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Introduction: Daratumumab (DARA) is a human CD38-targeting monoclonal antibody that induces deep clinical responses in MM pts through multifaceted mechanisms of action (MOA) including complement-dependent cytotoxicity, antibody-dependent cellular cytotoxicity, antibody-dependent cellular phagocytosis and induction of apoptosis. Flow cytometry analysis revealed a previously unknown immunomodulatory role of DARA, via T-cell induction expansion, T-cell activity enhancement, and reduction of immune suppressive cell populations including CD38+ myeloid-derived suppressor cells, CD38+ regulatory T cells (TRegs), and CD38+ regulatory B cells (BRegs). Next-generation mass cytometry (CyTOF), which allows high parameter evaluation of the immune system, was used to assess the effects of DARA alone or in combination on a more comprehensive profile of immune cell subpopulations. Methods: Relapsed/refractory MM pt samples from a subset of single agent studies; SIRIUS (32 pts; whole blood [WB] only; Lonial S et al. The Lancet, 2016) and GEN501 (5 pts; WB and bone marrow [BM], Lokhorst HM et al. NEJM, 2015) along with GEN503, a study of DARA plus lenalidomide and dexamethasone (9 pts; WB and BM; Plesner T et al. ASH 2015) were analyzed. Fluorochrome or metal-conjugated antibody panel stained samples were evaluated by flow cytometry or cytometry by time-of-flight (CyTOF®) platforms, respectively. FACS analyses were performed and analyzed by FACS Canto II flow cytometers and FACSDiva software. For CyTOF analysis, events were clustered by phenotype by a spanning tree progression of density normalized events (SPADE) algorithm, and each cluster was associated with an immune population via Cytobank® software. Differential analysis of population fractions and marker intensity, over time and between response groups, derived raw P values from t-tests and single cell level bootstrap adjusted P values corrected for multiple dependent hypothesis testing. Results were visualized using SPADE trees (Figure) and Radviz projections, a new method that allows for the comparison of populations and conditions while preserving the relation to original dimensions. Results: Flow cytometry and high-dimensional CyTOF analyses confirmed previous findings including higher CD38 expression on plasma cells compared with other immune populations of natural killer (NK), monocytes, B and T cells, and depletion of both plasma cells and NK cells upon DARA treatment. Interestingly, while NK cells were significantly reduced with DARA treatment, remaining active NK cells (CD16+CD56dim) demonstrated increased expression of activation markers CD69, CD25 and CD137 while also decreasing granzyme B and increasing naive marker CD27. Though functionality tests weren't performed, the ability to evaluate several markers simultaneously suggests these cells possess limited cytotoxicity. Additionally, these studies indicated depletion of CD38 positive immune suppressive subsets of Tregs and Bregs. CD38+ basophil reductions occurred independent of response and may provide insight to short-lived infusion related reactions. Several observations within the T-cell compartment were indicative of a DARA-mediated adaptive response in both WB and BM samples. T cells displayed increases in total numbers and shifted towards higher CD8:CD4 and effector:naïve ratios after 2 months of DARA treatment. Responders had higher expression levels of several activation markers including CD69 and HLA-DR along with increased production of cytolytic enzyme granzyme B in CD8+ T cells following DARA treatment. Interestingly, in the GEN503 sample set, pts who achieved a complete response presented with a distinct BM CD4 T-cell phenotype of high granzyme B positivity versus those that achieved a partial response or very good partial response. This observation suggests pts with an active immune phenotype may achieve deeper responses to DARA in combination with standard of care agents lenalidomide and dexamethasone. Conclusion: CyTOF analysis of pt samples from both single agent and combination DARA studies agree with flow cytometry and support the pharmacodynamics and immune modulatory MOA of DARA while providing additional insight into changes in T-cell subtypes and activation status. Future CyTOF analyses of clinical samples from phase 3 combination studies aim to confirm these observations and expand the understanding of the MOA of DARA. Disclosures Adams: Janssen Research & Development, LLC: Employment. Stevenaert:Janssen: Employment. Van der Borght:Janssen: Employment. Casneuf:Janssen R&D, Beerse, Belgium: Employment; Johnson & Johnson: Equity Ownership. Smets:Janssen: Employment. Bald:Janssen: Employment. Abraham:Janssen: Employment. Ceulemans:Janssen: Employment. Vanhoof:Janssen: Employment; Johnson & Johnson: Equity Ownership. Ahmadi:Janssen: Employment. Usmani:Onyx: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Array: Research Funding; BioPharma: Research Funding; Pharmacyclics: Research Funding; Takeda: Consultancy, Research Funding, Speakers Bureau; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Research Funding; Millenium: Membership on an entity's Board of Directors or advisory committees; Skyline: Membership on an entity's Board of Directors or advisory committees. Plesner:Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding. Lonial:Janssen: Consultancy; BMS: Consultancy; Merck: Consultancy; Novartis: Consultancy; Janssen: Consultancy; Onyx: Consultancy; Onyx: Consultancy; Millenium: Consultancy; Celgene: Consultancy; Novartis: Consultancy; BMS: Consultancy; Celgene: Consultancy. Lokhorst:Genmab: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding. Mutis:Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genmab: Research Funding; Celgene: Research Funding. van de Donk:Janssen: Research Funding; BMS: Research Funding; Amgen: Research Funding; Celgene: Research Funding. Sasser:Janssen Pharmaceuticals R&D: Employment; Johnson & Johnson: Equity Ownership.

Introduction: Daratumumab (DARA) is a human monoclonal IgG1κ CD38 antibody that functions through multifaceted mechanisms of action, including CDC, ADCC, ADCP, induction of apoptosis and immunomodulatory activity (Krejcik et al, Blood 2016;128:384-394). DARA was added to standard of care (SOC) regimens in two randomized, controlled, phase 3 trials in RRMM: POLLUX (DARA+lenalidomide/dexamethasone [DRd] vs lenalidomide/dexamethasone [Rd]) and CASTOR (DARA+bortezomib/dexamethasone [DVd] vs bortezomib/dexamethasone [Vd]). The addition of DARA to these regimens resulted in significant improvements in PFS (hazard ratio for PFS: 0.37 and 0.39 for POLLUX and CASTOR, respectively) and ORR (POLLUX: 93% DRd vs 76% Rd; CASTOR: 83% DVd vs 63% Vd) (Dimopoulos MA et al, N Engl J Med 2016; in press; Palumbo A et al, N Engl J Med 2016; in press). To determine the ability of DARA to drive deep clinical responses beyond complete response (CR), MRD was assessed in both POLLUX and CASTOR using next-generation sequencing (NGS) of B cell receptor (Ig). This is the first comprehensive and prospective study of MRD to date in randomized phase 3 clinical trials of RRMM patients and investigates the ability of DARA-containing regimens to drive deep responses in this challenging population. Methods: MRD was assessed in POLLUX (blinded to treatment group) at the time of suspected CR, and at 3 and 6 months post-suspected CR for patients who maintained this response. Similarly, in CASTOR, MRD was assessed for patients at the time of suspected CR (blinded to treatment group) and at 6 months and 12 months after first dose (at the end and 6 months after end of Vd background therapy, respectively). MRD was assessed on bone marrow aspirate samples prepared using Ficoll and evaluated by the ClonoSEQTM assay (Adaptive Biotechnologies, Seattle, WA, USA) at sensitivities of 0.01% (1 cancer cell per 10,000 nucleated cells or 10-4), 0.001% (10-5), and 0.0001% (10-6). The MRD negative rate per treatment arm was determined as the proportion of patients with negative MRD at any time point after the first dose and compared using the likelihood-ratio test. To allow for a stringent, unbiased evaluation of MRD in these studies, the entire intent-to-treat population was evaluated where patients were considered MRD positive if they had only MRD positive test result or had no MRD assessment. In POLLUX, 63% in DRd and 87% in Rd did not receive a MRD assessment, and for CASTOR, 76% in DVd and 87% in Vd were not assessed for MRD. Results: Median duration of follow-up was 13.5 months and 7.4 months in POLLUX and CASTOR, respectively. The addition of DARA to SOC regimens (Rd in POLLUX or Vd in CASTOR) resulted in significantly higher MRD negative rates at all three thresholds examined (10-4, 10-5, and 10-6; Table). Additionally, patients who achieved MRD negative status experienced fewer PFS events compared with MRD positive patients at a threshold of 10-5 (Figure). Among pts who achieved CR or better, the rate of MRD negativity was at least 3-fold higher across all sensitivity thresholds in the DARA + SOC treatment groups compared with SOC alone. In MRD positive patients, PFS was significantly longer in the DARA + SOC treatment groups compared with SOC alone treatment arms. Updated data from both studies will be presented at the annual meeting. Conclusion: These two studies represent the first randomized, controlled, prospective evaluation of MRD in the RRMM phase 3 clinical trial setting and demonstrate that DARA-containing therapies are able to drive patients to remarkably deep levels of clinical response. Regardless of the SOC component, DARA-containing regimens consistently demonstrated ≥3-fold increase in MRD negative rate compared with the control groups at all evaluated thresholds. Importantly, since patients who achieved MRD negative status demonstrated low PFS event rates, the deep clinical responses induced by DARA may lead to improved survival. Table MRD negative Rate Based on Threshold Used Table. MRD negative Rate Based on Threshold Used Figure Progression-free survival by MRD status in POLLUX and CASTOR Figure. Progression-free survival by MRD status in POLLUX and CASTOR Disclosures Avet-Loiseau: Celgene: Honoraria, Research Funding, Speakers Bureau; Amgen: Honoraria, Speakers Bureau; BMS: Honoraria, Speakers Bureau; Sanofi: Honoraria, Speakers Bureau; Janssen: Honoraria, Research Funding, Speakers Bureau. Casneuf:Janssen R&D, Beerse, Belgium: Employment; Johnson & Johnson: Equity Ownership. Chiu:Janssen: Employment. Moreau:Janssen: Honoraria, Speakers Bureau; Amgen: Honoraria; Novartis: Honoraria; Takeda: Honoraria; Bristol-Myers Squibb: Honoraria; Celgene: Honoraria. Plesner:Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding. Khokhar:Janssen: Employment. Qi:Janssen: Employment. Schecter:Janssen: Employment, Equity Ownership. Carlton:Adaptive Biotechnologies: Employment, Equity Ownership. Qin:Janssen: Employment. Liu:Janssen: Employment; J&J: Equity Ownership; Merck: Equity Ownership. Wu:Janssen: Employment. Zhuang:Janssen: Employment. Ahmadi:Janssen: Employment. Sasser:J&J: Equity Ownership; Janssen: Employment. San-Miguel:Amgen: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; MSD: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

Background: This EU PASS is an observational, non-interventional registry designed to characterize the safety profile of pomalidomide (POM) in the treatment of RRMM in a real-world setting. Objectives:To assess the incidence of adverse events (AEs) of special interest, including neutropenia, thrombocytopenia, venous thromboembolism, peripheral neuropathy, and second primary malignancies in patients (pts) with RRMM treated with POM according to current clinical practice. Methods: Pts with symptomatic RRMM were enrolled at the investigator's discretion and after the decision was made to treat with POM. Thromboprophylaxis was administered per local standard practice. AEs were graded according to National Cancer Institute-Common Terminology Criteria for Adverse Events (version 4). The study is ongoing and open for recruitment in centers across Europe. Results: As of June 2016, 218 pts across 100 institutions in 8 European countries were included in the safety population. At the time of this abstract, 153 pts (70.2%) were ongoing. Median age was 68 yrs (range, 37-88 yrs), with 39.9% of pts < 65 yrs, 32.6% between 65 and 75 yrs, and 27.5% ≥ 75 yrs; 56.9% were male. Median time from diagnosis was 4.7 yrs (range, 0.4-25.4 yrs). Median number of prior therapies was 3 (range, 0-10); 80.8% of pts had at least 3 prior lines. Most pts (95%) received prior lenalidomide (given in second line in 66.1% and in third line in 20.2%). Prior bortezomib was administered in 96.8% of pts (given in second line in 54.6% and in third line in 28.4%). Almost half of the pts (49.5%) had a good performance status (Eastern Cooperative Oncology Group performance status 0-1). In this analysis, median treatment duration was 12.9 wks (range, 0.7-87.9 wks). Overall, 50.9% of pts (n = 111) had grade 3-4 AEs. AEs of all grades occurred in 79.8% (n = 174). Neutropenia of all grades was only reported in 22% of pts (n = 48), and febrile neutropenia in 2.3% of pts (n = 5). Infections of all grades occurred in 44.5% of pts (n = 97); of those, 11.5% were pneumonia. Thrombocytopenia occurred in 7.8% (n = 17). Fatigue occurred frequently in 13.8% of pts (n = 30). There were some gastrointestinal disorders, such as diarrhea in 7.8% (n = 17), constipation in 6.4% (n = 14), and nausea in 6.0% (n = 13) of pts. Peripheral polyneuropathy was uncommon (3.2%; n = 8). Acute myocardial infarction and deep vein thrombosis were observed in 1 pt each, and 1 pt developed basal cell carcinoma. Conclusions: Results of this ongoing non-interventional study in RRMM on the use of POM in the real-world setting show a similar AE profile to that in the pivotal trial published by San Miguel in Lancet Oncology (2013). POM was generally well tolerated, and the overall safety profile is similar to that seen in pivotal trials. Updated data will be presented at the meeting. Disclosures Di Raimondo: ARIAD; Bristol-Myers Squibb; Novartis; Roche: Consultancy; Bristol-Myers Squibb; Celgene; Novartis: Speakers Bureau; Gilead Sciences: Other: Travel Expenses. Bentsen:Celgene, Teva: Other: Travel expenses. Kueenburg:Celgene International Sarl: Consultancy, Honoraria. Lersch:Celgene: Employment. Bacon:Celgene: Employment, Equity Ownership. Plesner:Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Introduction: FCR is still the standard frontline regimen for physically fit CLL pts without TP53 alteration. The international phase III CLL10 study demonstrated the inferiority of BR in comparison to FCR in this population. In order to evaluate long-term outcome and toxicity we performed an updated analysis after extended observation time. Methods and Patients: 561 pts were randomized as previously published (Eichhorst B et al., Lancet Oncol 2016). 282 and 279 pts were randomized to receive either 6 courses of FCR or BR respectively. After the end of treatment pts were followed 3-monthly for 2 years (yrs) and 6-monthly for 3 yrs, and afterwards annually until progressive disease (PD). After PD annual visits were documented either within the study or within the registry of the GCLLSG. Health related quality of life (HRQOL) was evaluated by using the EORTC C30 questionnaire, which was completed at baseline, after 3, 6 and 12 months and then annually until year 5. Results: After a median observation time of 58.2 months (mo) (range, 0 - 88 mo), the median progression-free survival (PFS) for FCR was 57.6 mo versus (vs) 42.3 mo for the BR arm [Hazard ratio (HR)=1.593 (95% confidence interval (CI), 1.271-1.996); p 65 yrs] (57.6 vs 42.3; p=0.134). Richter transformation (RT) was assessed as PD and occurred in 5 pts (1.8%) after FCR and 8 pts (2.9%) after BR. 77 (27.3%) pts following FCR and 108 (38.7%) following BR treatment received at least one subsequent therapy. BR was the most common second line therapy after prior FCR (42 pts), while BR re-exposure after frontline BR was performed in 31 pts. 33pts switched to FCR after BR first line therapy. So far, only 3 pts after FCR and 2 pts after BR received kinase inhibitors. 51 pts (18.1%) of the FCR arm and 54 (19.4%) pts of the BR arm deceased so far. Main cause of death in the FCR arm were secondary malignancies (14 pts; 5.0%), followed by CLL including RT (11 pts incl. 2 RT; 3.9%), infections (7 pts; 2.5%) and concomitant diseases (6 pts; 2.1%). In the BR arm CLL was the most common cause of death (15 pts incl. 6 pts with RT; 5.4%) followed by infections (12 pts; 4.3%) and concomitant disease and secondary malignancies (10 pts each; 4.3% each). Other causes of deaths were distributed similarly (incl. adverse events to frontline or relapse treatment or unknown). No differences in overall survival (OS) were observed (OS at 5 yrs, 80.9% for FCR vs 80.1% for BR; HR=1.108, 95% CI 0.755-1.627; p=0.599). The difference in OS for younger pts was statistically not significant (OS at 5-yrs 85.6% for FCR vs 81.1% for BR; p=0.119). Otherwise, 5-yrs OS was 78.8% for pts > 65 yrs receiving BR and 70.9% for those receiving FCR (p=0.238). Multivariate analysis identified treatment arm, male sex, high serum thymidine kinase (TK), del(11q) and unmuated IGHV status, but not age as independent prognostic factors for PFS. For OS only high serum TK and unmutated IGHV status were assessed as independent prognostic factors. Secondary neoplasia was documented in 49 (17.6%) of 279 FCR treated pts and 35 (12.5%) of 278 BR treated pts. No difference in the incidence rate of secondary solid tumors was observed between both arms (for FCR 28 (10.0%) and for BR 25 (9.1%)). Secondary MDS and/or AML occurred more frequently after FCR therapy (12 [4.3%] vs 2 [0.7%]), particularly in pts >65 yrs (6 [7.0%] vs 1 [0.9%]). 540 of 561 pts (96.3%) were evaluable for HRQOL analysis, 272 pts of the FCR and 268 pts of the BR arm. No differences between both arms were detected with respect to global health status or any functional or symptom scale. As compared to an age- and sex-matched normal population functional scale values were impaired mostly during treatment phase and symptom scales also during follow-up. However, after the end of therapy and during follow-up global health status was improved. Conclusion: Long-term follow-up data of the CLL10 study confirm the superiority of FCR regimen in young (≤ 65 yrs) and fit CLL patients. However, importantly these data support the recommendation of using BR in fit elderly pts. This particular group of pts had both a very good outcome after BR and compared to FCR a decreased risk of secondary MDS and/or AML. Disclosures Eichhorst: Novartis: Consultancy; Gilead: Consultancy, Research Funding, Speakers Bureau; Abbvie: Consultancy, Research Funding, Speakers Bureau; Janssen: Consultancy, Research Funding, Speakers Bureau; Celgene: Speakers Bureau; Roche: Consultancy, Research Funding, Speakers Bureau; Mundipharma: Consultancy, Research Funding, Speakers Bureau. Bahlo:F. Hoffman-La Roche: Honoraria, Other: Travel grant. Maurer:Mundipharma: Other: Travel grants. Kiehl:Roche: Consultancy, Other: Travel grants, Speakers Bureau. Fischer:Roche: Other: travel grants. Kneba:Amgen: Research Funding; Roche: Consultancy, Honoraria, Other: Travel grants, Research Funding; Gilead: Consultancy, Honoraria, Other: Travel grants, Research Funding; AbbVie: Consultancy, Honoraria, Other: Travel grants; Glaxo-SmithKline: Other: Travel grants; Janssen-Cilag: Consultancy, Honoraria, Other: Travel grants. Wendtner:Hoffmann-La Roche, Mundipharma, Janssen, Gilead, Abbvie, Servier, Morphosys: Consultancy, Other: Travle grants, Research Funding. Klapper:Roche, Novartis, Amgen, Takeda: Research Funding. Kreuzer:Roche Pharma GmbH and Mundipharma GmbH: Consultancy, Honoraria, Research Funding, Speakers Bureau; Gilead Sciences: Consultancy, Honoraria, Research Funding, Speakers Bureau. Böttcher:Celgene: Research Funding; Hoffmann-LaRoche: Honoraria, Other: Travel grants, Research Funding; AbbVie: Honoraria, Research Funding. Stilgenbauer:GSK: Consultancy, Honoraria, Other: Travel grants , Research Funding; AbbVie: Consultancy, Honoraria, Other: Travel grants, Research Funding; Amgen: Consultancy, Honoraria, Other: Travel grants, Research Funding; Novartis: Consultancy, Honoraria, Other: Travel grants , Research Funding; Genzyme: Consultancy, Honoraria, Other: Travel grants , Research Funding; Gilead: Consultancy, Honoraria, Other: Travel grants , Research Funding; Hoffmann-La Roche: Consultancy, Honoraria, Other: Travel grants , Research Funding; Celgene: Consultancy, Honoraria, Other: Travel grants , Research Funding; Genentech: Consultancy, Honoraria, Other: Travel grants , Research Funding; Mundipharma: Consultancy, Honoraria, Other: Travel grants , Research Funding; Pharmacyclics: Consultancy, Honoraria, Other: Travel grants , Research Funding; Janssen: Consultancy, Honoraria, Other: Travel grants , Research Funding; Sanofi: Consultancy, Honoraria, Other: Travel grants , Research Funding; Boehringer Ingelheim: Consultancy, Honoraria, Other: Travel grants , Research Funding. Fink:Celgene: Research Funding; Roche: Honoraria, Other: Travel grants; Mundipharma: Other: Travel grants; AbbVie: Other: Travel grants. Hallek:Celgene: Research Funding; Roche: Consultancy, Honoraria, Research Funding; Gilead: Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Genentech: Consultancy, Honoraria; GSK: Research Funding; Mundipharma: Research Funding; Janssen: Research Funding.

Introduction: For decades conventional skeletal survey (CSS) has been the standard imaging technique for the detection of bone impairment caused by Multiple Myeloma (MM). The development of cross-sectional imaging techniques like Whole Body Computed Tomography (WBCT) with or without Positron Emission Tomography (PET/PET-CT) and magnetic resonance imaging (MRI) led to the use of these techniques in several centers worldwide and to the implementation of their findings into the revised diagnostic criteria of MM. The aim of the current multicenter study was to compare sensitivity and prognostic significance of WBCT and CSS in patients with monoclonal plasma disorders. Methods: For the current retrospective analysis 308 patients with different stages of monoclonal plasma cell disorders from 8 centers were included. Compact discs with pseudonymized imaging data of WBCT and CSS were sent to the organizing center and uploaded to the picture archiving and communication system. In addition, questionnaires were completed by each center to collect clinical data of the patients. Image analysis was performed in consensus reading by three experienced radiologists (LAM, SD, VK) blinded to the clinical data of the patients. Osteolytic lesions in 19 pre-specified anatomic regions were characterized as "definitely present", "probably present", "probably absent" and "definitely absent". For the analysis only "definitely present" and "probably present" entries were counted as myeloma lesions. To define symptomatic or active versus smoldering myeloma based on the presence of bone lesions, findings of CSS were used to simulate the situation before the introduction of WBCT. For comparison of WBCT and CSS only those anatomic regions having valid measurements from both methods performed within 30 days without treatment in the meantime were considered. WBCT and CSS were tested for differences in detection sensitivity with the exact McNemar test. Results: Of 308 submitted datasets; 160 patients were without prior treatment and 56 of them had smoldering myeloma. Of 160 patients 80 (50%) had no osteolytic lesions on either CSS or WBCT and 33 (20.6%) showed osteolyses on both techniques. In 9 patients (5.6%) osteolyses were found on CSS but not in WBCT while 38 patients (23.8%) were positive on CT alone. Of note is that for some CT-scans, the upper extremities were not evaluable due to positioning during the examination. Odds ratio for osteolyses on WBCT compared to CSS was 4.22 (P < 0.0001). The regions where WBCT was most significantly superior in detecting osteolyses were the iliac bone, thoracic and lumbar spine as well as the ribs. CSS compared to WBCT revealed osteolyses in more patients only in the proximal extremities: for left humerus 10 (6.5%) versus 3 (1.9%), right humerus 12 (7.7%) versus 4 (2.6%), left femur 11 (7.1%) versus 7 (4.5%) and right femur 10 (6.5%) versus 8 (5.2%) respectively. Of 157 evaluable patients 63 (40.1%) showed osteoporosis (defined by radiological criteria) on both techniques while 29 (18.5%) had osteoporosis on WBCT only and 10 (6.4%) on CSS only (OR 2.90, P = 0.003). In the 56 patients with smoldering myeloma, with bone disease being defined according to the findings of CSS 12 (21.4%), were positive for osteolyses on WBCT (P = 0.0005). Conclusion: WBCT shows significantly more sites of bone destruction compared to conventional X-ray imaging particularly in skeletal regions composed primarily of trabecular bone while CSS shows more osteolyses in the appendicular skeleton - in part due to a limited field of view. More than 20% of patients with smoldering myeloma based on CSS have active disease that needs treatment based on WBCT. Therefore, WBCT is the current standard for the detection of osteolytic lesions in myeloma. The prognostic significance of WBCT and CSS findings for survival and correlation with clinical parameters will be reported at the meeting. Disclosures Hillengass: Celgene: Honoraria; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria; Novartis: Research Funding; Amgen: Consultancy, Honoraria; Sanofi: Research Funding. Plesner:Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding. Dimopoulos:Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Genesis: Consultancy, Honoraria. Goldschmidt:Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Chugai: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium: Membership on an entity's Board of Directors or advisory committees, Research Funding; Onyx: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees. Lentzsch:Celgene: Consultancy, Honoraria; BMS: Consultancy. Terpos:Takeda: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Genesis: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Novartis: Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Celgene: Honoraria.

ABSTRACT: An Ehrlich ascites tumour cell line (EHR2) was selected in vivo for resistance to mitoxantrone (MITOX). The resistant cell line (EHR2/MITOX) was 6123-, 33-, and 30-fold-resistant to mitoxantrone, daunorubicin, and etoposide, respectively, but retained sensitivity to vincristine. The resistant cells showed moderate sensitisation to mitoxantrone on treatment with verapamil or cyclosporin A. Compared with EHR2, the multidrug resistance-associated protein mRNA was increased 13-fold in EHR2/MITOX. Western blot analysis showed an unchanged, weak expression of P-glycoprotein. Topoisomerase IIα was reduced to one-third in EHR2/MITOX relative to EHR2 cells, whereas topoisomerase IIβ was present in EHR2 but could not be detected in EHR2/MITOX. In the resistant subline, net accumulation of MITOX (120 min) and daunorubicin (60 min) was reduced by 43% and 27%, respectively, as compared with EHR2. The efflux of daunorubicin from preloaded EHR2/MITOX cells was significantly increased. EHR2/MITOX microsomes had a significant basal unstimulated ATPase activity. The apparent K i value for vanadate inhibition of the ATPase activity in EHR2/MITOX microsomes was not significantly different from the K i value for P-glycoprotein-positive cells. However, whereas verapamil (50 μM) inhibited the ATPase activity of EHR2/MITOX microsomes, it stimulated the ATPase activity of microsomes derived from P-glycoprotein-positive cells. In conclusion, the resistance in EHR2/MITOX was multifactorial and appeared to be associated with: 1) a quantitative reduction in topoisomerase IIα and β protein; 2) reduced drug accumulation, probably as a result of increased expression of a novel transport protein with ATPase activity; and 3) increased expression of MRP mRNA.

A high-affinity receptor for urokinase-type plasminogen activator (uPAR) has been identified on the plasma membrane of a number of different cell types, and has been shown to be important for plasminogen activation, cell adhesion, and possibly signal transduction. uPAR and uPA cosediment with secretory vesicles and specific granules by subcellular fractionation and translocate to the plasma membrane upon activation of neutrophils. Here the subcellular distribution of uPAR and uPA is studied by electron microscopy of neutrophils using immunogold double labeling for uPAR and uPA and a set of markers for well-defined subtypes of granules: matrix metalloproteinase type-9 (MMP-9) for gelatinase granules, lactoferrin (LF) for specific granules, and myeloperoxidase (MPO) and neutrophil elastase (NE) for primary granules. With this technique uPAR colocalizes with uPA in 71% of labeled granules. In granules containing uPAR the degree of coexpression with MMP-9, MPO and NE was 19, 66, and 74%, respectively. In granules labeled for uPA the corresponding overlap with MMP-9, MPO and NE was 24, 64, and 51%, respectively. Low levels of co-localization were found for uPAR and LF (7%) and for uPA and lactoferrin (5%). The results indicate that uPAR and uPA are present in gelatinase granules and primary granules, but rarely in specific granules. The demonstration of uPAR and uPA in primary granules is of particular interest, and may indicate that uPAR and uPA participate in the activation of latent hepatocyte growth factor of neutrophils.

and showed evidence of disease in 145 (93%). After a median follow-up of 3.5 years, the median OS for the entire group was 7 months. Untreated and treated patients had median OS of 2 and 8 months, respectively. At least one previous line of therapy for MM and > 1 cytogenetic abnormality were independently associated with worse OS. The median OS for patients with 0, 1 and 2 of these risk factors were 25 months, 5.5 months and 2 months, respectively. Conclusion: Neurological manifestations observed in patients with MM should raise a suspicion of CNS involvement. The diagnosis of CNS MM should be based on imaging studies and CSF cytology and flow cytometry. Although prognosis is generally poor, especially in patients with a long history of chemotherapy and unfavorable cytogenetic profile, survival of individuals free from these negative prognostic factors can be prolonged due to administration of systemic treatment and/or radiotherapy. Prospective multi-institutional studies are warranted to improve the outcome of patients with CNS MM. PO-089 Improved tumor response and survival outcomes with post-transplant bortezomib (Btz) consolidation versus observation (Obs) alone in patients with newly diagnosed multiple myeloma (MM): Results from a randomized, open-label, multicenter, parallel-group phase 2 study O. Sezer, E. Terpos, R. Hajek, G. Sucak, S. Cagirgan, W. Linkesch, O.M. Akay, G. Armbrecht, T. Plesner, J.A. Snowden, H. Nahi, Z. Gulbas, A. Potamianou, C. Couturier, R. Olie, C. Feys, N. Allietta, M. Beksac Department of Hematology and Stem Cell Transplantation, Memorial Hospital, Istanbul, Turkey; Department of Clinical Therapeutics, University of Athens School of Medicine, Alexandra General Hospital, Athens, Greece; Internal Medicine and Hematooncology, Masaryk University Hospital Brno and Faculty of Medicine University Hospital Ostrava, Czech Republic; Department of Hematology, Faculty of Medicine, Gazi University, Ankara, Turkey; Department of Hematology and BMT Center, Izmir Medicalpark Private Hospital, Izmir, Turkey; Department of Hematology, Medical University Clinic, Medical University of Graz, Graz, Austria; Hematology Department, Eskisehir Osmangazi University, Eskisehir, Turkey; Centre for Muscle and Bone Research, Charite e Campus Benjamin Franklin, Berlin, Germany; Department of Hematology, Vejle Hospital, Vejle and IRS/ SDU, Denmark; Department of Haematology, Royal Hallamshire Hospital, Sheffield, UK; Haematology Centre, Karolinska University Hospital, Stockholm, Sweden; Bone Marrow Transplantation Department, Anadolu Health Center, Kocaeli, Turkey; Janssen-Cilag Pharmaceutical SACI, Athens, Greece; Janssen-Cilag, Issy-lesMoulineaux, France; Janssen-Cilag AG, Zug, Switzerland; Janssen Research & Development, Division of Janssen Pharmaceutica NV, Beerse, Belgium; Covance for Janssen-Cilag, Issy-les-Moulineaux, France; Department of Hematology, Ankara University, Ankara, Turkey Background: Induction therapy followed by high-dose chemotherapy and autologous stem cell transplantation (ASCT) is a standard frontline treatment for eligible MM patients (pts). PostASCT consolidation can improve depth of response and long-term outcomes. This phase 2 study evaluated the effects of Btz consolidation vs Obs alone on MM-related bone disease; pre-specified secondary endpoints of disease status (myeloma response), PFS, and OS are reported here. Methods: Eligible pts were adults with newly diagnosed MM who had achieved PR after single/double ASCT. Pts were randomized 1:1 (stratified by baseline bisphosphonate use and age) to receive 4 x 35-d cycles of Btz 1.6 mg/m2 IV on d 1, 8, 15, and 22, or Obs alone, followed by 18-mos’ follow-up for disease status and survival monitoring. Response was investigator-assessed per IMWG 2009 criteria. A last observation carried forward approach was used for missing assessments. PFS and OS were estimated using Kaplan-Meier analysis; exploratory p-values were determined using the log-rank test. Results: Between July 2009 and May 2012, 106 pts were randomized (52 Btz, 54 Obs). 2 pts were withdrawn prior to d 1, thus the modified intent-to-treat (mITT) population included 104 pts (51 Btz, 53 Obs): median age 58 vs 57 yrs; 71% vs 66% prior Btz-based induction. 41 (79%) and 46 (85%) pts completed the respective Btz consolidation and Obs phases as planned. CR+sCR rates at screening were 16% (Btz) and 15% (Obs). After the treatment/Obs phase, response rates were (Btz vs Obs): CR+sCR 22% vs 11%; VGPR 80% vs 68%; PD 8% vs 23%. PFS appeared longer with Btz consolidation vs Obs, both from randomization (median 44.9 vs 21.8 mos) and from first antiMM treatment (median 50.7 vs 35.0 mos) (Figure). After 21 (20%) deaths (11 Btz; 10 Obs), mean OS from randomization was 40.1 vs 36.6 mos and from first anti-MM treatment was 51.7 vs 47.1 mos; median OS was not reached in either arm at both time points. Pvalues for between-arm differences in response rates, PFS, and OS were all >0.05, likely due to small sample size. Conclusion: Compared with Obs alone, Btz consolidation was associated with trends for improved response depth and longer PFS and OS in newly diagnosed MM pts. Our response and PFS findings compare favorably with recent phase 3 data of Btz consolidation vs Obs in 15th International Myeloma Workshop, September 23-26, 2015 e129

to those in the overall population. STRATUS is a multicenter, single-arm, open-label, European phase 3b trial designed to further evaluate safety and efficacy of POM+LoDEX in a large pt population. This analysis examined pt outcomes by age. Patients and Methods: Pts with RRMM who had progressive disease (PD) during or within 60 days of their last line of Tx (refractory to last prior Tx) were included. Pts must also have experienced failure of bortezomib and lenalidomide Tx after 2 consecutive cycles of each (alone or in combination) and received adequate prior alkylator therapy. Pts with creatinine clearance 75 yrs) D1, 8, 15, and 22 of a 28-day cycle until PD or unacceptable toxicity. Thromboprophylaxis was required. Primary endpoint was safety; key secondary endpoints were overall response rate (ORR; partial response), duration of response (DOR), POM exposure, PFS, and OS. Outcomes were analyzed by pt age at baseline ( 65 vs > 65 yrs and 70 vs > 70 yrs). Results: As of Jan 23, 2015, 682 pts were enrolled. 676 received POM + LoDEX; 46% were 65 yrs, 54% > 65 yrs, 69% 70 yrs, and 31% > 70 yrs. Pts were heavily pretreated (median, 4 prior Tx). Median follow-up was 12.8 mos. Median relative dose intensity was similar across age groups (range, 0.92-0.93). Most frequently observed grade (Gr) 3/4 treatmentemergent adverse events (TEAEs) were neutropenia (47%-48%), anemia (29%-31%), thrombocytopenia (18%-25%), and infections (28%-34%). Discontinuation of POM due to TEAEs was infrequent: 65 yrs (4%), > 65 yrs (7%), 70 yrs (4%), and > 70 yrs (9%). Response rates ranged between 32% to 34% and median DOR ranged from 6.5 to 9.2 mos. Median PFS was similar regardless of age (range, 4.2-4.7 mos). Conclusion: POM+LoDEX was tolerable and demonstrated efficacy in RRMM pts regardless of age. Across age groups, the safety profile and relative dose intensity were similar; PFS and response rates were also similar and consistent with previous findings. Results support POM 4 mg as an effective starting dose and use of POM+LoDEX, regardless of age, in RRMM pts.

Of 560 consecutive, newly diagnosed untreated patients with B CLL submitted for chromosome study, G-banded karyotypes could be obtained in 480 cases (86%). Of these, 345 (72%) had normal karyotypes and 135 (28%) had clonal chromosome abnormalities: trisomy 12 (+12) was found in 40 cases, 20 as +12 alone (+12 single ), 20 as +12 with additional abnormalities (+12 complex ). Other frequent findings included abnormalities of 14q, chromosome 17, 13q and 6q. The immunophenotype was typical for CLL in 358 patients (CD5 + , Slg weak , mainly FMC7 − ) and atypical for CLL in 122 patients (25%) (CD5 − , or Slg strong or FMC7 + ). Chromosome abnormalities were found significantly more often in patients with atypical (48%) than in patients with typical CLL phenotype (22%) ( P complex , 14q +, del6q, and abnormalities of chromosome 17 were significantly more frequent in patients with atypical CLL phenotype, whereas +12 single was found equally often in patients with typical and atypical CLL phenotype. The cytomorphology of most of the +12 patients was that of classical CLL irrespective of phenotype. In univariate survival analysis the following cytogenetic findings were significantly correlated to a poor prognosis: chromosome 17 abnormalities, 14q +, an abnormal karyotype, +12 complex , more than one cytogenetic event, and the relative number of abnormal mitoses. In multivariate survival analysis chromosome 17 abnormalities were the only cytogenetic findings with independent prognostic value irrespective of immunophenotype. We conclude that in patients with typical CLL immunopenotype, chromosome abnormalities are somewhat less frequent at the time of diagnosis than hitherto believed. +12 single is compatible with classical CLL, and has no prognostic influence whereas chromosome 17 abnormalities signify a poor prognosis. In patients with an atypical CLL immunophenotype, chromosome abnormalities including +12 complex , 14q +, del 6q and chromosome 17 are found in about 50% of the patients, and in particular chromosome 17 abnormalities suggest a poor prognosis.

Summary The impact of first-line treatment with the anti-CD 20 chimeric monoclonal antibody rituximab in patients with warm-antibody reactive autoimmune haemolytic anaemia (WAIHA) is unknown. We report the first randomized study of 64 patients with newly diagnosed WAIHA who received prednisolone and rituximab combined (N = 32) or prednisolone monotherapy (N = 32). After 12 months, a satisfactory response was observed in 75% of the patients treated with rituximab and prednisolone but in a significantly smaller proportion (36%) of those given prednisolone alone (P = 0·003). Furthermore, relapse-free survival was significantly better after the combined therapy than after prednisolone monotherapy (P = 0·02). After 36 months, about 70% of the patients were still in remission in the rituximab-prednisolone group, whereas only about 45% were still in complete or partial remission in the prednisolone group. There was no significant difference between the two groups regarding adverse reactions to the studied medications. Likewise, serious adverse events were equally distributed, and no allergic reactions to rituximab were recorded. In conclusion, our data show that using rituximab and prednisolone combined rather than prednisolone alone as first-line treatment in WAIHA increases both the rate and the duration of the response.

Zoledronic acid (Zol) is frequently used for the treatment of bone disease in patients with multiple myeloma and breast cancer with metastasis to bone. Therefore, there is also an interest in finding the optimal dosing regimen to optimize effects, minimize side effects and reduce costs. In our phase II clinical trial we investigated the effect of Zol treatment on the serum levels of the bone markers collagen type 1 cross-linked C-telopeptide (CTX) and bone specific alkaline phosphatase (bALP) as well as on creatinine clearance (kidney function) in response to dosing and duration of treatment for each individual patient. Methods. We enrolled 30 multiple myeloma (MM) and 30 breast cancer (BC) patients whereof 10 of each had never received bisphosphonate and 20 had received at least six prior Zol treatments. Results. We found that Zol treatment strongly reduced CTX (Spearman's correlation, rs = −0.59, p = 0.0007) and bALP (Spearman's correlation, rs = −0.51, p = 0.0042) in MM patients while only CTX (Spearman's correlation, rs = −0.42, p = 0.024) was significantly affected in BC patients. Multiple linear regression analyses done on the entire cohort showed that the average time between each dose of Zol had the strongest impact on CTX (p < 0.001) and bALP (p = 0.011) levels while the total accumulated number of Zol infusions had a less pronounced effect on CTX levels (p = 0.015). In contrast, multiple linear regression analysis showed that the total number of Zol infusions had a strong negative impact on kidney function (p = 0.014) while the average time between each dose of Zol had no significant impact. Conclusion. Thus, if MM and BC patients are not treated regularly every month with Zol bone turnover is not fully suppressed, while prolonged treatment with zoledronic acid compromises kidney function. We believe that these data significantly contribute to the knowledge needed to find the optimal Zol treatment schedule.

CD38 is a type II transmembrane glycoprotein that is highly expressed in hematological malignancies, including multiple myeloma (MM). Therefore, CD38 is a promising target for antibody immunotherapy. Daratumumab is a human monoclonal antibody with broad-spectrum killing activity. In vitro, daratumumab induces anti-MM effects via complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis. Synergistic activity was demonstrated in vitro in combination with standard MM therapies, including bortezomib and lenalidomide. Protective bone marrow stromal cells did not influence daratumumab-induced CDC and ADCC, suggesting that daratumumab may have activity in the bone marrow microenvironment in vivo. Indeed, significant daratumumab-mediated tumor growth inhibition was shown in MM mouse xenograft models. A phase I/II clinical study is ongoing in patients with relapsed/refractory MM. This review discusses the preclinical pharmacology, pharmacokinetics and preliminary clinical efficacy of daratumumab and its potential in MM therapy.

The urokinase plasminogen activator (uPA) is a proteolytic enzyme which converts the proenzyme plasminogen to the active serine protease plasmin. A cell surface receptor for uPA (uPAR) is attached to the cell membrane by a glycosyl-phosphatidylinositol anchor. Binding of uPA to uPAR leads to an enhanced plasmin formation and thereby an amplification of pericellular proteolysis. We have shown previously that uPAR is expressed on normal blood monocytes and granulocytes, but is deficient on affected blood monocytes and granulocytes in patients with paroxysmal nocturnal haemoglobinuria (PNH), and that uPAR is present in plasma from these patients. In this study a newly established sensitive enzyme-linked immunosorbent assay (ELISA) has been applied for quantitation of uPAR in plasma. Unexpectedly, we found that uPAR is not only present in PNH plasma but also in plasma from healthy individuals. In 39 healthy individuals the mean plasma-uPAR value +/- SD was 31 +/- 15 pM, median 28 (range 11-108), and the corresponding value for six PNH patients was 116 +/- 67 pM, median 90 (range 61-228). The elevated uPAR-level in PNH patients was highly significant (Mann-Whitney test; P0.0001), and may possibly contribute to the propensity for thrombosis in PNH by inhibition of the fibrinolytic system. Binding of pro-uPA by uPAR in plasma may interfere with the appropriate binding of pro-uPA to cell-bound uPAR and therefore inhibit cell-associated plasmin generation and fibrinolysis. It is likely that the uPAR in normal plasma reflects the overall level of activity of the uPAR-mediated cell surface proteolysis. The present ELISA may be used for studies of uPAR levels in plasma from patients with conditions in which this activity might be increased, such as cancer and inflammatory disorders. Future studies will determine if uPAR in plasma is a parameter of clinical importance in these diseases.

Zoledronic acid (Zol) is used to treat bone disease in both multiple myeloma (MM) and breast cancer patients with bone metastasis (BC). However, bones of MM and BC patients show a difference in retention of the bisphosphonate used for bone scintigraphy. Therefore, we hypothesized that disease-specific factors may differently influence Zol retention in MM and BC patients. We tested this hypothesis in an investigator initiated phase II clinical trial in which we compared the whole-body retention (WBrt) of Zol in a cohort of 30 multiple myeloma (MM) and 30 breast cancer (BC) (20 Zol naive and 40 with six or more previous administrations). On average, 62% of the administered Zol was retained in the skeleton of both MM and BC patients and independently of the number of treatments. WBrt of Zol did not correlate with cross-linked C-telopeptide (CTX) levels, but linear regression analyses showed that WBrt of Zol correlated with bone-specific alkaline phosphatase (bALP) levels in BC (p = 0.001), and with CTX/bALP in Zol naive MM patients (p = 0.012). Especially in BC patients, WBrt correlated with age (p = 0.014) independently of kidney function. In MM patients WBrt was found to primarily correlate with the extent of bone disease (p = 0.028). Multivariate linear regression analyses of the entire cohort pointed out that WBrt of Zol was best predicted by age (p 0.000), osseous lesions (p 0.001), and the preceding Zol dosing (p 0.005) (r(2) = 0.97). Comparing bone scintigrams with CT/X-ray images showed a poor correlation between sites of active bone disease and binding of scintigraphy bisphosphonate in 36% of MM patients and in 13% of BC patients. We conclude that WBrt of Zol is primarily determined by two non-disease related factors and only one disease related, but that there may be differences in retention or drug delivery at individual sites of bone disease between MM and BC patients. In order to find the optimal dosing of Zol, these observations should be taken into account.

Abstract 1873 Background: Patients with multiple myeloma (MM) relapsed or refractory to current treatment options and ineligible for ASCT have a poor prognosis. Therefore new treatment options are highly needed for this patient population. Malignant MM cells exhibit very strong CD38 surface expression. Daratumumab is a human CD38 antibody with broad spectrum killing activity. Daratumumab mediates MM cell death via antibody dependent cellular cytotoxicity, complement dependent cytotoxicity and apoptosis. Daratumumab has been shown to inhibit growth of CD38-expression tumor cells in SCID mouse xenografts. We report the preliminary safety results from the ongoing first in man dose-escalation study (clin.trial.gov. NCT00574288). Methods: Patients (age > 18 yrs) ineligible for ASCT and relapsed or refractory to at least two different prior therapies receive 7 full and 2 pre-dose infusions of daratumumab (1st pre-dose day 1, 1st full dose day 2, 2nd pre-dose day 21, 2nd full-dose day 22 and thereafter weekly full-dose daratumumab). The study design encompasses a classic 3 + 3 dose escalation with the possibility to limit exposure to 1 patient in the first 2 cohorts pending safety data. The dose range is from 0.005 mg/kg to 24 mg/kg. The primary objective is to establish the safety profile and secondary objectives are to establish MTD and evaluate the efficacy. An independent Data Monitoring Committee evaluates the safety data for each cohort before dose-escalation. Results: Until May 31st, 2011 safety data for 20 patients, including dose range from 0.005 mg/kg up to 2 mg/kg cohort, were collected. Median age 62 yrs (42–76), F/M: 7/13, stage of MM (ISS): 1: 5 patients; 2: 9 patients; 3: 5 patients and 1 unknown. The most common AEs reported across all cohorts are pyrexia (35%), free hemoglobin (25%), anemia (25%), proteinuria (25%), cough (15%), dizziness (10%), flushing (10%), influenza-like-illness (10%), nausea (10%) hypo/hypertension (10%/10%), thrombocytopenia (10%), hemolysis (15%), lymphopenia (10%), arthralgia (10%), abdominal pain (10%) and chest pain (10%). Five SAE were assessed as related to daratumumab. One patient had anemia grade 3 and thrombocytopenia grade 4, one patient experienced cytokine release syndrome grade 2 and based on these DLT events 3 more patients were enrolled in the 0.1 mg/kg and 1.0mg/kg cohorts, one patient experienced a grade 3 bronchospasm 40 hours after end of trial drug infusion and one patient had AST elevation. All patients recovered after relevant treatment. Since implementation of relevant pre-medication and dilution of trial drug no severe infusion related AEs have been reported. The MTD has not been reached at the cut-off time point for this evaluation. Efficacy evaluation is ongoing and preliminary data will be presented at the meeting. Conclusion: Daratumumab has a favorable safety profile as monotherapy in patients with multiple myeloma in doses up to and including 2 mg/kg. The dose-escalation is ongoing and updated safety data will be presented at the meeting. When the MTD is established, an extension study with daratumumab as monotherapy will be conducted. Disclosures: Plesner: Genmab A/S: Consultancy. Lokhorst:Genmab: Consultancy. Valentin:Genmab A/S: Employment. Lisby:Genmab A/S: Employment. Richardson:Genmab: Consultancy.

Expression of the receptor for the urokinase type plasminogen activator (uPAR) has been studied by flow cytometry and immunohistology in normal blood and bone marrow cells, in vitro activated lymphoid cells, and tissue samples from reactive lymph nodes (n = 6), thymus (n = 2) and malignant lymphomas (n = 82), or leukemias (n = 32). HL-60 myeloid precursor cells and CD34-positive normal stem cells also were analyzed. In the normal cells, staining was confined to monocytes, macrophages, neutrophils, and myeloid precursors. No labelling was seen of normal or activated lymphoid cells. Purified CD34-positive hematopoietic progenitors were uPAR negative, but expressed uPAR during differentiation in short-term liquid culture stimulated in vitro by recombinant interleukin (IL)-1, IL-3, IL-6, granulocyte-macrophage colony stimulating factor (CSF), granulocyte-CSF, and stem cell factor. Enhanced uPAR expression was also seen in HL-60 cells after induction of differentiation with dimethyl sulfoxide or 1 alpha,25-dihydroxyvitamin D3. In lymphomas and leukemias, the staining pattern was similar to that seen in the normal cells with labelling of monocytic and myeloid that seen in the normal cells with labelling of monocytic and myeloid malignancies, but not of the neoplastic cells in B-cell or T-cell lymphomas or Hodgkin's disease. In conclusion, uPAR is a differentiation marker for myeloid and monocytic cells, and may act to facilitate migration of these cells in normal and pathologic conditions by cell-associated plasminogen activation. Whether expression of uPAR in myeloid and monocytic malignancies relates to their growth and behavior will be an important topic for investigations in the future.

Introduction : Daratumumab (DARA) is a human anti-CD38 IgG1κ monoclonal antibody that showed synergistic anti-tumor activity in combination with lenalidomide (LEN) in in vitro preclinical studies (van der Veer M. Haematologica 2011;96(2):284-90). DARA has shown favorable safety and robust efficacy as a single agent in patients with relapsed and refractory (RR) multiple myeloma (MM) (Lokhorst HM. J Clin Oncol 2014;32 Suppl:abstr 8513. Lonial S. J Clin Oncol 2015;33 Suppl:abstr LBA8512) and in combination with LEN/Dexamethasone (DEX) in patients with relapsed or RR MM (Plesner T. Blood 2014;124(21):84). This study assessed the updated safety and efficacy of DARA in combination with LEN/DEX following more than 12 months of exposure in patients with relapsed or RR MM. Methods : The study design of this ongoing, open-label phase 1/2 study of DARA in combination with LEN/DEX has been presented previously (Plesner T. Blood 2014;124(21):84). Briefly, the study comprised a 3 + 3 design dose escalation study (DARA 2-16 mg/kg + LEN/DEX; Part 1) and a cohort expansion study using the recommended phase 2 dose (DARA 16 mg/kg + LEN/DEX; Part 2). In Part 2, patients refractory to LEN were excluded and patients with ≥1 prior line of therapy were included. In Part 2, DARA 16 mg/kg was administered weekly during the first two 28-day cycles, every other week during Cycles 3 through 6, and monthly in Cycle 7 and beyond until disease progression or unacceptable toxicity. LEN 25 mg was administered orally on Days 1 through 21 of each cycle, and DEX 40 mg was given weekly. The primary objective was safety. Efficacy was evaluated per the International Myeloma Working Group criteria. The last patient was enrolled in August 2014. Results: Updated safety and efficacy results (data cut January 9, 2015) of DARA 16 mg/kg plus LEN/DEX in the expansion cohort (n = 32) are presented. The median (range) number of prior lines of therapy was 2 (1-3) and the median (range) duration of follow up was 7.8 (3.0-10.4) months. Eleven (34%) patients received prior LEN treatment. Six (19%) patients discontinued treatment due to either disease progression (n = 3), treatment-emergent adverse events (TEAE; 1 patient with gastric adenocarcinoma and 1 patient with laryngeal edema that was a grade 3 infusion-related reaction [IRR]), or physician decision (n = 1). The most common (>25%) TEAEs included neutropenia (81%), muscle spasms (44%), cough (38%), diarrhea (34%), fatigue and hypertension (28% each). Only 1 (3%) patient experienced febrile neutropenia (grade 1). Neutropenia was the most frequently (>25%) reported grade 3 or 4 TEAE (75%). Eighteen (56%) patients had IRRs and these were generally mild to moderate and occurred mostly during the first cycle. IRRs included cough (25%), allergic rhinitis, nausea, and vomiting (9% each), as well as dyspnea, nasal congestion, and hypertension (6% each). Two (6%) patients had grade 3 IRRs (laryngeal edema and hypertension). All patients with IRRs recovered and continued to receive treatment, with the exception of the patient with grade 3 laryngeal edema, who recovered but was discontinued from treatment per protocol. The overall response rate (Rajkumar SV. Blood. 2011;117:4691-5) was 88%, with 11 (34%) partial responses and 17 (53%) ≥very good partial responses (VGPRs) that included 7 (22%) stringent complete responses, 1 (3%) complete response, and 9 (28%) VGPRs. The median time to first and best response was 1 (95% confidence interval [CI], 0.9-1.9) month and 4.5 (95% CI, 1.9-5.6) months, respectively. The median duration of response was not reached, as 26 (93%) of 28 responders had not progressed or relapsed at the time of this analysis and remained on treatment at data cutoff. Importantly, responses deepened over time in 17 (61%) of 28 responders. The 12-month duration of response will be updated at the time of ASH. Conclusions : In the expansion cohort, the combination of DARA and LEN/DEX continues to be associated with a remarkably favorable benefit/risk profile for treatment of relapsed or RR MM. Deep and durable responses were maintained throughout the study. Additional updates on the safety and efficacy results of the expansion cohort with over 12 months of exposure will be presented at the time of the meeting. Disclosures Plesner: Roche and Novartis: Research Funding; Janssen and Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genmab: Membership on an entity's Board of Directors or advisory committees. Gimsing:Amgen: Honoraria. Laubach:Novartis: Research Funding; Onyx: Research Funding; Celgene: Research Funding; Millennium: Research Funding. Palumbo:Novartis, Sanofi Aventis: Honoraria; Celgene, Millennium Pharmaceuticals, Amgen, Bristol-Myers Squibb, Genmab, Janssen-Cilag, Onyx Pharmaceuticals: Consultancy, Honoraria. Lisby:Genmab: Employment. Basse:Genmab A/S: Employment. Wang:Janssen: Employment. Sasser:Janssen Pharmaceuticals: Employment. Guckert:Janssen: Employment. Yeh:Janssen: Employment. Ahmadi:Janssen: Employment. Lokhorst:Genmab: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Amgen: Honoraria. Richardson:Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Millennium Takeda: Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees.

Introduction: Daratumumab (DARA) is a novel human monoclonal antibody that targets CD38, a protein that is highly expressed on multiple myeloma (MM) cells. DARA acts through multiple immune effector-mediated mechanisms, including complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity, and antibody-dependent cellular phagocytosis. In two clinical studies (NCT00574288 [GEN501] and NCT01985126 [Sirius]) of DARA monotherapy in patients with relapsed and refractory MM, overall response rates were 36% and 29%, respectively. CD38 is highly expressed in myeloma cells but also expressed in lymphocytes and other immune cell populations. Therefore, the effects of DARA on immune cell populations and adaptive immune response pathways were investigated. Methods: The patient population investigated included treated subjects with MM that were relapsed after or were refractory to ≥2 prior therapies (GEN501) or had received ≥3 prior therapies, including a proteasome inhibitor (PI) and an immunomodulatory drug (IMiD), or were refractory to both a PI and an IMiD (Sirius). Patients assessed in this analysis were treated with 16 mg/kg DARA. When both studies were combined, median age (range) was 64 (31-84) years and median time from diagnosis was 5.12 (0.77-23.77) years. Seventy-six percent of patients had received >3 prior therapies and 91% were refractory to their last treatment. Clinical response was evaluated using IMWG consensus recommendations. Peripheral blood (PB) samples and bone marrow (BM) biopsies/aspirates were taken at prespecified time points and immunophenotyped by flow cytometry to enumerate various T-cell sub-types. T-cell clonality was measured by TCR sequencing. Antiviral T-cell response and regulatory T-cell (Treg) activity were analysed by functional in vitro assays. T-cell subpopulation counts were modelled over time with linear mixed modelling. Two group comparisons were performed using non-parametric Wilcoxon rank sum tests. Results: Data from 148 patients receiving 16 mg/kg DARA in GEN501 (n = 42) and Sirius (n = 106) were analyzed for changes in immune response. In PB, robust mean increases in CD3+ (44%), CD4+ (32%) and CD8+ (62%) T-cell counts per 100 days were seen with DARA treatment. However, responding evaluable patients (n = 45) showed significantly greater increases from baseline than nonresponders (n = 93) in CD3+ (P = 0.00012), CD4+ (P = 0.00031), and CD8+ (P = 0.00018) T cells. In BM aspirates the number of CD3+, CD4+, and CD8+ T-cells increased during treatment compared to baseline (the median percent increases were 19.95%, 5.66%, and 26.99% [n = 58]). Additionally, CD8+: CD4+ T-cell ratios significantly increased compared to baseline in both PB (P = 0.00017), and BM (P = 0.00016). T cell clonality, assessed by TCR sequencing, increased after DARA treatment compared with pretreatment (P = 0.049), with greater sums of absolute expansion in the repertoire (P = 0.037), as well as greater maximum expansion of a single clone (P = 0.048) in responders compared to nonresponders. Increased antiviral T-cell responses were observed post-DARA treatment, particularly in responders. Interestingly, a novel subpopulation of regulatory T cells was identified that expressed high levels of CD38. These cells comprised ~10% of all Tregs and were depleted by one DARA infusion. In ex vivo analyses, CD38+ Tregs appeared to be highly immune suppressive compared to CD38-Tregs. Conclusions: Robust T cell increases, increased CD8+: CD4+ ratios, increased antiviral responses, and increased T cell clonality were all observed after DARA treatment in a heavily pretreated, relapsed, and refractory patient population not expected to have strong immune responses. Improved clinical responses were associated with changes in these parameters. In addition, a sub-population of regulatory T cells expressing high CD38 levels was determined to be extremely immune suppressive and sensitive to DARA treatment. These data suggest a previously unknown immune modulatory role of DARA that may contribute to its efficacy, and a potential role for CD38 immune targeted therapies. We postulate that there are several distinct and complementary mechanisms that contribute to DARA's efficacy including increased antigen presentation through phagocytosis, targeting of immune suppressive Tregs, and increased adaptive immune responses. JK and TC contributed equally to this work. Disclosures Casneuf: Janssen: Employment. Verbist:Janssen: Employment. Bald:Janssen: Employment. Plesner:Genmab: Membership on an entity's Board of Directors or advisory committees; Roche and Novartis: Research Funding; Janssen and Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding. Liu:Janssen: Employment. van de Donk:Janssen Pharmaceuticals: Research Funding; Amgen: Research Funding; Celgene: Research Funding. Weiss:Janssen and Onclave: Research Funding; Janssen and Millennium: Consultancy. Ahmadi:Janssen: Employment. Lokhorst:Genmab: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Amgen: Honoraria. Mutis:Janssen: Research Funding; Genmab: Research Funding.

Abstract 475FN2 Background: An IMiDs® immunomodulatory agent, Len has a dual mechanism of action: its tumoricidal effect directly leads to tumor cell death, and its immunomodulatory effect may keep the tumor in remission. A phase 3, randomized, placebo (Pbo)-controlled trial, MM-015 compares MPR-R with fixed-duration MPR and MP induction in transplant-ineligible NDMM pts. Interim results showed unprecedented reduction in disease progression risk with MPR-R (Palumbo et al, IMW 2011); this analysis focuses on pts aged 65–75 yrs in whom the greatest benefit was observed. Methods: A total of 459 pts aged ≥ 65 yrs with NDMM were enrolled. Induction consisted of nine 28-day cycles of melphalan 0.18 mg/kg (D1-4), prednisone 2 mg/kg (D1-4), and Len 10 mg (D1-21) (MPR-R and MPR) or melphalan and prednisone with Pbo (MP). After induction, MPR-R pts received Len 10 mg (D1-21) maintenance until progression; MPR and MP pts received Pbo. Pts with progressive disease (PD) could enroll in an open-label extension phase to receive Len 25 mg (D1-21) ± dexamethasone 40 mg (D1-4, 9–12, and 17–20). This analysis includes data up to Feb 28, 2011 (median follow-up, 30 mos). Results: There were 116/152 (76%), 116/153 (76%), and 116/154 (75%) of MPR-R, MPR, and MP pts, respectively, aged 65–75 yrs. Nearly 50% had ISS stage III disease, > 40% had β2-microglobulin > 5.5 mg/L, and 40% had CrCL < 60 mL/min. With a median follow-up of 30 mos, MPR-R reduced progression risk by 70% and significantly prolonged median PFS (31 mos) vs MP (12 mos; hazard ratio [HR]: 0.30 [95% CI, 0.20–0.45]; P MPR-R significantly extended median PFS vs MPR (31 vs 15 mos; HR: 0.44 [95% CI, 0.30–0.66]; P 75 years old. Response rates and quality were similar. MPR induction alone provided a significant PFS benefit vs MP (15 vs 12 mos; HR: 0.64 [95% CI, 0.45–0.90]; P =.009). MPR induction had an acceptable safety profile, allowing the majority of pts to reach maintenance. During induction, discontinuation due to adverse events (AEs) occurred in 13% of MPR and 4% of MP pts. The most common induction grade (Gr) 4 hematologic AEs for MPR-R, MPR, and MP were neutropenia (39%, 29%, and 7%) and thrombocytopenia (33%, 12%, and 4%). Febrile neutropenia was low for all arms (1%, 1%, and 0%). The most frequent Gr 3/4 nonhematologic AE was bone pain (3%, 3%, and 4%). Len maintenance was generally well tolerated, with no evidence of cumulative toxicities. Only 5% of Len maintenance pts discontinued due to AEs. The most frequent Gr 4 hematologic AEs were thrombocytopenia (5%), neutropenia (4%), and anemia (3%); and Gr 3/4 nonhematologic AEs included bone pain (5%) and diarrhea (5%). Hematologic second primary malignancies (SPMs) during induction and maintenance were 3 (2 AML, 1 ALL), 1 (MDS), and 0 in the MPR-R, MPR, and MP arms, respectively. These corresponded to incidence rates (IRs) per 100 pt-yrs of 1.8, 0.7, and 0. Solid tumor SPMs occurred in 2 pts per arm, were heterogeneous and balanced across arms; IRs were 1.2, 1.4, and 1.6, respectively. Conclusions: Continuous Len treatment with MPR-R significantly reduced disease progression risk compared with MP and MPR in pts aged 65–75 yrs. MPR induction significantly extended PFS vs MP. To date, MPR-R has provided one of the longest median PFS (31 mos) among other available regimens (bortezomib, melphalan, prednisone: 24–27 mos; melphalan, prednisone, thalidomide: 28 mos; bortezomib, melphalan, prednisone, thalidomide: 37 mos). The PFS benefit in the landmark analysis supports continuous treatment to suppress disease in all patient age groups. Management of toxicities is essential to allow pts to continue therapy and receive full benefits. Although hematologic SPMs were imbalanced, the risk of PD/death clearly outweighs the risk of SPMs. MPR-R should be considered a standard of care in transplant-ineligible NDMM pts aged 65–75 yrs. Disclosures: Palumbo: Merck: Honoraria; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria. Off Label Use: Lenalidomide in newly diagnosed patients with multiple myeloma. Catalano:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Gisslinger:Celgene: Research Funding, Speakers Bureau; Novartis: Speakers Bureau; AOP-Orphan: Speakers Bureau. Wiktor-Jedrzejczak:Celgene: Honoraria; Janssen-Cilag: Honoraria; Novartis: Honoraria, Research Funding. Delforge:Celgene: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau. Weisel:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Van Droogenbroeck:Celgene: Consultancy; Novartis: Consultancy; BMS: Consultancy; Janssen Cilag: Consultancy. Cavo:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Millennium: Honoraria, Membership on an entity's Board of Directors or advisory committees. Blade:Celgene: Honoraria, Research Funding; Janssen: Honoraria, Research Funding. Beksac:Celgene: Honoraria, Speakers Bureau; Janssen Cilag: Honoraria, Speakers Bureau. Spicka:Celgene: Honoraria, Speakers Bureau; Janssen-Cilag: Honoraria, Speakers Bureau; Novartis: Honoraria. Yu:Celgene Corporation: Employment. Herbein:Celgene Corporation: Employment. Mei:Celgene Corporation: Employment. Jacques:Celgene Corporation: Employment. Dimopoulos:Celgene: Consultancy, Honoraria.

The arrival of the novel agents thalidomide, bortezomib, and lenalidomide has significantly changed our approach to the management of multiple myeloma and, importantly, patient outcomes have improved. These agents have been investigated intensively in different treatment settings, providing us with data to make evidence-based decisions regarding the optimal management of patients. This review is an update to a previous summary of European treatment practices that examines new data that have been published or presented at congresses up to the end of 2010 and assesses their impact on treatment practices. © AlphaMed Press., Advisory Board meeting supported by Janssen Pharmaceutical Companies of Johnson and Johnson in Europe, Middle East, and Africa (EMEA). Unrestricted educational grant provided by Janssen Pharmaceutical Companies of Johnson and Johnson in the EMEA to assist with editorial support.

Objectives: The aim of the study was to investigate the effect of bortezomib on osteoblast proliferation and differentiation, as well as on bone matrix deposition for the first time in bisphosphonate-naïve, previously untreated patients with myeloma. Methods: Twenty newly diagnosed patients received four cycles of bortezomib treatment, initially as monotherapy and then combined with a glucocorticoid from cycle two to four. Bone remodeling markers were monitored closely during treatment. Furthermore, the effects of bortezomib and a glucocorticoid on immature and mature osteoblasts were also studied in vitro. Results: Treatment with bortezomib caused a significant increase in bone-specific alkaline phosphatase and pro-collagen type I N-terminal propeptide, a novel bone formation marker. The addition of a glucocorticoid resulted in a transient decrease in collagen deposition. In vitro bortezomib induced osteoblast proliferation and differentiation. Differentiation but not proliferation was inhibited by glucocorticoid treatment. Conclusions: Bortezomib used as first-line treatment significantly increased collagen deposition in patients with multiple myeloma and osteolytic lesions, but the addition of a glucocorticoid to the treatment transiently inhibited the positive effect of bortezomib, suggesting that bortezomib may result in better healing of osteolytic lesions when used without glucocorticoids in patients that have obtained remission with a previous therapy. The potential bone-healing properties of single-agent bortezomib are currently being explored in a clinical study in patients who have undergone high-dose therapy and autologous stem cell transplantation.

Udgivelsesdato: 2009-Nov-16 Osteolytic lesions are a hallmark of multiple myeloma. They are due to the hyperactivity of bone resorbing osteoclasts and hypoactivity of bone forming osteoblasts, in response to neighbouring myeloma cells. This study identified a structure that deeply affects this response, because of its impact on the physical organisation of the myeloma cell microenvironment. The proximity between myeloma cells and osteoclasts or osteoblasts was shown to be conditioned by the recently discovered layer of flat cells that separates the osteoclasts and osteoblasts from the bone marrow, by forming a canopy over bone remodelling compartment (BRC). These canopies are frequently disrupted in myeloma, and this disruption correlates with increased proximity and density of myeloma cells. In vitro evidence indicates that this disruption may be due to direct contact between myeloma and BRC canopy cells. Importantly, this disruption and increased proximity and density of myeloma cells coincides with key myeloma-induced bone events, such as osteolytic lesions, impaired bone formation despite increased bone resorption, and fusion of myeloma cells with osteoclasts thereby forming myeloma-osteoclast hybrid cells. These findings strongly support a critical role of BRC canopies in myeloma-induced bone disease. BRC canopies could therefore be considered as a new therapeutic target.

This review presents an overview of the most recent data using the novel agents thalidomide, bortezomib, and lenalidomide in the treatment of multiple myeloma and summarizes European treatment practices incorporating these novel agents., The treatment of multiple myeloma (MM) has undergone significant developments in recent years. The availability of the novel agents thalidomide, bortezomib, and lenalidomide has expanded treatment options and has improved the outcome of patients with MM. Following the introduction of these agents in the relapsed/refractory setting, they are also undergoing investigation in the initial treatment of MM. A number of phase III trials have demonstrated the efficacy of novel agent combinations in the transplant and nontransplant settings, and based on these results standard induction regimens are being challenged and replaced. In the transplant setting, a number of newer induction regimens are now available that have been shown to be superior to the vincristine, doxorubicin, and dexamethasone regimen. Similarly, in the front-line treatment of patients not eligible for transplantation, regimens incorporating novel agents have been found to be superior to the traditional melphalan plus prednisone regimen. Importantly, some of the novel agents appear to be active in patients with high-risk disease, such as adverse cytogenetic features, and certain comorbidities, such as renal impairment. This review presents an overview of the most recent data with these novel agents and summarizes European treatment practices incorporating the novel agents.

Udgivelsesdato: 2009 The impact of chemotherapy resistant B cells in multiple myeloma (MM) needs to be evaluated by in vivo targeted therapy. Here we report the conclusions from a phase II randomized, placebo controlled trial adding fludarabine to the induction with cyclophosphamide-dexamethasone. Based on an interim toxicity and safety analysis, the trial was stopped following inclusion of 34 of a planned 80 patients due to a reduced number of patients (4/17) actually harvested in the experimental arm compared to the control arm (11/17; p lower than 0.05). In conclusion, the scheduled fludarabine dosage in 2 cycles combined with alkylating therapy impairs stem cell mobilization and standard therapy in young MM patients and should not be administrated up-front.

Udgivelsesdato: 2009-Jan During skeletal remodeling, pre-osteoclasts and pre-osteoblasts are targeted to critical sites of the bone to resorb and reconstruct bone matrix, respectively. Coordination of site-specific recruitment of these two cell types is a prerequisite to maintain the specific architecture of each bone within strict limits throughout adult life. Here, we determined that the bone marrow microanatomy adjacent to remodeling areas is a central player in this process. By using histomorphometry and multiple immunostainings, we demonstrated in biopsies exhibiting coupled bone resorption and formation that osteoclasts and osteoblasts on the bone surface were always covered by a canopy of flat cells expressing osteoblast markers. In contrast, in biopsies in which this canopy was disrupted, bone formation was deficient. Three-dimensional visualizations revealed that this canopy covered the entire remodeling site and was associated with capillaries, thereby forming a previously unrecognized microanatomical entity. Furthermore, pre-osteoclasts were positioned along these capillaries. These findings led to a model that implicates vasculature in the site-specific recruitment of osteoclasts and osteoblasts and embraces the current knowledge on the molecular mechanism of bone remodeling.

Blood mononuclear cells from 540 newly diagnosed, unselected patients with B-cell chronic lymphocytic leukemia (CLL) were examined by immunofluorescence flow cytometry for a panel of surface membrane markers, including IgM and IgD, the monoclonal antibodies anti-CD3, -5, -20, -21, -22, -FMC7, and, for the final 125 patients, anti-CD23. There were 503 CD5+ and 37 CD5- cases. In the CD5+ cases, the cells typically expressed IgM, IgD, CD20, CD21, CD22, and CD23. In univariate analysis, age, clinical stage, IgM-fluorescence intensity, CD23, and FMC7 had significant prognostic importance, with high IgM-fluorescence intensity, high FMC7, and low CD23 expression being associated with a short survival. There was no significant difference in survival between 351 cases expressing IgMD and 55 cases expressing IgM without IgD, or between kappa and lambda light chain monoclonal cases. CD20, CD21, and CD22 had no prognostic importance. In Cox multiple regression analyses, age, CD23, IgM-fluorescence intensity, and clinical stage (International Workshop System) had independent prognostic importance. Thus, besides clinical variables, CD23 and IgM intensity might be useful prognostic markers in the management of CD5+, B-cell CLL. The survival of CD5- patients was on the borderline of being significantly shorter than that of CD5+ patients. The majority of the CD5- cases were FMC7+, CD23-, had strong IgM fluorescence, and had splenomegaly.

Udgivelsesdato: Apr-2 Myeloma bone disease is due to bone degradation by osteoclasts, and absence of repair by bone forming osteoblasts. Recent observations suggest that the anti-myeloma drug bortezomib, a proteasome inhibitor, stimulates bone formation and may inhibit bone resorption. Here, we tested bortezomib on cultured osteoclasts in conditions mimicking the pulse treatment used in the clinic, thereby avoiding continuous proteasome inhibition and unselective toxicity. A 3h pulse with 25nM bortezomib followed by a 3-day culture in its absence markedly inhibited osteoclast activity as evaluated through bone resorption, TRAcP release, and RANKL-induced NF-kappaB translocation into nuclei, an event dependent on proteasomes and critical for osteoclast function. The effect on TRAcP was maximal during the first 24h post-pulse, and then tended to subside. Importantly, applying this pulse treatment to cultured myeloma cells drastically reduced their survival. We measured next the levels of two bone resorption markers in patients during the 3 days following five and seven therapeutic bortezomib administrations, respectively. These levels decreased significantly already 1-2 days after injection, and then increased, showing temporary inhibition of osteoclast activity and paralleling the in vitro effect on TRAcP. Our study demonstrates a direct inhibition of osteoclasts by bortezomib in conditions relevant to treatment of myeloma.

The human Sezary T-cell leukaemia line, HUT.78, represents a population of activated T cells, i.e. they are HLA-DR+ and IL-2R+. We have analysed the capacity of HUT.78 cells (1) to stimulate HLA-DR-specific T-cell lines or clones and (2) to be induced to synthesize IL-2 by anti-HLA-DR monoclonal antibodies. The results of our experiments show that HLA-DR molecules on HUT.78 cells can stimulate at least one HLA-DR-specific T-cell clone and can act as transmembrane signal transmitters.

The present work demonstrates that antibody-induced cross-linking of MHC class I antigens on Jurkat T lymphoma cells leads to a rise in intracellular calcium (Cai2+) and, in the presence of phorbol ester (PMA), to IL-2 production and IL-2 receptor expression. The rise in Cai2+ exhibited a profile very different from that obtained after anti-CD3 antibody-induced activation suggesting that activation signals are transduced differently after binding of anti-CD3 antibody and class I cross-linking, respectively. However, when Cai2+ was examined in individual Jurkat cells by means of a digital image processing system no differences were observed after cross-linking with anti-CD3 and anti-MHC class I antibodies, respectively. Two CD3-negative mutant lymphoma lines were nearly totally refractory to class I cross-linking. Taken together our results may indicate the existence of a functional linkage between the T cell receptor complex and MHC class I molecules.

We have tested a Quantitative Buffy Coat (QBC) instrument (Becton Dickinson, USA), and compared results obtained with this instrument to results obtained with standard methods in a haematology clinic. The basic principle of the method is a classical haematocrit centrifuge. The analysis provides a haematocrit value, platelet count, a total white blood count, and separates the white blood cells in granulocytes and mononuclear cells (lymphocytes plus monocytes). The instrument is easy to use but requires a trained observer. All results are available in 15 min. We have found the accuracy of the method good for all parameters. The precision of the instrument is good but for estimation of granulocytes and lymphocytes plus monocytes we did not find the high sensitivity claimed by the manufacturer (manufacturer's lower limit 0.02 x 10(9)/l; standard deviation for low levels of granulocytes: 0.300 x 10(9)/l and lymphocytes plus monocytes: 0.235 x 10(9)/l). A large fraction of samples (leukocytes 27%, platelets 40%) from a haematological clinic falls beyond the limits for reliable results set by the manufacturer, which reduces the utility of the instrument in such patients. Furthermore, in 21% and 10% of samples within the recommended range for leukocytes and platelets, respectively, QBC results could not be read owing to ill-defined boundaries. For granulocytes and lymphocytes plus monocytes 25% and 34% of the samples, respectively, could not be read owing to ill-defined boundaries. The instrument is not constructed to protect against blood contamination of the centrifuge and, therefore, the safety of the instrument is not satisfactory.

e144 PO-111 Appraisal of IgM Kappa/IgM Lambda variations using HevyLite after rituximab as consolidation therapy in patients with Waldenstrom’s Macroglobulinemia J.R. Eveillard, A. Achour, F. N’Go Sack, A. Tempescul, R. Le Calloc’h, A. Dagorne, B. Bendaoud, C. Berthou, S. Hillion, Y. Renaudineau Departement of Clinical Hematology, Brest University Hospital, Brest, France; Laboratory of Immunology and Immunotherapy, Brest University Hospital, Brest, France

Graduate School of Health Sciences, Kumamoto University; Department of Morphological and Physiological Sciences, Kumamoto University; Faculty of Life Sciences, Department of Neurology, Kumamoto University; Diagnostic Unit for Amyloidosis, Kumamoto University Hospital; Faculty of Life Sciences, Department of Hematology, Kumamoto University; Department of Surgical Pathology, Kumamoto General Hospital; Division of Informative Clinical Sciences, Kumamoto University Introduction: AL amyloidosis is a disease featuring deposition of Ig light chain (LC). Diagnosis is determined if the presence of LC in amyloid deposits is detected with anti-LC chain antibodies. Poor prognosis cases, like detected amyloid deposits in heart, require the prompt diagnoses. However, in several cases, the existence of LC is not detected within amyloid deposits with immunohistochemistry (IHC) using conventional anti-LC antibodies although monoclonal lambda light chain in serum or urine was proved. In such cases, we found deposition of particular light chain constant region (immunoglobulin light chain constant region 2: IGLC2) at amyloid tissues. Materials and Methods: Amyloid tissues were either stained with conventional anti-human lambda LC rabbit antiserum (DAKO PO-0130) or rabbit anti-IGLC2 antibodies (made by SIGMA ALDLICH). Deposited peptide within amyloid lesion was examined using Congo Red staining followed by combination of laser micro-dissection and mass-spectrometry combining (LC-MS/MS). Plasma cells were purified with CD138-immunomagnetic beads from bone marrow aspirated obtained from two cases. Recombination of LC DNA in purified plasma cells was analyzed using PCR and direct nucleic acid sequencing. Results and Discussion: LC-MS/MS analysis revealed the presence of IGLC2 in all cases without showing positive signal by conventional antibody. Recombination of IGLC2 was detected in plasma cells. Deposition of IGLC2 was confirmed by IHC using anti-IGLC2 antibodies. The present data imply that “IGLC2-related amyloidosis” might be overlooked by conventional IHC unless utilizing mass-spectrometry. Utilization of antibodies for IGLC2 should contribute to improve quality of diagnosis for AL-amyloidosis. We consider that investigation of IGLC2 is important for not only making diagnosis but also analyzing pathogenesis of AL-amyloidosis.

8590 Background: Residual therapeutic mAbs can be detected by assays intended to monitor clonal myeloma protein (M-protein). Daratumumab, a human anti-CD38 IgG1k mAb in MM clinical trials, has been...

Introduction: For physically fit CLL pts with low comorbidity burden FCR is the standard frontline regimen in advanced CLL. The CLL10 study, an international phase III study evaluated the efficacy and tolerance of BR in comparison to FCR in frontline therapy of fit pts without del(17p). Methods and Patients: 158 sites in five countries (Germany, Austria, Switzerland, Denmark and Czech Republic) registered 688 CLL pts for central screening including immunophenotyping, FISH, comorbidity burden and renal function. 564 CLL pts with CIRS score ≤ 6, creatinine clearance > 70 ml/min and without del(17p) were enrolled between 10/2008 and 6/2011. Pts were randomly assigned 1:1 to receive 6 courses of FCR (N= 284; F 25mg/m2 i.v. d1–3, C 250 mg/m2 i.v. d1–3, R 375 mg/m2 i.v. d 0 at first cycle and 500 mg/m2 d1 all subsequent cycles; q 28 days) or BR (N=280; B 90mg/m² i.v. d1+2, R 375 mg/m2 i.v. d 0 at first cycle and 500 mg/m2 d1 all subsequent cycles; q 28 days). A general prophylactic use of antibiotics or growth factors was not recommended. Three patients (2 FCR, 1 BR) were excluded because of deferred treatment. The median CIRS score was 2. There was no difference in median age (61.6 years (yrs) for all pts), but a significantly higher proportion of pts ≥ 70 yrs was included in the BR arm (22% vs 14%, p=0.020). Binet A was present in 22%, Binet B in 38 % and Binet C in 40 %. Unmutated IGHV status was not balanced between both groups (68% in BR versus 55% in FCR arm; p=0.003). All other characteristics showed no differences. The mean number of administered FCR courses was 5.27 courses vs 5.41 BR course (p=0.017). Results: The median observation time for all patients was 35.9 months (mo). 547 pts (FCR 274 ; BR 273) were evaluable for response and all pts (282 FCR ; 279 BR) included for progression-free survival (PFS) and overall survival (OS) analysis. The overall response rate in both arms was 97.8% (p=1.0). The complete response (CR) rate according to IWCLL and confirmed by central bone marrow immunohistology was 40.7% with FCR compared to 31.5% with BR (p=0.026). Four-colour-flow MRD data from peripheral blood (sensitivity 10-4) were available from 355 pts (185 FCR; 170 BR) at final staging. In the FCR arm 74.1% and 62.9% in the BR arm respectively of all evaluated pts were MRD negative (p=0.024). Bone marrow samples, available in 129 FCR and 98 BR pts, were MRD negative in 58.1% and 31.6% of pts, respectively (p 1 CIRS item. Multivariate analysis identified treatment arm, age ≥ 65 yrs, male sex, high serum TK, del(11q), absence of del(13q) and IGHV status as independent prognostic factors for PFS. No difference in OS was observed (at 36 mo 90.6% for FCR vs 92.2% for BR; HR=1.030, 95% CI 0.618-1.717; p=0.910). For OS male gender, age ≥ 60 yrs, high serum TK and IGHV status were assessed as independent prognostic factors. Severe neutropenia was more often observed in the FCR arm (87.7% vs 67.8%, p Conclusion: The final analysis of the CLL10 study shows that FCR remains the standard therapy in very fit CLL patients, because it yields higher CR rates, more MRD negativity and longer PFS in comparison to BR. However, in elderly fit pts high toxicity rates and infection rates result into dose reductions leading to similar efficacy between both arms. Elderly fit pts or pts with previous infections might benefit from BR as alternative regimen. Disclosures Eichhorst: Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding, Travel grant Other; Mundipharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Travel grant, Travel grant Other; GSK: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy. Off Label Use: The Combination of Bendamustine and Rituximab is not approved for frontline chemoimmunotherapy of CLL. Fink:Celgene: Other. Maurer:Mundipharma: Travel grant Other. Kiehl:Roche: Membership on an entity's Board of Directors or advisory committees. Gregor:Roche: Consultancy, Honoraria; Mundipharma: Consultancy, Honoraria. Trneny:Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Fischer:Roche: Other. Döhner:Roche: Research Funding. Kneba:Roche: Consultancy, Research Funding; Mundipharma: Consultancy, Research Funding. Wendtner:Mundipharma: Consultancy, Honoraria, Research Funding; Hoffmann-La Roche: Consultancy, Honoraria, Research Funding. Klapper:Hoffmann-La Roche: Research Funding; Takeda/Millenium: Research Funding. Kreuzer:Roche: Consultancy, Research Funding; Mundipharma: Consultancy, Research Funding. Stilgenbauer:Roche: Consultancy, Honoraria, Research Funding; Mundipharma: Consultancy, Honoraria, Research Funding. Böttcher:Roche: Honoraria, Research Funding, Travel grant Other. Hallek:Roche: Consultancy, Research Funding; Mundipharma: Consultancy, Research Funding.

Idiopathic thrombocytopenic purpura is an autoimmune disease which involves opsonization of platelets by autoantibodies directed against different surface glycoproteins, leading to their premature destruction by the reticuloendothelial system. Management of patients with refractory ITP is difficult. Recent studies have shown that rituximab, a chimeric anti-CD20 monoclonal antibody, is useful in the treatment of these patients, with overall response rates of about 50%. Most published reports have included a small number patients including case reports. The present study reports the results of a retrospective Danish multicenter study of rituximab in the treatment of adult patients with refractory ITP. Thirty-five patients (median age 52 years, range 17-82 years, 17 males) were included. One patient had immune thrombocytopenia and neutropenia. All patients had received prednisolone (Pred). Next to Pred, 25 patients had been treated with high-dose IgG, and in 16 patients a splenectomy had been performed. Sixteen patients had been treated with azathioprine. Other treatments included, e.g., cyclosporine, danazol, cyclophosphamide, vincristine, interferon, and dexamethasone. The patients were treated with a dose regimen of 375 mg/m2 i.v. approximately once weekly for 4 consecutive weeks. Six patients received a fixed dose of 500 mg disregarding their weight supplemented by 100 mg of methylprednisone i.v. or 50-100 mg of Pred given as premedication together with an antihistamine just before infusion of rituximab. The large majority of patients also received Pred and, in some cases, other concomitant immunosuppressive treatment during part of their rituximab treatment. A complete response (CR) was defined as a rise in the platelet count100 x 10(9)/L, a partial response (PR) as a rise in the platelet count50 x 10(9)/L, and a minor response (MR) as a rise in the platelet count50 x 10(9)/L. No response (NR) was defined as no increase in the platelet count. Because 4 patients were treated twice, a total of 39 outcomes of rituximab treatment were evaluated. Rituximab proved to be effective in 17 of 39 treatments [overall response 44% with 7 CR (18%) (1 patient showed a CR twice), 6 PR (15%), and 4 MR (10%)]. In 9/13 cases of CR or PR, the response (platelet level50 x 10(9)/L) was prompt, 1-2 weeks after the first infusion. The remaining patients responded 3-8 weeks later. Patients with CR and PR have been in remission for a median of 47 weeks. In general the side effects were few. In 2 cases, the treatment was stopped because of side effects either during or after the first infusion. Two fatal outcomes were recorded. A 71-year-old female with severe lung disease died 6 days after the first infusion of respiratory failure. The other patient, a 73-year-old man also with severe chronic obstructive lung disease, died of pneumonia approximately 13 weeks following the last rituximab treatment. It is concluded that rituximab may be a useful alternative therapy in patients with severe and symptomatic ITP refractory to conventional treatment.

Multiple myeloma is characterized by the accumulation of clonal malignant plasma cells in the bone marrow, which stimulates bone destruction by osteoclasts and reduces bone formation by osteoblasts. In turn, the changed bone microenvironment sustains survival of myeloma cells. Therefore, a challenge for treating multiple myeloma is discovering drugs targeting not only myeloma cells but also osteoclasts and osteoblasts. Because resveratrol (trans-3,4′,5-trihydroxystilbene) is reported to display antitumor activities on a variety of human cancer cells, we investigated the effects of this natural compound on myeloma and bone cells. We found that resveratrol reduces dose-dependently the growth of myeloma cell lines (RPMI 8226 and OPM-2) by a mechanism involving cell apoptosis. In cultures of human primary monocytes, resveratrol inhibits dose-dependently receptor activator of nuclear factor-κB (NF-κB) ligand–induced formation of tartrate-resistant acid phosphatase (TRACP)–positive multinucleated cells, TRACP activity in the medium, up-regulation of cathepsin K gene expression, and bone resorption. These inhibitions are associated with a down-regulation of RANK expression at both mRNA and cell surface protein levels and a decrease of NFATc1 stimulation and NF-κB nuclear translocation, whereas the gene expression of c-fms, CD14, and CD11a is up-regulated. Finally, resveratrol promotes dose-dependently the expression of osteoblast markers like osteocalcin and osteopontin in human bone marrow mesenchymal stem cells (hMSC-TERT) and stimulates their response to 1,25(OH)2 vitamin D3 [1,25(OH)2D3]. Moreover, resveratrol up-regulates dose-dependently the expression of 1,25(OH)2D3 nuclear receptor. Taken together, these results suggest that resveratrol or its derivatives deserve attention as potential drugs for treating multiple myeloma.