Abstract: Background Melphalan-based regimens, combined with Prednisone and Thalidomide (THAL)(MPT) or Bortezomib (V)(MPV), have been approved as standard therapy of non-transplant eligible newly diagnosed multiple myeloma (NDMM) patients. Similar to MPT and MPV also MP-Lenalidomide followed by Lenalidomide (LEN) maintenance (MPR-R) has a superior PFS compared to MP. We compared MPT followed by THAL maintenance (MPT-T) versus MPR-R in non-transplant eligible NDMM patients. Study design Patients were randomised to receive nine 4-weekly cycles of MPT (MEL 0.18 mg/kg days 1-4, PRED 2 mg/kg days 1-4 plus THAL 200 mg days1-28) followed by THAL maintenance 100 mg d1-28 until progression or nine cycles of MPR (MEL 0.18 mg/kg days 1-4, PRED 2 mg/kg days 1-4 plus LEN 10 mg days 1-21) followed by LEN maintenance 10 mg d1-21 until progression. In order to detect an improvement of progression free survival (PFS) with 90% power and a HR of 0.714 for patients receiving MPR-R, 668 patients had to be randomized. The study started in January 2009 and was closed for inclusion in October, 2012. This analysis is restricted to the first 560 eligible randomized patients, i.e. 280 per arm, including the minimum number of events (377) that were required for the primary endpoint. Results Patient characteristics are presented in table 1. The median follow up is 32.6 months. The results of FISH analysis on isolated plasma cells, being performed in 75% and 79% of patients treated with MPT-T and MPR-R respectively, are shown in table 1. Complete response (CR) rates on protocol were 10% (MPT-T) vs. 13% (MPR-R), (p=0.43), ≥ very good partial response (VGPR) rates were 49% vs. 44% (p=0.31), and ≥ partial response (PR) rates were 82% vs. 83% (p=0.91). The median PFS was 20 months (95% CI; 18-23) for MPT-T vs. 22 months (95% CI; 19-27) for MPR-R (HR = 0.86, 95% CI = 0.70-1.05, p-value adjusted for ISS =0.14). The median overall survival (OS) was 49 months (95% CI; 43-54) for MPT-T vs. 50 months (95% CI; 45-54) for MPR-R (HR = 0.79, 95% CI = 0.60-1.05, p-value adjusted for ISS =0.11). Del(17p) was associated with a lower PR rate (OR=0.45, 95% CI 0.22-0.95, p=0.003), worse PFS (HR=1.74, 95% CI 1.20-2.54, p=0.007) and decreased OS (HR=1.98, 95% CI 1.19-3.28, p=0.01). Grade ≥ 3 toxicity during induction and maintenance is presented in table 2. The dose intensity of THAL during induction was median 53% (mean 56%, SD 34%), vs. median 88% (mean 73%, SD 32%) for LEN during induction. There was a significantly higher discontinuation rate due to toxicity associated with THAL (28% within one year, and 58% within two years) vs LEN maintenance (10% within one year, and 16% within two years)(p 〈 0.001). The median duration of THAL maintenance was 5 months, vs. median 16 months of LEN maintenance. The incidence rates for second primary malignancies (SPM) were 3.3/100 patient years (MPT-T) and 2.4/100 patient years (MPR-R)(p=0.33), with 3 (MPT-T) and 6 (MPR-R) cases of AML/MDS and 18 (MPT-T) and 11 (MPR-R) solid tumors (excluding non-melanoma skin cancers). Detailed analyses will be performed in order to allow cross trial comparisons. Table 1 Demographics MPT - T MPR - R Total 280 280 Male/Female % 53/47 58/42 Median age [range] 72 [60-91] 73 [60-87] 〈 75 years % 64 59 ≥ 75 years % 36 41 ISS at randomization n (%) I 67 (24) 72 (26) II 137 (49) 131 (47) III 73 (26) 74 (26) Unknown 3 (1) 3 (1) LDH n (%) Normal 248 (89) 232 (83) Elevated 20 (7) 29 (10) Unknown 12 (4) 19 (7) FISH analysis on isolated plasma cells , n (%) 210 (75) 222 (79) 1q amplification 52 (37) 50 (32) t(4;14) 16 (11) 16 (9) del(17p) 22 (12) 16 (8) Table 2 Toxicity MPT - T MPR - R CTCAE grade (%) 3 4 3 4 During induction therapy Anemia 4 - 13 1 Thrombocytopenia 4 1 23 8 Neutropenia 20 5 43 21 Infections 17 3 16 3 Neuropathy 8 - - - VTE* 3 3 3 2 During maintenance therapy Anemia 1 1 1 - Thrombocytopenia 1 - 1 1 Neutropenia 3 - 8 3 Neuropathy 15 - 1 - VTE* 1 1 1 - Conclusions Treatment of elderly NDMM patients with MPT followed by THAL maintenance or MPR followed by LEN maintenance resulted in similar PFS. In addition, response rates and OS were not significantly different. Discontinuation of maintenance therapy due to toxicity was significantly higher for THAL versus LEN and SPM incidence rates similar across the groups. * Venous Thromboembolic Event including both deep venous thrombosis and pulmonary embolism This trial was registered as EudraCT 2007-004007-34 Disclosures Zweegman: 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; Janssen-Cilag: Membership on an entity's Board of Directors or advisory committees, Research Funding. Mellqvist:Celgene: Honoraria; Janssen-Cilag: Honoraria; Amgen: Honoraria; Mundipharma: Honoraria. Bos:Celgene: Research Funding. van de Donk:Janssen: Research Funding; Celgene: Research Funding. Sonneveld:Celgene: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Onyx: Honoraria, Research Funding; Millenium: Honoraria, Research Funding. Waage:Janssen-Cilag: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

Abstract: The outcome of multiple myeloma (MM) patients who are no longer responding to thalidomide, lenalidomide (LEN) and bortezomib (BORT) is very poor, with a median event-free survival of 5 months and median overall survival (OS) of 9 months (Kumar SK et al, Leukemia 2012; 26;149-157). We have previously shown in a small retrospective study that the combination of continuous low dose oral cyclophosphamide (endoxan) and prednisone combined with lenalidomide (REP) had remarkable activity in heavily pretreated LEN-refractory multiple myeloma patients (median 6 lines of previous chemotherapy) (vd Donk et al; Br J Haematol 2010;148(2):335-7). To determine the optimal dose of lenalidomide with continuous cyclophosphamide and prednisone, we initiated a prospective study to evaluate the maximum tolerated dose (MTD) of the REP regimen and to assess its efficacy and safety in LEN-refractory MM patients. Here we report safety and efficacy data from the phase 1 dose-escalation part of the REPEAT-study (NCT01352338). Patients and Methods Patients aged ≥ 18 years with LEN-refractory MM, ECOG-performance status 0-3 and adequate kidney, liver and hematologic function were included. Five dose levels were evaluated using a standard 3+3 design, based on dose-limiting toxicities (DLTs) occurring in cycle 1. Patients received LEN in doses ranging from 10-25 mg/day on days 1-21 of 28-day cycle, while oral cyclophosphamide (50 or 100 mg) and prednisone (20 mg) were given continuously. Therapy was continued until progression. The MTD for the phase 2 part is defined as the highest dose level with 0 or 1 DLT's observed in 6 patients. Results Up till now, 35 patients were enrolled (22 in phase 1 and 13 in phase 2) from August 2011 to June 2013. The phase 2 part is still recruiting and data are not evaluable yet. One patient in phase 1 was excluded because of study violation and is not included in the analysis. The median age of the 21 evaluable patients in phase 1 was 69 years (range 41-73); 76% were male. The median duration of the disease from diagnosis was 41 months (range 18-96), median number of prior therapies was 3 (range 2-6), and 12 patients (57%) had previously received autologous SCT. All patients were LEN-refractory, 19 (90%) had prior BORT treatment, and 16 (76%) had BORT-refractory MM. Fifty-five % of the patients were considered high risk by FISH. At the time of analysis, 16 of 21 patients in phase 1 have discontinued treatment because of disease progression (13), alternative treatment (allo-SCT) (1), or adverse events (2). The MTD was defined as LEN 25 mg days 1-21 of a 28-day cycle, combined with oral cyclophosphamide 50 mg and prednisone 20 mg continuously (dose level 4), based on three patients experiencing a DLT: two developed pneumonia (in dose levels 4 and 5; CTC grade 3), and one patient at dose level 5 experienced CTC grade 3 dyspnea. Neutropenia (18%) and thrombocytopenia (18%) were the most common grade 3 hematological adverse events (AEs), which were managed with growth factor support and/or dose modification. There were no grade 4 hematologic AEs. Grade 3 respiratory tract infections (29%) and grade 2 fatigue (19%) were the most common non-hematological AEs. Venous thromboembolism occurred in 1 patient. Figure 1 shows a waterfall plot of the responses of the patients that participated in the phase 1 part of the study. Overall response rate (≥ PR) was 67% with 6 out of 21 (29%) patients achieving at least VGPR. In addition 2 patients achieved MR (≥ MR: 76%). Median PFS and OS were 6.3 and 15.5 months respectively. Similar results were achieved in the subset of patients with LEN- and BORT-refractory disease. Interestingly, laboratory experiments with purified myeloma cells from these patients suggest synergism between LEN and cyclophosphamide. Conclusions The REP regimen induces high response rates and prolonged PFS and OS in LEN-refractory patients with acceptable toxicity. The MTD is defined as LEN 25 mg days 1-21 of a 28-day cycle, combined with oral cyclophosphamide 50 mg and prednisone 20 mg continuously. Phase 2 is enrolling patients and evaluates efficacy and safety of the REP regimen at the MTD. REP should be considered a valuable salvage option for LEN-refractory MM patients. We will present an updated follow-up at ASH. Disclosures: Sonneveld: Onyx: Research Funding; Millenium: Research Funding; Janssen-Cilag: Research Funding; Onyx: Honoraria; Celgene: Honoraria; Janssen-Cilag: Honoraria; Celgene: Research Funding. Lokhorst:Genmab A/S: Consultancy, Research Funding; Celgene: Honoraria; Johnson-Cilag: Honoraria; Mudipharma: Honoraria. van de Donk:Celgene: Research Funding.

Abstract: Introduction: Daratumumab (DARA)-based regimens are effective and well tolerated in both newly diagnosed and relapsed/refractory multiple myeloma (RRMM) patients. However, the prognosis for patients who have become refractory to proteasome inhibitors (PIs), immunomodulatory drugs (IMiDs) and DARA is poor. A better understanding of determinants of response and mechanisms of resistance to DARA may lead to new rationally designed treatment strategies. We therefore characterized the effect of DARA on the immune system in MM patients treated with DARA monotherapy in part A of the DARA-ATRA study (ClinicalTrials.gov NCT02751255). Methods: In part A of this prospective multicenter phase 2 trial, 63 DARA-naïve patients were treated with DARA monotherapy (16 mg/kg; approved schedule). Median number of prior lines of treatment was 4 (range 2-11), all patients were previously exposed to lenalidomide and a PI; 89% was refractory to an IMiD, 71% to a PI and 67% to both IMiD and PI. Bone marrow (BM) aspirates obtained at baseline (BL) and at progression (PD; primary refractory, or acquired resistance after prior response) were subjected to deep immune profiling using 28 surface- and intracellular proteins measured by flow cytometry (BL n=51, PD n=47). In a subset of these patients (BL n=39, PD n=33) mass cytometry (CyTOF) was also performed profiling 39 proteins. In addition, peripheral blood (PB) samples obtained at BL (n=44) and PD (n=37) were analyzed using CyTOF. Computational analyses of flow data were performed using UMAP and FlowSOM; CyTOF data were analyzed using SPADE and Freeviz. Statistical analyses included Wilcoxon rank-sum and signed-rank tests, Generalized Linear Mixed Models and ANOVA. Results: A partial response or better was achieved in 41% of patients treated with DARA monotherapy. We compared immune profiles of responding and non-responding (primary-refractory) patients. At BL, the percentages of MM-, T-, B- and NK cells in BM were similar between both groups. However, NK cells of non-responding patients had a lower proportion of CD16 + (P=0.029), and a higher proportion of TIM-3 + (P=0.010) and HLA-DR + (P=0.043) NK cells, suggesting an exhausted phenotype. Non-responders also had a higher proportion of TIM-3 + CD4 + (P=0.022) and TIM-3 + CD8 + T-cells (P=0.004), and a higher proportion of TIM-3 + regulatory T-cells (Tregs) (P=0.042). A higher proportion of TIM-3 + -NK cells, -T-cells, or -Tregs in BM was also associated with poor progression-free and overall survival (PFS; OS). In addition, clinical characteristics associated with poor PFS such as LDH (P=0.016), extramedullary disease (P=0.001) and (R-)ISS stage III (P=0.015) were associated with an increased number of phenotypically exhausted NK- and T-cells. Similar to prior studies, DARA treatment resulted in reduced levels of CD38 on all immune cell subsets and a marked decrease in Tregs, regulatory B cells (Bregs) and NK cells in both BM and PB. Upon acquired resistance, remaining NK cells displayed higher proportions of TIM-3 + (P=0.022), HLA-DR + (P=0.0007) and LAG-3 + (P=0.011), and lower proportions of CD16 + (P=0.002) (Figure 1), compatible with an exhausted phenotype. Furthermore, disease progression was associated with an increase in CD4 + and CD8 + terminally differentiated effector memory T-cells and a decrease in CD4 + and CD8 + central memory T-cells in BM. There was no significant change in the proportion of T-cells expressing immune checkpoint molecules. CyTOF analysis of BM and PB samples confirmed flow cytometric findings. Furthermore, data-driven analysis identified immune profiles specific to progression, including a significant decrease in the fraction of Granzyme B + NK cells, and increase in the fraction of Granzyme B + Bregs at the time of progression. Based on NK cell depletion and -exhaustion observed at PD, we hypothesized that NK cell repletion may restore DARA sensitivity. Indeed, in ex vivo experiments we show that DARA-resistance can be overcome by addition of healthy donor derived NK cells to MM cells obtained from DARA-refractory patients. Conclusion: Here we show that an increased proportion of NK cells with an exhausted phenotype is associated with primary and acquired DARA-resistance, which is in line with the important role of NK cells in DARA-mediated tumor cell elimination ex vivo. Future DARA-based treatment strategies may benefit from reinvigorating NK cells and restoring their cytotoxic capacities. Figure 1 Figure 1. Disclosures Zweegman: Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees. Minnema: Celgene: Other: Travel expenses; Alnylam: Consultancy; Cilag: Consultancy; Janssen: Consultancy; Kite/Gilead: Consultancy; BMS: Consultancy. Broyl: Celgene/BMS: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria. Levin: Roche, Janssen, Abbvie: Other: Travel Expenses, Ad-Board. Kersten: Kite/Gilead: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria; Roche: Honoraria; Takeda: Consultancy; BMS/Celgene: Consultancy; Miltenyi Biotech: Consultancy. Krevvata: Janssen: Current Employment. Casneuf: Janssen: Current Employment. Abraham: Janssen: Current Employment. Verona: Janssen: Current Employment. Smets: Janssen: Current Employment. Vanhoof: Janssen: Current Employment. Cortes-Selva: Janssen: Current Employment. van Steenbergen: Biolizard working for Janssen: Current Employment. Vieyra: Janssen: Current Employment. Sonneveld: Karyopharm: Consultancy, Honoraria, Research Funding; SkylineDx: Honoraria, Research Funding; Celgene/BMS: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding. Mutis: Janssen: Honoraria; Genmab: Research Funding; Takeda: Research Funding; Novartis: Research Funding; ONK Therapeutics: Research Funding. van de Donk: Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees; Janssen Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Cellectis: Research Funding; Servier: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding.