ALBERT

Description: Background: Minor histocompatibility antigens are antigenic peptides derived from normal cellular molecules which are presented in the context of major histocompatibility antigens (MHC class I and MHC class II). After an allogeneic hematopoietic stem cell transplantation (aHSCT) recipient-derived mHags can be recognized by T-cells of the transplanted immune system an mediate both a graft-versus-host disease and graft-versus- leukemia reaction. All mHags known to date have been identified by allo-reactive T-cells. Due to complex logistics needed for the establishment of such T-cell clones only a few mHags have been molecularly defined to date. Since patients with GvH develop antibodies against mHags and the presence of mHag antibodies has been shown to correlate with GvH and maintenance of remission [Miklos et al. (2005) Blood 105:2973-9] we set out to identify new mHags using SEREX the serological identification of antigens using recombinant expression cloning [Sahin et al. (1995) PNAS 92:11810-3]. Methods: Fibroblasts obtained by skin biopsies from patients with chronic GvH were propagated in vivo and used as a source to establish a cDNA library. cDNA was expressed by lambda phage in E.coli and expressed clones were screened for reactivity with antibodies in the serum of patients with chronic GvH. Positive clones were monoclonized and sequenced. The sequences were compared with known sequences using BLAST data bank. Reactive sera were compared with patient’s pre transplant serum and the serum from the donor to exclude pre-existing antibodies against (auto-)antigens expressed by the fibroblasts. Only neo-antigens recognized by the transplanted immune system were analyzed further. Results: cDNA libraries obtained from 7 patients with chronic GvH after HLA-identical stem cell transplantation were screened with the sera of these 7 patients by SEREX. Antibodies from one patient with chronic GvH were absent in the donor and the patients pre-transplant serum, reacted with c1ORF107. This constellation proves that the c1ORF107 antibodies in the patient’s serum developed after transplant and recognized c1ORF as a neo-antigen. The immunogenic c1ORF107 of the patient differed at previously described polymorphic position 275 (rs585627) with G in the recipient leading to glutamate and C in the donor leading to glutamine. The definition of the c1ORF107 epitopes eliciting a T-cell response by “reverse T-cell immunology” is currently underway. Conclusion: This study proves the principle that the analysis of the humoral immune response in patients with chronic GvH by SEREX allows for the definition of new mHags. SEREX in combination and followed by reverse T-cell immunology is a straight-forward approach which will expand considerably the number of molecularly defined mHags causing GvH in patients after allogeneic stem cell transplantation. Supported by Jose Carreras Foundation Germany

Description: Introduction. Allogeneic hematopoietic cell transplant (HCT) is an established treatment modality that is potentially curative for many patients with acute myeloid leukemia (AML). The development of reduced intensity conditioning (RIC) allows performing HCT in elderly and/or in heavily pretreated patients and in those with comorbidities precluding the use of standard myeloablative conditioning. Post-transplant relapse remains a challenge after RIC, particularly in patients with adverse prognosis factors.The so-called "sequential" transplant approach (e.g. FLAMSA regimen combining both intensive chemotherapy and RIC HCT within the same procedure) initially developed in patients with refractory AML, could be a promising strategy to improve disease control and decrease the risk of relapse in high-risk AML patients in complete remission (CR). Patients and methods. In the current study we analyzed transplantation outcomes in a cohort of 411 adults AML patients in CR at time of transplant, treated between 2002 and 2013. Patients received a "sequential" conditioning regimen based on Fludarabine 30 mg/m2/d, high-dose aracytine 1-2 g/m2/d, amsacrine 100 mg/m2/d for 5 days and after a 3 days rest, total body irradiation (TBI) 4Gy, cyclophosphamide 50-120 mg/kg, and antithymocyte globulin (ATG) for 2 to 3 days (TBI group, n=269 [65%]). In 142 (35%) patients, TBI was substituted by IV Busulfan 3.2 mg/kg/d for 2 days, or orally equivalent dose (Bu group). 323 patients (79%) had de-novo AML and 88 (21%) had a secondary AML (with prior myelodysplastic syndrome). At time of transplant, 300 (73%) patients were in CR1 and 111 (27%) in CR2. Cytogenetic study in de novo AML was favorable in 19 patients (6%), intermediate in 102 (32%) and poor in 41 (13%). Cytogenetic data were missing in 161 (50%). 104 (25%) patients received matched related donors (MRD) and 307 (75%) unrelated donor (URD) HCT. Majority of patients (94%) received mobilized peripheral blood stem cells graft. Results. Median follow-up of surviving patients was 28 months and median age at transplant was 54 years (18-76). ANC〉500/μL was achieved at a median of 17 (range, 9-74) days after HCT. Sixteen patients (4%) failed to engraft. Two year cumulative incidence of relapse (RI) and non-relapse mortality (NRM) were 22% (95%CI, 18-26%) and 22% (95%CI, 18-27%), respectively. The Kaplan-Meier estimate of overall (OS) and leukemia-free survival (LFS) at 2 years were 59% (95%CI, 54-65%) and 56% (95%CI, 50-61%), respectively. Acute GVHD (grade II-IV) occurred in 109 (28%) patients. The 2-year cumulative incidence of chronic GVHD was 31% (95%CI, 26-36), extensive in 17% (95%CI, 12-21). Two years RI, NRM, LFS and OS in TBI vs. Bu patients were 21.8% vs 21.7% (p=0.69), 29.4% vs 18.3% (p=0.008), 48.8% vs 59.6% (p=0.045) and 51.2% vs 64.0% (p=0.013), respectively. In multivariate analysis adjusted for variable with different distribution between Bu and TBI groups, the type of conditioning (TBI vs Bu) has no impact on RI, NRM, LFS and OS. Age over 55 at transplant was an independent adverse prognostic factor in multivariate analysis for NRM (hazard ratio (HR: 1.61, 95% CI: 1.00-2.61, p=0.05)), LFS (HR: 1.39, 95% CI: 1.00-1.92, p=0.05) and OS (HR: 1.55, 95% CI: 1.11-2.18, p=0.01). Being treated in an experienced center (defined as having including 10 or more transplants in the study) was associated with a significant lower RI (HR: 0.84, 95% CI: 0.75-0.93, p=0.001) and better LFS (HR: 0.91, 95% CI: 0.84-0.98, p=0.01) and OS (HR: 0.91, 95% CI: 0.84-0.98, p=0.02). Finally, transplantation from an URD was associated with a significant increase in NRM (HR: 2.11, 95% CI: 1.14-3.91, p=0.02). Of note, CR1 vs. CR2 and de novo vs. secondary AML had no impact on patients' outcome. Conclusions. These results in a rather large cohort of patients with AML suggest that a FLAMSA "sequential" regimen provided an efficient disease control in high-risk AML patients including in CR2 and secondary AML. Furthermore Busulfan and TBI based FLAMSA "sequential" regimens provide a similar outcome. These results should be confirmed in a multicenter well design randomized study. Disclosures Off Label Use: off-label drug use: antithymocyte globulin (ATG) for allo-SCT conditioning. Tischer:Sanofi-Aventis: Other: advisory board. Schmid:Neovii: Consultancy; Janssen Cilag: Other: Travel grand. Mayer:Janssen: Research Funding. Hallek:Pharmacyclics: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; Janssen: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; Boehringher Ingelheim: Honoraria, Other: Speakers Bureau and/or Advisory Boards; Mundipharma: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; Celgene: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; Gilead: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; Roche: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; AbbVie: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding.

Description: Background: Vitamin D3 deficiency impairs the rituximab-mediated antibody-dependent cellular cytotoxicity (ADCC) and the outcome of elderly patients with diffuse large B-cell lymphoma (Bittenbring et al., J Clin Oncol. 2014 Oct 10; 32(29):3242-8) treated with R-CHOP and of patients with follicular lymphoma (Kelly et al., J Clin Oncol. 2015 May 1; 33(1):1482-90. The aim of this study was to determine the optimal 25-OH-vitamin D3 (VD3) level for rituximab- and obinutuzumab-mediated ADCC. Methods: Ten individuals (5 males, 5 females; mean age: 67.7 years, range 41-79) without malignant disease or immunosuppression were included in this study after written informed consent. PBMC were isolated by density gradient centrifugation. CD16+ NK cells were separated by depleting all non-NK-cells magnetically. ADCC activity of the NK cells was tested against CD20-expressing Daudi cells without or after anti-CD20-antibody treatment with rituximab and obinutuzumab, respectively. Cytotoxic activity was assessed by LDH release by the target cells. NK cells were studied at four different VD3 serum levels: 1 (insufficient supply: 〈 20 ng/ml); 2 (lower normal range; 30 ng/ml), 3 (mid normal range; 65 ng/ml), and 4 (high normal range; 90 ng/ml). To achieve these levels the probands were substituted with cholecalciferol. Results: The median VD3 serum level before substitution was 10 ng/ml. The only formula for achieving predefined VD3 serum levels published by van Groningen et al. (Eur J Endocrinol. 2010; 162:805-11) failed to achieve the predetermined VD3 levels by far. Modifying this formula by using a multiplication factor of 1.8-2.2 we achieved the preset dose levels with high precision with median levels of 32.6 ng/ml, 66.4 ng/ml and 92.3 ng/ml aiming at dose levels of 30, 65 and 90 ng/ml, respectively. 2/10 test persons had starting VD3 levels above 30 ng/ml, but 6 of the remaining 8 individuals showed a significantly increased ADCC after VD3 substitution to level 2 (ranging from + 10 % to + 147 % increased Daudi lysis, p 〈 0.05). Further substitution to 65 ng/ml significantly increased ADCC activity in all 10 persons compared to the ADCC at 30 ng/ml (ranging from + 18.4 % to + 89.2 % increased Daudi lysis, p 〈 0.05). 8/10 individuals were further substituted to achieve 90 ng/ml. However, in 7/8 of these probands the NK-cell-mediated ADCC was significantly reduced compared to their values at 65 ng/ml (ranging from - 23.1 % to - 58.1 % decreased Daudi lysis, p 〈 0.05). The extent of the substitution-induced ADCC improvement varied individually and depended on the antibody concentration used to treat the target cells. Rituximab-mediated ADCC was significantly more affected by VD3 levels than obinutuzumab. Conclusion: Our study demonstrates for the first time that the ADCC of NK cells is optimal at median VD3 serum levels around 65 ng/ml. Our data strongly argue for a rapid vitamin D3 substitution before/at the beginning of R-CHOP treatment using the modified van Groningen formula. 65 ng/ml was chosen as the target VD3 level in the ongoing OPTIMAL〉60 study of the German High-Grade Non-Hodgkin Lymphoma Study Group (DSHNHL) in elderly patients with DLBCL. This study is supported by the Eva Mayr-Stihl-Stiftung (Waiblingen, Germany). Disclosures Pfreundschuh: Roche, Janssen, Celgene: Honoraria, Research Funding.

Description: Multiple myeloma (MM) is considered a chronic and incurable disease due to its highly complex and heterogeneous molecular abnormalities and the support from myeloma microenvironment factors. Macrophages are an abundant component of the stromal cell compartment and are believed to support proliferation, survival, and drug resistance of MM cells. Conversely, macrophages are key immune effector cells for the therapeutic effect of monoclonal antibodies and can directly eliminate tumor cells. However, myeloma-associated macrophages (MAMs) regularly fail to exert direct effector functions. Given their abundance in MM, an attractive therapeutic approach would be to stimulate their tumoricidal activity in order to promote antitumor immunity. Lenalidomide, an immunomodulatory agent that enhances antibody dependent cell mediated cytotoxicity (ADCC), has the potential to synergize with MOR202, an anti-CD38 monoclonal IgG1 antibody currently in phase I/IIa for the treatment of MM. Furthermore, vitamin D plays a key role in regulating effector functions of human macrophages. This is closely linked to the expression of the vitamin D-1-hydroxylase CYP27B1, which catalyzes the conversion of 25-hydroxy-vitamin D (25D) to the bioactive form 1,25-di-hydroxy-vitamin D (1,25D). We have previously shown, that vitamin D promotes tumoricidal activity of macrophages and improves the efficacy of rituximab-dependent cytotoxicity (Bruns et al., Sci. Transl. Med., 2015; Bittenbring et al., JCO, 2014). Therefore, we hypothesized that the combination of MOR202 with lenalidomide and MOR202 with 1,25D would enhance the MOR202- dependent macrophage-mediated effector functions against myeloma cells. Here we report that MAMs exhibit an altered vitamin D metabolism with a reduced expression of the vitamin D receptor (VDR) and CYP27B1, while the expression of CYP24A1, which catabolizes 1,25D, is increased. As a consequence MAMs cannot convert 25D into bioactive 1,25D. Given the importance of the vitamin D pathway for antibody mediated cytotoxicity, we screened several drugs for their ability to restore the vitamin D pathway in human macrophages. We found, by RNA-sequencing, that lenalidomide treatment modulates the phenotype of monocyte-derived macrophages and isolated MAMs, and that lenalidomide significantly increases the expression of the VDR, CYP27B1 and CAMP (cathelicidin antimicrobial peptide) in these macrophages. Furthermore, we demonstrate that isolated MAMs regularly fail to eliminate primary myeloma cells, and that the lack of effector functions can be overcome by treatment with lenalidomide and vitamin D. Moreover, we show that MOR202-dependent elimination of myeloma cells is enhanced by pre-treatment of isolated MAMs with lenalidomide and supplementation of vitamin D. In summary, these data show that the bioactive form of vitamin D (1,25D) is essential for the effector functions of MAMs and that the therapeutic activation of the vitamin D pathway by lenalidomide may restore their tumoricidal effector mechanisms. Furthermore, these findings highlight that stimulating the tumoricidal activity of MAMs, an abundant component of the stromal cell compartment, with lenalidomide and 1,25D is an attractive therapeutic approach in combination with the anti-CD38 monoclonal IgG1 antibody MOR202. Disclosures Bruns: Morphosys: Research Funding; Celgene: Research Funding.

Description: Introduction Relapse of non-Hodgkin lymphoma (NHL) following allogeneic stem cell transplantation (alloSCT) is a common occurrence associated with a poor outcome with limited treatment options. Donor lymphocyte infusions (DLI) are commonly employed in this setting in an attempt to exploit the allogeneic graft versus lymphoma (GVL) effect to induce remissions. There is however a paucity of data describing the efficacy of DLIs in inducing remissions in NHL subtypes and the risk of subsequently developing graft versus host disease (GVHD). We report here the largest series of patients with NHL receiving DLI for relapse after alloSCT. Methods Patients relapsing after an alloSCT for NHL and receiving DLI were identified on the EBMT database. Centres were invited to contribute additional data. 118 patients [follicular lymphoma (FL) n=28, diffuse large B cell lymphoma (DLBCL) n=28, T cell lymphoma (TCL) n=52 and mantle cell lymphoma (MCL) n=10] from 39 centres who received DLI as the only treatment for relapse were identified. Patients receiving DLI in combination with other therapy were excluded from this analysis. The median age at alloSCT was 50 (range 18-73) years. There were 81 male and 37 female patients who underwent an alloSCT with reduced intensity (RIC) (n=90) or myeloablative conditioning (MAC) (n=28) at a median of 2.4 (range 0.3-26.3) years from diagnosis. Allogeneic cells were provided from matched sibling (n=63), unrelated (n=47) or mismatched family (n=8) donors and 85 patients received either ATG/ALG or CAMPATH as part of GVHD prophylaxis. The median time from alloSCT to relapse was 3.8 months (range 18 days-67 months). Results Patients received a median of 1 (range 1-19) DLI at a median starting dose of 1.5 x106 CD3/kg (range 0.01-120x106 CD3/kg) at a median of 152 days (range 18-2136) post alloSCT. The median last dose of DLI was 10x106/kg (range 0.1-180x106/kg) given at a median of 353 days (range 41-2725) post alloSCT. The median time from relapse to the first DLI was 35 days (range 0-168). Acute GVHD and chronic GVHD prior to DLI was reported in 29% and 14% of patients, respectively. Of 93 evaluable patients 47 (51%) patients achieved a complete remission, 10 (11%) a partial remission, 13 (14%) stable disease and 23 (25%) had progressive disease following DLI. The median duration of response was 36 months (range 1-168). When analysed according to histology the overall response rate (ORR) (CR+PR) for FL was 84% (CR 68%), DLBCL 41% (32% CR), TCL 54% (46% CR) and MCL 86% (CR 71%). The median duration of responses for FL, DLBCL, TCL and MCL were 30 (range 2-150), 38 (4-76), 37 (range 1-168) and 50 months (13-116) respectively. With a median follow up of 77 months after the 1st DLI 36 (31%) patients remain in complete remission, 29 (25%) without any further therapy. 37 (35%) went on to receive additional antilymphoma therapy after the DLI. Of 17 patients with FL achieving a CR post DLI 12 (71%) remain in remission without further therapy at a median of 78 (9-158) months after DLI. Of 18 patients with TCL that achieved a CR with DLI, 15 (83%) remain in remission without further therapy at a median of 95 (range 42-161) months after DLI. Following the DLI 43 (36%) patients developed aGVHD (6 grade I, 13 grade II, 11 grade III, 9 grade IV, 4 grade unknown) and 33 (28%) developed cGVHD (11 limited, 20 extensive, 2 unknown). Following the first DLI the cumulative incidence of relapse was 31% (CI 22-41) at 4 years and of NRM 28% (CI 20-37). The 4-year PFS after 1st DLI was 39% (CI 30-50) and the OS, 44% (CI 35-54). Conclusions DLI induce significant rates of disease response in patients with NHL relapsing after alloSCT providing clear proof of principle of the allogeneic GVL effect. The rates of response are most impressive in FL and MCL. The majority of patients with TCL and FL that achieve a CR post DLI remain in remission with long term follow up. Acute and chronic GVHD is a significant complication of DLI. Disclosures Robinson: Sandoz: Speakers Bureau; Gilead: Honoraria, Speakers Bureau; Takeda: Consultancy, Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Speakers Bureau. Chalandon:Roche: Membership on an entity's Board of Directors or advisory committees, Other: Travel costs. Beelen:Medac: Consultancy, Other: Travel Support. Finke:Neovii: Consultancy, Honoraria, Other: travel grants, Research Funding; Medac: Consultancy, Honoraria, Other: travel grants, Research Funding; Riemser: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Other: travel grants, Research Funding. Tischer:Jazz Pharmaceuticals: Other: Jazz Advisory Board. Corradini:Amgen: Honoraria, Other: Advisory Board & Lecturer; Novartis: Honoraria, Other: Advisory Board & Lecturer; Abbvie: Honoraria, Other: Advisory Board & Lecturer; Sandoz: Other: Advisory Board; Sanofi: Honoraria, Other: Advisory Board & Lecturer; Gilead: Honoraria, Other: Advisory Board & Lecturer; Takeda: Honoraria, Other: Advisory Board & Lecturer; Roche: Honoraria, Other: Advisory Board & Lecturer; Janssen: Honoraria, Other: Lecturer; Celgene: Honoraria, Other: Advisory Board & Lecturer.

Description: Introduction: We recently showed that vitamin D deficiency leads to decreased overall survival of DLBCL-patients treated with rituximab-chemotherapy (Bittenbring et al, JCO, 2014). We hypothesized that rituximab-mediated NK cell-cytotoxicity is more effective at higher vitamin D levels. This was confirmed by vitamin D substitution of healthy volunteers, which increased their rituximab-mediated cytotoxicity in vitro against the Daudi lymphoma cell line. To unveil the molecular mechanisms behind this finding, resting NK cells before and after vitamin D supplementation were isolated from those volunteers and a whole transcriptome analysis was performed. Methods: We collected PBMCs from eight healthy volunteers with vitamin D deficiency before and after vitamin D substitution to 〉 30 ng/ml 25-OH vitamin D3. NK cells were isolated from PBMCs by magnetic depletion of all non-NK cells. Purity of the CD16+ cells was confirmed by flow cytometry. After isolating total RNA, we performed a microarray analysis using an Affymetrix Gene-Chip 2.0 ™. The signals were normalized using the LMA algorithm. For pathway analysis, gene set enrichment analysis (GSEA) was used. A two-step approach was chosen. Firstly, we separated 7.705 genes due to their involvement in the NK cell-mediated immune response according to the Gene Ontology database, irrespective of their differential expression. This dataset was used separately for specific analysis of the NK cell-cytotoxicity pathway to increase sensitivity. Secondly, the complete data set of 48.145 genes was used in an exploratory analysis in an attempt to screen for other dysregulated pathways involved in the immune response and vitamin D homeostasis. We used gene sets provided from the Molecular Signature Database. A significance level of 〈 0.05 for p and False Discovery Rate (FDR) was chosen. Real-time quantitative PCR was performed to confirm the results. Results: The NK cell-associated cytotoxicity pathway was found to be significantly upregulated after restoration of normal vitamin D levels in the specific analysis. The most significantly overexpressed genes in the gene set were five IFN-α subtypes (IFN-α2, IFN-α4, IFN-α6, IFN-α7, and IFN-α10) as well as IFN-κ. The exploratory analysis showed an upregulation of the response to type I interferon pathway and regulation of type I interferon mediated signaling pathway. The most upregulated genes in those pathways were again the IFN-α subtypes mentioned above. Other pathways involved in the immune response were found to be downregulated after vitamin D substitution, like interferon gamma response; cytokine production and chemotaxis. The common denominator of these pathways was the downregulation of three toll-like receptor genes (TLR-8, TLR-7, TLR-2). Conclusion: The increased expression of specific IFN-α subtypes could explain the increased rituximab-mediated NK cell-cytotoxicity after vitamin D substitution in deficient individuals. To the best of our knowledge, this is the first study to suggest a role for vitamin D in IFN-α regulation. TLRs are known to stimulate cytokine production in NK cells including IFN-α. It can be assumed, that the observed upregulation of IFN-α genes after vitamin D substitution leads to a negative feedback on positive regulators of cytokine production like TLR, causing their downregulation once vitamin D levels are restored. This implies a comprehensive role of vitamin D in IFN-α biosynthesis in human NK cells. Disclosures Stilgenbauer: AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Hoffmann La-Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Mundipharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmcyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Boehringer-Ingelheim: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genzyme: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Description: Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin's lymphoma. Approximately 60% of DLBCL patients can be cured using standard chemotherapy, including an anti-CD20 antibody (R-CHOP; rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone). However, 40% of DLBCL patients have refractory disease or experience relapse. This highlights the need for more effective therapies for this subset of relapsing or refractory patients. Tafasitamab (MOR208) is an Fc-enhanced, humanized, monoclonal anti-CD19 antibody, currently in clinical development in patients with relapsed or refractory (r/r) DLBCL in combination with either the immunomodulatory drug lenalidomide (LEN) (L-MIND study) or the chemotherapeutic agent bendamustine (B-MIND study). The B-lymphocyte antigen CD19 is broadly and homogeneously expressed throughout B-cell development and across different B-cell malignancies, including DLBCL. CD19 has been reported to enhance B-cell receptor signaling, which is assumed important for B-cell survival. This makes CD19 a promising therapeutic target in B-cell malignancies. Macrophages are abundant in the bone marrow stroma and lymph nodes of lymphoma patients. These lymphoma-associated macrophages (LAMs) support proliferation, survival, and drug resistance of lymphoma cells; therefore, serving a tumor-promoting function (Farinha et al, Blood, 2005). However, macrophages can also act as immune effector cells for antitumor effects exerted by therapeutic monoclonal antibodies, like tafasitamab. Given their abundance in DLBCL, an attractive therapeutic approach would be to stimulate their tumoricidal activity in order to promote antitumor immunity. Consequently, the immunomodulatory agent LEN has the potential to synergize with tafasitamab by enhancing antibody dependent cellular phagocytosis (ADCP). Here, we demonstrate that monocyte-derived macrophages and isolated LAMs from DLBCL patients can kill lymphoma cell lines and autologous lymphoma cells by tafasitamab-mediated phagocytosis. Tafasitamab-mediated ADCP activity was correlated with FcγRIII/FcγRI levels and inversely correlated with SIRPα expression on macrophage/effector cells (Spearman r between 0.51 and 0.62). Analyzing immune checkpoint receptor expression on LAMs of DLBCL patients, we found increased expression of VISTA, SLAMF7 and SIRPα compared with normal macrophages from healthy volunteers (VISTA: 2.8-fold, p=0.0006; SLAMF7: 2.9-fold, p=0.03; SIRPα: 1.8-fold, p=0.04). In contrast, we found no significant difference in FcγR expression (CD16: 1.4-fold, p=0.12; CD32: 1.3-fold, p=0.32; CD64: 1.2-fold, p=0.12). Moreover, pretreatment with lenalidomide showed a 3 to 5-fold enhanced tafasitamab-mediated cytotoxicity by macrophages and isolated LAMs (Figure 1A). Of note, the increased cytotoxicity was tafasitamab-specific and neither attributable to direct cytotoxic effects of LEN, nor increased CD19 expression on target cells. In addition, LEN treatment decreased the expression of SIRPα on macrophages (control vs. LEN: 3,117±652 MFI [mean fluorescent intensity] vs 2,327±610 MFI) (Figure 1B). This suggests that the CD47-SIRPα pathway may be suppressed by downregulation of SIRPα by LEN, contributing to the observed ADCP enhancement. These results suggest that tafasitamab leverages the presence of tumor-promoting LAMs as effector cells. The inherent ADCP activity of tafasitamab can be further improved by the immunomodulatory agent LEN, potentially via reduction of SIRPα levels on macrophages. Finally, our results provide a rationale for targeting the CD47-SIRPα axis that should be further explored in preclinical and clinical studies. Figure 1 Disclosures Endell: MorphoSys AG: Employment, Patents & Royalties. Boxhammer:MorphoSys AG: Employment, Patents & Royalties. Bruns:Morphosys AG: Research Funding.

Description: In addition to antimicrobial activity, macrophages regulate tissue development, remodeling, and repair. In terms of neoplastic growth, tumor-associated macrophages (TAMs) are even considered pro-tumorigenic based on their angiogenic and T-cell suppressive properties. In multiple myeloma (MM) macrophages represent an abundant component of the stromal cell compartment and are believed to support proliferation, survival, and drug resistance of MM cells. Those pro-tumorigenic functions are partly instructed by T-helper (TH)-2 cytokines such as interleukin-4 (IL-4) and IL-10, which skew TAM differentiation towards an alternatively activated, or so-called M2-like, phenotype. In contrast, M1 macrophages generally considered as potent effectors in response to microbial products or interferon-γ (IFN-γ), are characterized by superior antigen-presentation, abundant production of pro-inflammatory cytokines such as interleukin-12 (IL-12), and consequently, promote a polarized type I immune response against infections as well as malignant cells. Transcriptional regulation is a key determinant for macrophage polarization. The Ikaros (IKZF1) transcription factor is critical for lymphoid development and is found in all hematopoietic progenitors as well as in T-, B-, NK-cells and macrophages. Interestingly, IKZF1 is overexpressed in MM and selectively degraded by lenalidomide, which is approved in MM therapy. The potential role of IKZF1 in modulating macrophage polarization has not been elucidated yet. Here, we show that IKZF1expression is found highly elevated in M2-like macrophages and in MM TAMs. IKZF1 deletion in human macrophages by small interfering RNA (siRNA) or by lenalidomide yields an upregulation of M1-specific cytokines (IL-12 and IL-1b), chemokines (CXCL10 and CCL5), and costimulatory molecules (CD86 and CD40) and leads to a potent TH1-TH17 response. In fact, lenalidomide-pretreated macrophages display strong tumoricidal effects when co-cultured with MM cell lines as opposed to their untreated counterparts promoting MM proliferation and viability. Utilizing immunoprecipitation-sequencing (ChIP-Seq) we reveal that IKZF1 governs IRF4 and IRF5 expression in human macrophages. Recent studies demonstrate that IRF4 controls M2 macrophage polarization, while IRF5 regulates the M1 phenotype respectively. We could show that a lenalidomid-mediated M1-phenotype induction is efficiently abrogated by IRF4 overexpression or IRF5 silencing. Overall, these findings unravel a novel role for IKZF1 in orchestrating macrophage polarization via the IRF4/IRF5 pathway. Disclosures No relevant conflicts of interest to declare.

Description: PURPOSE of this analysis was to provide 10-year follow-up of the GCLLSG CLL3X trial which aimed at evaluating reduced-intensity conditioning (RIC) HSCT in patients with poor-risk CLL. DESIGN: The CLL3X trial included 100 patients (median age 53 (27-65) years), of whom 90 patients were allografted with blood stem cells from related (40%) or unrelated donors (60%) using fludarabine-alkylator-based RIC regimens. 24% had refractory CLL at HSCT, and 37% had a TP53 deletion and/or mutation. The 6-year follow-up of the trial including the observation that genetic risk factors such as TP53 lesions and SF3B1 and NOTCH1 mutations had no prognostic impact has been previously reported (Blood 2013;119:4851). Survival and relapse information was requested for all patients who underwent HSCT within the CLL3X trial in 9 German centres (the Canadian centre was unavailable for follow-up) and were alive at the 6-year follow-up. RESULTS: Follow-up information was received for 33/44 patients (75%) alive at the 6-year follow-up. Of these, 2 patients had experienced non-relapse mortality (NRM; 1 chronic GVHD, 1 secondary cancer) and another 2 had CLL recurrence 9.3 and 9.7 years post HSCT, respectively. With a median follow-up of survivors of 9.3 (0.6-15.5) years, 10-year NRM, relapse incidence (REL) event-free survival (EFS), and overall survival (OS) of all 90 patients allografted was 26%, 57%, 32%, and 51%, respectively. Absence of minimal residual disease (MRD) at the 12-month landmark post HSCT was highly predictive for an increased relapse risk (10-year REL 25% vs 80% if MRD was present at the 12-month landmark, p

Description: Introduction: Allogeneic hematopoietic stem cell transplantation (HCT) is a treatment for CLL that can give long disease control. Even with the availability of kinase and BCL2 inhibitors, HCT is still performed in fit patients (pts) with high-risk CLL. Almost exclusively, outcomes on matched related and unrelated donor transplantations in CLL have been published. Recently, mismatched related donors are gaining interest because of the better outcome of haploidentical HCT with post-transplantation cyclophosphamide (PTCY). Methods: All pts with CLL who received a first allogeneic HCT with a mismatched related donor and whose data were available in the EBMT registry were analyzed. Median values and ranges are reported for continuous variables and percentages for categorical variables. The probabilities of overall survival (OS) and progression-free survival (PFS) were calculated using the Kaplan-Meier method and the log-rank test for univariate comparisons. Relapse/progression and nonrelapse mortality (NRM) were analyzed together in a competing risk framework. Statistical analyses were performed using SPSS and R. Results: One-hundred-seventeen pts with CLL (74% males) underwent a mismatched related donor transplantation between 1984 and 2015 (1984-1999: 10, 2000-2004: 18, 2005-2009: 23, 2010-2016: 66). Median follow-up after HCT was 8 months (range 0-187 months). Median age at transplantation was 54 years (yrs) (range 27-71 yrs). Median time from diagnosis to HCT was 67 months (range 4-207 months). Eighteen pts (17%) had previously undergone autologous stem cell transplantation (ASCT). Disease status at HCT was CR in 16% of pts, PR in 39% and SD/PD in 45%. The Karnofsky score was known for 98 pts; 96% had a score of 70% or more at the time of HCT. Fifty-eight percent of pts received reduced-intensity conditioning, 42% myeloablative conditioning. Peripheral blood stem cells were used in 68% of pts, bone marrow in 32%. The HCT was sex matched in 41% of recipient-donor pairs. The relationship of the donor to the patient was known for 34 pts; in 53% the donor was a child, in 38% a sibling and in 6% a parent. Forty pts (38%) received PTCY as GVHD prophylaxis. In the other 77 pts various methods of T-cell depletion (TCD) were used, but not all methods were specified. At least 56% of those pts had in vivo TCD. For the whole cohort of pts OS at 2 and 5 yrs was 46% and 37%, respectively. PFS at 2 and 5 yrs was 38% and 30%, respectively. The use of PTCY did not have a significant impact on OS (49% vs. 42% at 2 yrs, 44% vs. 33% at 5 yrs, p=0.35) and PFS (45% vs. 31% at 2 yrs, 40% vs. 22% at 5 yrs, p=0.15). CI of NRM in the whole group at 2 and 5 yrs were 41% and 45%, respectively. CI of relapse at 2 and 5 yrs were 21% and 25%, respectively. The CI of NRM and relapse at 2 and 5 yrs were not statistically different in pts who received PTCY compared to other types of TCD (NRM: 38% vs. 45% at 2 yrs, 43% vs. 49% at 5 yrs, p=0.45; relapse: 17% vs. 25% at 2 yrs, 17% vs. 29% at 5 yrs, p=0.33). For the whole cohort, the incidence of acute graft-versus-host disease (aGVHD) at 100 days was 34% for grade II-IV and 16% for grade III-IV with a median time of onset of 23 days (range 4-57 days). Conclusions: Mismatched related donor HCT resulted in a 5-year PFS in 30% of the pts. This result seems only slightly inferior to matched donor transplant (5 yrs PFS 37%1). NRM was higher than expected in this cohort, but comparable to other studies on haploSCT with in vivo T-cell depleted grafts. In conclusion, a mismatched related donor HCT may be considered for high-risk chemoimmunotherapy-refractory or 17p deleted/TP53 mutated CLL pts without options for kinase and BCL2 inhibitor therapy. More data are needed to assess the value of PTCY for GVHD prophylaxis in this specific context. References: 1. Schetelig J, de Wreede L, Moreno C, et al. Risk factors for adverse outcome in patients with Chronic Lymphocytic Leukemia (CLL) undergoing Allogeneic Hematopoietic Cell transplantation (alloSCT): a Retrospective EBMT Analysis. Abstract WP024, EBMT meeting 2015. Figure 1 Figure 1. Disclosures Ciceri: MolMed SpA: Consultancy. Foà:Ariad: Speakers Bureau; Pfizer: Speakers Bureau; BMS: Consultancy; Celgene: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Gilead: Consultancy, Speakers Bureau; Janssen-Cilag: Consultancy, Speakers Bureau; Genetech: Consultancy; Roche: Consultancy, Speakers Bureau. Hallek:Mundipharma: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; F. Hoffmann-LaRoche: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; AbbVie: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; Janssen-Cilag: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; Gilead: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau. Schetelig:Sanofi: Honoraria. Kröger:Sanofi: Honoraria, Research Funding; Neovii: Honoraria, Research Funding; Riemser: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.