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Patients with multiple myeloma are at increased risk of vascular thromboembolic events (VTEs). This post hoc analysis evaluated VTEs in the randomised phase 2 GRIFFIN study (ClinicalTrials.gov Identifier: NCT02874742) that investigated lenalidomide/bortezomib/dexamethasone (RVd) ± daratumumab (D). Patients with newly diagnosed multiple myeloma who were eligible for autologous stem cell transplantation (ASCT) received D-RVd/RVd induction, high-dose therapy and ASCT, D-RVd/RVd consolidation and up to 2 years of lenalidomide maintenance therapy ± D. VTE prophylaxis was recommended (at least aspirin, ≥162 mg daily) in accordance with International Myeloma Working Group guidelines. In the safety population (D-RVd, n = 99; RVd, n = 102), VTEs occurred in 10.1% of D-RVd patients and 15.7% of RVd patients; grade 2-4 VTEs occurred in 9.1% and 14.7%, respectively. Median time to the first onset of VTE was longer for D-RVd versus RVd patients (305 days vs 119 days). Anti-thrombosis prophylaxis use was similar between arms (D-RVd, 84.8% vs RVd, 83.3%); among patients with VTEs, prophylaxis use at time of first VTE onset was 60.0% for D-RVd and 68.8% for RVd. In summary, the addition of daratumumab to RVd did not increase the incidence of VTEs, but the cumulative VTE incidence was relatively high in this cohort and anti-thrombotic prophylaxis use was suboptimal.

Introduction: DARA is approved across lines of therapy for multiple myeloma, including in combination with standard-of-care regimens for NDMM. The CXCR4 receptor antagonist plerixafor is used in conjunction with granulocyte colony-stimulating factor (G-CSF) to increase stem cell mobilization for autologous stem cell transplant (ASCT) and can be given by upfront decision or as a rescue strategy. The phase 2 randomized GRIFFIN study (NCT02874742) evaluates frontline DARA in combination with lenalidomide, bortezomib, and dexamethasone (D-RVd) in transplant-eligible NDMM. In the primary analysis, more pts undergoing stem cell mobilization/collection in the D-RVd group received plerixafor compared with the RVd group (69.5% [66/95] vs 56.3% [45/80]) (Voorhees PM, et al. Blood. 2020). The phase 2 MASTER study (NCT03224507) evaluates DARA plus carfilzomib, lenalidomide, and dexamethasone (D-KRd) in transplant-eligible NDMM (Costa LJ, et al. EHA Library. 2020). Here, we present a summary of stem cell mobilization, collection yields, and ASCT data following frontline DARA-based induction therapy in GRIFFIN and MASTER. Methods: Eligible pts had NDMM and were candidates for ASCT. In GRIFFIN, pts were randomized 1:1 to receive D-RVd or RVd. Pts received 4 induction cycles (21 days) of lenalidomide (R; 25 mg PO on Days 1-14), bortezomib (1.3 mg/m 2 SC on Days 1, 4, 8, and 11), and dexamethasone (d; 40 mg PO QW) ± DARA (16 mg/kg IV QW in Cycles 1-4). After Cycle 4, pts underwent stem cell mobilization with G-CSF ± plerixafor, per institutional standards; if unsuccessful, chemo mobilization was permitted. Pts then received ASCT and subsequently 2 consolidation cycles (21 days) of D-RVd or RVd followed by maintenance therapy with R ± DARA. In the single-arm MASTER study, pts received 4 D-KRd induction cycles, ASCT, and 0, 4 or 8 D-KRd consolidation cycles followed by maintenance therapy with R, based upon achievement of minimal residual disease-negativity. In each 28-day cycle, all pts received carfilzomib (20/56 mg/m 2 IV QW), R (25 mg PO on Days 1-21), d (40 mg PO or IV QW), and DARA (16 mg/kg IV QW for Cycles 1-2, Q2W for Cycles 3-6, and Q4W for Cycles 7+). Mobilization was with G-CSF ± plerixafor as per institutional standards. Results: In GRIFFIN, among 207 (D-RVd, n=104; RVd, n=103) randomized pts, 91.3% (n=95) of D-RVd pts and 77.7% (n=80) of RVd pts underwent stem cell mobilization; of those mobilized, 98.9% (n=94) and 97.5% (n=78) underwent ASCT, respectively. In MASTER, 123 D-KRd pts enrolled and at last follow-up, 91.1% (n=112) underwent stem cell mobilization; of those mobilized, 98.2% (n=110) completed ASCT. In GRIFFIN, 46.3% (n=81) of mobilized pts received plerixafor upfront (D-RVd, 51.6%, n=49; RVd, 40.0%, n=32), and 18.3% (n=32 pts) received rescue plerixafor (D-RVd, 20.0%, n=19; RVd, 16.3%, n=13). In MASTER, 70.5% (n=79) D-KRd pts received upfront plerixafor and 25.9% (n=29) received rescue plerixafor. Median CD34 + cell yield was 8.3 × 10 6/kg for D-RVd and 9.4 ×10 6/kg for RVd in GRIFFIN, 6.0 ×10 6/kg for D-KRd in MASTER, and was numerically higher for pts who received upfront plerixafor. Median days for stem cell collection was 1 for pts receiving RVd and 2 for those receiving D-RVd or D-KRd. Median transplanted CD34 + cell count was 4.2 ×10 6/kg for D-RVd and 4.8 ×10 6/kg for RVd in GRIFFIN, and 3.2 ×10 6/kg for D-KRd in MASTER. In GRIFFIN, 93.7% of D-RVd pts and 98.8% of RVd pts reached the minimum institutional CD34 + threshold to perform a single ASCT, which was comparable to results in MASTER (95.5% of D-KRd pts) after first mobilization attempt; 85.3% of D-RVd pts, 92.5% of RVd pts, and 79.5% of D-KRd pts collected 2 times the minimum threshold of stem cells. Additional data by upfront and rescue plerixafor strategies are shown in the Table. Conclusion: The addition of DARA to proteasome inhibitor/immunomodulatory drug/dexamethasone-based induction therapy has a modest impact on stem cell mobilization, with a lower yield of stem cells and higher median number of days required for collection. Nonetheless, pts were able to undergo transplantation, and most pts collected sufficient stem cells for 2 transplants. Pts who received plerixafor by an upfront decision had numerically higher stem cell yields than pts who received plerixafor by a rescue strategy. An upfront plerixafor strategy for pts receiving DARA-based quadruplet induction therapy should be considered with allowance for additional days of apheresis as needed. Figure 1 Figure 1. Disclosures Chhabra: GSK: Honoraria. Costa: Janssen: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria, Speakers Bureau; Karyopharm: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; BMS: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding, Speakers Bureau. Kaufman: BMS: Consultancy, Research Funding; Fortis Therapeutics: Research Funding; Roche/Genetech, Tecnopharma: Consultancy, Honoraria; Sutro, Takeda: Research Funding; Genentech, AbbVie, Janssen: Consultancy, Research Funding; Novartis: Research Funding; Incyte, celgene: Consultancy; Tecnofarma SAS, AbbVie: Honoraria; Janssen: Honoraria; Incyte, TG Therapeutics: Membership on an entity's Board of Directors or advisory committees; Heidelberg Pharma: Research Funding; Amgen: Research Funding. Sborov: Sanofi: Consultancy; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; SkylineDx: Consultancy; GlaxoSmithKline: Consultancy. Reeves: Incyte Corporation: Honoraria; Takeda: Honoraria; Bristol-Myers Squibb: Speakers Bureau; Pharma Essentia: Consultancy, Honoraria. Rodriguez: Karyopharm: Consultancy, Speakers Bureau; Oncopeptides: Consultancy, Honoraria; Amgen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau. Chari: Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Research Funding; Antengene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Shattuck Labs: Consultancy, Membership on an entity's Board of Directors or advisory committees; Secura Bio: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS/Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncopeptides: Consultancy, Membership on an entity's Board of Directors or advisory committees; Millenium/Takeda: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; Karyopharm: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; GlaxoSmithKline: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sanofi Genzyme: Consultancy, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Research Funding. Silbermann: Sanofi Genzyme: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Anderson: Celgene, BMS, Janssen, GSK, Karyopharm, Oncopeptides, Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Shah: Sutro Biopharma: Research Funding; Janssen: Research Funding; Indapta Therapeutics: Consultancy; CareDx: Consultancy; Sanofi: Consultancy; Kite: Consultancy; Poseida: Research Funding; Amgen: Consultancy; BMS/Celgene: Research Funding; Bluebird Bio: Research Funding; CSL Behring: Consultancy; GSK: Consultancy; Precision Biosciences: Research Funding; Teneobio: Research Funding; Oncopeptides: Consultancy; Nektar: Research Funding; Karyopharm: Consultancy. Bumma: Sanofi: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees. Holstein: Oncopeptides: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Genentech, GSK, Janssen, Secura Bio, Sorrento: Honoraria; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees. Jakubowiak: BMS: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Gracell: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees. Wildes: Carevive: Consultancy; Seattle Genetics: Consultancy; Sanofi: Consultancy; Janssen: Consultancy. Orlowski: CARsgen Therapeutics, Celgene, Exelixis, Janssen Biotech, Sanofi-Aventis, Takeda Pharmaceuticals North America, Inc.: Other: Clinical research funding; Asylia Therapeutics, Inc., BioTheryX, Inc., and Heidelberg Pharma, AG.: Other: Laboratory research funding; Asylia Therapeutics, Inc.: Current holder of individual stocks in a privately-held company, Patents & Royalties; Amgen, Inc., BioTheryX, Inc., Bristol-Myers Squibb, Celgene, Forma Therapeutics, Genzyme, GSK Biologicals, Janssen Biotech, Juno Therapeutics, Karyopharm Therapeutics, Inc., Kite Pharma, Neoleukin Corporation, Oncopeptides AB, Regeneron Pharmaceuticals, I: Membership on an entity's Board of Directors or advisory committees; Amgen, Inc., BioTheryX, Inc., Bristol-Myers Squibb, Celgene, EcoR1 Capital LLC, Genzyme, GSK Biologicals, Janssen Biotech, Karyopharm Therapeutics, Inc., Neoleukin Corporation, Oncopeptides AB, Regeneron Pharmaceuticals, Inc., Sanofi-Aventis, and Takeda P: Consultancy, Honoraria. Shain: Novartis Pharmaceuticals Corporation: Consultancy; Karyopharm Therapeutics Inc.: Honoraria, Research Funding; Janssen oncology: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sanofi Genzyme: Consultancy, Speakers Bureau; GlaxoSmithLine, LLC: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Adaptive Biotechnologies Corporation: Consultancy, Speakers Bureau; AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding. Cowan: Janssen: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding; Sanofi: Consultancy, Research Funding; Cellectar: Consultancy; Harpoon: Research Funding; GSK: Consultancy; Secura Bio: Consultancy; BMS: Research Funding; Nektar: Research Funding. Dholaria: Takeda: Research Funding; Jazz: Speakers Bureau; MEI: Research Funding; Angiocrine: Research Funding; Poseida: Research Funding; Celgene: Speakers Bureau; Pfizer: Research Funding; Janssen: Research Funding. Pei: Janssen: Current Employment, Current equity holder in publicly-traded company. Cortoos: Janssen: Current Employment, Current equity holder in publicly-traded company. Patel: Janssen: Current Employment. Bartlett: Janssen: Current Employment. Vermeulen: Janssen: Current Employment, Current equity holder in publicly-traded company. Lin: Janssen: Current Employment. Richardson: AstraZeneca: Consultancy; Regeneron: Consultancy; Celgene/BMS: Consultancy, Research Funding; Oncopeptides: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; AbbVie: Consultancy; GlaxoSmithKline: Consultancy; Karyopharm: Consultancy, Research Funding; Protocol Intelligence: Consultancy; Janssen: Consultancy; Sanofi: Consultancy; Secura Bio: Consultancy; Jazz Pharmaceuticals: Consultancy, Research Funding. Voorhees: Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: 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; Oncopeptides: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Secura Bio: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. OffLabel Disclosure: The specific regimen combination is not yet approved, but individual components are.

Introduction: DARA is approved for NDMM and previously treated MM. In the primary analysis of the phase 2 GRIFFIN trial (NCT02874742) in autologous stem cell transplant (ASCT)-eligible NDMM pts (median follow-up, 13.5 mo), DARA plus RVd (D-RVd) improved the rate of stringent complete response (sCR) by the end of post-ASCT consolidation versus RVd (42.4% vs 32.0%, 1-sided P=0.068) (Voorhees PM, et al. Blood. 2020). With longer follow-up (median, 27.4 mo), responses deepened and were improved for D-RVd versus RVd (sCR rate: 63.6% vs 47.4%, 2-sided P=0.0253), as did the MRD-negativity (10 -5) rate (62.5% vs 27.2%, P Methods: Pts with NDMM eligible for high-dose therapy (HDT) and ASCT were randomized 1:1 to receive RVd or D-RVd, stratified by ISS disease stage (I, II, or III) and creatinine clearance (30-50 or >50 mL/min). Pts received 4 RVd or D-RVd induction cycles, HDT, ASCT, 2 RVd or D-RVd consolidation cycles, and maintenance with lenalidomide (R) alone or with DARA (D-R) for 24 months. During induction and consolidation (21-day cycles), pts received R (25 mg PO on Days 1-14), bortezomib (1.3 mg/m 2 SC on Days 1, 4, 8, and 11), and dexamethasone (40 mg PO QW) ± DARA (16 mg/kg IV on Days 1, 8, and 15 of Cycles 1-4 and Day 1 of Cycles 5-6). During maintenance (Cycles 7-32; 28-day cycles), pts received R (10 mg PO on Days 1-21; if tolerated, 15 mg in Cycle 10+) ± DARA (16 mg/kg IV) Q8W (or Q4W per pt decision after protocol amendment 2) until disease progression or up to 24 months. The primary endpoint was sCR rate by the end of post-ASCT consolidation (tested at 1-sided α of 0.10). Responses were assessed per IMWG criteria by a validated computer algorithm. Key secondary endpoints included progression-free survival (PFS) and MRD negativity assessed by NGS at the minimum sensitivity threshold of 10 -5, at suspected complete response or better (≥CR), at the end of induction and consolidation, and after 12 and 24 months of maintenance, regardless of response. Secondary analyses were evaluated using 2-sided α of 0.05, not adjusted for multiplicity. Results: In total, 207 pts were randomized (D-RVd, n=104; RVd, n=103); baseline characteristics were well balanced. After 24 months of D-R or R maintenance therapy, the rate of sCR favored D-RVd versus RVd in the response-evaluable population (66.0% [66/100] vs 47.4% [46/97], 2-sided P=0.0096; Figure). In the intent-to-treat (ITT) population, MRD-negativity (10 -5) rates also remained higher for D-RVd versus RVd (64.4% [67/104] vs 30.1% [31/103], P3-fold higher for D-RVd versus RVd (44.2% vs 12.6%, P Conclusion: After 24 months of maintenance therapy, the addition of DARA to RVd induction and consolidation in conjunction with ASCT, followed by DARA plus R maintenance, continued to demonstrate deep and durable responses in pts with transplant-eligible NDMM, including sCR and MRD-negativity (10 -5 and 10 -6) rates. While this study was not powered for PFS, there is a positive trend towards improved PFS in the D-RVd group. No new safety concerns were observed with longer follow-up. These results support the use of D-RVd induction/consolidation and D-R maintenance in transplant-eligible NDMM pts. Figure 1 Figure 1. Disclosures Kaufman: Sutro, Takeda: Research Funding; Incyte, TG Therapeutics: Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Research Funding; Amgen: Research Funding; Tecnofarma SAS, AbbVie: Honoraria; Janssen: Honoraria; Fortis Therapeutics: Research Funding; Novartis: Research Funding; Incyte, celgene: Consultancy; Heidelberg Pharma: Research Funding; Roche/Genetech, Tecnopharma: Consultancy, Honoraria; Genentech, AbbVie, Janssen: Consultancy, Research Funding. Sborov: Sanofi: Consultancy; SkylineDx: Consultancy; GlaxoSmithKline: Consultancy; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees. Reeves: Bristol-Myers Squibb: Speakers Bureau; Incyte Corporation: Honoraria; Takeda: Honoraria; Pharma Essentia: Consultancy, Honoraria. Rodriguez: Janssen: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Karyopharm: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Oncopeptides: Consultancy, Honoraria. Chari: Oncopeptides: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Consultancy, Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Consultancy, Membership on an entity's Board of Directors or advisory committees; Shattuck Labs: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Research Funding; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Secura Bio: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sanofi Genzyme: Consultancy, Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Research Funding; Antengene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees; Millenium/Takeda: Consultancy, Research Funding; Janssen Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Research Funding; BMS/Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Silbermann: Janssen Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Sanofi Genzyme: Membership on an entity's Board of Directors or advisory committees, Research Funding. Costa: Pfizer: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria; BMS: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria, Speakers Bureau; Amgen: Consultancy, Honoraria, Research Funding, Speakers Bureau. Anderson: Celgene, BMS, Janssen, GSK, Karyopharm, Oncopeptides, Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Shah: GSK: Consultancy; Nektar: Research Funding; Kite: Consultancy; CareDx: Consultancy; CSL Behring: Consultancy; Indapta Therapeutics: Consultancy; Janssen: Research Funding; Poseida: Research Funding; Karyopharm: Consultancy; BMS/Celgene: Research Funding; Bluebird Bio: Research Funding; Oncopeptides: Consultancy; Teneobio: Research Funding; Sanofi: Consultancy; Precision Biosciences: Research Funding; Sutro Biopharma: Research Funding; Amgen: Consultancy. Bumma: Janssen: Membership on an entity's Board of Directors or advisory committees; Amgen: Speakers Bureau; Sanofi: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Holstein: Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Genentech, GSK, Janssen, Secura Bio, Sorrento: Honoraria; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Membership on an entity's Board of Directors or advisory committees, Research Funding. Jakubowiak: Abbvie: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Gracell: Membership on an entity's Board of Directors or advisory committees. Wildes: Carevive: Consultancy; Seattle Genetics: Consultancy; Sanofi: Consultancy; Janssen: Consultancy. Orlowski: Asylia Therapeutics, Inc., BioTheryX, Inc., and Heidelberg Pharma, AG.: Other: Laboratory research funding; CARsgen Therapeutics, Celgene, Exelixis, Janssen Biotech, Sanofi-Aventis, Takeda Pharmaceuticals North America, Inc.: Other: Clinical research funding; Asylia Therapeutics, Inc.: Current holder of individual stocks in a privately-held company, Patents & Royalties; Amgen, Inc., BioTheryX, Inc., Bristol-Myers Squibb, Celgene, Forma Therapeutics, Genzyme, GSK Biologicals, Janssen Biotech, Juno Therapeutics, Karyopharm Therapeutics, Inc., Kite Pharma, Neoleukin Corporation, Oncopeptides AB, Regeneron Pharmaceuticals, I: Membership on an entity's Board of Directors or advisory committees; Amgen, Inc., BioTheryX, Inc., Bristol-Myers Squibb, Celgene, EcoR1 Capital LLC, Genzyme, GSK Biologicals, Janssen Biotech, Karyopharm Therapeutics, Inc., Neoleukin Corporation, Oncopeptides AB, Regeneron Pharmaceuticals, Inc., Sanofi-Aventis, and Takeda P: Consultancy, Honoraria. Shain: AbbVie: 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, Speakers Bureau; Amgen Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sanofi Genzyme: Consultancy, Speakers Bureau; GlaxoSmithLine, LLC: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Adaptive Biotechnologies Corporation: Consultancy, Speakers Bureau; Janssen oncology: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Karyopharm Therapeutics Inc.: Honoraria, Research Funding; Novartis Pharmaceuticals Corporation: Consultancy. Cowan: BMS: Research Funding; Secura Bio: Consultancy; GSK: Consultancy; Harpoon: Research Funding; Cellectar: Consultancy; Sanofi: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Nektar: Research Funding. Pei: Janssen: Current Employment, Current equity holder in publicly-traded company. Cortoos: Janssen: Current Employment, Current equity holder in publicly-traded company. Patel: Janssen: Current Employment. Bartlett: Janssen: Current Employment. Vermeulen: Janssen: Current Employment, Current equity holder in publicly-traded company. Lin: Janssen: Current Employment. Richardson: Sanofi: Consultancy; Secura Bio: Consultancy; Regeneron: Consultancy; Karyopharm: Consultancy, Research Funding; Janssen: Consultancy; Protocol Intelligence: Consultancy; Celgene/BMS: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; GlaxoSmithKline: Consultancy; AstraZeneca: Consultancy; Oncopeptides: Consultancy, Research Funding; AbbVie: Consultancy; Jazz Pharmaceuticals: Consultancy, Research Funding. Voorhees: Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Oncopeptides: 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; Secura Bio: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. OffLabel Disclosure: The specific regimen combination is not yet approved, but individual components are.

We evaluated the effect of combining lenalidomide with therapeutic antibodies on antibody-dependant cell-mediated cytotoxicity (ADCC) of solid tumor cells, and the requirement for expression of natural killer (NK) cell-activating receptors and their solid tumor surface ligands. Twenty-three human tumor cell lines (colon, breast, lung, head and neck, ovary, and bone sarcoma) were analyzed. NK effector cells were isolated from healthy donors, pre-treated with and without lenalidomide, and incubated with antibody-coated tumor cells to determine ADCC. In blocking experiments, NK cells were pre-incubated with anti-DNAM-1 or anti-NKG2D antibodies, and target colorectal cells were pre-incubated with anti-CD155 (PVR), anti-MIC-A/B, or anti-ULBP 3 antibodies. Differences between groups were assessed using unpaired and paired Student's t test and one-way ANOVA. Lenalidomide enhanced NK cell-mediated ADCC of trastuzumab- and cetuximab-coated tumor cells. Activity against colorectal cancer cells was dependent on target antigen expression, but independent of KRAS status and FcγRIIIa genotype. The extent of ADCC and its enhancement by lenalidomide correlated with NK cell expression of NKG2D and DNAM-1, and tumor cell expression of PVR and MIC-A. Blocking of NKG2D and, to a lesser extent, DNAM-1 inhibited ADCC. Anti-MIC-A/B monoclonal antibody blocked natural cytotoxicity, but not ADCC. Lenalidomide enhances the ability of IgG1-isotype antibodies to mediate ADCC of solid tumor cells, the extent of which is largely dependent on NKG2D-NKG2D ligand interactions, but appears to be independent of MIC-A/B. This provides a rationale for exploratory clinical studies and an assessment of potential biomarkers predictive of clinical benefit.

Rituximab (RTX), a chimeric anti-CD20 antibody, is associated with direct induction of apoptosis and antibody-dependent cell-mediated cytotoxicity (ADCC) with clinical efficacy in mantle cell lymphoma (MCL). Lenalidomide (LEN), a novel immunomodulatory agent, sensitizes tumor cells and enhances ADCC. Our study attempted to elucidate the mechanism of LEN-enhanced RTX-mediated cytotoxicity of MCL cells. We found that LEN and RTX induced growth inhibition of both cultured and fresh primary MCL cells. LEN enhanced RTX-induced apoptosis via upregulating phosphorylation of c-Jun N-terminal protein kinases (JNK), Bcl-2, Bad; increasing release of cytochrome-c; enhancing activation of caspase-3, -8, -9 and cleavage of PARP. Meanwhile, LEN activated NK cells and increased CD16 expression on CD56(low)CD16(+) NK cells. Whole PBMCs but not NK cell-depleted PBMCs treated with LEN augmented 30% of RTX-dependent cytotoxicity. Daily treatment with LEN increased NK cells by 10-folds in SCID mice, and combination of LEN and RTX decreased tumor burden and prolonged survival of MCL-bearing SCID mice. Taken together, our study demonstrates that LEN plus RTX provides a synergistically therapeutic effect on MCL cells by enhancing apoptosis and RTX-dependent NK cell-mediated cytotoxicity and may be an optimal combination in the clinical trial of relapsed or refractory MCL.

Lenalidomide (Revlimid; CC-5013) and pomalidomide (CC-4047) are IMiDs proprietary drugs having immunomodulatory properties that have both shown activity in cancer clinical trials; lenalidomide is approved in the United States for a subset of MDS patients and for treatment of patients with multiple myeloma when used in combination with dexamethasone. These drugs exhibit a range of interesting clinical properties, including anti-angiogenic, anti-proliferative, and pro-erythropoietic activities although exact cellular target(s) remain unclear. Also, anti-inflammatory effects on LPS-stimulated monocytes (TNF-alpha is decreased) and costimulatory effects on anti-CD3 stimulated T cells, (enhanced T cell proliferation and proinflammatory cytokine production) are observed. These drugs also cause augmentation of NK-cell cytotoxic activity against tumour-cell targets. Having shown that pomalidomide confers T cell-dependent adjuvant-like protection in a preclinical whole tumour-cell vaccine-model, we now show that lenalidomide and pomalidomide strongly inhibit T-regulatory cell proliferation and suppressor-function. Both drugs inhibit IL-2-mediated generation of FOXP3 positive CTLA-4 positive CD25high CD4+ T regulatory cells from PBMCs by upto 50%. Furthermore, suppressor function of pre-treated T regulatory cells against autologous responder-cells is abolished or markedly inhibited without drug related cytotoxicity. Also, Balb/C mice exhibit 25% reduction of lymph-node T regulatory cells after pomalidomide treatment. Inhibition of T regulatory cell function was not due to changes in TGF-beta or IL-10 production but was associated with decreased T regulatory cell FOXP3 expression. In conclusion, our data provide one explanation for adjuvant properties of lenalidomide and pomalidomide and suggest that they may help overcome an important barrier to tumour-specific immunity in cancer patients.

The thalidomide analogue and immunomodulatory drug (IMiD) lenalidomide (CC-5013, REVLIMID) is emerging as a useful treatment for a number of cancers and has recently entered phase III trials for multiple myeloma. It has been suggested that the anti-tumor effect of lenalidomide is related to its anti-angiogenic potency. In this regard, we have previously shown that lenalidomide inhibits angiogenesis in both rat and human in vitro models but does not affect endothelial cell proliferation. We now show that oral administration of lenalidomide attenuates growth factor-induced angiogenesis in vivo; the rat mesenteric window assay was utilized to show that lenalidomide significantly inhibits vascularization in a dose-dependent manner. We also found that lenalidomide significantly inhibits growth factor-induced endothelial cell migration. This correlates with the inhibitory effect of lenalidomide on growth factor-induced Akt phosphorylation, thereby providing a potential mechanism for its anti-migratory and subsequent anti-angiogenic effects. These data further support the use of lenalidomide as an orally administered drug for the effective treatment of angiogenesis-dependent conditions, including cancer, and suggest a potential mechanism of action.

Background: Demonstrating improved overall survival (OS) with new multiple myeloma (MM) treatments is becoming difficult because of extended survival, so progression-free survival (PFS) is commonly used as a surrogate endpoint for OS. We evaluated PFS as a potential surrogate for OS by examining whether observed treatment effects on PFS are positively associated with treatment effects on OS in MM. Methods: A systematic literature review identified 21 randomized control trials reporting hazard ratios (HRs) for treatment effects on PFS and OS. Pearson's r estimated the relationship between HRs (HRPFS and HROS), and between log-transformed HRs (log(HRPFS) and log(HROS)). R2 values were estimated from linear regression models of the HR and the log(HR) relationships. Sensitivity and subgroup analyses examined the robustness of the HR findings. Results: Positive correlations were found between HRPFS and HROS (r = 0.82; p < 0.0001) and between log(HRPFS) and log(HROS) (r = 0.80; p < 0.0001). Linear regression models produced R2 values of 0.67 and 0.63 when regressing HROS on HRPFS, and log(HROS) on log(HRPFS), respectively. Sensitivity analyses supported the HR findings. Conclusion: This analysis provides evidence for a positive association between treatment effects on PFS and OS. Studies involving patient level data are necessary to confirm whether PFS is a valid surrogate for OS in MM.

Purpose: Lenalidomide has significant activity in myelodysplastic syndromes, multiple myeloma, and non-Hodgkin's lymphoma (NHL). In previous studies, natural killer (NK) cell expansion by lenalidomide was shown to enhance the cytotoxic effect of rituximab. This study assessed the ability of lenalidomide to enhance antibody-dependent cellular cytotoxicity (ADCC) in rituximab-treated NHL cell lines and primary tumor cells from patients with B-cell chronic lymphocytic leukemia (B-CLL) in vitro.Experimental Design: An in vitro ADCC system was used to assess the ability of lenalidomide to enhance human NK cell and monocyte function in response to rituximab.Results: Lenalidomide directly enhanced IFN-γ production via Fc-γ receptor-mediated signaling in response to IgG. It was also a potent enhancer of NK cell-mediated and monocyte-mediated tumor cell ADCC for a variety of rituximab-treated NHL cell lines in vitro, an effect that was dependent on the presence of antibody and either interleukin-2 or interleukin-12. Lenalidomide also enhanced the ability of NK cells to kill primary tumor cells derived from three patients with B-CLL who have been treated previously with fludarabine plus cyclophosphamide. Enhanced NK cell ADCC was associated with enhanced granzyme B and Fas ligand expression and could be inhibited by a granzyme B inhibitor and partially inhibited by antibody to FasL. Enhanced NK cell Fc-γ receptor signaling is associated with enhanced phosphorylated extracellular signal-related kinase levels leading to enhanced effector function.Conclusions: These findings suggest that lenalidomide has the potential to enhance the rituximab-induced killing of NHL cell lines and primary B-cell chronic lymphocytic leukemia cells via a NK cell-mediated and monocyte-mediated ADCC mechanism in vitro, providing a strong rationale for the combination of lenalidomide with IgG1 antibodies to target tumor-specific antigens in patients with cancer.

Lenalidomide (Revlimid) is approved for the treatment of transfusion-dependent patients with anemia due to low- or intermediate-1-risk Myelodysplastic Syndromes (MDS) associated with a del 5q cytogenetic abnormality with or without additional cytogenetic abnormalities, and in combination with dexamethasone for the treatment of multiple myeloma patients who have received at least one prior therapy. Previous reports suggest that lenalidomide is anti-angiogenic and this property appears to be related to efficacy in patients with MDS. We have investigated the effect of lenalidomide on the formation of microvessels in a novel in vitro angiogenesis assay utilizing human umbilical arterial rings and in a capillary-like cord formation assay using cultured primary endothelial cells. We found that lenalidomide consistently inhibits both sprout formation by arterial rings and cord formation by endothelial cells in a dose-dependent manner. We also found an inhibitory effect of lenalidomide on the associations between cadherin 5, beta-catenin and CD31, adherens junction proteins whose interaction is critical for endothelial cell cord formation. Furthermore, lenalidomide inhibited VEGF-induced PI3K-Akt pathway signaling, which is known to regulate adherens junction formation. We also found a strong inhibitory effect of lenalidomide on hypoxia-induced endothelial cell formation of cords and HIF-1 alpha expression, the main mediator of hypoxia-mediated effects and a key driver of angiogenesis and metastasis. Anti-metastatic activity of lenalidomide in vivo was confirmed in the B16-F10 mouse melanoma model by a >40% reduction in melanoma lung colony counts versus untreated mice. Our results suggest that inhibitory effects on microvessel formation, in particular adherens junction formation and inhibition of hypoxia-induced processes support a potential anti-angiogenic and anti-metastatic mechanism for this clinically active drug.

We have found that the synthetic compound CC-5079 potently inhibits cancer cell growth in vitro and in vivo by a novel combination of molecular mechanisms. CC-5079 inhibits proliferation of cancer cell lines from various organs and tissues at nanomolar concentrations. Its IC50 value ranges from 4.1 to 50 nmol/L. The effect of CC-5079 on cell growth is associated with cell cycle arrest in G2-M phase, increased phosphorylation of G2-M checkpoint proteins, and apoptosis. CC-5079 prevents polymerization of purified tubulin in a concentration-dependent manner in vitro and depolymerizes microtubules in cultured cancer cells. In competitive binding assays, CC-5079 competes with [3H]colchicine for binding to tubulin; however, it does not compete with [3H]paclitaxel (Taxol) or [3H]vinblastine. Our data indicate that CC-5079 inhibits cancer cell growth with a mechanism of action similar to that of other tubulin inhibitors. However, CC-5079 remains active against multidrug-resistant cancer cells unlike other tubulin-interacting drugs, such as Taxol and colchicine. Interestingly, CC-5079 also inhibits tumor necrosis factor-α (TNF-α) secretion from lipopolysaccharide-stimulated human peripheral blood mononuclear cells (IC50, 270 nmol/L). This inhibitory effect on TNF-α production is related to its inhibition of phosphodiesterase type 4 enzymatic activity. Moreover, in a mouse xenograft model using HCT-116 human colorectal tumor cells, CC-5079 significantly inhibits tumor growth in vivo. In conclusion, our data indicate that CC-5079 represents a new chemotype with novel mechanisms of action and that it has the potential to be developed for neoplastic and inflammatory disease therapy. (Cancer Res 2006; 66(2): 951-9)

Thalidomide, a drug originally used to treat morning sickness, was removed from the market place in the early 1960s after it was found to cause serious congenital birth defects. However, thalidomide has recently been investigated in a new light following its activity in a number of chronic diseases. Moreover, like thalidomide itself, its second-generation immunomodulatory drug (IMiD) analogues have been shown to act as powerful anticancer agents and are clearly active in the treatment of patients with relapsed multiple myeloma. These new drugs, in particular the second-generation IMiDs, lenalidomide (CC-5013, REVLIMID; Celgene Corp., NJ, USA) and CC-4047 (ACTIMID; Celgene Corp.), offer improvements over thalidomide (a first-generation IMiD) in terms of efficacy and safety in human studies. The key to the therapeutic potential of IMiDs lies in the fact that the drugs have multiple mechanisms of action, which may produce both anti-inflammatory and antitumour effects. These effects are probably contextual, depending both on the cell type and the stimulus involved. Mechanisms associated with IMiD activity include TNF-alpha-inhibitory, T cell costimulatory and antiangiogenic activities. Studies of the mechanisms of action of these drugs are ongoing and will facilitate the continued development of this class of compound in a number of diseases.

Thalidomide was originally used to treat morning sickness, but was banned in the 1960s for causing serious congenital birth defects. Remarkably, thalidomide was subsequently discovered to have anti-inflammatory and anti-angiogenic properties, and was identified as an effective treatment for multiple myeloma. A series of immunomodulatory drugs — created by chemical modification of thalidomide — have been developed to overcome the original devastating side effects. Their powerful anticancer properties mean that these drugs are now emerging from thalidomide's shadow as useful anticancer agents.

Background: In hematological malignancies lenalidomide (len) exerts tumoricidal and immunomodulatory effects and may also influence the tumor cell microenvironment by blocking the proangiogenic and proinvasive effects of growth factors and/or hypoxia. In solid tumor models len is antimetastatic but lacks overt tumoricidal activity. These observations led us to explore the effect of len on hypoxic solid tumor cells, in particular the expression of HIF-α and its upstream regulators p53 and HDM-2. Methods: Eighteen solid tumor cell lines were tested for the effect of len on the response to hypoxia (1 % O2, 5% CO2, 94% N2; Invivo2 400 hypoxic workstation). Cell lysates from three cell lines (HCT-15, MDA-MB-231 and SKOV-2) were assessed for HIF-1α, p53 and HDM-2 proteins by immunoblot. We also performed immunoprecipitation studies to explore the effect of len on the interaction of these proteins. The effect of len on HIF-1α was assessed in p53 siRNA transfected HCT-15 cells and on HIF-1 α mRNA in transfected Luc-HIF NIH 3T3 cells. Len effects on hypoxia-induced trans-well invasion of three cell lines were also assessed. Results: Len (1 µM) significantly inhibited hypoxia-induced HIF-1 a protein levels dose-dependently in the majority of tumor cell lines tested; breast (by 68%, MDA-MB-231; 65%, MCF-7), colorectal (76%, HCT-116; 65%, HCT-15; 82% HT-29), ovarian (71%, OVCAR-3; 66%, SKOV-3), renal (83%, 786-O; 57%, HEK293), prostate (54%, DU- 145) and pancreatic (45%, MiaPaca-3)(all p Conclusions: These results suggest that len inhibits the proinvasive hypoxic response in multiple, diverse tumor lines and may exert antimetastatic effects within the hypoxic solid tumor microenvironment. Citation Information: Clin Cancer Res 2010;16(14 Suppl):A5.

Abstract 2855 Poster Board II-831 Lenalidomide is an oral anti-proliferative and immunomodulatory drug. In combination with dexamethasone, it is indicated for the treatment of patients with multiple myeloma (MM) who have received at least one prior therapy. In this study we investigated direct antimyeloma effects of lenalidomide in vitro using a panel of human MM cell lines with various cytogenetic features and bone marrow cells from patients with active MM. We also assessed the effect of lenalidomide on expression of tumor suppressor and enhancer genes such as p21cip1, SPARC, ING1/4, p57kip2, p53, cyclin D1/2, IRF4/MUM1 and IRF8/ICSBP. At attainable plasma levels in treated patients, lenalidomide directly inhibited human MM cell proliferation. Lenalidomide strongly increased expression of tumor suppressor genes such as p21waf1/cip1, SPARC, IRF8, ING4 and p57kip2. In the MM cell lines tested, lenalidomide had partial but consistent inhibitory effects on expression of IRF4, an important MM survival factor. However, lenalidomide had no marked effect on expression of tumor enhancer genes such as VEGF, cyclin D1/2 and MAF or tumor suppressor genes such as ING1 and p53 in most lines of cells. This suggests that the antiproliferative effects of lenalidomide on MM cells may be related to the upregulation of some tumor suppressor gene expression. Statistical analyses show that the antiproliferative effect of lenalidomide is significantly correlated with the drug induced upregulation of SPARC and IRF8 expression (p=0.0016; p=0.012, respectively), but not with the drug induced changes of p21 (p> 0.05) and IRF4 expression (p> 0.05). Furthermore, the antiproliferative effect of lenalidomide was significantly correlated with the constitutive expression levels of cyclin D1 (p=0.021) and IRF4 (p=0.027), and inversely correlated with the constitutive level of cyclin D2 (p=0.041) in these MM cell lines. Using bone marrow myeloma cells from patients, we confirmed that the sensitivity of cells to lenalidomide was associated with SPARC and IRF8 upregulation and baseline levels of cyclin D1/2 and IRF4 expression. Using MM cell lines adapted to prolonged exposure (5 months) to lenalidomide, we found that cells became resistant to the drug in association with decreased baseline levels of cyclin D1 and IRF4. In conclusion, lenalidomide demonstrates direct inhibitory effects on proliferation various MM cells. These antimyeloma activities may help explain the clinical efficacy seen in patients treated with lenalidomide. Lenalidomide treatment of MM cells increased SPARC and IRF8 mRNA expression, whereas pre-treatment cyclin D1/2 and IRF4 mRNA levels were associated with increased sensitivity and may have prognostic potential for MM therapy with lenalidomide. Disclosures: Zhang: Celgene Corporation: Employment. Adams:Celgene Corporation: Employment. Kosek:Celgene Corporation: Employment. Schafer:Celgene Corporation: Employment. Bartlett:Celgene Corporation: Employment.

Abstract 4926 Introduction Despite recent advances in the treatment of Multiple Myeloma (MM) the disease remains incurable and initial response to therapy is followed inexorably by relapse, chemoresistance and death. Therefore, relapsed and refractory disease remains an area of high unmet need with an urgent need for new therapies that are able to exert significant effects on the tumor. Lenalidomide (Len), an oral anti-proliferative and immunomodulatory drug, has been approved in combination with dexamethasone (Dex) for previously treated MM. Early clinical results also suggest that pomalidomide (Pom) is effective in the treatment of relapsed and refractory MM, including patients who have previously been treated with Len. The achievable plasma levels of each agent and pharmacological activity in vitro suggest very little difference in terms of immune enhancing properties. In contrast, Pom is a more potent anti-proliferative agent. However, it is unknown how MM cell sensitivity to Pom might be affected by resistance to Len or how sensitivity to Len might be affected by prior treatment with Pom. The use of a cellular model mimicking the relapse/resistance issue found in the clinic can provide valuable information directly translatable to the treatment of patients. Methods In this study we have generated MM cell resistance by long term exposure to either Len or Pom at levels attainable in patient plasma. H929 and KMS-12-BM MM cell lines were exposed to Len or Pom at clinically achievable concentrations (1 μM Len or 0.1 μM Pom) for a continuous period of 5 months. Cell growth was measured by ATP assay. Resistance factors were calculated by dividing the compound growth IC50 in the long term treated cells by the growth IC50 in the parental cells. Results The continued culture of H929 and KMS-12-BM cells with Len resulted in Len resistance factors of >2270 and >47600, and Pom resistance factors of 725 and 280, respectively. Therefore, in the Len-resistant cells, cross-resistance to Pom was lower than resistance to Len. Conversely, the continued culture of H929 and KMS-12-BM cells with Pom resulted in Len resistance factors of >2270 and >47600, and Pom resistance factors of only 22 and 130, respectively. There was complete cross-resistance to Len in the Pom-resistant cells. Furthermore, the addition of Dex was able to improve the sensitivity of the resistant cells to Pom to levels that are attainable in the plasma of patients and far more than it improved sensitivity to Len. Thus, while Pom (and Pom/Dex) retained efficacy in Len-resistant MM cells, MM cells previously exposed to Pom became refractory to Len (and Len/Dex). Conclusions Recent clinical data suggests that Pom/Dex is active in relapsed/refractory MM patients who have previously been treated with Len/Dex. Our results provide mechanistic support for the importance of the anti-proliferative activity of Pom (and Pom/Dex) in this setting. However, our results also suggest that Len (and Len/Dex) may be less effective in patients who have previously been treated with Pom, especially in the context of relapsed/refractory disease where immune function is likely to get progressively weaker with successive lines of therapy and thereby less amenable to immune enhancing agents. Disclosures Adams: Celgene: Employment. Off Label Use: Pomalidomide is an anti-proliferative and immunomodulatory agent that is in clinical development for relapsed/refractory MM. Schafer:Celgene: Employment. Bartlett:Celgene: Employment.

Lenalidomide is an oral anti-angiogenic, anti-proliferative and immunomodulatory drug that is approved for the treatment of transfusion-dependent patients with anemia due to low- or intermediate-1-risk MDS associated with a del 5q cytogenetic abnormality with or without additional cytogenetic abnormalities, and in combination with dexamethasone for the treatment of previously treated multiple myeloma patients. Encouraging early results suggest a potential for clinical efficacy in B cell non-Hodgkin’s lymphoma (NHL). We have previously shown that lenalidomide can directly inhibit the proliferation of NHL cells and synergize with dexamethasone in inducing cell cycle arrest at the G1/S transition leading to caspase activation and apoptosis. In this study we have studied the potential for lenalidomide mediated growth inhibition, apoptosis and inhibition of angiogenic factors in NHL. We have assessed proliferation, expression of p21 and SPARC tumor suppressor genes and the production of the angiogenic factor vascular endothelial growth factor (VEGF) in six established NHL cells lines in vitro. The effect of lenalidomide on NHL cells was determined after 1–3 days using 3H-thymidine incorporation, microbead array and real time PCR. We found that lenalidomide displays anti-proliferative effects on Namalwa, Jeko-1 and Rec-1 cells when used alone and in combination with dexamethasone. However, several NHL cell lines, such as Granta-519, DB, and JVM-2, appear to be resistant. We also found that lenalidomide strongly inhibited VEGF production (but not mRNA levels) by the three sensitive lines (but not the three resistant lines) at much lower concentrations than required for inhibitory effects on proliferation. Excess recombinant human VEGF or neutralizing anti-VEGF antibody had no effect on Rec-1 proliferation suggesting that there is no connection between these two effects. Lenalidomide strongly increased the expression of tumor suppressor genes, such as p21cip/kip and SPARC (secreted protein acidic and rich in cysteine). In particular, elevation of SPARC mRNA correlated with the anti-proliferative effect of lenalidomide and was not seen in the resistant cells, whereas elevated p21 was observed in both sensitive and resistant (DB) cells. The effect of SPARC gene silencing on lenalidomide-induced effects is being assessed in siRNA studies. In conclusion, lenalidomide demonstrates anti-proliferative activity against certain sensitive NHL cells, particularly in combination with dexamethasone. Potent anti-VEGF activity of lenalidomide appears to be independent of proliferation and supports its anti-angiogenic potential. Lenalidomide-induced up-regulation of SPARC mRNA appears to correlate with sensitivity and may have biomarker potential in NHL.

Lenalidomide (Revlimid® is approved for the treatment of transfusion-dependent patients with anemia due to low- or intermediate-1-risk MDS associated with a del 5q cytogenetic abnormality with or without additional cytogenetic abnormalities, and in combination with dexamethasone is for the treatment of multiple myeloma patients who have received at least one prior therapy. Encouraging early results suggest a potential for clinical efficacy in B cell non-Hodgkin’s lymphoma (NHL). Potential mechanisms of action include anti-angiogenic, anti-proliferative and immunomodulatory activities. Lenalidomide has been shown to enhance Th1-type cytokines and T cell and NK cell activation markers in patients with advanced cancers. Furthermore, lenalidomide has been shown to enhance rituximab-mediated protection in a SCID mouse lymphoma model in vivo. We have utilized an in vitro ADCC system to assess the ability of lenalidomide to directly enhance human NK cell function in response to therapeutic antibodies, such as rituximab (chimeric anti-CD20 mAb). Isolated NK cells produced little or no IFN-γ in response to IgG and/or IL-2 or IL-12. However, pre-treatment of NK cells with lenalidomide greatly enhanced IFN-γ production by NK cells in a dose-dependent manner. In a functional ADCC assay, NHL cell lines (Namalwa, Farage & Raji) were pre-coated with rituximab and exposed to NK cells pre-treated with lenalidomide in the presence of either exogenous IL-2 or IL-12. After 4 hours in culture the viability of the tumor cells was assessed. Lenalidomide consistently and synergistically increased the killing of tumor cells in a dose-dependent manner and up to >4-fold compared to rituximab alone. Rituximab alone had only a small effect in this model and there was no killing of cells in the absence of rituximab. The presence of either exogenous IL-2 or IL-12 was required to see enhanced killing by lenalidomide. In cancer patients lenalidomide has been shown to increase serum IL-12 levels and is also known to induce IL-2 production by T cells in vitro. Potential mechanisms for enhanced ADCC include increased signaling through NK FCγ receptors and/or IL-2 or IL-12 receptors. However, we found that these receptors are unaffected by lenalidomide, although downstream effects on NK signaling pathways are likely and are being actively investigated. In conclusion, we have shown that lenalidomide strongly enhances the ability of rituximab to induce ADCC mediated killing of NHL cells in vitro. This provides a strong rationale for combination of these drugs in patients with NHL and CLL.

Inhibition of angiogenesis is currently perceived as one of the promising strategies in the cancer therapy. Thalidomide (Thalomid®) and its Immunomodulatory Drug (IMiD®) analogs have entered into clinical trials for the treatment of various types of cancers. Lenalidomide (Revlimid®, CC-5013) is currently being assessed in oncology clinical trials worldwide. In this study we have compared the anti-angiogenic mechanisms of thalidomide and lenalidomide in a variety of experimental systems representing distinct events of the angiogenic process. Endothelial cell (EC) proliferation is stimulated by growth factors. Neither thalidomide nor lenalidomide have an appreciable inhibitory effect on growth factor-induced proliferation of human umbilical vein endothelial cells (HUVEC). In both the HUVEC and B16-F10 mouse melanoma tube formation assays, lenalidomide appears to be at least 10-fold more potent than thalidomide, the latter indicating that effects extend to vessels lined by tumor cells. However, in endothelial cell migration assays, thalidomide is more active than lenalidomide; in both the HUVEC and B16-F10 monolayer scratch migration assays, thalidomide is clearly more potent than lenalidomide in preventing the migration of cells into the wounded/scratched region. In assays of HUVEC migration towards specific angiogenic factors, such as VEGF, bFGF, and TNF-α, thalidomide is also more potent than lenalidomide. Interestingly, assays where the whole physiological process of angiogenesis can be studied show differential sensitivities. Lenalidomide is more potent in the rat aorta assay, whereas thalidomide is more potent in the human umbilical explant assay. Mechanistic studies on signal transduction events triggered by VEGF show that both thalidomide and lenalidomide partially inhibit Akt phosphorylation in VEGF-induced HUVEC. Furthermore, inhibitory effects on the phosphorylation of Gab1, a scaffolding protein upstream of Akt activation, have been observed. In summary, our data indicate that both thalidomide and lenalidomide interfere with key events in the angiogenic process and that they can be differentiated qualitatively depending on which component part is studied.