Abstract: Pediatric ALL relapses after allogeneic stem cell transplantation display highly diverse, dynamic and patient-individual genetic lesions. Nine of 10 relapsing pediatric transplant recipients present with genetic alterations for which novel targeted therapies are available.

Abstract: Introduction: Proteasome inhibition is the backbone of various Multiple Myeloma (MM) treatment regimens, leading to durable responses and high quality remissions. However, under prolonged therapy patients eventually develop drug resistance and the underlying mechanisms have been poorly understood. Proteasome inhibitor resistant cell lines were generated by continuous exposure to proteasome inhibiting drugs. In these cell line models a number of variants within the PSMB5 gene were observed. This includes single point mutations, leading to conformational changes of the _5 subunit within the 20S proteolytic core of the proteasome, impairing its chymotryptic catalytic function and the binding of inhibitory drugs. However, no PSMB5 mutations could, so far, be identified in human disease, leaving the functional relevance of such mutations to be determined. Methods: We applied the MM Mutation Panel (M3P) and used the Personal Genome Machine (Life Technologies) to sequence CD138+ purified bone marrow plasma cells and peripheral blood germ line control from a MM patient in third relapse that had been previously treated by various proteasome inhibitor containing therapies (VTD-PACE, VCD, PAD-Rev). This patient was subsequently treated with a pomalidomide, bortezomib, adriamycin and dexamethasone combination therapy (Pom-PAD) and achieved a partial remission after 4 cycles of therapy. When being exposed to VCD at earlier relapse a complete remission was induced within 6 cycles of therapy, demonstrating excellent response to proteasome inhibition at this earlier disease stage. Results: We identified clonal missense mutations in this patient at known NRAS hotspot location (p.Gln61Arg) and in MAX (p.Arg35His). Most notably we found and validated four subclonal single point mutations in PSMB5, each of them in subclonal frequencies with variant reads (VR) ranging from 1.9%- 5.9% (average read depth 750X). All PSMB5 mutations occurred in a highly conserved region in exon 2 of the gene (p.Cys122Tyr, p.Met104Ile, p.Ala86Pro, p.Ala79Thr), with three of them being located within the proteasome inhibitor drug binding site. Our 400bp amplicon design allowed us to observe that each mutation identified in PSMB5 is exclusively present on a different sequencing read and no reads are shared between the mutations. This implies that the mutations are present on different subclones of the tumor, which means that, despite low VR frequencies of the single mutation, in sum more than a quarter of the whole tumor might be affected by mutated PSMB5. At a disease stage when the patient was well responsive to proteasome inhibitor treatment (at diagnosis and at first relapse), of note, none of the mutations in PSMB5 is detectable. Conclusion: Here we report the first in human PSMB5 mutation in a MM patient. These mutations evolved in parallel within different subclones of the disease under the selective pressure of bortezomib- containing treatment regimens, representing branching evolution. It is to speculate whether previous investigations negating the existence of PSMB5 mutations in proteasome inhibitor treated patients might not have sequenced deep enough or did set up a sensitivity cut-off too rigid to detect such subclonal mutations. Our finding suggests that the mutations identified may contribute to the development of proteasome inhibitor resistance, emphasizing the need for more detailed genomic characterization of tumors, including minor subclonal mutation detection. Functional analysis of the mutations is ongoing and results will be presented at the meeting. Figure Figure. Disclosures No relevant conflicts of interest to declare.

Abstract: We recently reported a truncating deletion in the NFKBIE gene, which encodes IκBε, a negative feedback regulator of NF-κB, in clinically aggressive chronic lymphocytic leukemia (CLL). Because preliminary data indicate enrichment of NFKBIE aberrations in other lymphoid malignancies, we screened a large patient cohort (n = 1460) diagnosed with different lymphoid neoplasms. While NFKBIE deletions were infrequent in follicular lymphoma, splenic marginal zone lymphoma, and T-cell acute lymphoblastic leukemia ( & lt;2%), slightly higher frequencies were seen in diffuse large B-cell lymphoma, mantle cell lymphoma, and primary central nervous system lymphoma (3% to 4%). In contrast, a remarkably high frequency of NFKBIE aberrations (46/203 cases [22.7%]) was observed in primary mediastinal B-cell lymphoma (PMBL) and Hodgkin lymphoma (3/11 cases [27.3%] ). NFKBIE-deleted PMBL patients were more often therapy refractory (P = .022) and displayed inferior outcome compared with wild-type patients (5-year survival, 59% vs 78%; P = .034); however, they appeared to benefit from radiotherapy (P = .022) and rituximab-containing regimens (P = .074). NFKBIE aberrations remained an independent factor in multivariate analysis (P = .003) and when restricting the analysis to immunochemotherapy-treated patients (P = .008). Whole-exome sequencing and gene expression profiling verified the importance of NF-κB deregulation in PMBL. In summary, we identify NFKBIE aberrations as a common genetic event across B-cell malignancies and highlight NFKBIE deletions as a novel poor-prognostic marker in PMBL.

Abstract: Hematological malignancies are successfully treated with chimeric antigen receptor (CAR) armed T cells. Despite the clinical success, CAR T cell therapy struggles still with some problems including the selection of tumor escape variants and on-target, off-tumor side reactions as well as massive cytokine release and uncontrollability of CAR T cell activity in the patients. In order to enable controllability of CAR T cells and to avoid unspecific side effects, we established a novel switchable, split and adaptable CAR platform technology, termed RevCAR system. The novel RevCARs lack the single chain variable fragment (scFv) commonly used as extracellular domain in conventional CARs. Instead of the scFv, RevCARs contain only a small peptide epitope as extracellular portion. This design reduces the CAR size, avoids unspecific antigen binding and prevents antigen independent tonic signaling caused by scFv dimerization. As RevCAR T cells do not recognize anything, they are per se inert. Only in the presence of a corresponding bispecific antibody based target module (RevTM) they can be specifically redirected to tumor cells. Therefor RevTMs consist of two scFvs. One recognizes the RevCAR peptide epitope and the other one simultaneously binds to a tumor associated antigen (TAA). By dosing of the RevTM, which has a very short half-life, the reactivity of RevCAR T cells can be switched on and off reversibly. Another advantage is that the RevCAR system can be flexibly adapted to any tumor antigen simply by exchanging the RevTM. Furthermore, the small RevCAR size is favorable for inserting more than one RevCAR in the same T cell thus facilitating the mode of gated targeting which is a highly attractive approach to minimize the risk for on-target, off-tumor toxicities against healthy tissues and to increase tumor specificity of conventional CAR T cells. For 'AND' gate targeting via the RevCAR system, two different RevCARs were constructed and expressed simultaneously in the same T cell. The two RevCARs differed with respect to the extracellular peptide epitope and the intracellular signaling domain. Moreover, the respective transmembrane domain was selected to isolate the respective RevCAR signal. The first RevCAR is designed to transmit the activation signal, the second RevCAR to deliver a costimulatory signal. For efficient RevCAR T cell activation, both RevCARs must be engaged via their respective RevTM which on the one hand binds to one of the two RevCAR epitopes and on the other hand to one of two TAAs expressed on the same target cell. Here, we present two RevCAR/RevTM systems for retargeting of AML cells as well as solid tumor cells including via gated targeting. In summary, we show proof of concept for a novel switchable RevCAR system that can be used for retargeting of AML cells as well as solid tumors. The novel modular RevCAR platform is characterized by small size, lacks unwanted tonic signaling effects, allows the control of RevCAR T cell activity, enables gated targeting strategies, and can be adapted to any tumor antigen and tumor type. Disclosures Koristka: Intellia Therapeutics: Employment. Bachmann:GEMoaB Monoclonals: Equity Ownership, Patents & Royalties.

Abstract: Deletion of the tumor suppressor gene TP53is significantly associated with an unfavorable clinical course of Multiple Myeloma (MM). In addition, point mutations that abrogate p53 function similarly shorten survival. Most recently 'double hit', bi-allelic TP53inactivated MM was identified as an ultimate high risk feature of MM, affecting 3.7% of newly diagnosed MM patients (NDMM, Walker et al., Leukemia 2018). For TP53genetic analysis we combined data from two targeted sequencing M3P cohorts with targeted sequencing and FISH data, one of NDMM (n=142, Kortüm et al. Blood Cancer J. 2016), and one of multirefractory patients (rMM, n=40; Kortüm et al. Blood 2016). We also included two independent cohorts with paired NDMM and rMM, one with mutational data (n=43; Corre J et al 2017) and one with paired FISH analysis (Merz M et al. Haematologica 2017). We confirmed an increase of mutations in TP53 (8% NDMM / 16.9% rrMM), as well as del17p (12.6% / 22%). Similarly, mono- (12.7% / 22.5%) and bi-allelic events (5.6% / 17.5%) both demonstrated a significant increase over time (Figure, top). Importantly we observed deletion after mutation and vice versa in our cohorts. Next we established an AMO-1 MM cell line model (TP53+/+) mimicking mono and bi-allelic TP53-inativation. After CRISPR/Cas9-mediated TP53destruction, we introduced a modified Sleeping Beauty (SB) vector with two separate expression cassettes (p53 wt / wt and mutant p53 (R282W or R175H). Functionality of the p53 system was confirmed using nutlin-3, as inhibitor of MDM2-p53 interaction, as described elsewhere. Results: Doxorubicin and Melphalan are commonly used compounds in the treatment of MM. The IC50 of Melphalan in AMO1 naïve cells was 5µM and 5.5 µM after the reintroduction of 2 copies of wt TP53 in the KO model, with cell viability at 10µM of 30% and 31% respectively, measured by AlamarBlue Assay. Strikingly, we observed significant resistance induction in our hemizygous systems (del/wt p53; cell viability at 10µM 60% and mut/wt (62%). This furthermore increased within our homozygous models (TP53 del/del (85%); TP53 mut/del 80%) (Figure, bottom). Similar results were observed under doxorubicin treatment. Remarkably, this effect was absent against proteasome inhibition. Conclusions Here we present first evidence of TP53 inactivation impacting drug response to Melphalan and Doxorubicin, which might lead to the clonal selection of MM subclones harboring increased risk. The fact, that response to proteasome inhibition was not affected in our model might, at least in part, might explain their ability to confine high risk in MM. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

Abstract: Background: The PTLD-1 trial has demonstrated the efficacy and safety of 4 cycles of weekly rituximab followed by 4 cycles of CHOP-21 + G-CSF in CD20-positive PTLD after solid organ transplantation. Median overall survival (OS) was 6.6 years, a clear improvement over the preceding rituximab monotherapy trials (2.4 years). However, response to rituximab induction predicted OS after completion of therapy. Based on the hypothesis that rituximab consolidation might be sufficient treatment for patients already in a complete response (CR) after rituximab induction, trial treatment was changed in 2007 through a protocol amendment introducing risk-stratified sequential treatment (RSST): rituximab consolidation for patients in CR after rituximab induction and R-CHOP-21 consolidation for all others. Methods: In this international, multicenter phase II trial (PTLD-1, 3rd amendment; NCT00590447), treatment-naïve adult solid organ transplant recipients diagnosed with CD20-positive PTLD were treated with rituximab (375 mg/m2 IV) on days 1, 8, 15 and 22. After restaging, patients in CR continued with four three-weekly courses of rituximab monotherapy while all others received 4 cycles of R-CHOP-21 + G-CSF. In case of disease progression during rituximab monotherapy R-CHOP was commenced immediately. The primary endpoint was treatment efficacy measured as response rates and response duration. Analysis was by intention to treat. This is the final analysis of 152 patients treated with RSST from 2007 to 2014 at centers in Germany (72), Belgium (36), France (24), Australia (7), Poland (7) and Italy (6) with a median follow-up of 4.5 years. The 70 patients treated with rituximab followed by CHOP-21 in the original PTLD-1 trial (median follow-up 5.1 years) served as a control population. Inclusion criteria and follow-up schedule were identical; there were no significant differences in the transplant- and lymphoma-related baseline factors listed below. Results: 115/152 patients were male. 69/152 were kidney, 40 liver, 18 lung, 15 heart, 5 heart/kidney, 3 kidney/pancreas and 2 heart/lung transplant recipients. Median age at diagnosis was 56 years. PTLD was late ( 〉 1 year after transplantation) in 120/152 (79%) of patients. 67/145 (46%) PTLD were EBV-associated. 130/152 patients had monomorphic, 20 polymorphic and 2 early lesion PTLD. The overall response rate (ORR) was 111/126 (88%, CR: 88/126 [70%]). Median duration of remission (DR) was not reached; the 3-year Kaplan-Meier estimate was 82% (compared to 71% in PTLD-1). In the intention-to-treat population (152 patients), the median time to progression (TTP) was not reached either. The 3-year Kaplan-Meier estimate was 78% (69% in PTLD-1). Median OS by intention-to-treat was 6.6 years (95% CI 5.5 - 7.6) with a 3-year estimate of 70% in comparison to 61% in PTLD-1. There was no significant difference in ORR, DR, TTP or OS between EBV-positive and EBV-negative PTLD. On the other hand, response to 4 applications of rituximab was a highly significant predictor of OS, TTP and progression-free survival (PFS) despite treatment stratification (all p 〈 0.001). 37/148 patients (25%) achieved CR with 4 cycles of rituximab and were allocated to rituximab consolidation. In this group, TTP in the intention-to-treat population was significantly longer than in the corresponding group in the PTLD-1 trial (37 patients versus 14 patients, p 〈 0.05). In the 111 patients allocated to R-CHOP consolidation, ORR was 78/92 (85%) with 55/92 (60%) complete remissions (89% and 60%, respectively, in PTLD-1). Median TTP was not reached, the 3-year estimate was 73% (69% in PTLD-1). In patients refractory to rituximab induction, the CR rate was 22/38 (58%) with R-CHOP compared to 3/11 (27%) in PTLD-1 with CHOP (p=0.07); median PFS was 1.4 years versus 0.3 years in PTLD-1, p 〈 0.05. The frequency of grade 3/4 leukopenia and infections was 63% and 34%, respectively. Treatment-related mortality occurred in 7%. Conclusions: This largest trial cohort in PTLD to date demonstrates for the first time that treatment stratification by response to rituximab induction is feasible, safe and effective. Rituximab consolidation in early rituximab responders results in significantly better disease control compared to CHOP consolidation. The addition of rituximab to CHOP chemotherapy improves outcome in patients refractory to rituximab monotherapy. Disclosures Trappe: Mundipharma: Research Funding; AbbVie: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Research Funding, Speakers Bureau; CSL Behring: Research Funding, Speakers Bureau. Zimmermann:Roche: Honoraria; Celgene: Other: Travel support. Morschhauser:Genentech Inc./Roche: Other: Advisory boards. Mollee:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Onyx: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Zaucha:Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Servier: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Speakers Bureau; Takeda: Speakers Bureau. Dührsen:Roche: Honoraria, Research Funding; Alexion Pharmaceuticals: Honoraria, Research Funding; Amgen: Honoraria, Research Funding. Hüttmann:Amgen: Research Funding; Roche: Research Funding. Salles:Calistoga Pharmaceuticals, Inc.; Celgene Corporation; Genentech, Inc.; Janssen Pharmaceutica Products, L.P.; Roche: Consultancy; Celgene Corporation; Roche and Gilead Sciences: Research Funding; Celgene Corporation; Roche: Speakers Bureau. Kliem:Astellas: Honoraria; Fresenius: Honoraria; Genzyme: Honoraria; Novartis: Honoraria; Roche: Honoraria; Raptor: Honoraria. Leblond:Janssen: Consultancy, Honoraria, Speakers Bureau; Mundipharma: Honoraria; Gilead: Consultancy, Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Speakers Bureau; GSK: Consultancy, Honoraria, Speakers Bureau. Choquet:Janssen: Consultancy; Roche: Consultancy.

Abstract: Background: So called triple-negative acute myeloid leukemias (AML) form a heterogeneous subgroup of intermediate-risk AML according to ELN criteria. Molecularly this group can be defined by the absence of internal tandem duplications (ITDs) in the fms related tyrosine kinase 3 gene (FLT3), wild type (wt) nucleophosmin gene (NPM1) and wt or single (homozygously or heterozygously) mutated CCAAT/enhancer binding protein α gene (CEBPA). To date, it remains unclear whether this group of patients benefits from allogeneic stem cell transplantation (alloSCT) as consolidation strategy in first complete remission (CR1). Aims: To evaluate the impact of alloSCT on the overall (OS) and relapse free survival (RFS) in patients with triple negative AML in first remission in comparison to post remission chemotherapy (PRT). Methods: We performed a subgroup analysis of 3041 AML patients aged 16-60 years who were enrolled into the AML 96 and the AML 2003 trials of the Study Alliance Leukemia (SAL). Selection criteria for this subgroup were NPM1 wt, negativity for FLT3-ITD and CEPBA double mutations, a karyotype that does not define the AML as favorable or adverse according to ELN criteria and the accomplishment of CR1. Status of molecular markers was evaluated with standard PCR techniques. Within the AML2003 trial, donor status was evaluated at study entry, making these data eligible for a donor-versus-no-donor analysis. Kaplan-Meier estimates were used to report on point estimates for survival probabilities. Multivariate Cox models were fitted to analyze the impact of alloSCT as time-dependent covariate. Age, gender, white blood cell count, lactate dehydrogenase, AML type (de novo, secondary AML following MDS or MPN, or therapy-related myeloid neoplasms) and ECOG performance status at diagnosis were selected as adjusting covariables. As-treated analyses used data from both trials, AML96 and AML2003. For these analyses alloSCT or PRT were entered as time-dependent covariates into extended Cox regression models. Survival outcomes were displayed with Simon-Makuch-plots. Results: In total, 497 patients (AML96: 217, AML2003: 280) with a median age of 47 years were evaluable for the analysis of OS from diagnosis. A total of 302 patients had reached CR1 and could be evaluated for RFS. In a multivariate donor-versus-no-donor analysis, OS of patients with a sibling donor was not significantly different to patients without a donor (HR 0.79, 95%CI 0.53 to 1.16, p=.2). Irrespective of whether the patient actually received alloSCT in CR1, the probability of OS at 5 years from study enrollment was 55% (95%CI, 45% to 67%) for patients with a sibling donor and 47% (95%CI, 40% to 54%) for patients without a donor. For RFS, the hazard ratio was 0.72 (95%CI, 0.5 to 1.05, p=0.09), with a trend in favor of better remission-control for patients with a sibling donor. At five years from CR1, RFS of patients with sibling donor was 48% (95%CI, 38% to 61%) compared to 36% (95%CI, 30% to 44%). However, the transplantation rate in the donor group was only 53% and 15% of patients in the no-donor group actually received alloSCT. Therefore, 'cross-over' effects lowered the power of donor-versus-no-donor analysis. In the multivariable as-treated analysis including patients from AML96 and AML 2003, OS and RFS of patients with alloSCT were significantly longer (OS: HR 0.58, 95%CI, 0.37 to 0.9, p=.02, RFS: 0.51, 95%CI, 0.34 to 0.76, p=0.001) compared to the PRT group. The probability of OS at 5 years from initiation of consolidation treatment (alloSCT vs. PRT) was 66% (95%CI, 57% to 76%) for patients who received alloSCT compared to 46% (95%CI, 38% to 55%) for PRT patients. The probability of RFS at 5 years from initiation of consolidation treatment was 55% (95%CI, 46% to 67%) for alloSCT patients and 31% (95%CI, 24% to 39%) for PRT patients. Conclusions: Due to cross-over effects which limit the power of the donor-versus-no-donor analysis we give more weight to the results of the as-treated analysis. This analysis suggests that eligible intermediate-risk AML patients with NPM1 wt and absent FLT3-ITD benefit from alloSCT in CR1. However, bias introduced by selection and confounding factors cannot be excluded for this type of analysis and could only be circumvented in randomized controlled trials. Disclosures Thiede: AgenDix: Employment, Other: Ownership. Rösler:Janssen: Consultancy, Other: Travel/Accommodation/Expenses. Middeke:Sanofi: Honoraria. Schetelig:Sanofi: Honoraria.

Abstract: Among evaluable patients with relapsed/refractory DLBCL who received blinatumomab 112 μg/d, overall response was 43% (CR was 19%). Blinatumomab continuous infusion was feasible with weekly stepwise dose escalation (9-28-112 μg/d) and dexamethasone prophylaxis.

Abstract: The common acute lymphoblastic leukemia antigen CD10 is a marker for several hematological malignancies, including acute lymphoblastic leukemia as well as T and B cell lymphomas, Burkitt lymphomas, and some solid tumors like renal cell carcinomas, pancreatic tumors and melanomas. Because of its tumor related expression pattern, CD10 is an attractive target for adoptively transferred T cells that are genetically modified to express chimeric antigen receptors (CARs). Recently, conventional CAR T cell therapy targeting CD19-positive hematological malignancies was clinically approved because of its impressive effectiveness in patients. However, CAR T cells can also cause severe side effects like on-target, off-tumor reactions, tumor lysis syndrome and cytokine release syndrome. Most critically, activity of conventional CAR T cells cannot be controlled, once they are applied in patients. As CD10 is also widely expressed on normal tissues, CAR T cell reactivity has to be controllable in order to stop CAR T cell therapy in case of on-target, off-tumor toxicities occur. Especially for this purpose, we have recently established a switchable, modular and universal CAR platform technology, named UniCAR system, which can be repeatedly turned on and off. In contrast to conventional CARs, that directly recognize a tumor-associated antigen (TAA) on the tumor cell surface via their extracellular single-chain variable fragment (scFv), the UniCAR system is structured in a modular manner of two components. The first component are T cells genetically engineered to express UniCARs and the second component are target modules (TMs). Most importantly, UniCARs cannot directly bind to a TAA because their extracellular scFv is directed against the peptide epitope E5B9 which is not present on the surface of living cells. Consequently, UniCAR armed T cells are per se inert. They can be redirected towards tumor cells only via a TM. TMs consist of a scFv targeting a TAA and the epitope E5B9 recognized by UniCARs allowing a cross-linkage of UniCAR T cells with tumor cells which results in T cell activation. As TMs have a very short half-life, UniCAR T cell activity can be controlled by dosing of the TM. Once the TM is administered, UniCAR T cells can be switched on, but once the TM injection is stopped and the TM is eliminated, UniCAR T cells are switched off immediately. Here, we show proof of concept for functionality of the UniCAR system targeting CD10-positive malignancies. Therefor, a novel anti-CD10 TM was constructed which is able to redirect UniCAR T cells to eliminate CD10-expressing tumor cells. In summary, we have established a universal, switchable, modular UniCAR platform technology that can be used to target CD10-positive malignancies. Disclosures Koristka: Intellia Therapeutics: Employment. Bachmann:GEMoaB Monoclonals: Equity Ownership, Patents & Royalties.