ALBERT

Description: The micro-environment in multiple myeloma (MM) is highly immunosuppressive with increased numbers of regulatory T cells (Tregs) and myeloid derived suppressor cells (MDSCs) favoring tumorcell survival and hampering immunotherapeutic strategies such as dendritic cell vaccination. Immunomodulatory drugs (IMiDs) are known to enhance T- and NK-cell function. In this study we evaluated the effects of low dose (0.5 microM) lenalidomide (Len) and pomalidomide (Pom) on the functionality of CD8+ and CD4+ T cells, MDSCs, Tregs and ex-vivo generated mononuclear derived dendritic cells (moDCs) obtained from MM patients after first autologous stem cell transplantation (ASCT). Peripheral blood mononuclear cell fractions were obtained by leukapheresis from 9 MM patients (age 29-62 years), in very good partial response (4/9) or complete response (5/9) after ASCT. The magnitude of cytokine release (mean +/- standard error of the mean, in ng/ml) by purified CD8+ T cells after 144 hours stimulation with anti-CD3/anti-CD28 coated microbeads was significantly increased after addition of Len and Pom to the culture medium, respectively : IFN-gamma (217.5 +/- 62.1 and 437.1 +/- 137.1** vs 66.4 +/- 21.0) , TNF-alpha (21.4 +/- 5.4 and 44.9 +/- 9.4*** vs 4.9 +/- 1.7) and IL-2 (5.3 +/- 2.7 and 12.7 +/- 6.6 vs 1.9 +/- 1.7 ng/ml) (** p〈 0.01, *** p〈 0.001). We also evaluated the number of different types of cytokines/chemokines on a per cell basis by intracellular flow cytometry staining for IFN-gamma, TNF-alpha, IL-2 and MIP-1beta and observed increased polyfunctionality of CD8+ and CD4+ T cells. After 72 h of stimulation with anti-CD3/CD28 microbeads the number of single, double, triple or quadruple functional CD8+ T cells increased from 5.96 %, 2.82 %, 0.1 %, 0 % (culture medium alone) to 9.68 %, 7.57 %, 0.41%, 0.03 % (Len) and 12.57 %, 8.96 %, 0.81 %, 0.03 % (Pom), respectively. A similar observation was made for CD4+ T cells. A significant percentage, median 5.7 % (4.0-7.2 %) of CD4+ CD25high CD127low (Tregs) was found in the CD4+ T cell population in 8 out of 9 patients, demonstrating the highly suppressive immune environment in myeloma patients even with low disease burden. Effector T cells (Teffs) were stimulated with anti-CD3/CD28 microbeads and cocultured at varying ratios with purified Tregs. After 144 h of coculture, Len and Pom reduced the suppressive effects of Tregs on Teffs proliferation and IFN-gamma and TNF-alpha production (see figure). A similar effect was observed for MDSC but did not reach statistical significance (data not shown). TriMix DCs (moDCs matured by electroporation with mRNA encoding TLR4, CD40L and CD70) and cytokine matured moDCs were cocultured with autologous CD4+ and CD8+ T cells and anti-CD3 microbeads. Adding IMiDs resulted in more polyfunctional CD4+ and CD8+ T cells with both types of DCs but effects were most pronounced with the TriMix variant. Our study shows that Len and Pom restore effector cell functions in myeloma patients with low tumor burden after ASCT. These findings provide a immunomechanistic explanation for IMiD-based maintenance therapy. They also offer a rationale to combine IMiD-based maintenance with immunotherapeutic approaches such as dendritic cell vaccination in this particular setting. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction Multiple myeloma (MM) is well-known for the development of drug resistance, leading to the need for multiple treatment lines at times of relapse or progression. Even then, most patients ultimately will succumb to this cancer. Therefore, there is a need for new therapeutic strategies to conquer this drug resistance. Lipidomics has recently gained more attention in the search for new cancer therapies. Lipids are mainly found in biological membranes and function as building blocks, but are also important metabolites that can influence energy, structure and signaling cascades. Lipid dysfunction has been correlated to other cancers, like prostate and breast cancer. In this study, we identified changes in lipid content in MM patients and further investigated this altered metabolism in vitro. Methods We performed a lipidomics assay to compare plasma from healthy volunteers to MM patients. For all in vitro experiments, we used four human MM cell lines (JJN3, OPM2, LP1, U266) and primary CD138+ patient samples, isolated by MACS. Differential mRNA expression (SMPD1 = acid sphingomyelinase, ASM) was measured by qRT-PCR and ASM protein levels by western blot. Exosomes were isolated by exoquick, while changes in secretion were measured by nanoparticle tracking analysis. Viability was measured by CellTiter Glo and apoptosis rates for melphalan, bortezomib and ASM inhibitor (amitriptyline) were measured by flow cytometry (annexinV-FITC/7-AAD staining). Drug efficacy of amitriptyline was also confirmed on primary CD138+ samples by CellTiter Glo. Results Lipidomics analysis revealed an increase in ceramides and a decrease in sphingomyelin. Therefore, we believe that the enzyme sphingomyelinase, which converts sphingomyelin into ceramide, is upregulated in MM, which we confirmed on primary CD138+ MM cells for ASM. We also observed an increase in SMPD1 expression by qRT-PCR and ASM levels by western blot after melphalan and bortezomib treatment. Furthermore, we also investigated effects of these drugs on exosome secretion, where we found an increase in the number of exosomes secreted, as well as higher ASM levels in these exosomes. U266-derived exosomes, containing high amounts of ASM, were able to transfer their resistance to ASM-low JJN3 cells as an increase in viability was measured. Inhibition of ASM by amitriptyline, combined with melphalan and bortezomib, increased apoptotic cell death by upregulating cleaved PARP and caspase 3 levels. Combination therapy of amitriptyline with melphalan and bortezomib was also successfully tested on primary CD138+ MM cells. Conclusion This study is the first to identify changes in sphingolipids in plasma of MM patients, where we matched the observed difference to an upregulation of ASM in MM cells. Standard-of-care drugs stimulated ASM expression and production, and cells were able to transfer their resistance to other cells by ASM-rich exosomes. Enzyme inhibition by amitriptyline, a cheap tricyclic antidepressant drug frequently used to combat neuropathic pain in MM, increased drug efficacy of standard-of-care drugs. This study therefore provides a rationale to add ASM-targeting drugs to current combination therapies in MM patients. Disclosures No relevant conflicts of interest to declare. OffLabel Disclosure: Amitriptyline is a tricyclic antidepressant, which is also often used to treat neuropathic pain in Multiple Myeloma patients. Here we describe additive effects of amitriptyline on bortezomib and melphalan treatment in MM.

Description: Introduction: CD38 is highly and uniformly expressed on myeloma cells (1). Daratumumab is a human anti-CD38 IgG1κ monoclonal antibody that has previously shown a favourable safety profile as a single agent in patients with relapsed and refractory (RR) multiple myeloma (MM) (2). This study further assesses the efficacy of Daratumumab in combination with Dexamethasone in heavily pre-treated myeloma patients that are refractory to Lenalidomide, Pomalidomide, and Bortezomib. Methods: This study is an ongoing, open-label phase II study of Daratumumab in combination with Dexamethasone (NCT02626481). Sixty-four, heavily pretreated Patients were recruited in thirty centres in France and Belgium from November 2015, to receive Daratumumab and Dexamethasone. Daratumumab 16 mg/kg was administered weekly during the first two 28-day cycles, every other week during Cycles three through six, and monthly in Cycle seven and beyond until disease progression or unacceptable toxicity. Patients were all refractory to Lenalidomide (Len), Pomalidomide (Pom) (defined by a progression within 60 days from last drug dosing) and Bortezomib (Bz) (defined by a progression within 6 months from last drug dosing). The primary objective was overall response rate as per the International Myeloma Working Group criteria. A planned safety and efficacy interim analysis was performed after the first 19 patients were enrolled. The last patient was enrolled on the 1stof August 2016. Results: Sixty-four patients were recruited onto the study. The median age (range) at screening was 61 (30-80). The median number (range) of prior lines of therapy was 6 (2-9). Sixty-seven percent of patient had previously received an autologous stem-cell transplant. At the time of screening, 20% of patients (n=13) had a t(4;14) and 12.5% (n=8) a del(17p). Planned interim analysis after the first 19 patients were enrolled did not find any unexpected toxicity. Safety and efficacy results (data cut May 15, 2016) of Daratumumab 16 mg/kg are presented here. No patient discontinued treatment due to Treatment Emergent Adverse Event such as infusion related reactions. Ten (15%) patients discontinued treatment due to disease progression after a median of one-cycle. The most common non-haematological TEAEs included infusion related (IRR, n=5, 8%), and fatigue (n=6, 9.3%). All patients with IRRs recovered and continued to receive treatment. Only six (9.5%) patients experienced hyperthermia. Thrombocytopenia and neutropenia were the most frequently reported grade 3 or 4 TEAE (11 and 5% respectively). Planned interim efficacy assessment showed a response rate (defined as a Partial Response (PR) or greater) in 3/19 patients at the end of the first cycle and 4/19 at the end of the second cycle, and a clinically relevant response (Stable Disease (SD) or greater) at the end of the second cycle for 11 of 19 patients, thus meeting the planed futility criteria and enabling the trial to go forward. As per the 15thof May, among the 40 evaluable patients (that had received at least 2 treatment cycles or progressed within the first) the overall response rate (3) was 25%, with eight (20%) partial responses (PR) and two (5%) very good partial responses (VGPRs) after a median of two cycles (range 1-5). An additional seven patients (17.5%) obtained a Minimal Response (MR) according to the EBMT criteria (4). This is consistent with prior results. Updated results will be presented at the time of ASH. Conclusions: Daratumumab in combination with Dexamethasone is a safe treatment option with a favourable benefit/risk profile for the treatment of triple relapsed or refractory (Len, Pom and Bz) myeloma patients. 1. Stevenson GT. CD38 as a Therapeutic Target. Mol Med. 2006;12(11-12):345-6. 2. Lokhorst HM, Plesner T, Laubach JP, Nahi H, Gimsing P, Hansson M, et al. Targeting CD38 with Daratumumab Monotherapy in Multiple Myeloma. N Engl J Med. 2015 Sep 24;373(13):1207-19. 3. Kyle R, Rajkumar S. Criteria for diagnosis, staging, risk stratification and response assessment of multiple myeloma. Leuk Off J Leuk Soc Am Leuk Res Fund UK. 2009 Jan;23(1):3-9. 4. Bladé J,et al. Criteria for evaluating disease response and progression in patients with multiple myeloma treated by high-dose therapy and haemopoietic stem cell transplantation. Myeloma Subcommittee of the EBMT. Br J Haematol. 1998 Sep;102(5):1115-23. Disclosures Boyle: Novartis: Honoraria; Pfizer: Honoraria; Takeda: Honoraria; Janssen: Honoraria. Leleu:Novartis: Honoraria; LeoPharma: Honoraria; Pierre Fabre: Honoraria; Amgen: Honoraria; Bristol-Myers Squibb: Honoraria; Takeda: Honoraria; Celgene: Honoraria; Janssen: Honoraria; TEVA: Membership on an entity's Board of Directors or advisory committees. Hulin:Amgen: Honoraria; Janssen: Honoraria; Bristol: Honoraria; celgene: Honoraria; takeda: Honoraria. Moreau:Takeda: Honoraria; Janssen: Honoraria, Speakers Bureau; Celgene: Honoraria; Novartis: Honoraria; Amgen: Honoraria; Bristol-Myers Squibb: Honoraria. Fohrer:amgen: Consultancy; celgne: Consultancy. Decaux:SIEMENS: Honoraria, Other: supply of free light chain assays , Research Funding; The Binding Site: Other: supply of free light chain assays , Research Funding. Avet-Loiseau:celgene: Consultancy; amgen: Consultancy; janssen: Consultancy; sanofi: Consultancy. Attal:celgene: Consultancy, Research Funding; amgen: Consultancy, Research Funding; janssen: Consultancy, Research Funding; sanofi: Consultancy. Facon:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: travel and expense, Speakers Bureau.

Description: Background Autologous stem cell transplantation (ASCT) is a frequent treatment option for various types of lymphoma. Few studies have addressed the occurrence of long-term morbidity and the quality of life (QoL) after ASCT. We analysed the overall survival (OS), the QoL and the impact of comorbidities before and after transplant in a consecutive cohort of lymphoma patients. Methods Patients transplanted in our center between 2003 and 2013 and with a minimum follow-up of 1 year were included. Survival was analysed by Kaplan-Meier methodology and the impact of prognostic factors was examined by standard multivariate analysis. Patients were inquired to complete the Self-reported Comorbidity Questionnaire (SCQ) as well as the EORTC QLQ-C30. Results of questionnaires were compared to a healthy reference population. Results A total of 85 lymphoma patients were identified with a median follow-up period of 76 months. The estimated OS probability was 62,7% (S.E. 5,5%) at 5 years and 60,8% (S.E. 5,6%) at 10,9 years. Indicators of a poor prognosis at time of transplantation were: age ≥ 60 years, high-intermediate to high-risk IPI for NHL and HCT-comorbidity index ≥ 3. Of long-term (〉 1 year) survivors 40 completed the EORTC QLQ-C30. They experienced lower cognitive and social functioning and reported more fatigue, dyspnea and financial difficulties, when compared to the reference population. A small proportion of patients reported significant complaints related to pain (22,5%) and fatigue (10%). Very long-term survivors (over 5 years post ASCT) had a better physical and role functioning, with less fatigue, dyspnea, insomnia and loss of appetite as compared to patients who were transplanted more recently. Patients with more self-reported comorbidities post-ASCT did significantly worse in terms of QoL after transplantation. They experienced worse physical, role, cognitive and social functioning, and had more complaints about fatigue, pain, dyspnea, diarrhea and insomnia. Conclusion The presence of comorbidities pre-ASCT, as determined by HCT-CI 〉3, was associated with a worse OS. Surprisingly, the overall long-term impact of ASCT on the QOL was limited with adverse effects only related to cognitive and social functions. These negative consequences also decreased over time. Finally, the occurrence of comorbidities after transplantation was associated with worse QoL. Disclosures No relevant conflicts of interest to declare.