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Proteasome inhibition is a highly effective treatment for multiple myeloma (MM). However, virtually all patients develop proteasome inhibitor resistance, which is associated with a poor prognosis. Hyperactive small ubiquitin-like modifier (SUMO) signaling is involved in both cancer pathogenesis and cancer progression. A state of increased SUMOylation has been associated with aggressive cancer biology. We found that relapsed/refractory MM is characterized by a SUMO-high state, and high expression of the SUMO E1-activating enzyme (SAE1/UBA2) is associated with poor overall survival. Consistently, continuous treatment of MM cell lines with carfilzomib (CFZ) enhanced SUMO pathway activity. Treatment of MM cell lines with the SUMO E1-activating enzyme inhibitor subasumstat (TAK-981) showed synergy with CFZ in both CFZ-sensitive and CFZ-resistant MM cell lines, irrespective of the TP53 state. Combination therapy was effective in primary MM cells and in 2 murine MM xenograft models. Mechanistically, combination treatment with subasumstat and CFZ enhanced genotoxic and proteotoxic stress, and induced apoptosis was associated with activity of the prolyl isomerase PIN1. In summary, our findings reveal activated SUMOylation as a therapeutic target in MM and point to combined SUMO/proteasome inhibition as a novel and potent strategy for the treatment of proteasome inhibitor–resistant MM.

Introduction The ReLApsE trial compared lenalidomide (LEN)/dexamethasone (DEX; Rd) re-induction, salvage high dose chemotherapy (HDCT), autologous stem cell transplantation (ASCT) and LEN maintenance with continuous Rd in relapsed multiple myeloma. Landmark (LM) analyses from salvage HDCT were performed due to the fact that ~30% of patients in the HDCT arm did not receive salvage HDCT/ASCT. These analyses showed a survival benefit in patients actually undergoing salvage HDCT/ASCT. Median PFS and OS from LM were 23.3 vs. 20.1 months (HR 0.74; p=0.09) and not reached vs. 57 months (HR 0.56; p=0.046) favoring the salvage HDCT/ASCT arm. Multivariate LM analyses showed significant associations of the salvage HDCT/ASCT arm with superior PFS (HR 0.6; p=0.01) and OS (HR 0.39; p=0.006). The present analysis aims to dissect treatment efficacy in relevant subgroups and provide clues for treatment stratification. Methods The ReLApsE trial (ISRCTN16345835) compared 3 Rd (LEN 25 mg, d1-21; DEX 40 mg, d1,8,15,22; 4 week cycles) re-induction cycles, HDCT (melphalan 200 mg/m2), ASCT and LEN maintenance (10 mg/d) until PD (arm B, n=139) with Rd until PD (arm A, n=138). Key inclusion criteria were 1-3 prior therapy lines, age ≤ 75, time to PD in case of front-line HDCT/ASCT (TTP1) ≥ 12 months and WHO PS ≤ 2. Exploratory subgroup analyses were performed in the ITT population for PFS/OS using an LM at HDCT (B; n=103) and the contemporaneous Rd cycle 5 (A; n=114). The median interval from randomization to LM was 117/122 days in arm B/A. Heterogeneity of treatment effect was assessed by cox regression with interaction term between treatment and subgroup factor. Results No significant differences in the PFS/OS benefit between arms were observed in subgroups according to baseline ISS (I/II/III; interaction p[i-p]=0.5/0.66), age (1; i-p=0.37/0.22), single vs. tandem front-line HDCT/ASCT (i-p=0.34/0.56), and TTP1 (12-24 vs. 24-48 vs. >48 months; i-p=0.91/0.21). The subgroups according to front-line HDCT/ASCT (yes/no) differed significantly with regard to PFS/OS benefit in arm B (i-p=0.006/0.001). A significant benefit was observed in patients with front-line HDCT/ASCT treated in arm B regarding PFS (HR 0.68, p=0.03; n=107[A]/98[B]) and OS (HR 0.43, p=0.009). Patients without front-line HDCT/ASCT constituted a very small subgroup with imbalances in baseline parameters adversely affecting arm B and had expectably inferior survival in arm B (PFS: HR 4.35, p=0.08; OS: HR 19.83, p=0.0078; n=7[A]/5[B]). The subgroup with baseline LDH upper limit of normal (ULN) differed significantly in PFS benefit in arm B (i-p=0.03) but not in OS benefit (i-p=0.46). Patients with LDHULN (PFS: HR 1.54, p=0.31; n=16[A]/18[B]). The subgroups according to baseline cytogenetic risk and R-ISS showed a trend towards a differential benefit in arm B regarding OS (i-p=0.05 and 0.09) but not PFS (i-p=0.5 and 0.88). Patients with low risk cytogenetics (i.e. absence of t(4;14), del17p, +1q>3 copies and t(14;16)) had significantly superior OS in arm B (HR 0.21; p=0.01; n=57[A]/35[B]), whereas patients with high risk cytogenetics had no significant difference in OS according to trial arm (HR 0.82, p=0.67; n=25[A]/28[B]). Patients with R-ISS I had significantly superior OS in arm B (HR 0.08; p=0.02; n=33[A]/25[B]), whereas no significant difference in OS according to trial arm was seen in patients with R-ISS II (HR 0.72, p=0.42; n=52[A]/43[B]) and R-ISS III (HR 0.65, p=0.6; n=3[A]/5[B]). Conclusions The ReLApsE trial is the first RCT of salvage HDCT/ASCT vs. continuous novel agent treatment. In the absence of a significant survival benefit for the primary endpoint, LM analyses indicated a significant PFS/OS benefit in patients actually undergoing HDCT/ASCT. No heterogeneity of treatment effect was observed according to ISS, age, renal function, response to re-induction, prior therapy lines, single vs. tandem front-line HDCT/ASCT, and TTP1. Subgroup effects regarding PFS and/or OS benefit from HDCT/ASCT were seen favoring patients with front-line HDCT/ASCT and patients with low risk according to LDH, cytogenetics and R-ISS. Disclosures Baertsch: Takeda: Consultancy; Novartis: Consultancy, Research Funding. Raab:Celgene: Consultancy, Honoraria; BMS: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding. Hillengass:Celgene: Consultancy, Honoraria, Other: Advisory Board, Research Funding; amgen: Consultancy, Honoraria, Other: Advisory Board; Novartis: Honoraria, Other: Advisory Board; Janssen: Honoraria, Other: Advisory Board; Takeda: Honoraria, Other: Advisory Board; BMS: Honoraria, Other: Advisory Board; Sanofi: Research Funding. Graeven:Roche: Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria. Fenk:Bristol-Meyers Squibb: Honoraria, Other: travel grant; Takeda: Honoraria; Amgen: Honoraria; Janssen: Honoraria; Celgene: Honoraria, Other: Travel grant, Research Funding. Haenel:Takeda: Honoraria; Novartis: Honoraria; Amgen: Honoraria; Roche: Honoraria. Scheid:Amgen: Honoraria; BMS: Honoraria; Celgene: Honoraria; Janssen: Honoraria; Novartis: Honoraria, Research Funding; Takeda: Honoraria, Research Funding. Salwender:Janssen: Honoraria, Other: travel support, Research Funding; Celgene: Honoraria, Other: travel suppport, Research Funding; Novartis: Honoraria, Other: travel suppport, Research Funding; Bristol-Myers Squibb: Honoraria, Other: travel suppport, Research Funding; Amgen: Honoraria, Other: travel suppport, Research Funding; Takeda: Honoraria. Weisel:Amgen, Celgene, Janssen, and Sanofi: Research Funding; Amgen, BMS, Celgene, Janssen, Juno, Sanofi, and Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen, BMS, Celgene, Janssen, and Takeda: Honoraria. Goldschmidt:Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Research Funding; Novartis: Honoraria, Research Funding; Mundipharma: Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Adaptive Biotechnology: Consultancy; ArtTempi: Honoraria; Bristol Myers Squibb: Consultancy, Honoraria, Research Funding; Chugai: Honoraria, Research Funding; Sanofi: Consultancy, Research Funding; Amgen: Consultancy, Research Funding.

Introduction Salvage high dose chemotherapy (HDCT) followed by autologous stem cell transplantation (ASCT) is used in fit patients with relapsed multiple myeloma (RMM) in clinical practice. However, the role of this approach in the era of continuous novel agent based treatment has not been defined in randomized trials. The ReLApsE trial compared lenalidomide/dexamethasone (Rd) re-induction, salvage HDCT/ASCT and lenalidomide (R) maintenance with standard continuous Rd in a randomized controlled multicenter trial. Methods Between 2010 and 2016, 282 patients were randomized of whom 277 constituted the intention-to-treat (ITT) population (arm B/A n=139/138). Arm B received 3 cycles of Rd (lenalidomide 25 mg, day 1-21; dexamethasone 40 mg, day 1, 8, 15, 22; 4 week cycles) re-induction, HDCT (melphalan 200 mg/m2), ASCT and R maintenance (10 mg daily) until progression (PD). Arm A was treated with Rd until PD. In both arms stem cells were harvested after the 3rd Rd cycle if no back-up transplant was available. Key inclusion criteria were 1-3 prior therapy lines, age ≤ 75 years, time to PD ≥ 12 months in case of front-line HDCT/ASCT and WHO PS ≤ 2. The primary endpoint was progression free survival (PFS). Secondary endpoints included overall survival (OS), response rates and toxicity. ISRCTN16345835, Eudra CT-No: 2009-013856-61. Results Arm B and A were balanced regarding age (median 61.3 vs. 62.2 years), ISS (I/II/III in 62.6/24.4/13% vs. 59.7/31/9.3%) and WHO PS (0/1/2 in 69.1/30.9/0% vs. 76.1/23.2/0.7%). Almost all patients had only 1 prior therapy line (arm B: 94.2% vs. arm A: 93.5%) and had received front-line HDCT/ASCT (92.8% vs. 94.2%). More patients in arm B had high risk cytogenetic aberrations (HR-CA; 42.9% vs. 31.6%) based on a higher frequency of t(4;14) (20.2% vs. 10.1%). The overall response rate (≥ partial response; ORR) for arm B and A was 77.9% and 74.6% (p=0.57) with 49.3% and 47.1% (p=0.81) achieving ≥ very good partial response as best response. Within a median follow up of 36.3 months, 183 PFS events and 76 deaths occurred. Median PFS in the ITT population was 20.7 months in arm B and 18.8 months in arm A without a statistically significant difference (HR 0.87; 95% CI 0.65-1.16; p=0.34). Median OS was not reached (NR) in arm B vs. 62.7 months in arm A (HR 0.81; 95% CI 0.52-1.28; p=0.37). In arm B, 41 patients (29.5%) did not receive the planned HDCT/ASCT. Thus, exploratory landmark (LM) analyses from HDCT and the contemporaneous Rd cycle 5 in arm A were performed (median interval from randomization to HDCT/Rd cycle 5: 117/122 days; n=103[B]/114[A]). They showed a trend towards superior PFS (23.3 vs. 20.1 months; HR 0.74; p=0.09) and significantly superior OS (NR vs. 57 months; HR 0.56; p=0.046) in arm B vs. A. Multivariate analyses revealed significant associations of treatment in arm B with superior LM PFS (HR 0.6; p=0.01) and LM OS (HR 0.39; p=0.006). Other factors in the LM multivariate models showing significant associations with survival were HR-CA (PFS, OS), number of prior therapy lines (PFS), and age (PFS). The ORR in arm B after HDCT/ASCT was significantly higher than in arm A after Rd cycle 5 (82.3% vs. 69.6%; p=0.04). Grade ≥3 adverse events were reported in 83% (arm B) and 74.5% (arm A; p=0.11). Grade ≥3 leukopenia/neutropenia was reported in 61.5 vs. 24.8% (p Conclusions This is the first RCT comparing salvage HDCT/ASCT with continuous novel agent based treatment. No significant PFS or OS difference was observed in the overall trial population. However, HR-CA were more frequent in the HDCT/ASCT arm and ~30% of patients did not receive the planned HDCT/ASCT. Landmark analyses from the time of HDCT indicate superior PFS and OS in patients actually undergoing salvage HDCT/ASCT. Salvage HDCT/ASCT was safe with an expected increase in hematological as wells as gastrointestinal toxicity but without treatment-related mortality in patients up to the age of 75 years in this multicenter trial. However, the number of patients not undergoing salvage HDCT/ASCT and the approval of more active Rd-based triplet regimens after the initiation of this trial prevents definite conclusions on the role of salvage HDCT/ASCT. Disclosures Goldschmidt: Amgen: Consultancy, Research Funding; Novartis: Honoraria, Research Funding; ArtTempi: Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Research Funding; Mundipharma: Research Funding; Takeda: Consultancy, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Bristol Myers Squibb: Consultancy, Honoraria, Research Funding; Adaptive Biotechnology: Consultancy; Chugai: Honoraria, Research Funding. Baertsch:Takeda: Consultancy; Novartis: Consultancy, Research Funding. Raab:Celgene: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding. Hillengass:Novartis: Honoraria, Other: Advisory Board; Sanofi: Research Funding; Takeda: Honoraria, Other: Advisory Board; Janssen: Honoraria, Other: Advisory Board; amgen: Consultancy, Honoraria, Other: Advisory Board; BMS: Honoraria, Other: Advisory Board; Celgene: Consultancy, Honoraria, Other: Advisory Board, Research Funding. Graeven:AbbVie: Honoraria; Roche: Membership on an entity's Board of Directors or advisory committees. Fenk:Takeda: Honoraria; Bristol-Meyers Squibb: Honoraria, Other: travel grant; Celgene: Honoraria, Other: Travel grant, Research Funding; Janssen: Honoraria; Amgen: Honoraria. Haenel:Novartis: Honoraria; Roche: Honoraria; Amgen: Honoraria; Takeda: Honoraria. Scheid:Novartis: Honoraria, Research Funding; Takeda: Honoraria, Research Funding; Janssen: Honoraria; Celgene: Honoraria; BMS: Honoraria; Amgen: Honoraria. Salwender:Celgene: Honoraria, Other: travel suppport, Research Funding; Janssen: Honoraria, Other: travel support, Research Funding; Novartis: Honoraria, Other: travel suppport, Research Funding; Takeda: Honoraria; Amgen: Honoraria, Other: travel suppport, Research Funding; Bristol-Myers Squibb: Honoraria, Other: travel suppport, Research Funding. Weisel:Amgen, Celgene, Janssen, and Sanofi: Research Funding; Amgen, BMS, Celgene, Janssen, and Takeda: Honoraria; Amgen, BMS, Celgene, Janssen, Juno, Sanofi, and Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Background: Isatuximab (ISA) is an anti-CD38 monoclonal antibody with multiple modes of action for killing tumor cells via direct tumor targeting and immune cell engagement. ISA, combined with bortezomib, has demonstrated strong potentiation in a multiple myeloma (MM) xenograft model (Clin Cancer Res 2014:20:4754). This supported evaluation of ISA with bortezomib combinations in pts with newly diagnosed multiple myeloma (NDMM) ineligible for transplant. In the initial cohort, ISA combined with bortezomib, cyclophosphamide, and dexamethasone (dex) was well tolerated with 73% of pts achieving very good partial response (VGPR) or better and 40% with complete response (CR) (Blood 2017; 130: 3160). The combination of bortezomib, lenalidomide, and dex (VRd) is also effective in NDMM (Lancet 2017:389:519-27). Here, we report initial data from a Phase Ib study of ISA plus VRd in pts with NDMM (NCT02513186). Methods: Pts with NDMM ineligible for transplantation were treated in 2 phases: induction and maintenance. Induction phase (four 6-week cycles [C]): ISA (10 mg/kg) on Day (D) 1, 8, 15, 22, 29 (C1), followed by D1, 15, 29 (C2-4); bortezomib (1.3 mg/m2) on D1, 4, 8, 11, 22, 25, 29, 32 (C1-4); lenalidomide (25 mg/day): D1-14 and D22-35 (C1-4); dex (20 mg/day): D1, 2, 4, 5, 8, 9, 11, 12, 15, 22, 23, 25, 26, 29, 30, 32, 33. Maintenance phase (4-week cycles): ISA (10 mg/kg) on D1, 15 (all cycles); lenalidomide (25 mg/day): D1-21 (all cycles); dex (40 mg): D1, 8, 15, 22 (all cycles), unless the pt was >75 years of age, then the dose was 20 mg. The primary objective was to evaluate safety and preliminary efficacy (overall response rate [ORR] and CR rate, [IMWG criteria]) of ISA plus VRd. Minimal residual disease (MRD) was evaluated using next generation sequencing (NGS) and flow cytometry (NGF) at a sensitivity of 10-6 in pts achieving VGPR or above. Here, we report results from a protocol-planned interim analysis. Results: All 22 pts were included in the safety analysis (pts who received ≥1 dose of ISA) and 14 were eligible for preliminary efficacy analyses (first 14 pts who completed the 4 induction cycles). Median age was 71 (range 63-77) years. At study entry, 6, 12, and 1 pt were International Staging System Stage I, II, and III, respectively. One pt had extramedullary plasmacytoma at baseline. At data cut-off (Mar 22, 2018), the median number of cycles was 5.5 (1-9). Three pts discontinued treatment (2 VGPR, 1 not efficacy-evaluable): 2 pts due to adverse event (AE); Grade (Gr) 3 infusion reaction (IR) (ISA-related; Gr 3 dyspnea, Gr 2 glottic edema, Gr 2 nasal edema, and Gr 2 generalized rash), and Gr 5 bacteremia (lenalidomide- and dex-related); and 1 pt withdrew consent; 19 (86%) pts are continuing treatment. Dose reduction of bortezomib, lenalidomide, and dex was required in 6 (29%), 4 (16%), and 5 (28%) pts, respectively. TEAEs occurred in 19 (86%) pts. Most frequent TEAEs (any Gr; excluding laboratory abnormalities) were constipation (10 pts [46%]), IRs and peripheral edema (9 pts [41%] each), asthenia, diarrhea, and peripheral sensory neuropathy (8 pts [36%] each), hypotension (7 pts [32%]), fatigue and respiratory tract infection (6 pts [27%] each), cough and dyspnea (5 pts [23%] each). Gr ≥3 AEs were reported in 10 (46%) and serious AEs (SAEs) in 4 (18%) pts. Treatment-related SAEs occurred in 2 (9%) pts (IR and pancreatitis). IRs were Gr 1/2 in all but 1 (5%) pt (Gr 3). Gr 3/4 laboratory hematologic abnormalities: lymphopenia (8/22), neutropenia (4/22), thrombocytopenia (4/22)VGPR, 1 partial response (PR) and 1 pt with stable diseaseMedian time to first response was 1.4 months (end of C1) and, with a median follow-up of 7.49 months (at cut-off date), no pt has progressed, with all except 3 pts continuing on therapy. Five (38.5%) of 13 pts achieved MRD-negative status (by NGF and NGS, or NGS only). Conclusion: These data suggest that ISA plus VRd followed by ISA plus Rd is well tolerated with a high ORR of 93%. All responders had VGPR or CR except 1 pt with PR. Quality of CR may have been underestimated due to ISA interference which could be resolved with an interference assay. Funding: Sanofi Disclosures Ocio: Janssen: Consultancy, Honoraria; AbbVie: Consultancy; BMS: Consultancy; Pharmamar: Consultancy; Takeda: Consultancy, Honoraria; Seattle Genetics: Consultancy; Amgen: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria; Sanofi: Research Funding; Mundipharma: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Array Pharmaceuticals: Research Funding. Rodriguez Otero:Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy; Janssen: Consultancy, Honoraria; Clínica Universidad de Navarra: Employment; Bristol Myers Squibb: Research Funding. Bringhen:Amgen: Honoraria, Other: Advisory Board; Celgene: Honoraria; Bristol-Myers Squibb: Honoraria; Janssen: Honoraria, Other: Advisory Board; Takeda: Consultancy. Oliva:Celgene: Honoraria; Amgen: Honoraria; Takeda: Honoraria. Attal:Amgen: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Janseen: Consultancy, Research Funding; Sanofi: Consultancy. Moreau: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; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees. Kanagavel:Sanofi: Employment, Equity Ownership. Fitzmaurice:Sanofi: Employment, Equity Ownership. Wu:Sanofi: Employment, Equity Ownership. Martinez Lopez:Janssen: Research Funding, Speakers Bureau; Bristol Myers Squibb: Research Funding, Speakers Bureau; Novartis: Research Funding, Speakers Bureau; Celgene: Research Funding, Speakers Bureau.

Introduction: Multiple myeloma (MM) cells strongly rely on the interaction with bone marrow mesenchymal stromal cells (BMMSCs) for proliferation and survival. We previously reported that MM cells influence the mitochondrial function and senescence of surrounding BMMSCs amongst others via NAD-dependent deacetylase sirtuin-3 (SIRT3) (Blood, 2014; BMC Cancer, 2015). In the further course we examined the function of SIRT3 in BMMSCs and possible underlying mechanisms of SIRT3 activation. Methods: In this study we transfected 2 healthy donor BMMSCs (HD-BMMSCs) for transient knock-down of SIRT3 using 4 different siRNAs (GeneSolution, Qiagen). We analysed mitochondrial membrane potential (ΔΨM) and reactive oxygen species (ROS) by FACS. Cell cycle analysis was performed using "cell cycle assay kit" (Abcam). Senescence was examined using FACS and senescence-associated β-galactosidase activity. Apoptosis was analysed using Annexin V-FITC Kit (Miltenyi) and protein expression was examined by western blot. In addition we analysed the influence of MCT transporter interaction on SIRT3 expression in 6 BMMSCs of myeloma patients (MM-BMMSCs) using α-cyano-4-hydroxycinnamic acid (α-CN). Results: SIRT3 knock-down in HD-BMMSCs induced 1.4- to 1.9-fold increase in ROS levels (p Conclusion: SIRT3 seems to be a major regulator of mitochondrial functions in BMMSCs. Thus, reduced expression of SIRT3 that was previously reported in MM-BMMSCs could be the reason for increased ROS levels, cell cycle arrest in S phase and premature senescence-like state of MM-BMMSCs. In addition, our data show that inhibition of MCT transporters in MM cells and MM-BMMSCs induces apoptosis in MM cells and inhibits increased expression of SIRT3 in MM-BMMSCs. Disabling of MCT transporter interaction could possibly inhibit sustained induction of mitochondrial stress response in MM-BMMSCs and circumvent induction of the premature senescence-like state. Disclosures Blau: MSD: Honoraria; JAZZ Pharma: Honoraria; Shire: Honoraria; Celgene: Honoraria, Research Funding; AMGEN: Honoraria; Janssen: Honoraria, Research Funding; Baxalta: Honoraria; BMS: Honoraria.

Introduction: DNMT3A mutation is one of the most common somatic mutations in acute myeloid leukemia (AML) patients with normal karyotype. The most frequent mutation is located at R882 codon in the methyltransferase domain. Because of prognostic significance and high stability during the disease evolution, DNMT3A mutations might represent highly informative biomarkers for prognosis and outcome of disease. Methods: Using allele-specific PCR with a Blocking reagent (ASB-PCR assay) for the quantitative detection of DNMT3A R882H mutation, we analyzed 350 follow-up samples from 28 AML patients in complete remission (CR) after induction and consolidation treatment and allogeneic stem cell transplantation (alloSCT). Seventeen patients included in the follow-up analysis harbored a NPM1 mutation. Using a well-established marker for the detection of minimal residual disease (MRD) allowed to estimate the stability of DNMT3A mutation in CR and complete molecular remission (molCR). In addition, we analyzed FLT3, IDH1, and IDH2 mutations in diagnostic and follow up samples and donor chimerism after alloSCT. Results: We found the persistence of DNMT3A R882H mutations in all patients in CR after standard therapy. On the contrary, after alloSCT, DNMT3A R882H mutation was not found in patients with CR and complete donor chimerism. In relapse of leukemia, an increasing of both NPM1 and DNMT3A mutated alleles were shown all cases. Conclusion: Persistence of DNMT3A mutation after standard chemotherapy could indicate the origin of mutation in the early pre-leukemic stem cells. The loss of correlation between NPM1 and DNMT3A in CR could be associated with leukemic clone evolution. It is impotant to note, that the removal of mutated leukemic stem cells after allo SCT indicates therapeutic options allo SCT for high risk AML patients. Increased of both DNMT3A and NPM1 mutated alleles in relapse indicates the presence of both mutations, at least partly, in the same leukemic clone. We conclude that quantitative detection of DNMT3A R882H mutations at different time points of AML disease could provide additional information about the role of mutations in development and progression of AML. Disclosures Blau: BMS: Honoraria; MSD: Honoraria; Celgene: Honoraria, Research Funding; AMGEN: Honoraria; JAZZ Pharma: Honoraria; Shire: Honoraria; Janssen: Honoraria, Research Funding; Baxalta: Honoraria.

Introduction: Over the last decade, the cyclin D1 (CCND1) c.870G>A polymorphism has been variously reported to confer risk for a large number of cancers. Deregulation of a D-group cyclin is a key feature of multiple myeloma (MM). Previously published meta-analysis shown a relationship between CCND1 c.870G>A and risk of t(11;14) in MM (Wienhold, 2013). Allogeneic stem cell transplantation (AlloSCT) is a potential curative treatment for MM patients. There are no data with regard to the role of CCND1 c.870G>A polymorphism in stem cells donors and outcome of transplantation in MM patients. Methods: In this study we developed an endonuclease restriction method to identify CCND1 c.870G>A polymorphism. Directly Sanger sequencing were used to confirm the results. Results: At first, we analyzed 130 MM patients and 100 healthy donors as a control. CCND1 c.870G>A polymorphism was strongly associated with the t(11;14)(q13;q32) (PA polymorphism 55 pairs of MM patients and their allogeneic stem cell donors, retrospectively. Interestingly, c.870G-genotype in related and unrelated donors was statistically correlated with poor outcome after AlloSCT (P Conclusion: Our data suggest the published data that constitutive genetic factor (CCND1 c.870 G>A polymorphism) is associated with a specific chromosomal translocation in patients with MM. Despite the fact that our group of patients after AlloSCT is small, we found a statistical association between the c.870G-genotype in donor and a poor prognosis after transplantation. Disclosures No relevant conflicts of interest to declare.

Introduction: Interaction of multiple myeloma (MM) cells with the surrounding bone marrow mesenchymal stromal cells (BMMSCs) is crucial for MM proliferation and survival. We have previously reported that in vitro cultured MM-BMMSCs display premature stress-induced senescence and constitutive changes in gene expression (Blood, 2013). Because mitochondrial oxidative stress could be the reason for senescence in MM-BMMSCs we investigated the metabolic interplay between MM-BMMSCs and MM cells. Methods: In this study we analyzed 51 patients with MM, 4 patients with monoclonal gammopathy of undetermined significance (MGUS) and 10 healthy donors. Investigations ofNAD-dependent deacetylase sirtuin-3 (SIRT3) expression and mitochondrial mass were performed with SYBR-Green Real-Time PCR and relative quantification by linear regression. In addition, 20 co-cultures of MM-BMMSCs and KMS12-PE MM cell line were carried out. After incubation for 72 h SIRT3 expression in MM-BMMSCs was analyzed. Moreover, reactive oxygen species (ROS) production and mitochondrial membrane potential (ΔΨM) in co-cultured KMS12-PE cells and MM-BMMSCs were investigated by FACS analysis. Results: MM-BMMSCs displayed a 2-fold decrease in SIRT3 expression and a 2-fold increase in mitochondrial mass compared to healthy donor BMMSCs (HD-BMMSCs). These changes were not detected in MGUS-BMMSCs, suggesting an association with disease progression. Furthermore, the increase in mitochondrial mass could be induced by oxidative stress in MM-BMMSCs and is a feature of senescent cells. Because SIRT3 is a major regulator of the mitochondrial function, reduced expression could account for accumulation of reactive oxygen species in MM-BMMSCs cultured in vitro. Interestingly we found a 4-fold upregulation of SIRT3 expression in MM-BMMSCs when co-cultured with KMS12-PE myeloma cells. This indicates that cell-cell-contact to MM cells influences the mitochondrial function in MM-BMMSCs and induces mitochondrial stress response. Moreover, we found decreased levels of ROS and ΔΨM in co-cultured KMS12-PE myeloma cells and MM-BMMSCs compared to cells cultured alone. Therefore, MM cells seem to modify the mitochondrial function and induce expression of antioxidants in MM-BMMSCs aimed at reducing ROS levels and increasing cell proliferation and survival pathways in both cell types. Conclusion: Our results indicate that MM cells influence the mitochondrial function of MM-BMMSCs. This interaction leads to reduced ROS levels in both cell types and could support their survival and growth. Moreover, the sustained induction of mitochondrial stress response could be the reason for premature senescence in MM-BMMSCs when separated from MM cells. Therefore, MM therapy outcome may be improved through the disabling of the metabolic interplay between MM cells and MM-BMMSCs. Disclosures No relevant conflicts of interest to declare.

Multiple myeloma (MM) is a B-cell malignancy characterized by accumulation of malignant plasma cells (PC) within the bone marrow. The bone marrow microenvironment such as bone marrow stroma cells (BMSC) supports MM disease progression, resistance to chemotherapy, protects the tumor cells against apoptosis and causes osteolytic bone disease and angiogenesis. The aim of this study was to identify constitutive genetic alterations in BMSC derived from patients with MM (MM-BMSC) in comparison to BMSC from healthy donors. For BMSC selection, mononuclear cells were isolated from fresh bone marrow aspirates and were further expanded in cell culture. We studied 25 MM patients and 5 healthy donors. Senescence status was determined in passage 1 of cell cultures. MM-BSMC displayed a considerably higher percentage of senescence cells (p A downregulation of CCNE1 (p=0,05), CDKN1B (p=0,29), and an upregulation of CCND1 (p=0,05), VCAM-1 (p=0,33), ICAM-1 (p=0,33), and IKK-alpha (p=0,05) was demonstrated. Furthermore, the expression profile of miRNAs, targeting the analyzed mRNA genes or correlating with senescence, was studied (miR-16, miR-221, miR-126, miR-223, miR-485-5p and miR-519d). For miRNA detection treatment with Poly(A)-Polymerase and cDNA-Synthesis with a Poly(T)VN-Adaptor primer were carried out following an amplification with an universal reverse primer corresponding to the adaptor sequence and a miRNA-specific primer. miR-16, miR-223, miR-485-5p and miR-519d were significantly upregulated, (p=0,02; p=0,004; p=0,02; and p=0,002, respectively), whereas miR-221 and miR-126 showed no considerable differences to BMSC obtained from healthy donors. Next we investigated incubation of immunmodulatory drug Lenalidomid in BMSC cultures. Cells were treated with 10 µM Lenalidomid over 72 hours and expression was normalized to a 0,01 % DMSO control. Significant difference in gene expression were visible for ICAM-1 (p=0,01). For CDKN1B (p=0,16) and VCAM-1 (p=0,12) we demonstrated changes in gene expression. Our results indicate aberrant expression of cell cycle and adhesion-related genes, such as CCNE1, CCND1 and CDKN1B VCAM-1, ICAM-1 and IKK-alpha in MM-BMSC as compared with healthy donors. Furthermore, we found significant upregulation of miR-16, miR-223, miR-485-5p and miR-519d. Further investigation are needed to determine molecular mechanisms in MM-BMSC and PC interaction that lead to constitutive changes in BMSC characteristics and gene expression patterns. Disclosures: No relevant conflicts of interest to declare.

Abstract 2048 Introduction: Organ siderosis (OS) occurs frequently after allogeneic stem cell transplantations and is associated with worse survival. Although commonly attributed to transfusions of erythrocyte sediments, we present the occurrence of OS in mice suffering from Graft-versus-Host-disease (GvHD) independent of transfusions. Methods: Prior to transplantation (day 0), C57BL/10-wildtype (wt) mice received treosulfan 2000mg/kg on day -3 to -1 and cyclophosphamide 200 mg/kg on day-1. Mice were transplanted with 5×106 bone marrow cells and 3×106 splenocytes of Balb/c (allogeneic) or C57BL/10 (syngeneic) origin. Mice were sacrificed on day+9. Liver iron content was determined by atomabsorption spectroscopy in graphit tubes. In addition, a semi-quantitative analysis of liver, kidney and spleen sections was performed in kinetic studies. Macroscopic and histological GvHD-Scores were assessed according to standard scores. Results: Treatment of mice without additional transplantation or transplantation of syngeneic cells did not increase the iron content of the liver compared to untreated mice (naïve: 6.7 +/− 1.3 μmol/g; chemotherapy only: 7.2 +/− 1.9; syngeneic: 7.2 +/− 2.1). In contrast, allogeneic transplantated mice suffering from GvHD developed a siderosis of the liver of 13.6 +/− 3.1 (p=0.01, Kruskal-Wallis test). Furthermore, the iron deposition was correlated with the macroscopic and histologic GvHD score. Conclusion: These results show the occurrence of OS in wildtype animals after allogeneic stem cell transplantation. Since the detection of iron deposition occurs early after transplantation, OS is unlikely due to increased intestinal iron absorption, but appears to be a consequence of blocked iron export out of hepatic epithelial cells and macrophages and potentially by redistribution of iron as a consequence of liver cell destruction. Further studies are needed to elucidate the role of regulatory proteins such as hepcidin and ferroportin for OS in the context of GvHD following stem cell transplantation. In view of GvHD-associated OS in transplanted patients occuring frequently independent of transfusions, the effect appears clinically relevant with treatment options remaining to be defined. Disclosures: No relevant conflicts of interest to declare.

Abstract 2370 Regardless of the treatment applied, the outcome for patients with refractory hematological malignancies is extremely poor. For haploidentical stem cell transplantation (haploSCT), which has been evaluated especially in high-risk AML patients lacking an HLA-identical donor, registry data show an overall survival below 10% for AML not transplanted in remission. In order to improve immune competence and to reduce the risk of relapse, we have combined the early transfer of NK cells with the haploSCT approach. PATIENTS AND METHODS: Twenty-five patients (AML = 16, ALL = 5, CML = 2, Hodgkin = 1, MDS = 1) received a haploSCT followed by transfer of purified CD56+CD3-NK cells at day +2. Conditioning consisted of total body irradiation, thiotepa, fludarabine, and OKT3. NK cells were isolated from the CD34- fraction using an automated two-step procedure of CD3+ depletion and subsequent CD56+ selection. No other immunosuppression was routinely delivered. RESULTS: Patients received a mean of 13.3 × 106 CD34+ cells/kg (range 6.12–26.97) with 2.89 × 104/kg (0.95-7.4) contaminating CD3+ cells. A mean of 9.8 × 106 CD56+CD3- NK cells/kg (range 1.61–32.2) was adoptively transferred. Most of the patients developed early GVHD of the skin (median onset day=12), which promptly resolved after short term treatment with steroids and CSA. The main cause of death consisted of infections (n = 10), chronic GvHD (n= 2), and relapse (n = 4). After a median follow-up of 1442 days (3.9 years) (range 0.1 to 7.1 years) 9 of 25 patients are alive and in complete remission resulting in a 2-year overall survival (OS) estimate of 29%. Out of 16 high-risk patients with AML, 10 had refractory or active disease prior to transplantation. For a matched pair analysis, these patients were matched for age, disease status, conditioning regimen and year of transplantation using the EBMT database. AML patients treated with haploSCT plus NK cell transfer had a superior 2-year overall survival (40 vs 11%, 95% CI) in comparison to matched controls with haploSCT only (p=0.02) CONCLUSIONS: HaploSCT plus NK cells is feasible and shows promising survival rates for a group of patients lacking other treatment options. Best results were achieved in patients with AML not only in remission but also with refractory disease. Disclosures: No relevant conflicts of interest to declare.

Hematogones are B-lymphocyte precursors found in large frequencies after chemotherapies. In this study, the frequency of CD10+CD19+ hematogones was analysed routinely prior and post allogeneic stem cell transplantation and compared with moelculargenetic data for donor chimerism and for clonal translocations. Because of similarities in morphology and immunophenotype with frequent expression of CD19, CD10 and TdT they may undistinguishable from malignant B-cell lymphoblasts. As one example underlying the diagnostic difficulties, one patient with thrombopenia day +60 after allogeneic stem cell transplantation is presented. A relapse of Richter’s syndrome was suspected. Investigations of bone marrow specimens revealed a mixed chimerism, the frequency of cells coexpressing CD10 and CD19 was 28%. However, a CD19-sorted chimerism revealed an almost complete donor chimerism. Donor-lymphocytes were administered to improve graft function. Afterwards, donor chimerism reached 100% and platelets reached normal values, but hematogones continued to exceed 5% in the following specimens. As a result of such cases, bone marrow specimens after allogeneic stem cell transplantation were systematically analyzed for the presence of hematogones. METHODS: Hematogones were analyzed by routine 2-color flow cytometry. Cells coexpressing CD10 and CD19 with lymphocytic light scatter properties were regarded as hematogones. Percentage of cells was determined on the basis on total events. 133 patients undergoing allogeneic stem cell transplantation for AML, ALL, CLL, MM, NHL or aplastic anemia from 2003 to 2008 and surviving more than 60 days after transplantation were included in the analysis. During follow-up, bone marrow specimens were collected 1, 2, 3 and 12 months and in patients with suspected relapse. In total, 446 bone marrow specimens prior (186 specimen) and after (260 specimen) transplantation were collected and reevaluated for the frequencies of hematogones. RESULTS: The frequency of hematogones exceeded 5% in 8 of 186 specimens prior but in 62 of 260 specimens after transplantation (4.3% and 23.8%, respectively; p DISCUSSION: In this study, varying patterns of early B-cell recovery after allogeneic stem cell transplantation were found. Presence of large numbers of hematogones may be misinterpreted as a relapse in patients with B-cell malignancies. Presence of cells coexpressing CD10 and CD19 should be regarded with caution and always be interpreted with moleculargenetic data. The physiological and clinical effects of early B-cell recovery after allogeneic stem cell transplantation remain to be investigated in more detail.

NK-cells have been shown to play a pivotal role in haploidentical hematopoietic cell transplantation (HHCT) for engraftment, GvL effects and to combat infectious complications. Different strategies have been employed to hasten NK-cell recovery after HHCT. Here we compare the immune recovery of 17 patients after CD34 selected HHCT receiving additional adoptive CD3-depleted CD56-enriched NK cells 2 days after HHCT (adoptive NK-cells), with 18 patients receiving CD3/CD19 depleted grafts (CD3/CD19) for HHCT. Transplantations were performed at two different institutions with a median follow-up of >1 year. Conditioning consisted of 12 Gy TBI, thiotepa (10mg/kg), fludarabine (150 mg/m2) and OKT3 (day −4 to +2) in the group receiving CD34 selected grafts and adoptive NK-cell transfusions. All patients in the CD3/CD19 group received conditioning with fludarabine (150–200 mg/m2), thiotepa (10 mg/kg), melphalan (120 mg/m2) and OKT-3 (day −5 to +14). No postgrafting immunosuppression was used in both groups. Seven out of the 17 patients in the adoptive NK-cell group received IL-2 activated NK cells. Median age was 37 years in the adoptive NK-cell group compared to 40 years in the CD3/CD19 group. Diagnoses in the adoptive NK-cell group included AML (n=10), ALL (n=3), CML (n=2), and Hodgkin’s disease (n=1) and MDS (n=1). Diagnoses in the CD3/CD19 group were AML (n=10), ALL (n=5), NHL (n=1), CML (n=1) and multiple myeloma (n=1). The grafts contained a median of 12.5x10E6 CD34+ cells/kg and 1.1×10E4 CD3+ cells/kg in the CD34 selected group versus 9.2×10E6 CD34+cells/kg and 2.3×10E4 CD3+cells/kg in the CD3/CD19 group. The number of transferred CD56+ cells was 8.3×10E6/kg in the adoptive NK-cell group and 7.2×10E7/kg cells in the CD3/CD19 group. Hematopoietic recovery was similar in both groups. Among the patients receiving adoptive NK-cells we observed a striking difference in immune recovery between the patients receiving IL2-activated and those treated with non-activated NK cells: patients receiving activated NK cells showed significantly lower numbers of NK- and T cells during the first months post transplant (p=

Pattern recognition receptors such as Toll-like-receptors (TLR) and NOD proteins bind specifically to microbial components and activate innate immune responses. Reducing the bacterial load by antibiotic treatment has been shown to ameliorate the severity of graft-versus-host-disease (GvHD) after allogenic stem cell transplantation. The impact of bacteria and TLRs on the induction of T-cell-alloreactivity and GvHD was investigated in a murine transplantation model. C57B/10 wild-type (wt) mice served as recipients of MHC-mismatched Balb/c grafts after conditioning with treosulfan and cyclophosphamide. Analyses of the gut microbiota in fecal samples showed a pronounced increase of luminal load of both gram-positive and gram-negative bacteria within 11 days after stem cell transplantation. The increase in the Escherichia coli load was significantly correlated with the severity of graft-versus-host-disease and with decreased survival rates. Analysis of the kinetics of fecal E. coli load and the histologial and clinical GvHD scores revealed that the increase of the fecal E. coli load is detected simultanously to the onset of clinical overt GvHD. Because TLR 2 and 4 represent the major receptors for gram-positive and gram-negative bacteria, respectively, the incidence and severity of GvHD was investigated in TLR2, TLR4 and TLR2/4 deficient mice on a C57B/10 background. The onset of GvHD was delayed in recipient mice lacking TLR2 and both TLR2 and 4 (p

BACKGROUND: Treosulfan is increasingly used in clinical conditioning regimens because of its myeloablative and immunosuppressive effects and the low hepatotoxicity compared to busulfane. The myeloablative and immunosuppressive characteristics of treosulfane was investigated in a murine MHC mismatch transplantation model. METHODS: C57BL/10 (H-2Db) female mice were treated with treosulfan (2000 mg/kg) on day -3 to -1 and increasing doses of cyclophosphamide (without, 100 or 200mg/kg) at day -1, or 3000 mg/kg treosulfan with and without 200 mg/kg cyclophosphamide at day -1. Some mice were not transplanted to examine the myeloablative effect of the regimens, the rest of mice were transplanted with 10 × 10^6 marrow cells from Balb/c mice (H-2Dd) donors and monitored for donor-type chimerism determined by flow cytometry. RESULTS: Mice treated with treosulfan and cyclophosphamid without transplantation died, indicating the myeloablative effect of the regimens. Ca. 50% of mice treated with 3000 mg/kg treosulfan and 200 mg/kg cyclophosphamide at day -1 died. Analysis of bone marrow cells in the remaining mice showed a complete rejection at day 55. Approximately 90% of the recipients of three daily doses of treosulfan and increasing doses of cyclophosphamide survived until the end of experiment (day +100). All hosts engrafted when 200 mg/kg cyclophosphamide was applied. 85% of mice engrafted after 100mg/kg cyclophosphamide. 100% of grafts were rejected in the group with treosulfan alone (2000 mg/kg). CONCLUSION: A chemotherapeutic regimen for achieving a long term survival in a MHC-mismatch situation without additional need of antibodies was established in mice. While investigated for the practicability in an animal model as alternative to TBI, the high rate of engraftments in a MHC mismatch situation makes the chemotherapeutic regimen suitable for clinical situations.

Background: In vivo T cell depletion with ATG or Alemtuzumab is effective to reduce the incidence of graft-versus host disease (GVHD) caused by alloreactive T cells. However, there is also a potential impact of these substances on the function of natural killer (NK) cells who are the predominant cells in peripheral blood in the early phase after hematopoietic stem cell transplantation (HSCT) and mediate beneficial graft-versus-tumor activity. Using a novel flow cytometric assay, which detects the lytic granule membrane protein CD107a as a marker for NK cell degranulation, we investigated the effect of T cell depletion with ATG and Alemtuzumab on NK cell function in the early phase after HSCT. Methods: PBMCs of 34 patients (pts) at day +30 after allogeneic HSCT and of 16 healthy donors were coincubated at 37°C for 3 h with the NK sensitive cell line HL60. In each tube, containing 400μl effector/target suspension (2x106 cells), 20μl of PE-Cy5 conjugated anti-CD107a monoclonal antibody was added prior to incubation. After the first 1 h 10μl of the secretion inhibitor 2 mM monensin was added. At the end of coincubation cells were stained with mAbs (CD56, CD3) for flow cytometry. The percentage of CD107a expressing NK cells was assessed and the absolute number of degranulating NK cells/μl was calculated. Results: Treatment Characteristics: Fourteen pts received ATG, ten pts were treated with Alemtuzumab and ten patients did not receive T cell depletion. The source of donor was: MRD 12 and MUD 22. NK cell count: The median NK cell count was: 250/μl in healthy individuals, 250/μl in pts without T cell depletion, 400/μl in pts with ATG and 100/μl in pts receiving Alemtuzumab (p Conclusion: With a new and feasible method we were able to quantify and characterize tumor reactive NK cells after HSCT. We found that NK cell mediated cytotoxicity towards tumor targets is influenced by the type of T cell depletion: The NK cell activity in patients receiving Alemtuzumab was considerably reduced whereas ATG had only moderate impact on the NK cell activity in the early phase after HSCT.

BACKGROUND: Intestinal Graft-versus-Host disease is a frequent and often lethal complication after allogenic stem cell transplantation. Since NOD2 polymorphisms have been recognized as potential triggers of severe intestinal GvHD in humans, we have developed murine transplantation models to investigate the role of different pattern recognition receptors (PRR) in GvHD and GvL. Here we report our results on the role of TLR2 and TLR4 for the induction of GvHD. METHODS: Severity of GvHD in wildtype (wt) C57B/10 (H-2Db), TLR2−/−, TLR4−/−, and combined TLR2−/−TLR4−/− C57B/10 mice was investigated. Mice received treosulfan 2000 mg/kg from day -3 to -1 and cyclophosphamide 200 mg/kg day -1 prior to injection of 10×10^6 H-2Dd BM cells and 5×10^6 splenocytes (SC). Survival and GvHD score were assessed daily. Engraftment was determined every 2 weeks in pB and at the end of the experiments in bone marrow by flow cytometry. T cell alloreactivity in GvH direction was assessed by MLR using splenocytes as stimulators from PRR-deficient mice or wt as control and CFSE-staining as read-out. The relevance of PRR ligands for the enhancement of GvH alloreactivity was determined by addition of lipid A, lipopetides, or CpG. RESULTS: in vivo data: The transfer of 10×10^6 BMC + 5×10^6 SC induced a severe GvHD in all wt recipients, leading to death of 90% of the animals within 20 days. Recipient mice lacking either TLR2 or TLR4 showed only a slightly and not significantly decreased GvHD lethality. In recipients lacking both PPRs, i.e. TLR2 and TLR4, GvHD was generally milder and the majority (60%) of the animals survived until day 20 (p CONCLUSION: Our in vivo and in vitro data consistantly show that bacterial components are involved in triggering GvH alloreactivity via different types of PPRs. Binding of bacterial substances to TLR2 and TLR4 leads to activation of the immune system and subsequent induction of GvHD. Our data provide an experimental basis for the development of strategies for modulation of the intestinal gut flora by selective gut decontamination and/or probiotic regimens to prevent GvHD in humans.