ALBERT

Description: Abstract 3511 Introduction: In order to allow a better understanding of acute myeloid leukemia (AML) and develop more promising therapeutic strategies the establishment of functional and reproducible in vivo models is widely pursued. Of available model systems, xenografts in immunodeficient mice reproduce the clinical situation best. Here, we performed extensive analysis of AML engraftment in NOD/SCID-IL2-receptor-gamma-chain−/− (NSG) mice comparing tail vein versus intratibial injection and growth behavior of AML patient-derived bone marrow versus peripheral blood cells. Furthermore, tumor growth characteristics in the murine host were correlated with the disease stage and the molecular risk factor profile of the individual donors. Methods: Bone marrow and peripheral blood cells from 17 AML patients were injected intratibially into NSG mice (n=4–8/patient, 82 mice in total). As controls, 14 mice received bone marrow from three different donors and 5 mice were mock-injected. Tumor growth was monitored via a) determination of overall survival, b) fluorescence-based in vivo imaging (IVI, Kodak FX, Alexa750 labeled anti-human CD45 or CD33 and c) confirmation of IVI data by histological and immunohistochemical examination of bone marrow and spleen. When highly positive IVI signals and/or the overall condition of individual mice indicated enlarged tumor burden, the respective animals were sacrificed and the human AML cells transferred to another animal. In parallel the engraftment pattern of AML cells 2–4 weeks posttransplant was correlated with clinical disease activity, application route and origin of the particular tumor cells. Results: Patients included in the present study represent multiple different French-American-British (FAB) subtypes, various karyotypes and molecular features in terms of the mutational status of NPM1 and FLT3. All patient-derived specimens were capable of recapitulating the disease in NSG mice at 4–6 weeks after transplantation. Over a period of 13 months 12 out of 17 xenografts could be passaged once and 9 at least twice. Up to six passages were performed for an individual AML xenograft. In contrast, engraftment of healthy donor bone marrow cells could be determined merely until day 56 after implantation. The human bone marrow cells of the healthy donors did not engraft in serial passages. Mean survival time of AML bearing animals ranged between 21 and 82 days for a respective xenograft. No differences could be determined between engraftment capacities of peripheral blood or bone marrow cells of one patient. Neither karyotype, FAB classification nor leucocyte count or the percentage of monomorphic blasts in the bone marrow seemed to have an impact on engraftment capacity in the murine organism. However, mice bearing AML xenografts with mutations in FLT3 as well as in NPM1 showed particular short overall survival times and high tumor cell engraftment determined by IVI. This phenomenon became more obvious along the different passages. The intratibial approach proved to be superior in comparison to the intravenous application as cells of an individual patient engrafted faster when injected directly into the bone marrow microenvironment. Determination or tumor load via IVI permits to closely monitor not only the growth behavior but also the homing characteristics of the human cells over time. The positive IVI signals in bone marrow and spleen could be confirmed by histological examination as well as by immunohistochemistry specific for human CD45 and CD33. Conclusions: Our xenografts show a close resemblance to the AML-disease regarding the level of dissemination and organ involvement. Collection of whole-body IVI data proved to be a time- and animal-saving analysis that allows to closely monitor AML growth. As AML is characterized by an increasing number of molecular subtypes with completely different therapeutic options it seems to be extremely worthwhile to develop patient derived xenograft models representing as many AML subtypes as possible. Our results suggest that this model reflects the heterogeneity and important clinical characteristics of the disease, and thus may serve as a tool for preclinical drug testing and investigation of the pathophysiology of AML. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3924 Targeted therapies in terms of monoclonal antibodies have become standard in the treatment of various lymphomas. Albeit being more specific than conventional therapy, the used antibodies target surface receptors both present on polyclonal and monoclonal hematopoietic cells. Due to its specificity for the malignant B-cell clone the B-cell receptor (BCR) is an ideal therapeutic target in lymphoma therapy. Moreover, using peptides has several advantages over whole antibodies: reduced immunogenicity, better epitope mimicry and tissue penetration, easier synthesis and more favourable pharmacokinetics (no uptake into the reticulo-endothelial system). Peptides mimicking the epitope recognized by lymphoma BCRs have therefore been praised as promising therapeutic tools for years (Lam, West J Med., 1993) but a proof-of-concept has only been published recently in mice bearing subcutaneous A20 lymphoma (Palmieri et al., Blood, 2010). In the current study, we have established a human cell line-derived disseminated Burkitt′s lymphoma model (SUP-B8) in NOD/SCID mice by intravenous injection. Our active principle was the tetramerized BCR binding peptide YSFEDLYRRGGK-biotin (termed T-peptide, Renschler et al., PNAS, 1994) which was applied intravenously on day (d) 12, 14, 16 and 19 after injection of the tumor cells, respectively. The therapeutic efficacy was evaluated in comparison to several control groups (tetramerized control peptide (termed C-peptide, RDYSYERLFGGK-biotin), vehicle (0.8% ACN in water, 200μl/d) and untreated animals). Tumor cell engraftment was monitored via daily surveillance of disease symptoms, FACS (anti-human lambda, CD19, anti-murine CD45) and fluorescence-based in vivo imaging system (FI, Kodak FX, Alexa750 labeled anti-human CD45) on days 12 and 21. SUP-B8 engrafted predominantly in the bone marrow (BM, take rate = 100%) and marrow infiltration increased in untreated mice between start and end of therapy from 1 ± 0.4% (d 12) to 39.8 ± 9.4% (d 21). Other sites of engraftment were subcutis (38%) and spleen (8%). The examined compounds were well tolerated in tumor-bearing mice, no acute toxicity could be observed and maximal body weight loss was below 15%. Treatment of xenograft mice with the tetramerized BCR-binding peptide significantly reduced bone marrow infiltration compared to controls (T-peptide 8.1 ± 4.6%, C-peptide: 32.8 ± 8%, p=0.037, vehicle: 30.5 ± 7.9%, p=0.029). Considering the short half-life of uncoupled peptides and the injection schedule every second day, this is a remarkable reduction. For further optimization of this promising therapeutic approach we plan to couple peptides to effector molecules via acid labile linkers; this is based on the evidence that confocal imaging of Burkitt lymphoma cell lines showed the processing of specific BCR binding peptides in acidic organelles of the cell. In summary, we conclude that BCR targeted peptide-based therapy is a feasible method with remarkable therapeutic results in vivo and future studies will focus on coupling specific peptides to appropriate effector molecules or combinational therapeutic approaches using conventional chemotherapeutics. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2141 Acute myeloid leukemia (AML), one of the most common leukemia in adults, is characterized by the accumulation of abnormal white blood cells in the bone marrow. Even with modern advances in therapeutics and molecular diagnostics, the majority of AML patients die from their disease. Angiogenesis contributes to the development of hematologic malignancies, although its role has not been as clearly defined in hematologic malignancies as in solid tumors. In the present study we have determined antitumoral activity of two well-known inhibitors of angiogenesis. Sorafenib, a small molecule inhibitor affecting inter alia the VEGF pathway (100mg/kg/day (d); applied 12 consecutive days) and Cetuximab, a chimeric antibody against human EGFR (30 mg/kg/d; applied once weekly for 3 weeks) were evaluated in comparison to Etoposide, a topoisomerase II inhibitor (24mg/kg/day; applied 3 consecutive days) as a well established compound in AML treatment regimens in comparison with a control group. Equal parts of HL-60 cells were injected intravenously and into the peritoneal cavity of NOD/SCID mice and respective therapies were started 14 days after implantation. Tumor growth was monitored by a) daily monitoring of AML symptoms, and b) weekly fluorescence-based in vivo imaging (FI) using a Alexa750-labeled anti-human CD45 antibody and c) verification of the FI data by histological examination of bone marrow and spleen at the end of the study. Tumor inhibition was calculated as the proportional reduction of mean AML cell infiltration at the respective compartment of the test- compared to the control-group (in %). HL-60 cells engrafted predominantly in bone marrow (BM; take rate = 100%), but were as well detectable in the spleen (30%). At the respective doses and schedules the examined compounds were well tolerated in tumor-bearing mice. No acute toxicity could be observed and maximal body weight loss was below 15%. Tumor development was clearly reduced by Cetuximab (reduction of 53% vs control), albeit to a lesser extend then Sorafenib (reduction of 99% vs control), which induced a complete remission within the treatment period. Treatment of Etoposide induced no markable tumor growth inhibition (reduction of 10% vs control). Thus, HL-60 cells engrafted in NOD/SCID mice representing a valuable in vivo model for AML which exhibits high reproducibility and take-rates in relevant compartments closely mimicking the clinical situation. Collection of whole-body FI data proved to be a time- and animal-saving analysis that allows to closely monitor AML growth. With regard to the demographic development, AML will be more and more a disease of the elderly. Thus, development of new therapeutic options compared to high-dose chemotherapy will be highly required. As the VEGF and EGFR pathways are closely related, further investigations will include the evaluation of potentially synergistic effects in combination of Sorafenib with Cetuximab in human disseminated AML xenograft models. Disclosures: No relevant conflicts of interest to declare.

Description: Background Appropriate animal models for hematological malignancies are highly attractive, because they allow the study of the tumor biology and underlying disease mechanisms. They also constitute a major prerequisite for rapid bench-to-bedside translation of investigational anticancer therapies. To validate our multiple myeloma patient (pt)-derived xenograft (MM PDX) model (Schueler et al, Expert Opin Biol Ther, 2013), we systematically analyzed a panel of MM PDX with regard to their sensitivity towards standard of care treatment and compared these data with the pts' clinical outcome. Methods Bone marrow (BM) cells of 11 MM pts were implanted intratibialy (i.t.) into 103 NOD/Shi-scid/IL-2Rγnull (NOG) mice (n= 6-18 / pt sample). Mice were treated according to pts' therapy with VCD (Bortezomib, Cyclophosphamide, Dexamethasone), or to evaluate additional treatment options with Rd (Lenalidomide, Dexamethasone). Tumor growth and antitumoral activity in mice were assessed in tumor-bearing mice and compared to untreated control mice as well as to pts' response. Tumor growth in the mouse model was monitored by whole-body fluorescence-based in-vivo-imaging (IVI) using CF750-labeled α-HLA ABC antibody before and during treatment as well as 24h after last treatment cycle as described (Schueler J. PLOSone 2013). Mock-injected animals served as negative controls. Engraftment of human MM cells in murine organs was confirmed by flow cytometry and patho-histological analyses (immunostaining) at the end of the study. Results The pt cohort included a typical MM clientele for referral centers, with a median age of 75 years (range 56-85), median BM infiltration of 80% (20-90), and high- and standard-risk cytogenetics in 5 and 6 pts, respectively. All pts had advanced disease with Durie&Salmon stage III and active/symptomatic MM. All pts received VCD after diagnosis and BM sampling. MM cell engraftment could reliably be determined from experimental day 10 on in all 11 MM PDX models, at all assessed sites, namely within the BM, spleen and peripheral blood (PB) of recipient mice. Individual pt samples displayed distinct tumor growth patterns in vivo. Fluorescence intensity of engrafted murine organs ranged from 2- to 15-fold compared to mock injected control mice. Mean IVI signals in BM of recipient mice were 10-fold higher as compared to spleen signals, qualifying the BM niche as the preferred homing localization of pts' MM cells. Of note, both injected and non-injected BM sites were infiltrated by MM cells 10 days after tumor cell injection. Engraftment of human MM cells in the respective murine organs was confirmed by flow cytometry (HLA ABC, CD138, CD38) and histology and verified MM engraftment via both methods, confirming prior reports (Schüler PLOSone 2013; Groen Blood 2012;120:e9-16, Overdijk MAbs. 2015;7:311-21). The murine engraftment capacity was independent of MM type, disease stage, BM infiltration and cytogenetics of the donor pt. VCD was applied to 9 different MM PDX models and induced partial remission (PR; defined as at least 50% reduction of murine tumor load in BM, spleen and/or PB) in 5 out of 9 tested MM PDX models, whereas 2 cases each showed stable disease (SD) or progression (PD). The response rates in the mouse avatars mirrored the clinical outcome of the respective MM pts in 8/9 cases; only one MM pt showed serological and clinical PR, whereas the corresponding mice displayed SD. Rd induced PR in 1 and PD in a second MM PDX model, underlining the feasibility of MM PDX for drug screening approaches. Conclusions Due to the complex tumor biology, murine models of MM are still challenging. Our data support the preclinical rationale to use i.t.-injected NOG mice, since they closely resemble clinical MM with respect to symptoms, disseminated disease sites and response to anticancer treatment. Possible applications for the MM mouse avatars include development of new anticancer drugs as well as definition of biomarker strategies and selection of treatment options for individual pts with relapsed/refractory MM. The data of our preclinical study may serve as a useful future strategy to guide treatment decisions in refractory pts. The suitability as a drug development tool will be additionally determined performing treatment experiments with novel agents, e.g. elotuzumab or daratumumab. Disclosures Schueler: Oncotest GmbH: Employment. Klingner:Oncotest GmbH: Employment.

Description: Abstract 3010 Introduction: In order to allow a better understanding of multiple myeloma (MM), the establishment of functional and reproducible in vivo models is widely pursued. Of available model systems, xenografts in immunodeficient mice reproduce the clinical situation advantageously. Here, the engraftment capacity of MM patient-derived bone marrow cells implanted into NOD/SCID-IL2-receptor-gamma-chain-/- (NSG) mice was meticulously investigated. Material and Methods: Bone marrow cells from 7 MM patients were injected intratibialy into NSG mice (n=5/patient). As controls, 5 mice received healthy donor bone marrow and 5 mice were mock-injected. Tumor growth was monitored via a) daily MM-symptom acquisition, such as hind limb paresis, apathy and consistent foot dragging, b) FACS (human HLA-A,B,C; CD138; CD45; CD38) and c) fluorescence-based in vivo imaging (FI, Kodak FX, Alexa750 labeled anti-human CD138, CD38, CD45 and HLA-ABC) in bone marrow, peripheral blood, spleen and lymph node sites of the respective animals. Results: There were significant differences in engraftment capacity, persistence of human cells and expression of selected markers between bone marrow of MM patients and healthy donors: 1.) infiltration of the spleen and lymph nodes was exclusively detected in NSG-mice bearing patient-derived MM cells, whereas cells of healthy donors were - if detected - exclusively found within the murine bone marrow; 2.) mean FI-areas in the bone marrow of MM-patient-derived injected mice were significantly increased as compared to mice bearing bone marrow cells of healthy donors (p=0.006); 3.) patient-derived MM cells expressed CD138, CD38 and HLA-ABC. In contrast, bone marrow cells of healthy donors expressed exclusively CD45 and CD138. The CD138 cell population determined by FACS in patients' bone marrow cells (before NSG-injection) decreased from a median of 11.3% to 0.8% 56 days after implantation (in NSG mice), either due to preferably CD138-negative plasma cell engraftment or the CD138 loss within the murine environment as previously described. Fifty-six days after implantation, patient-derived MM cells could be detected in all animals via FACS-analysis. Follow-up analyses by FI confirmed, that bone marrow engraftment was prominent and observed in all (35/35) NSG mice, albeit also in others organs. Patient-derived MM cells within the bone marrow could be detected in parallel via FACS- and FI-analyses in 10 NSG mice and within the peripheral blood in 12 NSG mice (total of 35 mice being examined). Maximal bone marrow-, peripheral blood- and spleen-engraftment numbers in NSG mice were as high as 4%, 25% and 52%, respectively, suggesting that in peripheral blood- and spleen-sites, MM-cell engraftment could even surmount that of bone marrow-sites. Spleen and other organ involvement observed in our xenografts have been confirmed in previous murine MM-models (Murillo et al. Clin Cancer Res, 2008), postulating that similarly to spleen-colony-forming-cells in hematopoiesis, spleen and other sites serve as fertile tumor engraftment locations.Differences in engraftment capacity and expression pattern between respective patient-derived MM specimen were evident, but did not strikingly correlate with MM-patients' characteristics, such as MM-subtypes, disease stage or expression pattern of the primary material; this observation also well correlating with previous reports (e.g. (Pilarski et al. Blood, 2000). Conclusions: Murine MM-models have shown to be exceedingly challenging in their ability to induce valid and trustworthy MM-patient-derived cell engraftment; here our NSG model suggest to harbor MM-cells. Our data demonstrates that intratibially-injected NSG mice mimic the clinical MM disease with respect to the disseminated nature of the disease and the indispensable engraftment of clonogenic plasma cells into the bone marrow. Collection of whole-body FI data proved to be a time- and animal-saving analysis that allows to closely monitor MM growth. Disclosures: No relevant conflicts of interest to declare.

Description: Many efforts are made to treat acute leukemia effectively, but still; new treatment options are urgently needed. In the past, mainly chemically induced and cell line-derived mouse models were used to evaluate the efficacy of new drugs. The high failure rate of 96% in early clinical development illustrates the urgent need for more predictive preclinical models as a major prerequisite for rapid bench-to-bedside translation of investigational anticancer therapies. Transplantable patient-derived xenograft (PDX) models of leukemia offer a strong preclinical tool for drug screening and biomarker development as they represent the complex clinical tumor heterogeneity and molecular diversity of human cancer. Between 2011 and 2015 we collected 91 samples of peripheral blood (PB) or bone marrow (BM) from patients diagnosed with acute myeloid or lymphoblastic leukemia (AML, ALL) and injected them intratibially (i.t.) into NOG (NOD/Shi-scid/IL-2Rγnull) mice (n=1-5/patient sample). Infiltration of human leukemic cells was determined by flow cytometry in murine PB, BM and spleen. If more than 5% of hCD45+, hCD33+, hCD34+, hCD38+, and/or HLA-ABC+ cells were detectable in one sample, it was classified as engrafted. Results were confirmed by histo-pathological examination. FISH analyses confirmed the cytogenetical concordance with the donor patient where available. 8 models were treated with cytarabine and results were compared to patient´s outcome and treatment experiments using cell line derived models of AML. Whole exome sequencing analyses of the transplantable models are underway for a deeper characterization of the respective leukemic clone which adapted to the murine microenvironment. Specimens of 44 female and 47 male patients (median age: 59 years, median BM infiltration: 39%) were collected. The donor patient cohort covered a broad range of different molecular subtypes: amongst others 5 MLL-rearrangements, 1 MLL-Deletion, 2 t(8;21) translocations, 20 cytogenetically normal and 7 complex karyotypes were included. Up to now 12 PDX models were in passage (P) 5 and higher, where P1 represents first implantation of human cells into the murine BM. 15 PDX models grew in P3 - P4, 21 in P2, 36 in P1 and 7 samples showed no engraftment. Engraftment capacity of the leukemic cells did not correlate significantly with any of the patient characteristics. BM engraftment ranged from 30% - 80%. Spleen and PB depicted 5 - 30% of leukemic cells. Infiltration rate in different organs and immuno-phenotypic characteristics of the human cells were specific for a defined model and preserved during serial transplantation. Take rates within one mouse cohort in serial transplantation were ≥98% for all transplantable PDX, similar to cell line derived models thereby qualifying the PDX approach as a suitable preclinical platform. Overall survival (OS) in P1 ranged from 52 to 〉 310 days (d). Models which could be serially transplanted showed model specific median OS ranging from 32 - 150 d. Comparable cell line derived models depicted median OS of 13 - 45 d. Of note, cell line derived models (KG-1, NOMO-1, MOLM-13, THP-1, MV4-11 or HL60, n ≥ 10 mice/cell line) induced hind limb paresis in all recipient mice. These symptoms could not be observed in PDX and most important are not part of the clinical picture. Cytarabine induced a significantly prolonged OS in 8/8 tested PDX models. Respective donor patients showed hematologic response under cytarabine based therapy highlighting the excellent predictivity of the in vivo platform. In contrast none of the investigated cell line derived models showed sensitivity towards cytarabine, although representing similar subtypes of AML as the investigated PDX models. Taken together a constantly expanding panel of well characterized AML/ALL PDX covering a broad range of different subtypes of the disease is available for drug development and tumorbiology studies. The comparison with cell line derived in vivo models revealed significant advantages of the PDX approach as the latter represents the molecular diversity more in detail, mimics the clinical signs of leukemia more realistic and most important mirrors sensitivity towards standard of care in a direct comparison with the donor patient´s clinical outcome. Therefore, we strongly believe that the AML/ALL PDX platform is a robust and predictive tool to address translational challenges in oncology research. Disclosures Oswald: Oncotest GmbH: Employment. Klingner:Oncotest GmbH: Employment. Lenhard:Oncotest GmbH: Employment. Schueler:Oncotest GmbH: Employment.

Description: Patient-derived xenografts (PDX) have become reliable tools for preclinical research for many types of cancer. We recently reported the establishment of 4 human diffuse-large B-cell lymphoma (DLBCL) PDX in mice arising from patient prostate carcinoma tissue (Wetterauer,C. et al. 2015). All PDX were EBV associated and of ABC subtype as determined by the new algorithm of Choi et al. 2009. In contrast, none of the donor patients showed clinical or histological evidence of lymphoma disease. To address the question why lymphoma engraftment takes place in immunodeficient mice whereas patients do not show clinical evidence of disease, we compared growth behavior of the DLBCL PDX in 4 different mouse strains harboring different immunological deficiencies. Furthermore using 3 different implantation routes the dissemination behavior of these models into different immunological niches was evaluated. Results were compared to a similar study carried out with an EBV negative DLBCL PDX of a secondary, cerebral lymphoma (ABC-subtype). None of 4 EBV+ DLBCL PDX showed tumor growth after implantation in NMRI nu/nu (B- cells+, T cells-, NK cells+, n=5 mice/PDX) or 1147-F mice (B cells-, T cells+, NK cells+ n=5 mice/PDX). Subcutaneous implantation into NOD/SCID mice (B cells-, T cells-, NK cells+ n=5 mice/PDX) revealed tumor growth for 2 of 4 lymphomas displaying take rates of 80% (4 out of 5 mice) and 100% (5 out of 5 mice) within one model. Infiltration of murine NK cells in tumor tissue was determined by IHC (analyses ongoing). All 4 EBV+ lymphoma models showed stable growth in NOG mice (B cells-, T cells-, NK cells- n= 10 - 12 mice/PDX) with a take rate of 100% and an average passaging time of 20 days, ranging from 14 to 26 depending on the specific model. Taken together these results indicate that tumor growth of EBV+ lymphomas in mice depends on the NK cell activity as well as the B- and T cells status of the recipient mouse. These cell populations control tumor engraftment in mice, which might explain the absence of lymphoma disease in donor patients. In contrast the PDX of a clinically apparent EBV- DLBCL patient was able to circumvent NK cell block in NMRI nu/nu mice and showed similar take rates (100%, 5 out of 5 engrafted mice) but prolonged passaging times (35 days) as compared to NOG (26 days). Comparison of tumor cell engraftment after subcutaneous (s.c.), intravenous (i.v.) and intratibial (i.t.) injection of tumor cells in NOG mice revealed disseminated tumor growth for the EBV- DLBCL PDX exclusively when injected i.v. or i.t.. After subcutaneous implantation of lymphoma material, no tumor cells could be detected by flow cytometry or IHC in lymph nodes, blood, spleen, brain, liver, lung or bone marrow of recipient mice. However, lymphoma cells could be detected in liver and bone marrow of all (n= 8) i.v. and i.t. injected mice. Currently we are analyzing the dissemination behavior of the EBV+ lymphomas after injection into the different immunological niches as described above. In summary these data highlight the importance of the tumor microenvironment in lymphoma engraftment and dissemination in vivo. Accordingly we could show for our EBV associated lymphoma models that NK-, T- and B cell population control engraftment in vivo and are possibly involved in controlling EBV+ transformed lymphatic cells in patients. Furthermore, we showed that injection into the biologically relevant niche enables lymphoma cells to form multiple tumor nodules in distinct organs in vivo. These models contribute to a better understanding of the interplay between lymphoma cells and their microenvironment. They will thereby facilitate the discovery of new targets for innovative anti-lymphoma treatment strategies. Disclosures Tschuch: Oncotest GmbH: Employment. Klingner:Oncotest GmbH: Employment. Lenhard:Oncotest GmbH: Employment. Haapaniemi:Biositehisto Oy: Employment. Schueler:Oncotest GmbH: Employment.