are the rates of adherence/compliance, how satisfied are patients with their treatment, what patient-related factors lead to treatment discontinuation? Can long-term treatment response be predicted in a real-world setting?

Results: Data on diagnostic monitoring, treatment setting and treatment duration with Dasatinib as well as baseline characteristics and safety data of the first 150 patients will be presented.

Conclusion: DasPAQT is intended to provide insight into the routine health care management of CML-patients treated with Dasatinib and its related outcomes. The factors that CML patients and treating physicians may encounter in a real-world setting will be observed and analyzed to understand the benefits and effectiveness of Dasatinib CML treatment.

Disclosure: Hans Tesch: Employment or Leadership Position: Niedergelassener Onkologe im Centrum für Hämatologie und Onkologie Bethanien Frankfurt.; Advisory Role: Beratungstätigkeit u.a. Novartis, Roche, Bristol-Myers Squibb; Financing of Scientific Research: u.a. Novartis, Roche, Bristol-Myers Squibb; Expert Testimony: Ja, Bristol-Myers SquibbTilmann Steinmetz: Employment or Leadership Position: Geschäftsführer X-Med GmbH, Leitung: Onkologie-Köln.; Advisory Role: Amgen; BMS, Celgene, Hexal-Sandoz, Medice, Novartis; Janssen-Cilag; Pharmacosmos; Roche; Vifor; Ariad; Stock Ownership: Kommanditist von GermanOncology; Financing of Scientific Research: KV-No, Privatpatienten; Expert Testimony: Untersuchungen Studienleitung IIT/NIS/Register: Amgen, Celgene, Novartis, Vifor; Other Financial Relationships: Reisekosten: Alexion, Amgen, Bayer, Celgene, Janssen-Cilag, Novartis, Sanofi, Vifor; Immaterial Conflict of Interests: Mitgliedschaften: DGHO, ESMO, DGPM, BNHO, NIONo (Vorsitz).

P213 – R-EFECT: A multicenter, retrospective evaluation of CML therapy in Austria assessing the impact of early treatment response on patient outcomes in a real-life setting

Petzer A.1, Herndlhofer S.2, Weltermann A.3, Sliwa T.4, Schmidt S.5, Greil R.6, Wölfler A.7, Wiesholzer M.8, Dormann C.1, Thaler J.9, Tinchon C.10, Ruckser R.11, Lang A.12, Hänig J.13, Winiger I.13, Muenchmeier N.13, Sperr W.R.2

1Barmherzige Schwestern Hospital Linz, Department of Medical Oncology, Hematology and Gastroenterology, Linz, Austria, 2Medical University of Vienna, Department of Internal Medicine I, Division of Hematology, Vienna, Austria, 3Krankenhaus der Elisabethinen Linz, Department of Medicine I – Hematology with Stem Cell Transplantation, Hemostasis and Medical Oncology, Linz, Austria, 4Hanusch Hospital Vienna, Department of Medicine III: Hematology Oncology, Vienna, Austria, 5University of Innsbruck, Internal Medicine V: Hematology-Oncology, Innsbruck, Austria, 6Paracelsus University Hospital Salzburg, Department of Medicine III, Salzburg, Austria, 7Medical University of Graz, Division of Hematology, Department of Internal Medicine, Graz, Austria, 8University Hospital St. Poelten, Karl Landsteiner University of Health Sciences, Department of Internal Medicine I, St. Poelten, Austria, 9Klinikum Wels-Grieskirchen, Department of Internal Medicine IV, Wels, Austria, 10LKH Leoben-Eisenerz, Department for Hematology and Oncology, Leoben, Austria, 11Donauspital/ Sozialmedizinisches Zentrum Ost, 2nd Medical Department, Vienna, Austria, 12LKH Feldkirch, Department of Internal Medicine, Feldkirch, Austria, 13Novartis Pharma GmbH, Oncology, Vienna, Austria

Introduction: Data from randomized clinical trials indicate that CML patients (pts) reaching early molecular response at 3 months (EMR; < 10% BCR-ABL/ABL) achieve better OS and benefit from a reduced risk of disease progression.This study has aimed to evaluate the impact of an early treatment response (ETR) in daily clinical practice by analyzing patient data available at CML treatment centers in Austria.

Methods: 12 participating centers were asked to retrospectively document data of CP-CML pts that were diagnosed between January 2004 and July 2010 and treated frontline with a TKI outside of a clinical trial. A minimum of 5 years of follow-up was required unless an earlier time of death was known. The term “ETR” was defined as EMR (< 10% BCR-ABL/ABL) or optimal cytogenetic response at 3 months according to ELN 2013 criteria (i.e. MCyR; Ph+ =35%). Additionally, if neither BCR-ABL/ABL IS values nor cytogenetic response data were available, pts were scored for ETR using raw BCR-ABL/ABL values. This was supported by a subgroup analysis which demonstrated a good comparability between raw and IS values.

Results: Of a total of 211 documented pts 35 were excluded due to missing or unevaluable response data at 3 months, resulting in an analysis cohort of 176 pts. Median age at diagnosis was 56 years, with 40.3% being female. ETR was achieved by 77.3% of pts and associated with a higher 5 year OS (92.5%; p = 0.02) and PFS (95.6%; p = 0.06) with no early deaths by 12 months compared to ETR negative (ETR-) pts with an OS of 77.5% and a PFS of 87.5% and 2 early deaths by 12 months, respectively. At the last visit (median follow-up was 94.5 months and similar in both subgroups) differences in OS and PFS were even more pronounced between ETR+ and ETR- pts (OS 88.1% vs. 67.5%, p = 0.003; PFS 92.6% vs. 84.2%, p = 0.055). Interestingly, the majority of pts reaching ETR had low and intermediate SOKAL scores at diagnosis (low/int/high: 52.9%/34.1%/12.9%), whereas pts failing ETR had predominantly intermediate or high SOKAL scores (20.0%/52.0%/28.0%). Pts reaching ETR were less likely to be switched from imatinib to another TKI (76.7% vs. 55.3% still on imatinib at last visit; p = 0.02).

Conclusion: These data from a real life setting support the findings from randomized trials demonstrating that ETR is associated with superior PFS and OS. Pts failing ETR should be closely monitored and treated properly according to available guidelines.

Disclosure: Andreas Petzer: Advisory Role: Novartis, BMS, Pfizer, Ariad; Financing of Scientific Research: Novartis, BMS, Ariad; Expert Testimony: NovartisWolfgang Sperr: Financing of Scientific Research: Novartis

P214 – Standard of care of patients with CML treated in community based oncology group practices in Rhineland-Palatinate (Germany)

Weide R.1, Rendenbach B.2, Grundheber M.3, Burkhard O.4, Behringer J.5, Maasberg M.6, Ehscheidt P.7, Strehl J.W.8, Hansen R.9, Feiten S.10

1Praxisklinik für Hämatologie und Onkologie, Koblenz, Germany, 2Gemeinschaftspraxis für Hämatologie, Onkologie und Nephrologie, Trier, Germany, 3Onkologische Schwerpunktpraxis, Trier, Germany, 4Internistische Gemeinschaftspraxis Hämatologie, Onkologie, Palliativmedizin, Worms, Germany, 5Onkologische Schwerpunktpraxis, Speyer, Germany, 6Gemeinschaftspraxis für Hämatologie und Onkologie, Mayen, Germany, 7Praxis für Hämatologie und Onkologie, Neuwied, Germany, 8Schwerpunktpraxis Hämatologie und Internistische Onkologie, Altenkirchen, Germany, 9Schwerpunktpraxis für Hämatologie und Onkologie, Kaiserslautern, Germany, 10Institut für Versorgungsforschung in der Onkologie, Koblenz, Germany

Introduction: Significant progress has been made in CML-therapy since the introduction of imatinib and other tyrosine kinase inhibitors (TKI) into clinical care. The aim of this study was to assess diagnosis, treatment and outcome of CML-patients who received their treatment in community based oncology practices in Rhineland-Palatinate and whether European LeukemiaNET-guidelines were followed.

Methods: All Ph-/BCR-ABL-positive CML-patients who were treated between 12/2001-12/2015 in 9 oncology group practices were analysed retrospectively concerning diagnosis, treatment and outcome according to European LeukemiaNET-guidelines. Data were collected from patient files into a central data base and analysed statistically with SPSS.

Results: 264 patients (pts) with a median age of 60 (18-90) were analysed. 126 (48%) were female, 138 (52%) were male. At initial diagnosis bone marrow biopsy was performed in 213 pts (81%). Cytogenetics was applied in 204 pts (77%) (38% in blood, 56% in bone marrow). FISH-analysis was used in 155 pts (59%) (33% in blood, 36% in bone marrow). PCR-testing to detect a BCR-ABL1-rearrangement was applied in 200 pts (76%) (52% blood, 37% bone marrow). 258 pts (98%) were in chronic phase, 5 (2%) in accelerated phase and 1 (0.4%) in blast crisis at diagnosis. EUTOS score could be calculated in 131 pts (50%). 20% were high risk, 80% low risk. 252 pts (95%) received some form of TKI-therapy. Out of 416 TKI-therapies 308 (74%) were PCR-based monitored, 148 (36%) were monitored by cytogenetics. First line treatment was imatinib in 201 pts (80%), 51 pts (20%) received a second generation TKI. Second line treatment consisted of dasatinib in 59%, nilotinib in 32%, imatinib in 6% and bosutinib in 3%. Third line treatment was nilotinib in 56%, dasatinib in 35%, ponatinib in 6% and imatinib in 3%. 62 pts (23%) were treated within a study protocol. 13 pts (5%) received an allogeneic transplantation. Overall survival probability was 88% after 5 years and 72% after 10 years. Disease specific survival was 95% after 5 years and 86% after 10 years.

Conclusion: The overwhelming majority of CML-patients treated in oncology group practices receive standard of care as suggested by European LeukemiaNET-guidelines. Overall survival in routine care is comparable to international studies.

Disclosure: No conflict of interest disclosed.

P215 – Treatment results of 13 years CML therapy in a private office. A contribution of the German CML Alliance

Kämpfe D.1, Haverkamp T.2, Heil G.3, Schulte C.4, Tesch H.5, Stein H.6, Die Deutsche CML-Allianz

1Praxis für Hämatologie / Onkologie, Lüdenscheid, Germany, 2MVZ Dr. Eberhard & Partner Dortmund (ÜBAG), Dortmund, Germany, 3Klinik für Hämatologie und Onkologie am Klinikum Lüdenscheid – Märkische Gesundheitsholding GmbH und Co KG, Lüdenscheid, Germany, 4Institut für Hämatopathologie, Hamburg, Germany, 5Centrum für Hämatologie und Onkologie Bethanien, Frankfurt / Main, Germany, 6Pathodiagnostik Berlin, Berliner Referenzzentrum für Lymphom- und Hämatopathologie, Berlin, Germany

Introduction: Many patients with CML are diagnosed and treated in private offices of hematologists/oncologists in Germany. In registry studies it was found that only about one quarter of these patients are included in the context of clinical studies. Here we present the analysis of a complete, non-selected data set of all patients with CML disease, who have been treated by one doctor in a private office during the last 13 years.

Methods: During the period from 07/2003 to 03/2016, all patients diagnosed with CML were identified and their therapy, response and survival were analyzed. The analysis was based on medical reports, laboratory data and prescription data of these patients. Demographics and treatment outcomes of these patients were compared with data from studies (CML IV, IRIS).

Results: Altogether 51 patients were treated with CML in this period, 33 patients were newly diagnosed (50 Ph+, 1 Ph- CML). 48 (96%) patients were in the CP, 2 (4%) patients in AP at diagnosis (ID 01/97…03/2016 with median age of 62 (22 ... 88) years). One patient was autologous and another patient was allogeneic transplanted before the evaluation period (4%). 5 (10%) patients had at least one additional neoplasia.

During the follow-up period (median: 5.5 (0 ... 20.5) years), two patients had an acceleration during therapy, both reached subsequent CPs under treatment again. A 67 year old woman with ovarian cancer died in a blast crisis.

Analysis of 3.785 mo. of therapy split up in 65% IMA±IFN, 12% IFN±HU, 6% NIL±IFN, 6% no treatment (5% TFR, 1% break) and 2% DAS (other therapies remaining).

With 12 (24%) patients one or more mutational analysis of the bcr-abl gene were performed. With 3 (6%) patients there were mutations found, one with T315I mutation.

With patients with a delayed start therapy with TKI (> 90 after ID) fewer reached a molecular response (eg. MMR 72 vs. 90%, p < 0.05) and they required a significantly longer time to reach these responses (median time to MMR 126 vs. 15 months, p < 0.05). Until now a total of 5 (10%) patients achieved a treatment free remission (TFR) successfully after achieving a deep and stable molecular remission (median TFR 38 (8 ... 98) months).

Conclusions: Comparable treatment results can be achieved for patients with CML in a private office as well as in studies with a very similar survival rate. Small differences could be easily explained by different age (older patients in office) and comorbidities (unselected patient cohort).

Disclosure: Dietrich Kämpfe: Immaterial Conflict of Interests: logistische Unterstützung durch die Firma ARIAD Pharmaceuticals Inc.Harald Stein: No conflict of interest disclosed.

Posterdiskussion – Myelodysplastisches Syndrom, sonstige Hämatologie

P216 – Highly variable Separase activity patterns in bone marrow of patients with myelodysplastic syndrome and acute myeloid leukemia

Ruppenthal S.1, Prinzhorn W.1, Kleiner H.1, Lammer F.1, Nowak D.1, Hofmann W.-K.1, Fabarius A.1, Seifarth W.1

1Department of Hematology and Oncology, University Medical Centre, Mannheim, Germany

Introduction:ESPL1/Separase, an endopeptidase, is a key player for centrosome duplication and separation of sister chromatids in anaphase of mitosis. Overexpression and deregulated proteolytic activity of Separase is associated with the occurrence of supernumerary centrosomes, chromosomal missegregation and aneuploidy, as frequently observed in human cancers. Increased Separase proteolytic activity in a small subpopulation of tumor cells may serve as a driver of tumor heterogeneity and clonal evolution in human hematopoietic disorders. Recently, we have shown that in CML patients Separase activity was highly increased when compared to healthy donors. No data are currently available on Separase activity in myelodysplastic syndromes (MDS) and MDS-related secondary acute myeloid leukemia (sAML). Therefore we set out to measure Separase activity in respective clinical bone marrow (BM) samples.

Methods: Density gradient centrifugation using Ficoll-Paque was performed to separate mononuclear cells from BM specimen of healthy donors and patients with MDS and AML. Separase activity in living cells was measured by fluorescence-activated cell sorting (FACS) employing a rhodamine 110 (Rh110)-conjugated Rad21 cleavage site peptide as intracellular substrate. The number of Separase-active cells and the intercellular Separase activity distribution (expressed as SAD-value) were calculated for each sample.

Results: We have analyzed 33 BM samples derived from patients with MDS (n = 18, median age 67.4 years, range 27-85), with AML (n = 11, median age 61.3 years, range 25-73) and healthy donors (n = 4, median age 57 years, range 26-76). A tendency to increased numbers of Separase-active cells in MDS and AML (5.3 and 6.3 vs. 3.8 in healthy donors) and a higher variation of SAD values within the MDS group (inter-sample distribution 8.4, range 6.6-15.0) and AML group (inter-sample distribution 11.5, range 6.5-18) compared to healthy donors (inter-sample distribution 4.5, range 9.1-13.6) was observed. A significantly higher SAD value in sAML group was detected when compared to primary AML and MDS (sAML vs. MDS, p = 0.0345; sAML vs. AML, p = 0.0124).

Conclusion: We observed an increasing occurrence of highly variable Separase activity patterns concurring with malignancy. This high variability may reflect the transformation process from MDS into sAML. To corroborate this hypothesis with respect to clinical data, a higher number of patients and controls will be investigated.

Disclosure: No conflict of interest disclosed.

P217 – Recurrent mutations, expression analysis and functional characterization of cohesin subunits in myelodysplastic syndromes and acute myeloid leukemia

Abolfathi M.1, Schroeder T.2, Kartal-Kaess M.1, Bochtler T.1, Roßberg A.1, Jauch A.3, Haas R.2, Krämer A.1

1Clinical Cooperation Unit Molecular Hematology/Oncology, German Cancer Research Center (DKFZ) and Department of Internal Medicine V, University of Heidelberg, Heidelberg, Germany, 2Department of Hematology, Oncology and Clinical Immunology, Heinrich-Heine-University, Düsseldorf, Germany, 3Institute of Human Genetics, University of Heidelberg, Heidelberg, Germany

Myelodysplastic syndromes (MDS) are hematopoietic stem cell disorders that are associated with bone marrow failure and transformation into acute myeloid leukemia (AML). An increasing list of genes including the cohesin complex subunits STAG2, RAD21, SMC1 and SMC3A has been described to be mutated in both MDS and AML. Whether cohesin mutations contribute to leukemogenesis via interference with gene expression or chromosomal stability remains controversial. In this study, we analyzed a panel of 63 genes for mutations in 63 MDS blood or bone marrow samples using a targeted re-sequencing approach. STAG2 was mutated in only 3/63 (4.8%) of samples. No mutations in other cohesin components were found. On the other hand, STAG2 expression was lost in 18 out of 74 (24.3%) AML samples due to STAG2 mutations in 20% (2/10) and promoter methylation in 58.3% (7/12) of cases. In addition, we used CRISPR/Cas9 genome editing to knock out STAG2 in diploid, chromosomally stable HCT116-p53+/+ and HCT116-p53-/- cells. Whereas loss of STAG2 led to alterations in gene expression profiles in both cell lines, chromosome aberrations were only induced in the HCT116-p53-/- background. We conclude that the expression of STAG2 is lost in about one quarter of AML cases, frequently as a consequence of promoter methylation. Depending on the genetic background, both disturbed gene expression and aneuploidy are associated with loss of STAG2.

Disclosure: No conflict of interest disclosed.

P218 – Molecular tracking of somatic mutations in MDS patients with monosomy 7 receiving azacytidine

Dierks S.1, Martin R.1, Shumilov E.1, Bacher U.1, Ganster C.1, Shirneshan K.1, Flach J.1, Haase D.1

1Universitätsmedizin Göttingen, Hämatologie und Medizinische Onkologie, Göttingen, Germany

Introduction: Myelodysplastic syndromes (MDS) are heterogeneous hematopoietic stem cell disorders with clonal cytogenetic anomalies in ~50% of cases. In addition to characteristic cytogenetic aberrations like del(5q) or monosomy 7 (-7), recent studies detected somatic molecular mutations in 80-90% of MDS patients (pts). Demethylating agents e.g. 5-azacytidine (AZA, Vidaza®) are an effective option for high-risk MDS pts, especially if they have a monosomy 7. However, response to AZA is achieved in a part of pts only and remains temporary. Still the molecular mechanisms that underlie response as well as primary/secondary resistance are poorly understood.

Methods: Sanger sequencing (comprising 17 recurrently mutated genes) was used to identify mutations in high-risk MDS patients with -7 either as isolated or as combined aberration. Subsequently, peripheral blood samples of 2 patients with an isolated -7 were retrospectively analyzed from time points before, during and after AZA therapy by next generation sequencing (NGS) (sensitivity: ~5-10%).

Results: Both patients with a sole -7 were subjected to AZA therapy. Initial mutations were found in ASXL1, RUNX1 and U2AF1 (pt #1) and SRSF2, IDH2 and RUNX1 (pt #2), respectively. Retrospective NGS analyses before, during and after therapy sensitively detected and allowed reconstruction of molecular evolution of subclones disappearing or emerging during or after AZA. During follow-up Patient #2 acquired a transient JAK2mut as well as mutations in NOTCH1 and CUX1 after AZA was stopped. Thus, certain subclones with mutations in genes like RUNX1, which were associated with -7, responded well to AZA and disappeared, whereas other mutations in genes like CUX1 (Tumor suppressor gene on chr. 7q22.1) emerged after AZA discontinuation. Recently, a third pt with -7 has been identified and will likewise be treated and monitored.

Conclusion: The progression of certain clones under AZA may be associated with the emergence of new subclones and devolution of previously existing responsive mutations. Sensitive molecular follow-up analyses of these mutation patterns by NGS may improve the understanding and prediction of therapy response and resistance to demethylating agents in pts with -7. Therefore, improved surveillance of the molecular course under therapy using NGS technology may pave the way to more individualized therapeutic strategies for high-risk MDS pts.

Disclosure: No conflict of interest disclosed.

P219 – Deferasirox in the routine-treatment of MDS patients with chronic iron overload: interim results from the non-interventional, prospective study EXSEPT

Nolte F.1, Schumann C.2, Bueckner U.3, Schmidt B.4, Hebart H.5, Rubanov O.6, Kühn R.-B.7, Kreil S.2, Metzgeroth G.2, Johrs C.8, Albrecht S.8, Boch T.2, Hofmann W.-K.2

1St. Hedwig Krankenhaus, Berlin, Germany, 2University Hospital Mannheim, Medical Faculty Mannheim of the University of Heidelberg, Mannheim, Germany, Department of Hematology and Oncology, Mannheim, Germany, 3Office-based Hematology/Oncology specialist, Bochum, Germany, 4Office-based Hematology/Oncology specialist, Munich, Germany, 5Stauferklinikum Schwaebisch Gmuend, Internal Medicine, Mutlangen, Germany, 6Office-based Hematology/Oncology specialist, Hameln, Germany, 7Office-based Hematology/Oncology specialist, Oldenburg, Germany, 8Novartis Pharma GmbH, Nuremberg, Germany

Introduction: Myelodysplastic disorders (MDS) are oligoclonal stem cell disorders characterized by peripheral cytopenias and an increased risk for progression to acute myeloid leukemia. The majority of patients depends on regular red blood cell (RBC) transfusions during the course of their disease putting them at risk for transfusional iron overload (tIO). The iron chelator deferasirox (DFX) is approved for the treatment of tIO.

Methods: The non-interventional study EXSEPT included patients (pts) with IO treated with DFX. We performed a subgroup interim analysis in pts with MDS and tIO. DFX was prescribed according to market authorization. Pts were enrolled between 2010 and 2014 with a follow-up of 2 years. Efficacy was calculated by change in in serum ferritin (SF) in pts for whom SF values were available at 24 mo after DFX treatment start (n = 49). Safety was evaluated in pts with at least one follow-up visit after dosing of DFX (n = 280).

Results: 280 MDS pts were enrolled with a median age of 74 yrs (2-93 yrs). Most pts were classified as having either low or int-1 risk according to the IPSS Score (n = 167; 60%). At baseline transfusion frequency was 2-4 RBC per mo in most pts (57%). Mean daily starting dose of DFX was 15.6 mg/kg (SD 6.6) and 169 pts (60%) remained on the selected starting dose. Dose adjustments were done in 111 (40%) pts with dose increases reported in 65 pts (23%), mainly due to an initially planned dose escalation strategy (n = 45; 46%). Insufficient responses led to dose increase in 27 pts (28%). Mean SF levels declined from 2093 µg/mL at baseline (SD 1091) to 1751 µg/mL at 24 months (SD 1179), i.e. total mean change of -441 µg/mL (SD 1364). Premature discontinuation occurred in 195 pts (70%) after a mean time of 253 days (SD 177). Reason for discontinuation was death in 59 pts and adverse events (AEs) in 44 pts. AEs were reported in 249 pts; 32% of the AEs occurred within the first 3 mo. Most frequently reported AEs were decrease in renal creatinine clearance (73 pts; 26%), increase in blood creatinine (53 pts; 19%); fatigue (20 pts; 7%), diarrhea (42 pts; 15%) and nausea (28 pts; 10%).

Conclusions: Our data confirm the efficacy of DFX in reducing SF. Safety profile and discontinuation rates were as previously observed. While AEs remain a major reason for discontinuation, especially for older patients, dose adjustment, i.e. dose escalation strategies could be considered for improvement of treatment tolerability.

Disclosure: Florian Nolte: Advisory Role: Ja; Financing of Scientific Research: Ja; Expert Testimony: JaW.-K. Hofmann: Advisory Role: Ja; Financing of Scientific Research: Ja; Expert Testimony: Ja

P220 – The relevance of the international prognostic scoring system (IPSS) for patients with myelodysplasia (MDS) at higher age

Steinmetz H.T.1, Wahdat R.1, Haastert B.2, Sauer A.3, Lathan B.4, Lerchenmüller C.5, Tesch H.6, Germing U.7, Schmitz S.1

1Outpatient clinic, hematology + oncology, Cologne, Germany, 2mediStatistica, Neuenrade, Germany, 3Outpatient clinic, hematology + oncology, Potsdam, Germany, 4Outpatient clinic, hematology + oncology, Dortmund, Germany, 5Outpatient clinic, hematology + oncology, Münster, Germany, 6Outpatient clinic, hematology + oncology, Frankfurt a.M., Germany, 7University Hospital, hematology + oncology, Düsseldorf, Germany

The IPSS was developed to estimate the prognosis of patients (pts) with MDS in a cohort of 816 pts of academic centers with a median age of 69 years (y). It was an aim of the regular care MDS-registry to describe the age distribution of MDS in regular care and the meaning of the IPSS in various classes of age.

Methods: Pts with written informed consent could be included if a bone marrow biopsy has been performed. They were eligible if basic data and the quarterly course of the disease were documented. Statistical analysis: Data were described overall and stratified by age classes. Corresponding global tests were performed (Chisquare, Kruskal-Wallis). Survival was estimated by Kaplan-Meier curves stratified by IPSS and age being compared by a bivariate Cox regression model.

Results: Between July 2009 and March 2016 (81 months) 2,118 pts from 90 institutions mainly outpatient practices, were documented and eligible. Median age of 843 (39.8%) women and 1,275 (60.2%) men were 74.9y (min-max: 26.5 - 94.2). The duration of observation, frequencies of IPSS, need for transfusions (Tx) prior to diagnosis (d), and the Charlson comorbidity index (CCI) are given in the table.


Higher age and IPSS risk were significantly associated with the risk of death, an additional interaction between age and IPSS risk was significant (p = 0.0198, Cox model).


Conclusion: Prognostic properties of IPSS seem to be lower in higher age groups, which are more prevalent in the MDS pts in the German regular care registry. So it will be important to look for additional prognostic factors for older pts.

Supported by an unrestricted grant from Celgene and Novartis.

Disclosure: Hans Steinmetz: Employment or Leadership Position: CEO X-Med GmbH und Onkologie Köln; Advisory Role: Amgen; BMS, Boehringer-Ingelheim, Celgene, Hexal-Sandoz, Medice, Novartis; Janssen-Cilag; Pharmacosmos; Vifor; Stock Ownership: Kommanditist von GermanOncology; Financing of Scientific Research: KV-No, Privatpatienten; Expert Testimony: Studienleitung IIT/NIS: Amgen, Celgene, Novartis, Vifor. Studienarzt für Studien folgender Sponsoren: AIO, Amgen, BMS, Celgene, IoMedico, Janssen-Cilag, Kompetenznetz Maligne Lymphome und KN Akute Leukämien, Lilly, Merck, Novartis, Pfizer, Pharmacosmos; Other Financial Relationships: Reisekosten: Alexion, Amgen, Bayer, BMS, Celgene, Janssen-Cilag, Novartis, Sanofi, Vifor; Immaterial Conflict of Interests: Mitgliedschaften: DGHO, ESMO, DGPM, BNHO, NIONoStephan Schmitz: Employment or Leadership Position: CEO X-Med GmbH und Onkologie Köln; Advisory Role: Amgen; BMS, Boehringer-Ingelheim, Celgene, MSD, Novartis; Janssen-Cilag; Stock Ownership: Kommanditist von GermanOncology; Financing of Scientific Research: KV-No, Privatpatienten; Expert Testimony: Studienarzt für Studien folgender Sponsoren: AIO, Amgen, BMS, Celgene, IoMedico, Janssen-Cilag, Kompetenznetz Maligne Lymphome und KN Akute Leukämien, Lilly, Merck, Novartis, Pfizer; Other Financial Relationships: Reisekosten: BNHO,; Immaterial Conflict of Interests: Mitgliedschaften: DGHO, ESMO, ASCO, DGPM, BNHO, NIONo.

P221 – Successful management of Thrombotic thrombocytopenic purpura in early pregnancy with maternal and fetal survival

Voskova D.1, Greul R.1, Lenger D.1, Hellmich U.2, Siedler D.3, Fridrik M.A.1, Fuchs D.1

1Kepler Universitätsklinikum, Klinik für Interne 3 - Schwerpunkt Hämatologie und Onkologie, Linz, Austria, 2Kepler Universitätsklinikum, Klinik für Interne 2 - Schwerpunkt Nephrologie, Endokrinologie/Diabetologie, Rheumatologie, Hepatologie, Linz, Austria, 3Kepler Universitätsklinikum, Klinik für Gynäkologie, Geburtshilfe und gynäkologische Endokrinologie, Linz, Austria

Thrombotic thrombocytopenic purpura is a rare disease and usually presents with microangiopathic hemolytic anemia and thrombocytopenia (1). Pregnancy is a known risk factor for a first occurrence of TTP, but TTP in early pregnancy is extremely rare. The best course of management is unclear (2).

We present the case of a nulliparous, 28-year-old women who presented with microangiopathic hemolytic anemia and severe thrombocytopenia (15 G/l) in the 10th week of gestation. TTP was confirmed by ADAMTS13 levels (< 1%) and detectable antibodies. Bone marrow biopsy was unremarkable and the pregnancy was intact.

The patient was treated with plasma exchange and steroids (prednisone 1mg/kg/day). Although she developed an anaphylactic reaction to plasma exchange after three sessions, ADAMTS 13 levels had improved and thrombocytes were normalized. She was continued on prednisone (tapered and discontinued in gestational week 30) and enoxaparine 40mg/day. She delivered a healthy male infant per caesarean section in the 37+4 week of gestation.

There are fewer then 30 published cases of TTP in early pregnancy in the literature. Maternal survival was excellent, but fetal survival remained poor in the majority of published cases.

We could show that TTP in early pregnancy can be successfully managed with plasma exchange with fetal and maternal survival.


1 Moschcowitz E.: An acute febrile pleiochromic anemia with hyaline thrombosis of the terminal arterioles and capillaries: an undescribed disease. Arch Intern Med. 1925 Jan 1;36(1):89-93.

2 Scully M, Thomas M, Underwood M, Watson H, Langley K, Camilleri RS, et al.: Throm-botic thrombocytopenic purpura and pregnancy: presentation, management, and sub-sequent pregnancy outcomes. Blood. 2014 Jul 10;124(2):211-219.

Disclosure: No conflict of interest disclosed.

P222 – Aortic thrombosis leading to the diagnosis of Paroxysmal Nocturnal Hemoglobinuria (PNH)

Alashkar F.1, Schemuth H.2, Herich-Terhürne D.1, Dührsen U.1, Röth A.1

1Universitätsklinikum Essen, Westdeutsches Tumorzentrum, Klinik für Hämatologie, Essen, Germany, 2Universitätsklinikum Essen, Institut für Diagnostische und Interventionelle Radiologie und Neuroradiologie, Essen, Germany

Introduction: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare clonal blood disorder of the hematopoietic stem cells characterized by chronic, uncontrolled complement-mediated intravascular hemolysis and platelet activation resulting in thrombophilia with thromboembolic events (TEs) and cytopenia due to bone marrow failure. Eculizumab, an anti-C5 monoclonal antibody, has been proven to reduce PNH-related complications, including TEs.

Patient / Results: In Sep 2010 a 57-year-old female patient was referred to a general hospital for investigation due to recurrent, unexplainable abdominal painful crises being evident since 2008. Initially, the diagnosis of a perforated sigma diverticulitis with an abdominal abscess was made, and a sigma resection was performed. However, abdominal crises continued by Oct and Dec 2010 and further invasive work-up showed gastritis. Surprisingly, an endoscopic ultrasound of the stomach indentified a partial thrombosis of the thoracic and abdominal aorta (hemoglobin (Hb) 11.4g/dL, platelets (PLTs) 129/nL, lactate dehydrogenase (LDH) 287U/L). The medical history was otherwise unremarkable for TEs, thus, phenprocoumon was initiated and switched to acetylsalicylic acid by Aug 2013, as the aortic thrombus underwent regressive changes in the follow-up CT-scan. In Sep 2013 abdominal painful crises relapsed, and even a papillotomy was performed due to the impression of a papillary sclerosis with mild dilation of the ductus choledochus. With the development of a progressive thrombocytopenia (70/nL), the diagnosis of PNH was made by flow-cytometry.

The patient was referred to our centre in Dec 2013 and treatment with eculizumab was initiated because of symptomatic PNH (thrombosis, abdominal crises). By the time of presentation, laboratory evaluation revealed a mild hemolytic anemia (11.1g/dL) with an increased LDH (1167U/L), an increase of D-dimer (1mg/L), and a PNH clone size of 63% (Gran., FLAER). Until now, eculizumab is well tolerated and no further abdominal crises or TEs were observed (Hb 9.9g/dL; PLTs 59/nL; LDH 295U/L; D-dimer 0.28mg/L).

Conclusion: In PNH up to 40% of the patients develop TEs and in 21% of the cases, TEs precede the diagnosis of PNH. Abdominal pain, dyspnea or chest pain are possible predictors for TEs. Screening for thrombophilia should include PNH diagnostics by flow-cytometry in patients with thromboembolic complications and evidence of hemolysis, atypical thrombotic locations or unexplained thromboses.

Disclosure: Ferras Alashkar: Other Financial Relationships: Reisekostenerstattungen: Alexion PharamaceuticalsAlexander Röth: Advisory Role: Alexion Pharamaceuticals, Novartis, Roche; Financing of Scientific Research: Alexion Pharamaceuticals; Expert Testimony: Alexion Pharamaceuticals, Geron; Other Financial Relationships: Reisekostenerstattungen: Alexion Pharamaceuticals

P223 – Infection control with granulocyte transfusions in very severe aplastic anemia (VSAA)

Farsijani N.M.1, Dührsen U.1, Röth A.1

1Uniklinik Essen, Hämatologie, Essen, Germany

Introduction: Aplastic anemia commonly results from auto-reactive T-cell-mediated hematopoietic stem cell (HSC) suppression, mandating intensive immunosuppressive therapy (IST) with cyclosporine A and antithymocyte globulin when HSC transplantation is not feasible. Good IST response rates up to 80% are hampered by fatal infections due to neutropenia and therapy-related immunosuppression. To replenish the neutropenic immune system granulocyte substitution with allogeneic granulocytes has been introduced since the 1960s. However, lack of sufficiently powered clinical trials has rendered this treatment approach controversial and rarely used.

Patient/Results: A 72-year-old woman was diagnosed with VSAA and subsequently treated with IST in 10/2014. 2 weeks after IST initiation she was admitted to the hospital with neutropenic fever and a CRP value of 14.7 mg/dl. Broad spectrum anti-infective therapy was initiated but despite the use of several different agents including piperacillin/tazobactam, clarithromycin, meropenem, aciclovir, voriconazole, and liposomal amphotericin B (LAMB), fevers aggravated with rising CRP values up to 33.0 mg/dl. Chest computer tomography (CT) revealed a solitary infiltrate in the left lower pulmonary lobe, highly suspicious of invasive aspergillosis. With lack of other treatment options allogeneic granulocyte transfusions were initiated starting in 12/2014. Additionally to conventional anti-infective therapy with meropenem, clarithromycin, and LAMB, 4 granulocyte transfusions were carried out every other day. Per transfusion the total PMNC number ranged from 11.97*10^9 to 73.61*10^9 with an average PMNC number of 50.88*10^9. This led to a significant increase in PMNC blood counts, and associated with falling CRP values down to 6.9 mg/dl and resolution of fever. 7 days after the last transfusion CRP values rose again without change in conventional anti-infective medication, so that granulocyte transfusions were re-initiated. This led to an immediate response with persistent CRP values < 5 mg/dl and hospital discharge. Control chest CT confirmed resolution of the pulmonary infectious focus.

Conclusion: Granulocyte transfusions represent a valuable treatment option in refractory neutropenic infections, warranting reliable randomized trials. Especially in the setting of VSAA where neutrophil recovery after ISH is anticipated granulocyte transfusions should be considered when other anti-infective treatments have failed.

Disclosure: Navid Farsijani: No conflict of interest disclosed.Alexander Röth: Advisory Role: Alexion, Novartis; Expert Testimony: Alexion

P224 – Care situation of patients with iron deficiency/iron deficiency anaemia in Germany

Hubmann M.1, Maier D.2, Rosmolen J.C.3, Dietzfelbinger H.1

1Internistische Praxis für Hämatologie und Onkologie, Herrsching, Germany, 2emphasis – Institut für Marktforschung im Gesundheitswesen GmbH, München, Germany, 3Vifor Pharma Deutschland GmbH, München, Germany

Introduction: Iron deficiency (ID) is one of most severe and important nutritional deficiencies worldwide and is thought to be the most common cause of anaemia (approx. 80%). Despite its possible severe clinical sequelae, ID is often underdiagnosed and undertreated. A market research study examined how often practising physicians in Germany diagnose ID/iron deficiency anaemia (IDA), its main symptoms, diagnostics and treatment.

Methods: Between 09/2015 and 03/2016, 827 general practitioners and internists were asked in a semi-structured interview how they diagnose and treat ID/IDA. They answered 22 detailed questions based on specific patient data from the previous quarter. Data evaluation was anonymised and performed for Germany as a whole.


Patient structure: The respondents treat a mean of 27.9 ID/IDA patients (median: 20; range: 1-400) per quarter. The most common cause of ID/IDA was cited as hypermenorrhoea (30%), followed by gastroenterological disorders (21%), chronic heart failure (10%), renal failure (10%) and cancers (9%).

Symptoms: ID/IDA patients most often report exhaustion (93%), fatigue (92%) and impaired capacity (85%).

Diagnostics: Most respondents cited Hb (95%) and serum ferritin values (87%) as standard parameters in ID diagnosis. 40% cited transferrin saturation (TSAT) and 28% C-reactive protein (CRP) as standard diagnostics.

Therapy: 91% of asymptomatic ID/IDA patients usually undergo initial oral therapy, whereas ? of symptomatic patients are given i.v. iron. In severe chronic diseases, such as inflammatory bowel disease (IBD) or cancers, i.v. iron is often first-line treatment (56/43%, respectively). Reasons for switching from oral to i.v. therapy include intolerance and insufficient response to oral therapy (95%) or an urgent need for iron (89%).

Conclusions: In practice, severe chronic diseases, as well as hypermenorrhoea, are relevant causes of ID/IDA. In primary diseases often accompanied by a chronic inflammatory reaction (IBD, renal failure, heart failure, cancers), ID is treated slightly more often with i.v. iron products. The diagnostics cited are often insufficient to confirm ID (most respondents did not use CRP and TSAT as standard diagnostics). The decision on whether to use oral or i.v. iron replacement was apparently guided more by ID/IDA symptoms than by pathophysiological considerations.

Disclosure: Max Hubmann: No conflict of interest disclosed.Hermann Dietzfelbinger: Other Financial Relationships: Sponsoring für Herrschinger Hämato-Onkologie-Symposien

P225 – c-Cbl regulates c-Mpl receptor trafficking and its internalization

Saur S.J.1, Märklin M.1, Ganser M.1, Kanz L.1, Kopp H.-G.1, Müller M.1

1Universitätsklinikum Tübingen, Medizinische Klinik und Poliklinik, Hämatologie und internistische Onkologie, Tübingen, Germany

Megakaryopoiesis is controlled by a variety of hematopoietic growth factors to maintain physiological levels of circulating platelets. Thrombopoietin (TPO) signalling via its receptor c-Mpl is a key regulator of megakaryopoiesis. Consequently, TPO/c-Mpl signalling needs to be tightly regulated to maintain physiological megakaryopoiesis. One of the most effective mechanisms to permanently disable activated signalling proteins is by targeted degradation via lysosomes or proteasomes. Previous studies have identified c-Cbl as an E3 ligase responsible for the ubiquitination of c-Mpl in cell lines. In this study, we investigated the mechanisms of TPO-mediated c-Mpl degradation in primary mouse cells.

In order to determine the potential role of c-Cbl in murine megakaryopoiesis we used a conditional PF4-Cre c-Cbl knockout (ko) mouse model to specifically delete c-Cbl in the megakaryocytic lineage. Megakaryocytes were generated in vitro by culturing bone marrow from WT and conditional c-Cbl ko lines for 72 hrs in the presence of rmTPO.

Conditional c-Cbl ko mice showed significant bone marrow megakaryocyte hyperplasia. Platelet counts were significantly elevated and platelets were of smaller size in c-Cbl ko mice as compared to control mice. We furthert demonstrate that there were more young platelets produced within a 24 h period in the c-Cbl ko mice although the half-life of platelets was similar in the both cohorts. C-Cbl ko mice showed a severe defect in thrombus formation as assessed in an in vivo thrombus formation model with Fe3Cl. Although TPO plasma levels were increased in the c-Cbl ko mice, there was no difference in liver mRNA levels in the two cohorts. We found that c-Cbl ko mice express more c-Mpl on protein and mRNA levels compared with wild type control. In addition, c-Mpl surface expression was reduced and internalization of the receptor was significantly impaired after TPO stimulation in c-Cbl ko mice. After incubating platelets in vitro with TPO, we found c-Cbl ko platelets to show a severe TPO uptake defect compared with wild type control platelets.

Taken together, we demonstrated that c-Cbl ablation leads to a reduced c-Mpl surface expression and an impaired internalization, which results in increased plasma TPO levels culminating in increased megakaryopoiesis. These data enhance our understanding of the regulation of TPO signalling and the physiological role of c-Cbl in the megakaryocytic lineage.

Disclosure: No conflict of interest disclosed.

P226 – Evaluation of the XN Series Bodyfluid Software with special consideration of oncological samples

Hughes D.1, Stamminger G.1

1Labor Chemnitz, Chemnitz, Germany

Objective: To evaluate the Body fluid software of the Sysmex XN analyser series and its capability in accurately screening samples containing suspected tumor cells

Methods: A total of 118 serous body fluid samples were analysed over a period of 4 months on the XN 1000 (Sysmex) analyser using the body fluid software. Samples comprised of 46 ascites (A) and 42 pleural (P) fluid (collected in EDTA tubes) and 30 CSF samples obtained from in-house patients, all of which were analysed within 2 hours of receipt. Manual cell differential, evaluation of cell changes and screening of tumor cells was performed on each sample following cytospin and staining. The ‘high fluorescent’ cell count (HF) and the pre-determined cut off value of =100µl by the manufacturer were then compared against microscopic examination.

Results: Following microscopic examination 13 samples (P = 8, A = 3, CSF = 2) were manually flagged as having suspected tumor cells, 8 of which were immunohistochemically confirmed by the pathology department as malignant. Five samples including both CSF samples were not examined by the pathologist but were from patients with already confirmed malignancy. Of these 8 samples 4 (50%) had an average HF value of 33µl. A further 2 samples which were above the cut-off were found to only strongly reactive in nature. The remaining two samples which incidentally had very high cell counts (>1500µl) had HF values above the cut-off. Furthermore 9 samples which had a count greater than 100µl but no tumor cells and also examined by the pathologist were found to be reactive in nature. The high HF count was due a high number of benign mesothelial cells which are also highly fluorescent.

Conclusion: In high concentrations the body fluid software can be relied upon in screening samples which may contain tumor cells. However at lower counts its capability is questionable. The cut-off of 100µl may allow an unsatisfactory number of samples to slip the net. By reducing the cut-off value to 33µl for example this raises the question of whether the body fluid software leads to any added benefit in screening samples for tumor cells. With lowered cut-off values the number of samples which require microscopy would only be marginally reduced.

Disclosure: No conflict of interest disclosed.

Posterdiskussion – Multiples Myelom 1

P227 – Influence of Histone Deacetylase Inhibitors (HDACis) on the expression of adhesion molecules and modern target structures in Multiple Myeloma (MM)

Müller S.1, Senger J.2, Waldschmidt J.M.1, Wider D.1, Thomsen A.3, Ihorst G.4, Duyster J.1, Hug M.J.5, Jung M.2, Wäsch R.1, Engelhardt M.1

1Universitätsklinikum Freiburg, Department für Hämatologie, Onkologie und Stammzelltransplantation, Freiburg i. Br., Germany, 2Institut für Pharmazeutische Wissenschaften, Alber-Ludwigs-Universität, Freiburg i. Br., Germany, 3Universitätsklinikum Freiburg, Department für Radiologische Diagnostik und Therapie, Freiburg i. Br., Germany, 4Universitätsklinikum Freiburg, Studienzentrum, Freiburg i. Br., Germany, 5Universitätsklinikum Freiburg, Klinikumsapotheke, Freiburg i. Br., Germany

Background: During the last decades the vital role of the bone marrow (BM) has increasingly been explored to elucidate the progression and drug resistance in MM, this leading to advances of treatment strategies to modulate the interaction between malignant plasma cells (PCs) and the microenvironment. We here examined the influence of HDACis on the BM niche in a novel 3D co-culture.

Methods: The cells are seeded in conical microwells and their proliferation and viability are assessed. These properties are compared both in presence or absence of stroma and 2D cultures. Cells are incubated with HDACis, their synergisms with established antimyeloma drugs and stroma effects upon the treatment can be visualized via flow-cytometry and viability assays. The expression levels of relevant surface molecules (CXCR4, CD38, SLAM-F7, CD138) in primary MM cells and MM cell lines (MMCLs) are determined using FACS-analysis.

Results: Inside the microcavities, PCs showed a decreased cell proliferation rate compared to 2D cultures. Cell growth and expression pattern of surface proteins were further influenced by the presence of BM stroma cells (BMSCs), leading to a decrease in CXCR4 but persistence of CD138 levels. Our current results suggest evident differences regarding growth and treatment response in our 3D versus 2D culture systems, which we currently validate using HDACi as a novel substance class in MM. The novel selective HDAC-6 inhibitor JS28 was selected from more than 20 compounds due to trypsin-dependent in vitro HDAC-inhibition assay results. The phenotypic HDACi effect was determined with assessment in 2 MMCLs U266 and RPMI 8226: treatment of MM cells were performed with use of panobinostat or JS28 for 48 hours, either alone or combined with bortezomib, to test synergism. The calculated combination indices (CI) in both MMCLs were < 1 (=synergistic) within a range of approximately EC40 to EC55 for both combined treatment schedules.

Conclusions: Our current data underline the importance of accurate 3D co-culture models to mimic in vivo proliferation and drug resistance and to predict later clinical treatment success more effectively. So far, there is insufficient data that conclusively unravel the complex interactions between HDACis, their different subtype selectivity and the increasing number of treatment options in MM. In this context, our ongoing investigations will focus on expression of adhesion molecules, their targeting and their alteration by HDACis.

Disclosure: No conflict of interest disclosed.

P228 – Single cell subfractions in the bone marrow vary in their impact on myeloma growth and display potential targets to antagonize MM-stromal protection

Waldschmidt J.M.1, Wider D.1, Müller S.1, Follo M.1, Klein C.1, Thomsen A.R.2, Herget G.3, Südkamp N.P.3, Wäsch R.1, Duyster J.1, Engelhardt M.1

1Universitätsklinik Freiburg, Dept. Hämatologie, Onkologie und Stammzelltransplantation, Freiburg, Germany, 2Universitätsklinik Freiburg, Dept. Strahlentherapie, Freiburg, Germany, 3Universitätsklinik Freiburg, Dept. Orthopädie und Traumatologie, Freiburg, Germany

Introduction: The bone marrow (BM) as a specific tumor microenvironment crucially contributes to quiescence, drug resistance and ultimately to progression of multiple myeloma. However, little is known about the distinct role of niche cell subfractions and their protective impact on myeloma. Our focus here was to more closely study the cellular composition and function of the BM niche by utilizing a novel bone-derived in vitro 3D co-culture platform.

Methods: This adhesion-independent three-dimensional co-culture model consisted of an agarose matrix interlayer containing 100 microwells/cm². Each microwell was 1.5mm in depth and permeable for oxygen and cytokines, but not for BMSCs. MMCL and primary BM patient (pt) cells were utilized with and without (w/o) HS-5 vs. M210B4 stromal support. Analyses included trypan blue, Annexin/PI, MTT, FACS, cell cycle analyses and H2B-mCherry/cytochrome c-GFP assays (Udi, BJH 2013). To examine the effect of distinct BM niche cell populations on MM growth, niche cell subsets from C57BL6J mice were acquired, digested and FACS-sorted to collect cell subfractions of mesenchymal stem and progenitor cells (MSPC), endothelial cells, osteoblasts, premature CD146+ MSPCs (PaS) and CXCL12-abundant reticular cells (CaRs).

Results: Pt samples after 7 days (d) of culture benefitted from both murine M210-B4 and human HS-5 co-culture. FACS-sorting of murine bone and BM cells led to valid subset separation, illustrated by the multipolar morphology of CD31+ endothelial cells and CD31-, CD45-, Sca1+ MSPCs with fibroblast-like bipolar appearance, whereas PaS cells remained undifferentiated and positive for CD146. We observed that C57BL6J-derived murine cells differed in terms of their growth support for OPM-2 cells after 6d of co-culture: MSPCs from murine BM were more beneficial than those derived from murine bone. BM-MSPCs also induced stronger support than premature MSPCs and CXCL12-abundant reticular cells, commonly considered to be crucial mediators of adhesion in the murine niche.

Conclusions: We present a 3D culture model which reflects stromal protection of MM cells and may prove more reliable for ex-vivo drug screening and longitudinal studies. To overcome the limitation of a murine co-culture model, current analyses focus on the generation of human BM cell subsets from both MM pts and control patients suffering from orthopedic diseases.

Disclosure: No conflict of interest disclosed.

P229 – Lenalidomide enhances MOR202 dependent macrophage-mediated effector functions via the vitamin D pathway

Bruns H.1, Busch L.1, Böttcher M.1, Mougiakakos D.1, Bittenbring J.T.2, Nolting J.3, Bisht S.3, Büttner M.4, Rehli M.5, Wimmer J.5, Volmer D.6, Beier F.7, Gezer D.7, Neumann F.2, Bach C.1, Balzer H.1, Moi S.1, Brossart P.3, Mackensen A.1

1Medizinische Klinik 5, Universitätsklinikum Erlangen-Nürnberg, Erlangen, Germany, 2Medizinische Klinik 1, Saarland University Medical School, Homburg, Germany, 3Medizinische Klinik III, Klinik für Hämatologie, Onkologie und Rheumatologie, Bonn, Germany, 4Institut für Pathologie, Erlangen, Germany, 5Department of Internal Medicine III, Hematology and Oncology, University Hospital of Regensburg, Regensburg, Germany, 6Institute of Bioanalytical Chemistry, Saarland University, Saarbrücken, Germany, 7Department of Oncology, Hematology and Stem Cell Transplantation, RWTH Medical School, Aachen, Germany

The bone marrow niche plays a critical role in determining the fate of malignant plasma cells in multiple myeloma (MM). Macrophages are an abundant component of the stromal cell compartment and are believed to support survival and drug resistance of MM cells. Conversely, macrophages are key immune effector cells for the therapeutic effect of monoclonal antibodies. However, myeloma-associated macrophages (MAMs) regularly fail to exert direct effector functions. Lenalidomide, an immunomodulatory agent that enhances antibody dependent cell mediated cytotoxicity (ADCC), has the potential to synergize with MOR202, an anti-CD38 monoclonal IgG1 antibody currently in phase I/IIa for the treatment of MM. Furthermore, vitamin D plays a key role in regulating effector functions of human macrophages. This is closely linked to the expression of the vitamin D-1-hydroxylase CYP27B1, which catalyzes the conversion of 25-hydroxy-vitamin D (25D) to the bioactive form 1,25-di-hydroxy-vitamin D (1,25D). We have previously shown, that vitamin D promotes tumoricidal activity of macrophages and improves the efficacy of rituximab-dependent cytotoxicity. Therefore, we hypothesized that the combination of MOR202 with lenalidomide and MOR202 with 1,25D would enhance the MOR202- dependent macrophage-mediated effector functions against MM cells.

Here we report that MAMs exhibit an altered vitamin D metabolism with a reduced expression of the vitamin D receptor (VDR) and CYP27B1. As a consequence MAMs cannot convert 25D into bioactive 1,25D. Given the importance of the vitamin D pathway for antibody mediated cytotoxicity, we screened several drugs for their ability to restore the vitamin D pathway in human macrophages. We found, by RNA-sequencing, that lenalidomide treatment modulates the phenotype of macrophages and isolated MAMs, and that lenalidomide significantly increases the expression of the VDR and CYP27B1. Furthermore, we demonstrate that isolated MAMs regularly fail to eliminate primary MM cells, and that the lack of effector functions can be overcome by treatment with lenalidomide and vitamin D. Moreover, we show that MOR202-dependent elimination of MM cells is enhanced by pre-treatment of isolated MAMs with lenalidomide and supplementation of vitamin D.

In summary, these data show that vitamin D is essential for the effector functions of MAMs and that the therapeutic activation of the vitamin D pathway by lenalidomide may restore their tumoricidal effector mechanisms.

Disclosure: Heiko Bruns: Expert Testimony: Finanzierung des Projektes von Morphosys und CelgeneAndreas Mackensen: No conflict of interest disclosed.

P230 – Long term in vitro survival of plasma cells derived from bone marrow of multiple myeloma patients

Waechter M.1, Nogai A.1, Kühnel A.1, Wulf-Goldenberg A.2, Kunitz A.1, Blau O.1, Schmidt-Hieber M.3, Pezzutto A.1, Jehn C.1, Vuong L.1, Doerken B.1, Blau I.W.1

1Charité University Berlin, Department of Hematology, Oncology and Tumorimmunology, Berlin, Germany, 2Berlin-Buch GmbH, Experimental Pharmacology & Oncology, Berlin, Germany, 3Helios Kliniken Berlin, Hematology, Berlin, Germany

Introduction: Multiple Myeloma (MM) is a neoplastic B-cell malignancy strongly regulated by bone marrow microenvironment. In vitro co-culture models or in vivo systems using immune deficient mice are normally used for clarification of specific interactions of MM-cells and BMSC and to identify new therapeutic targets.

Unfortunately, mesenchymal stem cells are a very rare cell population in bone marrow aspirates (< 0,01%) and their expansion can take several weeks of in vitro culture. MM cells on the other hand are more frequent but don’t survive in culture, separated from their supportive microenvironment.

The purpose of our study was to establish a culture system capable of keeping primary MM cells alive over a period of several weeks and thus making it possible to use them together with the expanded MSCs in co-culture or animal trials.

Methods: Mononuclear cells (MNCs) were separated from bone marrow aspirates by Ficoll density gradient centrifugation and characterized by FACS analysis for detecting the size of the MM cell population (CD45-/CD138+). Weekly culture monitoring was done by cell counting using hemocytometer, FACS analysis and Trypan blue staining. After 4 or 5 weeks of culture, cytospins were performed and staining with the Hemacolor staining kit (MERCK) followed by cytomorphological inspection. The monoclonality of the MM cell population was proved by detecting intracellular light chain expression using Simultest anti Kappa/ anti Lambda (BD). The vitality of the MM cells was tested by Annexin staining using the Annexin V-FITC Kit (miltenyi Biotec) as recommended by the manufacturer.

Results: Long term cultures of bone marrow MNCs derived from 8 Patients with MM were established and maintained for several weeks. The size of MM cell population in bone marrow aspirates of patients varied between below 10% and more than 50% of the MNCs. Proportion of dead cells (trypan blue positive cells) sustained during the culture period at about 20%. At the end of the culture up to 85% of the MM cells presented as alive in the Annexin test. Both the expression of CD138+ and light chain type were held steady.

Conclusions: MM cells are able to survive in vitro together with all their accessory cell types present in the MNC fraction of bone marrow aspirate over a period of several weeks. This makes it possible to apply MSCs and MM cells from the same donor simultaneously for co-culture or animal trials.

Disclosure: Marlies Waechter: No conflict of interest disclosed.Igor Wolfgang Blau: Expert Testimony: Grant Celgene

P231 – RalA and RalB are RAS-independent targets in multiple myeloma cells

Seibold M.1, Stühmer T.1, Schmiedl N.1, Mottok A.2, Rosenwald A.2, Chatterjee M.1, Einsele H.1, Bargou R.C.3, Steinbrunn T.1

1Universitätsklinikum Würzburg, Medizinische Klinik und Poliklinik II, Würzburg, Germany, 2Universität Würzburg, Pathologisches Institut, Würzburg, Germany, 3Comprehensive Cancer Center Mainfranken, Würzburg, Germany

Introduction: Ral has been branded as a putative effector pathway downstream of oncogenic RAS in several cancer entities, and may promote proliferation, survival and drug resistance of multiple myeloma (MM) cells. We used shRNA-mediated knockdown of RalA and RalB isoforms to appraise their role as potential therapeutic targets.

Methods: First, we performed immunohistochemical staining of primary bone marrow trephines of MM patients and Western blotting in MM cell lines to evaluate Ral protein expression. Next, knockdown of RalA or RalB was achieved with transient or stable transfection of MM cell lines by electroporation and the effect on cell survival and apoptosis was measured with flow cytometry using annexin V-APC/propidium iodide staining. To test potential dependence of Ral on oncogenic KRAS or NRAS, Ral pulldown assays were applied.

Results: Whereas RalA was prominently expressed in the majority of primary MM cells and MM cells lines, RalB showed modest and heterogeneous expression levels. No obvious correlation with oncogenic RAS mutations could be observed. Abrogation of RalA by shRNA-mediated knockdown impaired MM cell survival in two thirds of the tested cell lines, whereas depletion of RalB did not induce relevant levels of apoptosis in the majority of cells.

Surprisingly, Ral activity was proven to be independent of oncogenic KRAS or NRAS mutations at positions 12 or 61.

Conclusion: Ral may provide a potential therapeutic target in multiple myeloma independent of the presence of oncogenic RAS mutations.

Disclosure: No conflict of interest disclosed.

P232 – Cyclin D1 C.870G>A polymorphism in patients with multiple myeloma, impact of donor Cyclin D1 C.870G>A polymorphism on the outcome after transplantation

Puckert F.M.1, Blau I.W.1, Kühnel A.1, Nogai A.1, Rieger K.1, Kunitz A.1, Hemmati P.1, Arnold R.1, Pezzutto A.1, Doerken B.1, Blau O.1

1Charité University Berlin, Department of Hematology, Oncology and Tumorimmunology, Berlin, Germany

Background: Deregulation of cyclin D-group is considered an important factor in the pathogenesis of a large number of cancers. Since overexpression of cyclin D disturbs the G1/S transition in the cell cycle, it is considered one of the early key molecular events of cancerogenesis. Previously published data shown a relationship between cyclin D1 (CCND1) c.870G>A polymorphism and risk of t(11;14) in patients with multiple myeloma (MM). Little is known about the prognostic impact of CCND1 c.870G>A polymorphism in MM patients. Moreover, there is no evidence about prognostic impact of the donor CCND1 c.870G>A polymorphism on the outcome after allogeneic stem transplantation (alloSCT) in MM patients.

Methods: Peripheral blood samples from 250 MM patients were analyzed. Age of patients ranged from 58 to 85 (at median, 57). In addition, samples from 75 pairs MM patients and stem cells donors were studied. To identify CCND1 c.870G>A polymorphism, PCR assay with endonuclease restrictions was developed.

Results: In all MM patients, CCND1 c.870G>A polymorphism was strongly associated with the t(11;14)(q13;q32) (P < 0.001). In MM patients after alloSCT, c.870G-genotype in donors cells was statistically correlated with poor outcome after transplantation (P < 0.01). Moreover, only patients transplanted with “G-Genotype” donors developed secondary cancer after AlloSCT (P < 0.01). No secondary tumors were diagnosed in the group of MM patients received grafts from “A-, AG-genotype” donors.

Conclusions: Our results suggest the published data that constitutive genetic factor (CCND1 c.870 G>A polymorphism) is associated with a specific chromosomal translocation in patients with MM.

Moreover, we could propose significant impact of genetic polymorphism in donor cells on the outcome after stem cell transplantation.

Disclosure: No conflict of interest disclosed.

P233 – Analysis of metabolic factors in modulation of interaction between plasma cells and mesenchymal stromal cells in multiple myeloma patients in vitro

Kühnel A.1, Waechter M.1, Kunitz A.1, Nogai A.1, Blau O.1, Pezzutto A.1, Doerken B.1, Blau I.W.1

1Charité University Berlin, Department of Hematology, Oncology and Tumorimmunology, Berlin, Germany

Introduction: Within the progression and proliferation of multiple myeloma (MM), bone marrow mesenchymal stromal cells (BMMSCs) represent a crucial component in creating a tumor supportive MM microenvironment. Regarding the increasing interest in cancer-metabolism, proliferating cancer cells demand elevated nutrient supply, resulting in an anabolic metabolism described as ‘Warburg effect’. Various GLUT transporters as well as the proliferator-activated receptor-? coactivator 1a (PGC-1a) and the central growth pathway PI3K/AKT/mTOR are upregulated in MM, contributing to an anabolic state. In the present study, we investigated the role of BMMSCs regarding the Warburg effect by identifying metabolic key molecules in a co-culture system.

Methods: BMMSCs from patients (MM-BMMSCs, n = 25) and donors (HD-BMMSCs, n = 5) were isolated and co-cultured with KMS12-PE and JJN-3 cells. Protein levels of GLUT1, GLUT4, PGC-1a and PI3K of mono-and co-cultured cells were detected via western blotting. Pyruvate-kinase activity and Lactate-dehydrogenase activity were measured using customary kits (Pierce™).

Results: We have found the increasing of GLUT4 expression in the co-culture of KMS12-PE with MM-BMMSCs compared to mono cell culture. However the expression GLUT4 in JJN-3 showed no significant differences between mono and co-culture. Protein expression of GLUT1 was increased in co-cultures of KMS12-PE and JJN-3 as compared with mono cell culture.

Conclusion: Our data suggest that BMMSCs from MM patients can modify expression level of metabolic factors in cell line. This in vitro study confirms the hypothesis that the hematopoietic microenvironment supporting the survival and proliferation of MM cells.

Disclosure: Aline Kühnel: No conflict of interest disclosed.Igor Wolfgang Blau: Expert Testimony: Grant Celgene

P234 – Potent in vitro and in vivo effects of polyclonal anti-human-myeloma globulins

Schieferdecker A.1,2, Shoshani O.3,4, Westner B.5,6, Zipori D.3, Fehse B.1, Kröger N.1, Ayuk F.1

1Department of Stem Cell Transplantation, University Medical Center Hamburg-Eppendorf, Hamburg, Germany, 2Department of Oncology and Hematology with section Pneumology, Hubertus Wald Tumorzentrum / UCCH, University Medical Center Hamburg-Eppendorf, Hamburg, Germany, 3Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel, 4San Diego Branch, Ludwig Institute for Cancer Research, La Jolla, United States, 5Neovii (formerly Fresenius) Biotech GmbH, Gräfelfing, Germany, 6Acino AG, Miesbach, Germany

Introduction: Polyclonal anti T lymphocyte globulins (ATG) are used in allogeneic stem cell transplantation to prevent graft versus host disease and have been reported to kill human myeloma cells in vitro and in vivo. We reasoned that polyclonal anti-human-myeloma globulins (AMG) might improve antimyeloma effects without increasing toxicity.

Methods: AMG were produced by immunizing rabbits with human myeloma cell lines RPMI-8226 (AMG-8226) or KMS-12-BM (AMG-12-BM). In vitro complement-dependent and -independent cytotoxicity of ATG and AMG were compared in myeloma cells (RPMI-8226, KMS-12-BM, OPM-2), primary T cells and non-hematopoietic cells (Hacat and Panc-1). The cytotoxicity assays were analysed by flow cytometry after staining with 7AAD. Combination effects of the polyclonals with bortezomib or melphalan were analysed using Calcusyn®. For analyzing the binding capacity of hematopoetic specific antibodies of ATG and AMG an absorption assay was used. In vivo effects were studied in a xenograft NOD-SCID mouse model. Therefore mice were subcutaneously injected with MM1S myeloma cells and thereafter treated with ATG, AMG-8226 or AMG-12-BM.

Results: Both AMG demonstrated stronger cytotoxicity against myeloma cells compared to ATG. In primary T cells AMG-8226 exerted greater complement-dependent cytotoxicity than ATG, whereas complement-independent cytotoxicity did not differ. Effects on non-hematopoietic cell lines were also similar. Competitive blocking assays revealed four fold more antibodies against CD38 in AMG-8226 compared to ATG. Low concentrations of AMG-8226 and ATG enhanced ADCC. At higher concentrations, ATG had stronger inhibitory effects on ADCC compared to AMG-8226. Combinations of ATG and AMG-8226 with melphalan or bortezomib showed additive to synergistic cytotoxic effects on myeloma cells. In line with in vitro data, mouse experiments with the MM.1S myeloma cell line showed stronger antitumor effects for both AMGs compared to ATG.

Conclusion: Our data show more potent antimyeloma effects of AMG compared to ATG with no increase in toxicity and may lay the ground for the development of polyclonal antibodies for the treatment of multiple myeloma.

Disclosure: Aneta Schieferdecker: No conflict of interest disclosed.Francis Ayuk: Financing of Scientific Research: Neovii (formerly Fresenius) Biotech GmbH

P235 – MOR202 alone and in combination with pomalidomide or lenalidomide in relapsed or refractory multiple myeloma: data from clinically relevant cohorts of a phase I/IIa study

Raab M.S.1, Chatterjee M.2, Goldschmidt H.1, Agis H.3, Blau I.W.4, Einsele H.2, Engelhardt M.5, Ferstl B.6, Gramatzki M.7, Röllig C.8, Weisel K.9, Jarutat T.10, Weinelt D.10, Boxhammer R.10, Winderlich M.10, Peschel C.11

1University Hospital Heidelberg, Department of Medicine V, Heidelberg, Germany, 2University Hospital of Wuerzburg, Department of Internal Medicine II, Wuerzburg, Germany, 3University Hospital of Internal Medicine – AKH Wien, Department of Medicine I, Vienna, Austria, 4Charité Campus Benjamin Franklin, Department of Internal Medicine III, Berlin, Germany, 5Medical University Hospital, Hematology & Oncology Department, Freiburg, Germany, 6Friedrich-Alexander-University Erlangen-Nuremberg, Department of Internal Medicine 5 - Hematology and Oncology, Erlangen, Germany, 7University Hospital Schleswig-Holstein Division of Stem Cell Transplantation and Immunotherapy, Department of Medicine, Kiel, Germany, 8University Hospital Carl Gustav Carus, Department of Medicine I, Dresden, Germany, 9University Hospital of Tuebingen, Department of Hematology, Oncology, Immunology, Rheumatology and Pulmonology, Tuebingen, Germany, 10MorphoSys AG, Martinsried, Germany, 11Technical University of Munich, Department of Internal Medicine III, Munich, Germany

Introduction: MOR202, a HuCAL-derived, human IgG1 CD38 monoclonal antibody, induces potent antibody-dependent cellular cytotoxicity and antibody-dependent cellular phagocytosis. Preclinical models show high activity of single-agent MOR202 and synergy in combination with immunomodulatory drugs (IMiDs), lenalidomide (LEN) or pomalidomide (POM). Unlike other CD38 antibodies, MOR202 does not induce complement-dependent cytotoxicity, thought to be a major contributor to infusion-related reactions (IRRs). The primary objectives of this study were to evaluate the safety, maximum tolerated dose (MTD) and recommended phase II dose of MOR202 in patients with relapsed or refractory multiple myeloma (R-R MM).

Methods: This is an interim analysis of a multicenter, dose-escalation phase I/IIa study of MOR202. Preliminary safety and efficacy data from 3 cohorts of patients treated with MOR202 alone or with an IMiD are presented: MOR202 4, 8 and 16 mg/kg weekly; MOR202 8 or 16 mg/kg weekly with either LEN or POM. All patients in these cohorts received prophylactic low dose dexamethasone.

Results: As of January 29, 2016, 20 patients had been treated: 11 with MOR202 alone, 5 with MOR202+LEN and 4 with MOR202+POM. Patients receiving MOR202 alone or with POM were relapsed or refractory to prior bortezomib and LEN with a median of 4 prior regimens. Patients given MOR202 with LEN had a median of 2 prior regimens, mainly bortezomib, cyclophosphamide and autologous stem cell transplant. The MTD has not been reached. MOR202 alone or with an IMiD was well tolerated with mainly hematological toxicity. No MOR202-related study discontinuations or deaths were recorded. A 2-hour MOR202 infusion was feasible in all patients. Only 1/20 patients had an IRR (grade 1). Responses were reported in 8/18 evaluable patients: 3/10 patients (1 very good partial response [VGPR] and 2 partial responses [PR]) in the MOR202 alone cohort, 3/4 patients (all PR) in the MOR202+LEN cohort and 2/4 patients (1 complete response and 1 VGPR) in the MOR202+POM cohort. At the time of analysis, 7/8 responses were ongoing, with the longest duration >10 months in a patient given MOR202 alone. Preservation of high CD38 levels on MM cells under MOR202 therapy was shown.

Conclusions: In this analysis, MOR202 given as a 2-hour infusion showed excellent infusion tolerability and overall safety profile. Promising preliminary efficacy and long-lasting tumor control was seen for MOR202 +/- IMiDs in patients with R-R MM.

Disclosure: Marc Raab: Advisory Role: Novartis; Amgen; Celgene; MorphoSys; Financing of Scientific Research: Novartis; Amgen; Celgene; MorphoSys; Expert Testimony: Novartis; MorphoSys; Other Financial Relationships: Travel, Accomodation, Expenses: Novartis; Amgen; Celgene; MorphoSysChristian Peschel: Advisory Role: Bristol-Myers Squibb; Financing of Scientific Research: Bristol-Myers Squibb; Amgen; MorphoSys; Other Financial Relationships: Travel, Accomodation, Expenses: Bristol-Myers Squibb; Amgen

P236 – High-dose therapy (HDT) with melphalan and autologous stem cell transplantation (ASCT) for patient older than 70 years with multiple myeloma – feasible therapy or significant risk of complications?

Brockhoff H.1, Meyer zum Büschenfelde C.2, Salwender H.3

1Asklepios Campus Hamburg, Hamburg, Germany, 2Asklepios Klinik Altona, Hämatologie/ Onkologie, Hamburg, Germany, 3Asklepios Klinik Altona, Hamburg, Germany

Introduction: HDT-ASCT is the standard treatment for patients with multiple myeloma younger than 65. The treatment numbers of older patients (>70 years) with HDT-ASCT grew steadily in the last years. However, there is only a small number of studies yielding insufficient data regarding overall-survival (OS) and risk assessment for these patients.

Methods: We retrospectively analyzed a group of 62 patients – median age at ASCT: 71.3 years – with multiple myeloma who underwent HDT-ASCT treatment at the Asklepios Klinik Altona Hospital between 2004 and 2013. The primary aim was to explore OS and treatment related mortality (TRM). Furthermore, we analyzed potential risk factors such as melphalan-dosage, chronic renal failure (Durie-Salmon stage A/B), tandem-therapy, use of new drugs in induction therapy and response after induction therapy. For survival analysis we used the Kaplan-Meier method.

Results: TRM was 0%. The median OS was 51.9 months. Patients who received 200mg/m² melphalan at least once had a longer median OS compared to patients who received a lower dosage (85.3 v. 41.7 months; p = 0.01). Chronic renal failure significantly limited OS (33.7 v. 57.8 months; p = 0.007). Tandem therapy (single: 43.5 v. tandem: 55.9 months; p = 0.39), induction therapy with new drugs (new drugs: 73.9 v. no-new drugs: 55.9 months; p = 0.32) and response after induction therapy (complete remission/ very good partial remission: 73.9 v. partial remission/minimal remission/stable disease: 49.1 months; p = 0.23) could not be identified as significant risk factors in our analysis.

Conclusion: 0% TRM in our patient group shows that older individuals generally tolerate the toxicity of HDT with melphalan well. When patients older than 70 years, without chronic renal failure, are treated with HDT-ASCT, they should receive a 200mg/m² melphalan-regime to achieve better OS. The median OS of 51.9 months is comparable with the standard treatment for multiple myeloma for older patients (>65 years) e.g. Revilimid+ Dexamethasone (RD) or Velcade+ Melphalan+ Prednisolone (VMP). However, as the administration time for HDT-ASCT is much shorter than RD/ VMP, adverse medical effects of long time glucocorticoid treatment should be considered when choosing a therapy. A dual approach with first-line HDCT-ASCT and RD/VMP in refractory myeloma should be explored in future studies for patients >65years. This could lead to a more economical treatment with a higher quality of life for patients.

Disclosure: No conflict of interest disclosed.

P237 – Standard Operating Procedures (SOPs) of centers of excellence for the diagnosis, treatment and follow-up of multiple myeloma patients: similarities and differences

Scheid C.1, Engelhardt M.2, Goldschmidt H.3, Einsele H.4, Starbatty B.5, Bischoff M.6, Glossmann J.-P.7, Skoetz N.5, Centers of excellence working group SOP

1Universität zu Köln, Klinik I für Innere Medizin, Köln, Germany, 2Uniklinik Freiburg, Klinik I für Innere Medizin, Freiburg, Germany, 3Universitätsklinikum Heidelberg, Hämatologie, Onkologie, Rheumatologie, Heidelberg, Germany, 4Universitätsklinik Würzburg, Medizinische Klinik und Poliklink II, Würzburg, Germany, 5Centrum für Integrierte Onkologie (CIO) KölnBonn, Koordinationsstelle der AG SOP, Köln, Germany, 6Uniklinik Freiburg, Institut für Medizinische Biometrie und Informatik, Freiburg, Germany, 7Centrum für Integrierte Onkologie (CIO) KölnBonn, Köln, Germany

Introduction: Currently 13 centers of excellence exist, all funded by the German Cancer Aid (DKH). It is mandatory for them to provide up-to-date standard operating procedures (SOPs). Usually, SOPs are developed in one center and consist of clinical pathways which reflect recommendations given by current evidence-based guidelines. In addition, latest research results from clinical trials and specific in-house features are integrated to adapt the recommendations on center-specific needs. If questions of interest may not be covered by guidelines, interdisciplinary experts from the center develop a new SOP. To harmonize methods and evidence processing, the working group SOP has developed a common standard for SOPs (SOP handbook ccc-netzwerk.de).

The objective of this project is to describe the process of developing one common network SOP for multiple myeloma.

Methods: Four centers (Freiburg, Heidelberg, Köln/Bonn, Würzburg) with high expertise in treatment of myeloma patients indicated interest in developing the network SOP and provided center-specific SOPs. The co-ordinating office of the working group SOP (funded by the DKH, No. 111493) collated all suggestions and recommendations in one clinical pathway to be integrated in the network SOP, in line with the SOP handbook. During that process, a number similarities and discrepancies both in format and content were identified.

Results: As the four SOPs differ in their extent and deepness, recommendations are given in various levels of detail. Most of the SOPs describe quite similar diagnostic procedures for myeloma, local standards exist for the usage of computer tomography or MRT. All the SOPs recommend to treat the patient within a clinical trial, latest at relapsed stage. As some of the SOPs directly link to a clinical study register, this information is more up-to-date compared to specific mentioned trials that might be closed before the SOP is updated. As the updating process varies across these SOPs, some also include drugs which were recently approved, as other don’t. The network SOP will be finalized soon and be freely available as a clinical pathway on the CCC network website.

Conclusions: The collation of the suggestions and recommendations from four highly experienced centers is a first step in identifying nationwide standards and differences. Harmonization of these recommendations for the network SOP will identify evidence-base gaps and will lead to a standard in Germany for improved myeloma treatment.

Disclosure: Christof Scheid: No conflict of interest disclosed.Nicole Skoetz: Employment or Leadership Position: Wissenschaftliche Koordinatorin AG SOP des Netzwerks onkologischer Spitzenzentren

P238 – Systematic classification of death causes in multiple myeloma patients

Haas E.-M.1, Löpprich M.2, Lücke S.3, Kunz C.3, Pritsch M.1, Knaup-Gregori P.2, Hillengass J.1, Goldschmidt H.1,4, Mai E.K.1

1Department of Hematology and Oncology, University Hospital of Heidelberg, Heidelberg, Germany, 2Institute of Medical Biometry and Informatics, University of Heidelberg, Heidelberg, Germany, 3Division of Biostatistics, German Cancer Research Center, Heidelberg, Germany, 4National Center for Tumor Diseases (NCT), Heidelberg, Germany

Introduction: Causes of death (COD) in multiple myeloma (MM) patients and their relation to MM or therapeutic side effects have not been systematically evaluated yet.

Methods: A hierarchically structured COD classification was built according to the Qualitative Content Analysis. A superordinate system of categories distinguished (1) MM-dependent, (2) MM-independent, (3) not attributable to (1)/(2) and (4) unknown COD. The MM-dependent COD were further subdivided into (1A) MM progression-related, (1B) therapy-related and (1C) not attributable to (1A)/(1B). A subordinate system defined COD on four levels of different specificity applying the MedDRA terminology (Figure 1).


COD of 483 deceased patients, from a cohort of 818 MM patients who had received upfront high-dose therapy, were assessed with the constructed classification. Competing-risks analyses were conducted subsequently on the whole cohort.

Results: 80.8% of the COD (n = 483) were MM-dependent, 1.7% MM-independent, 7.0% not attributable to (1)/(2) and 10.6% unknown. Among the MM-dependent COD, 41.8% were MM progression-related, 19.5% therapy-related and 38.7% not attributable to (1A)/(1B). The most common therapy-related COD were sepsis (27.6%), pulmonary sepsis (15.8%) and pneumonia (11.8%). In competing-risks analyses known adverse prognostic factors were associated with an increased risk for MM progression-related death. In contrast, multivariate competing-risks analyses demonstrated that low platelet counts (LPC, < 150/nl; HR = 2.87, p = 0.01) and renal impairment (RI, serum creatinine = 2mg/dl; HR = 2.13, p = 0.11) are associated with an increased risk for therapy-related death.

Conclusions: The majority of MM patients died MM-dependent, and among those cases one fifth were related to therapeutic side effects. LPC and RI constitute important risk factors for therapy-related death, reflecting an impaired ability to tolerate MM therapy. Our classification has proved to be reliable and its application in future registries and prospective trials might ensure a reliable collection of data on COD in MM patients.

Disclosure: Eva-Maria Haas: Other Financial Relationships: Reisekostenerstattung: Bristol-Myers SquibbElias Mai: Other Financial Relationships: Reisekostenerstattung: Janssen-Cilag, Celgene, Onyx und Mundipharma

P239 – Bortezomib (BOR)-Thalidomide-Dexamethasone (VTD) as induction treatment for newly diagnosed Multiple Myeloma (MM) is associated with a lower rate of Second Primary Malignancies (SPMs) compared to Thalidomide-Dexamethasone (TD)

Brioli A.1,2, Pezzi A.2, Mügge L.-O.1, Derudas D.3, Petti M.C.4, Zannetti B.A.2, Ferrara F.5, Rocchi S.2, Nobile F.6, Baraldi A.7, Musto P.8, Lanza F.9, Mancuso K.2, Canepa L.10, Catalano L.5, Lazzaro A.11, Pinotti G.12, Boccadoro M.13, Hochhaus A.1, Cavo M.2

1Universitätsklinikum Jena, Klinik für Innere Medizin II, Hämatologie und Internistische Onkologie, Jena, Germany, 2Bologna University School of Medicine, Seragnoli Institute of Hematology, Bologna, Italy, 3GIMEMA, Italian Myeloma Network, Cagliari, Italy, 4GIMEMA, Italian Myeloma Network, Roma, Italy, 5GIMEMA, Italian Myeloma Network, Napoli, Italy, 6GIMEMA, Italian Myeloma Network, Reggio Calabria, Italy, 7GIMEMA, Italian Myeloma Network, Alessandria, Italy, 8GIMEMA, Italian Myeloma Network, Rionero in Vulture, Italy, 9GIMEMA, Italian Myeloma Network, Cremona, Italy, 10GIMEMA, Italian Myeloma Network, Genova, Italy, 11GIMEMA, Italian Myeloma Network, Piacenza, Italy, 12GIMEMA, Italian Myeloma Network, Varese, Italy, 13GIMEMA, Italian Myeloma Network, Torino, Italy

Introduction: The availability immunomodulatory drugs, IMiDs, and proteasome inhibitors, PI, for the front line treatment of Multiple Myeloma (MM) has significantly improved patients’ outcomes, however concerns have been raised regarding the possibility that IMiDs might increase the risk of developing SPMs. Conversely the use of PI in elderly newly diagnosed MM (NDMM) patients (pts) was not associated with an increased risk of SPMs, while the full impact of an upfront treatment containing both a PI and an IMiD in association with high dose Melphalan (HDM) still has to be investigated. To address this issue, we have evaluated the incidence of SPMs in the GIMEMA 26866138-MMY-3006 multicentre phase III study that compared BOR, thalidomide and dexamethasone (VTD) versus TD as induction before, and consolidation after, a double course of HDM.

Methods: Of the 480 transplant eligible NDMM pts enrolled, 474 received assigned treatment. Data on the incidence of SPMs are available for 299 pts (63%, 148 VTD and 151 TD).

Results: The median follow up was 73 months. 25/299 pts (8%) developed a SPM: 7 (2%) hematologic and 18 (6%) non hematologic. The median time from trial entry to development of the SPM was 36 months (range 8.4-69.0). The number of pts developing a SPM was lower for VTD (5%) compared to TD (11%, p = 0.068). Among pts developing a SPM, the proportion of solid malignancies was similar between treatment arms (75% and 71% in VTD and TD, respectively). Similarly, hematologic SPMs were 25% for VTD and 29% for TD. Overall the incidence rate (IR) of developing a SPM was 1% at 1 year and 9.9% at 6 years (yrs). This incidence was significantly lower for pts in VTD arm compared with patients in TD (6% vs 13% at 6 yrs, p = 0.037). When looking at the IR of solid tumors only 5% of VTD-treated pts developed a solid SPM, as compared to 9.6% in TD; similarly, less hematologic SPMs were observed in the BOR arm (1% vs 4% at 6 yrs for VTD and TD, respectively). When the analysis was performed according to SPMs type, no statistical significance could be demonstrated.

Conclusions: Our data compare favorably with data previously reported on the incidence of developing SPMs in NDMM treated with BOR and Melphalan frontline. With a follow up of 6 years, we were able to confirm that treatment with PI is associated with a low risk of developing SPM. Our data also suggest that treatment with PI might decrease the risk of developing a SPM compared to IMiDs based treatment.

Disclosure: Annamaria Brioli: Financing of Scientific Research: Celgene, JanssenMichele Cavo: Financing of Scientific Research: Celgene, Janssen

P240 – Does renal failure affect outcome after autologous stem cell transplantation in patients with multiple myeloma?

Antlanger M.1, Lamm W.2, Reiter T.2, Porpaczy E.2, Minichsdorfer C.2, Rabitsch W.2, Gisslinger H.2, Agis H.2, Krauth M.-T.2

1Medizinische Universität Wien, Innere Medizin III, Abt. für Nephrologie, Wien, Austria, 2Medizinische Universität Wien, Wien, Austria

Introduction: Renal impairment (RI) is frequent in patients with Multiple Myeloma (MM) and has been proven to be a prognostic factor regarding overall survival (OS). MM patients with impaired renal function often fail to qualify for high-dose chemotherapy and are excluded from autologous stem cell transplantation (ASCT), since a higher transplant-related mortality has been postulated. However, it remains unclear whether these historical inferior outcome data still hold true in times of modern, immuno-chemotherapeutical therapy regimen.

Methods: 195 MM patients (median age 54 years) who had undergone ASCT between 1999 and 2015 were analyzed. Renal function at the time of diagnosis and transplantation was assessed by eGFR (MDRD). Kaplan-Meier curves and log-rank tests were used for OS and progression-free survival (PFS) calculation.

Results: Estimated mean overall survival from the time of diagnosis was 93 months (90% CI: 77-109). No difference was found when comparing patients who never had renal impairment with those who presented with renal failure at diagnosis and improved throughout therapy course as well as with those whose renal function was always impaired (Fig 1).


Estimated mean PFS was 83 months (CI 12-61). Again, renal impairment did not result in reduced survival (Fig 2).


Conclusions: In this retrospective analysis, a relatively large cohort of MM patients who had undergone ASCT was analyzed regarding outcome according to renal function. Our data show that neither OS nor PFS after ASCT were negatively impacted by mild to moderate RI. Since exclusion from ASCT results in shorter survival per se, it therefore seems to be of pivotal clinical importance that patients with MM and RI should rather be evaluated pro-actively for high-dose immuno-chemotherapy including ASCT than excluded from these therapy regimen.

Disclosure: No conflict of interest disclosed.

P241 – Outpatient care of multiple myeloma patients in the western Ruhr area. A retrospective assessment from 3 hemato-oncological practices of related size in Essen, Ratingen and Bottrop

Rudolph R.1, Hannig C.2, Langer W.3

1Hämato-Onkologische Gemeinschaftspraxis Dres. Rudolph & von Verschuer, Essen, Germany, 2Schwerpunktpraxis für Hämatologie und Internistische Onkologie, Bottrop, Germany, 3Krebszentrum Ratingen, Ratingen, Germany

A total of 227 patients were acquired, 86 in Essen, 82 in Bottrop and 61 in Ratingen. 63 pts. were < 65 years old, 73 pts. 65 - 75 years, 91 pts. > 75 years. 114 pts. were female,113 male.

Sort of therapy in 2012: watch & wait 174 pts., Bortezomib-based schedules 70 pts., immunomodulatory therapy 53 pts., cytostatic-based therapy 21 pts., high-dose therapy with stem cell transplantation 11 pts., radiotherapy 10 pts., dexamethasone 4 pts..

Supportive treatment in 2012: Bisphosphonates 104 pts., red cell concentrate 36 pts., pain management 36 pts., antibiotics 23 pts., erythropoietin 22 pts., herpes prophylaxis 21 pts., heparin 14 pts., antibiotic prophylaxis 8 pts., radiotherapy 7 pts., ASS 6 pts., platelet concentrate 4 pts., resection of extramedullary lesions 1 pts., denosumab 1 pts..

Co-morbidity in 2012: Arterial vascular disease 140 pts., renal failure 60 pts., diabetes 46 pts., respiratory disorder 22 pts., degenerative skeletal disorder 29 pts., osteoporosis 18 pts., venous vascular disease 17 pts., status post solid tumors 17 pts., peripheral neuropathy 10 pts., solid tumors 10 pts.,osteonecrosis of the jaw 7 pts., dementia 6 pts., endocrinological disorder 6 pts., status post traumatic fractures 5 pts., status post severe infections 5 pts., metabolic disease 5 pts., depression 4 pts., amyloidosis 2 pts., neuropsychiatric disorder 2 pts., status post hematologic malignancies 2 pts., benign hematologic disease 1 pts., disorder of the sensorium 1 pts..

Conclusions: Considering a myeloma incidence rate of 3 - 6 / 100.000 persons and a 5-year prevalence of 17.000 (Germany 2010) outpatient care of 227 patients by 3 hemato-oncological practices within 1 year is strikingly high. Myeloma patients of all stages got initial diagnosis and first as well as following treatment. All approved agents were applied in an outpatient setting (except for high-dose therapy) including multimodal treatments in co-operation with complementary facilities. The schedules applied were conform to latest guidelines and considered latest study results, co-morbidity was carefully regarded. Supportive treatment was fully realized on an outpatient basis. The quality of outpatient care of myeloma patients is to classify as high.

Disclosure: Roland Rudolph: Expert Testimony: Fa. Celgene, MünchenWerner Langer: Expert Testimony: Fa. Celgene, München

Posterdiskussion – Lymphome

P242 – Functional identification of novel molecular dependencies in Cyclin D1 driven lymphoma

Ehrenfeld S.1,2, Veratti P.1,2, Schneider D.1,2, Duyster J.1,2, Miething C.1,2

1Universitätsklinik Freiburg, Department of Medicine I Hematology, Oncology and Stem-Cell Transplantation, Freiburg, Germany, 2DKTK, Standort Freiburg, Freiburg, Germany

Mantle cell lymphoma (MCL) presents as a highly disseminated B-cell malignancy with short responses to current therapies and a great need for new therapeutic strategies. The PI3K pathway has emerged as a promising therapeutic target, as MCL cell lines and patients have shown substantial response rates to rapamycin and analogs. We have developed a new mouse model for MCL using Eµ-myc transgene mice that overexpress the MCL hallmark lesion Cyclin D1 together with the reverse tet transactivator. By deriving primary tumor cell lines from diseased mice, we were able to generate a powerful platform for inducible in vitro screening approaches. We performed a functional shRNA-based genetic screen targeting the PI3K pathway to identify novel molecular dependencies in Cyclin D1 driven lymphomagenesis.

Using a two colored, antibiotic selectable and tet-inducible retroviral shRNA expression vector system, a library composed of 4 shRNA subpools targeting more than 300 different genes within the PI3K pathway was introduced into murine MCL cells by low-titer retroviral infection. Subsequently shRNA expression was induced by addition of doxycycline and shRNA representation was deconvoluted by NGS to compare variation between knockdown and control cells.

Our screen identified a range of proteins that significantly impaired cell proliferation and cell viability. Among these, we found multiple targets within the PI3K pathway and the molecular dependency on this pathway was in line with the observed high sensitivity of these cells towards pharmacological mTOR inhibition. Their mode of action and their impact on the PI3K pathway signaling cascade is currently investigated in more detail. Interestingly, a shRNA targeting the anti-apoptotic protein Bcl-2 showed highly selective depletion in the MCL cells. Since MCL patients have shown particular sensitivity towards a novel Bcl2 inhibitor (ABT-199), we are also further exploring the effects of Bcl2 knockdown in Cyclin D1 overexpressing lymphoma.

In summary, our shRNA screening approach represents a powerful tool to functionally select novel targets involved in cell proliferation and cell viability, enabling the identification of a range of target genes connected to the PI3K pathway in MCL cells. The newly identified targets will be further investigated in murine and human MCL cells to characterize their role in Cyclin D1-driven lymphomagenesis, as well as their potential as therapeutic targets.

Disclosure: No conflict of interest disclosed.

P243 – Clinical and molecular characterization of leukemic follicular lymphomas

Viardot A.1, Estenfelder S.1, Tausch E.1, Schrell S.1, Stilgenbauer S.1

1Universitätsklinikum Ulm, Klinik für Innere Medizin III, Ulm, Germany

Introduction: Leukemic variants of follicular lymphoma are rare; the clinical significance is unknown.

Methods: Chart review of patients with histologically proven follicular lymphoma (FL) and lymphocytosis > 5/nl at diagnosis, treated with chemoimmunotherapy between 2003 and 2015 at a single institution.

Results: We identified 23 patients with leukemic FL at initial diagnosis. The median age was 53 years (range 22-74), 70% of patients were younger than 60 years. The initial lymphocyte count was 21.5/nl (range 5.6-154/nl). The initial histopathological diagnosis was FL Grade 1 (65%), Grade 2 (22%) and Grade 3a or composite lymphoma (13%). In 20/22 patients, immunophenotyping showed CD10 expression. FISH analysis was available in 16/23 patients: the translocation t(14;18)(q32;q21) was detectable in all but 2 patients with tetraploidy on chromosome arm 18q21. The most frequent additional aberrations was gain on 18q21 in 9/16 cases. IGHV status in all analyzed six patients was mutated (homology between 85.5 to 92.7%). The follicular lymphoma international prognostic index was high (median 3), because of stage 4 (100%), elevated LDH (48%), decreased hemoglobin level (43%) and involvement of more than 4 lymph node areas (83%). On the other hand, bulky disease was rare and most patients (16/23) had lymph nodes smaller than 3cm. All patients received chemoimmuntherapy immediately after diagnosis (14 CHOP, 7 Bendamustin, 1 FM and 1 CHOEP, combined with anti-CD20 antibody, respectively). Fifteen patients relapsed after a median time of 16 months. The majority had a nodal relapse; only five patients were leukemic again. The median progression free survival was 14.5 months. Nine patients received autologous stem cell transplantation, and three patients allogeneic stem cell transplantation. With only three deaths (transplant-associated in one patient), the overall survival appeared not dismal.

Conclusion: Leukemic FL is a rare variant with typical clinical and molecular characteristics. Patients tend to be younger, have a higher IPI and relapse early after standard chemoimmunotherapy compared to nodal FL. However, overall survival may not be inferior, possibly due to intensive treatment e.g. autologous stem cell transplantation.

Disclosure: Andreas Viardot: Advisory Role: Amgen, Janssen, Gilead, BMS; Financing of Scientific Research: Amgen, Roche, Pfizer, Janssen; Other Financial Relationships: Takeda, JanssenStephan Stilgenbauer: No conflict of interest disclosed.

P244 – Ibrutinib as first line therapy in a case of Bing-Neel Syndrome

Fellas G.1, Kiehl M.1, Hopfer O.1

1Klinikum Frankfurt (Oder), Medizinische Klinik I, Frankfurt Oder, Germany

Bing-Neel Syndrome is the rare CNS manifestation of Waldenström macroglobulinemia. In up to 36% of cases it is the first manifestation of the disease and it can present as either a diffuse or tumoral form. Until recently the treatment of this rare entity was based on chemotherapeutics with a good blood-brain penetration, radiation and autologous stem cell transplantation. Ibrutinib, a Bruton’s tyrosine kinase inhibitor, showed efficiency in the treatment of Waldenström macroglobulinemia and good blood-brain penetration. Based on these observations, ibrutinib was used earlier for refractory/relapsed patients with Bing-Neel Syndrome and showed very good results (Cabannes-Hamy A. et al. AJH, 2016).

We report here on a case of Bing-Neel syndrome (MYD88L265P) that was treated with ibrutinib 420mg/d. After 4 weeks of therapy a very good response was seen (MRI). No adverse events were noted and the patient’s symptoms have subsided.

Ibrutinib has shown a very good efficacy as first line therapy in this case extending its use for the treatment of Bing-Neel Syndrome.


Disclosure: No conflict of interest disclosed.

P245 – BARs (B-cell receptor antigens for reverse targeting): a novel and ultimately specific treatment concept for B-cell neoplasms

Bewarder M.1, Thurner L.1, Fadle N.1, Kemele M.1, Regitz E.1, Neumann F.1, Körbel C.2, Laschke M.2, Preuss K.-D.1, Pfreundschuh M.1

1Saarland University Medical School, Dept. Internal Medicine I, José Carreras Center for Immuno- and Gene Therapy, Homburg, Germany, 2Saarland University Medical School, Clinical and Experimental Surgery, Homburg, Germany

Introduction: The major task of a B-cell receptor is the binding and internalization of its antigenic target, and its processing for antigen presentation to T-cells. Chronic antigenic stimulation has been discussed to play a role in the pathogenesis of malignant B-cell lymphomas. We therefore systematically searched for the antigenic targets of BCRs from various B-cell neoplasms.

Methods: Recombinant BCRs were expressed as recombinant Fabs (rFabs) based on corresponding pairs of genomic VH+VL regions from lymphoma biopsies. Whenever possible, “natural” Fabs (nFabs) were also obtained by papain digestion of fresh or cultured lymphoma cells. Both nFab and rFab were used to screen for binding to recombinant protein macroarrays.

Results: Two antigens (hyperphosphorylated paratarg-7 and sumoylated HSP-90 which are modified in patients compared to healthy controls) are targets of paraproteins from (depending on ethnicity) 30-50% of all MM patients; the BCR from 67% of patients with primary CNS lymphoma target hyperglycosylated neurabin, 26% of the BCR from ABC-type DLBCL target hypophosphorylated ARS2 and 45% of all MCL BCR target LRPAP1; optineurin is BCR target of 12% follicular lymphomas and various autoantigens have been identified as targets of roughly 30% of all CLL cases. For all autoantigens binding to its specific BCR, rapid internalization and induction of proliferation was demonstrated. Most importantly, BCR-specific cytotoxicity of recombinant pseudomonas-exotoxin conjugated ARS2 against an ABC-DLBCL cell line with a ARS2 specific BCR was demonstrated in vitro and in vivo after establishment of lymphomas in SCID beige mice.

Conclusions: Assuming that only a minority of BCR targets have been identified to date, the prevalence of posttranslationally modified autoantigens strongly supports a role of chronic antigenic stimulation in many B-cell neoplasms. Due to the predominance of a single or few BCR antigens in each malignant B-cell entity studied, BARs represent an attractive and novel therapeutic concept for a broad spectrum of B-cell neoplasms and are the first therapeutic approach in oncology that targets exclusively the malignant cells. BARs can be used for conjugation with toxins, radionuclides and small molecules as well as for bispecific constructs (e. g. with CD3 or CD16) and CAR T-cells, the toxicity of which should be drastically reduced due to the ultimate specificity of BARs that spares normal B-cells.

Supported by Wilhelm-Sander-Stiftung

Disclosure: No conflict of interest disclosed.

P246 – Subgroup analyses of diffuse large B-cell lymphoma and indolent lymphoma cohorts from a phase IIa study of single-agent MOR208 in patients with relapsed or refractory non-Hodgkin’s lymphoma (R-R NHL)

Buske C.1, Jurczak W.2, Zinzani P.L.3, Gaidano G.4, Goy A.5, Provencio M.6, Nagy Z.7, Robak T.8, Maddocks K.9, Ambarkhane S.10, Winderlich M.10, Endell J.10, Blum K.A.9

1Institute of Experimental Cancer Research, Comprehensive Cancer Center Ulm, University Hospital Ulm, Ulm, Germany, 2Jagiellonian University, Department of Hematology, Kraków, Poland, 3University of Bologna, Institute of Hematology “L. e A. Seràgnoli”, Bologna, Italy, 4Amedeo Avogadro University of Eastern Piedmont, Division of Hematology, Department of Translational Medicine, Novara, Italy, 5John Theurer Cancer Center, Hackensack University Medical Center, Hackensack, United States, 6University Hospital Puerta De Hierro, Department of Medical Oncology, Madrid, Spain, 7Semmelweis University, First Department of Internal Medicine, Budapest, Hungary, 8Medical University of Lodz, Department of Hematology, Lodz, Poland, 9The Ohio State University Comprehensive Cancer Center, Department of Internal Medicine, Division of Hematology, Columbus, United States, 10MorphoSys AG, Martinsried, Germany

Introduction: CD19 is a potential B-cell tumor target. A phase I study showed MOR208, an Fc-engineered, humanized CD19 antibody, to be safe and well-tolerated with encouraging single-agent activity in patients with chronic lymphocytic leukemia. This analysis of our phase IIa study focused on the preliminary efficacy and safety of MOR208 in patients with diffuse large B-cell lymphoma (DLBCL) and indolent (i)NHL.

Methods: Patients with R-R NHL progressing after at least one prior rituximab-containing therapy were eligible. Treatment comprised single-agent MOR208, 12 mg/kg IV, weekly, for 8 weeks (2 cycles), which could continue for 4 additional weeks in patients with at least stable disease after 2 cycles. MOR208 maintenance could be given every 2 or 4 weeks until progression in patients with complete (CR) or partial response (PR) at 12 weeks. The primary endpoint was overall response rate (ORR).

Results: 92 patients were enrolled including 35 with DLBCL and 45 with iNHL. ORR was 26% in DLBCL (9/35; 2 CRs and 7 PRs), and 27% in iNHL (12/45; 5 CRs and 7 PRs). In evaluable patients (those completing =2 cycles, with a response assessment) the ORR was 36% in DLBCL (9/25), and 30% in iNHL (12/40). Median duration of response was 13.7 months for DLBCL (1.2-26; 3 ongoing and >20 months in remission) and 8.4 months for iNHL (2.5-20.3; 6 ongoing, including 1 patient >20.3 months in remission). In DLBCL responders, 5/9 were rituximab-refractory (either no response or response lasting < 6 months to a previous rituximab-containing therapy), of which 2 patients had a response duration of >19.8 months and 1 other of >1 year under MOR208 treatment. The incidence of grade =3 hematologic treatment-emergent adverse events was low (DLBCL: 9/35, 26%; iNHL: 4/45, 9%), including neutropenia, anemia and thrombocytopenia in 14%, 9% and 6% of patients with DLBCL and 4%, 0% and 0% of patients with iNHL. Infusion-related reactions were seen in 3/35 (9%) and 4/45 (9%) patients in DLBCL and iNHL cohorts; all were grade 1-2 except for one grade 4 dyspnea. There were no treatment-related deaths and no trend towards late toxicity.

Conclusions: Single-agent MOR208 was associated with a promising ORR including CRs and long-lasting responses in DLBCL and iNHL cohorts, including in patients with rituximab-refractory disease. These efficacy data, and the excellent safety and tolerability profile of MOR208 further justify the development of MOR208 as part of a combination therapy for B-cell NHLs.

Disclosure: No conflict of interest disclosed.

P247 – Reality of care in Primary Central Nervous System Lymphoma. A retrospective survival analysis of 192 patients at a German University Medical Center

Schlosser T.1,2, Illerhaus G.1,3, Schäfer H.S.1

1Universitätsklinikum Freiburg, Department Innere Medizin, Klinik für innere Medizin 1, Freiburg, Germany, 2Universitätsklinikum Leipzig AöR, Klinik und Poliklinik für Gastroenterologie und Rheumatologie, Leipzig, Germany, 3Klinikum Stuttgart, Klinik für Hämatologie, Onkologie und Palliativmedizin, Stuttgart, Germany

Introduction: Primary CNS lymphoma (PCNSL) is a highly aggressive non-Hodgkin lymphoma with affection of the neuroaxis. Because of its rarity, studies report about very small numbers of patients often treated within prospective trials. These investigations are often biased by inclusion criteria such as performance status and age. Little is known about the reality of treatment of PCNSL patients in its entirety and only few institutions worldwide provide sufficient data for a broad single-center experience.

Methods: A retrospective analysis of 192 consecutive immunocompetent patients with PCNSL seen between 1991 and 2013 at the Department of Hematology and Oncology of Freiburg University Medical Center (Germany) was performed. All ages and every performance status were included to obtain real-life data about the provided healthcare. Kaplan-Meier-estimator was used for survival estimations. Uni- and multivariable analyses were performed to identify predictors for overall survival (OS).

Results: The estimated median overall survival (OS) was 31 months (CI 20-65 months). After one, three and five years 64% (CI 58-71%), 48% (CI 41-56%) and 44% (CI 37 52%) of the patients were alive. Age, performance status, LDH serum level, affection of deep brainstructures, Cerebrospinal fluid (CSF) protein level and CSF cell count were significantly associated with OS in univariable testing. Multivariable Cox modelling revealed age, performance status and LDH serum level as independent prognostic factors for OS. The median event-free survival (EFS) time was 10 months (95% CI 7-17 months).

Conclusion: Our data confirm known clinical prognostic factors. OS rates are at least comparable if not better than previous reports. In this health service research analysis we report on impressive OS rates in a “real-life” PCNSL patient cohort representing high level of care in these patients at Freiburg University Medical Center.

Disclosure: No conflict of interest disclosed.

P248 – Multicenter case series of primary adrenal lymphoma

Majidi F.1, Martino S.1, Haase M.2, Chortis V.3,4, Arlt W.3,4, Spyroglou A.5, Beuschlein F.5, Fassnacht M.6, Kanji A.7, Habra M.A.7, Schott M.2, Haas R.1, Gattermann N.1

1Heinrich-Heine University Düsseldorf, Dept. of Hematology, Oncology and Clinical Immunology, Düsseldorf, Germany, 2Heinrich-Heine University Düsseldorf, Dept. of Endocrinology, Düsseldorf, Germany, 3University of Birmingham, Institute of Metabolism and Systems Research, Birmingham, United Kingdom, 4Birmingham Health Partners, Centre for Endocrinology, Diabetes and Metabolism, Birmingham, United Kingdom, 5Ludwig-Maximilians-Universität München, Medizinische Klinik IV, München, Germany, 6Julius-Maximilian-Universität Würzburg, Medizinische Klinik I, Würzburg, Germany, 7University of Texas, MD Anderson Cancer Center, Houston, United States

Introduction: Primary adrenal lymphoma (PAL) is an extremely rare malignant condition with poor response to therapy and unfavorable prognosis. It is defined as histologically proven lymphoma that involves one or both adrenal glands and presents with no prior history of lymphoma.

Methods: We collected 19 patients with PAL from five different centers in Europe and the USA and retrospectively analyzed clinical and pathological features.

Results: Median age at diagnosis was 67 (range: 35-82) years. Males were more frequently affected than females (ratio 5:1). The most common symptoms at diagnosis were back pain (29.2%), upper abdominal pain (20.8%) and B symptoms (47%, including weight loss in 20.8%). In 2 patients PAL was detected incidentally. Median survival was 8 (1-132) months. Median level of LDH was 542 U/L (210-6515). Bilateral adrenal involvement was detected in 61% and correlated with significantly shorter overall survival: median 3.5 mo. (1.4-11) versus 57 mo. (8-132), with and without bilateral involvement, respectively (P = 0.04). Adrenal insufficiency was diagnosed in 26% of patients, all with bilateral involvement, but had no significant effect on overall survival. In patients with unilateral involvement, the left and right adrenals were affected with similar frequency and resulted in similar clinical picture and prognosis. Mean tumor size (largest diameter) was 9.2 cm (4-16). We found no prognostic impact of tumor size. On histopathology, 17 cases (89%) were diffuse large B cell lymphomas. The remaining 2 cases were a large T cell lymphoma and a NK/T cell lymphoma. Ki67 index was higher than 75% in all 8 cases evaluated. CNS involvement was present in 26%, surprisingly without significant impact on prognosis. In 5 patients another extranodal involvement was detected in lung, kidney (2), duodenum or stomach. Of 13 patients receiving immuno-chemotherapy according to the R-CHOP protocol, 5 obtained complete remission and one achieved partial remission.

Conclusion: According to our data, involvement of both adrenal glands is the most important prognostic factor in primary adrenal lymphoma. Up to now, R-CHOP has been the treatment of choice, but the majority of patients fail to respond. Investigations into the molecular pathology of PAL are required to improve our understanding of this rare lymphoid malignancy. It remains to be seen whether modern lymphoma treatments targeting the B cell receptor pathway can improve the outlook of patients with PAL.

Disclosure: No conflict of interest disclosed.

P249 – Decreased ZEB1 expression in Sézary syndrome and its impact on apoptosis and DNA damage response

Gand C.1, Grabarczyk P.1, Hirt C.1, Cabron A.-S.1, Przybylski G.2, Izykowska K.2, Weissmann R.3, Kuß A.W.3, Schmidt C.A.1

1Universitätsmedizin Greifswald, Innere Medizin C Hämatologie und Onkologie, Greifswald, Germany, 2Polish Academy of Sciences, Institute of Human Genetics, Poznan, Poland, 3Universitätsmedizin Greifswald Institut für Humangenetik, Interfakultäres Institut für Genetik und funktionelle Genomforschung, Ernst-Moritz Arndt Universität Greifswald, Greifswald, Germany

Introduction: Sézary syndrome is a rare leukemic form of cutaneous T-cell lymphoma. During the last decades many studies revealed through cytogenetics, comparative genomic hybridization (CGH) and next generation sequencing (NGS) analysis numerous genomic alterations, which are often associated with complex rearrangements. Therefore, a major defect in DNA damage repair mechanisms is probably responsible for the chromosomal instability. The zinc finger, homeodomain transcription factor ZEB1 was recently found to be involved in DNA damage response through Checkpoint kinase 1 (CHK 1). We and others have shown ZEB1 loss as one of the most frequent genetic alterations seen in Sézary syndrome samples. We therefore analyzed the effects of ZEB1 downregulation in T-cell lines focusing particularly on DNA damage response.

Methods: To examine the functional consequences of reduced ZEB1 expression, CD4 T-cells from healthy donors and the T-cell lymphoma cell lines Jurkat and HuT 78 were treated with ZEB1 specific siRNAs. Knockdown efficiencies were verified by RT-PCR and Western Blot experiments. Annexin V/ 7AAD double-staining was used for apoptosis detection. Furthermore a ZEB1 deficient Jurkat cell line was established through transduction of ZEB1 specific lentiviral shRNA constructs. DNA damage was induced by Etoposid, Camptothecin and UV-C light. The amount of DNA damage was analyzed through ?-H2A.X staining.

Results: An increased spontaneous apoptosis rate was not observed upon siRNA mediated ZEB1 knockdown in healthy T-cells or in the T-cell lines HuT 78 and Jurkat. Furthermore, no influence on chemoresistance upon Camptothecin and Etoposide treatment was found. Interestingly, an increased DNA damage rate could be demonstrated upon treatment with UV-C light, Camptothecin and Etoposide in Jurkat cells that were transduced with ZEB1 specific shRNAs compared to scrambled and non-transduced controls.

Conclusion: Jurkat cells with reduced ZEB1 expression were prone to DNA-damaging stimulants. Therefore the frequent ZEB1 deletion in Sézary syndrome might be involved in the accumulation of genetic alterations in this disease. These results provide a promising basis for further investigations on the underlying mechanisms of the complex genetic alterations seen in this T-cell lymphoma.

Disclosure: No conflict of interest disclosed.

P250 – A small cell variant of anaplastic large cell ALK positive primary cutaneous T-lymphoma

Vergoulidou M.1, Decker T.2, Grobe N.1

1Dietrich Bonhoeffer Klinikum, Innere Medizin 1, Neubrandenburg, Germany, 2Dietrich Bonhoeffer Klinikum, Institut für Pathologie, Neubrandenburg, Germany

T-lymphomas are rare entities. We report a case of a small cell variant ALK-positive anaplastic large cell lymphoma (ALCL). A 56-year-old male patient presented with multifocal, partially ulcerating skin infiltrates around the right lower and upper eyelid. Biopsy revealed small cell blast morphology. Immunochemistry was negative for CD79a, CD20, CD3, CD4 and CD7 but positive for CD2, cytoplasmatic ALK1 and CD30. CT imaging revealed left axillary lymphadenopathy, so left axillary lymph node dissection was carried out, without histological evidence of lymphoma. Bone marrow biopsy showed no lymphoma infiltration. Patient’s history was remarkable for multiple sclerosis, urothelial carcinoma with excision before ten years and chronic anal fistula. Patient was treated with multi-agent chemotherapy (CHOP) and had complete remission of the skin infiltrates.

Primary cutaneous (c-ALCL) and systemic ALK-positive ALCL have identical histopathological findings, further identification is based on clinical findings. Prognosis differs significantly, c-ALCL has 90% long-term survival and only up to 10% extra-cutaneous manifestations. Systemic ALCL small-cell variant has 70% 5-year survival, may present skin infiltrates and presents up to 20% bone marrow infiltration. Our patient fulfilled rather the c-ALCL criteria but due to multifocal skin infiltrates and al least radiologically shown extra-cutaneous manifestation i.e. axillary lymphadenopathy, he received chemotherapy with good tolerability and complete remission.

To our knowledge, small cell variant of p-ALCL or systemic ALCL is extremely rare, with only one published case series and sets a diagnostic as well as therapeutical challenge.

Disclosure: No conflict of interest disclosed.

P251 – ALK-negativ anaplastic large cell-lymphoma – a supprising diagnosis in a patient with low grade prostatic cancer: a case report

Piribauer M.1, Weiss H.1, Tomka M.1, Siebert F.1

1Krankenhaus der Barmherzige Brüder, Innere Medizin, Hämato-Onkologie, St. Veit/Glan, Austria

Introduction: Prostatic cancer is the most frequent tumor in males.

Anaplastic large cell-lymphoma (ALCL) are a rare hematological malignancy and a distinct subtype of mature T-Cell lymphomas. We presenting a 72year-old patient with liver and lung lesions where the histological investigation shows an ALK- negative ALCL four years after diagnosis of a low grade prostatic cancer.

Case report: A 72year-old male patient was admitted to our hospital due to newly diagnosed lesions in the liver and lung. Four years before he had radical prostatectomy and lymph node removal because of prostatic cancer. Histologic Adenocarcinoma, Gleason Score 3+4/7, pT2c, N0, R0. The histological investigation of tissue probe from the bronchoscopy could not fount malignant cells. Only cytological investigation shows cells of an adenocarcinoma, G2. So the next procedure was a puncture of one liver leasion. Histological shows now a solid growing cancer, the tests of immunohistochemistry were outstanding. In the meantime the patient general condition and dyspnea worsened. With the suspicion of a lung cancer (NSE with 32, 6 µg/l elevated (normal range: < 18,3 µg/ml) PSA was in normal range), we started chemotherapy with cisplatin and vinorelbine. After one cycle the patient shows a clinical benefit, dyspnea improved. Days later, the result of immunohistochemistry shows no polymorphic carcinoma and no markers for prostatic cancer. Negative were ALK 1and TIA 1 and CD 20. Positive was CD 30 and the T-cell markers CD3 and CD5. Now the diagnosis of an ALK-negative Anaplastic T-cell-lymphoma, IPI-score high risk, was found. Chemotherapy was switched to COMP-scheme. The patient tolerated chemotherapy well, the dyspnea improved rapidly. Now, four cycles was administrated (planned six cycles), the patient are in well condition.

Conclusions: To our knowledge, is this the first case of an ALK- negative ALCL in a male patient with low grade prostatic cancer. After literature search, ALCLs were often described in female patients with breast implantants, some in pancreatic, lung and skin and rarely in leukemia. Among all systemic ALCLs, those that are ALK negative constitute 15-50% of cases. The prognosis is poorer than in ALK-positive ALCLs. The recommended chemotherapy is anthracycline based. It usually involves lymph nodes at diagnosis (49%) and, less frequently, extranodal sites (20%). This case also underlines the importance to puncture lesions for histological investigation in unclear cases.

Disclosure: No conflict of interest disclosed.

P252 – Allogeneic stem cell transplantation in refractory Hodgkin’s lymphoma after CR with Nivolumab

Fellas G.1, Kiehl M.1, Hopfer O.1

1Klinikum Frankfurt (Oder), Frankfurt Oder, Germany

Hodgkin’s lymphoma is a hematologic malignancy, characterized by the presence of the Reed-Sternberg cells within an inflammatory infiltrate. Although it is a highly curable disease, approximately 10-15% of cases are primary refractory to the convenient therapies with subsequently poor outcomes.

Recurrent chromosome 9p24.1 amplification in Reed-Sternberg cells results in overexpression of the PD-1 ligands (PDL1 and PDL2) with subsequent T cell exhaustion. Recently, Nivolumab, a novel fully human monoclonal IgG4 antibody directed against PD-1, showed very good remission rates in patients with refractory disease.

We report the cases of two previously heavily treated young patients with refractory Hodgkin’s lymphoma. Both patients were treated with Nivolumab 3mg/kg every 2 weeks. One patient showed primarily a good response but he was progressive after 9 cycles and the therapy was discontinued. However, the other patient reached a complete response (CR) after 8 cycles of Nivolumab therapy and was able to proceed to consolidating allogeneic stem cell transplantation. After 200 days the patient is still in CR without any significant complications.

The response to treatment was very different raising the issue of response prediction. In the post-transplant period there was seen no increased acute GvHD risk, stressing the concept of PD-1 blockade before and possibly also after allogeneic transplantion in r/r Hodgkins lymphoma.

We could show here that PD-1 check point blockade can lead to a complete remission (CR) in previously refractory Hodgkin’s lymphoma and that subsequent consolidating allogeneic stem cell transplantation is possible with acceptable adverse events.


Disclosure: No conflict of interest disclosed.

P253 – A comprehensive analysis of seasonality patterns of incidence and mortality in Hodgkin lymphoma across geographic regions

Borchmann S.1, Müller H.1, Engert A.1

1Uniklinik Köln, Klinik I für Innere Medizin, Köln, Germany

Introduction: The goal of this analysis is to provide a comprehensive analysis of seasonal variation in incidence and mortality risk of HL with a focus on latitudinal differences.

Methods: HL cases diagnosed between 1973 and 2012 in the 18 Surveillance, Epidemiology, and End Results (SEER) registries were eligible (n = 50,179). After exclusion of cases with partly missing data (n = 8,700) and the SEER registries “Alaska natives” (n = 17) and “Rural Georgia” (n = 57) due to insufficient case numbers, 41,405 cases were included. Seasonality of incidence was analyzed for all cases and predefined subgroups using cosinor analysis. The overall mortality risk following a HL diagnosis in winter (Sep.-Feb.) vs. summer (Mar.-Aug.), after correction for known risk factors, was compared using a Cox proportional-hazards model. The interaction between seasonal mortality differences and latitude was analyzed.

Results: HL shows a seasonal incidence pattern with a peak around March and a trough around September (p < 0.001). Only cases of the mixed cellularity (p < 0.001), nodular sclerosis (p < 0.001) and lymphocyte depleted subtype (p = 0.002) showed a seasonal incidence pattern. The estimated amplitude of seasonal differences between the peak and the trough of monthly incidence was particularly high for the age groups 20-29, 30-39 and 60-69, with amplitudes of 0.121, 0.114 and 0.096 respectively, coinciding with age groups of increased HL incidence. Cases diagnosed at lower (< 38.05°N) latitudes showed a decreased seasonality of incidence with an amplitude (0.055) compared to cases at higher (=38.05°N) latitudes (0.102) (p = 0.023 for difference).

The risk of dying within the first 3 years after a HL diagnosis in winter is increased at higher (=38.05°N) latitudes (HR: 1.082 [1.009;1.161], p = 0.027), whereas no seasonal difference in mortality was observed for cases diagnosed at lower (< 38.05°N) latitudes (HR: 0.990 [0.926;1.059], p = 0.772).

Conclusion: Increased seasonality of incidence at higher latitudes and the interaction between higher latitudes and increased mortality after a HL diagnosis in winter show the influence of latitude on these patterns. As latitude is closely linked to seasonal variations in Vitamin D serum levels, a protective effect of Vitamin D in HL is a possible explanation. Further evidence, especially on the direct association between Vitamin D levels and the clinical course of HL, needs to be collected to improve the understanding of the role of Vitamin D in HL.

Disclosure: No conflict of interest disclosed.

P254 – Metamorphosis of a lymphoma – from extranodal marginal zone lymphoma over a diffuse large B-cell lymphoma to Hodgkin lymphoma – a case presentation

Potenberg J.1, Reyher-Klein S.2

1Onkologisches Zentrum Ev. Waldkrankenhaus, Berlin, Germany, 2Pathologisches Institut am Ev. Waldkrankenhaus, Berlin, Germany

Introduction: The prognosis of localized gastric malt lymphoma is apparently good. We present here a case of lymphoma whose dangerousness has been judged differently during treatment and finally took a fatal outcome.

Methods: To provide a high quality of our diagnostic and treatment we try to include our patients in studies and obtain a second opinion in difficult cases.

Results: The previously healthy patient (born 1961) was diagnosed in January 2011 with a gastric malt lymphoma, stage E II. The patient was included in a study performed by the German Gastrointestinal Study Group. After eradication of H. pylori, radiotherapy including tomotherapy of the stomach was performed and remission was achieved. May 2013 the lymphoma inthe stomach recurred. The biopsy showed now areas of diffuse large cell b-cell lymphoma. 6 cycles of R-CHOP 14 were given and a complete remission was obtained.

November 2015 the patient developed multiple lymphomas. An axillary node was exstirpated and the diagnosis was Hodgkin lymphoma with nodular sclerosis. Chemotherapy with BEACOPP escalated was applied. During therapy a further progress was noted. The therapy was switched to DHAP. During the second cycle of DHAP there was again progression. Brentuximab-vedotin was given. After the first cycle the patient got pneumonia and died despite the treatment in an intensive care unit.

Conclusions: A case is presented here that showed a development from extranodal marginal zone lymphoma over a diffuse large B-cell lymphoma to Hodgkin lymphoma. Despite the wealth of opportunities our patient died of the lymphoma.

Disclosure: No conflict of interest disclosed.

P255 – Unusual presentation of Hodgkin’s lymphoma

Dickmann J.R.M.1, Kerstan H.2, Heine M.3, Morche M.4, Austein T.1

1St. Bernhard Hospital, Department of Haematology and Oncology, Brake, Germany, 2St. Bernhard Hospital, Department of Gastroenterology, Brake, Germany, 3Institute of Pathology, Bremerhaven, Germany, 4Institute of Radiology Jade Weser, Brake, Germany

Introduction: Lymphomas typically present with peripheral lymph node swelling. Occasionally they may manifest within other lymph node regions or organs1. We report an unusual case of Hodgkin’s lymphoma affecting the skin and colon.

Case: A 42-year-old man presented to our department with general decline, night sweats and dyspnoea. Examination found generalised lymphadenopathy and a right pectoral skin exanthema. Laboratory data showed a normocytic anaemia (Hb 7,7 g/dl, MCV 78fl) with a leuko- and thrombocytosis. A colonoscopy found numerous polyps in the sigmoid colon. Histologically these contained atypical CD30+ cells.

Skin, lymph node and bone marrow biopsies were all consistent with a diagnosis of Hodgkin’s lymphoma. Radiological investigations suggested the involvement of the lung parenchyma. Due to the patient’s poor physical condition, we started chemotherapy with ABVD. After improvement of the patient’s condition, we escalated the therapy and he achieved a complete remission after four cycles of BEACOPP.


Discussion: Hodgkin’s lymphoma is rarely found to manifest in the gastrointestinal tract. Unfortunately the impact on prognosis is not known. There are a few cases described in the literature of primary gastrointestinal Hodgkin’s lymphoma2 presenting with a stricture or ulceration. Our patient gave no history of gastrointestinal symptoms and our findings were unexpected. More data is needed to ascertain what impact this manifestation has on the prognosis for patients.

Conclusion: When patients with a suspected diagnosis of lymphoma present with a severe anaemia, endoscopic investigations may be considered to rule out gastrointestinal involvement.


1 Austein T, Demme B: Uncommon recurrence of follicular lymphoma, Eur J Haematol. 2012 Jan;88:91.

2 Sharma S. et al. Primary Intestinal Hodgkin´s Lymphoma: An Uncommon Presentation. J Lab Physicians. 2013 Jul;5:124–126.

Disclosure: No conflict of interest disclosed.

P256 – HIV-patient with advanced Hodgkin Lymphoma, hyperinflammation and limited NSCLC

Mueller M.1, Rittweger M.1, Weber C.1, Lindner A.1, Arasteh K.1

1Klinik für Infektiologie und Gastroenterologie, Vivantes Auguste-Viktoria-Klinikum, Berlin, Germany

Introduction: We report a case of a HIV-positive patient with classical Hodgkin-Lymphoma (HL), severe hyperinflammation and a limited NSCLC.

Method: Case report.

Results: A 66y male patient with recently diagnosed HIV-infection (CD 95/µl, 33%, viral load 8600 c/ml), a history of syphilis and tobacco use of 60 packyears, was admitted in our hospital because of fever, encephalopathy, pancytopenia and hypoalbuminemia. The seriously ill patient was transferred to the ICU with hyperinflammation. 6 of 8 criteria for hemophagocytic lymphohistiocytosis (HLH) were positive (Henter 2007).

A classical EBV-associated Hodgkin Lymphoma was diagnosed with bone marrow, liver and lymph node involvement. CT-scan and biopsy revealed also a peripheral lung tumour (large-cell neuroendocrine carcinoma).

Patient recovered quickly after one cycle of ABVD (dose-adjusted because of hyperbilirubinemia). A complete remission of the HL was achieved after 5 cycles of BEACOPP-baseline and PET-CT showed also a partial remission of the NSCLC. A HSV2 encephalitis were treated successfully and the NSCLC was resected subsequently (pT2b,N1,V1,R0,G3). After tumour board discussion 4 cycles of Cisplatin/Etoposid (dose reduction because of thrombocytopenia and febrile neutropenia) were applied. He died unexpectedly shortly after the last cycle. Autopsy revealed heart failure.


-In HIV-positive patients with fever of unknown origin HLH due to Hodgkin-Lymphoma should be ruled out.

-Second neoplasia in an immunosuppressed patient is not a contraindication for curative treatment.

Disclosure: No conflict of interest disclosed.

Posterdiskussion – Allogene Stammzelltransplantation 1

P257 – Reconstitution of CD8+ Treg after allogeneic stem-cell transplantation

Holderried T.A.W.1, Sauerborn P.1, Kim H.-J.2, Cantor H.2, Wolf D.1, Brossart P.1

1University Hospital Bonn, Department of Hematology, Oncology and Rheumatology, Bonn, Germany, 2Harvard Medical School, Department of Cancer Immunology and Virology, Boston, United States

Graft-versus-Host Disease (GvHD) is a potentially life-threatening complication after allogeneic stem-cell transplantation (allo-SCT), limiting the therapeutic success of this paradigmatic immunotherapeutic approach to treat hematological diseases. CD4+ regulatory T cells (Treg) have been shown to be beneficial in the prevention and/or treatment of GvHD while sparing the Graft-versus-Leukemia (GvL) effect. The contribution of regulatory CD8+ T cell-mediated immune responses in GvHD, however, has not been studied so far. Recent studies have identified a small subset of IL-15 dependent CD8+ Treg that are essential for maintenance of self-tolerance and which is characterized by the co-expression of CD44, CD122 and Ly49. The differentiation and the immune-regulatory function of CD8+ Treg depend on the transcription factor Helios.

Here we have identified the early development of CD8+ Treg after allogeneic stem-cell transplantation. We defined the human homologue of murine CD8+ Treg using the killer cell immunoglobulin-like receptor (KIR), the functional homologue of the murine Ly49 receptor, as a surface marker on CD8+ T cells to determine the human regulatory CD8+ T cell subset. Similar to murine CD8+ Treg, the cells express CD44, CD122 as well as the transcription factor Helios. In vitro assays showed responsiveness of KIR+CD8+ cells to IL-15 and suppression of CD4+ TFH cell responses. Assessment of KIR+CD8+ T cells in patients after allo-SCT revealed the presence of CD8+ Treg as early as day +30 after transplantation. The proportion of the CD8+ Treg subset increases thereafter and reaches a peak around day +90. This T cell population has a stable expression of the surface markers CD44 and CD122 whereas the transcription factor Helios is significantly up-regulated after allo-SCT when compared to healthy donors.

Taken together, we have identified that Helios+KIR+CD8+ Treg population recover early after allo-SCT. Our findings implicate an important role of CD8+ Treg during regeneration of the immune system in these patients and may control development of GvHD. Systematic quantification of KIR+CD8+ T cells in patients after allo-SCT may provide a deeper insight into the functional involvement of this Treg population in the pathogenesis of acute and chronic GvHD. These data will then set the stage for potential immune-modulatory strategies specifically activating or expanding CD8+ Treg to prevent and/or treat GvHD.

Disclosure: No conflict of interest disclosed.

P258 – Trephine biopsies are preferable to isolate mesenchymal stromal cells after allogeneic hematopoietic stem cell transplantation

Krüger T.1, Middeke J.M.1, Stölzel F.1, Mütherig A.1, List C.1, Brandt K.1, Heidrich K.1, Döpper J.1, Teipel R.1, Francke S.1, Müller K.1, Kräter M.1, Dhawan A.1, Ordemann R.1, Schuler U.1, Oelschlägel U.1, Wermke M.1,2, Wehner R.3, Schmitz M.3,4,5, Bornhäuser M.1,4,5, von Bonin M.1,5,6

1Medizinische Klinik und Poliklinik I, Universitätsklinikum Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany, 2Universitäts Krebs Centrum (UCC), Early Clinical Trial Unit (ECTU), Universitätsklinikum Carl Gustav Carus, Dresden, Germany, 3Institut für Immunologie, Medizinische Fakultät, Technische Universität Dresden, Dresden, Germany, 4Nationales Centrum für Tumorerkrankungen (NCT), Partnerstandort Dresden, Dresden, Germany, 5Deutsches Konsortium für Translationale Krebsforschung (DKTK), Standort Dresden, Dresden, Germany, 6Deutsches Krebsforschungszentrum (DKFZ), Heidelberg, Germany

Introduction: Mesenchymal stromal cells (MSCs) substantially participate in maintaining hematopoiesis as self-renewing progenitors of several different stromal cells of the bone marrow microenvironment and as part of the hematopoietic stem cell (HSC) niche. Because bone marrow stromal cells remain host-derived after allogeneic stem cell transplantation (SCT) it is suggested that the HSC niche could be a target of Graft versus Host Disease (GvHD). Indeed myelosuppression has been repeatedly described in the context of GvHD and in particular a low platelet count was proposed as an indicator of GvHD severity. Hence MSCs as part of the HSC niche may play a key role in development and modulation of GvHD associated myelosuppression. Therefore, it would be clinically relevant to investigate these cells in this context. However, bone marrow aspirates usually used to isolate MSCs fail to reliably recover sufficient amounts of cells after allogeneic SCT.

Methods: Bone marrow aspirates (5-10ml) and trephine biopsies were performed within routine diagnostics after allogeneic SCT. Both, aspirates and trephine biopsies were processed using a collagenase based protocol. The colony-forming unit – fibroblast (CFU-F) assay was used to functionally detect MSCs. MSC frequency was determined by limiting dilution assay. Remaining cells were seeded for expansion.

Results: Limiting dilution assay of 21 pairs of aspirates and trephine biopsies revealed a significant higher MSC frequency in trephine biopsies (trephine biopsy: 1 MSC in 1.5×104 ± 2.7×104 TNCs; aspirates: 1 MSC in 8.97×105 ± 8.27×105 TNCs, p = 0.0002). Furthermore, expansion of MSCs isolated from trephine biopsies was more reliable and led to a significantly higher yield at passage 0 compared to aspirate derived MSCs.

Conclusion: Collagenase digestion of trephine biopsies is a suitable method to reliably isolate sufficient numbers of MSCs after allogeneic SCT which now allows the examination of their role in GvHD-mediated myelosuppression.

Disclosure: No conflict of interest disclosed.

P259 – Chip-based digital PCR: an accurate and sensitive method for routine chimerism monitoring after hematopoietic stem cell transplantation

Gourri E.1, Schanz U.2, Frey B.M.1, Gassner C.1

1Blood Transfusion Service Zurich, Swiss Red Cross, Department of Molecular Diagnostics and Research & Development (MOC), Zurich-Schlieren, Switzerland, 2University Hospital Zurich, Department of Hematology, Zurich, Switzerland

Introduction: After hematopoietic stem cell transplantation (HSCT) recipient and donor populations of leukocytes cohabit in the peripheral blood of the patient, resulting in “chimerism”. Monitoring chimerism is crucial to follow the outcome of the disease and to make informed clinical decision concerning further therapeutic interventions. Current chimerism monitoring is performed using Short-Tandem-Repeats analysis (STR) and quantitative real-time PCR (RT-PCR). However, both methods suffer from limited sensitivity and reproducibility. Digital PCR (dPCR) represents a new alternative for accurate and reproducible chimerism monitoring.

Methods: Chip-based Quantstudio 3D chip-based dPCR (Applied Biosystems, Reinach, Switzerland) allows for SNP detection in approximately 20’000 separate RT-PCRs per sample, with statistical presence of only 0, 1, or 2 DNA molecules per each 865 pL reaction well. Fluorescence of each well is separately measured and delivers absolute counts of the two different alleles. Original samples were analyzed for their SNP genotypes by PCR using Sequence Specific Priming (PCR-SSP).

Results: Using artificial DNA mixes, we estimated our limit of detection to range at approximately 0.5% for the minor DNA. Results within the same samples were highly homogeneous using six different SNP-Taqman assays for evaluation. Typing samples of Instand’s external proficiency testing (EPT) for post-HSCT chimerism, was performed on one pre-HSCT patient and donor sample each and on five post-HSCT samples, using only two different SNP-Taqman assays. Instand honored our result quality with 20/20 points. On 15 DNA sample triplets, consisting of one pre-HSCT, one donor and one post-HSCT specimen each, dPCR results were consistent with previous STR results in 11 out 15 triplets. Discrepancies were only observed for 4 triplets, where chimerism was estimated to range below 5% by STR analysis, a method with known limited sensitivity (Stahl et al., Exp Hematol, 2015 Jun).

Conclusion: According to the high performance of our method in the Instand EPT and based on the results of 15 patient/donor/post-HSCT sample triplets, chip-based dPCR appears as an accurate and routinely applicable technique for exact chimerism determination with excellent sensitivity.

Disclosure: No conflict of interest disclosed.

P260 – IL17 as a marker for acute GVHD

Wolff D.1, Schmitt T.1, Wölfinger P.1, Bode S.1, Theobald M.1, Wagner E.M.1

1UCT Universitätsmedizin Mainz, Hämatologie, internistische Onkologie, Pneumologie, Mainz, Germany

Introduction: Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative option for patients suffering from AML. Despite of the beneficial graft versus leukemia effect the Graft versus host disease (GVHD) as a dysregulation of the donor immune cells is one of the major complications after HSCT and causes morbidity and mortality. Major players in this immune reaction are donor T cells (CD8 and CD4 T cells), but the mechanism is not fully understood. Especially the role of CD4 T cells remains unclear. Here we investigate on IL17-producing CD4 T- cells in the course of GVHD in our patients after allogeneic HSCT. IL17 producing cells are controversial in literature, their impact in development of GVHD is under discussion.

Methods: We analyzed PBMC´s from patients after allogeneic HSCT and dose reduced conditioning regimens with multicolor FACS-staining for CD3, CD4, CD8, and regulatory T-cells (CD25, CD127neg, FoxP3) as well as intracellular IL17. IL17 production was measured with and without stimulation with PMA. Our patient cohort consists of 21 patients with acute GVHD, 13 patients with chronic muco-cutaneous GVHD and 5 healthy controls. For 3 patients with overlap syndrome we collected blood samples in the time course of GVHD and correlated our findings with clinical outcome and response to treatment.

Results: At onset of GVHD we found no significant differences in the absolute and relative amounts of CD3, CD4, CD8 and Treg counts in our patients with acute or chronic GVHD. Also the unstimulated baseline secretion of IL17 was similar in patients with acute or chronic GVHD compared to healthy controls. In contrast, after stimulation with PMA the increase in IL17 positive T cells was significantly higher in patients with acute GVHD compared with patients with chronic GVHD or healthy controls. Especially in patients with severe gastrointestinal GVHD we found highest percentages of IL17-producing CD4-T cells. In patients with overlap syndrome, these amounts of IL17 positive CD4-Tcells correlates with treatment refractory persisting GVHD.

Conclusion: In patients after allogeneic stem cell transplantation the increase in IL17 producing CD4 T-cells after PMA-stimulation correlates with acute GVHD and treatment response. This biomarker should be investigated in a prospective manner to diagnose GVHD and could guide immunosuppressive treatment.

Disclosure: No conflict of interest disclosed.

P261 – Results of sequential therapy in the setting of unmanipulated HLA-haploidentical transplantation utilizing post-transplantation high-dose cyclophosphamide in the treatment of high-risk AML and MDS

Prevalsek D.1, Fritsch S.1, Hubmann M.1, Zoellner A.-K.1, Köhnke T.1, Engel N.1, Bücklein V.L.1, Schulz C.1, Mumm F.1, Berking S.1, Ledderose G.1, Stemmler H.-J.1, Spiekermann K.1, Hiddemann W.1, Hausmann A.1,2, Tischer J.1

1Klinikum der Universität München – Campus Grosshadern, Medizinische Klinik und Poliklinik III, München, Germany, 2Klinikum München-Schwabing, Medizinische Klinik I, München, Germany

Hematopoietic stem cell transplantation offers a curative treatment option for patients suffering from high-risk AML and MDS, but the availability of HLA-matched donors might be limited.

To evaluate the feasibility and outcome of sequential therapy in the context of unmanipulated HLA-haploidentical hematopoietic stem cell transplantation (haplo-HSCT) utilizing post-transplantation cyclophosphamide (PTCY) in patients with high-risk, relapsed and refractory AML/MDS, we retrospectively evaluated the course of 64 patients (AML n = 61, MDS n = 3; median age: 50 years) transplanted between 2009 and 2015 at our center. Disease was advanced in 55 patients including 24 patients with relapse after a first allogeneic transplantation. 54 patients presented with active disease at time of haplo-HSCT while 9 patients were in CR. All patients received sequential therapy combining cytoreductive chemotherapy (clofarabine n = 34; FLAMSA n = 25; FLAG-Ida n = 2; others n = 3) and reduced-intensity conditioning (RIC) which was started after three days of rest thereafter. Conditioning was drug-based in the majority of patients (n = 42). As backbone of post-grafting immunosuppression high-dose cyclophosphamide was given on day + 3 and +4 in all patients. Bone marrow was the graft source in 37 patients.

One graft rejection was observed. Neutrophil/platelet engraftment was achieved in 58/49 patients, respectively. Acute GvHD grade II-IV occurred in 19 patients (30%) while it was severe (grade III-IV) in only 3 (5%). Chronic GvHD was most frequently mild (n = 9) to moderate (n = 8); one severe chronic GvHD occurred. Severe (grade III-IV) mucositis, hemorrhagic cystitis and hand-foot syndrome/rash were observed in 10, 5 and 2 patients, respectively; no patient developed VOD. Kidney failure requiring hemodialysis occurred in 7 patients. CMV reactivation was observed in 59%, while only one patient developed CMV disease (pneumonia) and no patient developed PTLD. Probable or proven (n = 2) invasive aspergillosis was diagnosed in 10 patients. One-year non-relapse mortality was 27.5%. After a median follow up of 21 months (range: 3-64), estimated two-year OS and DFS were 39%, both.

In summary, sequential therapy in the setting of TCR haplo-HSCT using PTCY is well tolerated with low rates of GvHD and acceptable NRM in high-risk AML/MDS, while providing an effective anti-leukemic activity in advanced disease. Thus, we suggest that donor availability can be expanded in patients with high-risk AML/MDS.

Disclosure: No conflict of interest disclosed.

P262 – Graft-versus-leukemia effects in T-prolymphocytic leukemia: evidence from minimal residual disease kinetics and T cell receptor repertoire analyses

Sellner L.1, Brüggemann M.2, Schlitt M.2, Knecht H.2, Herrmann D.2, Reigl T.3, Krejci A.3, Bystry V.3, Darzentas N.3, Rieger M.4, Dietrich S.1, Zenz T.1, Ho A.D.1, Kneba M.2, Dreger P.1

1Universitätsklinikum Heidelberg, Medizinische Klinik V, Heidelberg, Germany, 2Universitätsklinikum Schleswig-Holstein, Kiel, Germany, 3Central European Institute of Technology, Brno, Czech Republic, 4Onkologische Schwerpunktpraxis Darmstadt, Darmstadt, Germany

Introduction: Allogeneic stem cell transplantation (alloSCT) may provide long-term disease control in T-prolymphocytic leukemia (T-PLL). However, direct evidence of graft-versus-leukemia (GVL) activity in T-PLL is lacking. We investigated GVL by correlating minimal residual disease (MRD) kinetics with immune modulatory interventions and T cell receptor (TCR) repertoire diversity after alloSCT.

Methods: The study sample consisted of 10 patients who received alloSCT for T-PLL at the University of Heidelberg between 2007 and 2015. Quantitative MRD monitoring was performed using clone-specific real-time quantitative PCR (RQ-PCR) of clonal TCR beta (TRB) and/or gamma gene rearrangements. TCR repertoire diversity was analyzed longitudinally by next-generation sequencing (NGS) on Illumina’s MiSeq platform.

Results: The 3-year relapse-free and overall survival was 48% (95%CI 16-80%) and 58% (95%CI 27-90%), respectively. All patients had a cytological complete response (CR) after alloSCT (5 unrelated, 4 related, 1 haploidentical). 2 patients died early because of acute GvHD, and one had no MRD marker, leaving 7 patients for MRD monitoring. Of these, 3 were MRD- at alloSCT, whereas 5 patients remained or became MRD+ early after alloSCT. In all of these 5 patients, immunosuppression tapering (3) or DLI (2) resulted in significant reduction of MRD levels (range 1-3 log) and was accompanied by cGvHD in 3 patients. However, durable MRD- was obtained in only 2 patients (alive 86+ and 12+ months post transplant). The TRB repertoire of the three patients with the longest follow-up (up to six years) was interrogated longitudinally using NGS. In all patients, MRD decline was reproducibly associated with a shift from a clonal, T-PLL-driven profile to a polyclonal signature which largely corresponded to the donor TCR repertoire and receded with increasing MRD levels. Notably, there was no obvious correlation of GVL-induced MRD decline with emergence of dominant T cell clonotypes that could explain a clonal GVL effect.

Conclusions: This study provides first direct evidence for GVL activity in T-PLL, even though it appears to be often only limited or transient. Moreover, GVL in T-PLL does not seem to be driven by the emergence of novel dominant T cell clones but is rather relying on poly- or oligoclonal T cell responses. Nonetheless, alloSCT in T-PLL is a valuable treatment option, and further evaluation of MRD monitoring is warranted to optimize patient care after alloSCT.

Disclosure: No conflict of interest disclosed.

P263 – Proteasome inhibitors and immunomodulatory drugs improve survival in relapsed or progressive multiple myeloma after allogeneic hematopoietic cell transplantation

Schneidawind C.1, Duerr-Stoerzer S.1, Faul C.1, Kanz L.1, Weisel K.1, Bethge W.1, Schneidawind D.1

1Department of Medicine II, Eberhard Karls University, Tübingen, Germany

Introduction: Multiple myeloma is the most common indication for autologous hematopoietic cell transplantation (HCT) but transplantation from an allogeneic donor is controversial and reserved for high-risk patients with refractory, relapsed or progressive disease.

Methods: We report a retrospective single center analysis of 41 consecutive patients that underwent allogeneic HCT for the treatment of multiple myeloma from 2001 to 2015.

Results: We identified and analyzed 41 consecutive patients with a median age of 53 years (range, 35-67) transplanted for the treatment of relapsed (n = 11), progressive (n = 27) or refractory (n = 3) multiple myeloma. Median time from diagnosis of multiple myeloma to allogeneic HCT was 34 months (range, 2-142). Median time from first autologous HCT to allogeneic HCT was 21 months (range, 1-107). The number of patients transplanted from matched unrelated donors (MUD, n = 20) was high compared with matched related donors (MRD, n = 13) or mismatched unrelated donors (MMUD, n = 8). 3-year event-free survival (EFS) and overall survival (OS) of the whole cohort were 15% and 51%, respectively. Allogeneic HCT after a second high-dose chemotherapy with autologous stem cell support (n = 18) was associated with a decreased 3-year EFS (6% vs. 24%, p = 0.04) and OS (35% vs. 64%, p = 0.09). Cumulative incidence of non-relapse mortality (NRM) at 3 years was 20%. However, the 3-year cumulative incidence of relapse or progression adjusted for NRM as competing risk was 65%. In case of relapse or progression after allogeneic HCT, the treatment with proteasome inhibitors or immunomodulatory drugs significantly improved survival (1-year OS 79% vs. 29%, p = 0.001). Importantly, the administration of immunomodulatory drugs and/or proteasome inhibitors was well tolerated. We did not observe acute graft-versus-host disease (GVHD) =2 but limited and extensive chronic GVHD occurred in 5 (25%) and 3 (15%) patients that received a lenalidomid-containing regimen.

Conclusions: Our data suggest that proteasome inhibitors and immunomodulatory drugs are important for the management of relapsed and progressive multiple myeloma following allogeneic HCT. The incorporation of these agents into transplant protocols might improve outcomes and refine the role of allogeneic HCT for the treatment of multiple myeloma as a platform for long-term disease control.

Disclosure: No conflict of interest disclosed.

P264 – Central nervous system complications after allogeneic hematopoietic cell transplantation – a significant cause of morbidity and mortality

Simon C.1, Zierhut M.2, Mirza N.1,3, Faul C.1, Korn A.4, Vogel W.1, Rammensee H.-G.3, Kanz L.1, Bethge W.A.1, Haen S.P.1,3

1Medizinische Universitätsklinik Tübingen, Abteilung II für Onkologie, Hämatologie, Immunologie, Rheumatologie und Pulmologie, Tübingen, Germany, 2Universitätsaugenklinik Tübingen, Tübingen, Germany, 3Interfakultäres Institut für Zellbiologie, Abteilung Immunologie, Tübingen, Germany, 4Universitätsklinik für Neuroradiologie, Tübingen, Germany

Introduction: Central nervous system (CNS) complications after allogeneic hematopoietic cell transplantation (HCT) can represent diagnostic and therapeutic challenges since differential diagnosis between malignant, inflammatory, circulatory or infectious causes is difficult and time-consuming but immediate precise therapy is mandatory to prevent permanent neurological damage.

Methods: Out of 1,204 patients undegroing allogeneic HCT at our institution between 2003 and 2015, we identified 102 patients (8.6%) with post-transplantation CNS complications. The patients (38 women and 64 men, median age 55 years, range 19-75 years) received HCT due to acute myeloid (n = 49) or lymphoblastic (n = 24) leukemia, chronic lymphocytic (n = 2), myelomonocytic (n = 2) or myeloid (n = 1) leukemia, lymphoma (n = 10), myelodysplastic (n = 8) or myeloproliferative (n = 5) syndromes, as well as severe aplastic anemia (n = 1). Conditioning was performed using reduced intensity (n = 61) or myeloablative (n = 41) regimens.

Results: CNS complications comprised bleeding (n = 23) or thromboembolic (n = 11) events, infections (n = 19), toxic leukoencephalopathy (n = 10), inflammatory changes without detection of underlying cause (n = 11), CNS relapse (n = 10), secondary intracerebral tumors (n = 3), posterior reversible encephalopathy syndrome (n = 4), seizures (n = 3), migraine (n = 2), degenerative Parkinson-like syndrome (n = 1) or neurological and psychiatric syndromes without detection of a clear etiology (n = 5). Median time between HCT and onset of CNS symptoms was 4.6 months (range, 0-155 months). Treatment was guided by the respective diagnosis and included systemic or intrathecal chemotherapy, anti-infective or anti-inflammatory drugs, anticoagulation and recanalization leading to resolution of neurological symptoms (n = 31), amelioration (n = 15), stabilization (n = 28) or disease progression and death (n = 28). Cumulative incidence of CNS disease-related deaths was 11% and 21% at 100 days and 12 months, respectively (1% and 2% for the complete patient cohort of 1,204 patients). Median overall survival was 13.8 and 6.4 months after HCT and onset of CNS complications, respectively.

Conclusions: CNS manifestations account for a significant proportion of HCT-related complications resulting in significant mortality and morbidity with 70% patients experiencing permanent neurological damage. Therefore, precise and prompt diagnosis is essential to guide therapy and can be sometimes challenging.

Disclosure: No conflict of interest disclosed.

P265 – Antibody protection against measles, mumps and rubella depends on underlying disease and conditioning regimen in patients given allogeneic hematopoietic cell transplantation

Bögeholz J.1, Manz M.G.1, Schanz U.1, Müller A.M.S.1

1Universitätsspital Zürich, Division of Hematology, Zürich, Switzerland

Introduction: Following allogeneic hematopoietic cell transplantation (HCT) the immune system is severely compromised due to pharmacological immunosuppression and disruption of lymphoid tissues by conditioning and donor T cell alloreactivity. Guidelines recommend re-vaccinations against certain pathogens, however, live-vaccines should be avoided in the early post-HCT period. Here, we studied the dynamics of antibody (AB) titers against measles, mumps, and rubella (MMR) during the first 3 years (y) post-HCT.

Methods: We retrospectively analyzed serial AB titers in 79 patients who underwent allogenic HCT from HLA-matched related donors from 2009-2014 at our center. AB titers against MMR were measured prior to HCT, at 6 months (m), 1, 2, and 3y post-HCT.

Results: Most patients had protective AB titers (measles 94%, mumps 85%, rubella 92%) prior to HCT. Although only recipients of seropositive donors were included in the analysis AB protection was lost in a substantial proportion of patients post-HCT (protection @2y in 79% for measles, 50% for mumps 50%, 76% for rubella). The proportion of patients who retained protective AB titers at 1y post-HCT was higher in recipients of mobilized peripheral blood compared with bone marrow (BM) grafts (measles p = 0.01, mumps = 0.02, rubella p = 0.13). For rubella, absolute AB titers were available. Figure 1 displays the decline of rubella AB concentrations @6m, 1y, 2y, and 3y post-HCT. Patients with lymphoid malignancies, MAC regimen, ongoing GVHD and pharmacological immunosuppression had a steeper decline of rubella AB titers.


Conclusion: Despite donor seropositivity there is a marked decline of AB titers post-HCT with loss of protection in a substantial proportion of patient. Surprisingly, BM grafts did not provide better AB protection post-HCT, despite their higher content of plasma cells. Together with the observations that (i) patients with lymphoid malignancies (who have received (B-) lymphocyte targeted therapies prior to HCT) had lower AB levels, while (ii) those given RIC have higher AB levels our data suggest that residual host plasma cells significantly contribute to AB production during the first years post-HCT.

Disclosure: No conflict of interest disclosed.

P266 – Allogeneic stem cell transplantation for secondary and therapy-related acute myeloid leukemia: a single center analysis of long-term outcome

Hemmati P.1, Pfeifer K.1, Vuong L.1, Jehn C.1, Terwey T.1, le Coutre P.1, Dörken B.1, Arnold R.1

1Charité – Universitätsmedizin Berlin, Medizinische Klinik m.S. Hämatologie, Onkologie und Tumorimmunologe, Berlin, Germany

Introduction: Acute myeloid leukemia (AML) evolving from myelodysplastic syndrome (MDS) or myeloproliferative neoplasia (MPN) as well as therapy-related AML represent distinct entities in the current WHO classification and are characterized by an inferior outcome compared to de novo AML. This is mainly due to unfavorable cytogenetics, older age and/or comorbidities as well as poor response to induction therapy. For the majority of patients, allogeneic stem cell transplantation represents the only option to achieve long-term disease control and definitive cure.

Patients and methods: With regards to long-term outcome, we retrospectively analyzed 204 patients with secondary AML, i.e. AML following MDS/MPN, therapy-related AML, or MDS RAEB-II transplanted at our center between 1996 and 2015. Cytogenetic risk was categorized according to the SWOG/ECOG classification and was favorable (N = 3; 2%), intermediate (N = 94; 46%), unfavorable (N = 84; 41%), or unknown/undetermined (N = 23; 11%). At the time of alloSCT, 98 patients (48%) were in complete hematologic remission (CHR), whereas 106 patients (52%) had active disease. Standard myeloablative conditioning (MAC) was used in 41 patients (20%), whereas reduced intensity conditioning (RIC) was applied in 163 patients (80%). Grafts were from related (N = 51; 25%) or unrelated (matched: N = 112; 55% or mismatched: N = 41; 20%) donors.

Results: The median follow-up of the surviving patients was 46 (5-24) months. At 1, 3, 5, and 10 years after alloSCT, disease-free survival (DFS) of the entire cohort was 50%, 38%, 36%, and 27%. At the same time points, the cumulative incidence of relapse (CI-R) or non-relapse mortality (CI-NRM) was 30%, 37%, 37%, and 40% or 20%, 25%, 27%, and 33%, respectively. Univariate analyses revealed a number of factors associated with inferior outcome, e.g. poor-risk cytogenetics, the presence of tAML, the use of reduced-intensity conditioning, and comorbidities, whereas age, donor type (unrelated versus unrelated), and remission status had no significant impact on overall results.

Conclusions: Our results indicate that alloSCT represents an important treatment option for patients with secondary and therapy-related AML. However, relapse rates approach 40% at 10 years, which prompts the development of novel strategies to prevent disease recurrence. Furthermore, NRM remains high. Therefore, patients must be carefully selected for transplantation.

Disclosure: No conflict of interest disclosed.

P267 – BKV disease during the first 100 days after allogeneic hematopoietic stem cell transplantation – results of a screening program and retrospective analysis

Posdzich P.1, Herling M.1, Chemnitz J.1, Leitzke S.1, Di Christanziano V.2, Höller K.2, Kaiser R.2, Scheid C.1, Holtick U.1

1Klinik I für Innere Medizin, Universität Köln, Köln, Germany, 2Institut für Virologie, Universität Köln, Köln, Germany

Introduction: BK virus (BKV) is a human polyomavirus often acquired in childhood which can reactivate after allogeneic hematopoietic stem cell transplantation (HSCT). BKV reactivation may present in different ways from asymptomatic viruria to hemorrhagic cystitis or nephritis.

Methods: We evaluated 377 patients (159 male, 218 female, median age 52 years) who were transplanted at the University of Cologne between 2008 and 2013 to assess incidence and risk factors for BKV disease after allogeneic HSCT. All 377 patients were screened routinely for BKV in urine and serum. We defined BKV disease as hemorrhagic cystitis (HC) with BK virus >100.000/ml in urine, hematuria (2+ until 4+ in dipstick test) and negative urine microbiology. To find risk factors for BKV disease, the impact of conditioning, CMV status, graft-versus-host disease (GvHD), underlying disease, donor mismatch and stem cell source was analyzed.

Results: According to our definition, BKV cystitis occurred in 37 of 377 patients (9,8%) in the first 100 days after HSCT. In 14 of these 37 patients (37,8%) BKV was also detected in serum. BKV was detected in the urine of 194 patients (51,4%) at any time during the screening program, but only 19,1% developed hemorrhagic cystitis. Sixty-two percent of patients suffering from BKV disease were female, 37,8% male. BKV disease was diagnosed in 13,3% of patients not in complete remission as opposed to 5,4% of patients in complete remission prior to conditioning (p = 0,010). BKV disease was less common in matched related or unrelated (7,7%) as compared to mismatched or haplo-identical donors (16,7%; p = 0,012).

Acute GvHD grade II to IV was described in 122 of 377 patients (32,36%). Of them, 13,9% suffered from BKV cystitis in comparison to 7,84% with no or grade I acute GvHD, which did not reach statistical significance. We could not detect any correlation between BKV disease and administration of cyclophosphamide, total body irradiation or anti-thymocyte globuline. There was no association with the graft source. BKV disease was no predictor for non-relapse mortality, overall and relapse-free survival.

Conclusion: BKV disease is a common complication after allogeneic HSCT. The virus can be found in the urine of about half of our transplanted patients, but only a small proportion developed an HC. State of remission before conditioning and donor mismatch are associated with the development of BKV disease, which had no impact on survival in our cohort.

Disclosure: No conflict of interest disclosed.

P268 – High CD34+ cell dose is associated with increased early mortality after allogeneic hematopoietic stem cell transplantation

Neumann K.1, Lange S.1, Kragl B.1, Glass Ä.2, Große-Thie C.1, Wittke C.1, Freitag S.1, Henze L.1, Kleine H.-D.3, Junghanss C.1

1Department of Medicine, Clinic III – Hematology/Oncology/Palliative Care, Rostock University Medical Center, Rostock, Germany, 2Institute for Biostatistics and Informatics in Medicine and Ageing Research, Rostock, Germany, 3Seracell Stammzelltechnologie GmbH, Rostock, Germany

Introduction: Survival after allogeneic hematopoietic stem cell transplantation (HSCT) is influenced by a variety of recipient- and graft-related factors as well as conditioning regimen. Whereas some research groups point out higher mortality rates with increased CD34+ cell doses, others do not. We aimed at determining the impact of CD34+ cell dose on early mortality after allogeneic HSCT in a retrospective cohort analysis.

Patients and methods: Data sets of 436 consecutive patients who underwent allogeneic HSCT at our center from 1998 to 10/2015 were evaluated regarding their early mortality rates (d+30, d+60). Cox regression analysis comprised the variables viable CD34+ cell dose, recipient sex, age, body mass index (BMI), HSCT indication, conditioning intensity, stem cell source, post-transplant immunosuppression and donor type.

Results: Median recipient age at time of HSCT was 49 (15-76) years. Indications for HSCT comprised AML/MDS (44.5%), ALL (8.3%), CML (10.1%), NHL (15.1%), CLL (4.4%), Hodgkin’s disease (0.7%), multiple myeloma (8.0%) and other diseases (8.9%). Conditioning was carried out employing myeloablative (27.6%) and reduced intensity or non-myeloablative (72.4%) regimens with treosulfan/fludarabine-based conditioning being most common (53.2%). Matched unrelated donors (54.2%) and matched related donors (33.1%) accounted for the most frequent donor types. Patients received a median CD34+ cell dose of 4.4×106/kg (0.4×106-22×106/kg). Grafts originating from bone marrow (n = 72) and peripheral blood (n = 364) contained a median CD34+ cell dose of 2.0×106/kg and 5.0×106/kg (p < 0.001). Mortality at d+30 and d+60 amounted to 6.9% and 10.7%, respectively.

In multivariable analysis, cell numbers < 2.0×106 (14.0%) and even < 1.0×106 CD34+ cells/kg (2.3%) were not found to negatively impact early mortality. Conversely, patients receiving =9.0×106 CD34+ cells/kg (7.1%) experienced significantly increased mortality at d+30 (p = 0.027) and d+60 (p = 0.041).

Additionally, myeloablative conditioning proved to be associated with significantly higher early mortality compared to reduced intensity or non-myeloablative regimens (d+30: p = 0.005; d+60: p = 0.037).

Conclusions: Early post-transplant mortality seems to be increased following allogeneic HSCT of viable CD34+ cell doses =9.0×106 CD34+ cells/kg.

Disclosure: No conflict of interest disclosed.

P269 – Overall and median survival of patients after allogenic stem cell transplantation with conditioning by treosulfan and fludarabin

Freitag S.1, Kragl B.1, Wittke C.1, Brueckner F.1, Gläser D.1,2, Große-Thie C.1, Henze L.1, Hilgendorf I.1,3, Gläser H.1, Freund M.1, Junghanss C.1

1Department of Medicine, Clinic III – Hematology/Oncology/Palliative Care, Rostock University Medical Center, Rostock, Germany, 2Clinic of Internal Medicine III – Hematology/Oncology/Hemostaseology, Palliative Care, Complementary Medicine, Klinikum Südstadt Rostock, Rostock, Germany, 3Clinic of Internal Medicine II – Division of Hematology and Internal Oncology, Jena University Hospital, Jena, Germany

Introduction: Allogeneic stem cell transplantation is frequently applied in variable hematological diseases. During the last decade applied conditioning regimens have shifted from myeloablative to non-myeloablative and reduced intensity conditioning regimen (RIC). Treosulfan (Treo) and fludarabine (Flud) + antithymocyte globulin (ATG) based regimens have been evaluated successfully in several phase-II- studies. One large phase-III-study in AML is ongoing. Here we report on a large cohort of stem cell transplantation (SCT) recipients that received Treo/Flud conditioning before SCT.

Method: We analyzed consecutive allo-SCT patients in the time period from 2/1998 to 4/ 2016 who received conditioning therapy using Treo/Flud based regimens in regards to patient characteristics and overall survival.

Results: A total of 240 patients were enrolled. The patient population consisted of 137 (57%) males and 103 (43%) females with a median age of 52 years (range: 16-76 years). Underlying diseases were: acute leukemia 99 (41%), lymphoma 42 (17%), chronic leukemia 38 (16%), plasmacell disorders 22 (9%), MDS 20 (8%), and other 19 (8%). Total Treo doses were median 42g/m² (30g/m²-50g/m²) usually split in 3-5 single doses on d-6 to d-2. Flud total dose was in general 150/m² divided onto 5 days. GVHD prophylaxis was in general cyclosporine A/ MTX based. Unrelated alloSCT recipients received in addition antithymoglobulin. Most transplants were matched related (71/29%) and matched unrelated (163/68%). Early mortality rates were as follows: 30d: 12 (5%), 60d: 23 (9%), 100d: 39 (16%). The 1 year and 5 years survival rates were 62% and 45% respectively. The median overall survival for all patients was 589 days (range: 10- 5901d). Overall survival varied based on underlying disease status.

Conclusion: In our large but heterogenous cohort Treo/Flud based conditioning is associated with moderate early mortality rates compared to historical myeloablative conditioning regimen.

Disclosure: No conflict of interest disclosed.

P270 – Are the polyomaviruses BK and JC associated with opportunistic infections, graft versus host disease or worse outcome in adult patients receiving their first allogenic stem cell transplantation with low dose alemtuzumab?

Schneidewind L.1, Neumann T.2, Knoll F.2, Zimmermann K.3, Smola S.1, Krüger W.2

1Universitätsklinikum des Saarlandes, Institut für Virologie, Homburg/Saar, Germany, 2Universitätsmedizin Greifswald, Klinik für Innere Medizin C, Hämatologie/Onkologie, Greifswald, Germany, 3Universitätsmedizin Greifswald, Friedrich Löffler Institut für Medizinische Mikrobiologie, Greifswald, Germany

Introduction: Polyomaviruses BK (BKPyV) and particularly also JC (BKPyV) are important pathogens in allogenic stem cell transplantation. Their association with other opportunistic infections like CMV reactivation and acute or chronic GvHD is controversial discussed. However, there is a lack of data for adult allogenic stem cell transplantation and especially for JCPyV. We conducted a retrospective study of adult patients receiving their first allogenic stem cell transplantation with low dose alemtuzumab at the University Hospital Greifswald.

Methods: Data from all patient records (64) of adult patients undergoing their first allogenic stem cell transplantation and receiving low dose alemtuzumab for GvHD prophylaxis from 03/2010, when quantitative PCR for the BKPyV and JCPyV was established, to 12/2014 were collected including outpatient data of our clinic. Follow up time was two years after infusion of the stem cell product.

Results: We found acute leukaemia being the most frequent underlying disease (45.3%), conditioning included myeloablative (67.2%) and non-myeloablative protocols (32.8%). All patients received alemtuzumab 10 mg at day -2 (respectively 20 mg in case of mismatch) as GvHD prophylaxis before transplantation. On the whole, infectious complications occurred rarely when receiving low dose alemtuzumab, e. g. BKPyV associated hemorrhagic cystitis was diagnosed in 10 patients (15.6%). There was no significant association of BKPyV or JCPyV with CMV reactivation, EBV reactivation, HHV-6 or parvovirus B19 infection, if they required treatment. Interestingly, there was a significant association of BKPyV associated hemorrhagic cystitis with toxoplasmosis (p = 0.013). Additionally, there was a significant link with BKPy and JCPy viruria at the same time point with toxoplasmosis (p = 0.047). In this study BKPyV and JCPyV were not associated with acute or chronic GvHD. Furthermore, BKPyV or JCPyV were not associated with relapse or death.

Conclusion: In our study of adult allogenic stem cell transplant recipients receiving low dose alemtuzumab for GvHD prophylaxis we found a low frequency of viral reactivations and no association of BKPyV or JCPyV with severe viral infections or GvHD. Only the association of both polyomaviruses with toxoplasmosis was significant. This is a novel and interesting finding. Prospective studies with drug monitoring and monitoring of the immune system are necessary to confirm these findings for this kind of population.

Disclosure: Laila Schneidewind: Expert Testimony: Autorin wird aktuell mit einem Forschungsstipendium der Monika Kutzner Stuftung unterstützt.William Krüger: No conflict of interest disclosed.

P271 – Second allogeneic hematopoietic cell transplantation to treat relapsed acute leukemia

Hagmaier V.1, Schneidawind C.1, Duerr-Stoerzer S.1, Faul C.1, Kanz L.1, Bethge W.1, Schneidawind D.1

1Department of Medicine II, Eberhard Karls University, Tübingen, Germany

Introduction: Allogeneic hematopoietic cell transplantation (HCT) is the treatment of choice for high-risk myeloid and lymphoid leukemias. Relapse after allogeneic HCT is associated with a dismal prognosis and further therapeutic options are limited. One potential curative approach is a second allogeneic HCT. However, there is no consensus about optimal transplant modalities and suitable patients.

Methods: We performed a retrospective analysis of our institutional database to evaluate risk factors that influence survival after a second allogeneic HCT for the treatment of relapsed acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL).

Results: We identified 40 patients (f = 13, m = 27) with a median age of 41 years (range, 18-65) that received a second allogeneic HCT to treat relapsed AML (n = 29) or ALL (n = 11) at our institution from 2002 to 2015. At time of second HCT, 42% of patients were in complete remission (CR). Grafts from either matched related (MRD, n = 3), matched unrelated (MUD, n = 12), mismatched related (MMRD, n = 22) or mismatched unrelated donors (MMUD, n = 3) were used. 3 patients received an allograft from the same donor as at primary HCT. Current overall survival (OS) is 15/40 patients with a median follow-up of 8 months (range 1-156) of patients alive resulting in a Kaplan-Meier estimated 3-year event-free survival (EFS) and OS of 33%. Causes of death were relapse (n = 13), infections (n = 7) and others (n = 5). Cumulative incidence of non-relapse mortality (NRM) and relapse at 3 years was 29% and 44%, respectively. Early transplantation within one year after the previous allogeneic HCT showed a similar outcome compared with later HCT (3-year OS 30% vs. 33%, p = 0.41). Likewise, relapse within 6 months after the first allogeneic HCT was not associated with a significantly reduced 3-year OS after the second allogeneic HCT (30% vs. 35%, p = 0.33). However, ALL patients showed a decreased 3-year OS (13% vs. 39%, p = 0.05). Moreover, we found that a second allogeneic HCT in CR translated into a significantly improved 3-year OS when compared to patients with active disease (51% vs. 21%, p = 0.02).

Conclusions: A second allogeneic HCT is a curative treatment option for relapsed acute leukemias in selected patients. Prior salvage chemotherapy resulting in CR is an important prerequisite for long-term disease-free survival.

Disclosure: No conflict of interest disclosed.

Posterdiskussion – Immuntherapie

P272 – Fluorescence based reporter cells rapidly identify and distinguish functional Chimeric Antigen Receptors (CARs)

Rydzek J.1, Jutz S.2, Leitner J.2, Wallstabe L.1, Nerreter T.1, Huppa J.B.3, Einsele H.1, Steinberger P.2, Hudecek M.1

1Universitätsklinikum Würzburg, Medizinische Klinik II, Hämatologie, Würzburg, Germany, 2Medizinische Universität Wien, Institut für Immunologie, Zentrum für Pathophysiologie, Infektiologie und Immunologie, Wien, Austria, 3Medizinische Universität Wien, Institut für Hygiene und Angewandte Immunologie, Zentrum für Pathophysiologie, Infektiologie und Immunologie, Wien, Austria

Introduction: Adoptive immunotherapy with chimeric antigen receptor (CAR)modified Tcells is under intense preclinical and clinical investigation involving a rapidly increasing spectrum of new CAR designs and target antigens. Here, we present a novel reporter cell line that enables highthroughput testing and identification of functional receptor designs to inform the selection of CARs for indepth preclinical validation and clinical translation.

Methods: Triple parameter reporter (TPR) cells were derived from Jurkat cells and modified with three inducible reporter genes, each expressing a distinct fluorophore under control of the Tcell transcription factors NF?B, NFAT or AP1. TPR cells were transduced with CAR constructs, cocultured with target antigenexpressing stimulator cells and reporter geneactivation was analyzed by flow cytometry. Stimulator cells with membranebound antiCD3 scFv that triggers CD3 on TPRs were used as positive control and reference.

Results: We transduced TPR cells with CD19 and ROR1specific CARs that contained a signaling module of CD3zeta and 41BB (2nd generation CAR) and cocultured them with CD19 and ROR1expressing stimulator cells respectively. We detected specific and rapid reporter gene induction mediated by NF?B and NFAT as early as 9 hours and peaking between 24 to 48 hours of coculture. We then compared the activation signal that was obtained with CARs incorporating CD28 or 41BB alone (2nd generation CAR) or in tandem (3rd generation CAR). Interestingly, we detected stronger induction of NF?B in CARs providing 41BB rather than CD28 costimulation at 24 hours. However, the inclusion of tandem CD28 and 41BB costimulation in 3rd generation CARs did not result in stronger or more durable activation, consistent with our observation that primary Tcells expressing the 3rd generation CD19 or ROR1CAR did not confer superior effector functions in vitro and in vivo.

Conclusions: Our data demonstrate the potential of TPR cells to evaluate CARs based on the induction of key Tcell transcription factors. We are currently integrating TPRs into the testing of novel CAR designs with enhanced stimulatory and costimulatory capacity to rapidly identify the most potent formats from a large pool of constructs. We are confident our TPR test system will accelerate preclinical development and facilitate clinical translation of CARs with optimal antitumor function and safety profile for hematologic and solid malignancies.

Disclosure: No conflict of interest disclosed.

P273 – Ex vivo analysis of the tumor antigen-specific T cell repertoire in a melanoma patient

Lennerz V.1, Schroers B.1, Luebcke S.1, Fatho M.1, Kukla K.1, Brettschneider J.1, Woelfel C.1, Pagel S.1, Zhao F.2, Echchannaoui H.1, Theobald M.1, Schadendorf D.2, Paschen A.2, Woelfel T.1

1University Medical Center (UMC) Mainz, Medical Department 3, University Cancer Center (UCT), Research Center for Immuntherapy (FZI) of the Johannes Gutenberg-University and German Cancer Research Consortium (DKTK), partner site Frankfurt/Mainz, Mainz, Germany, 2University Hospital, University Duisburg/Essen, Department of Dermatology, and German Cancer Research Consortium (DKTK), partner site Essen/Düsseldorf, Essen, Germany

Introduction: In patient Ma-Mel-86 surviving metastatic disease for approximately seven years, four melanoma lines were derived from distinct lymph node metastases occurring in the years 1, 3, 4 and 7. The melanoma lines exhibited various immune escape phenotypes including partial or complete HLA class I deficiency. Blood-derived melanoma-reactive CD8+ abTCR+ T cell clones were found to recognize more than seven non-mutated shared or mutated neoantigens in HLA-restricted and more than 2 surface membrane proteins in HLA-independent manner. In a next step, we have investigated how the melanoma-reactive T cell repertoire evolved during the course of the disease.

Methods: The TCRVb chains of 18 T cell clones with known and unknown specificities were sequenced and served as target sequences. Each 3.6 µg of genomic DNA (~ 5.5×10e5 cells) from 2 PBMC samples collected in year 1 (sample 1) and year 3 (sample 2) were subjected to TCRVb high-throughput sequencing using the ImmunoSEQ Assay (Adaptive Biotechnologies, Seattle, USA). Sample 2 had been collected six months after the first of two HLA class I-negative lymph node metastases had emerged.

Results: Of 18 target TCRVb sequences, only 2 were found in sample 1. They were expressed by HLA-restricted T cell clones recognizing a mutated neoantigen and a yet unknown antigen, respectively. Both TCR were also detectable in sample 2 indicating that they were expressed by memory T cells. In sample 2, altogether 15/18 target TCRVb sequences were detectable. Among these were 6 TCRVb sequences assigned to HLA-independent T cells. TCRs against known HLA-restricted mutated and shared antigens were detected at low (< 0,01%) to intermediate (0,01-0,1%) frequencies, whereas HLA-independent TCRs were detected at intermediate to high frequencies (>1%). Only the above mentioned HLA-restricted TCR against a yet unknown antigen was detectable at high frequencies in both samples.

Conclusions: With disease progression, the patient’s anti-melanoma T cell repertoire became considerably broader. Two TCRs were assigned to long-term memory T cell responses. Our data indicate that, as soon as HLA-deficient metastases appeared, a dominant HLA-independent anti-melanoma T cell repertoire evolved.

Disclosure: No conflict of interest disclosed.

P274 – Immunization with a solid nanoscopic imiquimod suspension enhances induction of CTL responses

Aranda Lopez P.C.1, Denny M.2, Hartmann A.-K.3, von Stebut-Borschitz E.4, Stassen M.3, Schild H.3, Theobald M.1, Langguth P.2, Radsak M.P.1

1Johannes Gutenberg University Medical Center, Third Department of Medicine, Mainz, Germany, 2Johannes Gutenberg University, Biopharmaceutics and Pharmaceutical Technology, Mainz, Germany, 3Johannes Gutenberg University Medical Center, Institute for Immunology, Mainz, Germany, 4Johannes Gutenberg University Medical Center, Dermatology, Mainz, Germany

Introduction: Transcutaneous immunization (TCI) is a novel vaccination strategy to induce strong therapeutic cytotoxic T-lymphocyte (CTL) responses by directly targeting skin-resident professional antigen-presenting cells (APC). This vaccination approach is very promising to overcome current limitations of standard vaccination approaches that are mostly effective in prophylaxis, but not in the treatment of diseases. In this context, we have developed a TCI method based on a synthetic TLR7 agonist imiquimod. We generated a freeze-dried solid suspension of crystalline imiquimod (IMI-Sol), which upon shear force (i.e. spreading on the skin) transforms to a liquid phase that permeates into the skin.

Methods: Mice were shaved on their backs and immunized on two consecutive days either with the commercially available Aldara® together with SIINFEKL peptide or with IMI-Sol followed by application of SIINFEKL in officinal cremor basalis. Induction of immune response was characterized by analysis of peptide-specific T-cells and in vivo cytolytic activity. Concerning the signalling cascade we immunized transgenic mouse strains, deficient for various signalling molecules as well as mastcell-deficient mice. To evaluate inflammation in the skin, we treated mice either two times (standard protocol) or four times (psoriasis like protocol) and measured epidermal thickness compared to untreated controls.

Results: Comparing IMI-Sol vaccination with the commercially available Aldara® we found superior vaccination capacity of IMI-Sol. Using TLR7-/- or MyD88-/- mice, we confirmed that the induction of CTL responses with IMI-Sol was completely dependent on the TLR/MyD88 pathway. In contrast to Aldara® treatment, CTL responses induced by IMI-Sol are mastcell as well as IL-1 receptor independent. Analysing inflammation in the skin revealed increased epidermal thickness after IMI-Sol using our standard protocol (2 treatments), whereas comparable skin reactions were observed in a psoriasis like experiment (4 treatments).

Conclusions: All together the development of revised preparations and the deeper understanding of the underlying mechanisms may harbour the key for the rationale design of a next generation transcutaneous vaccination platform that can be used for the treatment of persistent infections and cancer.

Disclosure: No conflict of interest disclosed.

P275 – Construction of an HLA-independent single-chain T cell receptor (scTCR) against TRP2

Wölfel M.1, Wölfel C.1,2,3, Paschen A.4,5, Wölfel T.1,3,6, Theobald M.1,2,3, Echchannaoui H.1,2,3

1University Medical Center (UMC) of the Johannes Gutenberg-University, Internal Medicine III, Mainz, Germany, 2University Cancer Center (UCT), Mainz, Germany, 3German Cancer Research Consortium (DKTK), partner site Frankfurt/Mainz, Mainz, Germany, 4University Hospital, University Duisburg/Essen, Dermatology, Essen, Germany, 5German Cancer Research Consortium (DKTK), partner site Essen/Düsseldorf, Essen, Germany, 6Research Center for Immunotherapy (FZI), Mainz, Germany

Introduction: From the peripheral blood of melanoma patient Ma-Mel-86 with clinically evident HLA I- metastases we have isolated HLA-independent CD8+ aßT cell receptor (TCR)+ cytolytic T cells recognizing TRP2 as an intact molecule on the cell surface of autologous melanoma cells. Their TCR chains have been cloned and proved to be functional in a retroviral bicistronic double-chain construct (dcTCR). We intended to design a single-chain (sc)TCR construct to reduce the risk of mispairing with endogenous TCRs upon adoptive transfer and thereby enhance both safety and efficacy.

Methods: The scTCR was constructed in analogy to the procedure described by Knies et al. (Oncotarget 7:21119, 2016). It consists of the TRP2-recognizing variable (V) domains of the original dcTCR covalently bridged with a 19mer Glycine/Serine-rich linker (Li) and murine (mu) constant a and ß domains (Va-Li-Vß-muCß-muCa). Non-native cysteine residues were introduced into the murine C regions to generate a second disulfide bond between the C regions and thus minimize mispairing with endogenous TCRs. Finally the scTCR construct was cloned into retroviral vector pMX-DEST via Gateway recombination. Functional testing was performed with IFN? ELISPOT and standard 51Cr release assays.

Results: Jurkat-76 cells lacking endogenous TCR and peripheral blood lymphocytes (PBL) from healthy donors were transduced with pMX constructs encoding the anti-TRP2 dcTCR and the anti-TRP2 scTCR. scTCR and dcTCR surface expression was confirmed by flow cytometry with an antibody against the murine aßTCR constant region. Successfully transduced PBL were expanded by weekly restimulations with TRP2+ Ma-Mel-86 cells. Both CD4+ and CD8+ T cell subsets expressing the scTCR recognized Ma-Mel-86 cells as well as HEK293Tcells that were HLAI/II- due to TALEN-mediated knockout of the genes encoding B2M (ß2 microglobulin) and CIITA (HLA class II transactivator) and that were transfected with titrated amounts of TRP2-encoding cDNA. In comparative functional analyses the scTCR and the dcTCR constructs were equivalent and recognized both murine and human TRP2.

Conclusions: The anti-TRP2 scTCR construct specifically recognized TRP2 in an HLA-independent fashion. It was equivalent to the parental dcTCR construct in ELISPOT and lysis assays. With respect to potential therapeutic applications of HLA-independent TCR against tumor-associated surface molecules, scTCR constructs form a basis for versatile and modular applications.

Disclosure: No conflict of interest disclosed.

P276 – Glyco- and Fc engineering of IgA antibodies for cancer immunotherapy improved pharmacokinetics & myeloid effector cell engagement

Kretschmer A.1, Lohse S.2, Meyer S.3, Meulenbroek L.A.P.M.3, Jansen J.H.M.3, Möginger U.4, Sondermann P.5, Kolarich D.4, Leusen J.H.W.3, Valerius T.1

1Division of Stem Cell Transplantation and Immunotherapy, 2nd Department of Medicine, UKSH and CAU, Kiel, Germany, 2Institute of Virology, Saarland University Medical Center and Saarland University Faculty of Medicine, Homburg/Saar, Germany, 3Laboratory for Translational Immunology, University Medical Center Utrecht, Utrecht, Netherlands, 4Department of Biomolecular Systems, MPI for Colloids and Interfaces, Potsdam, Germany, 5SppreMol GmbH, Martinsried, Germany

Introduction: Antibodies of IgA isotype play an important role in bridging adaptive and innate immunity. FcaRI (CD89) - dependent engagement of myeloid cells appears to be crucial to activate effector mechanisms like phagocytosis or antibody-dependent cell-mediated cytotoxicity (ADCC). Recently, there has been increasing evidence that myeloid cells constitute important effector cells in cancer and cancer immunotherapy. Thus, we decided to develop production and purification technologies for recombinant IgA antibodies, which demonstrated in vivo efficacy in syngeneic and xenogeneic tumor models. Here, we describe an Fc engineering approach to further improve their immunotherapeutic potential.

Methods: Recombinant IgA antibodies against the epidermal growth factor receptor (EGFR) were produced by co-transfecting CHO-K1 cells with vectors encoding the variable region of Cetuximab and Iga heavy and ? light chain constant regions, respectively. An Fc engineered IgA2m(1) antibody was generated by mutating two N-glycosylation sites (166 and 337) and by removing two free cysteines (311 and 471). The resulting antibody variant was compared to wild type IgA2 regarding biochemical characteristics as well as Fab- and Fc- mediated effector functions. Additionally, serum half-life and in vivo efficacy in a xenogeneic FcaRI- transgenic tumor model were evaluated.

Results: Rational engineering of the constant regions of an IgA2m(1) antibody resulted in monomeric IgA molecules with improved biochemical characteristics, identical Fab- and Fc- mediated effector functions, but with significantly lower levels of terminal galactose. This molecule demonstrated lower asialoglycoprotein-receptor (ASGPR) binding and subsequently improved pharmacokinetics in mice. Compared to wild type IgA, this novel molecule displayed enhanced therapeutic efficacy in different in vivo models, which required human FcaRI-dependent myeloid effector cell engagement.

Conclusion: These results demonstrated that glyco- and Fc engineering of antibodies from the IgA isotype directed against EGFR leads to an improvement of their immunotherapeutic efficacy by overcoming of some limitations of wild type IgA antibodies (e.g. stability, pharmacokinetics, in vivo efficacy). Thus, these results promote the concept of FcaRI- dependent engagement of myeloid effector cells as a promising approach for antibody-based tumor immunotherapy.

Disclosure: No conflict of interest disclosed.

P277 – Targeting cancer metabolism for the improvement of checkpoint therapy

Thiel A.1, Singer K.1, Renner-Sattler K.1, Kreutz M.1

1Universitätsklinikum Regensburg, Medizinische Klinik lll, Regensburg, Germany,

A major breakthrough in cancer immunotherapy was the discovery of immune checkpoint proteins and the development of specific inhibitors. Especially blocking PD-1 and PD-L1 has been shown to be associated with an enhanced overall survival in metastatic disease of various tumour entities. However only a limited patient cohort shows sufficient response to the therapy. Therapy failure does not inevitably correlate with the threshold of PD-L1 expression on the tumour in the non-responding patient-population. The reasons for the low response rates need to be elucidated.

It has been demonstrated that tumour metabolites, in particular lactic acid suppress the anti-tumor immune response and moreover can skew immune cells to a regulatory phenotype. To investigate the impact of lactic acid and regulatory immune cells on checkpoint inhibition we established an in-vitro co-culture model of B16-F10 melanoma cells and 2C TCR transgenic T cells. The B16-F10 melanoma cells were transduced to express the K(b)-binding peptide SIYRYYGL which is recognized by high-affinity 2C TCR transgenic T cells.

We herein demonstrate the differential impact of lactic acid and regulatory immune populations on checkpoint inhibition with anti-PD-1 and anti-PD-L1 in-vitro.

The overall objective of our work is the development of combination therapies targeting cancer metabolism for improvement of checkpoint therapy.

Disclosure: No conflict of interest disclosed.

P279 – Glykolipid antigen-specific invariant natural killer T cells for protection from graft-versus-host disease

Schmid H.1, Duerr-Stoerzer S.1, Schneidawind C.1, Kanz L.1, Salih H.1, Savage P.2, Schneidawind D.1

1Department of Medicine II, Eberhard Karls University, Tübingen, Germany, 2Department of Chemistry and Biochemistry, Brigham Young University, Provo, United States

Introduction: Invariant natural killer T (iNKT) cells are potent regulators of immune responses in both humans (TCRa Va24-Ja18) and mice (TCRa Va14-Ja18). This particular thymus-derived T-cell subset is restricted to the MHC-I-like and lipid antigen-presenting molecule CD1d. Glycolipids such as a-GalCer induce a strong activation and proliferation of iNKT cells through T-cell receptor signaling. We recently showed in murine studies that adoptively transferred iNKT cells of either donor or third party mice protect from lethal graft-versus-host disease (GVHD) through a robust expansion of donor CD4+FoxP3+ regulatory T cells (Tregs) while preserving graft-versus-tumor (GVT) effects. Immune polarization of iNKT cells is critical for their immunoregulatory function upon adoptive transfer. Since iNKT cells constitute less than 0.5% of human peripheral blood mononuclear cells (PBMCs), in vitro expansion with their glycolipid ligands is required before these cells can be used for cytotherapy.

Methods: We tested the impact of three different glycolipids (a-GalCer, PBS57, PBS44) on human iNKT-cell proliferation, subset expansion and immune polarization by flow cytometry, intracellular cytokine staining and ELISA.

Results: Three weeks of cell culture and autologous restimulation with either a-GalCer, PBS57 or PBS44 resulted in a robust proliferation of iNKT cells from human PBMCs. We did not find significant differences in iNKT-cell expansion (1500x vs. 1727x vs. 1772x, p = 0.49) and purity (33% vs. 38% vs. 39%, p = 0.24) with the three different glycolipids tested in our study. Importantly, we observed a growth advantage of the CD4+ subset resulting in an outgrowth from 14% (day 0) to 87% (day 21) of all iNKT cells (p < 0.001). IL-4 secretion of culture expanded human iNKT cells was comparable in the three groups (18% vs. 24% vs. 31%, p = 0.68).

Conclusions: All glycolipids induce a robust proliferation of human iNKT cells in vitro. Interestingly, we found a preferential expansion of CD4+ iNKT cells secreting the Th2 cytokine IL-4. This is critical since our previous murine studies indicate that the CD4 subset and Th2 phenotype are associated with immune tolerance and protection from GVHD through Treg expansion.

Disclosure: No conflict of interest disclosed.

P280 – Influence of cytoreductive and immunmodulatory drugs on antibody-based approaches in AML

Krupka C.1,2, Lindl B.1,2, Platzer J.1,2, Brauneck F.1,2, Kischel R.3, Kufer P.3, Lichtenegger F.S.1,2, Köhnke T.1,2, Rothe M.1,2, Deiser K.1,2, Augsberger C.1,2, Altmann T.1,2, Spiekermann K.1,4, Hiddemann W.1,4, Subklewe M.1,2,4

1LMU Munich; Klinikum der Universität München, Department of Internal Medicine III, München, Germany, 2Helmholtz Zentrum München, Clinical Cooperation Group Immunotherapy, Munich, Germany, 3AMGEN Research (Munich) GmbH, Munich, Germany, 4German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Germany

Previously, we demonstrated that the CD33/CD3 BiTE® antibody construct AMG 330 is able to recruit residual autologous T cells, resulting in effective cytotoxicity against primary AML cells. Based on these data, a phase I study in relapsed/refractory AML has been initiated (NCT02520427). Clinical experience in ALL has shown a clear correlation of leukemic burden and a cytokine release syndrome (CRS) during treatment with blinatumomab. A cytoreductive phase prior to antibody therapy might be beneficial to reduce the severity of the CRS. Furthermore antibody mediated unwanted toxicity is treated with steroids (dexamethasone, DEX) or tocilizumab (TOC, IL-6R antibody). However, little is known about the relevance of these drugs on effector cell function. Therefore we evaluated the influence of cytoreductive- (azacythidine, AZA; decitabine, DEC; cytarabine, ARA-C; hydroxyurea, HU) and immunmodulatory drugs (DEX, TOC) on antibody-mediated T-cell function. T cells and AML cells were cocultured with or without AMG 330 for 3-7 days. T cells were incubated with the drug prior to coculture (AZA, DEC, ARA-C, HU) or simultaneously added (DEX, TOC). Lysis of AML cells and T-cell proliferation were assessed by flow cytometry. Pretreatment of T cells with ARA-C completely abrogated T-cell function (% lysis: untreated(UT) 98.0 vs 20 µM 4.2) and proliferation. AZA and DEC impaired AMG 330-mediated cytotoxicity (% lysis UT vs AZA at 1, 5, 10 µM: 99.8 vs 99.2 vs 52.1 vs 28.6; UT vs DEC at 0.3, 2, 5 µM: 78.9 vs 57.9 vs 48.6 vs 5.8) and T-cell proliferation in a concentration dependant manner. In contrast, pretreatment with HU had no effect on T-cell function (% lysis: UT vs HU at 10, 100 µM: 100 vs 100 vs 97.3) and proliferation. The addition of DEX to primary AML cultures considerably impaired T-cell proliferation (fold change T cells: UT 12.9 vs DEX 1.3) and IFN? secretion (pg/ml: UT: 813 vs DEX: 64). TOC had no negative effect on T-cell proliferation (fold change T cells: UT: 5.0 vs TOC: 4.9). Similarly, secretion of IFN? (pg/ml: UT: 712 vs TOC: 839) was not affected. In summary we could show that in contrast to HU, pretreatment with ARA-C, AZA and DEC negatively influenced T-cell function. Furthermore, in comparison to TOC the use of DEX considerably decreased AMG 330 mediated T-cell proliferation and cytokine secretion. As most antibody based immunotherapeutic strategies depend on effector cell function, our data support the use of HU and TOC for combinatorial approaches.

Disclosure: Christina Krupka: Expert Testimony: wissenschaftliches Projekt finanziell unterstützt durch AMGEN Research Munich GmbHMarion Subklewe: Advisory Role: AMGEN Research (Munich); Expert Testimony: wissenschaftliches Projekt finanziell unterstützt durch AMGEN Research Munich GmbH

P281 – Long term efficacy of WT1 peptide vaccination in patients with WT1 expressing AML /MDS and solid malignancies

Lukas K.1, Scheibenbogen C.2, Asemissen A.M.3, Busse A.1, Ochsenreither S.1, Blau I.1, Baldus C.1, Thiel E.1, Keilholz U.4, Letsch A.1

1Charité, Campus Benjamin Franklin, Med. Klinik III, Berlin, Germany, 2Charité – Universitätsmedizin Berlin (CVK), Institute of Medical Immunology, Berlin, Germany, 3UKE Hamburg, Hematology, Oncology and Bone Marrow Transplantation, Hamburg, Germany, 4Charite-Unversitätsmedizin, Charité Comprehensive Cancer Center, Berlin, Germany

Immunotherapy is a novel strategy for generating antitumor immunity as part of cancer treatments. The present study aims to assess the feasibility, immunogenicity and clinical effects of WT1 peptide vaccination based on the growing evidence that Wilms´ tumor protein 1 (WT1) is a promising tumor antigen for the development of universal cancer vaccines. In this light, we conducted two separate phase-II proof of principle vaccine trial in patients with active AML/MDS and with WT1 overexpressing solid tumors. Here we concentrate on patients remaining on vaccination for at least one year in order to analyze characteristics for long-term vaccination success.

The vaccination schedule was identical for both trials: 0.2mg HLA-A*0201-restricted WT1.126-134 peptide admixed with 1 mg KLH (day 3), and 62.5µg GM-CSF (day 1-4) every two weeks x 4 s.c./i.d., followed by either biweekly or every four weeks for 10 months and at increased intervals of up to 3-monthly thereafter. The two trials included patients with active AML and high-risk MDS (n = 20), patients with AML in high risk complete remission (n = 9), as well as with mostly heavily pretreated ovarian (n = 7), thyroid (n = 2), breast (n = 1), gastric (n = 1), and larynx cancer (n = 1), astrocytoma (n = 1) and mesothelioma (n = 4). Standard criteria were used for response assessment. Patients remained on study in absence of limiting toxicity and disease progression.Toxicity to treatment was mild , and no patient went off protocol for adverse events or consent withdrawal.

Eleven patients remained on study for more than 1 year with disease control, including 3/19 patients with active AML, 4/9 with AML in complete remission, 2/7 patients with ovarian cancer and 2/4 with mesothelioma. Of these 11 patients four remained on study for more than 3 years, including 3 patients with AML (75, 70+ and 114+ months) and one mesothelioma patient (84+ months).

We could demonstrate remarkable longterm responses induced by WT1 vaccination in patients with AML and solid tumors. Further vaccine development, especially in combination with checkpoint inhibitors seem promising. Overall, no correlation between prior treatment and duration on vaccine was observed, since 2/4 patients in long-term disease control had previously aggressive chemotherapy. Of the solid tumor patients, chemotherapy-pretreated ovarian cancer and mesothelioma appear to be sensitive to WT1 vaccine, although other histologies cannot be excluded due to low patient numbers.

Disclosure: Kaja Lukas: No conflict of interest disclosed.Anne Letsch: Financing of Scientific Research: BMS,Merck, Novartis; Expert Testimony: Celgene, Novartis

P282 – Mobilization of tissue-resident memory cells by the granulocyte colony-stimulating factor (G-CSF)

Olfe L.1, Asadi K.1, Becker S.1, Mensen A.1, Szyska M.2, Tietze-Bürger C.3, Scheibenbogen C.1, Schetelig J.4, Dörken B.5, Arnold R.5, Na I.-K.1,2,5

1Institute for Medical Immunology, Charité CVK, Berlin, Germany, 2Experimental and Clinical Research Center (ECRC), Berlin, Germany, 3Charité Stem Cell Facility, Berlin, Germany, 4Universitätsklinikum Carl Gustav Carus, Technische Universität, Dresden, Germany, 5Department of Hematology, Oncology and Tumorimmunology, Charité, Berlin, Germany

Introduction: Infectious complications are a major cause of mortality after allogeneic hematopoietic stem cell transplantation, which may largely reflect an impairment of immune memory function. Donor immune cells as part of the peripheral blood (PB) stem cell transplant contribute to an immediate protection. Subset distribution as well as homing potential and functionality of the transplant’s memory cells influence the outcome of the patient’s immune reconstitution. Therefore, we are analyzing the impact of G-CSF exposure on PB memory composition and functionality.

Methods: We analyzed peripheral blood mononuclear cells (PBMCs) of adult healthy stem cell donors before and after four-day subcutaneous application of G-CSF in order to quantitatively, phenotypically and functionally analyze memory subsets via multicolor flow cytometry. In parallel, we studied bone marrow (BM) resident memory cells in femur heads from patients undergoing hip replacement surgery.

Results: In our preliminary data, we could show that the absolute numbers of memory T and B cells in PB increased up to fivefold after treatment with G-CSF. We could find a higher proportion of CD69+ memory CD4 T cells (0.7 ± 0.3% vs. 2.2 ± 0.3%) and memory CD8 T cells (2.4 ± 0.8% vs. 6.9 ± 0.7%) after treatment with G-SCF. In contrast, CLA-expressing memory CD4 T cells (20.1 ± 2.9% vs. 11.2 ± 0.8%) and memory CD8 T cells (17.2 ± 4.3% vs. 10.1 ± 3.4%) declined. CD103 and CRTH2 expressing memory T cells increased, whereas the percentage of alpha4beta7 expression was similar before and after G-CSF. Compared to PB samples, BM memory cells exhibited a higher percentage of CD69 and CRTH2 expression, while CLA expression was negligible. Number of samples are still limited (n = 5) and significances are not achieved so far.

Conclusion: Our paired analysis of donor PBMCs before and after mobilization identifies newly recruited memory subsets and potentially enables the delineation of their tissue origin. Based on this detailed characterization of the apheresis product in respect to the memory composition and functionality, we are currently studying the fate of the transferred memory cell subsets in the patients in order to analyze which human memory subsets and survival niches are able to promote long-term immune memory.

Disclosure: No conflict of interest disclosed.

P283 – A prospective multicenter safety surveillance study with a 5% intravenous human immunoglobulin (IVIg) preparation, in patients suitable for IVIg treatment, with or without premedication

Peinert S.1, Abenhardt W.2, Fenchel K.3, Hübner A.4, Reichert D.5, Scheidegger C.6, Schmidt A.7, von Wussow P.8, Galic M.9

1Onkologie Westerstede Aurich Rhauderfehn, Rhauderfehn, Germany, 2Onkologie Elisenhof, Munich, Germany, 3MVZ Saaletal, Saalfeld/Saale, Germany, 4Schwerpunktpraxis für Hämatologie und Onkologie, Bayreuth, Germany, 5Onkologie Westerstede Aurich Rhauderfehn, Westerstede, Germany, 6Nordbadpraxis, München, Germany, 7Onkologie Schmidt, Cottbus, Germany, 8Facharzt für Innere Medizin, Hannover, Germany, 9Kedrion International, Wien, Austria

Introduction: We investigated the tolerability of a human immunoglobulin preparation (Ig VENA® 50g/l for infusion) in patients with primary immunodeficiencies or secondary immunodeficiencies e.g. after cytotoxic treatment for haemato-oncologic malignancies; and with autoimmune diseases like idiopathic thrombocytopenic purpura (ITP) and chronic inflammatory demyelinating polyneuropathy (CIDP). The nature and frequency of adverse drug reactions (ADR) were recorded under routine clinical conditions or with premedication administered prior to infusions. The study was conducted over a four year period.

Methods: A multicenter, prospective, non-interventional study conducted in Germany. Subjects received 5% IVIg according to the prescribing information. Dosage, tolerability, any premedication, and adverse events were recorded. Dose regimen was at the investigator´s discretion.

Results: 202 patients (120 f, 82 m) received a total of 1,218 infusions. IVIg was used as replacement therapy in immunodeficiency syndromes in 92.6% of patients (n = 187/202), and for immunomodulation in 7.4% of patients (n = 15/202), with a mean total dose per infusion of 15.9 g and 31.2 g, respectively. Across all indications the tolerability of IVIg infusions was judged by the physicians as very good or good in 99.2% (n = 1,208/1,218; very good n = 972/1,218, good n = 236/1,218) of the infusions even in the absence of premedication in 92% of infusions. Where premedication was given, mainly antihistamines and steroids were used. In total 8 non-serious ADRs were reported in 7 patients. None of the patients who had received premedication had an ADR; there were no serious ADRs reported during the study period.

Conclusions: Treatment with 5% IVIg demonstrated an excellent tolerability profile when used under routine clinical conditions and in the absence of premedication, in the vast majority of infusions.

Disclosure: Stefan Peinert: Financing of Scientific Research: Honorarium received from Kedrion international; Expert Testimony: Kedrion Internaional was the study sponsorMaja Galic: Employment or Leadership Position: Medical Director at Kedrion International; Expert Testimony: Kedrion Internaional was the study sponsor

P284 – Programmed death ligand 1 serum levels may reflect immunosuppression in patients with advanced renal cell cancer under treatment with a gene-modified allogeneic tumor cell vaccine (RCC26 (IL-7/CD80))

Flörcken A.1,2, Panzer I.1,2, Kopp J.3, Dörken B.1,2, Blankenstein T.4,5, Pezzutto A.6, Westermann J.1,2

1Charité University Medicine, Campus Virchow-Klinikum, Department of Hematology, Oncology, and Tumor Immunology, Berlin, Germany, 2Labor Berlin Charite´ Vivantes GmbH, Berlin, Germany, 3Charité University Medicine Berlin, Campus Berlin-Buch, Experimental and Clinical Research Center, Berlin, Germany, 4Max Delbrück Center for Molecular Medicine, Berlin, Germany, 5Charité University Medicine Berlin, Campus Benjamin Franklin, Institute of Immunology, Berlin, Germany, 6Charité University Medicine Berlin, Campus Benjamin Franklin, Department of Hematology, Oncology, and Tumor Immunology, Berlin, Germany

Introduction: Tumor-induced immunosuppression is a hallmark of cancer and a major challenge for immunotherapy. We have previously reported on a clinical phase-I trial with an allogeneic, HLA-A2-matched RCC cell line transfected with IL-7 and CD80 as a vaccine in RCC patients (Westermann et al. 2011), showing severe immunosuppression at baseline. Interestingly, an association with the nuclear factor-kappa B (NF-kB) signaling pathway could be established, implying that suppression of NF-kB may play a key role for tumor-induced immunosuppression in RCC patients (Flörcken et al. 2015).

Methods: To further understand why the allogeneic gene-modified (IL-7/CD80 co-transfected) renal cell cancer vaccine failed to induce clinically relevant TH-1-polarized immune responses, PD-L1 serum levels were analyzed both in patients that had been treated within the vaccination study (n = 6) and in healthy donors (n = 10). Additionally, PD-L1 serum levels were correlated with clinical and immunological data from our previous studies.

Results: Before vaccination, highly elevated levels of soluble PD-L1 were measured in RCC patients (mean sPD-L1 in ng/ml =1.68) as compared to healthy controls (mean sPD-L1 in ng/ml = 0.56) (p < 0.001). Interestingly, the vaccine was able to reduce soluble PD-L1 levels in RCC patients (mean sPD-L1 before vaccination:1.68 ng/ml, after vaccination: 1.35 ng/ml, n.s.), however normal soluble PD-L1 serum levels were not achieved. Furthermore, reduction of PD-L1 serum levels was associated with a longer PFS, although this correlation did not reach statistical significance.

Conclusion: To the best of our knowledge, this is the first report on soluble PD-L1 serum levels during treatment with a gene-modified cancer vaccine. Our data suggest that measurement of PD-L1 in cancer patients might be a useful tool for both the quantification of immunosuppression and the monitoring of immune responses within immunotherapy trials.

Disclosure: Anne Flörcken: Financing of Scientific Research: Bayer Healthcare, Pfizer Pharma; Other Financial Relationships: Pfizer Pharma, Bayer HealthcareJörg Westermann: No conflict of interest disclosed.

P285 – The deubiquitinase inhibitor b-AP15 and its effect on phenotype and function of monocyte-derived dendritic cells

Altdoerfer V.1, Kropp K.1, Haen S.P.1, Grünebach F.1, Rittig S.M.1, Kanz L.1, Salih H.R.1,2, Dörfel D.1,2

1Eberhard Karls University, Department of Hematology and Oncology, Tübingen, Germany, 2Clinical Collaboration Unit Translational Immunology, German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Partner site Tübingen, Tübingen, Germany

Introduction: The therapeutic efficacy of proteasome inhibition is well established in multiple myeloma and mantle cell lymphoma, which has led to its approval and incorporation in treatment algorithms for these malignancies. The first-in-class drug bortezomib impairs proteasome function by inhibition of the 20S core particle. However, proteasome function can also be targeted by inhibition of the 19S deubiquitinase (DUB) activity. Recently, b-AP15 was identified as a novel small molecule DUB inhibitor that inhibits growth of cancer cells in preclinical analyses (D´Arcy et al., Nat. Med. 2011). As bortezomib impairs several immune properties of monocyte-derived dendritic cells (DCs), in the current study we analyzed the influence of b-AP15 on DC phenotype and function.

Methods: DCs were generated from PBMC of healthy donors. Plastic adherent monocytes were cultured in RP10 medium supplemented with granulocyte macrophage-colony-stimulating factor (100 ng/mL) and interleukin-4 (20 ng/mL). The medium was replenished with cytokines every other day and different concentrations of b-AP15 (10 nM, 100 nM, 500 nM, 1000 nM) were added to the culture on day 6. IL-10- (10 ng/mL) and bortezomib-treated (10 ng/mL) DCs served as controls. For maturation, lipopolysaccharide (LPS; 100 ng/mL) was added on day 6 and DCs were harvested on day 7 for immunophenotyping and functional analyses.

Results: Immunophenotyping on day 7 revealed a clear downregulation of CD1a, CD83, CD86 and CD80 by bortezomib and IL-10 in mature DCs compared to untreated controls. In contrast, no influence of b-AP15 on these immunoregulatory molecules and maturation markers was observed upon exposure to b-AP15 compared to the untreated control. In line with differential effects of the two proteasome inhibitors on the immunophenotype, b-AP15 did also not impair DC migration and stimulatory capacity, while comparable effects of both compounds on apoptosis, susceptibility to NK cell killing and metabolic acticitiy were observed with various mantle cell lymphoma cell lines.

Conclusion: In summary, our results suggest that b-AP15 mediates similar antitumor efficacy as bortezomib while preserving the immunostimulatory capacity of DCs, indicating that b-AP15 may be exquisitely suitable for combinatory treatment approaches.

Disclosure: No conflict of interest disclosed.

Posterdiskussion – Lungentumoren

P286 – Combining anti-VEGF therapy and immune checkpoint inhibitors improves outcome in small cell lung carcinomas

Meder L.1,2, Schuldt P.2, Vlasic I.1,3, Volz C.1,2, Golfmann K.1,2, Zaplatina A.1,2, Florin A.2,4, Tharun L.2,4, Büttner R.2,4, Reinhardt C.1,2,3, Ullrich R.1,2

1University Hospital Cologne, Department of Internal Medicine I, Cologne, Germany, 2University Hospital Cologne, Lung Cancer Group Cologne, LCGC, Cologne, Germany, 3University of Cologne, Cluster of Excellence in Cellular Stress Responses in Aging-associated Diseases, CECAD, Cologne, Germany, 4University Hospital Cologne, Institute of Pathology, Cologne, Germany

Metastatic lung cancer is the leading cause of smoking- and cancer-related deaths worldwide. SCLC is the most aggressive lung cancer and is driven by lesions in RB1 and TP53. SCLC accounts for approximately 14% of lung cancers with a median survival of 9 months, diagnosed at late stage. Thus, SCLC is usually not treated by surgery but with radiation- or chemotherapy.

Results from KEYNOTE-028 were presented on the ASCO conference 2015. Here, programmed death ligand 1 (PD-L1) positive late stage SCLC patients received high-affinity, humanized monoclonal programmed death receptor 1 (PD-1) antibody pembrolizumab (MK-3475). They showed response rates of up to 25% which is remarkable for SCLC, since targeted therapy for this tumor entity are rare.

However, most SCLC patients harbor a primary resistance or acquire resistance during treatment by an immune suppressive microenvironment or other resistance mechanisms triggered by the tumor cells themselves. Thus, there is a critical need to combine immune checkpoint inhibitors with other therapies to overcome these resistances.

We implemented a combined therapy concept including anti-VEGF and anti-PD-L1 monoclonal antibody therapy for SCLC in mouse models. As a read out we used X-ray computed tomography, flow cytometry and end point immunohistochemistry.

We found significantly increased progression-free survival (PFS) upon anti-VEGF and anti-PD-L1 mono-therapy, which was prolonged upon combined therapy. Moreover, we identified in murine SCLC tumors which acquired resistance upon anti-PD-L1 mono-therapy, a significant increase in PD1+/TIM3+ cytotoxic T cells. This exhausted T cell phenotype was significantly reverted upon combined anti-VEGF and anti-PD-L1 therapy. Furthermore, anti-VEGF mono-therapy provided an increased metastatic potential to SCLC cells which was significantly reduced upon combination with anti-PD-L1 therapy.

Taken together, there is evidence for the benefit of implementing combined anti-angiogenic and anti-immune checkpoint therapy approaches in the clinic in order to overcome acquired resistances. However, it remains a compulsive issue in SCLC to improve not only the PFS but also the the overall survival and to prolong the duration of therapy responses.

Disclosure: No conflict of interest disclosed.

P287 – MYSTIC – A Phase III, randomized, open-label study of Durvalumab (MEDI4736) in combination with Tremelimumab or Durvalumab alone versus platinum-based chemotherapy in first-line treatment of patients with advanced stage IV non small cell lung cancer (aNSCLC): A German update

von Pawel J.1, Fischer J.2, Alt J.3, Rittmeyer A.4, Wehler T.5, Laack E.6, Griesinger F.7, Schneider C.P.8, Panse J.9, Dieing A.10, Rawluk J.11, Serke M.12, Kropf-Sanchen C.13, Lerchenmüller C.14, Bohnet S.15, Bischoff H.16, Nusch A.17, de Wit M.18, Rupprecht M.19, Alt A.20, McIntosh S.21, Rizvi N.A.22, Seggewiß-Bernhardt R.23

1Asklepios Klinik Gauting GmbH – Betriebsstätte Gauting, Gauting, Germany, 2Lungenkrebszentrum Klinik Löwenstein, Löwenstein, Germany, 3Universitätsmedizin der Johannes Gutenberg-Universität Mainz, Medizinische Klinik III, Mainz, Germany, 4Fachklinik für Lungenerkrankungen Immenhausen, Immenhausen, Germany, 5Universitätsklinikum des Saarlandes, Homburg/Saar, Germany, 6Hämato-Onkologie Hamburg, Hamburg, Germany, 7Pius-Hospital Oldenburg, Oldenburg, Germany, 8Zentralklinik Bad Berka GmbH, Bad Berka, Germany, 9Uniklinik RWTH Aachen, Aachen, Germany, 10Vivantes Klinikum Am Urban, Berlin, Germany, 11Universitätsklinikum Freiburg, Freiburg, Germany, 12Lungenklinik Hemer, Hemer, Germany, 13Universitätsklinikum Ulm, Ulm, Germany, 14Praxis GEHO Münster, Münster, Germany, 15Universitätsklinikum Schleswig-Holstein, Lübeck, Germany, 16Thoraxklinik Heidelberg gGmbH, Heidelberg, Germany, 17Praxis für Hämatologie und internistische Onkologie, Velbert, Germany, 18Vivantes Klinikum Neukölln, Berlin, Germany, 19AstraZeneca GmbH, Medical Affairs, Wedel, Germany, 20AstraZeneca GmbH, Clinical Operations, Wedel, Germany, 21AstraZeneca Ltd., Global Medicines Development, Macclesfield, United Kingdom, 22Columbia University Hospital, New York, United States, 23Universitätsklinikum Würzburg, Würzburg, Germany

Introduction: Standard of care (SoC) therapy in 1st-line NSCLC is a platinum-based regimen for EGFR- and ALK-Wildtype-patients (pts). However, durable benefit is rare owing to most developing resistance to chemotherapy. Currently, immune checkpoint inhibitors are a highly promising new approach. Preclinical data suggest that blocking both, the inhibitory CTLA-4 as well as PD-1 receptors or the ligand, PD-L1, is non-redundant and has a synergistic effect to restore the immune-mediated antitumor response through T-cells. Durvalumab is a selective, high affinity human IgG1 monoclonal antibody (mAb) that blocks PD-L1 binding to PD-1 and CD80, and Tremelimumab is a selective human IgG2 mAb inhibitor of CTLA-4. Durvalumab monotherapy has already shown durable responses in NSCLC and Durvalumab plus Tremelimumab has shown encouraging clinical activity and a manageable safety profile in pts with NSCLC. A comprehensive clinical development program of Durvalumab +/- Tremelimumab in NSCLC is underway. MYSTIC (NCT02453282) is a randomized, open-label, multicenter, global, Phase III study to determine the efficacy and safety of Durvalumab plus Tremelimumab or Durvalumab monotherapy versus SoC platinum-based doublets in the 1st-line treatment of pts with NSCLC. In Germany, 21 sites are participating in the trial.

Methods: In MYSTIC, immunotherapy- and chemotherapy-naïve pts with NSCLC who are wild-type for EGFR and ALK will be randomized (1:1:1) to receive Durvalumab (20 mg/kg IV every 4 weeks for up to 12 months) plus Tremelimumab (1 mg/kg IV every 4 weeks for up to 4 doses); Durvalumab monotherapy (20 mg/kg IV every 4 weeks for up to 12 months); or SoC chemotherapy. Stratification factors are PD-L1 status and histology. The primary endpoint is progression-free survival (PFS). Secondary endpoints will further assess objective response rate, duration of response, proportion of pts alive and progression free at 12 months, time from randomization to second progression, and overall survival; safety (CTCAE v4.03) and tolerability; health-related quality of life; pharmacokinetics; and immunogenicity. Exploratory outcomes include analysis of potential biomarkers of response to treatment, a comprehensive analysis of the tumor immune activation landscape by IHC, as well as tumor mutation load, are assessed on a biopsy at trial entry. Recruitment is ongoing. To date, 95 pts out of 675 have been randomized in Germany since First Subject In was on Oct 2015.

Disclosure: No conflict of interest disclosed.

P288 – Anti angiogenic treatment induces tumor cell invasion and metastasis via EphA2 signaling in NSCLC

Volz C.1, Zaplatina A.1, Siobal M.1, Chatterjee S.1, Schöttle J.1, Meder L.1, Florin A.2, Koker M.1, Buettner R.2, Miao H.3,4, Wang B.3,4, Hallek M.1, Acker-Palmer A.5, Heukamp L.C.2, Thomas R.K.2,6, Ullrich R.1

1University Hospital Cologne, Department of Internal Medicine I, Center for Molecular Medicine and Center for Integrated Oncology, Cologne, Germany, 2University Hospital Medical School, Institute of Pathology, Cologne, Germany, 3Rammelkamp Center for Research, MetroHealth Medical Center, Case Western Reserve University School of Medicine, Cleveland, United States, 4Case Western Reserve University School of Medicine, Department of Pharmacology and Oncology, Cleveland, United States, 5University of Frankfurt, Institute for Cell Biology and Neuroscience, Frankfurt, Germany, 6University of Cologne, Medical Faculty, Department of Translational Genomics, Cologne, Germany

VEGF / VEGFR-2 signaling is one of the main driving forces during tumor angiogenesis. However, clinical findings have shown that VEGF / VEGFR-2 targeted therapies provide only little efficacy in tumor shrinkage or benefit in survival. Moreover, recent preclinical studies reported that VEGF targeted therapies conditioned the tumor cells to aggressive invasiveness and metastasis. Using a spheroid invasion model we found that drug-induced inhibition of VEGFR2 on tumor cells drives tumor cell migration. Biochemically, we found that VEGFR2 inhibition on tumor cells induces serine phosphorylation of EphA2. Drug-induced inhibition of tumor VEGFR2 releases a newly identified EphA2/VEGFR2 heterocomplex, thereby allowing AKT to phosphorylate Serine 897 of EphA2. This VEGFR2 inhibition induced EphA2-Serine 897-phosphorylation mediates an EMT-like transition. Selective genetic modeling of the Serine 897 of EphA2 or drug-induced inhibition of EphA2 abrogates the formation of metastases in vivo upon VEGFR2 inhibition. In summary, our findings indicate that VEGFR2 targeted therapy conditions VEGFR2-positive NSCLC to Serine 897 EphA2-dependent aggressive tumor growth and metastasis. These data shed light on the molecular mechanisms explaining the limited efficacy of VEGFR2 targeted anti-angiogenic treatment in lung cancer patients.

Disclosure: No conflict of interest disclosed.

P289 – Results from a multi-center, open-label, phase II study investigating the combination of RAD001 (everolimus) with paclitaxel and carboplatin in first line treatment of patients with advanced (stage IV) large cell lung cancer with neuroendocrine differentiation (LCNEC)

Engel-Riedel W.1, Grohé C.2, Kropf-Sanchen C.3, von Pawel J.4, Gütz S.5, Kollmeier J.6, Eberhardt W.7, Christopoulos P.8, Nimmrich I.9, Sieder C.9, Baum V.9, Serke M.10, Thomas M.8

1Kliniken der Stadt Köln, Lungenklinik, Köln, Germany, 2Evangelische Lungenklinik Berlin, Berlin, Germany, 3Universitätsklinikum Ulm, Ulm, Germany, 4Asklepios Fachkliniken München-Gauting, München-Gauting, Germany, 5Evangelisches Diakonissenkrankenhaus Leipzig, Leipzig, Germany, 6Helios Kliniken Berlin, Berlin, Germany, 7Universitätsklinikum Essen, Essen, Germany, 8Thoraxklinik Heidelberg, Heidelberg, Germany, 9Novartis Pharma GmbH, Nürnberg, Germany, 10Lungenklinik Hemer, Hemer, Germany

Introduction: Large cell neuroendocrine carcinoma of the lung (LCNEC) make up approximately 3% of all lung cancers. These tumors in general have a bad prognosis and currently there are only very limited treatment options, including platinum derivatives and etoposide.The PI3/AKT/mTOR pathway is known to be dysregulated in neuroendocrine tumors (NETs). As the mTOR inhibitor RAD001 (everolimus) already has proven effectiveness in different types of NETs including carcinoids and pancreatic NETs, we tested whether RAD001 might be also an effective treatment option in advanced LCNEC patients

Methods: In the presented Phase II trial, daily RAD001 was combined with carboplatin and paclitaxel in patients with histologically confirmed stage IV LCNEC according to WHO criteria. Further inclusion criteria were measurable disease according to RECIST 1.1 and adequate bone marrow, renal, and liver function. Main exclusion criteria were symptomatic CNS metastases and prior treatment for advanced LCNEC. Enrolled patients received daily everolimus in combination with 4 cycles of carboplatin and paclitaxel, followed by RAD001 maintenance therapy. The primary objective was to evaluate the efficacy of this treatment by assessing the proportion of progression-free patients after three months of treatment.

Results: Ten German trial sites enrolled altogether 49 patients to the trial (mean age: 62 ± 9 years; 71% men). The primary endpoint (progression-free at month 3) was achieved by 24 patients (49%), assessed by an independent central imaging reviewer. Further endpoints were an overall response rate (ORR) until month 3 of 45%, a disease control rate (DCR) until month 3 of 73.5%, a median progression-free survival (PFS) of 4.3 months, and a median overall survival (OS) of 9.8 months. At least one toxicity occurred in 86% of all enrolled patients with grade 3/4 toxicities in 51%. Most frequent toxicities were diarrhea, fatigue, anemia, and neutropenia.

Conclusions: The presented results show that a combined therapy of carboplatin and paclitaxel with the mTOR inhibitor RAD001 is an alternative treatment option for LCNEC patients. When comparing to other trials, the effectiveness is comparable to a treatment regimen of cisplatin and etoposide.

Disclosure: Walburga Engel-Riedel: Advisory Role: Advisory Boards; Financing of Scientific Research: Lilly, Boehringer-Ingelheim, Novartis, RocheMichael Thomas: Financing of Scientific Research: Astrazeneca, Roche, Novartis, Pfizer, MSD, BMS, Lilly, Boehringer

P290 – Economic burden of clinical trials in lung cancer in a German Comprehensive Cancer Center

Kron F.1, Kostenko A.1, Scheffler M.1, Glossmann J.1, Fischer R.1, Michels S.1, Nogova L.1, Hallek M.1, Zander T.1, Wolf J.1, Lung Cancer Group Cologne

1Uniklinik Köln, Klinik I für Innere Medizin, Köln, Germany

Introduction: Recent success of individualized lung cancer therapy has triggered fundamental changes in clinical research strategies. To date there is a strong focus on early proof of concept trials in genetically preselected, often rare, patient subgroups. From a hospital management perspective, this analysis focuses on the economic burden caused by such trials for advanced lung cancer patients in a German Comprehensive Cancer Center (CCC).

Methods: Clinical and economic data from trials for advanced lung cancer (LC), pharma-sponsored trials (PhST) as well as investigator initiated trials (IIT), conducted between 2011 and 2015 at the Center for Integrated Oncology (CIO) Cologne, were analyzed using a profit-center calculation model.

Results: 161 patients were enrolled in 27 clinical trials including 2,173 trial visits in total (PhST: 1,319, IIT: 854). We have identified `trial visit` as a key economic parameter determining costs and payments. In comparison of two groups (A=3; B>3 patients enrolled) we found negative profit margins of €-1,444 per patient (PP) in A and positive in B (€217). Significant differences were also found between PhST and IIT (p = 0.009) concerning the number of visits PP, not, however, with regard to duration of treatment PP. Additionally, sub analysis show structural differences in cost composition by conducting PhST and IIT.

Conclusion: Individualized, genomically targeted LC therapies goes along with an increase of early phase trials in rare genetic subgroups and, consequently, low patient recruitments. For trials with low patient numbers and IIT, it is difficult to break even with the costs. These observations demonstrate the need for CCC to thoroughly recalculating the costs of such trials in order to receive adequate, cost-covering compensation by pharmaceutical companies. They also underline the need for novel structures in public funding for investigator-initiated clinical trials in precision medicine.

Disclosure: No conflict of interest disclosed.

P291 – Detection of an EGFR kinase domain duplication in a lung adenocarcinoma patient by liquid biopsy using hybrid capture based next generation sequencing

Wiest G.1, Kohlhäufel M.2, Müller J.3, Lakis S.3, Wesseler C.1, Mariotti E.3, Zacherle T.3, Leenders F.3, Gloeckner C.3, Heuckmann J.M.3, Menon R.3, Heukamp L.3

1Asklepios Klinikum Harburg, Atemwegs-, Lungen- und Thoraxklinik, Hamburg, Germany, 2Klinik Schillerhöhe, Abteilung für Pneumologie und Thoraxonkologie, Gerlingen, Germany, 3NEO New Oncology, Cologne, Germany

Introduction: EGFR kinase domain duplications are a rare but targetable alteration, driving lung cancer. Only limited patient data has been published on the detection of the kinase duplications. This molecular alteration has been shown to lead to the activation of EGFR and is thought to be predictive for response to treatment with EGFR inhibitors. Using a hybrid-capture based next-generation sequencing method, we describe here the initial detection of the EGFR kinase domain duplication in an FFPE sample from the primary tumor (NEOplus) and the subsequent detection in a liquid biopsy (NEOliquid) sample

Method: A 72-year-old patient was diagnosed with a lung adenocarcinoma. Initial routine tests were negative for genomic alterations in known targetable lung cancer genes. Therefore, the patient was treated with 3 cycles of chemotherapy, but the patient continued to progress.

Recently a tissue sample and a liquid biopsy was analyzed by NEO New Oncology (Cologne, Germany) using NEOplus and NEOliquid. The assay uses a hybrid-capture based next-generation sequencing assay that covers clinically relevant genomic alterations, such as point mutations, small insertions and deletions, selected gene fusions and copy number alterations within a panel of more than 90 genes for FFPE material or 30 genes for liquid biopsies.

Results: Hybrid capture-based NGS assay were able to identify an EGFR kinase domain duplication. Based on the identification of the duplication, the patient was put on second line treatment with the EGFR inhibitor Afatinib. Despite a response of the primary tumor and the metastasis in lymph nodes and adrenal gland, a recent scan revealed that the metastasis in the brain and liver progressed. The discordance in response might depict the tumors heterogeneity or drug transportation and metabolism to the metastatic foci.

Conclusion: Here we describe the detection of a rare EGFR kinase domain duplication using the NEO assay. Interestingly, when treated with Afatinib, some of the patient´s distal metastasis continued to progress. Novel technologies capable of detecting rare genomic alterations, in the routine setting, further stresses on the urgent need to develop and identify drugs to treat patients harboring these rare mutations.

Disclosure: No conflict of interest disclosed.

P292 – Implementation of the lung-cancer biology and outcome (LuCa-BiO) study integrating routine clinical data, quality of life assessment and liquid biopsy biobanking

Schlenk R.F.1, Schmidtke-Schrezenmeier G.2, Neagoie A.M.1, Schmelzle B.1, Nagel G.3, Buske C.4, Wessendorf S.5, Schwänen C.5, Hetzel M.6, Liewald F.7, Brinkmann F.8, Ott G.9, Sträter J.10, Hamel T.1, Kuhn P.11, Welke C.11, Lang G.12, Möller P.13, Marienfeld R.13, Döhner H.1, Krof-Sanchen C.2, Bullinger L.1

1Universitätsklinikum Ulm, Innere Medizin III, Ulm, Germany, 2Universitätsklinikum Ulm, Innere Medizin II, Ulm, Germany, 3Epidemiologie und Biometrie, Epidemiologie, Ulm, Germany, 4Universität Ulm, Experimentelle Tumorforschung, Ulm, Germany, 5Klinikum Esslingen, Innere Medizin, Esslingen, Germany, 6Krankenhaus vom Roten Kreuz Bad Cannstatt, Pneumologie, Internistische Intensivmedizin, Beatmungsmedizin und Allgemeine Innere Medizin, Stuttgart, Germany, 7Klinikum Esslingen, Esslingen, Germany, 8Diakonie-Klinikum Stuttgart und OSP Stuttgart e.V., Stuttgart, Germany, 9Robert-Bosch-Krankenhaus, Pathologie, Stuttgart, Germany, 10Institut für Pathologie Esslingen, Esslingen, Germany, 11Comprehensive Cancer Center Ulm, Ulm, Germany, 12Universitätsklinikum Ulm, Thoraxchirurgie, Ulm, Germany, 13Universitätsklinikum Ulm, Pathologie, Ulm, Germany

Background: Despite several approaches in prevention and early detection of lung cancer the prognosis is still poor. Recently, emerging data on the molecular background of lung cancer introduced several new approaches in the systemic therapy especially in patients with advanced stages. The aim of the LuCa-BiO protocol (ClinicalTrials.gov: NCT02613637) is to set up a multicenter registry including clinical, molecular and quality of life data to allow implementation of focussed interventional clinical programs and healthcare research.

Methods: We set up a multicenter registry with a validated and certified web-based registration platform separating personal patient-data for the trusted third party from pseudonymized data for the liquid-biopsy lab, the quality of life assessment center as well as for the clinical data center and protocol coordination. Sites receive at the same time an easy to handle registration documentation sheet containing all relevant information. Clinical data are extracted from cancer registry datasets required by law with regular updates. Material from diagnostic specimens is available at the participating centers. Quality of life (QoL) is assessed by the EORTC QLC-C30 and LC 13 questionnaires.

Results: The trial started in May 2014 with an exploratory set-up phase with three clinical centers. Until May 2016, 400 patients were registered and for the analysis the first 242 patient with available cancer registry data were selected. Clinical data and liquid biopsies are available from all included patients, QoL data in 188 patients. Median age at registration was 67 years (range, 31-89 years), 62% of the patients were male, 82% were registered at initial diagnosis and 18% later on; 16% had small cell lung cancers (SCLC) and 84% Non-SCLC with 57% adenocarcinoma and 31% squamous-cell carcinoma. UICC staging at diagnosis was IA 18%, IB 14%, IIA 8%, IIB 4%, IIIA 14%, IIIB 12% and IV 29%. QoL data revealed at diagnosis a median global status of QoL 75% (range, 14-100%), a median function-related QoL of 76% (range, 7-100%) a median symptom-oriented QoL of 79% (range, 21-100%) and a median overall QoL of 58% (16-100%).

Conclusions: This first data from our LuCa-BiO study in lung cancer patients show the feasibility of a multicenter approach combining cancer registry data with QoL assessment and liquid biopsy biobanking. Molecular and treatment data are currently updated.

Disclosure: No conflict of interest disclosed.

P293 – Exploratory analysis of efficacy by histology and frontline therapies in a nonsquamous non-small cell lung cancer (NSCLC) subgroup in REVEL: A randomized phase III study of ramucirumab (RAM) plus docetaxel (DOC) vs DOC plus placebo (PBO) for second-line treatment of stage IV NSCLC

Schuette W.1, Reck M.2, Kimmich M.3, Schumann C.4, Paz-Ares L.5, Garon E.6, Pérol M.7, Zimmermann A.8, Lee P.9

1Krankenhaus Martha-Maria Halle-Dölau, Klinik für Innere Medizin II, Halle (Saale), Germany, 2LungenClinic Grosshansdorf, Airway Research Center North (ARCN), Großhansdorf, Germany, 3Abt. Pneumologie und Pneumologische Onkologie Klinik Schillerhöhe, Zentrum für Pneumologie, Beatmungsmedizin und Thoraxchirurgie, Gerlingen, Germany, 4Klinik für Pneumologie, Thoraxonkologie, Schlaf- und Beatmungsmedizin, Kempten, Germany, 5University Hospital Virgen del Rocío, Medical Oncology, Seville, Spain, 6David Geffen School of Medicine at UCLA, Translational Research in Oncology-US Network, Los Angeles, United States, 7Département de Cancérologie Médicale Centre Léon-Bérard, Lyon, France, 8Eli Lilly and Company, Indianapolis, United States, 9Eli Lilly and Company, Bridgewater, United States

Introduction: The phase III trial REVEL (NCT01168973) led to FDA and EMA approval of RAM + DOC for metastatic NSCLC patients with disease progression on/after standard platinum-based frontline chemotherapy (pbFCT). Pemetrexed (PEM) is standard of care in nonsquamous NSCLC within pbFCT. An exploratory efficacy analysis of RAM + DOC for nonsquamous NSCLC, including adenocarcinoma, is reported.

Methods: Frontline therapy for nonsquamous patients was defined as PEM induction therapy ± any PEM maintenance therapy (PEM IT) and non-PEM induction therapy ± non-PEM maintenance therapy (non-PEM IT). Nonsquamous patients received DOC (75 mg/m2) + RAM (10 mg/kg; n = 465) or DOC + PBO (n = 447) after disease progression on pbFCT. Endpoints were overall survival (OS), progression-free survival (PFS) and objective response rate (ORR). OS and PFS were analyzed using the Kaplan-Meier method and the Cox proportional hazard model; ORR was analyzed using Cochran-Mantel-Haenszel test.

Results: Of 912 patients with nonsquamous NSCLC (73%), the majority were adenocarcinoma (79%; N = 725). Baseline characteristics and post-study therapy use in nonsquamous patients were balanced between PEM IT and non-PEM IT subgroups. Maintenance therapy use was higher for patients with prior PEM IT (37.9%) than non-PEM IT (13.7%). RAM + DOC efficacy for adenocarcinoma [OS = 11.2 months (HR, 95% CI: 0.83, 0.69-0.99); PFS = 4.5 months (HR, 95% CI: 0.75, 0.64-0.88); ORR = 19%] was consistent with nonsquamous [OS = 11.1 months (HR, 95% CI: 0.83, 0.71-0.97); PFS = 4.6 months (HR, 95% CI: 0.77, 0.67-0.88); ORR = 22%].

Results for RAM + DOC vs DOC + PBO were: PEM IT: OS = 11.8 months vs 9.0 months (HR, 95% CI: 0.779, 0.62-0.98); non-PEM IT: OS = 11.0 months vs 9.9 months (HR, 95% CI: 0.855, 0.68-1.07); PEM IT: PFS = 5.1 months vs 3.7 months (HR, 95% CI: 0.691, 0.56-0.85); nonPEM IT: PFS = 4.5 months vs 3.5 months (HR, 95% CI: 0.772, 0.63-0.94); PEM IT: ORR = 20.0% vs 14.9%; non-PEM IT: ORR: 24.0% vs 14.3%.

Conclusions: In nonsquamous NSCLC, RAM + DOC improved OS, PFS and ORR regardless of prior PEM or non-PEM treatment. Favorable efficacy was seen for NSCLC histological subtypes, including adenocarcinoma.

Disclosure: Wolfgang Schuette: Advisory Role: Yes; Financing of Scientific Research: YesPablo Lee: Employment or Leadership Position: Yes; Stock Ownership: Yes

P294 – Expression of the orphan G-protein-coupled receptor GPR19 in different lung cancer entities

Gerlach L.1, Kaemmerer D.2, Sänger J.3, Schulz S.1, Lupp A.1

1Universitätsklinikum Jena, Institut für Pharmakologie und Toxikologie, Jena, Germany, 2Zentralklinik Bad Berka, Klinik für Allgemein- und Viszeralchirurgie, Bad Berka, Germany, 3Labor für Pathologie und Zytologie Bad Berka, Bad Berka, Germany

Introduction: Lung cancer is one of the leading causes of tumor-related death worldwide, not least since for most of the entities no efficient standard therapy is available so far. Therefore, the discovery of new markers to open up additional treatment options is urgently needed. Recently it has been shown that the mRNA of the orphan G-protein-coupled receptor (GPCR) GPR19 is highly expressed in small cell lung cancer (SCLC). Thus, the aim of the present study was to investigate the expression of this receptor at the protein level by means of immunohistochemistry not only in SCLC, but also in other lung cancer entities using a novel rabbit anti-GPR19 antibody.

Methods: Overall, 320 samples from 132 lung cancer patients (typical carcinoid [TC]: n = 21; atypical carcinoid [AC]: n = 25; SCLC: n = 42; adenocarcinoma: n = 22; squamous cell carcinoma: n = 22), comprising both primary tumors and metastases, were evaluated for the GPR19 expression by means of immunohistochemistry. The immunohistochemical stainings were evaluated using the Immunoreactive Score (IRS), comprising values from 0 to 12 points, and correlated to clinical data. Tumors with IRS values = 3 IRS points were considered positive.

Results: With the only exception of the squamous cell carcinomas, the GPR19 could be detected at a high frequency in all lung cancer entities investigated. It was present in 95% of the TC, in 96% of the AC, and in 91% of the SCLC and of the adenocarcinomas. In contrast, only 23% of the squamous cell carcinomas were GPR19 positive. Also with respect to the intensity of expression, squamous cell carcinomas displayed distinctly lower IRS values as compared to the other tumor entities and there was also a significant difference between the values of the adenocarcinomas and those of the SCLC (median IRS values: TC: 8.0, AC: 7.6, SCLC: 9.0, adenocarcinomas: 6.1; squamous cell carcinomas: 1.5). Between primary tumors and metastases no significant difference in GPR19 expression was observed.

Conclusions: Because of the high incidence and intensity of expression in TC, AC and SCLC, the GPR19 may serve as a good target for diagnostics and therapy especially of lung tumors with neuroendocrine background.

Disclosure: No conflict of interest disclosed.

P295 – Clinical research platform into molecular testing, treatment and outcome of non-small cell lung carcinoma patients (CRISP): A prospective German registry in stage IV NSCLC – AIO-TRK-0315

Griesinger F.1, Eberhardt W.E.E.2,3, Marschner N.4, Jänicke M.5, Spring L.5, Sahlmann J.5, Karatas A.6, Hipper A.6, Sebastian M.7, Thomas M.8,9

1Pius-Hospital Oldenburg, University of Oldenburg, Universitätsklinik Innere Medizin-Onkologie, Oldenburg, Germany, 2University Hospital Essen, Department of Medical Oncology, Essen, Germany, 3Ruhrlandklinik and University Duisburg-Essen, West German Cancer Centre, Essen, Germany, 4Praxis für interdisziplinäre Onkologie und Hämatologie, Freiburg, Germany, 5iOMEDICO, Freiburg, Germany, 6AIO-Studien-gGmbH, Berlin, Germany, 7Klinikum der J.W. Goethe-Universität Frankfurt, Medizinische Klinik II Hämatologie/Onkologie, Rheumatologie, Infektiologie, HIV, Frankfurt am Main, Germany, 8Thoraxklinik am Universitätsklinikum Heidelberg, Heidelberg, Germany, 9German Center for Lung Research (DZL), Heidelberg, Germany

Background: Treatment in NSCLC is quickly evolving. Whether outcome and PRO data generated in clinical trials with narrow inclusion and exclusion criteria will hold up in the routine practice is of high interest, especially due to the increasing costs of new drugs. Therefore registry data are of increasing importance to patients, physicians and third parties. This is why we have started a prospective, clinical registry for patients with metastatic non-small cell lung cancer.

Methods: The purpose of CRISP is to set up a national clinical research platform to document representative data on molecular testing, sequences of therapies and other treatment modalities, course of disease in patients with advanced or metastatic NSCLC not amenable to curative treatment. A particular focus is on molecular biomarker testing of patients before the start of first-line treatment. PRO assessment will provide large-scale data on quality of life and anxiety/depression for real-life patients in routine practice. In addition, two questionnaires (concerning individual quality of life and patient-caregiver communication) will be validated in German patients with metastatic NSCLC. Furthermore CRISP will set up a decentral tissue annotation for future collaborative, investigational scientific biomarker testing. This study will be carried out in up to 150 representative cancer centers in all therapeutic sectors in Germany. More than 5000 patients will be recruited and followed up to a maximum of 3 years, respectively until death. The first patients have been included as of December 2015.

Results: Preliminary data will be presented at the meeting in terms of molecular test rates, demographic data as well as treatment stratification in the 1st line setting.

Conclusion: The registry CRISP will be the first to present representative real life data, covering all treatment settings of patients with NSCLC in Germany.

ClinicalTrials.gov Identifier: NCT02622581. CRISP is supported by Grants from AstraZeneca, Boehringer Ingelheim, Bristol-Myers Squibb, Celgene, Merck Sharp & Dohme, Novartis, and Pfizer.

Disclosure: No conflict of interest disclosed.

P296 – Intercalated TKI and chemotherapy induction in EGFR mt+ NSCLC stages IIIA to IV OMD: report of 5 cases and phase II study

Griesinger F.1, Lüers A.1, Roeper J.1, Falk M.2, Conradi I.S.1, Reinhardt M.3, Kluge A.4, Willborn K.5, Prenzel R.6, Scriba D.7, Henke R.-P.8, Hallas C.2, Netchaeva M.1, Tiemann M.2

1Pius-Hospital Oldenburg, University of Oldenburg, Hämatolologie/Onkologie, Innere Medizin-Onkologie, Oldenburg, Germany, 2Hämatopathologie Hamburg, Pathologie, Hamburg, Germany, 3Pius-Hospital Oldenburg, Nuklearmedizin, Oldenburg, Germany, 4Pius-Hospital Oldenburg, Radiologie, Oldenburg, Germany, 5Pius-Hospital Oldenburg, University of Oldenburg, Strahlentherapie und Radioonkologie, Medizinische Physik, Oldenburg, Germany, 6Pius-Hospital Oldenburg, Innere Medizin, Pneumologie, Gastroenterologie, Oldenburg, Germany, 7Pius-Hospital Oldenburg, Thorax- und Gefäßchirurgie, Oldenburg, Germany, 8Pathologisches Institut Oldenburg, Pathologie, Oldenburg, Germany

Background: EGFR TKI treatment is standard of care in patients with stage IV NSCLC carrying an activating EGFR mutation. Induction concepts in locally advanced EGFR mt+ NSCLC including TKI have not been studied extensively. Recently new focus has been shed on intercalated regimens of chemotherapy and TKI, showing improved PFS as well as OS. This concept was used as induction regimen in 5 patients with activating EGFR mutation in stages IIIA and IIIB.

Methods: Patients were diagnosed and worked up according to standard imaging, histology and immunohistology methods. EGFR, KRAS, BRAF, ALK and P53 mutation analysis were performed with standard procedures as described by Halbfass et al. 2013. Remission induction was measured by RECIST 1.1, regression grading by Junker criteria.

Results: 3 f never or light smokers (pt #1, 3, 5), 59, 63, 62 y.o. and 2 m never or light smoker (pt# 2and 4) 57 and 70 y.o. had adenocarcinoma of the lung, 2 with L858R (#1,2)and 3 with E 19 Del (#3-5). 4/5 patients (#1-4) carried a a p53 mutation, case #1 a disruptive, cases #2-4 a non disruptive mutation. Stages were IIIB (#1-3), IV OMD with 2 metastases in 1 vertebra (#4) and IIIA (#5). Induction therapy was started with gefitinib 250 mg/die p.o. (#1,4,5) or erlotinib 150 mg/die p.o. days -12 to -1 (#3,4) in order to prove responsiveness of the tumour to EGFR-TKI. On day 0 partial response or no progression was achieved in all 5 pts. Therapy was continued with 3 cycles of Docetaxel 75 mg/m2 d1 and Cisplatin 50 mg/m2 d 1 and 2 qd22 or Paclitaxel 200 mg/m2 + Carboplatin AUC 6.0 d1 in combination with Gefitinib 250 mg / die or Erlotinib 100 mg/ die d4-19. PR was was achieved after 2 cycles in all pts. All 5 pts were resected and regression grade IIB or III was observed in mediastinal lymph nodes and primary tumor in pts #1-4, only IIA and I in pt #5. 4/4 pts with prior N2 or N3 LN received postoperative radiotherapy. Patients #1, 3, 5 are in CR, patient 2 and 4 developed one isolated CNS metastasis which has been stereotactically irradiated. Patients’ survival times are 10+, 13+, 41+, 7+, 5+ months respectively.

Conclusion: Intercalated TKI treatment is a promising treatment choice in patients with EGFR mt+ locally advanced NSCLC. A phase II trial (NeoIntercal) trial is currently under way in 9 German centers in stages II and III using Gefitinib in combination with induction taxane based chemotherapy, supported by AIO, ASTRA Zeneca and the University of Oldenburg.

Disclosure: Frank Griesinger: Advisory Role: Pfizer, Astra, Boehringer, Novartis, Roche, Celgene, MSD, BMS, Clovis; Financing of Scientific Research: Pfizer, Astra, Boehringer, Novartis, Roche, Celgene, MSD, BMS, Clovis; Expert Testimony: ASTRAMarkus Tiemann: Advisory Role: Pfizer, Astra, Boehringer, Novartis, Roche, Celgene, MSD, BMS; Honoraria: Pfizer, Astra, Boehringer, Novartis, Roche, Celgene, MSD, BMS; Expert Testimony: Astra Zeneca

P297 – Non-Small Cell Lung Cancer (NSCLC) - treatment research and treatment reality in oncology practices (adjuvant therapy)

Wilhelm S.1, Hutzschenreuter U.2, Innig G.3, Göttel R.4, Tessen H.-W.5, Projektgruppe Internistische Onkologie (PIO)

1Onkologische Schwerpunktpraxis, Güstrow, Germany, 2Onkologische Schwerpunktpraxis, Nordhorn, Germany, 3Onkologische Schwerpunktpraxis, Rheine, Germany, 4rgb Onkologisches Management GmbH, Sarstedt, Germany, 5Onkologische Kooperation Harz, Goslar, Germany

Introduction: According to S3 guidelines, patients with a NSCLC stage II or IIIA are to receive a cisplatin-based adjuvant combination chemotherapy for 4 cycles within 60 days after a R0 resection (recommendation level A). Stage IB requires an individual therapy decision. Patients with an adjuvant chemotherapy in phase-III trials lived a median of 66-94 months.

Methods: Since 2003, 89 oncological practises from 16 federal states have been documenting 2,864 disease histories of patients with a non-small cell lung cancer within the framework of the Project team of Internal Oncology (PIO). 2,723 of the cases with a total of 6,135 therapies could be evaluated in the registry ONCOReg. 409 (15.0%) patients received an adjuvant chemotherapy, 305 (74.6%) intravenous vinorelbine/cisplatin. Detailed results are available for 291 patients from 42 practises.


Patients’ characteristics

Gender: 209 (71.8%) male; 82 (28.2%) female

Age at initial diagnosis (median): 63 (41-78) years; 8 (2.8%) > 75 years; 54 (18.6%) > 70 years

Previous therapy: 7 (2.4%) patients

UICC: 53 (18.2%) I; 126 (43.3%) II; 106 (36.4%) III; 6 n.s. (T1-2, Nx)

Histology: 138 (47.4%) adenocarcinoma; 146 (50.2%) squamous cell carcinoma; 7 others

Smokers/Non-smokers: 99 (34.0%)/107 (36.8%)/85 n. s.


Period from surgery to start of therapy (median): 42 (15-191) days

Number of cycles (median): 4 (1-6)

End of therapy: 180 (61.9%) as scheduled; 42 (14.4%) toxicity; 24 (8.2%) refusal; 14 (4.8%) hospitalization; 31 others

Radiotherapy: 62 (21.3%)

The median monitoring period covers 19.4 (0.3-111.1) months. Distant metastases have been reported for 82 patients so far, a local recurrence for 34 patients and a recurrent neoplasm for 8 patients. 51 (62.8%) patients received a chemotherapy due to recurrences or distant metastases. The 1st-line therapy for them consisted mainly in platinum derivatives (28 (54.9%), taxanes (17 (33.3%) und antimetabolites (15 (29.4%).


The median disease-free survival is at 32.2 months. UICC I: months not reached; II: 38.4 months; III: 23.0 months). The median overall survival has not been reached so far. The 3-year survival rate is at 70%. UICC I: not reached (3-year survival 81%); II: 91.6 months (70%); III: 56.2 months (65%). Smokers not reached (64%); non-smokers 75.1 months (71%)

Conclusions: The adjuvant therapy of the NSCLC is performed in compliance to the approved guidelines in treatment research.The survival data reflect the existing data.

Disclosure: Stefan Wilhelm: Employment or Leadership Position: Praxisinhaber- Gemeinschaftspraxis; Advisory Role: Lilly, Roche, Sozialgutachten, Gerichte; Financing of Scientific Research: Amgen, Medac, Lilly, Mundipharm, Roche, Boehringer Ingelheim, GlaxoSmithKline, Novartis, AstraZenecaHans-Werner Tessen: No conflict of interest disclosed.

P298 – Successful intercalating multimodal treatment strategy in a patient with advanced adenocarcinoma of the lung harboring the uncommon complex EGFR mutation L833V/H835L

Frille A.1, Sändig I.2, Wirtz H.1

1Universitätsklinikum Leipzig, Pneumologie, Leipzig, Germany, 2Universitätsklinikum Leipzig, Institut für Pathologie, Leipzig, Germany

Introduction: According to international guidelines for the treatment of advanced non-small cell lung cancer (NSCLC), sensitizing mutations of the epidermal growth factor receptor (EGFR) are predictive for response to the EGFR tyrosine kinase inhibitors (TKI) gefitinib erlotinib and afatinib in terms of improved response rate, progression-free survival, quality of life, and even overall survival. However, efficacy of those TKI for uncommon mutations in the EGFR gene may be distinct from common mutations like exon 19 deletions and L858R.

Case: A 63-year-old male Caucasian never-smoker in a good performance status was diagnosed with a poorly differentiated adenocarcinoma of the right lower lobe of the lung. Fluorodeoxyglucose positron emission tomography (FDG-PET) revealed in addition hypermetabolic subcarinal, paratracheal, and supraclavicular lymph nodes on the right side being suspicious for malignancy. The initial clinical TNM classification was cT2a cN3 cM0 (stage IIIB). A complex mutation of L833V and H835L in exon 21 of the EGFR gene was detected from the tumor specimen by nucleic acid amplification via polymerase chain reaction followed by Sanger sequencing of EGFR from exon 18 to exon 21. Common drug-sensitive mutations including exon 19 deletions and L858R were not found. An oral regimen of the TKI gefitinib 250 mg per day was started. Since eight weeks of TKI treatment showed a stable disease, we decided to perform an intercalating chemotherapy containing cisplatin/pemetrexed over four courses leading now to a partial response of the pulmonary target lesion. Chemotherapy was then switched to second generation TKI afatinib 40 mg per day for six weeks resulting in a further partial response. After multidisciplinary discussion, the patient then underwent curative surgery with resection of right lower lobe and systemic lymphadenectomy revealing a postoperative ypT1b pN2 cM0, L1, V0 Pn0, R2 (supraclavicular lymph nodes). After recovery form surgery, an adjuvant concurrent chemoradiotherapy of the supraclavicular and mediastinal lymph nodes was performed and one course of cisplatin/pemetrexed was administered. Last restaging showed no evidence of suspicious hypermetabolic lesions in FDG-PET.

Conclusion: Evidence predicting the efficacy of TKI for uncommon complex mutations of the EGFR is often lacking. Patients with a very good performance status despite an advanced N3-disease may need an intense discussion about individual multimodal treatment plan.

Disclosure: No conflict of interest disclosed.

P299 – Adenocarcinoma in pleural tissue in a patient after double lung transplantation: the question of tumor origin and therapeutic considerations

Ufen M.-P.1, Janssen C.1, Müller L.1

1Onkologie Unter Ems, Leer, Germany

Introduction: Lung transplantation may help to improve the prognosis of patients with end-stage lung diseases. The pre-interventional screening of donor and recipient organs is responsible for the successful outcome of lung transplantation, for example by detection of a malignant tumor in patients with severe pulmonary comorbidities. Nevertheless, the occurrence of a lung cancer after lug transplantation is rarely reported in literature.

Case report: We report on the case of a 61 years old male patient with an double lung transplantation due to an combined pulmonary fibrosis and emphysema (CPFE) with a history of heavy smoking (50 py). Immunosuppression was performed with everolimus, MMF and steroids. A decortication of the left lower lobe were carried out 11 months post transplantation due to a persistent pleural effusion. The histological examination of pleural biopsies showed a low differentiated adenocarcinoma (K-RAS-mutated), classified as cTx cNx pM1a (PLE). Pathologic work-up failed to detect a definite allocation of primary tumor. Staging diagnostics by PET-CT and intestinoscopy provided no evidence for an other primary. Consequently a pulmonary origin must be supposed, although no tumor was found in the donor lung. A pathologic examination of the explanted lung has not been performed.

The pleural carcinomatosis defined an advanced disease, therefore a systemic treatment in palliative intention was carried out with pemetrexed only because of a reduced physical condition based on a pulmonary embolism. The first restaging after two months of treatment showed no evidence of disease except a small pleural effusion, which cannot be examined by taking a sample.

Conclusions: Although the primary tumor cannot be determined without any doubt-a origin in the donor or recipient lung must be supposed. In accordance with the transplant centre, a malignancy of the explanted lung seems to be more likely due to the smoking history. In future we would recommend further pathological examination of the explanted organ to solve the question of origin. Remarkably, there is no definitive tumor manifestation detectable under the therapy with pemetrexed and everolimus. The addition of a m-TOR Inhibitor may have influenced the treatment with pemetrexed and represents potentially a novel therapeutic concept in lung cancer.

Disclosure: No conflict of interest disclosed.

P300 – Bioluma: Biomarkers for nivolumab and evaluation of nivolumab plus ipilimumab in lung cancer – A phase II trial of nivolumab in combination with ipilimumab to evaluate efficacy and safety in lung cancer and to evaluate biomarkers predictive for response to immune checkpoint inhibition

Fischer R.N.1, Abdulla D.1, Michels S.1, Nogova L.1, Brandes V.1, Scheffler M.1, Schäfer S.2, Scheel A.2, Thurat M.1, Vehreschild M.3, Thomas R.4, von Bergwelt-Baildon M.5, Büttner R.2, Wolf J.1

1Uniklinik Köln, Klinik I für Innere Medizin, Lunge Cancer Group Cologne, Köln, Germany, 2Uniklinik Köln, Institut für Pathologie, Köln, Germany, 3Uniklinik Köln, Klinik I für Innere Medizin, German Centre for Infection Research, Köln, Germany, 4Universität Köln, Institut für Translationale Genomik, Köln, Germany, 5Uniklinik Köln, Klinik I für Innere Medizin, Interventionelle Immunologie, Köln, Germany

Introduction: Checkpoint inhibition is a promising strategy in the treatment of various cancer entities. The anti-PD-1 antibody nivolumab recently was approved for secondline treatment in squamous and non-squamous non-small cell lung cancer (NSCLC). In small cell lung cancer (SCLC), preliminary data from ongoing clinical trials show promising results even in platinum-refractory subjects. However, the achievement of response rates of approximately 20% implicates the unmet need to further enhance therapeutic activity of checkpoint inhibition. In addition, strategies to better define patients responding to checkpoint inhibition before therapy, i.e. to identify predictive biomarkers need to be developed. Here, we present the ongoing clinical trial BIOLUMA which aims to evaluate efficacy and safety of adding ipilimumab to nivolumab in the treatment of nivolumab-refractory NSCLC patients and to evaluate efficacy and safety of upfront combination therapy of nivolumab and ipilimumab followed by nivolumab monotherapy in subjects with SCLC. In addition, a broad biomarker program is included in order to identify biomarker predictive for response and resistance to checkpoint inhibition.

Methods: BIOLUMA is a multicentric non-randomised phase II trial in patients with non-squamous NSCLC and patients with SCLC after failure of platinum-based first-line therapy. In 12 German trial centres, NSCLC patients are treated with nivolumab until disease progression and subsequently receive a combination therapy of nivolumab and ipilimumab. SCLC patients receive four cycles of nivolumab in combination with ipilimumab and subsequent nivolumab monotherapy.

Primary endpoint for the NSCLC cohort is ORR after addition of ipilimumab to nivolumab treatment. For the SCLC cohort, primary endpoint is ORR of the upfront combination therapy nivolumab and ipilimumab.

In addition, extensive analysis of tumor biopsy material, peripheral blood and gut microbiome is performed. Tumor cells and tumor microenvironment are characterized by histology and immunohistochemistry. The role of specific somatic mutations and overall mutational load is analyzed by whole genome or whole exome sequencing (WGS, WES), neoepitope prediction and modeling of HLA-processing. Cellular and soluble blood components are assessed by FACS and ELISA-assays. Intestinal microbiome samples are analyzed by deep-sequencing.

Disclosure: Rieke Fischer: Advisory Role: Teilnahme an Advisory Boards Bristol-Myers Squibb; Financing of Scientific Research: Honorare von Bristol-Myers Squibb für Vorträge, Teilnahme an ADJürgen Wolf: Advisory Role: Teilnahme an Advisory Boards Bristol-Myers Squibb; Financing of Scientific Research: Honorare von Bristol-Myers Squibb für Vorträge, Teilnahme an AD

Posterdiskussion – Kolorektale Karzinome

P301 – SATB1 as a therapeutic target candidate in colorectal cancer: preclinical studies

Frömberg A.1, Rabe M.1, Linnebacher M.2, Aigner A.1

1Universität Leipzig, Medizinische Fakultät, Rudolf-Boehm-Institut für Pharmakologie und Toxikologie, Selbständige Abteilung Klinische Pharmakologie, Leipzig, Germany, 2University of Rostock, Department of General, Thoracic, Vascular and Transplantation Surgery, Rostock, Germany

Introduction: The Special AT-rich Binding Protein 1 (SATB1) influences the expression of multiple genes on an epigenetic level, by acting as a chromatin organizer. Importantly, SATB1 overexpression has been described several human cancers and connected to carcinogenesis. The expression of SATB1 has also been found to correlate with tumor progression and is associated with poor prognosis.

The functional relevance of SATB1, its possible mechanisms of action and its potential as a target for new therapeutic strategies in colorectal cancer (CRC) were largely unknown and subject of this study.

Methods: RNAi-mediated gene knockdown of SATB1 was employed in primary and in established colorectal cancer cell lines. Proliferation and colony formation assays, flow cytometry analyses of cell cycle and determination of apoptosis induction upon SATB1 knockdown were performed. By qRT-PCR and Western blotting, SATB1 knockdown effects on multiple important (proto-)oncogenes and other molecules involved in cell cycle, EMT and cell adhesion were analysed. An in vivo study using a nanoparticle-based platform for siRNA delivery was performed in a tumor xenograft model.

Results: SATB1 knockdown led to markedly reduced SATB1 levels and caused inhibition of proliferation, deceleration of cell cycle progression and pro-apoptotic effects. Further analyses revealed effects of SATB1 on multiple signaling pathways influencing e.g. EMT, apoptosis and ErbB receptor expression. In an s.c. tumor xenograft model in athymic nude mice, stable SATB1 knockdown cells showed markedly reduced tumor formation.

The therapeutic potential of SATB1 inhibition was further explored in vivo in CRC xenograft bearing mice treated with nanoparticles for siRNA delivery. A marked inhibition of tumor growth was observed in the siRNA treatment group.

Conclusions: Our results indicate an important and complex role of SATB1 in the tumorigenesis of colorectal cancer. We establish SATB1 inhibition or siRNA-mediated knockdown as a promising target for pharmacological intervention.

Disclosure: No conflict of interest disclosed.

P302 – Impact of early tumour shrinkage (ETS) on overall survival (OS) in patients with RAS wild-type (WT) metastatic colorectal cancer (mCRC) receiving first-line treatment in three randomised panitumumab trials: An exploratory study-level meta-analysis

Karthaus M.1, Rivera F.2, Valladares-Ayerbes M.3, Gallego J.4, Koukakis R.5, Demonty G.6, Douillard J.-Y.7

1Städtisches Klinikum München, Klinikum Neuperlach, Munich, Germany, 2Hospital Universitario Marqués de Valdecilla, Santander, Spain, 3Virgen del Rocio Hospital, Seville, Spain, 4Hospital General Universitario de Elche, Elche, Spain, 5Amgen Ltd, Uxbridge, United Kingdom, 6Amgen (Europe) GmbH, Zug, Switzerland, 7Institut de Cancérologie de l‘Ouest (ICO) René Gauducheau, St Herblain, France

Introduction: Tumour shrinkage is an important aim of treatment in patients with mCRC as it can provide relief of tumour-related symptoms and increase the chance of resection. ETS has been associated with improved OS and can also provide an early indication of treatment sensitivity.) A meta-analysis was conducted to analyse the impact of ETS on OS in patients with RAS WT mCRC receiving first-line treatment in three randomised panitumumab trials.

Methods: Three randomised, first-line panitumumab trials have reported ETS and OS data: PRIME – phase III, panitumumab + FOLFOX4 vs FOLFOX4 (NCT00364013); PEAK – phase II, panitumumab + mFOLFOX6 vs bevacizumab + mFOLFOX6 (NCT00819780); PLANET – phase II, panitumumab + FOLFOX4 vs panitumumab + FOLFIRI (NCT00885885). This retrospective study-level meta-analysis aimed to estimate the effect of ETS =20% vs < 20% and ETS =30% vs < 30% at week 8 on OS in patients with RAS WT mCRC receiving first-line treatment (overall) in these studies. Meta-analysis techniques (fixed-effects modelling [unconditional maximum likelihood method] and random-effects modelling [DerSimonian and Laird modelling methods]), were used to pool study-level data using the inverse-variance of each trial as the weight.

Results: Data from a total of 641 patients with RAS WT mCRC who were evaluable for OS and ETS in these studies, were included in the meta-analysis. Achievement of ETS =20% vs ETS < 20% (hazard ratio [HR]: 0.45 [95% confidence intervals [CI]: 0.37-0.54) and ETS =30% vs ETS < 30% (HR: 0.46 [95% CI: 0.38-0.55]) was associated with improved OS. Results from the individual studies are included in the table.


Conclusions: This exploratory study-level meta-analysis of pooled data from three first-line panitumumab trials (PRIME, PEAK and PLANET) suggests that ETS (=20% or =30% at week 8) is associated with improved OS and hence may be a useful early indicator of treatment benefit.

Disclosure: Meinolf Karthaus: Advisory Role: Consulting/advisory activities for Amgen and Roche; Financing of Scientific Research: Honoraria from Amgen and Roche; Other Financial Relationships: Travel and accomodation from Amgen and RocheJean-Yves Douillard: Advisory Role: Consulting/advisory activities for Amgen, Bayer, Roche and Merck; Financing of Scientific Research: Honoraria from Amgen, Bayer, Roche and Merck; Expert Testimony: Research funding from Merck Serono; Other Financial Relationships: Travel, accomodation and expenses from Amgen, Bayer, Roche and Merck

P303 – An exploratory analysis evaluating the effect of sequence of biologic therapies on overall survival (OS) in patients (pts) with RAS wild-type (WT) metastatic colorectal carcinoma (mCRC)

Karthaus M.1, Sobrero A.2, Douillard J.-Y.3, Rivera F.4, Forget F.5,Valladares-Ayerbes M.6, Demonty G.7, Guan X.8, Peeters M.9

1Städtisches Klinikum München, Klinikum Neuperlach, Munich, Germany, 2IRCCS Ospedale San Martino IST, Genova, Italy, 3ICO R. Gauducheau, St. Herblain, France, 4Hospital Universitario Marqués de Valdecilla, Santander, Spain, 5Centre Hospitalier de l’Ardenne, Libramont, Belgium, 6Virgen del Rocio Hospital, Seville, Spain, 7Amgen (Europe) GmbH, Medical Development, Zug, Switzerland, 8Amgen Inc., Biostatistics, Thousand Oaks, United States, 9Antwerp University Hospital, Edegem, Belgium

Introduction: Addition of epidermal growth factor receptor inhibitors (EGFRi) or vascular endothelial growth factor inhibitors (VEGFi) to chemotherapy in the first-line setting provides clinical benefit to pts with RAS WT mCRC, vs chemotherapy alone. The choice of first-line biologic therapy may impact on mCRC biology and sensitivity to subsequent treatments, as suggested by preclinical studies, but the optimal treatment sequence has not yet been assessed in a randomised prospective clinical trial. An exploratory analysis of OS for pts with RAS WT mCRC receiving either first-line panitumumab (pmab) plus second-line VEGFi, or firstline bevacizumab (beva) and second-line EGFRi was conducted.

Methods: Pt-level OS data from three randomised mCRC trials was used for this exploratory retrospective analysis. PEAK (NCT00819780) was a phase II study of first-line pmab+mFOLFOX6 vs beva+mFOLFOX6; PRIME (NCT00364013) was a phase III study of first-line pmab+FOLFOX4 vs FOLFOX4 alone and study 181 (NCT00339183) was a phase III trial of second-line pmab+FOLFIRI vs FOLFIRI alone. OS was analysed for all pts with RAS WT mCRC treated with either first-line pmab (PEAK and PRIME) plus secondline VEGFi, or firstline beva (PEAK and 181) and secondline EGFRi.

Results: Of the pts included in the analysis (n = 104), 66 received first-line pmab and secondline VEGFi and 38 were treated with first-line beva plus second-line EGFRi. At the time of analysis, 63.6% and 92.1% of pmab?VEGFi and beva?EGFRi pts, respectively, had died. OS HRs comparing the pmab?VEGFi and beva?EGFRi treatment sequences are reported in the table. Overall, pooled analysis of PEAK+PRIME pts vs PEAK+181 pts showed a median OS of 36.8 (95% CI: 30.3-43.8) vs 27.8 (95% CI: 24.2-35.6) months for pmab?VEGFi vs beva?EGFRi treatments (HR for OS [95% CI]: 0.65 [0.42-1.03]).


Conclusions: This exploratory analysis may suggest a trend towards improved OS for pts with RAS WT mCRC treated with first-line pmab+chemotherapy followed by second-line VEGFi vs those treated with firstline beva plus second-line EGFRi, although numbers were small. A larger, prospective randomised trial is needed to confirm the appropriate sequence of EGFRi/VEGFi in these pts.

Disclosure: Meinolf Karthaus: Advisory Role: Consulting/advisory activities for Amgen and Roche; Financing of Scientific Research: Honoraria from Amgen and Roche; Other Financial Relationships: Travel and accommodation from Amgen and RocheMarc Peeters: Advisory Role: Consultancy/advisory roles for Amgen; consultancy/symposia participation for Merck Serono; Expert Testimony: Research Funding from Amgen

P304 – Regorafenib in previously treated metastatic colorectal cancer (mCRC): Analysis of age subgroups in the open-label phase 3b CONSIGN trial

Kasper S.1, van Cutsem E.2, Ciardello F.3, Ychou M.4, Seitz J.-F.5, Hofheinz R.-D.6, Arriaga Y.E.7, Verma U.7, Garcia-Carbonero R.8, Grothey A.9, Miriyala A.10, Kalmus J.10, Kappeler C.10, Falcone A.11, Zaniboni A.12

1Universitätsklinikum Essen, Westdeutsches Tumorzentrum, Essen, Germany, 2University Hospital Leuven, Leuven, Belgium, 3Second University of Naples, Naples, Italy, 4Institut Regional du Cancer de Montpellier, Montpellier, France, 5Aix-Marseille University, Marseille, France, 6Universitätsklinik Mannheim, Mannheim, Germany, 7University of Texas Southwestern Medical Center, Dallas, United States, 8Hospital Universitario Doce de Octubre, Madrid, Spain, 9Mayo Clinic, Rochester, United States, 10Bayer Pharma AG, Berlin, Germany, 11University of Pisa, Pisa, Italy, 12Fondazione Poliambulanza, Brescia, Italy

Introduction: In the phase 3 CORRECT trial, regorafenib improved survival vs placebo in treatment-refractory mCRC. The large, single-arm, phase 3b CONSIGN trial (NCT01538680) was designed to provide continued access to regorafenib for patients with mCRC and to further characterize regorafenib safety. In CONSIGN, adverse events (AEs) and progression-free survival (PFS) were consistent with results reported in phase 3 trials. We performed a subgroup analysis of CONSIGN to assess outcomes by age.

Methods: Patients with mCRC who progressed after standard therapies and had ECOG PS 0-1 received regorafenib 160 mg daily for the first 3 weeks of each 4-week cycle until disease progression, death, or unacceptable toxicity. The primary endpoint was safety. PFS, assessed per investigator, was the only efficacy measure collected.

Results: A total of 2872 patients were assigned to treatment (< 65: n = 1720; =65: n = 1152); n = 1713 and n = 1151, respectively, were evaluable for safety. ECOG PS 0/1 were 49%/51% (< 65) and 44%/55% (=65). The median (range) treatment duration was 2.5 months (0.03–30.4) and 2.3 months (0.03–28.5) in patients < 65 and =65, respectively; mean (SD) percent of planned dose was 76% (20) and 74% (20), respectively. Most patients experienced a regorafenib-related AE (< 65: 91%; =65: 92%; Table). AEs led to treatment discontinuation in 25% (< 65) and 26% (=65) of patients. Treatment-emergent NCI-CTCAE v4.0 hepatic grade =3 laboratory toxicities (< 65; =65) included increased bilirubin (14%; 12%), increased AST (8%; 5%), and increased ALT (7%; 4%). Estimated median PFS (95% CI) in the < 65 group was 2.7 months (2.6, 2.8) and in the =65 group was 2.6 months (2.5, 2.7).


Conclusions: The safety profile of regorafenib was generally comparable in patients <65 and =65 years of age. PFS was similar across age subgroups.

Disclosure: Stefan Kasper: Advisory Role: Amgen, Bayer, BMS, Celgene, Lilly, Merck, MSD, Roche, Sanofi.; Financing of Scientific Research: Amgen, Lilly, Merck, Sanofi; Expert Testimony: Celgene, Merck; Other Financial Relationships: Amgen, Lilly, Meck, Sanofi, Roche, Bayer (Travel)Alberto Zaniboni: No conflict of interest disclosed.

P305 – Outcome of patients with KRAS exon 2 wildtype (KRAS-wt) metastatic colorectal carcinoma (mCRC) with cetuximab-based first-line treatment in the non-interventional study ERBITAG and impact of comorbidity and age

Sahm S.1, Göhler T.2, Hering-Schubert C.3, Janssen J.4, Neumann U.P.5, Schwittay M.6, Zahn M.-O.7, Stenzel K.G.8, Steinbach-Büchert A.K.8, Overkamp F.9

1Ketteler Krankenhaus, Offenbach, Germany, 2Onkozentrum Dresden/Freiberg, Dresden, Germany, 3St. Georg Klinikum, Eisenach, Germany, 4Onkologie Westerstede Aurich Rhauderfehn, Westerstede, Germany, 5Universitätsklinik der RWTH, Aachen, Germany, 6Tumorzentrum Leipziger Land, Groitzsch, Germany, 7Onkologische Kooperation Harz, Goslar, Germany, 8Merck Serono GmbH, Darmstadt, Germany, 9Oncologianova GmbH, Recklinghausen, Germany

Background: Cetuximab in combination with irinotecan- or oxaliplatin-based chemotherapy has shown to increase ORR, PFS, OS of KRAS-wt mCRC patients (pts). ERBITAG aimed to evaluate safety and efficacy of cetuximab in combination with various first-line chemotherapy regimens in pts with unresectable KRAS-wt mCRC.

Methods: KRAS-wt pts on a cetuximab-based first-line treatment with written informed consent could be enrolled in this prospective, non-interventional study. Primary endpoint was ORR, secondary endpoints were amongst others PFS, OS, TTF, and resection rate of liver metastasis. Comorbidities were documented and evaluated by the Charlson Comorbidity Index (CCI).

Results: 817 eligible KRAS-wt mCRC pts were enrolled at 144 sites across Germany, documentations for 456 pts were finalised and evaluated. The median age was 65 [27–87] yrs, with 51.5% = 65 yrs, 34.0% > 65–75 yrs, and 14.5% > 75 yrs. ECOG performance status was 0, 1, 2, or missing in 34.4%, 49.6%, 8.8%, and 7.2% of pts, respectively. CCI was 0 in 54.4%, and = 1 in 45.6%. Resection of liver and/or lung metastases was done in 17.3% of pts, 13.4% were R0 resected. For pts with liver limited disease resection rate and R0-rate were 29.3% and 23.8%, respectively. Pts with CCI 0 had no different outcome regardless of age (Tab.1). Pts with CCI = 1 and > 75 yrs had a lower ORR and decreased TTF, pts > 65–75 yrs had only a decreased TTF as compared to the = 65 yrs age group (Tab. 1).

Conclusions: In this large observational trial outcomes (ORR and PFS) of KRAS-wt mCRC pts on a cetuximab-based first-line treatment were comparable to those reported in pivotal trials. Pts older than 75 yrs without comorbidities (CCI = 0) showed no difference to younger pts in ORR, PFS, and TTF. Pts > 75 yrs with CCI = 1 had a significant lower ORR and decreased TTF.


Disclosure: Stephan Sahm: Stock Ownership: Fresenius Medical Care; Merck KGaA; Financing of Scientific Research: Fresenius Medical Care; Expert Testimony: Merck Serono (Institution); Roche (Institution) Friedrich Overkamp: Advisory Role: Merck Serono; Financing of Scientific Research: Merck Serono

P306 – The colorectal carcinoma – treatment research and treatment reality in oncology practices (Anti-VEGF or Anti-EGFR therapies)

Tessen H.-W.1, Rubanov O.2, Grundeis M.3, Teich M.3, Elsel W.4, Schlichting A.5, Valdix A.6, Projektgruppe Internistische Onkologie (PIO)

1Onkologische Kooperation Harz, Goslar, Germany, 2Onkologische Schwerpunktpraxis, Hameln, Germany, 3Onkologische Schwerpunktpraxis, Chemnitz, Germany, 4Onkologische Schwerpunktpraxis, Glauchau, Germany, 5rgb Onkologisches Management GmbH, Sarstedt, Germany, 6Onkologische Schwerpunktpraxis, Schwerin, Germany

Introduction: For the palliative therapy of the colorectal carcinoma there are potent drugs available such as the cytostatics 5-FU, irinotecan, oxaliplatin and capecitabine. In 01/2005, the monoclonal antibody bevacizumab was approved, cetuximab in 06/2004, panitumumab in 12/2007 (cetuximab and panitumumab for carcinoma with wild-type Ras). As of 02/2013, aflibercept can be administered in combination with FOLFIRI after an oxaliplatin-based treatment. How are these substances applied / combined in everyday life?

Methods: Since 2003, 124 oncology practices in 16 federal states have been documenting 9,301 disease histories of patients with a colorectal carcinoma (CRC) as part of the Project team of Internal Oncology (PIO). 8,687 cases thereof with a total of 19,589 therapies were analysed in the registry ONCOReg. Distant metastases were present in 5,691 patients during the course of the disease.

Results: For 5,606 patients, 14,053 palliative therapies have been documented so far.


1,026 (18.0%) of the patients had a secondary metastases resection, 2.5% of the patients an interventional treatment for metastases.


The median progression-free survival in the complete registry is at 9.9 or 6.4 months for the 1st- and 2nd-line therapy, the median overall survival at 25.0 and 15.3 months. Patients with a secondary metastases resection survived 50.5 months, patients without a secondary metastases resection 24.8 months (p < 0.00001). Patients who were treated with aflibercept and had received a metastases resection survived 54.5 months.Conclusion: The median overall survival of 25.0 months from the start of the 1st-line therapy reflects the targeted treatment of the colorectal carcinoma in the practices. Patients with a secondary metastases resection had a significantly prolonged overall survival (54.5 months with an aflibercept-containing therapy).Current data will be presented.

Disclosure: No conflict of interest disclosed.

P307 – Treatment and outcome of patients with metastatic colorectal cancer (mCRC) in routine care 1995 - 2015

Weide R.1, Feiten S.2, Chakupurakal G.1, Friesenhahn V.2, Kleboth K.2, Köppler H.1, Lutschkin J.2, Thomalla J.1, van Roye C.1, Heymanns J.1

1Praxisklinik für Hämatologie und Onkologie, Koblenz, Germany, 2Institut für Versorgungsforschung in der Onkologie, Koblenz, Germany

Introduction: Evaluation of treatment strategies and outcome of patients with mCRC who received their treatment in an oncology group practice by 5 oncologists between 06/95 - 04/15. Comparison of different treatment periods (1995 - 2004 versus 2005 - 2015).

Methods: All consecutive patients with mCRC who were diagnosed between 06/95 - 04/15 were analysed retrospectively concerning treatment and outcome. Data were collected from patient files into a data base and analysed statistically using SPSS.

Results: 526 patients were analysed. Median age was 66 (28 - 89). 45% were female, 55% were male. KRAS-mutation status was known in 25%; 44% were mutated, 56% showed a KRAS-wildtype. In patients diagnosed between 2007 and 2015 (n = 148) KRAS-mutation status was known in 75%; 48% were mutated, 52% wildtype. First line therapy consisted of 5-FU+folinic acid-regimens in 75%; FOLFIRI was applied in 14%, FOLFOX in 25%, CAPIRI in 2% and CAPOX in 4%. Bevacizumab was used in 17%, an Anti-EGFR-antibody in 1%. Second line therapy consisted of 5-FU+folinic acid-regimens in 64%; FOLFIRI was applied in 21%, FOLFOX in 28%, CAPIRI in 2% and CAPOX in 7%. Bevacizumab was used in 22%, an Anti-EGFR-antibody in 4%. Third line therapy consisted of 5-FU+folinic acid-regimens in 44%; FOLFIRI was applied in 23%, FOLFOX in 15%, CAPIRI in 7% and CAPOX in 6%. Bevacizumab was used in 18%, an Anti-EGFR-antibody was used in 18% as well. 18 patients (3%) received regorafenib as third and further line therapy. 4% of patients received best supportive care only. The median overall survival is 23.5 months (1.4 - 193.4+). In patients >75 years median overall survival is 22.6 months (2.8 - 163.1+). The comparison of different treatment periods (1995 - 2004 versus 2005 - 2015) revealed a substantial difference in survival (22.3 months versus 27.8 months).

Conclusion: Modern cytoreductive therapy leads to an improvement in overall survival in patients with mCRC who receive routine care. Substantial improvement has been achieved during the last ten years compared to 1995 - 2004.

Disclosure: No conflict of interest disclosed.

P308 – Impact of surgical resection of liver metastases on outcome of patients with KRAS-wildtype exon 2 (KRAS-wt) metastatic colorectal carcinoma (mCRC) treated with a cetuximab-based first-line therapy – Analysis of survival times in relation to secondary resection in the German non-interventional study ERBITAG

Neumann U.P.1, Göhler T.2, Hering-Schubert C.3, Janssen J.4, Sahm S.5, Schwittay M.6, Zahn M.-O.7, Stenzel K.G.8, Overkamp F.9

1Universitätsklinik der RWTH Aachen, Klinik für Allgemein-, Viszeral- und Transplantationschirurgie, Aachen, Germany, 2Onkozentrum Dresden/Freiberg, Dresden, Germany, 3St. Georg Klinikum Eisenach, Eisenach, Germany, 4Onkologie Westerstede Aurich Rhauderfehn, Westerstede, Germany, 5Ketteler Krankenhaus, Offenbach, Germany, 6Tumorzentrum Leipziger Land, Groitzsch, Germany, 7Onkologische Kooperation Harz, Goslar, Germany, 8Merck Serono GmbH, Darmstadt, Germany, 9Oncologianova GmbH, Recklinghausen, Germany

Background: Cetuximab in combination with irinotecan- or oxaliplatin-based chemotherapy has shown to increase amongst other efficacy parameter the R0 resection rate of liver metastases (LM) of primary irresectable KRAS-wt mCRC patients (pts). The non-interventional study ERBITAG aimed to evaluate safety and efficacy of cetuximab in combination with various first-line chemotherapy regimens in pts with unresectable KRAS-wt mCRC.

Methods: KRAS-wt pts on a cetuximab-based first-line treatment with written informed consent could be enrolled in this prospective, non-interventional study. Primary endpoint was ORR, secondary endpoints were amongst others PFS, OS, TTF, and resection rate of liver metastasis. Here we update the interim analysis in terms of the outcome of pts according to secondary resection of LM.

Results: 456 KRAS-wt mCRC pts out of 817 recruited KRAS-wt pts were evaluable for this interim analysis. Location of metastasis was liver, lung, lymph nodes, and peritoneum in 70.6%, 23.5%, 22.4%, and 17.8% of pts, respectively. 39.7% of pts had liver-limited disease (LLD). 79.2% had surgery of the primary tumor and 15.6% had a resection of metastases before enrolment. The median [range] age was 65 [27–87] yrs, ECOG performance status was 0, 1, 2, or missing in 34.4%, 49.6%, 8.8%, and 7.2% of pts, respectively. Resection of LM and/or lung metastases was done in 17.3% of pts, 13.4% were R0 resected. For LLD-pts resection rate and R0-rate were 29.3% and 23.8%, respectively. Intraoperative and postoperative complications were observed in 1.1% and 12.4% of pts with resection of metastasis, respectively. In 4.5% of pts a revision surgery was necessary. Median PFS was 20.3 and 9.5 months for pts with and without resection of LM, respectively (p < 0.0001). Median OS was 42.0 and 18.2 months for pts with and without resection of LM, respectively (p < 0.0001). For LLD-pts median PFS was 20.5 and 11.9 months and median OS 42.0 and 18.7 months for pts with and without resection of LM, respectively(p < 0.0001 each).

Conclusions: Pts with resection of LM had a significant longer PFS and OS compared to pts without resection.

Disclosure: Ulf Neumann: No conflict of interest disclosed.Friedrich Overkamp: Advisory Role: Merck Serono; Financing of Scientific Research: Merck Serono

P309 – Mutation profiling and consecutive treatment decisions under knowledge of mutation results in patients with metastatic colorectal cancer (mCRC) in real world evidence (RWE) of oncological practices – first results of the GO-KOLORAS registry study

Lipp R.1, Freigang F.1, Brecht P.1, Schwaner I.2, Steinmetz T.3, Schulte C.4, Tiemann M.5

1GermanOncology GmbH, Hamburg, Germany, 2Onkologische Schwerpunktpraxis Kurfürstendamm, Berlin, Germany, 3Gemeinschaftspraxis für Hämatologie und Onkologie, Köln, Germany, 4Gemeinschaftspraxis für Hämatologie und Onkologie, Dortmund, Germany, 5Institut für Hämatopathologie, Hamburg, Germany

Introduction: The GO-KOLORAS registry study started in October 2015 and investigate the mutation profilings of several markers in tumor samples of colorectal cancer analysed by Next-Generation-Sequencing (NGS) and the subsequent decisions of treatments for 1° and 2°line based on the mutation reports under conditions of RWE in German oncological practices.

Methods: From October 2015 data of the first n = 78 (of planned 1.440 patients) with mCRC were sampled from more than 20 oncological practices in Germany. The NGS examinations of KRAS, NRAS, AKT 1, APC, BRAF, CTNNB1, PIK3CA, PTEN, SMAD4, TP53 and MSS were performed centrally in formalin-fixed paraffin-embedded tumor samples and reported to the oncologists at the point of a decision for a 1° or 2°line. All data of mutation profilings and clinical as well as health economic data were captured by a web-based documentation software. Clinical data of the GO-KOLORAS trial were compared with historical data of 342 patients with mCRC, who started 1° or 2°line therapies in the same oncological practices between 2014 and 2015.

Results: Of the first patients with mCRC in the GO-KOLORAS trial (34% women, 66% men, mean age 68 years) the NGS analyses showed the following portions of mutations: KRAS 40,0%, NRAS 6,7%, AKT 1 2,2%, APC 80,0%, BRAF 8,9%, CTNNB1 2,2%, PIK3CA 20,0%, PTEN 4,4%, SMAD4 20,0%, TP53 80,0%. Additionally we found an instability in MSS in 2,2%. The total rate of RAS wildtype was 53,3% (in subgroups WT for KRAS 60,0%, for NRAS 93,3%). In the WT group (1° and 2°line) the portions of targeted therapies with Cetuximab/Panitumumab could be increased by 9,3% in the 1° and by 7,2% in the 2°line compared to historical data from 2014–2015. Updated data regarding the treatments and analyses of marker profiling groups will be presented at the meeting.

Conclusions: NGS with a broad marker profiling could provide essential informations about RAS status and additional profilings regarding further markers. The GO-KOLORAS registry will investigate the subsequent decisions of the oncologists for 1° and 2°line therapies in mCRC based on the NGS reports with a broad marker profiling under conditions of RWE. First data showed a slight increase of targeted therapies in 1° and 2° line, which may be caused in the contemporary submission of the NGS results, but up to now no significant difference to the historical data from 2014–2015.

Disclosure: Rainer Lipp: Advisory Role: Beratertätigkeiten bei Omnicare; Financing of Scientific Research: Vortragshonorar von Omnicare; Expert Testimony: Forschungsunterstützung von OmnicareMarkus Tiemann: No conflict of interest disclosed.

P310 – Impact of prophylactic treatments of cetuximab-based skin reactions in patients with metastatic colorectal carcinoma (mCRC). Interim analysis of the German non-interventional study ERBITAG

Sahm S.1, Göhler T.2, Hering-Schubert C.3, Janssen J.4, Schwittay M.5, Zahn M.-O.6, Stenzel K.G.7, Steinbach-Büchert A.K.7, Overkamp F.8

1Ketteler Krankenhaus, Offenbach, Germany, 2Onkozentrum Dresden/Freiberg, Dresden, Germany, 3St. Georg Klinikum Eisenach, Eisenach, Germany, 4Onkologie Westerstede Aurich Rhauderfehn, Westerstede, Germany, 5Tumorzentrum Leipziger Land, Groitzsch, Germany, 6Onkologische Kooperation Harz, Goslar, Germany, 7Merck Serono GmbH, Darmstadt, Germany, 8Oncologianova GmbH, Recklinghausen, Germany

Background: Prophylactic treatments are widely used to ameliorate skin reactions induced by EGFR inhibition. Randomised studies have shown a positive impact of prophylactic antibiotics.

Methods: Patients (Pts) with KRAS-wt and later RAS-wt mCRC treated with a first-line chemotherapy regimen plus cetuximab with written informed consent were eligible for this prospective, non-interventional study. Physicians were requested to complete a questionnaire and document any applied prophylactic and reactive skin toxicity treatment for every pt. Different prophylactic treatment regimens were categorized in 5 groups: systemic antibiotics (SA), skin care without antibiotics or corticosteroids (SC), other topical treatments (OT) (e.g. antibiotics or corticosteroids), any prophylaxis (P), and no prophylaxis (NP).

Results: Data from 497 pts at 178 centers were finally collected and evaluable at data cut off. For all reported skin reactions the maximum NCI-CTCAE grade per patient and prophylactic treatment group was evaluated (table). Although none of the prophylactic regimens were significant different from NP, SA showed a numerically lower rate of all skin reactions by 8.1% (p = 0.06) and of rash acneiforme by 5.3% (p = 0.134) versus NP. 96.2% of reactive treatments of skin toxicities were done without consultation of a dermatologist and the medication was topical or systemic in 60.9% and 32.3%, respectively. Response to reactive treatment was complete remission or significant improvement of the skin reactions in 68.6% (topical) and 66.0% (systemic).

Conclusions: Pts given prophylactic SA showed numerically fewer grade 3–4 skin reactions in comparison to NP (7.6% vs. 15.7%), but without significance. An important reason for failing significance may be the low number of pts receiving prophylactic SA (only in 18.5% of pts). Reactive treatment (systemic or topical) of skin toxicities led to an improvement in the majority of pts.


Disclosure: Stephan Sahm: Stock Ownership: Fresenius Medical Care; Merck KGaA; Financing of Scientific Research: Fresenius Medical Care; Expert Testimony: Merck Serono (Institution); Roche (Institution)

Friedrich Overkamp: Advisory Role: Merck Serono; Financing of Scientific Research: Merck Serono

P311 – Simultaneous diagnosis of aggressive B-NHL and colon-cancer: Case report and review of the literature

Chitic A.P.1, Sprey C.1, Meyer R.1, Schulte E.1, Hindahl H.1, Bolder U.1, Luckhaupt H.1

1St Johannes Hospital Dortmund, Department of Internal Medicine II, Department of Surgery and Department of ENT, Dortmund, Germany

Introduction: Treating patients according to guidelines is standard of care. However, the simultaneous diagnosis of two malignant diseases usually is not covered by these guidelines. We report on an older patient, diagnosed with B-NHL and colon cancer.

Case: A 85 years old woman, presented with intermittent abdominal pain and obstipation. She was in very good performance status (ECOG 1) without relevant co-morbidities besides a known hypercholesterinemia.

Upon clinical examination, our patient revealed moderate pain in the right lower abdominal quadrant, without muscular defense. An abdominal ultrasound showed splenomegaly and para-aortic lymphadenopathy. CT-scan confirmed splenomegaly and generalized lymphadenopathy. In addition, an infiltrative process of the coequal region was found. Colonoscopy exhibited a stenosing ulcerative mass in the cecum, with the typical histological features of a colon adenocarcinoma. A supraclavicular lymph node was surgically extracted, and a diffuse large B cell lymphoma was histologically confirmed. Bone marrow involvement was ruled out by flow cytometry and histology.

While having extended evidence supported guidelines and therapy algorithms for both these pathologies, none of them provides recommendations for cases with simultaneous tumors of different histology. In addition, the enlarged abdominal lymph-nodes could have been manifestations of either disease. After discussion in our local tumorboard and intensive discussions with the patient and her family, we decided treating the lymphoma first, and initiated a therapy with elderly R-CHOP-14. This therapy was very well tolerated and a restaging after four cycles chemotherapy documented not only regression of all lymphoma manifestations but also of the cecal mass. After six cycles, the patient was in complete remission with the lymphoma. She subsequently underwent surgical resection of the colon carcinoma in curative intention.

Conclusion: A better documentation in a registry of these patients with two oncological diseases would help us to observe how other colleges in such a situation are handling. In our patient with a stage III A Lymphoma and a curative treatable colon carcinoma (postoperative stage II UICC), was this strategy the only chance to cure the patient. An interesting side effect is the partially response of the colon cancer under lymphoma therapy.

Disclosure: No conflict of interest disclosed.

P312 – Development of a standardised and reliable slice culture system for gastrointestinal cancers

Soennichsen R.1,2, Kallendrusch S.1, Hennig L.1,2, Koerfer J.1,2, Merz F.1, Winter K.1, Haehnel S.1, Kaiser N.1, Richter C.1, Monecke A.3, Wittekind C.3, Hoffmeister A.4, Jansen-Winkeln B.5, Gockel I.5, Aigner A.6, Bechmann I.1, Lordick F.2

1University of Leipzig, Institute of Anatomy, Leipzig, Germany, 2University Hospital Leipzig, University Cancer Center Leipzig, Leipzig, Germany, 3University Hospital Leipzig, Institute of Pathology, Leipzig, Germany, 4University Hospital Leipzig, Department of Gastroenterology and Rheumatology, Section of Interdisciplinary Endoscopy and Sonography, Leipzig, Germany, 5University Hospital Leipzig, Department of Visceral, Transplant, Thoracic and Vascular Surgery, Leipzig, Germany, 6University of Leipzig, Rudolf-Boehm-Institute for Pharmacology and Toxicology, Clinical Pharmacology, Leipzig, Germany

Introduction: Gastric and colon cancers are among the most prevalent malignant tumours and have a high mortality rate. Gastric cancer in particular is characterized by heterogeneity, low overall survival and high rates of non-responders. Despite recent advances in medical and multidisciplinary treatment, developing individual therapeutic strategies remains a challenge. There is an ongoing need for developing assays to predict treatment response of individual patients and tumours.

Methods: Our group succeeded to cultivate tissue slices from primary gastric carcinomas (Koerfer J et al., 2016). To implement this model for clinical applications a fast and objective readout is necessary. We therefore standardised the established slice culture system for gastric and colon cancer and optimised a histology based readout.

A triplicate slice approach was used to address the problem of tumour heterogeneity. Cytotoxic drugs (oxaliplatin, cisplatin, 5-FU, docetaxel) were added to culture medium for 48–72 hours after a resting period. Analysis was done using ImageJ (ImageJ, Schneider et al., 2012). In short, paraffin sections were prepared and stained with different immunofluorescence dyes such as Hoechst 33342, Ki67 and AE 1+3. Images of three slices were pooled and modified with a standardised, automated workflow in ImageJ. The mean value of positive pixels per condition was calculated and results were correlated with manual cell counting and experimental conditions.

Results: Our standardised experimental setup in combination with an automated, pixel-based readout represents a promising option to analyse human tumour derived tissue of gastrointestinal cancers. Values obtained by using the ImageJ workflow correlated with manual cell counting of four observers (n = 48): r = 0.841 and no significant difference was found between normed values of both methods within observers (n = 12): p = 0.885, p = 0.885, p = 0.977and p = 0.707.

Conclusions: We report results of our standardised slice culture readout system to assess response of human gastric and colon cancer tissue to chemotherapy ex vivo. This novel method offers the opportunity to study tumour biology and drug resistance within a model closer to its original environment compared with other models. It is a first step towards an assay that might enable the prediction of individual tumour response prior to therapy.

Disclosure: Rasmus Soennichsen: No conflict of interest disclosed.Florian Lordick: Advisory Role: Amgen, Biontech, Boston Biomedical, Ganymed, Lilly, MSD, Nordic, Roche, Taiho; Financing of Scientific Research: Vortragshonorare: Amgen, Celgene, Roche, Lilly; Expert Testimony: Böhringer-Ingelheim, GSK, Fresenius Biotech; Immaterial Conflict of Interests: Reisekostenunterstützung: Amgen, Bayer, MSD, Roche, Taiho

Fortbildung – Myelodysplastisches Syndrom

V313 – WHO classification 2016 for the myelodysplastic syndromes (MDS): Meaning for the diagnostic work-up

Germing U.1, Strupp C.1, Nachtkamp K.1, Aul C.2, Giagounidis A.3, Gattermann N.1

1Heinrich-Heine University Düsseldorf, Department of Hematology, Oncology and Clinical Immunology, Düsseldorf, Germany, 2St Johannes Hospital Duisburg, Department of Hematology, Oncology and Clinical Immunology, Duisburg, Germany, 3Marienhospital Düsseldorf, Department of Hematology and Oncology, Düsseldorf, Germany

A refinement of the classification of the MDS has been proposed by the WHO working group in 2016: 1) definition of the ring sideroblastic MDS types including SF3B1 mutation as a classifier for patients with less than 15% ring sideroblasts (RS) and the reintroduction of a group of MDS with multilineage dysplasia and RS, 2) inclusion of MDS with del(5q) and one additional non chromosome 7 anomaly in to the MDS del(5q) group, 3) replacement of the term RCUD by MDS with single lineage dysplasia (MDSSLD), RARS by MDSSLDRS (with Ringsideroblasts), RCMD by MDS multilineage dysplasia (MDSMLD), and MDSMLDRS, RAEB by MDSEB, and 4) definition to assess the medullary blast count, namely the blasts should be based on all nucleated cells neglecting the amount of erythroid cells. Based on centrally diagnosed 3190 patients in the Düsseldorf registry, we validated the new proposals. 256 patients were diagnosed as MDSSLD (8%), 978 MDSMLD (30,6%), 227 MDSSLD RS (7,1%); 321 MDSMLD RS (10,1%), 159 MDS del(5q) (5%), 481 MDSEB 1 (15,1%), 620 MDSEB 2 (19,5%), and 148 MDS-U (4,6%). 344 patients (16,5% of the non RAEB types) changed the category, mainly moving from RCMD to MDSMLD-RS, RCUD and RCMD to MDS del(5q). Median survival times of the refined groups differed from more than 63 months in the MDSSLD (RS) groups, 36 months in the MDSMLD (RS) groups, 76 months of the MDS del(5q) group and 21 and 11 months in the MDSEB 1 and 2 groups, respectively. The difference between the groups with regard to the risk of AML evolution also differed significantly. No major changes were made with regard to the MDS-U categories, including patients with MDSSLD and pancytopenia, MDSSLD and MDSMLD with 1% peripheral blasts and MDS without clear dysplasia but specific chromosomal aberration. In summary, the proposals of the WHO group for the classification of MDS are thoughtful, taking into account biologic parameters of the diseases, a more precise wording, to some extend pragmatic and feasible.

Disclosure: No conflict of interest disclosed.

V314 – MDS: Standards of therapy in 2016

Platzbecker U.1, Deutsche MDS und EMSCO

1Universitätsklinikum Dresden, Medizinische Klinik und Poliklinik I, Dresden, Germany

The heterogeneity of MDS manifests in the individual patient as a disease ranging from an indolent condition with a considerable life expectancy to forms approaching the aggressiveness of acute myeloid leukemia (AML). A risk-adapted treatment strategy is therefore mandatory for a disease showing such a highly variable clinical course. During the past decades treatment has been stratified according to the International Prognostic Scoring System (IPSS) risk score; i.e. into “lower-risk” MDS (low/int-1, LR-MDS) where correction of cytopenia was the main objective and “higher-risk” MDS (int-2/high, HR-MDS) where the reduction or delay of progression or AML evolution and prolonged survival was the objective.

Erythropoiesis-stimulating agents (ESAs) constitute the first line treatment except for transfusion dependent patients with a 5q deletion, where lenalidomide can induce higher response rates. Furthermore, results of second line treatments of anemia are modest, and most of the patients will eventually require repeated RBC transfusions, subsequently leading to iron overload. In this situation, the role of iron chelation therapy requires further studies, but it is usually recommended in highly transfused patients with an expected prolonged survival or when scheduled for allogeneic HSCT. In case of thrombocytopenia, TPO-R agonists might be effective, while HMAs may be useful in selected cases. Intensive treatment approaches are hampered by the advanced age of most MDS patients, which potentially results in an unacceptable adverse event rate. This is especially true for allogeneic hematopoietic stem cell transplantation, which, despite curative potential, is selected as an option by only a minority of patients. Recent developments with hypomethylating agents have broadened the therapeutic armamentarium and these newer treatments, although not potentially curative, can lead to durable responses in many MDS patients. Most importantly, new treatment approaches are currently explored inside clinical trials including inhibitors of the TGF-beta pathway and combinations of IMIDs, ESAs or other epigenetic drugs with HMAs. In the interest of our patients we hope that these efforts will extend our therapeutic armamentarium in the near future.

Disclosure: Uwe Platzbecker: Advisory Role: Celgene, Amgen, Janssen, Novartis; Financing of Scientific Research: Celgene, Amgen, Janssen, Novartis; Expert Testimony: Celgene, Amgen, Janssen, Novartis

V316 – When hypomethylating agents fail

Pfeilstöcker M.1

1Hanusch Hospital, 3rd Dept Medicine, Vienna, Austria

Introduction: Hypomethylating Agents (HMA) are treatment standard for high risk MDS patients not eligible for allogeneic transplantation. Clinical benefit and improved overall survival (OS) was shown in trials and registries. However up to 40% of patients do not respond and most responders progress beyond 1–2 years. Outcome after HMA failure is poor.

Methods: Possible strategies addressing HMA failure are reviewed.

Results: Restarting HMA in former responders has dismal outcomes, therefore HMA should be maintained in responders. Data on subsequent treatment with Decitabine after Azacytidine (Aza) failure are limited, showing only short OS. New HMAs currently being tested are Guadecitabine and Sapacitabine. Standard chemotherapy after HMA failure is not recommended, first line data in MDS and second line use in AML show inferior results. Newer chemotherapy agents such as Vosaroxazin might provide benefit in elderly patients, a trial in high risk MDS is ongoing. Genetically heterogeneous MDS provides a plethora of potential targets for treatment approaches: among others early data with immune checkpoint inhibitors and modulators of apoptosis merit further study. Randomized phase 3 data are scarce, only the ras mimetic Rigosertib was investigated after HMA failure randomized against best supportive care. The primary endpoint OS was not met, but benefit in primary HMA failure and very high risk patients was seen, a respective confirmatory trial is ongoing. Studies of combination treatment with the aim of improving monotherapy results have not yet been successful in phase 3. A trial testing Aza in combination with Lenalidomide or Vorinostat was negative, due to increased toxicity and failure to deliver full planned doses. For further progress in this field identification of druggable targets common to all or many MDS patients would be beneficial: the splicing apparatus could represent a new target option. Predictive markers for specific agents need to be identified, also using molecular data from NGS involving computer models. Probably also the design of clinical trials needs to be reconsidered.

Conclusions: There is still need for improvement in HR MDS therapy where no standard approach for the relapsed setting is available, therefore patients should be included in clinical trials whenever possible. Those that become eligible for transplant with first line treatment should be reevaluated in this respect, as this still represents the only curative treatment option.

Disclosure: Michael Pfeilstöcker: Financing of Scientific Research: Celgene, Janssen, Novartis

Fortbildung – Chronische lymphatische Leukämie

V319 – Treatment failure: toxicities, clonal evolution and Richter´s transformation

Steurer M.1

1Medizinische Universität, Innsbruck, Austria

Treatment failure in oncology has often been reduced to drug resistance of the malignant clone. However, other aspects such as toxicity may also limit the antineoplastic activity of a specific treatment modality. In the field of CLL potent immuno-chemotherapeutic regimens and the recent advent of novel agents have improved our therapeutic armamentarium but have also introduced distinct toxicities that have to be dealt with. Moreover, they may also impact on the biology of the disease in terms of clonal evolution and/or Richter´s transformation.

In this educational overview three defined aspects of treatment failure, i.e. toxicities, clonal evolution and Richter´s transformation, will be discussed – in particular with focus on novel agents.

Disclosure: Michael Steurer: Advisory Role: Roche, Janssen-Cilag, Gilead; Financing of Scientific Research: Roche, Janssen-Cilag, Gilead; Expert Testimony: Roche, Janssen-Cilag, Gilead

Fortbildung – Sarkome 2016 – was gibt es Neues?

V321 – Metastatic soft tissue sarcomas in 2016 - update and current clinical trials

Kasper B.1

1Universität Heidelberg, Universitätsmedizin Mannheim, Interdisziplinäres Tumorzentrum Mannheim (ITM), Sarkom Zentrum, Mannheim, Germany

Soft tissue sarcomas (STS) are a heterogeneous group of tumours arising mainly from the embryonic mesoderm comprising more than 50 different histological entities exhibiting great differences in terms of clinical behavior, pathogenesis and genetics. In up to 50% of patients distant metastases will occur and the median overall survival for patients with advanced disease is approximately 12 months. As effective targeted treatments are scarce, doxorubicin and ifosfamide – being used for more than 30 years now – still remain the backbone of systemic chemotherapy. In most cases, patients with advanced STS have a poor prognosis and the primary goal of treatment is disease control and palliation. Recently, trabectedin, pazopanib and eribulin have been introduced beyond first line therapy and have enriched the therapeutic armamentarium significantly. New data and clinical trials of interest will be presented here.

Regarding trabectedin – approved in Europe in 2007 – a large phase III study in the USA comparing trabectedin versus DTIC stressed its sustained activity in STS patients. Even though the primary endpoint, overall survival, was not reached, the results led to global FDA registration in October 2015 for the treatment of patients with leiomyosarcomas and liposarcomas. The anti-angiogenic compound pazopanib has been tested in a large EORTC phase III trial (PALETTE) demonstrating a significant advantage regarding PFS prolongation of about three months in favour of pazopanib versus placebo for certain STS subtypes excluding liposarcomas. This study led to its approval in USA, Europe and Japan in 2012. The phase III trial of eribulin met its primary endpoint of an overall survival benefit of two months (13.5 versus 11.5 months) in favour of eribulin compared to DTIC in pretreated patients with advanced leiomyosarcomas or adipocytic sarcomas. The final approval of eribulin in early 2016, however, was restricted to pretreated liposarcoma patients. Olaratumab, a fully human anti-PDGFRa monoclonal antibody, has been tested in a phase II trial in combination with doxorubicin; olaratumab is the first agent added to doxorubicin to improve overall survival (25.0 versus 14.7 months) in advanced/metastatic STS patients in a randomized setting. A worldwide, placebo-controlled phase III study (ANNOUNCE) finalized recruitment. Another interesting candidate being tested in a phase II/III study in liposarcoma patients is selinexor, a nuclear exportin protein 1 inhibitor.

Disclosure: No conflict of interest disclosed.

V322 – Bone Sarcoma- therapeutic concepts

Dirksen U.T.1

1University Hospital Muenster, Paed Haem Onc, Muenster, Germany

Patients with newly diagnosed localized bone sarcoma are treated with a combination of multiagent cytotoxic chemotherapy and local control measures (surgery, radiation, or surgery with radiation) directed against the primary tumor. Cooperative group clinical trials such as EURAMOS-1, Euro E.W.I.N.G.- 99 and AEWS0031 provided evidence for the use of multidrug treatment. Results of the trials are presented and discussed.

Patients with more advanced disease (either with metastasis at presentation or with recurrence) require newer approaches and clinical trials are ongoing of promising agents. Several studies focus on the introduction of a personalized medicine approach. Aim of these studies is to identify targets on the individual tumor in order to provide a personalized medicine recommendation. Pilot studies are ongoing and results are pending. Furthermore, phase II trials on PARP inhibitors, tyrosine kinase inhibitors, monoclonal antibodies, check- point inhibitors and other immunomodulators are tested. Results on completed studies will be presented and discussed.

Disclosure: No conflict of interest disclosed.

V323 – Proton therapy – chances and limits?

Geismar D.1,2, Timmermann B.1,2

1West German Proton Therapy Center, Essen, Germany, 2Clinic for Particle Therapy, University Hospital, Essen, Germany

Protons are particles with a positive charge, inertia and energy depended range in tissue. In comparison to photons, protons have totally different physical properties. Energy output of protons after entry into the tissue is low at first and only rises abruptly after almost complete deceleration in the Bragg-Peak. Dose to normal tissue proximal and especially distal the treatment volume can be reduced.

Therefore, proton beam therapy is of increasing interest in multimodality cancer care and gives the opportunity for improved local control by dose escalation or the reduction of treatment related toxicity. This is relevant especially for patients with tumors which are located in close proximity of critical structures or in particular sensitive tissues and indicate high treatment doses, like many sarcomas.

For chordoma and chondrosarcoma of the skull base it was possible to show that by dose-escalated radiotherapy with protons local control could be improved with acceptable toxicity. Due to increasing treatment capacities proton therapy is becoming more widely available for this patient group. Treatment concepts for soft tissue sarcomas and bone sarcoma of the skull base, the spine, and the retroperitoneal region are under evaluation and are increasingly available for patient treatment.

The West German Proton Therapy Center Essen (WPE) started treatments for patients in Mai 2013. Meanwhile more than 160 sarcoma patients were treated. 61% of the patients were children (age < 18 years). Current early prospective data suggest a good feasibility and tolerable side effects, while applying high treatment doses in difficult regions and sensitive normal tissue.

Disclosure: No conflict of interest disclosed.

Fortbildung – Melanom

V327 – Circulating tumor DNA as biomarker for disease activity and response to treatment in BRAF V600E mutant malignant melanoma

von Bubnoff D.1, Follo M.2, Graf E.3, Pfeifer D.2, Duyster J.2, Meiß F.1, von Bubnoff N.2

1Universitätsklinikum Freiburg, Klinik für Dermatologie und Venerologie, Freiburg, Germany, 2Universitätsklinikum Freiburg, Klinik für Innere Medizin I, Hämatologie, Onkologie und Stammzelltransplantation, Freiburg, Germany, 3Institut für Medizinische Biometrie und Statistik, Freiburg, Germany

Introduction: Presence of BRAF V600E mutation in malignant melanoma tissue predicts for response to BRAF inhibitor treatment. However, this tumor specific mutation currently can not be used to monitor response to treatment or to predict relapse. Available biomarkers such as LDH or S100B exhibit limited sensitivity and specificity to predict response or progression in melanoma. We hypothesized that free plasma circulating tumor (ct)DNA containing BRAF V600E predicts outcome in BRAF V600E mutant melanoma.

Methods: We developed a hydrolysis probe based, Locked Nucleic Acid assay to detect BRAF V600E and wild type ctDNA by droplet digital PCR on a BioRad QX100 system. Reaction sensitivity was 0.01% with high specificity. We analysed 107 plasma samples from 38 patients (pts) with stage III or stage IV BRAF V600E positive melanoma. The cohort included 30 unselected pts with 83 retrospective samples, 6 pts with baseline samples from a prospective trial (DRKS00009507), and 2 pts with 16 retrospective and 2 baseline samples from the prospective study. Results were correlated with LDH, S100 and imaging data.

Results: Of 34 stage IV pts, 26 tested positive for BRAF V600E ctDNA at least once. Of 8 negative pts, 3 were in CR, 3 had stable disease, and 2 were negative baseline samples of the prospective trial. All 4 stage III pts were in CR, and 2 tested positive for BRAF V600E ctDNA at least once. Positive pts had a mean of 9 BRAF V600E copies/ml plasma (stage III; range: 1–17) and 483 copies/ml plasma (stage IV; range: 0,1–16.388). BRAF V600E ctDNA copies were strongly correlated with both LDH and S100 levels. Dynamic changes of BRAF V600E ctDNA correlated with disease course and response to treatment. A negative to positive conversion or increase was associated with progression (11 pts) or stable disease (1 pt) but not with response, whereas a positive to negative conversion or decrease of mutant BRAF copies correlated with response (10 pts) or mixed response (1 pt), but not with stable disease or progression.

Conclusion: Dynamic changes in BRAF V600E ctDNA indicated response or progression in BRAF V600E mutated melanoma and correlated with LDH and S100. Based on these results, we have set up a prospective trial to determine the detection rate of BRAF V600E ctDNA in stage III and IV melanoma, and to analyze the predictive value of dynamic changes of BRAF ctDNA versus LDH and S100 with respect to clinical endpoints.

Disclosure: Dagmar von Bubnoff: No conflict of interest disclosed.Nikolas von Bubnnoff: Financing of Scientific Research: Fa. Novartis, Fa. BMS; Expert Testimony: Fa. Novartis

Fortbildung – Blut ist ein besonderer Saft – Transfusionsmedizin

V329 – Choose wisely – Individualized and economic indications for platelet transfusions

Schäfer-Eckart K.1

1Klinikum Nürnberg Nord, Medizinische Klinik 5, Nürnberg, Germany

About 500 000 platelet units are transfused in Germany every year. But platelet products are a limited resource and their use has to be judicious, especially as it has been assumed, that about 30–40% are not indicated according to national guidelines.

To achieve not just a more economic but also an individual transfusion approach for patients with hypoproliferative thrombocytopenia three questions have to be answered.

1. What kind of platelet concentrate is appropriate for the individual patient?

60% of all platelet units used are single donor apheresis platelets, 40% are pooled platelet concentrates. While one platelet apheresis yields in 2 platelet concentrates, a pooled platelet concentrate can be produced with 4 whole blood donations. It has to be defined which patients really need an apheresis concentrate and for which the more economic and “donor sparing” pooled concentrate is appropriate.

2. What is the optimal platelet dose? A randomized study has shown, that the median platelet dose could be reduced to a dose below the national specification (1,1 x 1011 vs 3 x 1011) without a higher risk of severe blee-ding.

3. Is a prophylactic transfusion strategy necessary for every patient with hypoproliferative thrombocytopenia? About 75% of all platelet products are used for hematological and oncological patients. For patients with hypoproliferative thrombocytopenia a prophylactic platelet transfusion strategy with a morning platelet trigger below 10/nl has been defined as standard practice by national and international guidelines. In recent years this practice has been challenged and a therapeutic platelet transfusion strategy has been tested in clinically stable patients. Two randomized studies compared these strategies. The prophylactic transfusion strategy has been shown to be more save in patients with acute leukemia. On the other side after autologous blood stem cell transplantation a therapeutic transfusion strategy could reduce the number of transfused units by about 30% and is not resulting in more severe bleeding.

These questions will be discussed in detail.

Disclosure: No conflict of interest disclosed.

Wissenschaftliches Symposium – Kontroversen in der Therapie des kolorektalen Karzinoms

V335 – Big data in the management of CRC. What is the benefit of extensive genomic analysis of the tumor?

Wicki A.1,2

1Universitätsspital Basel, Onkologie, Basel, Switzerland, 2Universität Basel, Dept. Biomedizin und Dept. Klinische Forschung, Basel, Switzerland

The term big data describes the gathering of structured and unstructured information, and the subsequent mining of large data silos. In cancer medicine, the largest data hub that has been tackled in the last few years is the tumor genome. Based on the genomic profile of more than 4000 patients with colorectal cancer, a new consensus classification of CRC has been proposed. In addition, the number of public institutions and private vendors that offer sequencing services is steeply increasing. In this presentation, I want to explore perspectives that are offered by extensive sequencing of colorectal cancer. What sampling is adequate? How do we deal with tumor heterogeneity? Are there patients who generally benefit from such an approach and to whom should we offer sequencing? Can we use sequencing to guide targeted and immune therapy? And finally, how can we best make use of sequence data in our daily practice?

Disclosure: Andreas Wicki: Expert Testimony: Piqur Therapeutics, Basel, Switzerland; Actelion Ltd, Allschwil, Switzerland

Fortbildung – Nierenzellkarzinom

V336 – Pathology and pathophysiology of renal cell carcinomas

Haitel A.1

1Institut für Klinische Pathologie der Medizinischen Universität Wien, AKH, Wien, Austria

The recently published WHO classification of Tumors of the Urinary System1 includes many different types of Renal Cell Carcinoma:

  1. Clear cell renal cell carcinoma

  2. Multilocular cystic renal neoplasm of low malignant potential

  3. Papillary renal cell carcinoma

  4. Hereditary leiomyomatosis and renal cell carcinoma-associated renal cell carcinoma (HLRCC)

  5. Chromophobe renal carcinoma

  6. Collecting duct carcinoma

  7. Renal medullary carcinoma

  8. MiT familily translocation renal cell carcinomas

  9. Succinate dehydrogenase-deficient renal cell carcinoma

  10. Mucinous tubular and spindle cell carcinoma

  11. Tubulocystic renal cell carcinoma

  12. Acquired cystic disease-associated renal cell carcinoma

  13. Clear cell papillary renal cell carcinoma

  14. Renal cell carcinoma, unclassified

All these tumors show different morphology but also different immunohistochemical staining results. In addition they differ in prognosis and genetic profile.

Clear cell renal cell carcinoma (ccRCC) shows VHL mutation in sporadic as well as hereditary ccRCC. This results in the loss of von Hippel-Lindau protein function which leads to progression and metastases. In addition they frequently show loss of heterocygosity (LOH)3p, which harbors VHLgene and at least four further tumor suppressor genes.

The genetic profile of papillary renal cell carcinomas (pRCC) differs completely, as they show gains of Chromosome 7, 17 and loss of Y. In addition ca. 13% of sporadic pRCC and all pRCC of patients with hereditary pRCC show MET alteration. Sometimes pRCC show ALK (anaplastic lymphoma kinase) rearrangements.

A distinct and very aggressive type of pRCC is that of HLRCC, associated with a germ line mutation of the fumarate hydratase gene located on Chormosome1.

Chromosomes 1 and 17 are involved in translocation RCCs of the MiT family, too. TFE3 is located on Chromosom X and the two most common RCCs of this group are t(X;1)(p11.2;q21) which fuses PRCC and TFE3 genes and the t(X;17)(p11.2;q25) which fuses ASPL and TFE3, the same fusion found in alveolar soft part sarcomas. The latter are more likely to present with metastases.The translocation TFEB t(6;11) seems to be more indolent, only few cases are presented in the literature with metastases.

Collecting Duct Carcinoma, also called Bellini duct carcinoma, is a very aggressive tumor. It shows LOHs on multiple chromosomes, but also trisomies and amplifications of Her2/neu have been reported.


1 WHO Class. of Tum. of the Urin. Syst. and Male Genital Org., Moch et al., Lyon, 2016.

Disclosure: No conflict of interest disclosed.

V337 – S3 guideline for renal cell carcinoma – current treatment algorithm

Grünwald V.1

1MHH, Hannover, Germany

Introduction: The past decade of medical treatment in renal cell carcinoma (RCC) has been dominated by targeted therapies, which is a milestone in RCC treatment. An overall survival of 30 months remains the benchmark within clinical trials with the sequential use of the currently available therapies. The development of novel agents during the years have led to a more diverse environment and rendered the need for an update of the current S3 guideline in RCC.

Methods: A systematic review for medical treatment of RCC was performed (from January 2013 on) and the evidence was graded according to SIGN criteria. Recommendation were given after consensus was achieved. Specifics on methodology can be found at: http://leitlinienprogramm-onkologie.de/Leitlinien.7.0.html.

Results: The addition of cabozantinib and nivolumab to the armentarium has added value to the armentarium to treat RCC. Recommendations for their use within the context of the current landscape are given.

Conclusions: After a fascinating decade of drug development in RCC, novel agents can be still added to the treatment algorithm.

Disclosure: Viktor Grünwald: Employment or Leadership Position: keins; Advisory Role: BMS, Novartis, Pfizer; Financing of Scientific Research: BMS, Novartis, Pfizer

Freier Vortrag – Immuntherapie solider Tumoren

V339 – Immune-related gene expression signatures as predictive biomarkers for outcome after concurrent chemoradiation in patients with locally advanced oropharyngeal carcinomas

Heß A.-K.1, Joehrens K.2, Keilholz U.3, Rieke D.3, Weichert W.4, Balermpas P.5, Roedel C.5, Mairinger F.D.6, Hummel M.2, Augstein P.3,7, Budach V.1,7, Tinhofer I.1,7

1Charité – Universitätsmedizin Berlin, Radioonkologie und Strahlentherapie, Berlin, Germany, 2Charité – Universitätsmedizin Berlin, Pathologie, Berlin, Germany, 3Charité Comprehensive Cancer Center, Berlin, Germany, 4Technische Universität München, Pathologie, München, Germany, 5DKFZ Heidelberg, Frankfurt, Germany, 6Universitätsklinikum Essen, Pathologie, Germany, 7DKFZ Heidelberg, Berlin, Germany

Introduction: The extent of immune-cell infiltration has previously been linked to the efficacy of concurrent chemoradiation (CRTX) in locally advanced squamous cell carcinoma of the head and neck. We established immune-related gene expression signatures in oropharyngeal carcinoma (OPC) patients from the ARO04–01 phase III trial as candidate predictive biomarker for cisplatin (CDDP)- and mitomycin C (MMC)-based CRTX. Potential overlap between the immune signatures of CRTX efficacy and the previously reported ‘inflamed’ signature of pembrolizumab activity was determined.

Methods: Immune profiles were established from archival tumor specimens of OPC patients treated in the ARO04–01 study with concurrent MMC-CTRX (N = 24) or CDDP-CTRX (N = 23) were included. The CDDP-CTRX immune profile was validated in further 36 tumor specimens from the ARO04–01study (N = 7) and routine-care patients (N = 29). FFPE-extracted RNA was analyzed using the PanCancer Immune Profiling Panel on the NanoString nCounter® system. Gene expression signatures predictive for 3yr-OS were established by Spearman Rank correlation and hierarchical cluster analysis. Survival estimates according to the immune signatures were determined by Kaplan-Meier-analysis. The potential interference with the HPV status was evaluated in multivariate Cox regression models.

Results: Signature 1 showed a strong positive association with OS (P < 0.001) and PFS (P = 0.006) after CDDP-CRTX but not MMC-CRTX. Its positive predictive value for 3yr-OS was 87.5%, and the negative predictive value was 100%; AUC = 0.97 [95% CI, 0.89–1.0]. Signature 1 was enriched for genes of the adaptive immune system, mainly affecting T-cell receptor and PD-1 signaling. A partial overlap with genes from the ‘inflamed’ pembrolizumab signature was observed. In contrast, signature 2 which showed a significant association with outcome for both CRTX regimens was enriched for genes of the innate immune system, regulating Toll-like receptor, TNF receptor and NFkappaB signaling. Despite a higher prevalence of signature 1 in HPV+ tumors, its predictive value for OS and PFS after CDDP-CRTX was independent of the HPV status.

Conclusions: We demonstrate for the first time an impact of adaptive immunity and PD-1 signaling on the efficacy of CDDP-CRTX which may be important for inclusion of PD-1/PD-L1 inhibitors into curative treatment settings. The NanoString technology for immune gene expression analysis should provide a useful tool for patient stratification.

Disclosure: No conflict of interest disclosed.

V340 – PD-L1: a novel prognostic biomarker in head and neck squamous cell carcinoma

Müller T.1, Brägelmann J.2, Dietrich D.1, Perner S.1, Kristiansen G.1, Bootz F.3, Brossart P.2

1Universitätsklinikum Bonn, Institut für Pathologie, Bonn, Germany, 2Universitätsklinikum Bonn, Medizinische Klinik III, Bonn, Germany, 3Universitätsklinikum Bonn, Klinik und Poliklinik für Hals-Nasen-Ohrenheilkunde, Bonn, Germany

Background and Aims: PD-1/PD-L1 pathway seems to play a pivotal role in T cell downregulation during immune response to external antigens and self-antigens to prevent tissue damage and limit autoimmunity. Today there is no data available about PD-L1 expression in HNSCC, which induced us to analyze PD-L1 expression in HNSCC.

Methods: Two independent cohorts of HNSCC (n1 = 98, n2 = 195) in a TMA format were analysed by immunohistochemistry (PD-L1, clone EPR1161 2 antibody) and evaluated by two observers. PD-L1 expression was scored semiquantitatively as negative vs. low vs. high. Statistical analysis was conducted with SPSS.

Results: High expression levels of PD-L1 were detected in 15.3% of tumor cases in the training cohort and 27.7% of tumor specimens in the test cohort. Kaplan-Meier analysis demonstrated a significant prognostic value of PD-L1 in both cohorts (training cohort: p < 0.004; test cohort: p < 0.001), which was confirmed in a multivariate analyses encompassing recognized prognostic factors like tumor stage, nodal status, distant metastases, lymphatic invasion, vascular invasion , grading ,extracapsular expansion and surgical margin status (p = 0.02; HR = 2.926 [95%CI = 1.183 - 7.235]). Moreover strong PD-L1 expression was associated with the presence of distant metastases in the test cohort (p = 0.025).

Conclusion: In summary we identified PD-L1 as expressed in HNSCC, which may provide a rational basis for anti PD-1/PD-L1 therapy in HNSCC patients. Additionally, it may serve as a multivariate prognostic biomarker in HNSCC, which deserves further study.

Disclosure: Tim Müller: No conflict of interest disclosed.Peter Brossart: Advisory Role: BMS, MSD, Roche; Stock Ownership: Immatics; Financing of Scientific Research: BMS, MSD, Roche

V341 – Impact of nivolumab versus docetaxel on disease-related symptoms in patients with advanced non-squamous NSCLC from CheckMate 057

Horn M.1, Gralla R.J.2, Spigel D.R.3, Bennett B.4, Taylor F.4, Penrod J.R.5, DeRosa M.4, Dastani H.5, Strycker Orsini L.5, Reck M.1

1Lungen Clinic Grosshansdorf, Airway Research Center North (ARCN), Grosshansdorf, Germany, 2Albert Einstein College of Medicine, Jacobi Medical Center, Bronx, United States, 3Sarah Cannon Research Institute, Medical Oncology, Nashville, United States, 4Adelphi Values, Boston, United States, 5Bristol-Myers Squibb, Princeton, United States

Introduction: Nivolumab, a fully human IgG4 programmed death-1 immune checkpoint inhibitor, is approved in the US and EU for patients with previously treated advanced/metastatic NSCLC, based on results of 2 phase 3 trials: CheckMate 017 (NCT01642004) in squamous (SQ) NSCLC and CheckMate 057 (NCT01673867) in non-SQ (NSQ) NSCLC. We assessed the impact of nivolumab versus docetaxel on disease-related symptoms using the Lung Cancer Symptom Scale (LCSS) in patients with advanced non-SQ NSCLC from CheckMate 057.

Methods: LCSS was evaluated every other cycle (Q4W) for nivolumab and every cycle (Q3W) for docetaxel for the first 6 mo on treatment, every 6 wk thereafter, and at 2 post-treatment follow-up visits. The LCSS has two components: the Average Symptom Burden Index (ASBI; based on 6 symptoms: anorexia, fatigue, cough, dyspnea, hemoptysis, and pain) and the 3-Item Global Index (3-IGI; symptom distress, interference with activities, and health-related quality of life [HRQoL]). Changes from baseline in LCSS scores and time to first deterioration (TTD) in symptoms were analyzed.

Results: By wk 12, 17.8% (52/292) of nivolumab patients showed clinically meaningful symptom improvement (based on minimally important difference [MID]) compared with 19.7% (57/290) of docetaxel patients. Numerical differences in mean changes from baseline in the ASBI and 3-IGI favoring nivolumab appeared at the first common assessment (wk 12) and continued throughout the assessment period. At common assessments with >10 patients in each arm (wk 12–48), statistically significant differences favoring nivolumab were noted for the ASBI at wk 12, 24, 30, and 42, and for the 3-IGI at wk 24 and 30. At =1 common assessments, there were statistically significant differences favoring nivolumab for 5 of 6 symptoms (anorexia, fatigue, dyspnea, hemoptysis, and pain) and 2 of 3 items of the 3-IGI (HRQoL and symptom distress). Nivolumab versus docetaxel had a significantly longer TTD (based on MID) for the ASBI (HR = 0.65; 95% CI, 0.49–0.85) and 3-IGI (HR = 0.63; 95% CI, 0.48–0.82), with Kaplan-Meier curves separating between arms at ˜2 months. A similar pattern occurred for TTD for individual ASBI symptoms and 3-IGI items.

Conclusions: In CheckMate 057, patients with previously treated advanced non-SQ NSCLC had significantly lower symptom burden and better HRQoL while treated with nivolumab compared with docetaxel. Nivolumab- versus docetaxel-treated patients also demonstrated significantly longer TTD.

Disclosure: Marlitt Horn: No conflict of interest disclosed.Martin Reck: Advisory Role: Consulting/advisory role: Roche, Lilly, BMS, MSD, Astra Zeneca, Pfizer, Boehringer-Ingelheim, Celgene; Other Financial Relationships: Speakers Bureau: Roche, Lilly, BMS, MSD, Astra Zeneca, Pfizer, Boehringer-Ingelheim, Celgene

V342 – Local treatment of tumors or metastases in combination with systemic ipilimumab immunotherapy prolongs overall survival in patients with advanced malignant melanoma

Theurich S.1,2, Rothschild S.I.3, Hoffmann M.4, Fabri M.4, Sommer A.4, Garcia-Marquez M.2, Thelen M.2, Schill C.5, Merki R.5, Schmid T.6, Koeberle D.6, Zippelius A.3, Baues C.7, Mauch C.4, Tigges C.8, Kreuter A.8, Borggrefe J.9, Schlaak M.4, von Bergwelt-Baildon M.2

1Uniklinik Köln, Klinik I für Innere Medizin, Köln, Germany, 2Uniklinik Köln, Interventionelle Immunologie Köln, Klinik I für Innere Medizin, Köln, Germany, 3Universitätsspital Basel, Klinik für Innere Medizin, Onkologie, Basel, Switzerland, 4Uniklinik Köln, Klinik für Dermatologie und Venerologie, Köln, Germany, 5Kantonales Spital Aarau, Aarau, Switzerland, 6St. Claraspital Basel, Basel, Switzerland, 7Uniklinik Köln, Klinik für Strahlentherapie, Köln, Germany, 8Helios-Klinik St. Elisabeth, Klinik für Dermatologie und Venerologie, Oberhausen, Germany, 9Uniklinik Köln, Institut für Radiologie, Köln, Germany

Background: Immune checkpoint inhibition with ipilimumab has revolutionized cancer immunotherapy and significantly improved outcomes of patients with advanced malignant melanoma. Local peripheral treatments (LPT) such as radiotherapy or electrochemotherapy have been shown to modulate systemic immune responses. Preliminary data have raised the hypothesis that the combination of systemic immune checkpoint blockade with LPT could lead to improved clinical outcomes.

Patients and methods: Clinical data of consecutively treated melanoma patients at four cancer centers in Germany and Switzerland were analyzed. Patients either received ipilimumab or ipilimumab and LPT if indicated for local tumor control. Additional immune assessments were performed in order to identify tumor-specific immune responses during the combination treatment.

Results: A total of 127 melanoma patients were analyzed who either received ipilimumab (n = 82) or ipilimumab+LPT (n = 45). We found that the addition of LPT to ipilimumab significantly prolonged median overall survival (OS) (93 versus 42 weeks, p = 0.0028). As a potential immunological correlate, we identified significantly increased melanoma-specific T-cell responses following ipilimumab+LPT in one exemplary patient. Interestingly, adverse immune-related events were not significantly increased by the combination treatment. In a multivariable Cox regression analysis we demonstrate that the effect of added LPT on OS remained significant, after adjusting for BRAF status, tumor stage, tumor burden and CNS metastases (adjusted HR = 0.56, 95% CI = 0.31 to 1.01, p = 0.05).

Conclusion: Our data suggest that the addition of LPT to ipilimumab is safe and effective in patients with metastatic melanoma irrespective of adverse disease characteristics. We hypothesize that enhancement of tumor-specific immune responses is most likely the underlying mechanism and that this combinatory approach warrants prospective validation.

Disclosure: No conflict of interest disclosed.

V343 – Correlation of somatic mutational load as determined by panel based hybrid capture sequencing assay with tumor response to immunotherapy

Griesinger F.1, Mariotti E.2, Menon R.2, Müller J.2, Lakis S.2, Walsh N.3, Grohe C.4, Crown J.3, Heuckmann J.M.2, Heukamp L.2

1Universitätsklinik Innere Medizin-Onkologie, Klinik für Hämatologie und Onkologie, Oldenburg, Germany, 2NEO New Oncology, Cologne, Germany, 3University College Dublin, Dublin, Ireland, 4Evangelische Lungenklinik Berlin, Berlin, Germany

Background: The use of immune checkpoint inhibitors in the field of cancer immunotherapy is revolutionizing cancer treatment. However, only a subset of tumors are responding, and powerful predictors for response are missing. Recent publications have emphasized on the importance of mutational load to identify patients who are likely to benefit from ‘mono’ or ‘combinational’ immunotherapy. Therefore, the use of a hybrid capture based next generation sequencing assay, like NEOplus, capable of reliably detected somatic mutational load in the diagnostic setting could support patient stratification for the treatment with immune checkpoint inhibitors.

Method: Here, we describe an in silico analysis on the correlation of exonic territory and predictive power for response to immune checkpoint inhibitors based on published data. Furthermore, a broad range of tumor samples derived from multiple indications with corresponding patient response data were retrospectively analyzed for mutational load using a hybrid-capture based next-generation sequencing assay that covers clinically relevant genomic alterations in a panel of more than 90 genes, including point mutations, small insertions and deletions, selected gene fusions and copy number alterations.

Results: We were able to show correlations between exonic territory covered by next-generation sequencing and predictive power for response to immune checkpoint inhibitors. In addition, panels covering only a selected sub-fraction of the human exome are able to reliably detect somatic mutational load in the analyzed tumor samples.

Conclusion: Correlations between mutational load and patient response to immunotherapy were drawn in order to categorize patients who would most likely benefit from treatment.

Disclosure: Frank Griesinger: No conflict of interest disclosed.Lukas Heukamp: Employment or Leadership Position: NEO New Oncology; Stock Ownership: Stock ownership at NEO New Oncology

V344 – Efficacy, safety and predictive biomarker results from a randomized phase II study comparing atezolizumab vs docetaxel in patients with advanced NSCLC (POPLAR)

Schulz C.1, Spira A.I.2, Park K.3, Mazieres J.4, Vansteenkiste J.5, Ballinger M.6, Waterkamp D.6, Fehrenbacher L.7

1Universitätsklinikum Regensburg, Klinik und Poliklinik für Innere Medizin II, Bereich Pneumologie, Regensburg, Germany, 2Virginia Cancer Specialists Research Institute, Fairfax, United States, 3Samsung Medical Centre, Division of Hematology/Oncology, Seoul, Korea, Republic of, 4Toulouse University Hospital, Toulouse, France, 5University Hospitals KU Leuven, Leuven, Belgium, 6Genentech, Inc., South San Francisco, United States, 7Kaiser Permanente Medical Center, Vallejo, United States

Introduction: In a Phase Ia clinical trial, atezolizumab (anti-PDL1) showed single-agent activity in NSCLC patients (pts) with the objective response rate (ORR) associated with PD-L1 expression on tumor-infiltrating immune cells (IC) and/or tumor cells (TC). Here we report results from the first randomized study comparing atezolizumab vs docetaxel (doc) in unselected NSCLC pts with subgroup analyses by PD-L1-expression.

Methods: 287 previously-treated NSCLC pts were stratified by PD-L1 IC status, histology (squamous vs nonsquamous) and number of prior lines of therapy (1 or 2) and randomized (1:1) to 1200 mg IV q3w atezolizumab (n = 144) or 75 mg/m2 IV q3w doc (n = 143). PD-L1 expression was centrally evaluated using an immunohistochemistry (IHC) assay based on the SP142 antibody; pts were scored as IC 0, 1, 2 or 3 and TC 0, 1, 2, or 3. The objectives of this study were to evaluate the efficacy and safety of atezolizumab as compared with doc. The primary efficacy endpoint was overall survival (OS) and secondary endpoints were progression-free survival (PFS) and ORR. These endpoints will be assessed in both PD-L1-selected and the ITT patient populations. OS and PFS will be compared between treatment arms using the log-rank test; Kaplan-Meier methodology will be used to estimate median OS and PFS for each treatment arm and construct survival curves. Cox regression proportional hazard models will be used to estimate hazard ratios. An estimate of ORR (overall response of partial or complete response per RECIST v1.1) and its 95% CI will be calculated using the Clopper-Pearson method for each treatment arm. The incidence and severity of adverse events will be recorded. Enrollment was completed in March 2014. An analysis of the primary endpoint was performed in March 2015, with a minimum duration of follow-up of ˜ 11.5 mo. Results from the OS, PFS and ORR analyses for both the overall population and subgroups by IC and TC status will be presented. Demographic and safety data will be summarized.

Disclosure: Christian Schulz: Advisory Role: Beratertätigkeiten für: AstraZeneca, Boehringer, BMS, Lilly, Novartis, Roche.; Financing of Scientific Research: Vortragshonorare von den Firmen: AstraZeneca, Boehringer, Celgene, MSD, Pfizer, Roche.; Other Financial Relationships: Reisekostenunterstützung von den Firmen AstraZeneca, Boehringer, BMSLouis Fehrenbacher: No conflict of interest disclosed.

Freier Vortrag – Epigenetik

V345 – EZH2 mutations in myelodysplastic/myeloproliferative neoplasms

Rinke J.1, Müller J.2, Bläß M.F.3, Chase A.4, Schäfer V.1, Winkelmann N.1, Haferlach C.5, Cross N.C.4, Hochhaus A.1, Ernst T.1

1Abteilung Hämatologie und Internistische Onkologie, Klinik für Innere Medizin II, Universitätsklinikum Jena, Jena, Germany, 2Friedrich Schiller Universität Jena, Institut für Molekulare Zellbiologie, Jena, Germany, 3Klinik für Anästhesiologie und Intensivmedizin, Universitätsklinikum Jena, Jena, Germany, 4Wessex Regional Genetics Laboratory, Salisbury District Hospital, Salisbury, United Kingdom, 5MLL Münchner Leukämielabor, Munich, Germany

Introduction: Mutations in the epigenetic regulator gene EZH2 are frequently observed in myelodysplastic/myeloproliferative neoplasms (MDS/MPN; 10–13%). Here, we sought to genetically characterize a cohort of 40 EZH2-mutated MDS/MPN patients, to investigate mutational patterns associated with EZH2. Additionally, we analyzed clonal hierarchies of found mutations and evaluated how EZH2 mutations might influence genome-wide gene expression.

Methods: The cohort included 40 MDS/MPN patients (male, n = 22; median age, 76 yrs; range, 61 to 88 yrs) with EZH2 mutations. Targeted deep next-generation sequencing (NGS) was used to analyze a panel of 30 commonly mutated genes in myeloid disorders. Clonal hierarchies were investigated in three patients using cryopreserved CD34+ cells to grow granulocyte-macrophage colonies which were analyzed using Sanger sequencing after DNA extraction and whole genome amplification. Stable shRNA-mediated down regulation of EZH2 in MOLM-13 was achieved via lentiviral transduction and confirmed by western blot analysis. Total RNA was used for genome wide expression analysis of cell lines to detect EZH2 targets, and compare whether these targets are differentially expressed in a subset of our cohort compared to EZH2-wildtype MDS/MPN patients.

Results: The NGS screen revealed a complex pattern of mutations with a total of 187 individual mutations. In addition to EZH2 mutations, 23 patients showed cooperating TET2 mutations (58%), followed by RUNX1 (40%), ASXL1 (33%), CBL (25%), ZRSR2 (20%), SRSF2 (15%), NRAS (15%) and STAG2 (15%). The majority of patients with mutations in TET2, RUNX1 and/or ASXL1 carried frameshift, nonsense or splice site mutations in these genes (83%, 81%, 92%, respectively). Colony assays showed a complex mutational hierarchy with various related subclones and the presence of a dominant clone. EZH2 mutations seemed to be an early event in leukemogenesis followed by SETBP1 mutations. Expression data is currently being analyzed.

Conclusion: Here we have shown that EZH2 mutations are frequently associated with TET2, RUNX1 and ASXL1 mutations in MDS/MPN. EZH2 and U2AF35 mutations are mutually exclusive with fewer than expected SRSF2 mutations. Further unraveling mutational patterns and associations will help to better define disease entities and aid prognostic and therapeutic efforts. As EZH2 is an early event in leukemogenesis, it could be used for disease monitoring and may serve as a suitable drug target in the future.

Disclosure: No conflict of interest disclosed.

V346 – Good response to epigenetic treatment with azacitidine in patients with myelodysplastic syndroms and chronic myelomonocytic leukemia is associated with clonal stabilization

Speith J.1, Rinke J.1, Schäfer V.1, Gawlitza A.1, Waldau A.1, Hochhaus A.1, Ernst T.1

1Abteilung Hämatologie und Internistische Onkologie, Klinik für Innere Medizin II, Universitätsklinikum Jena, Jena, Germany

Introduction: High-risk patients with MDS and CMML who do not qualify for an allogeneic stem cell transplantation can be treated with the demethylating substance azacitidine (Vidaza®). However, prediction of response to epigenetic therapy is difficult since the mechanism of action has not been elucidated sufficiently. Here, we aim to investigate the kinetics and evolution of molecular mutations in patients with MDS and CMML during treatment with azacitidine to get more insight into the molecular effect of this demethylating substance.

Methods: We performed systematical mutational analysis of a clinically well-characterized cohort of patients with MDS-RAEB-I/II (n = 10) and CMML-I/II (n = 5) who responded well under treatment with azacitidine defined as a decrease of blasts < 5% in MDS or < 10% in CMML. Serial bone marrow samples at diagnosis and best clinical response were investigated by targeted deep next-generation sequencing (NGS) for a panel of 30 commonly mutated genes in myeloid disorders. Constitutional DNA obtained from buccal swabs was investigated to check somatic origin of mutations.

Results: A total of 24 somatic mutations were identified in 8/10 MDS and in 5/5 CMML patients at diagnosis. Recurrently affected genes were TET2 (n = 5), RUNX1 (n = 4), EZH2 (n = 3), SRSF2 (n = 3), DNMT3A (n = 2), ASXL1 (n = 2), and KRAS (n = 2). Nearly 50% of patients (7/15) had = 2 oncogenic mutations. A total of 23 somatic mutations were identified at the time of best response during treatment with azacitidine. Regarding mutation kinetics, 19/27 clones (70%) have already been observed at diagnosis and showed a complete stabilization of quantitative mutation load in 5 patients, 5/27 subclones (19%) decreased or disappeared (KRAS, n = 2; TP53, ASXL1, SRSF2) in five patients and 3 clones (11%) developed (KRAS, n = 2; ETV6) in three patients under epigenetic treatment. 3/5 patients with a clonal stabilization harbored TET2 mutations.

Conclusion: In the majority of patients we were able to show that a response or remission of the disease under treatment with azacitidine was associated with a stabilization of the clonal architecture rather than a decrease or eradication of mutant subclones. Almost all patients with TET2 mutations achieved a clonal stabilization.

Disclosure: No conflict of interest disclosed.

V347 – Targeted sequencing reveals an accumulation of alterations in epigenetic regulators in relapsed ALL patients: data of the GMALL registry

Neumann M.1,2,3, Bastian L.1, Schlee C.1, Ortiz Tanchez J.1, Vosberg S.2,3,4, Rani James A.1,2,3, Schröder M.P.1, Schwartz S.1, Gökbuget N.5, Hoelzer D.5, Graf A.6, Krebs S.6, Blum H.6, Hecht J.7,8, Brüggemann M.9, Greif P.A.2,3,4, Baldus C.D.1,2,3

1Charité – Universitätsmedizin Berlin (CBF), Hämatologie und Onkologie, Berlin, Germany, 2German Cancer Research Center (DKFZ), Heidelberg, Germany, 3German Cancer Consortium (DKTK), Heidelberg, Germany, 4Ludwig-Maximilians-University, Department of Internal Medicine 3, Munich, Germany, 5Goethe University Hospital, Department of Medicine II, Hematology/Oncology, Frankfurt/M, Germany, 6Ludwig-Maximilians-University, Laboratory for Functional Genome Analysis, Gene-Center, Munich, Germany, 7The Barcelona Institute of Science and Technology, Centre for Genomic Regulation (CRG), Barcelona, Spain, 8Universitat Pompeu Fabra (UPF), Barcelona, Spain, 9University Hospital Kiel, Department of Hematology and Oncology, Kiel, Germany

Introduction: Targeted therapies based on molecular alterations are not established in standard treatment of ALL (with the exception of Phpos-ALL). The example of Ph-like-ALL with proven efficacy of tyrosine kinase inhibitors underline the potential of identification of targetable lesions. Comprehensive analysis of mutational lesions in adult ALL is limited. Herein, we investigated the mutational spectrum in the routine diagnostic setting of the GMALL.

Patients and methods: Over the period of 12 months, we prospectively investigated gDNA samples of 85 ALL patients (Phpos-BCP-ALL n = 23, Phneg-BCP-ALL n = 36, pro B-ALL n = 10, T-ALL n = 11, B-ALL n = 5). In addition, samples from 52 Phneg BCP-ALL patients were examined at first diagnosis and relapse. The GMALL gene panel comprised 206 genes with a target region of 1.05 Mbp (SureSelect, Agilent). Sequencing was performed on an Illumina HiSeq1500 (100bp paired-end) with 16 samples/lane and an average read depth of ~600 reads/base. The turn-around-time was 4–6 weeks with incoming samples as the most limiting factor.

Results: In Phneg-BCP-ALL (n = 98), the mutational spectrum was similar to childhood ALL, with the most alterations in KRAS (18%), PAX5 (16%), TP53 (9%), JAK1/2 (9%) and NRAS (8%). In T-ALL, NOTCH1 (6/11) and PHF6 (6/11) showed the most hits. Overall, 46% of all patients showed alterations in epigenetic modifiers.

More than half of the patients (28/52, 54%) gained mutations in relapse, among those were known relapse related mutations like TP53 (n = 3) or CREBBP (n = 3). Ten patients (19%) acquired mutations in histone methylases or demethylases (KMT2D, SETD2, KMT2C, KDM6A, KDM5A). Notably, none of the 52 patients lost an existing mutation at first diagnosis in one of these genes.

In one patient with a second relapse after blinatumumab therapy, we detected a FLT3 mutation in the tyrosine kinase domain. In experimental therapy with sorafenib, this patient showed a blast clearance in the peripheral blood and recovering of platelet count for a period of two months.

Conclusions: The mutational screening using this GMALL gene panel at first diagnosis of an ALL is feasible. BCP-ALL shows a high rate of alterations in epigenetic modifiers at initial diagnosis and an increased accumulation in relapsed patients. Besides these, a number of detected alterations might offer therapeutic possibilities in relapsed or MRD-positive patients. It remains challenging to translate these findings into clinical practise.

Disclosure: No conflict of interest disclosed.

V348 – The Allelic Ratio (AR) of DNMT3A R882 mutations (Mut) determined by digital droplet PCR (ddPCR) is a potential prognostic factor in Acute Myeloid Leukemia (AML)

Grimm J.1, Dick T.1, Bill M.1, Jentzsch M.1, Schulz J.1, Schmalbrock L.1, Bonifacio L.1, Knyrim M.1, Schubert K.1, Cross M.1, Pönisch W.1, Vucinic V.1, Behre G.1, Franke G.-N.1, Niederwieser D.1, Schwind S.1

1Abteilung Hämatologie und Internistische Onkologie, Universitätsklinikum Leipzig, Leipzig, Germany

In AML the AR of various mut (e.g.FLT3-ITD) has been shown to impact on clinical outcome. However, little is known about DNMT3A R882mut in this respect. The highly sensitive & specific ddPCR may represent a feasible tool for AR assessment.

AML patients (pts) treated at our institution between 2000 & 2015 with DNMT3A R882H or R882Cmut & diagnostic (n = 33) &/or follow-up (n = 20) cDNA samples were analyzed by ddPCR with a duplex assay measuring DNMT3Awildtype (wt) & mut. Absolute copy numbers of DNMT3Amut/DNMT3Awt defined the AR. Samples with an AR< 0.0001 or < 3 positive (+) droplets were considered negative (-) according to manufacturer’s recommendations. Mut in NPM1, CEPBA & presence of FLT3-ITD & -TKD were assessed. For pts receiving hematopoietic stem cell transplantation (HSCT; n = 27) data on bone marrow chimerism were collected.

We identified 35 pts (median age 63 years [y], range 29–87y) with a DNMT3A R882H (71.4%) or R882Cmut (28.6%). All mut were heterozygous. At diagnosis pts with a DNMT3A R882mut AR>1 (31.4%, median AR 0.92) more often had secondary AML (P = 0.06), were less frequently NPM1mut (P = 0.03), had fewer platelets (P = 0.08), more blasts in peripheral blood (P = 0.04) & had shorter overall survival (P = 0.005, Figure1A).

Of 15 pts with available cDNA up to 30 days before HSCT (pre-HSCT) 13 pts (86.7%) remained DNMT3A R882mut + & the AR declined in all pts (median AR 0.14, median AR reduction 67.3%, P < .001).

28 days after HSCT cDNA of 5 pts was available. One patient was DNMT3A R882mut - & is in continuous remission. 3 of 4 DNMT3A R882mut + pts (AR range 0.0001–0.51) experienced relapse after HSCT. Notably in 1 of those pts the DNMT3A R882mut AR increased by 2 log-levels between day 28 & 56 post-HSCT while the bone marrow total chimerism remained at 100% (Figure1B).

Despite the limited number of pts our data suggest an association of the DNMT3A R882mut AR with biological features & prognosis in AML. We show that ddPCR is an eligible method to determine the DNMT3A R882mut AR & we aim to include the AR assessment in clinical trials to further investigate its prognostic influence & potential role as minimal residual disease marker after HSCT.


Disclosure: No conflict of interest disclosed.

V349 – Synergistic inhibition of chromatin modifiers reverses a leukemogenic gene expression program in NPM1 mutant leukemia

Kühn M.W.M.1,2, Song E.2, Feng Z.2, Sinha A.2, Chen C.-W.2, Deshpande A.J.2, Cusan M.2, Farnoud N.R.2, Koche R.P.2, Bradner J.E.3, De Stanchina E.4, Vassiliou G.S.5, Hoshii T.2, Armstrong S.A.2,6

1Universitätsmedizin der Johannes Gutenberg-Universität Mainz, Medizinische Klinik III, Mainz, Germany, 2Memorial Sloan Kettering Cancer Center, Cancer Biology & Genetics Program, New York, United States, 3Dana Farber Cancer Institute, Harvard Medical School, Boston, United States, 4Memorial Sloan Kettering Cancer Center, Antitumor Assessment Facility, New York, United States, 5Wellcome Trust Sanger Institute, Hinxton, Cambridge, United Kingdom, 6Dana Farber Cancer Institute, Harvard Medical School, Pediatric Oncology, Boston, United States

Introduction: Aberrant HOX expression is commonly found in NPM1 mutant (NPM1mt) AML and FLT3 is concomitantly mutated in ~60% of these cases. Little is known how these cells maintain aberrant gene expression. Since MLL and DOT1L control HOX expression in other settings, we investigated potential dependencies of NPM1mt AML on these chromatin modifiers and delineated their potential role in regulating leukemogenic gene expression.

Methods: CRISPR-Cas9 genome editing across exons encoding specific MLL protein domains was used in a negative selection screen to assess dependencies of NPM1mt AML. This concept was extended with inhibitors of the menin-MLL interaction (MI2–2; MI503). Dependencies on DOT1L were explored using the inhibitor EPZ4777. Target validation was performed in a human xenograft and two conditional murine knock-in models of Npm1mt AML (Npm1CA/+Flt3ITD/+; Npm1CA/+RosaSB/+).

Results: CRISPR-Cas9 negative selection screening revealed NPM1mt OCI-AML3 cells to be exceptionally dependent on the menin binding site in MLL. Pharmacological menin-MLL inhibition resulted in dramatic HOX and MEIS1 suppression, differentiation induction, and profound growth inhibition in human OCI-AML3 and murine Npm1mt AML cells. Of interest, ectopic expression of Meis1, Hoxb4, or Hoxa9-Meis1 rescued the antiproliferative drug effects. In vivo MI503 treatment resulted in HOX and MEIS1 suppression, reduction of leukemia burden, and a significant survival advantage of the NPM1mt AML xenograft and the Npm1CA/+RosaSB/+ model. Since MEIS1 was most dramatically suppressed, we assessed drug treatment effects on the MEIS1 target FLT3 that was also substantially downregulated. H3K79me2 levels across the HOX locus were also reduced upon MI503 treatment and pointed to DOT1L as having a role in HOX gene regulation. EPZ4777 treatment resulted in HOX, MEIS1, and FLT3 downregulation, cell growth inhibition, and profound differentiation of NPM1mt AML blasts. Next, we investigated effects of combined menin-MLL and DOT1L inhibition and found superior suppression of HOX and FLT3, superior differentiation effects, and synergistic growth inhibition. Combinatorial drug treatment further reduced leukemia initiating potential compared to single drug treatment in vivo.

Conclusion: MLL and DOT1L are chromatin regulators that control leukemogenic gene expression in NPM1mt AML. Combinatorial small-molecule inhibition represents a therapeutic opportunity that should soon be ready for clinical assessment.

Disclosure: Michael Kühn: No conflict of interest disclosed.Scott Armstrong: Advisory Role: Consultant for Epizyme, Inc. and Imago Biosciences.

V350 – Combined pharmacological DNMT and HDAC inhibition in AML cells: Integrative transcriptome and methylome analyses reveal synergistic gene downregulation associated with gene body demethylation

Schlosser P.1,2, Blagitko-Dorfs N.2, Greve G.2,3, Pfeifer D.2, Lübbert M.2

1Medical Center – University of Freiburg, Institute for Medical Biometry and Statistics, Freiburg, Germany, 2Medical Center – University of Freiburg, Department of Hematology, Oncology and Stem Cell Transplantation, Freiburg, Germany, 3Faculty of Biology, University of Freiburg, Freiburg, Germany

Background: Transcriptome-wide studies have shown that HMAs can downregulate mRNAs. Recently, Yang et al.showed that gene body methylation can be strikingly reversed by DAC in cancer cells, with downregulation of genes regulated by c-MYC, implicating gene body methylation as a novel drug target. Therefore, we asked on a transcriptome- and methylome-wide level whether HMA-induced reversal of gene body methylation occurs in AML, and whether this is associated with gene repression.

Materials and methods: U937 cells were treated with 3×24h pulses of DAC (50nM), alone or in combination with the HDACi panobinostat (Pano, 8nM) or Valproic acid (VPA, 1mM) for 24h. Transcriptome and methylome profiles (biological triplicates) were generated using Affymetrix Human Gene 2.0 ST and Infinium HumanMethylation450 BeadChip arrays. Data were analyzed using R package RnBeads applying standard methods.

Results: Transcriptome analyses revealed that DAC, Pano or VPA alone induced 207, 267 and 179 transcripts, with downregulation of 28, 45 and 22 transcripts, respectively (FDR< 0.01; =2-fold change). When DAC was combined with either Pano (D+P) or VPA (D+V), a striking synergistic effect was observed for upregulated transcripts. Remarkably, a synergistic effect on downregulated transcripts was even stronger. As expected, DAC treatment resulted in extensive DNA demethylation: 295.6K CpGs became >10% demethylated (FDR< 0.01) The ratio of transcripts mapping to promoter- vs. gene body-demethylated CpGs doubled from up- to downregulated transcripts (all 3 treatments). Of the 667 transcripts downregulated by D+P, the 227 gene-body demethylation-associated transcripts included various putative or bona fide oncogenes (such as c-MYC) and, notably, many epigenetic modifiers. Gene ontology analysis revealed an enrichment for genes involved in RNA processing and transcription validated for several genes encoding histone methylation “writer” enzymes by RT-qPCR.

Conclusions: The combination of a HMA with either of two HDACi resulted not only in synergistic gene reactivation but also in massive, synergistic downregulation of genes including oncogenes and histone modifying enzymes, which was associated predominantly with gene body DNA demethylation. These findings have implications for the mechanisms of action of combined epigenetic treatment and for a better understanding of clinical responses in trials where this approach is tested, such as the randomized DECIDER AML trial.

Disclosure: Pascal Schlosser: No conflict of interest disclosed.Michael Lübbert: Financing of Scientific Research: Novartis, Janssen-Cilag; Expert Testimony: Janssen-Cilag

Expertenseminar – Patienten mit ZNS-Lymphomen

V352 – Primary central nervous system lymphoma in the elderly

Illerhaus G.1

1Klinikum Stuttgart, Hämatologie, Onkologie und Palliativmedizin; Stuttgart Cancer Center, Stuttgart, Germany

Patients older than 60 years account for 50% of all PCNSL cases. Although elderly patients are able to tolerate aggressive systemic chemotherapy, they have an inferior prognosis compared with younger patients and are more seriously affected by iatrogenic toxicity, especially neurological side-effects following whole brain radiotherapy. Therefore, elderly patients represent a unique and vulnerable treatment subgroup. An US registry study of 579 elderly patients diagnosed with PCNSL in the 1990s revealed that the median survival was only 7 months and WBRT alone was the most common treatment modality (46%). The addition of rituximab to high-dose methotrexate (HD-MTX)-based chemotherapy has shown a significant survival improvement in the IELSG-trial prospective, however, this trial was not particularly designed for elderly patients.

Based on a systematic review of studies focusing on elderly PCNSL patients, there is only one completed randomized phase II trial for elderly patients, which suggests that a combination of HD-MTX with vincristine, procarbazine, and cytarabine is more effective than HD-MTX with temozolomide regarding response; however, difference in progression free survival were only marginal and no rituximab was used in this study. Data from the recently completed multicenter German PRIMAIN study strengthen the evidence that immunochemotherapy with HD-MTX combined with an oral alkylating agent (e.g. procarbazine) together with rituximab is a feasible and effective treatment and could provide a benchmark for future comparative studies. Still, treatment-associated morbidities and treatment delays are major issues that need to be addressed in future trials e.g. by increasing rituximab density in the beginning and introduction of better tolerable novel agents. To reduce the risk of relapse, immuno-modulatory agents (e.g. lenalodomide) could be attractive options to be tested as maintenance treatment. High-dose chemotherapy followed by autologous stem cell transplantation (HDT-ASCT) has been shown to be feasible and effective in younger patients. A pilot study investigating HDT-ASCT feasibility in elderly patients is currently ongoing.

In summary, HD-MTX with oral alkylating agents (e.g. procarbazine or temozolomide) and rituximab are advisable and should be offered to elderly and frail patients. The role of HDT-ASCT in fit elderly patients is under investigation. More prospective trials designed for elderly PCNSL patients are needed.

Disclosure: Gerald Illerhaus: Financing of Scientific Research: Riemser; Expert Testimony: Riemser

Freier Vortrag – Akute myeloische Leukämie – Therapie 2

V353 – The new WHO classification (2016) defines a revised diagnostic approach to myeloid neoplasms with erythroid precursors comprising = 50% of bone marrow nucleated cells: Applicability and clinical impact

Haferlach T.1, Kern W.1, Perglerova K.2, Haferlach C.1

1MLL Münchner Leukämielabor GmbH, München, Germany, 2MLL 2, Paríkova, Czech Republic

Introduction: The 2016 revision to WHO classification of myeloid neoplasms (Arber et al., BLOOD, prepub 11.04.2016) revises AML with = 50% bone marrow erythroid precursors and =20% myeloid blasts among non-erythroid cells. In the new classification the denominator for determining the percentage of myeloid blasts is total bone marrow cells. Thereby, erythroid leukemias showing blast cells ‹20% of the total cells are now classified as MDS. Further, cases with NPM1mut, CEBPAdmut, RUNX1mut all qualify for genetically defined entities.

Patients and methods: We here investigated 190 cases of AEL (WHO 2008/FAB AML M6) all diagnosed in our laboratory between 2005–2016.

Results: Following WHO 2016 we first subtracted cases with NPM1 (n = 28), CEBPAdm (n = 2) and RUNX1 (n = 16) mutations (46/190, 24%). We then separated cases according to BM blasts and found 35% (51/144) cases with =20% total blasts. 18/51 (35%) classify as “myelodysplasia-related changes” due to their cytogenetics and/or multilineage dysplasia. The remaining 33 (65%) cases with =20% of total blasts now are AEL. The remaining 93/144 (65%) cases showed < 20% total blasts and are now MDS. Within these 48/93 cases (52%) show “myelodysplasia-related changes”. We now compare new AEL (n = 33) and new MDS (n = 45). Both new subgroups show a normal karyotype in 73% each. In AEL and MDS the following mutations were observed at not significantly different frequencies: MLL-PTD (39%, 26%), ASXL1 (18%, 16%), FLT3-ITD (17%, 4%), TET2 (0%, 20%), SF3B1 (0%, 13%). Only IDH1 showed a statistically different distribution (AEL 27%, MDS 0%, p = 0.046). Using univariate Cox regression analysis, in all 144 patients only cytogenetics classified as “myelodysplasia-related changes” (p = 0.013; HR 3.1) and TP53 mutation (p = 0.001; HR 6.4) showed significant negative prognostic impact. We did not find any effect for BM blasts %. In multivariate analysis only mutated TP53 showed a significant and poor outcome (p = 0.01 HR 7.5). Regarding survival we did not find significant differences between AEL (n = 15; 2-yrs OS: 64%) and new MDS cases (n = 24; 2-yrs OS: 66%).

Conclusions: AEL/AML M6 were newly defined in WHO 2016. Based on 190 patients according to WHO 2008, we find that 35% of patients remain AML and 65% of patients are MDS now. Genetics and outcome seem to be quite overlapping. Patients with the respective morphological features should be treated according to genetics rather than number of myeloid blasts in the BM.

Disclosure: Torsten Haferlach: Employment or Leadership Position: Geschäftsführer MLL Münchner Leukämie LaborClaudia Haferlach: Employment or Leadership Position: Geschäftsführerin MLL Münchner Leukämie Labor

V354 – A Fc-optimized FLT3 antibody for elimination of minimal residual disease (MRD) in patients with AML

Dörfel D.1,2, Körner S.1,2, Nübling T.1,2, Bamberg M.3, Bühring H.-J.1, Hofmann M.3, Grosse-Hovest L.4, Aulwurm S.4, Bethge W.1, Kanz L.1, Jung G.3, Salih H.R.1,2

1Department of Hematology and Oncology, Eberhard Karls University, Tübingen, Germany, 2Clinical Collaboration Unit Translational Immunology, German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Partner site Tübingen, Tübingen, Germany, 3Department of Immunology, Eberhard Karls University, Tübingen, Germany, 4Synimmune GmbH, Tübingen, Germany

In the majority of patients with AML, a morphological complete remission (CR) can be achieved by chemotherapeutic treatment. However, minimal residual disease (MRD) remains detectable in many patients. Recently, persistance of MRD defined e.g. by detection of NPM1-mutated transcripts (mutNPM1) was identified as powerful prognostic parameter for relapse (e.g., Ivey et al, NEJM 2016). Here we report our clinical observations upon application of a novel Fc-optimized antibody (4G8-SDIEM, FLYSYN) directed to the receptor tyrosine kinase FLT3 that is expressed on the surface of AML cells. Our antibody was developed with the aim to eliminate MRD after conventional therapy and to achieve this goal with less toxicity (i.e. GvHD) than donor lymphocyte infusion (DLI) and allogeneic stem cell transplantation (SCT). After preclinical characterization, which revealed its profound ability to mediate ADCC against AML cells (Hofmann et al, Leukemia 2012), the antibody was produced in a university-owned GMP-unit and applied on a compassionate need basis to 8 AML patients (male, 4; female, 4; median age, 60 years, (range 42–76)) in CR with persistant or increasing MRD (mutNPM1, n = 5; CBFB-MYH11 n = 1, AML-ETO1 n = 1, KITD816V n = 1). 5 patients had undergone SCT, 3 had refused or were unfit for SCT. FLT3 surface expression on leukemic blasts was confirmed by FACS in 6/8 patients. The antibody was applied in a dose of 25 mg total, and patients were then followed for clinical status and MRD levels. No relevant side effects (in particular no hematological toxicity) were observed. The serum half-live was found to range from 39–166 hours. In 3 patients (notably all with mutNPM1 as MRD marker), the single application of 4G8SDIEM (in 2 cases subsequently followed by treatment with one dose of DLI) resulted in rapid elimination of MRD. One patient that had received DLI died of complications due to GvHD and liver toxicity after reaching and maintaining a molecular CR. The other two patients (1 with, 1w/o DLI) remain in molecular CR for at present 44 and 42 months after 4G8-SDIEM application without further specific treatment. These results imply that 4G8SDIEM might be capable to reduce or eliminate MRD after conventional therapy at the cost of very few side effects. Based on these observations, a phase I/II trial enrolling AML patients ineligible for SCT in CR with detectable mutNPM1 is presently submitted to the regulatory authorities and envisaged to start recruitment in July 2016.

Disclosure: No conflict of interest disclosed.

V355 – Long-term outcome of sorafenib treatment in FLT3-ITD-positive acute myeloid leukemia patients relapsing after allogeneic stem cell transplantation

Metzelder S.K.1, Schroeder T.2, Lübbert M.3, Götze K.4, Scholl S.5, Ditschkowski M.6, Meyer R.G.7, Dreger P.8, Basara N.9, Fey M.10, Salih H.11, Finck A.3, Finke J.3, Pabst T.10, Giagounidis A.12, Neubauer A.1, Burchert A.1

1Department of Hematology/Oncology/Immunology, University Hospital Marburg, Marburg, Germany, 2University of Duesseldorf, Medical Faculty, Department of Hematology, Oncology and Clinical Immunology, Düsseldorf, Germany, 3University of Freiburg, Department of Hematology/Oncology, Freiburg, Germany, 4Dept. of Internal Medicine III, Technische Universität München, München, Germany, 5Abteilung Hämatologie/Onkologie, Universitätsklinikum Jena, Jena, Germany, 6Department of Bone Marrow Transplantation, West German Cancer Center, University Hospital Essen, Essen, Germany, 7St.-Johannes-Hospital Dortmund, Klinik für Innere Medizin II, Dortmund, Germany, 8Department of Medicine, University of Heidelberg, Heidelberg, Germany, 9Malteser Krankenhaus St. Franziskus-Hospital, Medizinische Klinik I, Flensburg, Germany, 10Department of Medical Oncology, Inselspital and University of Bern, Bern, Switzerland, 11Department of Hematology/Oncology, Eberhard Karls-University, Tübingen, Germany, 12Marien Hospital Düsseldorf, Klinik für Onkologie, Hämatologie und Palliativmedizin, Düsseldorf, Germany

In acute myeloid leukemia (AML) the presence of an internal tandem duplication (ITD) in the FLT3 gene predicts a poor prognosis. We recently reported on 65 FLT3-ITD positive AML patients, who were given sorafenib monotherapy for relapsed or refractory disease, including 29 patients, who relapsed after allogeneic stem cell transplantation (allo-SCT) (Metzelder et al. 2012). The long-term efficacy of sorafenib monotherapy in this situation of ultra-poor risk AML is ill defined. Here we report on the long-term follow-up of these 29 patients. The median follow up in this cohort was 6.4 years (range, 4–8.6). Six of the 29 patients (21%) are still alive. Excluding one patient who received a 2nd allo-SCT after developing sorafenib resistance, five of the 29 patients experience long-lasting remissions with a 5-year overall survival of 17%. After a median treatment duration of 29 months (range, 18–39) sorafenib treatment was stopped in four patients. One of these patients relapsed eleven months after stopping sorafenib but is currently in complete molecular remission 56 months after restarting sorafenib. The other three patients are still in treatment free remission for 31, 37 and 59 months. For the first time we show not only that sorafenib monotherapy can induce durable remissions in a selected group of relapsed ITD positive AML patients after allo-SCT but also that stopping sorafenib after prolonged treatment is possible with a realistic chance of curation.

Disclosure: No conflict of interest disclosed.

V356 – Persistence of driver mutations during complete remission associates with shorter survival and contributes to the inferior outcomes of elderly patients with acute myeloid leukemia

Rothenberg-Thurley M.1, Amler S.2, Görlich D.2, Sauerland M.C.2, Schneider S.1, Konstandin N.P.1, Schaaf S.3, Nazeer Batcha A.M.3, Bräundl K.1, Ksienzyk B.1, Zellmaier E.1, Mansmann U.3,4, Fiegl M.1, Subklewe M.1, Bohlander S.K.5, Faldum A.2, Hiddemann W.1,4, Spiekermann K.1,4, Braess J.6, Metzeler K.H.1,4, AMLCG

1Medizinische Klinik III, Klinikum der Universität München, München, Germany, 2Institut für Biometrie und Klinische Forschung, Universität Münster, Münster, Germany, 3Institut für medizinische Informationsverarbeitung, Biometrie und Epidemiologie, LMU München, München, Germany, 4Deutsches Konsortium für Translationale Krebsforschung (DKTK), Heidelberg, Germany, 5Department of Molecular Medicine and Pathology, University of Auckland, Auckland, New Zealand, 6Klinik für Onkologie und Hämatologie, Krankenhaus Barmherzige Brüder, Regensburg, Germany

Introduction: AML patients (pts) in complete morphological remission may harbor persistent preleukemic clones, which might be a source of leukemic relapse. Previous studies demonstrated an adverse outcome of pts with persistent mutations in remission (Klco et al., JAMA 2015; PMID: 26305651). We studied persistence of driver mutations in remission and outcomes in a cohort of uniformly treated AML patients.

Patients and methods: We studied 108 adult AML pts (median age, 56 years [y]; range 20–80y) who received intensive induction chemotherapy in the multicenter AMLCG-2008 trial, and reached either complete remission (CR) (74%) or CR with incomplete blood count recovery (CRi) (26%). In bone marrow (BM) or blood (pB) specimens obtained at diagnosis (BM, n = 96; pB, n = 12) and during first remission (BM, n = 102; pB, n = 6), we analyzed 68 recurrently mutated genes by multiplexed amplicon sequencing.

Results: At diagnosis, 426 mutations were detected in 42 genes (median, 4 mutations per patient; range, 0–10). In the paired remission samples, 61 mutations were still present in 38/107 pts (36%), while 69 pts (64%) had no persistent mutation. Persistence of mutations during morphological remission was most commonly observed for DNMT3A (23/37 pts with mutations at diagnosis), TET2 (8/13), SRSF2 (5/8) and ASXL1 (5/12). Mutations in other genes including NPM1, FLT3, WT1, and NRAS were cleared in the remission samples.

Pts with =1 persisting mutations in remission were older (median, 63y) than pts without any persisting mutation (median, 48y; p < 0.001). Persistence of =1 mutation in remission, in contrast to complete mutation clearance, associated with shorter relapse-free survival (RFS; Fig. 1A) and overall survival (OS; Fig. 1B).

In multivariate analyses adjusting for age, ELN genetic risk groups, and remission status (CR vs. CRi), detection of =1 persisting mutation remained associated with inferior RFS (HR, 2.1; P = 0.02) and OS (HR, 2.5; P = 0.02).

Conclusion: Detection of persisting mutations during first CR/CRi is common in older AML pts. Mutation persistence associates with shorter RFS and OS and might contribute to the inferior outcomes of elderly AML pts.


Disclosure: No conflict of interest disclosed.

V357 – BCR-ABL+ acute myeloid leukemia: a rare and intriguing entity – a retrospective analysis of clinical and molecular features

Neuendorff N.R.1, Burmeister T.1,2, Dörken B.1,2, Westermann J.1,2

1Charité Universitätsmedizin Berlin, Klinik für Hämatologie, Onkologie und Tumorimmunologie, Campus Virchow Klinikum, Berlin, Germany, 2Labor Berlin Charité-Vivantes GmbH, Berlin, Germany

Introduction:BCR-ABL+ AML is a rare subgroup of AML, likely to be included in the new WHO classification as a provisional entity. The distinction between BCR-ABL+ AML and CML blast crisis (CML-BC) is often challenging. We analysed all cases published since 1975 and from our institution regarding clinical and molecular features for a better characterization and distinction from CML-BC.

Methods:BCR-ABL+ AML cases were obtained by a Pubmed literature search and from our institutional database. For comparability, cases were (if necessary) re-classified according to the latest WHO classification or, in the case of AUL/MPAL, according to EGIL and/or WHO classification. AUL/MPAL cases were excluded from the analysis, likewise those with an antecedent myeloproliferative disorder and cases with 100% BCR-ABL+ metaphases (published before 1990), if no additional features were given to distinguish AML from CML-BC. Finally, 126 cases of de novo BCR-ABL+ AML were analysed.

Results: According to WHO 2008, 38% of the cases can be categorized as “AML-NOS”, 32.5% as “AML with myelodysplasia-related changes” and 23.8% as “AML with recurrent genetic aberrations” (mostly CBF leukemia). In a substantial portion, co-expression of lymphoid markers was present, not fulfilling the criteria for classification as “MPAL/AUL”. Basophilia was seen in only 5.6% of cases, splenomegaly in 16.7%. BCR-ABL transcripts (p190 and p210) were equally distributed. According to ELN criteria (irrespective of BCR-ABL), 21.4% belonged to the “favorable”, 39.7% to the “intermediate-I/II” and 30.2% to the “adverse” risk group (8.7% could not be classified due to a lack of data). Remarkably, long-term survival without allogeneic stem cell transplantation was reported within the favorable risk group.

Conclusions: To the best of our knowledge, our study provides the most comprehensive overview on BCR-ABL+ AML published so far. With regard to our database with 126 de novo BCR-ABL+ AML we have developed a diagnostic algorithm including patients´medical history, basophilia, splenomegaly, the type of BCR-ABL transcript, the percentage of BCR-ABL+ metaphases in the bone marrow and cytogenetics. Furthermore, our data suggest the prognosis of BCR-ABL+ AML is rather determined by the specific genetic background than by BCR-ABL itself. Finally, we conclude that treatment with tyrosine kinase inhibitors remains an individual decision, which may be particularly appropriate beyond first line therapy.

Disclosure: Nina Neuendorff: Other Financial Relationships: Stipendium der Firma MedacJörg Westermann: Advisory Role: Novartis, BMS, Celgene; Financing of Scientific Research: Novartis, BMS, Celgene; Expert Testimony: Novartis

V358 – Real life experience with ATRA-arsenic trioxide based regimen in acute promyelocytic leukemia – results of the prospective German intergroup Napoleon registry

Platzbecker U.1, Lengfelder E.2, Götze K.3, Röllig C.1, Kramer M.1, Sauer M.1, Albers N.4, Heuser M.5, Link H.6, Schäfer-Eckart K.7, Martin S.8, Krause S.9, Hänel M.10, Bochtler T.11, Jakob A.12, Reichle A.13, Biersack H.14, Baldus C.15, Görner M.16, Basara N.17, Mezger J.18, Naumann R.19, Kürschner D.20, de Wit M.21, Nolte F.22, Scholl S.23, Ringhoffer M.24, Tischler H.J.25, Greiner J.26, Kremers S.27, Machherndl-Spandl S.28, Koller E.29, Wattad M.30, Aschauer G.31, Radsak M.32, Kubin T.33, Salih H.34, Wolf D.35, Thiede C.1, Serve H.36, Dührsen U.37, Hochhaus A.23, Brümmendorf T.38, Bornhäuser M.1, Döhner H.39, Ehninger G.1, Schlenk R.F.39

1Universitätsklinikum Carl Gustav Carus, Medizinische Klinik und Poliklinik I, Dresden, Germany, 2Universitätsklinikum Mannheim, III. Medizinische Klinik, Mannheim, Germany, 3Klinikum rechts der Isar der Technischen Universität München, III. Medizinische Klinik, München, Germany, 4Gesellschaft für Medizinische Innovation – Hämatologie und Onkologie mbH (GMIHO), Berlin, Germany, 5Medizinische Hochschule Hannover, Klinik für Hämatologie, Hämostaseologie, Onkologie und Stammzelltransplantation, Hannover, Germany, 6Westpfalz-Klinikum GmbH, Medizinische Klinik I, Kaiserslautern, Germany, 7Klinikum Nord, Medizinische Klinik V, Nürnberg, Germany, 8Robert-Bosch-Krankenhaus Stuttgart, Abteilung Hämatologie, Onkologie und Palliativmedizin, Stuttgart, Germany, 9Universitätsklinikum Erlangen, Medizinische Klinik V, Erlangen, Germany, 10Klinikum Chemnitz, Klinik für Innere Medizin III, Chemnitz, Germany, 11Universitätsklinikum Heidelberg, Medizinische Klinik V, Heidelberg, Germany, 12Ortenau Klinikum, Offenburg, Germany, 13Universitätsklinikum Regensburg, Klinik für Hämatologie und Internistische Onkologie, Regensburg, Germany, 14Universitätsklinikum Lübeck, Medizinische Klinik I, Lübeck, Germany, 15Charité Berlin, Campus Benjamin Franklin, Medizinische Klinik mit Schwerpunkt Hämatologie, Onkologie, Berlin, Germany, 16Klinikum Bielefeld Mitte, Klinik für Hämatologie, Onkologie und Palliativmedizin, Bielefeld, Germany, 17Malteser Krankenhaus St. Franziskus-Hospital, Medizinische Klinik I, Flensburg, Germany, 18St. Vincentius Kliniken, Medizinische Klinik II, Karlsruhe, Germany, 19Gemeinschaftsklinikum Mittelrhein, Klinik für Innere Medizin, Koblenz, Germany, 20MVZ Mitte und MVZ Delitzsch, Delitzsch, Germany, 21Vivantes Klinikum Neukölln, Klinik für Hämatologie und Onkologie, Berlin, Germany, 22St. Hedwig-Krankenhaus, Klinik für Innere Medizin, Berlin, Germany, 23Universitätsklinikum Jena, Klinik für Innere Medizin II, Jena, Germany, 24Städtisches Klinikum Karlsruhe, Medizinische Klinik III, Karlsruhe, Germany, 25Johannes Wesling Klinikum Minden, Zentrum für Innere Medizin, Minden, Germany, 26Diakonie-Klinikum Stuttgart, Medizinische Klinik II, Stuttgart, Germany, 27Caritas-Krankenhaus Lebach, Innere Medizin, Lebach, Germany, 28Krankenhaus der Elisabethinen Linz, Interne Abteilung, Linz, Austria, 29Hanuschkrankenhaus Wien, III. Medizinische Abteilung, Wien, Austria, 30Kliniken Essen Süd, Ev. Krankenhaus Essen-Werden, Zentrum für Innere Medizin – Hämatologie, internistische Onkologie u. Stammzelltransplantation, Essen, Germany, 31Krankenhaus der Barmherzigen Schwestern, Abteilung für Innere Medizin I, Linz, Austria, 32Klinikum der Johannes Gutenberg Universität Mainz, III. Medizinische Klinik und Poliklinik, Mainz, Germany, 33Klinikum Traunstein, Innere Medizin, Abteilung Hämato-Onkologie, Traunstein, Germany, 34Universitätsklinikum Tübingen, Department für Innere Medizin, Innere Medizin II, Tübingen, Germany, 35Universitätsklinikum Bonn, Medizinische Klinik und Poliklinik III, Bonn, Germany, 36Universitätsklinikum der J.W. Goethe-Universität Frankfurt, Medizinische Klinik II, Frankfurt, Germany, 37Universitätsklinikum Essen, Klinik für Hämatologie, Essen, Germany, 38Universitätsklinikum Aachen, Medizinische Klinik IV, Aachen, Germany, 39Universitätsklinikum Ulm, Innere Medizin III, Ulm, Germany

Standard therapy of acute promyelocytic leukemia has long time relied on the combination of All-trans-retinoic acid (ATRA) and chemotherapy. The introduction of arsenic trioxide (ATO) in APL treatment has allowed obtaining similarly high remission and even superior survival rates coupled with significantly reduced myelosuppression. Recent results of the APL0406 trial by the GIMEMA-AMLSG-SAL study groups showed that the combination of All-trans retinoic acid (ATRA) and arsenic trioxide (ATO) is superior to standard ATRA and chemotherapy (CHT) in front-line therapy of low/intermediate risk acute promyelocytic leukemia (APL). The translation of these results into clinical practice for APL patients in Germany are uncertain also given the fact that ATO is not approved by the European Medical Agency for front-line therapy in APL. In order to provide evidence and a reflection of the clinical reality of APL patient care in Germany an intergroup APL registry (NAPOLEON) has been initiated recently by German AML study groups. Eligibility is defined as age of at least 18 years and newly diagnosed or relapsed APL. Here we report the first series of patients prospectively enrolled into this registry. The study is conducted in accordance with the Declaration of Helsinki, received IRB approval by all participating centers and is registered at ClinicalTrials.gov (NCT02192619).

As of 1st of April 2016 a total of 84 patients have been included into the study with a median age of 58 years (range 22–87). All had newly diagnosed APL (100%) with 62% (n = 52) being of low/intermediate risk according to the Sanz score. Out of these patients 75% (n = 39) received an ATO-ATRA based induction regimen followed by a median of 4 courses of ATO-ATRA consolidation (according to the APL 0406 study). Of 36 patients evaluable for response to induction therapy, 36/36 (100%) patients achieved a complete remission (CR) after ATO-ATRA. Early death rate within 30 days of therapy was 0%. After a median follow-up of 12 months, the event-free survival, cumulative incidence of relapse and overall survival at 12 months for these patients were 100%, 0% and 100%, respectively. Therapy was well tolerated and no new safety signals have been obtained.

These real world data from a prospective German registry provide further evidence for the safety and anti-leukemic efficacy of ATO-ATRA in low/intermediate risk APL. These results further support ATO-ATRA as the new standard of care in this clinical setting.

Disclosure: Uwe Platzbecker: Expert Testimony: Research grant by TEVA PharmaceuticalsRichard Schlenk: Expert Testimony: Research grant by TEVA Pharmaceuticals

Fortbildung – Multiples Myelom

V359 – The new definition of multiple myeloma

Ludwig H.P.1

1Wilhelminen Cancer Research Institute, Center for Oncology, Haematology, Vienna, Austria

Symptomatic multiple myeloma usually evolves from a precursor state, namely from monoclonal gammopathy of undefined significance (MGUS) or from smoldering multiple myeloma (SMM). Several risk factors and algorithms for identifying patients at higher risk for transformation from MGUS and SMM to symptomatic myeloma have been developed. In addition, to these parameters, the following criteria for ultra-high-risk for rapid progression to symptomatic multiple myeloma have been established: SMM with bone marrow plasma cell infiltration of =60%, or with a free light chain ratio of =100, or with presence of >1 focal bone lesion of =5mm detected by MRI scan. Patients with these characteristics have a transformation rate after 2 years of 62–95%, 72–76% and 55–62%, respectively. These new criteria are now, in conjunction with the traditional CRAB criteria, summarized under the acronym SLiM CRAB.

The high progression rate of patients with ultra-high-risk SMM prompted the International Multiple Myeloma Working Group to designate these patients ‘early myeloma’ in which active anti-myeloma therapy should be initiated. This recommendation is based on the results of a small prospective randomized trial in patients with high risk SMM. Patients were assigned either to active therapy with lenalidomide-dexamethasone given for nine cycles followed by lenalidomide maintenance therapy or to control (Quiredex trial, Mateos MV et al., NEJM 2013). Results showed a significant increase of PFS and OS in the active therapy cohort. However, due to some limitations of this trial confirmation of these results in a study applying modern imaging technology such as whole body CT, or MRI, or PET/CT and free light chain monitoring of patients (which was not available in the Quiredex trial) seems desirable. Indeed, several trials presently are ongoing or planned in this patient population, but only one compares early treatment with an untreated control group.

Although these recommendations for initiation of early therapy in patients with ultra-high-risk SMM have been widely distributed, clinical reasoning should be the basis for decision making in those patients. Given the substantial number of ultra-high-risk SMM patients with =1 small focal lesion, or a free light chain ratio of =100 not having progressed after 2 years (38–45%, and 24–28%, respectively), a watch-and-wait strategy with short follow up intervals may be a reasonable alternative for carefully selected patients.

Disclosure: Heinz Ludwig: Financing of Scientific Research: Vortragshonorare und Honorare von Advisory Boards; Expert Testimony: Takeda

V360 – First-line therapy

Einsele H.1

1University Hospital Würzburg, Internal Medicine II, Würzburg, Germany

In younger patients 70–75 years in Germany, we generally evaluate the suitability of the patient for high dose chemotherapy and autologous stem cell transplantation. If there are no significant comorbidities the patient – preferentially in a clinical trial – will be treated with high dose chemotherapy and autologous stem cell transplantation. The induction treatment should avoid the use of melphalan and long term exposure to IMiDs to allow a sufficient collection of stem cells. The induction treatment prior to stem cell collection should include at least one of the novel agents, currently the combination of two novel agents is tested to increase response quality prior to stem cell transplantation. The currently most successful induction treatment for patients undergoing autologous stem cell transplantation is the combination of carfilzomib, revlimid and dexamethasone which in studies in the US has been shown to be associated with low toxicity and often induces a VGPR or even complete remission already prior to transplantation.

Currently there is a trend to go for tandem transplantation because several studies and also meta-analyses have shown that especially in high risk patients progression free and overall survival is significantly prolonged after tandem transplantation.

Several study groups have shown that in patients with a suboptimal response after stem cell transplantation, consolidation can improve response quality but also improve progression and overall survival. Thus, consolidation treatment after autologous stem cell transplantation is increasingly used and already standard in France.

Allogeneic stem cell transplantation is only offered as first line treatment in patients with ultra high risk criteria like patients with 17p deletion, extramedullary disease or high serum LDH. For patients who are not suitable for a stem cell transplantation there are currently two treatment regimens that are widely applied. One is the combination of bortezomib with melphalan/prednisolone mostly applied over 9 cycles or the combination of revlimid and dexamethasone according to the first study administered until progression.

Disclosure: Hermann Einsele: Advisory Role: Janssen, Celgene, Amgen, Novartis; Financing of Scientific Research: Janssen, Celgene, Amgen, Novartis; Expert Testimony: Janssen, Celgene Amgen

V361 – Therapy of relapsed and relapsed refractory multiple myeloma

Driessen C.1

1Kantonsspital St. Gallen, St. Gallen, Switzerland

Current advances in the drug treatment of multiple myeloma (MM) allow delivering active therapy to the majority of MM patients. However, despite a consistently increasing proportion of patients with high quality remissions after first line therapy, most MM patients ultimately relapse and require subsequent treatments for relapsed and relapsed/refractory MM. The recent approval of next generation drugs like carfilzomib, ixazomib, panobinostat, daratumumab and elotuzumab have led to an unprecedented complexity of therapeutic options in this setting. The increasing clonal diversity, genetic complexity and redundant signaling especially in R/R MM highlight the ubiquitin proteasome system and the immunophenotype as key therapeutic targets, which are conserved in MM requiring advanced line treatment. We shall here briefly review the biological principles of R/R MM and then relate the mechanisms of action, clinical data and biological-pharmacological properties of available drugs to this biology. Based on this, we shall attempt to rationally position currently available options for drug therapy of R/R MM with regard to sequence, combinations and patient as well as disease characteristics.

Disclosure: Christoph Driessen: Advisory Role: Celgene, Amgen; Financing of Scientific Research: Celgene, Amgen, Bristol-Meyers Squibb, Novartis, Takeda, Mundipharma; Expert Testimony: Amgen, Novartis, Celgene, Mundipharma

Fortbildung – Pankreaskarzinom

V367 – Evidence-based treatment options in metastatic pancreatic adenocarcinoma

Böck S.1

1Klinikum der Universität München – Campus Großhadern, Medizinische Klinik und Poliklinik III und Comprehensive Cancer Center, München, Germany

Since its introduction in the late 90ies, the nucleoside analogue gemcitabine has become the main backbone for systemic treatment of pancreatic ductal adenocarcinoma. For patients with metastatic disease, a large number of phase III trials investigating gemcitabine plus a second drug failed to show an improvement with regard to overall survival. The first trial that found a statistically significant, albeit clinically marginal, survival benefit was the PA.3 study that compared single-agent gemcitabine to gemcitabine in combination with the oral anti-EGFR tyrosine kinase inhibitor erlotinib. In that study, the benefit of adding erlotinib was mainly restricted to patients that developed skin rash during treatment with the EGFR-targeting agent.

More recently, the global phase III MPACT study showed a significant increase in response rate, progression-free and overall survival for the combination of gemcitabine with nab-paclitaxel in comparison to gemcitabine alone. Based on these data, nab-paclitaxel received regulatory approval for the first-line treatment of metastatic pancreatic cancer in 2014. With the clinical investigation of FOLFIRINOX (5-fluorouracil [5-FU], folinic acid [FA], oxaliplatin and irinotecan) a gemcitabine-free regimen was introduced in the clinic based on the French randomized phase III Prodige 4 - ACCORD 11/0402 study comparing FOLFIRINOX to gemcitabine in a selected patient population (e. g. ECOG performance status 0 to 1, Bilirubin < 1.5 x ULN, no significant cardiovascular co-morbidities) with metastatic pancreatic adenocarcinoma: FOLFIRINOX significantly increased response rate, progression-free and overall survival and – of note – also quality of life. Both regimens (FOLFIRINOX and gemcitabine + nab-paclitaxel) are currently investigated also in the adjuvant, neoadjuvant and locally advanced disease settings. Up to now, there is no evidence for the efficacy of other molecular targeted drugs than erlotinib in the 1st-line treatment of metastatic pancreatic adenocarcinoma; clinical studies using checkpoint inhibitors are currently ongoing.

During the last years increasing evidence supporting the use of 2nd-line chemotherapy after failure of 1st-line gemcitabine-based treatment was generated: two randomized phase III studies were able to find an improvement in overall survival with the use of 5-FU/FA either in combination with oxaliplatin (CONKO-003) or nanoliposomal irinotecan (NAPOLI-1) compared to a reference arm with 5-FU/FA only.

Disclosure: Stefan Böck: Advisory Role: Baxalta, Celgene; Financing of Scientific Research: Celgene, Roche; Expert Testimony: Celgene, Roche, Clovis Oncology

Wissenschaftliches Symposium – Wie teuer dürfen Arzneimittel sein?

V370 – Costs of drugs in oncology. The cases of chronic myelogenous leukemia (CML) and immunotherapy

Hochhaus A.1

1Universitätsklinikum Jena, Abt. Hämatologie/Onkologie, Jena, Germany

The development of BCR-ABL tyrosine kinase inhibitors (TKIs) has revolutionized CML therapy and allowed patients over the past 15 years to live healthy and productive lives. Imatinib is considered to be the most successful targeted anticancer agent yet developed given its substantial efficacy in treating CML and other malignant diseases. In addition, it set the stage for a series of ATP competing drugs in oncology.

This dramatic clinical benefit has come at a high cost to individual patients and health care systems. The arrival of generic imatinib has the potential to markedly impact the cost of care for CML. For most European Union (EU) member countries, the imatinib patent for CML will expire in 2016. Loss of patent exclusivity opens the market to competition from multiple manufacturers. Physicians’ willingness to prescribe generic drugs is related to patient benefit, considering potential differences in safety and efficacy of the generics. In some countries, anecdotal concerns have been raised that the bioavailability and potency of generic imatinib is not equivalent to the branded drug; however, this may not be applicable to the EU.

The use of generic imatinib should also be discussed in the context of improved efficacy and tolerability of second generation TKIs. Reduction of the rate of early transition into advanced disease, faster elimination of the leukemic burden and earlier access to discontinuation attempts based on faster achievement of deep molecular responses are arguments in favor of the use of branded second generation drugs. A shorter treatment duration with best available drugs may help to reduce treatment costs as well.

In addition to targeted therapy, immunotherapy is experiencing a fulminant renaissance with the introduction of antibodies and small molecules activating the patient’s immune system against tumor neoantigens. These new treatments could radically change oncology, but the question remains whether immunotherapies will be affordable for the treatment of all patients with approved indications.

What is a fair price for a cancer drug? Certainly a price that would maintain reasonable profits to drug companies, but remain affordable to patients and to the health care systems. Not all cancer drugs are alike, and those with marginal value will not command high prices. The long-term value of individual immunotherapies remains to be seen and with the development of multiple options prices may reach reasonable levels.

Disclosure: Andreas Hochhaus: Employment or Leadership Position: UK Jena; Financing of Scientific Research: Novartis, BMS, Pfizer, Incyte (Ariad); Expert Testimony: Novartis, BMS, Pfizer, Incyte (Ariad)

V371 – Current prices of innovative drugs are too high

Ludwig W.-D.1

1HELIOS Klinikum Berlin-Buch, Hämatologie, Onkologie, Tumorimmunologie, Palliativmedizin, Berlin, Germany

Rapid progress in the fields of tumor biology, genetics, and immunology have greatly improved our understanding of the mechanisms driving tumor formation and growth, and have spurred the development of a large number of innovative cancer drugs and promising drug-based treatment strategies (e.g., immunotherapy). Simultaneously, the launch prices of new anticancer drugs, often with only marginal benefits for their approved indications, have increased over time and their use has contributed to the rising cost of cancer care. Today, about one tenth of the public expenditures for cancer in Europe (about 6–8% of the health care budget) are attributable to the costs of cancer drugs. Cancer drug prices have almost doubled from a decade ago, and nowadays the average price of one year treatment with a cancer drug or combination therapies in oncology may exceed € 100,000 per person. A recent survey of the prices of cancer drugs in high-income countries in Europe has disclosed marked variations in ex-factory prices of originator cancer drugs with Germany, Switzerland, and Sweden showing price data in similarly high ranges. In Germany, anticancer drugs represent the drug class with the highest level of spending in 2015 for the statutory health insurances: approx. 5 billions € only for outpatients. This strong upward trend in pricing of cancer drugs represents a significant challenge for our health-care system. Therefore, diverse measures have been proposed to agree upon drug prices according to their real benefits and to optimize reimbursement of cancer drugs. These include cost-effectiveness thresholds, pricing negotiations based on the early benefit assessment of new drugs within the framework of the act on the reform of the market for medicinal products (AMNOG), and programs recently introduced to help physicians, payers, and patients understand the value of new cancer drugs and thus make better choices about their use. The pros and cons of these initiatives will be briefly discussed. In order to ensure affordable drug-based treatment of cancer in the near future additional measures have to be taken which combine the development and availability of promising new cancer drugs with the sustainability of national health systems.

Disclosure: No conflict of interest disclosed.

Fortbildung – Noch kurativ oder schon palliativ? Ethik in der Onkologie

V374 – Indication in advanced cancer. Conceptual analyses and findings from empirical research

Schildmann J.1

1Ruhr Universität Bochum, Institut für Medizinische Ethik und Geschichte der Medizin, Bochum, Germany

There is considerable variation in decisions whether to offer patients with advanced cancer further cancer specific treatment or not. This variation cannot be fully explained by differences concerning the clinical situation or patients’ preferences. In this presentation I will firstly analyse the concept of “indication” and explore value related aspects of indication as one possible explanation for the variation observed in clinical practice. In a second step there will be a summary of findings from empirical research on factors possibly relevant for decisions about treatment in advanced cancer. In the final part I will discuss strategies which may support ethically informed decisions about the offering or limiting of cancer specific treatment in patients with advanced cancer.


Emanuel EJ, Young-Xu Y, Levinsky NG et al. Chemotherapy use among Medicare beneficiaries at the end of life. Ann Intern Med 2003;138:639–643.

Schildmann J, Tan J, Salloch S, Vollmann J: “Well, I think there is great variation...”: a qualitative study of oncologists´ experiences and views regarding medical criteria and other factors relevant to treatment decisions in advanced cancer. Oncologist 2013; 18:90–96.

Disclosure: No conflict of interest disclosed.

V375 – Early integration of palliative care!?

Lordick F.1

1University Medicine Leipzig, University Cancer Center Leipzig (UCCL), Leipzig, Germany

7 years ago ASCO released a statement that was adopted by the Arbeitsgemeinschaft Palliativmedizin of the German Cancer Society: “Palliative cancer care is the integration into cancer care of therapies that address the multiple issues that cause suffering for patients and their families and impact their life quality. Effective provision of palliative cancer care requires an interdisciplinary team that can provide care in all patient settings, including outpatient clinics, acute and long-term care facilities, and private homes. Changes in current policy, drug availability, and education are necessary for the integration of palliative care throughout the experience of cancer, for the achievement of quality improvement initiatives, and for effective palliative cancer care research. The need for palliative cancer care is greater than ever notwithstanding the strides made over the last decade. Further efforts are needed to realize the integration of palliative care in the model and vision of comprehensive cancer care by 2020.”The publication of the German multidisciplinary and multiprofessional evidence-based and expert-consented (S3) “Guideline for Palliative Care of Patients with Incurable Cancer” is an important step into this direction. Another milestone shall be the planned law for improving hospice and palliative care in Germany (Hospiz- und Palliativgesetz HPG) which was passed in November 2015.In the past, research data in the field of palliative care were scarce. Actions were taken on a more intuitive and empirical basis. Meanwhile, important research results have been published elucidating especially the role of early integration of palliative care into routine oncology. This raises the question who should deliver palliative care to cancer patients. First and foremost, we need to better understand the real burden on our patients, their preferences and care needs during the course of an incurable disease. This is why we started a multidisciplinary research program in the scientific working group (Sektion B) of the German Cancer Society. Eventually, we need to tailor “who shall provide” and “how to deliver” optimized palliative care. The current understanding is that every physician who cares for patients with severe and incurable diseases should have been trained and should be able to deliver general aspects of symptom relieve and palliative care while for complex palliative counceling and care the specialized palliative care physician is needed.

Disclosure: Florian Lordick: Advisory Role: Amgen, Biontech, Boston Biomedical, Ganymed, Lilly, MSD, Nordic, Roche, Taiho; Financing of Scientific Research: Vortragshonorare: Amgen, Celgene, Roche, Lilly; Expert Testimony: Böhringer-Ingelheim, GSK, Fresenius Biotech,; Immaterial Conflict of Interests: Reiseunterstützung: Amgen, Bayer, MSD, Roche, Taiho

V376 – More than quality of life assessment: How to value the usefulness of palliative care interventions in patients suffering from cancer

Alt-Epping B.1

1Universitätsmedizin Göttingen, Göttingen, Germany

Palliative Care has emerged from a „philosophy based on attitudes and skills“ (Saunders 2001) to an independent medical field with an according, framing, structural context. Since then, at the latest, similar standards, medical and economical, have to be applied on palliative care institutions (wards, consultation / liaison services, outpatient clinics, or specialized palliative home care) like on institutions and structures in any other medical field. The question, though, what characterizes “good” palliative care, how palliative care may be of use, and for whom, and how its usefulness can be determined appropriately, is much more complex to answer in palliative care compared to other „curative“ or interventional fields in medicine.

This congess contribution describes the multifaceted therapeutic aims in palliative care (including the patient´s perceptions as well as the family environment´s expectations), the problems to assess these therapeutic aims and the therapeutic effectiveness of palliative care interventions in an operationalizable manner (and thereby challenging current „quality of life“ concepts), and the resulting ethical implications on increasingly claimed standard value rating and benchmarking processes that have now reached the field of palliative care.

Disclosure: No conflict of interest disclosed.

V377 – Communication with patients and caregivers as preparation for palliative care

Hübner J.1

1Deutsche Krebsgesellschaft, Berlin, Germany

More and more, early integration of palliative care becomes an accepted part of cancer care. After removing essential doubts (increasing of anxiety, destroying hope) by clinical studies and with the growing evidence on the advantages with respect to symptom management, the question is open as to how early integration may be addressed and integrated in daily communication with the patients.

This task is demanding as it is more than just transmitting bad news. Hope might be an essential issue in early integration. The fundament of hope depends on the patient´s individual situation of disease as well as his/her perspectives of life. Additional considerations may be valuable. The needs of patients with advanced cancer are autonomy, individuality, self esteem, authenticity, appreciation.

The essential message to convey could be:

· We respect your needs and values.

· The physician who is responsible for you is reliably at your side.

· Its is you, whom we put in the center, your preferences and your aims.

· This attendance is a comprehensive – Survival, quality of life and meaning are important goals.

Disclosure: Jutta Hübner: Employment or Leadership Position: Deutsche Krebsgesellschaft; Advisory Role: Verschiedene Sozialgerichte

Fortbildung – Schwangerschaft und Krebs

V381 – Fertility after radio-chemotherapy

Dirksen U.T.1, IGHG

1Universitätsklinikum Münster, Münster, Germany

Five-year survival of childhood, adolescent and young adult cancer currently exceeds 70%. Late health outcomes research has demonstrated that a significant proportion of survivors experience chronic health sequelae. A prominent concern among young cancer survivors, which may causes profound emotional distress, is the increased risk of gonadotoxicity following specific cancer treatments. In female survivors, the risk of premature ovarian insufficiency (POI) from primary gland failure (premature menopause), is associated with infertility, but also with other serious sequelae secondary to estrogen deficiency, such as osteoporosis, cardiovascular disorders, impaired wellbeing and compromised sexual health.. In male survivors impaired spermatogenesis (ISG) results in reduced or lost fertility. Testosterone deficiency causes delayed or arrested puberty and a range of adverse somatic and psychological consequences in adults. Evidence-based guidelines are the backbone for a counseling interview at the time of diagnosis and a well-directed surveillance. The International Guideline Harmonization Group organized an endeavor to critically examine evidence and harmonize long-term follow-up guidelines. The working groups comprised experts from nine countries, chosen in view of their expertise and publications in endocrinology, andrology, pediatric and adolescent hematology/oncology, urology, gynecology, radiation oncology, psychology, epidemiology and guideline methodology. Systematic literature research was performed on publications from 1999 to 2014. Published data on the impact of single chemotherapeutic agents on female of male fertility was of limited quality. Definitions: POI was defined as amenorrhea < 40 years and two independent elevated FSH levels. ISG as azo- oligospermia (15 x 106 spermatozoa/ml) and/or low inhibin B- levels. POI-risk is increased after very high dose alkylating agents. The POI-risk following radiotherapy to the ovaries is age dependent. The sterilizing dose is 20.3Gy in infants, 18.4Gy at age 10 years, and 16.5Gy at age 20 years. Male infertility is associated with the treatment of high dose alkylating agents, total body irradiation and radiotherapy involving the testes of 2–3Gy. Following from this, an European consortium is making an attempt to gain more evidence- based data on the impact of systemic treatment on fertility under consideration of patient- derived individual hereditary risk factors.

Disclosure: No conflict of interest disclosed.

Freier Vortrag – Mammakarzinom

V384 – Determinants of long term quality of life in breast cancer patients: Results from the clinical cancer registry of the OCC Stuttgart

Meisner C.1, Heidemann E.2, Rössle S.2, Henke D.1, Brinkmann F.2

1Universitätsklinikum Tübingen, Institut für Klinische Epidemiologie und angewandte Biometrie, Tübingen, Germany, 2Onkologischer Schwerpunkt Stuttgart (OSP), Stuttgart, Germany

Introduction: Health related quality of life (QoL) of breast cancer patients after curative surgical treatment is an important endpoint in clinical trials but also in health services research. 2711 patients of the four breast cancer centers of the OCC Stuttgart were supported by a special service during a symptom oriented follow-up care program. These patients were asked to participate on periodic surveys for QoL. Aim of this paper was to describe long-term course of QoL and determine predictors of QoL in breast cancer patients.

Methods: A total of 2711 breast cancer patients were tested with the QoL instrument EORTC QLQ-C30. The patients were contacted by the special service by mail every 3 months. All domains of EORTC QLQ-C30 were estimated at different time intervals during their follow-up care up to five years and compared with a reference group of healthy German women.

Results: The breast cancer patients reported worse mean scores in all domains of EORTC QLQ-C30 at time of diagnosis. The functional scores increase in the first year of follow-up but not reaching the level of the healthy controls. Only marginal increases were seen for the functional scores of most of the patients after one year of follow-up care. The symptom scores decrease in the first year of follow-up. The scores for nausea/vomiting and appetite loss reach the level of the healthy controls. The greatest deficits are reported for fatigue, pain, dyspnea and insomnia also in long term. The differences between patients and healthy controls were smaller for age groups >60 years. Therapy-related and tumor biological factors had only marginal influence on QoL.

Conclusions: Data collection and analysis of quality of life data is an integral part of the routine in the Oncological Comprehensive Center Stuttgart. Data on the course of the domains of QoL according to EORTC QLQ-C30 for short and long term and analyses for determinants of QoL will be presented. They show a huge variability.

Key Words: Breast cancer · Quality of Life · Follow-up

Disclosure: No conflict of interest disclosed.

V385 – Patient-reported outcomes of 905 patients with breast cancer two years after start of curative systemic treatment

Marschner N.1, Trarbach T.2, Dörfel S.3, Meyer D.4, Müller-Hagen S.5, Zaiss M.6, Boller E.7, Kruggel L.7, TMK Registry Group

1Praxis für interdisziplinäre Onkologie & Hämatologie, Freiburg i.Br., Germany, 2Klinikum Wilhelmshaven gGmbH, Wilhelmshaven, Germany, 3Onkozentrum Dresden/Freiberg, Dresden, Germany, 4OSP Göttingen Dres. Meyer / Ammon / Metz, Göttingen, Germany, 5Hämatologisch-Onkologischer Schwerpunkt, Hamburg, Germany, 6Praxis für interdisziplinäre Onkologie & Hämatologie, Müllheim, Germany, 7iOMEDICO AG, Freiburg i.Br., Germany

Introduction: Patients (Pts) with localized, operable breast cancer have to deal with the psychological strain of having cancer as well as with side effects during therapy. However, what is their situation 2 years (yrs) after start of the systemic antineoplastic treatment? MaLife prospectively investigates Patient-reported-outcomes (PROs) to better understand the real-life situation of these pts during and after treatment.

Patients and methods: MaLife is conducted with the Tumour Registry Breast Cancer II (TMK II), an open, prospective, multicenter, observational study of pts recruited at the start of (neo)adjuvant or palliative systemic therapy in Germany. Besides documentation of medical data pts regularly receive a set of questionnaires for 5 yrs. Here, data on 905 pts recruited from more than 100 sites and their PROs (FACT-G, EORTC-QLQ-BR23, FACT-Taxane, HADS-D and specifically developed questionnaires) are presented.

Results: Median age at start of systemic therapy was 56 yrs. Endocrine therapy (ET) was documented for 58% of pts, 93% of pts received chemotherapy. For all pts observed = 2 yrs, questionnaire return rate was 78% 2 yrs after start of therapy. For 5% of pts relapse or death was documented and therefore questionnaire shipping was stopped. 2 yrs after start of therapy the global quality of life slightly improved (FACT-G, +4%), mainly because of an increase in the physical and functional well-being subscores (+12% each). Future perspective increased by 15% (EORTC-QLQ-BR23 subscale). Systemic therapy side effects (EORTC-QLQ-BR23 subscale) and gastrointestinal symptoms, i.e. obstipation and lack of appetite (specific items), decreased by at least 10%. Pts still reported about neuropathic symptoms (FACT-Taxane, mean change from start of therapy to 2 yrs: +9%), pain in neck, shoulders, back and hips (specific items, +9–11%) and problems in memorizing (specific item, +12%). Pts receiving ET were affected by increased bone pain (specific item, +18%). 31% and 30% of pts showed elevated anxiety at start of therapy and after 2 yrs, respectively. Elevated depression was reported by 29% and 23% of pts at start of therapy and after 2 yrs, respectively (HADS-D).

Conclusions: Although 2 yrs after start of therapy pts report better physical and functional well-being and fewer side effects, they still report symptoms of polyneuropathy, pain in the upper body and problems in memorizing.

Disclosure: No conflict of interest disclosed.

V386 – Influence of arm crank ergometry on development of lymphedema in breast cancer patients after axillary dissection

Schmidt T.1, Berner J.2, Jonat W.2, Weisser B.3, Röcken C.1, Mundhenke C.2

1Krebszentrum Nord, Universitätsklinikum Schleswig-Holstein, Kiel, Germany, 2Klinik für Gynäkologie und Geburtshilfe, Universitätsklinikum Schleswig-Holstein, Kiel, Germany, 3Institut für Sportwissenschaft, Christian-Albrechts-Universität, Kiel, Germany

Introduction: Breast cancer-related lymphedema of the arm can be a long-term sequela of medical treatment and is associated with reduced physical functioning and quality of life. In the past physical activity has been proscribed for women with breast-cancer-related lymphedema. The purpose of this study was to assess the safety and efficacy of an arm crank ergometer (ACE) in breast cancer patients with axillary dissection.

Method: In a randomized, controlled clinical intervention trial twice-weekly supervised ACE-training was compared with usual care (UC) in 49 breast cancer patients after medical treatment over 12 weeks. Endpoints were change in body composition of the upper extremity measured by bioelectrical impedance analysis (BIA), arm volume (arm circumference), muscular strength (NM), quality of live (QL) (EORTC QLQ C30+BR23) and fatigue (MFI 20) before and after 12 weeks.

Results: During the intervention no exacerbate was detected. Patients in the ACE improved significantly in lean body mass (LBM) (OP-arm p: 0.017; control-arm p: 0.004), skeletal muscle mass (ACE p: 0.049; UC p: 0.385) and in a significant decrease in body fat (ACE p: 0.009; UC p: 0.393). In the ACE, as well the UC group a significant increase of the armpit circumference was detected during the training period. The magnitude of the gain was higher in the UC. In all other measured regions of the arm a significant decrease of circumference was seen in both groups. As compared with the control group, the ACE-group had a greater improvement in muscular strength of the upper extremity (ACE p: n.s.; UC p: n.s.). In both groups a trend for improvement of QL and general fatigue (ACE p: n.s.; UC p: n.s.) was seen. In the ACE-group a significant improvement in physical functioning (ACE p: 0.39; UC p: 0.355) and a decrease in physical fatigue (ACE p: 0.001; UC p: 0.519) were detected.

Conclusion: The results confirm the safe execution of the training with an ACE and highlight improvements in strength, QL and reduced symptoms.

All authors declare no conflict of interest.

Disclosure: No conflict of interest disclosed.

V387 – Mcl-1 inhibition as a novel approach for personalized breast cancer therapy

Bashari M.H.1,2, Malvestiti S.1, Fan F.1, Vallet S.1, Sattler M.3, Cardone M.H.4, Opferman J.T.5, Jäger D.1, Podar K.1

1National Center for Tumor Diseases (NCT), University of Heidelberg, Medical Oncology, Heidelberg, Germany, 2Faculty of Medicine, Universitas Padjadjaran, Department of Pharmacology and Therapy, Bandung, Indonesia, 3Dana-Farber Cancer Institute, Harvard Medical School, Medical Oncology, Boston, United States, 4Eutropics Pharmaceuticals, Inc., Cambridge, United States, 5St. Jude Children’s Research Hospital, Memphis, United States

Introduction: Despite significant therapeutic improvements over the last two decades, breast cancer (BC) remains the leading cause of cancer-related mortality in women. Anti-apoptotic Bcl-2 family members including Bcl-2, Bcl-xL and Mcl-1 are frequently upregulated in solid and hematologic malignancies and thereby violate the programmed cell death. Based on our own and other studies, Mcl-1 levels, in particular, are consistently elevated in Her2-positive and triple negative (TN)BC and correlate with drug resistance and poor prognosis. Mcl-1 therefore represents a promising target for BC therapy.

Methods: The anti-BC cell-activity of the novel small molecule Mcl-1 inhibitor EU5346 was evaluated alone or in combination with other antiapoptotic agents, hormone therapy, targeted therapy, or chemotherapy using proliferation and spheroid forming assays as well as immunoblotting. Synergistic activity of combination therapies was determined by the Chou-Talalay method. Protein level-based activity of EU5346, the anti-Bcl-2 inhibitor ABT-199 and the anti-BCL-xL inhibitor WEHI-539 was verified in Mcl-1?/nullversus Mcl-1wt/wt Murine Embryonic Fibroblasts.

Results: Using protein profiling and ONCOMINE analysis of our own and publically available data showed that Mcl-1 is consistently overexpressed not only in Her2-positive and TN, but also Luminal A BC cells, while Bcl-2 and Bcl-xL expression is variable. As expected, anti-BC activity of ABT-199 and WEHI-539 in Her2-positive, TN, and Luminal A BC cells was based on Bcl-2 and Bcl-xL protein levels. In order to predict the IC50 of EU5346 in BC cells we additionally developed a mathematical scoring system on the protein levels of Bak, Bim, and NOXA. Synergistic anti-BC activity of low dose EU5346 with ABT-199 or WEHI-539 was observed only in those cells expressing Bcl-2 or Bcl-xL, respectively. Similarly, low dose EU5346 also induced significant anti-BC activity in Luminal A cells, when combined with tamoxifen; in Her2-positive BC cells, when combined with trastuzumab; and in TNBC cells, when combined with paclitaxel. Finally, EU5346 overcame resistance to paclitaxel in paclitaxel-resistant TNBC cells.

Conclusions: Our data further support a key role of Mcl-1 in the survival and drug resistance of BC cells. Importantly, they provide the preclinical framework for the personalized, clinical use of Mcl-1-inhibitor-based drug combinations with antiapoptotic, antihormonal or chemotherapies to improve patient outcome in BC.

Disclosure: Muhammad Bashari: Expert Testimony: MHB received research support from EUTROPICSKlaus Podar: No conflict of interest disclosed.

V388 – Novel oncogenic roles of the transcriptional regulator EVI1 in ER- breast carcinoma

Wang H.1,2, Schäfer T.1, Konantz M.1, Braun M.3, Perner S.4,5, Varga Z.6, Moch H.6, Reich S.2, Fehm T.7, Kanz L.2, Schulze-Osthoff K.8,9, Lengerke C.1,2,10

1University Hospital Basel, Department of Biomedicine, Basel, Switzerland, 2University of Tuebingen Medical Center II, Department of Hematology & Oncology, Tuebingen, Germany, 3University Hospital of Bonn, Department of Prostate Cancer Research, Institute of Pathology, Bonn, Germany, 4University of Luebeck, Institute of Pathology, Luebeck, Germany, 5The Leibniz Research Center Borstel, Borstel, Germany, 6University Hospital Zurich, Institute of Surgical Pathology, Zurich, Switzerland, 7University Hospital Duesseldorf, Women´s Hospital, Duesseldorf, Germany, 8University of Tuebingen, Interfaculty Institute of Biochemistry, Tuebingen, Germany, 9German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Germany, 10University Hospital Basel, Clinic for Hematology, Basel, Switzerland

Introduction: Ecotropic viral integration site-1 (EVI1) is a transcription factor, particularly known for its role in acute myeloid leukemia aggressiveness. Aberrant EVI1 expression is also detectable in several solid tumors. Here, we explore EVI1 in breast carcinoma and find that high EVI1 expression particularly impacts prognosis and biology of the estrogen receptor negative (ER-) subset.

Methods: EVI1 expression was analyzed by qRT-PCR, immunoblot and respectively immunohistochemistry in 8 human BC cell lines and a tissue microarray (TMA) containing 608 patient samples. EVI1 expression was modulated in BC cell lines and primary cells using lentiviral vectors and effects analyzed in cell cycle, BrdU, apoptosis, tumor sphere, migration and in vivo tumorigenicity (zebrafish and mouse) xenotransplantation assays and molecularly by microarray gene expression and ChIP assays.

Results: EVI1 expression was detectable in ~80% of BC samples, independent of their ER status; however, high expression associated with shorter overall survival in ER- (p = 0.028) but not ER+ (p = 0.307) patients. Functionally, EVI1 downregulation impaired BC cell proliferation by inducing a G0/G1 arrest and enhancing apoptosis. Furthermore, EVI1 knockdown suprpessed tumor sphere formation and migration of BC cells. Gain-of-function studies using EVI1 overexpression induced opposite phenotypes confirming these data. Moreover, EVI1 knockdown suppressed in vivo tumorigenicity of human BC cells xenotransplanted in fish and mice. Interestingly, treatment with ß-estradiol rescued growth and in vivo tumorigenicity of ER+ (but not ER-) EVI1 knockdown BC cells. Molecularly, EVI1 induced the MAPK-pathway and the expression of GPCR signaling associated genes such as KISS1 which could be identified by ChIP as a direct EVI1 target. In line, stimulation with the KISS1 ligand Kisspeptin induced MDAMB231 cell migration, and functionally rescued migration in EVI1 knockdown ER- but not ER+ BC cells.

Conclusion: EVI1 expresses in a majority of breast carcinoma samples independent of their ER status. However, in the presence of estrogen, EVI1 selectively impacts the biology of ER- tumors where it drives cell growth via MAPK and migration via KISS1. Although not selectively marking breast cancer stem cells (CSCs), EVI1 expression appears to co-regulate this compartment. Prospective studies are needed to analyze the importance of EVI1 as a biomarker in breast carcinoma.

Disclosure: No conflict of interest disclosed.

V389 – The role of FGFR1 signaling in breast cancer to induce tumor angiogenesis

Golfmann K.1, Meder L.1, Schuldt P.1, Ullrich R.1,2

1University of Cologne, Cancer Therapy and Molecular Imaging, Clinic I for Internal Medicine and Centre for Molecular Medicine Cologne (CMMC), Cologne, Germany, 2University Hospital of Cologne, Center for Integrated Oncology, Cologne, Germany

The phase I study of BGJ398, a potent, selective pan-FGFR inhibitor, demonstrated no clinical efficacy in breast cancer harboring amplification of FGFR1. In contrast, Lucitanib and also Dovitinib, dual FGFR and VEGFR inhibitors, demonstrated promising results in patients with FGFR1 amplified breast cancer. Hence, FGFR1 amplification seen in breast cancer may not be as much of a driver event in these patients. We here aim to decipher the role of FGFR1 for tumor angiogenesis and to identify a possible feedback mechanism in breast cancer.

Preclinical study with BGJ398, Dovitinib and BAY1 (a selective FGFR inhibitor from BAYERHealthcare) on FGFR1 amplified breast cancer cell lines. Copy number and expression profile were prior tested. Multiplex ELISA, Western Blot analysis and knockdown experiments were performed to unravel the molecular downstream pathway. All in vivo studies were conducted with an orthotopic breast cancer model and tumor growth was measured by BLI_µCT.

Breast cancer cell lines MDA-MB-134, CAL-120 and JIMT-1, carrying the amplified FGFR1 gene, were not sensitive to selective FGFR1 inhibition in vitro and knockdown of FGFR1 with shRNA does not repressed cellular growth. Nevertheless, BGJ398 and BAY1 effectively inhibited phosphorylation of FGFR1 and downstream MEK/ERK signalling without deregulating the AKT pathway. We demonstrated that FGFR1 amplified breast cancer cells upregulate VEGF-A expression and secretion under FGF-2 stimulation via activation of PI3K/AKT signalling cascade. Only a combination of FGFR1 and PI3K- inhibition causes significant VEGF-A reduction. We also demonstrated that FGFR1 overexpression was positively correlated with vessel density and tumour growth in vivo. Finally, Dovitinib treatment, in contrast to BGJ398, resulted in significant tumour growth inhibition in FGFR1-amplified orthotopic breast cancer xenografts

The results underscore the angiogenic role of FGFR1-mediated signaling in breast cancer. They provide important insights into FGFR1 signaling and a possible feedback mechanism in FGFR1 amplified breast cancer patients.

Disclosure: No conflict of interest disclosed.

Freier Vortrag – Niedrigmaligne B-Zell-Lymphome – Klinik

V390 – Two years Rituximab maintenance vs. observation after first line treatment with bendamustine plus rituximab (B-R) in patients with mantle cell lymphoma: first results of a prospective, randomized, multicenter phase 2 study (a subgroup study of the StiL NHL7-2008 MAINTAIN trial; Clinicaltrials.gov Identifier: NCT00877214)

Rummel M.1, Knauf W.2, Goerner M.3, Söling U.4, Lange E.5, Hertenstein B.6, Eggert J.7, Schliesser G.C.8, Weide R.9, Blumenstengel K.10, Detlefsen N.11, Hinke A.12, Kauff F.13, Barth J.13, Studiengruppe indolente Lymphome (StiL)

1Justus-Liebig-Universität, Universitätsklinik, Med. Kl IV, Gießen, Germany, 2Onkologische Praxis, Frankfurt, Germany, 3Städtisches Klinikum, Bielefeld, Germany, 4Onkologische Praxis, Kassel, Germany, 5Evangelisches Krankenhaus, Hamm, Germany, 6Klinikum Bremen Mitte, Hämatologie und Onkologie, Bremen, Germany, 7Onkologische Praxis, Moers, Germany, 8Onkologische Praxis, Gießen, Germany, 9Praxis für Hämatologie und Onkologie, Koblenz, Germany, 10Praxis für Hämatologie und Onkologie, Eisenach, Germany, 11Justus-Liebig-Universität, Universitätsklinik, Gießen, Germany, 12WiSP, Wissenschaftlicher Service Pharma GmbH, Langenfeld, Germany, 13Justus-Liebig-Universität, Universitätsklinik, Hämatologie, Med Kl. IV, Gießen, Germany

Introduction: Rituximab maintenance is part of a standard treatment approach for follicular lymphoma. In mantle cell lymphoma (MCL), however, it is not yet common practice. In this study we compared the effect of rituximab maintenance vs observation after first-line treatment with B-R in patients with previously untreated MCL.

Methods: Patients were required to have stage II (with bulky disease >7 cm), III, or IV disease for registration in this study. Primary endpoint was progression free survival (PFS). Secondary endpoints included response rates, overall survival (OS), time to progression, event free survival, toxicity. Patients were treated with up to 6 cycles of B-R plus 2 additional cycles rituximab. 122 Patients who have responded to B-R were then randomized to either rituximab maintenance (375 mg/m2 every 2 months for a total of 2 years) or observation only.

Results: A total of 118 patients were evaluable for the analysis, 58 (49%) were randomized to maintenance with rituximab and 60 (51%) to observation, respectively. Patient characteristics were comparable for both groups. Median patient age was 71 years, median time of observation was 57,7 months at the time of this analysis (April 2016).

No statistical significant differences in PFS between both arms could be observed (p = 0.211, 49 events, HR 0.70, 95% CI 0.40 - 1.22). The median PFS for R maintenance was 74.8 months (95% CI 65.4 - nyr). For the observation arm the median PFS was 54.7 months (95% CI 40.1. - n. y. r.). The results for overall survival showed no difference (p = 0.338, 27 events, HR 1.45, 95% CI 0.68 - 3.10) with a median of nyr for both arms.

Conclusions: After a median observation time of 4.8 years from 1st cycle B-R, the results are yet inconclusive. Up to date we were not able to demonstrate a statistical evidence supporting the benefit of R maintenance after B-R in the treatment of patients with MCL. Longer follow-up is needed before final results can be presented.

Disclosure: Mathias Rummel: Advisory Role: Mundipharma, Roche; Financing of Scientific Research: Mundipharma, RocheJürgen Barth: Financing of Scientific Research: Mundipharma, Roche

V391 – Single-agent Ibrutinib is efficacious and well tolerated in Rituximab-refractory patients with Waldenström’s Macroglobulinemia (WM): Initial results from an international, multicenter, open-label phase 3 substudy (iNNOVATE™)

Buske C.1, Trotman J.2, Tedeschi A.3, Matous J.V.4, Macdonald D.5, Tam C.6, Tournilhac O.7, Ma S.8, Oriol A.9, Heffner L.T.10, Shustik C.11, Garcia-Sanz R.12, Cornell R.F.13, Fernández de Larrea C.14, Castillo J.J.15, Granell M.16, Kyrtsonis M.-C.17, Leblond V.18, Symeonidis A.19, Singh P.20, Li J.20, Graef T.20, Bilotti E.20, Treon S.15, Dimopoulos M.A.21, on behalf of the iNNOVATE™ investigators

1Comprehensive Cancer Center Ulm, Institute of Experimental Cancer Research, University Hospital of Ulm, Ulm, Germany, 2Concord Hospital, University of Sydney, Concord, Australia, 3Niguarda Ca’ Granda Hospital, Milan, Italy, 4Colorado Blood Cancer Institute, Denver, United States, 5Dalhousie University, Halifax, Canada, 6Peter MacCallum Cancer Centre & St. Vincent’s Hospital, Melbourne, Australia, 7Hématologie Clinique Adulte et Thérapie Cellulaire, CHU Clermont-Ferrand, Clermont-Ferrand, France, 8Robert H. Luri Comprehensive Cancer Center, Feinberg School of Medicine, Northwestern University, Chicago, United States, 9Institut Català d’Oncologia, Hospital Germans Trias i Pujol, Barcelona, Spain, 10Winship Cancer Institute of Emory University, Atlanta, United States, 11Royal Victoria Hospital at McGill University Health Centre, Montreal, Canada, 12Hospital Universitario de Salamanca, Salamanca, Spain, 13Vanderbilt-Ingram Cancer Center, Nashville, United States, 14Hospital Clínic de Barcelona, IDIBAPS, Barcelona, Spain, 15Dana-Farber Cancer Institute, Boston, United States, 16Hospital Sant Pau, Barcelona, Spain, 17National and Kapodistrian University of Athens, School of Medicine, Athens, Greece, 18Hôpital Pitié-Salpêtrière APHP, UPMC Université Paris, Paris, France, 19University of Patras Medical School, Patras, Greece, 20Pharmacyclics LLC, an AbbVie Company, Sunnyvale, United States, 21National and Kapodistrian University of Athens School of Medicine, Athens, Greece

Introduction: Single-agent ibrutinib (ibr) demonstrated durable responses in previously treated patients (pts) with WM (Treon, NEJM 2015). We report efficacy and safety of ibr in WM pts refractory to their last rituximab-containing therapy (relapse after < 12 months or failure to achieve at least a minor response).

Methods: Pts with confirmed diagnosis of WM, rituximab refractory, symptomatic disease requiring treatment (Kyle 2003), platelets =50,000 cells/mm3, and absolute neutrophil count =750 cells/mm³ received oral ibr 420 mg daily continuously until progressive disease (PD) or unacceptable toxicity. Study endpoints included PFS, ORR, OS, and hemoglobin (Hgb) improvement.

Results: Among 31 treated pts, median age was 67 years (range, 47–90); 42% had a high International Prognostic Score System for WM, with a median of 4 prior therapies (68% with =3 prior therapies) and ECOG 2 in 19%. In addition to rituximab, the most common prior therapies were corticosteroids and alkylating agents (81% each). Median Hgb and IgM at baseline were 10.3 g/dL and 3830 mg/dL, respectively. Any-grade adverse events (AEs; >15%) included diarrhea (42%); upper respiratory tract infections, hypertension, increased bruising (23% each); nausea, thrombocytopenia, neutropenia (19% each); and pyrexia, arthralgia, back pain (16% each). Common grade =3 AEs included neutropenia (13%), hypertension (10%), anemia and diarrhea (6% each). Serious AEs occurred in 29% pts. Four pts discontinued ibr—2 due to PD and 2 due to an AE (gastrointestinal AL amyloidosis and diarrhea); 27 (87%) continue ibr. No IgM flare, atrial fibrillation or major bleeding occurred. Four of 5 pts had no need for plasmapheresis for disease control at the end of cycle 1. ORR at a median follow-up of 12.7 months was 84% (major response rate [MRR; =partial response] of 68%), and the estimated 1-year PFS rate was 93%. Baseline median Hgb increased to 12.8 g/dL at Week 49. Baseline median IgM declined by >50% after 1 cycle, with continued improvement over time (median 920 mg/dL at Week 49). Initial analysis of 23 baseline samples identified MYD88MUT/CXCR4WT in 16 pts and MYD88MUT/CXCR4WHIM in 6 pts. ORR and MRR were 88% vs. 83% and 75% vs. 83%, respectively. One of the 23 pts had MYD88WT/CXCR4WT with a best response of stable disease.

Conclusions: Single-agent ibr is highly active (84% ORR) and well tolerated in the rituximab-refractory WM population and presents an attractive treatment option in this patient population.

Disclosure: Christian Buske: Employment or Leadership Position: N/A; Advisory Role: Janssen, Roche, Gilead; Stock Ownership: N/A; Honoraria: N/A; Financing of Scientific Research: Roche, Celgene, Janssen; Expert Testimony: Roche, Janssen; Other Financial Relationships: Travel, accommodations, expenses: Roche, Celgene, Janssen; Immaterial Conflict of Interests: N/AMeletios Dimopoulos: Employment or Leadership Position: N/A; Advisory Role: Janssen, Celgene, Onyx, Amgen, Novartis; Stock Ownership: N/A; Honoraria: N/A; Financing of Scientific Research: Janssen, Celgene, Onyx, Amgen, Novartis; Expert Testimony: Genesis Pharma; Other Financial Relationships: N/A; Immaterial Conflict of Interests: N/A

V392 – Treatment and outcome patterns in patients with relapsed Waldenström Macroglobulinemia from a large observational Pan-European data platform

Buske C.1, Sadullah S.2, Kastritis E.3, Tedeschi A.4, Garcia-Sanz R.5, Bolkun L.6, Leleu X.7, Willenbacher W.8, Hajek R.9, Minnema M.C.10, Cheng M.11, Graef T.11, Dimopoulos M.A.3, on behalf of the European Consortium for Waldenström’s Macroglobulinemia (ECWM)

1University Hospital Ulm, Ulm, Germany, 2James Paget University Hospital, Norfolk, United Kingdom, 3National and Kapodistrian University of Athens, Athens, Greece, 4Niguarda Ca’ Granda Hospital, Milan, Italy, 5Complejo Asistencial Universitario de Salamanca, Salamanca, Spain, 6Medical University Hospital of Bialystok, Bialystok, Poland, 7Hopital de La Milétrie, CHU, Poitiers, France, 8Innsbruck Medical University, Innsbruck, Austria, 9University Hospital of Ostrava, Ostrava, Czech Republic, 10University Medical Center Utrecht, Utrecht, Netherlands, 11Pharmacyclics LLC, an AbbVie Company, Sunnyvale, United States

Introduction: Data on treatment choices and their outcome in patients (pts) outside clinical trials are lacking for Waldenström macroglobulinemia (WM). The project goal was to generate real-world data on diagnosis, treatment patterns, and outcomes for WM over a decade in a large pan-European effort.

Methods: Retrospective record reviews were completed for pts who fit the following criteria: confirmed WM, symptomatic disease at treatment initiation, frontline (FL) treatment started between Jan 2000 and Dec 2010, at least 1 salvage regimen, and availability of clinical/biologic evaluation at diagnosis/initial therapy. Endpoints included initial/subsequent treatment choices, progression-free survival (PFS), and overall survival (OS).

Results: Charts were reviewed for 368/155 pts across second line (2L)/third line (3L). Median age at treatment initiation was 63 yr (range, 29–89). Reasons for initiating treatment were cytopenias (77%; anemia [75%]), constitutional symptoms (56%), IgM-related symptoms (55%), and organomegaly (26%). Monotherapy use fell from 35% in FL to 21%/22% in 2L/3L. Age =70 was associated with greater FL use of monotherapy (40%) vs combination therapy (29%). Combination therapy with rituximab increased from 36% in FL to 63%/56% in 2L/3L. Across all lines, rituximab and cyclophosphamide were the most common agents, excluding steroids, used as monotherapy or in combinations. Median PFS decreased from 31 mo for FL to 24/16 mo for 2L/3L. Median PFS varied by country and choice of agents but was similar for pts < 50 and =50 yr. Median PFS improved for pts who received rituximab in 2L vs pts who did not (26 vs 19 mo, P = 0.014). PFS was similar in 3L with regard to rituximab use (15 vs 16 mo, P = 0.69). FL rituximab did not affect median PFS of subsequent lines (2L: 24 vs 23 mo, P = 0.87; 3L: 13 vs 16 mo, P = 0.12). Median OS was not reached but was significantly lower in pts =75 yr (70 mo; P < 0.0001) or in pts with high-risk IPSSWM risk score (91 mo; P = 0.0014). No difference in OS was observed in pts with FL rituximab compared to pts not treated with FL rituximab. Other malignancies were reported in 14% after treatment for WM.

Conclusions: For European WM pts, monotherapy is widely used, even at first relapse, with notable differences between countries. Rituximab use was associated with improvement in median 2L PFS. This large dataset will promote understanding of WM treatment practices and survival outside of a clinical trial setting.

Disclosure: Christian Buske: Advisory Role: Janssen, Roche, Gilead; Financing of Scientific Research: Roche, Celgene, Janssen; Expert Testimony: Roche, JanssenMeletios Dimopoulos: Advisory Role: Janssen, Celgene, Onyx, Amgen, Novartis; Financing of Scientific Research: Janssen, Celgene, Onyx, Amgen, Novartis; Expert Testimony: Genesis Pharma

V393 – IgH-based Next-Generation Sequencing for MRD detection in follicular lymphoma

Herzog A.1, Knecht H.1, Herrmann D.1, Unterhalt M.2, Hiddemann W.2, Kneba M.1, Brüggemann M.1, Hoster E.2, Pott C.1

1Medizinische Klinik II, Universitätsklinikum Schleswig-Holstein, Campus Kiel, Kiel, Germany, 2Medizinische Klinik und Poliklinik III, Klinikum der Universität München, München, Germany

Introduction: MRD monitoring is of great value to determine the risk of relapse in patients with follicular lymphoma (FL). However, MRD assessment is restricted to those 50%-65% of patients harbouring a PCR detectable t(14;18) rearrangement as marker for sensitive RQ-PCR. Next generation sequencing (NGS) of IGH might overcome limitations of current PCR based approaches. Therefore we applied an optimized IGH-based NGS approach to identify tumor-specific IGH (V-(D)-J) clonotypes and tested the reliability for quantitation of MRD in FL patients.

Methods: We selected 94 diagnostic (PM) with 166 follow-up (FU) samples from 86 FL patients of the German Low-Grade Lymphoma Study Group (GLSG) according to the presence of a clonal IGH rearrangement detectable by consensus PCR. Paired-end libraries were prepared and sequenced on Illumina MiSeq (2 x 250bp). FU samples were spiked with predefined copy numbers of internal reference DNA to enable assessment of MRD by normalisation of NGS reads after sequencing. Identification of IGH clonotypes and quantitation of MRD was done with the bioinformatic analysis pipeline Arrest/Interrogate (Bystry et al. 2016, under revision). For validation of NGS results t(14;18) RQ-PCR from 15 patients (18 PM and 35 FU) and IGH ASO-PCR from 15 patients (15 PM and 31 FU) was performed.

Results: Of 30 FL Patients investigated by RQ-PCR in parallel, NGS identified an IGH clonotype in 26 PM. In the 4 NGS negative samples RQ-PCR demonstrated low level MRD below 10-3. 24/57 FU samples were MRD+ and 16 MRD- by both methods. 17 FU were MRD+ by either RQ-PCR (n = 14) or NGS (n = 3). 9 of the NGS negative samples showed very low-level RQ-PCR MRD (10-5 ). MRD results determined by NGS were well correlated to RQ-PCR values (r2 = 0.599, p < 0.000001).In additional 56 Patients without RQ-PCR assays but qualitative detection of clonal IGH rearrangements using GeneScan, NGS identified an IGH clonotype in 52/56 (92.8% ) of the FL patients. 26/93 FU samples were MRD+ and 67 MRD-. 14 were MRD+ by NGS only, while in 2 samples only GeneScan showed MRD+.

Conclusions: The application of IGH-NGS showed robust identification of clonotypes in FL patients allowing reliable and sensitive MRD assessment. However, lymphoma infiltration of PM and somatic mutation of IGH are critical points for identification of the tumor-specific clonotypes by NGS. IGH-NGS has a great potential to complement current MRD methods allowing MRD assessment in the majority of patients.

Disclosure: No conflict of interest disclosed.

V394 – 1st-line treatment of patients with mantle cell lymphoma: Treatment reality and first outcome data from the German prospective TLN Registry

Knauf W.1, Bertram M.2, Fietz T.3, Kirste T.4, Grugel R.5, Schnitzler M.5, Abenhardt W.6, TLN Registergruppe

1Onkologische Gemeinschaftspraxis, Frankfurt a.M., Germany, 2Hämatologisch-Onkologischer Schwerpunkt, Hamburg, Germany, 3Praxis für Innere Medizin, Hämatologie und Onkologie, Singen (Hohentwiel), Germany, 4Praxis für interdisziplinäre Onkologie & Hämatologie, Freiburg i.Br., Germany, 5iOMEDICO, Freiburg i. Br., Germany, 6MVZ Onkologie im Elisenhof, München, Germany

Introduction: Amongst non-Hodgkin lymphomas (NHL), the mantle cell lymphoma (MCL) represents a rather more aggressive type with poor prognosis. For the young patients (pts) guidelines recommend a dose-intensified immuno-chemotherapy followed by autologous stem-cell transplant (ASCT) as first-line treatment (Dreyling, 2014, 2016). However, the majority of pts are elderly (>65 years), and therefore the choice of chemotherapy is influenced significantly by the pts’ general state of health. While clinical trials are restricted to highly selected pts, clinical registries offer insight into real-life treatment and effectiveness.

Methods: The open, prospective, clinical registry on lymphoid neoplasms (TLN Registry, ClinicalTrial.gov NCT00889798) documents the treatment of pts with lymphoid B-cell neoplasms by German office-based haematologists. Pts are followed for 5 years (yrs). Data regarding pts and tumour characteristics, co-morbidities, all systemic treatments and response rates, date(s) of progression(s) and date of death are recorded. Between 2009 and 2014, a total of 3,795 pts have been recruited by 122 study sites.

Results: Of 1,078 pts with low-grade NHL recruited between 04/2009 and 02/2014 at the start of 1st-line therapy, 139 were diagnosed with / treated for MCL.

Pts were median 72yrs old (range 31–92yrs, 74% older than 65yrs), 68% male, 82% presented with tumour stage III/IV (Ann Arbor), 6% with ECOG=2 and 71% with co-morbidities (7% moderate/severe renal disease, 15% diabetes, 48% hypertension, 7% heart failure, 4% heart infarction).

61% were treated with bendamustine+rituximab (BR), 18% received rituximab+cyclophosphamide+doxorubicine+vincristine+prednisone (R-CHOP); other regimens were used in individual cases only (each =2%).

Objective response rate (ORR) as assessed by the local site was 87% with the clinical (unconfirmed) complete remission rate of 46%.

With a median follow-up of 39 months (mth), prospective median progression free survival (PFSREG) is 62mth (95%-CI 44mth-NA) and 3yrs PFS rate is 65% (95%-CI 54%-73%;). 3yrs overall survival rate (OS) is 76%, while median OS is not reached. 25% of pts have received a 2nd-line therapy. Overall, 13% of pts have been lost to follow-up.

Conclusion: In office-based practices in Germany, pts with previously untreated MCL are predominantly treated with BR. First outcome data show that the effectiveness (ORR, PFS and OS) for unselected pts in routine practice is similar to pts treated in trials.

Disclosure: Wolfgang Knauf: Advisory Role: Advisory Board Roche, Mundipharma; Financing of Scientific Research: Vortragshonorare Roche, Mundipharma; Other Financial Relationships: Reisekosten Roche, MundipharmaWolfgang Abenhardt: Employment or Leadership Position: Ärztlicher Geschäftsführer der MVZ; Advisory Role: Advisory Board bei AMGEN, BMS, Celgene, Gilead, Janssen, MSK, Novartis, ROCHE; Other Financial Relationships: Unterstützung des Münchner Lymphom Workshops durch AMGEN, Celgene, Gilead, ROCHE

V395 – CLL2-BIG – a sequential treatment regimen of Bendamustine followed by GA101 and Ibrutinib followed by Ibrutinib and GA101 maintenance in patients with Chronic Lymphocytic Leukemia (CLL): Interim results of a phase II-trial by the German CLL Study Group

von Tresckow J.1, Cramer P.1, Bahlo J.1, Engelke A.1, Langerbeins P.1, Fink A.-M.1, Klaproth H.2, Tausch E.3, Fischer K.1, Wendtner C.-M.1,4, Kreuzer K.-A.1, Stilgenbauer S.3, Böttcher S.5, Eichhorst B.1, Hallek M.1

1Uniklinik Köln, Klinik I für Innere Medizin/Zentrum für Integrierte Onkologie/Deutsche CLL Studiengruppe, Köln, Germany, 2Hämatologische/Onkologische Praxis Dr. Schmidt/Klaproth, Neunkirchen, Germany, 3Uniklinik Ulm, Klinik III für Innere Medizin, Ulm, Germany, 4Klinikum Schwabing, Klinik für Hämatologie, Onkologie, Immunologie, Palliativmedizin, Infektiologie und Tropenmedizin, München, Germany, 5Universitätsklinikum Schleswig-Holstein, Campus Kiel, Klinik für Innere Medizin II, Kiel, Germany

Introduction: Treatment with anti-CD20 antibodies and targeted drugs show promising efficacy and tolerability in CLL.

With the CLL2-BIG trial, a novel combination regimen according to the “sequential triple-T” concept of a tailored and targeted treatment aiming at total eradication of minimal residual disease (MRD) was designed.

Methods: Physically fit and unfit CLL-patients were recruited according to a predefined allocation for the two strata of first-line and relapse/refractory treatment.

Patients with high tumor burden received two cycles of bendamustine debulking before administration of six cycles of induction treatment with obinutuzumab and ibrutinib. Maintenance therapy with ibrutinib and obinutuzumab every three months followed until achievement of MRD-negative complete remission or up to 24 months.

The primary endpoint is overall response rate (ORR) at the end of induction; secondary endpoints include ORR after debulking and safety parameters.

Results: 66 patients were included, thereof 31 first-line and 35 relapsed/refractory with a median number of one prior therapy (range 1–5). The median age was 66.5 (36–85) years and the median CIRS score was 2.5 (0–11). 10 patients had a del(17p).

46 patients received debulking treatment including four patients who stopped study therapy after the first course of bendamustine.

17 patients responded to debulking; in eight patients a (clinical) complete and in nine patients a partial remission was assessed. 21 patients showed a stable disease, six patients progressed.

In 20 patients debulking was omitted due to contraindications or low tumor burden.

Obinutuzumab was administered to 62 patients of whom 42 received debulking. Occurrence of infusion related reactions (IRR) related to obinutuzumab is shown in table 1.


In general, the combination of obinutuzumab and ibrutinib was well tolerated and SAEs were in line with the known toxicity profiles of both drugs.

Conclusion: A chemotherapy reduced and targeted regimen is investigated. Our data suggest that bendamustine debulking prior to obinutuzumab might reduce the occurrence of IRRs as compared to historical data [Goede V., 2014].

Disclosure: Julia von Tresckow: Advisory Role: Janssen-Cilag; Expert Testimony: Janssen-Cilag, F. Hoffmann-LaRocheMichael Hallek: Advisory Role: AbbVie, Amgen, Celgene, F. Hoffmann-LaRoche, Gilead, Janssen-Cilag, Mundipharma; Expert Testimony: AbbVie, Amgen, Celgene, F. Hoffmann-LaRoche, Gilead, Janssen-Cilag, Mundipharma

Fortbildung – Migrationsanämien

V396 – “Migration anemias“- an overview

Cario H.1

1Klinik für Kinder- und Jugendmedizin, Universitätsklinikum, Ulm, Germany

The recent dramatically increased migration of people from the Middle East and Africa raises particular challenges for all parts of the society including the healthcare system. Diseases which previously did not seem to play role in Germany are brought into the focus of healthcare providers in all medical fields including hematology, facing apparently new, widely unknown or neglected red blood cell disorders. However, due to previous migration processes many disorders here summed up as “migration anemias” have actually been present as serious medical problem in Germany already for longtime. This particularly concerns the hemoglobinopathies, i.e. thalassemias and sickle cell disease, representing the most common monogenic inherited disorders worldwide, together with another “migration anemia”, G6PD deficiency. As compared to other Central European countries, Germany relatively late experienced migration from countries where these disorders are endemic. Rather few patients have been distributed over the whole country. Until the late 1990s, almost exclusively children and adolescents were affected. The heterogeneous origin of patients and various socioeconomic factors did hamper the formation of effective patient organizations. All these factors contributed to a rather low awareness of these disorders leading to their only marginal role in the medical education but also in daily medical practice.

Major new aspects in the current situation concern the sudden increase of the number of patients with “migration anemias” needing regular care, many of them affected by severe prior complications. Transfusion therapy is frequently complicated by previous alloimmunization. Excessive iron overload, but also “typical” but “forgotten” symptoms of disorders like thalassemia are now challenging the medical staff. The central allocation of refugees in Germany leads to their presentation to healthcare providers who did not have any contact to patients with “migration anemias” in the past but now have to care for them. At present, the previously claimed centralization of patients with these disorders is not feasible. It has to be replaced by professional networks of pediatric and adult hematologists providing access to adequate medical care for all patients with “migration anemias”, based on existing jointly developed guidelines of DGHO and GPOH and including necessary diagnostic procedures and consultation of specialists experienced in the care of these disorders.

Disclosure: No conflict of interest disclosed.

V397 – Non-transfusion-dependent thalassemia (NTDT) - therapy

Dickerhoff R.1

1Uni-Klinik Düsseldorf, Pädiatrie Hämatologie/Onkologie, Düsseldorf, Germany

Non-transfusion-dependent-thalassemia used to be called Thalassemia Intermedia, because its severity was generally thought to be in between the asymptomatic carriership (Thal. Minor) and the transfusion dependent Thalassemia Major. As NTDTpatients got older it became apparent that this entity can present with a larger spectrum of problems than previously thought and very often morbidity in NTDT is more pronounced than in Thalassemia Major. Also, a wide variety of mutations (both a and ß chain mutations) have been found to cause NTDT. Many NTDT patients go undiagnosed for a long time as they present by hemoglobin analysis as heterozygous ß thalassemias – and nobody thinks of the existence of so called dominant ß thalassemia or looks for a triplication of alpha globin genes accompanying a ß globin mutation .

NTDT children with growth failure, widening of marrow space( prominent maxillae)or permanent hemoglobin levels below 6 g/dl need regular transfusions. As patients get older the effects of inefficient erythropoiesis become noticeable: increasing splenomegaly, iron overload (even without transfusions) and extramedullary hematopoiesis (EMH), most often parallel to the spinal column. Splenomegaly , unless extreme or accompanied by severe pancytopenia, should be treated with chronic transfusions as splenectomy carries a high risk for thrombosis. In EMH with neurological symptoms low dose radiation can rapidly decrease the EMH volume while in EMH without neurological impairment transfusions or hydroxycarbamide are indicated to decrease inefficient erythropoiesis. After puberty iron overload has to be looked for and treated with chelation. Transfusions are also necessary in patients with pulmonary hypertension, in some patients with severe HbH disease (non-deletional HbH disease), sometimes during pregnancy, infection or in patients who develop thrombosis despite anticoagulation. Symptomatic cholelithiasis requires cholecystectomy. Leg ulcers are very painful and heal slowly, even with dedicated wound care. Osteopenia and osteoporosis cause very distressing bone pain in many NTDT patients. In some NTDT patients hydroxyurea is useful to decrease transfusion needs.

Each NTDT patient is unique and his or her clinical problems have to be evaluated individually. The challenge begins with a correct diagnosis and goes on with a very individualized treatment.

Disclosure: No conflict of interest disclosed.

V398 – Sickle cell disease

Corbacioglu S.1

1Universitätsklinikum Regensburg, Päd. Hämatologie, Onkoloige und Stammzelltransplantation, Regensburg, Germany

Hemoglobinopathies are the commonest, life-threatening, monogenic disorders in the world. Over 300.000 newborns with sickle cell disease (SCD) are born annually in Africa and Asia, 100.000 affected patients in the United States. In Europe the prevalence is 15 from 100.000 and there are approximately 3000 recorded SCD patients in Germany, with many additionally unregistered and refugees from high prevalence countries. SCD is overtaking haemophilia and cystic fibrosis in certain parts of Europe.

The average life expectancy is between 40 to 50 years. In developing countries, the mortality is between 50%-80% during infancy. SCD is a progressively debilitating and chronic multi-organ disease with a 30% to 50% incidence of disability and unemployment. At 45 years 24% of SCD patients will have developed a stroke, 30% develop silent strokes leading to cognitive impairment in childhood and an impaired quality of life. SCD is the leading cause of stroke in children and adolescents. SCD is a major public health concern. From 1989 through 1993, an average of 75,000 hospitalizations due to SCD occurred in the United States, costing approximately $475 million. Next to future gene therapy which remains disappointing to date, hematopoietic stem cell transplantation (HSCT) is currently the only curative option for SCD. But only 20% have a matched donor available (MUD). This is reflected in the different transplant registries where despite its prevalence only 1200 HSCTs are reported with 75% from matched sibling donors (MSD). Therefore 80% receive currently symptomatic treatment. But medical treatment options besides HSCT cannot cure the disease but only delay the onset of SCD related complications. Several surveys among SCD patients and parents showed that 62% accepted a transplant related mortality of >10% and 30% a TRM >30%. GF >10% was acceptable to 64% and >30% for 41%. Chronic GVHD was unacceptable to the majority (80%). Cord blood and haplo-SCT using post-cyclophosphamide (POST-CY) failed in prospective trials due to high graft failure (GF) rates. T-depleted haploidentical HSCT is a well established transplant modality which not only offers a donor for everybody but is also associated with a very low incidence of GvhD. In summary SCD is one of the most prevalent and devastating haematological diseases, barely known in Germany, for which standard of care is insufficient and novel therapeutic options need to be explored in well controlled prospective trials.

Disclosure: No conflict of interest disclosed.

Freier Vortrag – Myelodysplastisches Syndrom – experimentell

V401 – Erythroferrone (ERFE) and Growth Differentiation Factor 15 (GDF15) are highly differentially overexpressed in CD71 positive Erythroprogenitor cells of patients with Myelodysplastic Syndromes and associated with survival

Mossner M.1, Stöhr A.1, Jann J.C.1, Nolte F.2, Nowak V.1, Obländer J.1, Pressler J.1, Xanthopoulos C.1, Palme I.1, Baldus C.D.3, Schulze T.J.4, Boch T.1, Metzgeroth G.1, Neumann M.3, Hofmann W.K.1, Nowak D.1

1Medizinische Fakultät Mannheim der Universität Heidelberg. III. Medizinische Klinik, Hämatologie und Onkologie, Mannheim, Germany, 2St. Hedwig Krankenhaus, Berlin, Germany, 3Charité, Campus Benjamin Franklin, Berlin, Germany, 4Institut für Transfusionsmedizin und Immunologie, Medizinische Fakultät Mannheim der Universität Heidelberg, Mannheim, Germany

Introduction: Erythroferrone (ERFE) is a regulator of iron homeostasis in the context of hematopoietic stress and erythropoietin (EPO) stimulation as it induces increased iron availability by downregulation of hepcidin. Growth differentiation factor 15 (GDF15) also plays an active but yet to be established role in signaling during ineffective erythropoiesis. In order to investigate potentially disturbed molecular roles of ERFE and GDF15 in Myelodysplastic Syndromes (MDS), we analyzed their gene expression levels in highly purified CD71 positive bone marrow cells derived from MDS patients and healthy controls and correlated the differential expression data with clinical parameters and survival.

Methods: CD71+ erythroprogenitor cells of a total of n = 148 MDS, n = 18 sAML patients and n = 35 healthy individuals were isolated immunomagnetically via MACS columns (Miltenyi). After total RNA extraction using the AllPrep DNA/RNA Mini kit (Qiagen), cDNA was transcribed from RNA via Quantitect cDNA synthesis kit (Qiagen). Subsequently, ERFE and GDF15 expression was quantified from cDNA by quantitative PCR.

Results: As compared to unselected BM or other specific differentiation lineages, ERFE was almost exclusively expressed in the erythropoietic CD71+ compartment. ERFE expression in the CD71+ subset revealed a highly significant overexpression in MDS IPSS-low/int-1-risk (fold change (FC) = 4.3, p < 0.0001), IPSS-int-2/high-risk (FC = 6.23, p < 0.0001) and sAML (FC = 6.69, p < 0.0001) relative to healthy controls. As compared to healthy controls, GDF15 showed a mean FC = 66 (p < 0,0001). ERFE expression showed no correlation with clinical laboratory parameters. GDF15 levels were positively correlated with plasma iron levels, transferrin saturation and endogenous EPO levels. Both high ERFE and GDF15 expression levels in CD71+ progenitor cells were significantly associated with superior overall survival (ERFE: p = 0.0007, GDF15: p = 0.0001).

Conclusion: Highly aberrant overexpression of ERFE and GDF15 in CD71+ erythropoietic progenitor cells suggests an important role for these genes in the dysfunctional erythropoiesis of MDS. The relationship of high ERFE and GDF15 expression with superior survival, especially in low risk MDS patients with no apparent coherence to other established clinical markers warrants further pursuit of ERFE and GDF15 expression profiles in CD71+ BM cells of MDS patients as a possible independent prognostic marker and therapeutic target.

Disclosure: No conflict of interest disclosed.

V402 – Bone homeostasis is altered in a mouse model of myelodysplastic syndrome

Weidner H.1, Rauner M.2, Bulycheva E.1, Khandanpour C.3, Bornhäuser M.1,4, Hofbauer L.C.2,4, Platzbecker U.1,4

1Department of Medicine I, Technical University, Dresden, Germany, 2Department of Medicine III, Technical University, Division of Endocrinology, Diabetes, and Bone Diseases, Dresden, Germany, 3Department of Hematology, West German Cancer Center, University Hospital Essen, University Duisburg-Essen, Essen, Germany, 4German Cancer Consortium (DKTK), partner site Dresden and German Cancer Research Center (DKFZ), Heidelberg, Germany

Introduction: Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal diseases that mainly occur in the elderly and are characterized by an ineffective hematopoiesis leading to cytopenias. Increasing evidence suggests the bone microenvironment as a critical modulator to influencing the progression of MDS. However, the underlying mechanisms are not fully understood. NUP98-HOXD13 (NHD13) mice develop MDS-like symptoms at an age of 6 months, including anemia and an increased number of blasts in the bone marrow. Thus, it may be used as model to study bone homeostasis in the setting of MDS. Here, we assessed bone homeostasis in NHD13 mice before (2 month-old) and after (6 month-old) the onset of MDS.

Methods: To investigate the bone phenotype we used micro-computed tomography of the femurs of 2- and 6-month-old male NHD13 and wild-type (WT) mice. Bone samples were histologically stained for the osteoclast marker tartrate-resistant acid phosphatase. In addition, serum was analyzed by ELISAs to assess the bone turnover markers C-terminal telopeptide of type I collagen (CTX-I) and procollagen type I propeptide (P1NP).

Results: Two-month-old NHD13 mice had already developed leukopenia in the absence of other cytopenias and showed alterations of bone homeostasis. The trabecular bone volume [-26%; P < .05] and trabecular number [-20%; P < .01] were reduced in the femurs of NHD13 compared to WT mice, but the bone resorption marker CTX-I was not changed. Interestingly, the bone formation marker P1NP was increased [3-fold, P < .001]. At the age of 6 months the NHD13 mice had developed anemia indicated by a reduced hematocrit [-17%; P < .001]. The bone volume was unchanged but the trabecular architecture was altered compared to WT mice. Similar to the 2-month-old NHD13 mice, the number of trabeculae was reduced in the 6-month-old NHD13 mice [-15%; P < .001] whereas their thickness was increased [+18%; P < .01]. The histology of NHD13 mice indicated an increased number of osteoblasts [1.5-fold; P < .01] and also the bone formation marker P1NP was elevated [+66%; P < .05] compared to WT mice. Although NHD13 mice had fewer osteoclasts [-77%; P < .01], the bone resorption marker CTX-I was increased by 32% [P < .05].

Conclusion: NHD13 mice show an altered bone homeostasis before and after the onset of MDS. In summary, our study indicates that already minimal alterations in the hematopoietic system have an impact on the bone microenvironment, thus influencing bone homeostasis.

Disclosure: No conflict of interest disclosed.

V403 – Iron-overload links to genetic instability in myelodysplastic syndromes

Westhofen G.1, Ganster C.1, Beyer F.2, Rassaf T.3, Al-Ali H.K.4, Stuhlmann R.5, Glass B.5, Bacher U.1, Brümmendorf T.2, Germing U.6, Gattermann N.6, Haase D.1

1University Medicine Göttingen, Clinics of Hematology and Medical Oncology, Specialized Diagnostic Laboratories INDIGHO, Göttingen, Germany, 2RWTH Aachen University Hospital, Department of Haematology, Oncology, Haemostaseology, and Stem Cell Transplantation, Aachen, Germany, 3Heinrich-Heine University Düsseldorf, Department of Cardiology, Pneumology, Angiology, Düsseldorf, Germany, 4University Hospital of Leipzig, Leipzig, Germany, 5Asklepios Klinik St. Georg, Hamburg, Germany, 6Heinrich-Heine University Düsseldorf, Department of Hematology, Oncology, and clinical Immunology, Düsseldorf, Germany

Introduction: As numerous experimental as well as clinical studies have shown, iron overload (IO) results in not only a reduced overall survival, but also decreases the leukemia free survival in patients suffering from myelodysplastic syndromes (MDS), yet the related pathomechansims remain unclarified. The aim of our study was therefore to examine and identify associations between genetic instability, oxidative stress and elevated iron parameters in patients suffering from MDS.

Methods: 55 patients (pts) with proven MDS have been included in our study, dividing the cohort into two groups according to their serum ferritin (SF) levels, using 275µg/l as a cut-off value for normal vs. elevated SF. All patients were investigated for oxidative stress markers (plasma nitric oxide metabolites; DNA double-stranded breaks (DSB) in CD34+ cells, examined by ?H2AX immunofluorescent staining), replicative stress markers (alteration of telomere length (TL) in granulocytes and lymphocytes), and markers for genetic instability (cytogenetic alterations investigated by chromosome banding, SNP-array- and FISH-analyses; molecular mutations, identified by Sanger-sequencing and NGS).

Results: Pts. with elevated SF displayed more DSB per CD34+ cell than pts with normal SF levels (median 5.65 [range 2.1–10.8] vs. 1.9 [0.5–6.8], p = 0.016) .Cytogenetic analyses showed an increase in size of the total genomic aberration with elevated SF (median 0 Mbp [0–155] vs. 33.66 Mbp [0–247.9], p > 0.05). TL was significantly reduced in granulocytes of pts with high SF compared to patients with normal SF (median -1.611kb [-4.062–1.311] vs. 0.480kb [-3.127- 5.318], p = 0.006); TL in lymphocytes was not influenced by SF. Not only elevated SF, but also the number of erythrocyte concentrates transfused in the past 3 months showed these correlations with genetic instability markers.

Conclusions: Our study, as best to our knowledge is the first to explicitly investigate the link between IO and genome stability, revealed an association of distinct markers for genetic instability with elevated SF and transfusion dependency. Furthermore we were able to show a negative effect of iron at much lower SF levels than previously assumed, starting at the upper normal level of 275µg/l in our clinical chemistry lab. These results further support the thesis of IO being causally related to disease progression and transformation in MDS and should be taken into account for future diagnostics and therapeutic decision-making.

Disclosure: Gina Westhofen: No conflict of interest disclosed.Detlef Haase: Financing of Scientific Research: Novartis; Expert Testimony: Novartis

V404 – Cytomorphological and molecular background of isolated Y-loss in myelodysplastic syndromes

Ganster C.1, Bacher U.1, Germing U.2, Shumilov E.1, Strupp C.2, Shirneshan K.1, Dierks S.1, Stuhlmann R.3, Glass B.3, Bäsecke J.4, Simon-Becker S.5, Sievers B.6, Martin R.1, Flach J.1, Harder L.7, Haase D.1

1Universitätsmedizin Göttingen, Klinik für Hämatologie und Medizinische Onkologie, Göttingen, Germany, 2Universitätsklinikum Düsseldorf, Klinik für Hämatologie, Onkologie und Klinische Immunologie, Düsseldorf, Germany, 3Asklepios Klinik St. Georg, Klinik für Hämatologie, Onkologie und Stammzelltransplantation, Hamburg, Germany, 4St. Josefs-Hospital, Onkologie und Hämatologie, Cloppenburg, Germany, 5MVZ Alsfeld, Alsfeld, Germany, 6Onkologische Schwerpunktpraxis, Hildesheim, Germany, 7Institut für Tumorgenetik Nord, Kiel, Germany

Introduction: Loss of the Y chromosome (LOY) is a frequent aberration in myelodysplastic syndromes (MDS), observed as a single aberration in 3–4% of male MDS patients (pts.). In previous studies we could show that LOY is not only a phenomenon of higher age in MDS, but may also be a marker for a malignant clone. MDS pts. with isolated LOY have a good prognosis with a very low risk for leukemic transformation. To facilitate an unambiguous discrimination between age-related and clonal LOY, the aim of this study was to identify molecular mutations or cytomorphological features typical for MDS pts. with isolated LOY.

Methods: We compared the mutational profile (17 myeloid genes) between 17 pts. with isolated LOY and two pts. with LOY and trisomy 15 and a control group of 197 pts. with MDS and sAML and other cytogenetic abnormalities or normal karyotype. In addition, bone marrow aspirates of 41 pts. with isolated LOY were cytomorphologically evaluated according to the standards of the Duesseldorf MDS registry.

Results: The most frequently mutated gene in molecularly analyzed pts. (LOY in =50% of metaphases in proven MDS or in =75% in suspected MDS) was TET2 (n = 7/19). Further mutations affected CBL (n = 2/15), ZRSR2 (2/15), ASXL1 (1/19) and SRSF2 (1/19). Overall, the proportion of pts. with molecular mutations was lower in the subgroup with isolated LOY compared to the control group (53% vs. 72%). MDS according to WHO criteria (2008) was cytomorphologically confirmed in 36/41 pts. (19x LOY in =75% of metaphases, 22x in < 75%). Besides a bone marrow blast count of < 5% in 40/41 cases, a heterogeneous distribution of MDS subtypes was observed. Peripheral blood counts [Hb: mean 10.4 vs. 9.7 g/dL; WBC: 4.9 vs. 6.0×10(9)/L, thrombocytes: 163 vs. 198×10(9)/L] and dysplasia of the individual cell lines (erythro-, granulo-, megakaryopoesis) did not differ significantly between the groups with a LOY clone size =75% and < 75%.

Conclusion: MDS with clonal LOY seems to comprise a heterogeneous mixture of cytomorphologic subtypes and to be outlined by marked heterogeneity of the molecular mutation patterns. However, MDS pts. with LOY mostly show absence of marrow blast increase =5% and of molecular high risk markers (mutations in TP53, RUNX1, EZH2, ASXL1). Molecular MDS-associated mutations are less frequent in this cytogenetic subgroup as compared to overall MDS. These results are in line with the known favorable prognosis of these pts. according to the IPSS and IPSS-R.

Disclosure: No conflict of interest disclosed.

V405 – Development of RAS-induced zebrafish leukemia models

Alghisi E.1, Konantz M.1, Mione M.2, Lengerke C.1,3

1Universitätsspital Basel, Department für Biomedizin, Basel, Switzerland, 2Centro di Biologia Integrata, University of Trento, Trento, Italy, 3Universitätsspital Basel, Abteilung für Hämatologie, Basel, Switzerland

Background: The zebrafish has emerged as a versatile novel experimental model for studies on developmental hematopoiesis and leukemogenesis. Several oncogenes involved in human leukemia have been successfully overexpressed in zebrafish.

Aims: We aim to use the zebrafish model to investigate cooperating molecular events involved in leukemia initiation, progression and chemo-resistance.

Methods: We took advantage of the Gal4/UAS binary system to overexpress human oncogenic HRAS in zebrafish hematopoietic cells using specific promoters (fli.1, pu.1, runx.1, mpeg1). Blood cell development was studied at different stages by real-time PCR analysis of hematopoietic gene expression, flow cytometry, immunohistochemistry and blood smear morphological assessment. Small molecule library screens are performed on live HRAS overexpressing transgenic larvae and effects assessed by microscopy.

Results: Different phenotypes were observed depending on the promoter driving the oncogene expression. HRAS induction via the early hematopoietic promoter fli.1 affected primitive hematopoiesis inducing a myelo-erythroid proliferation and delayed erythrocyte maturation (Alghisi et al. 2013). Alternatively, HRAS expression driven by runx1, pu.1 and mpeg1 did not affect primitive hematopoiesis and allowed studies at later developmental stages. After 1 month, runx1-HRAS fish displayed a cellular expansion of hematopoietic stem/progenitor cells (HSPC) in the kidney marrow (KM), the zebrafish definitive hematopoietic compartment. KM cytospin preparation and flow cytometric analysis confirmed high numbers of undifferentiated cells, indicating that HRAS-overexpression induced HSPC proliferation and impaired differentiation capacity. Similarly, mpeg1-HRAS fish also show increased numbers of blood progenitors in the KM and abnormal gene expression of progenitor markers. Using fli.1-HRAS fish and an FDA approved library comprising around 2000 compounds we have set up an in vivo screen to identify drugs that can suppress the HRAS-mediated hematologic phenotype.

Conclusion and outlook: We have established a zebrafish model of HRAS induced hematologic disease. We are currently further investigating this model using in vitro colony forming assays, serial re-transplantations and exploring effects of HRAS in cooperation with other oncogenes. Active compounds identified in the small molecule screen shall be presented at the meeting and further tested for their efficacy in adult disease models.

Disclosure: No conflict of interest disclosed.

V406 – Frequency and clonogenic capacity of the rare CFU-F subpopulation of Mesenchymal Stem Cells (MSC) is significantly reduced in Myelodysplastic Syndromes (MDS) and Acute Myeloid Leukemia (AML)

Weickert M.-T.1, Garz A.-K.1, Zwick A.1, Ziegenhain C.2, Grath S.3, Strunk D.4, Enard W.2, Peschel C.1, Oostendorp R.1, Götze K.S.1

1Technische Universität München, III. Med. Klinik, München, Germany, 2Faculty of Biology, Ludwig-Maximilians University Munich, Anthropology and Human Genomics Department, München, Germany, 3Faculty of Biology, Ludwig-Maximilians University Munich, Evolutionary and Functional Genomics, München, Germany, 4Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Institut für Experimentelle und Klinische Zelltherapie, Salzburg, Austria

Crosstalk of hematopoietic stem cells (HSC) with stromal niche cells is crucial for regulation of HSC function. The stromal niche is derived from mesenchymal stem cells (MSC), giving rise to adipocytes, osteoblasts and chondroblasts. Dsyregulation of niche integrity contributes to leukemogenesis and alterations in MSC function play a prominent role in myelodysplastic syndromes (MDS). We developed a flow cytometric sorting protocol to isolate the rare population of MSC by surface marker expression to study unmanipulated MSC. We asked whether certain MSC subpopulations are dysregulated in MDS and acute myeloid leukemia (AML) and how this may influence HSC behavior. MSC were isolated by flow cytometric sorting from whole bone marrow from patients with newly diagnosed MDS, AML or healthy individuals. Cells were sorted by negativity for CD31/CD235a/CD45 and positivity for CD271. Two distinct populations were identified within the CD31-/CD235a-/CD45-/CD271+ gate: CD271+/CD73+/CD105+/CD90+ (termed +/+) and CD271+/CD73-/CD105-/CD90- (termed ±) cells. To determine CFU-F frequency, cells were seeded in 24-well-plates and formation of CFU-F was observed every second day. CFU-F formation was only observed in the subpopulation of +/+ cells but not in the +/- population. MDS and AML samples showed a slightly reduced +/+ population frequency compared to healthy samples. However, CFU-F capacity in the +/+ population was dramatically impaired in MDS. Proliferation of CFU-F was strongly reduced in MDS samples and only CFU-F forming cells from healthy controls could be expanded subsequently. Differentiation into adipogenic and osteogenic lineages was altered in MDS and AML compared to healthy MSC. Co-culture