Abstract: Background: The genetic hallmark of BL is MYC gene rearrangement. However, due to histopathological, immunohistochemical and cytogenetic similarities, differentiation from Diffuse Large B-Cell Lymphoma (DLBCL) remains challenging in some cases. Although being suspected to negatively influence outcomes, detection of high expression of BCL2 and MYC (double expressor) as well as rearrangements of MYC and BCL2 and/or BCL6 (double/triple hit) did not lead to confirmation of different therapeutic approaches in DLBCL yet. The recent update of the World Health Organization (WHO) classification of lymphoid neoplasms has thus established a category termed High-Grade B-Cell Lymphoma (HGBL), defined by a distinct morphology and/or presence of double/triple hit status. While BL displays favorable outcomes in clinical routine upon treatment with intensive immunochemotherapeutic regimens, clear evidence regarding the optimal therapeutic approach for HGBL remains limited and the diagnostic discrimination of BL and HGBL can be challenging, too. Moreover, data on the frequency of revisions of the diagnosis of BL to DLBCL or HGBL and its impact on treatment and outcome are sparse. Methods: All consecutive patients managed with histopathological suspected diagnosis of BL upon preliminary assessment at the reporting institution between December 2010 and July 2020 were identified. Final diagnosis of the respective aggressive B-Cell lymphoma was grouped according to the most recent WHO classification and divided into four subgroups: a) BL, b) revision from DLBCL to BL, c) DLBCL, d) HGBL. General patient characteristics as well as response to treatment in Computed Tomography (CT) or Positron Emission Tomography (PET) (evaluation by individual investigator), information regarding survival supported by the register of deaths in North-Rhine Westphalia and the date of last follow-up were collected from individual patient files. Results: Overall, we identified n=66 patients with suspected diagnosis of BL within the preliminary assessment. Final histopathological results confirmed n=31 patients as BL (Group A), while n=23 patients were described as showing features of both BL and DLBCL (Uncertain & Grey-zone, Group D). Additionally, in n=12 patients the final diagnosis was revised either from DLBCL to BL (n=9, Group B) or B-ALL/BL to DLBCL (n=3, Group C). Regarding the reference histopathological reviews (RHR) and the latest version of the WHO classification of lymphoid neoplasms, all patients from Group C and Group D were finally classified as either DLBCL (n=12) or HGBL (n=14). In total, n=13 patients had revision of the final diagnosis, either upon receipt of RHR (n=8) or repeated biopsy upon progressive disease (PD) (n=5). When comparing patients with revised diagnosis due to PD or RHR, a median of six (range 4-8) and two (range 1-2) cycles of systemic therapy were applied prior to revision, respectively. Regarding outcomes, 2/5 patients (40%) with revised diagnosis upon PD and 7/8 patients (87,5%) with revised diagnosis upon RHR were alive at the time of last follow-up (median follow-up 15,07 months), respectively (Fig 1). Patients suffering from BL (Group A) displayed a significantly increased progression-free survival (PFS) compared to all other groups (p=0.034). In contrast, patients whose diagnosis was revised from DLBCL to BL after initiation of treatment with DLBCL protocols had an inferior PFS than patients initially diagnosed with BL (p=0.045) that was comparable to patients with the final diagnosis of DLBCL or HGBL. Two-year overall survival was generally favorable in all groups, ranging from almost 60% (HGBL) to 86,2% (BL) without showing statistically significant differences (p=0.2). Conclusion In conclusion, timely and precise histopathological diagnostic procedures seem to play a critical role for optimizing treatment strategies of BL, HGBL and high-risk DLBCL upfront. Since administration of BL treatment protocols seems feasible in clinical routine and may result in favorable results not only in BL but as well in HGBL, these results might encourage its upfront usage in doubtful situations regarding histopathological results. However, with only a small number of patients being evaluable in the present analysis, controlled clinical trials are necessary to confirm these trends. Figure 1 Figure 1. Disclosures Heger: Novartis: Research Funding; Gilead: Other: Travel funding. Gödel: Gilead: Other: Personal fees / travel support; Novartis: Other: Travel support. Balke-Want: Novartis: Research Funding. Simon: Gilead: Other: Travel support. Bröckelmann: BMS: Honoraria, Research Funding; Takeda: Honoraria, Research Funding; MSD: Research Funding; BeiGene: Research Funding. Von Tresckow: Amgen: Consultancy, Honoraria; AstraZeneca: Honoraria, Other: Congress and travel support; AbbVie: Other: Congress and travel support; BMS-Celgene: Consultancy, Honoraria, Other: Congress and travel support; Kite-Gilead: Consultancy, Honoraria; MSD: Consultancy, Honoraria, Other: Congress and travel support, Research Funding; Novartis: Consultancy, Honoraria, Other: Congress and travel support, Research Funding; Pentixafarm: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Other, Research Funding.

Abstract: Burkitt lymphoma (BL) and diffuse large B‐cell lymphoma (DLBCL) are aggressive B‐cell non‐Hodgkin lymphomas (B‐NHL) with a generally favorable prognosis after immunochemotherapy. The outcome of BL is superior to DLBCL. In 2016, a distinct group of lymphomas displaying characteristics of both BL and DLBCL (high grade B‐cell lymphoma, HGBL) was introduced into the WHO classification. Histopathological discrimination of BL, DLBCL, and HGBL may be challenging. Data on the frequency of histopathological difficulties resulting in revision of the final diagnosis of BL/DLBCL/HGBL and its impact on the prognosis are limited. Methods We assessed histopathological features and clinical outcomes of 66 patients with suspected diagnosis of BL at the reporting institution between 2010 and 2020. Results The median age was 51 years (range 19‐82) and final histopathological diagnosis revealed BL (n = 40), DLBCL (n = 12), or HGBL (n = 14). Patients with DLBCL and HGBL were either treated with DLBCL‐directed (83.3% and 35.7%) or BL‐directed (16.7% and 64.3%) protocols. Patients in whom diagnosis was revised from DLBCL to BL after initiation of DLBCL‐directed treatment had a significantly inferior progression‐free survival (PFS) than patients initially diagnosed with BL (p = 0.045), thus resembling rather the prognosis of DLBCL/HGBL. There was no difference between patients with DLBCL and HGBL, respectively, regarding PFS and OS (p = 0.38 and p = 0.27). Conclusion These results suggest that timely and precise histopathological diagnosis as well as reference histopathological review of the underlying lymphoma is critical to determine up‐front treatment strategies. Consequently, selection of more aggressive treatment protocols in case of difficulties with discrimination between DLBCL/HGBL/BL may be a reasonable approach.

Abstract: Introduction: Anti-CD 19 directed CAR-T cell therapy has shown very promising effects in treatment of diffuse-large B-cell lymphoma (DLBCL). However, a substantial fraction of patients does not respond to treatment or relapses after approximately three to six months [Schuster, NEJM, 2017; Neelapu, NEJM, 2017]. Resistance mechanisms against anti-CD19 directed CAR-T cell therapy have so far only been described in B-cell acute lymphoblastic leukemia (B-ALL) and involve mutations, splice variants or even loss of CD19 [Sotillo, Cancer Discovery, 2015] . To further elucidate mechanisms of acquired resistance in DLBCL, we obtained matched pair tumor samples from four patients before and after treatment with CTL019 treated within the phase II JULIET trial. Methods: We performed whole exome sequencing in one patient (#UKK2) employing one matched normal sample that has been sequenced at a coverage of 130x and one pre-treatment as well as two post-treatment samples that were all sequenced at a coverage of 160x. Whole exome sequencing data were used to reconstruct clonal evolution of DLBCL after treatment with CTL019 as has been described previously [Herling, Nature Communications, 2018]. Furthermore, we obtained biopsies at relapse or disease progression including matched normal samples from three more patients. Whole exome sequencing from the three missing patients as well as 3´RNA sequencing from all four patients' samples are ongoing and results will be updated for presentation at the meeting. Results: The first analyzed patient, #UKK2, is a 67 year old male patient, who had chemotherapy refractory disease after treatment with R-CHOP, R-DHAP and Pixantrone. He showed predominantly pelvic lesions including infiltration of the iliopsoas muscle. The patient achieved a complete response at month 3 (Figure 1A) after CTL019 transfusion. At month 5 he developed new cutaneous lesions on his left thigh (Figure 1B, posttreatment sample 1) as well as new muscle infiltrations on his right quadriceps femoris muscle (posttreatment sample 2) and rectus abdominis muscle. Pathological examination confirmed relapse of CD19 positive DLBCL in both posttreatment samples. Whole exome sequencing from a biopsy obtained before study entry and the above mentioned posttreament biopsies showed clonal evolution of a single missense mutation in PTPRA (N297D). PTPRA encodes for the receptor protein-tyrosine phosphatase alpha, a receptor phosphatase involved in activation of c-Src via dephosphorylation of an inhibitory tyrosine at position 530 (Y530) [Mustelin, Science Signaling, 2002 and Gut, International Journal of Oncology, 2017]. On the other hand, c-Src is an onco-protein known to cause an invasive phenotype and metastasis in different tumor entities [Ishizawar, Cancer Cell, 2004] . All other mutations observed were either already clonal before CTL019 treatment or gained clonality in only one posttreatment biopsy (Figure 1C). Conclusion: To our knowledge, this is the first report identifying clonal evolution during CD19 redirected CAR-T cell therapy in r/r DLBCL involving PTPRA. We hypothesize that this PTPRA mutation contributes to an invasive phenotype and extranodal lymphoma growth pattern allowing the lymphoma to escape from the pattern allowing the lymphoma to escape from the CAR-T cell attack. Genomic workup of patients with pre- and posttreatment biopsies and functional investigation of this hypothesis is ongoing and will be presented. Disclosures Balke-Want: Novartis: Honoraria. Borchmann:Novartis: Consultancy, Honoraria.

Abstract: Background CD19 redirected chimeric antigen receptor (CAR) T-cell therapy has proven efficacy in relapsed or chemotherapy-refractory (r/r) aggressive B-cell non-Hodgkin lymphoma (B-NHL). However, targeting a single B-cell antigen leads to selective pressure with potential antigen-escape and subsequent relapse. A tandem CAR targeting CD20 and CD19 (pLTG1497) has been developed to overcome this limitation. Preclinical evaluation showed improved anti-lymphoma activity. Thus, we initiated a first-in-human, phase I clinical study of autologous pLTG1497-transduced CAR T-cells (MB-CART2019.1) in r/r B-NHL patients. Aims In this phase I prospective multi-center trial (NCT03870945) we aimed to evaluate the maximum tolerated dose (MTD) of MB-CART2019.1 in adult patients with CD20 and CD19 positive r/r B-NHL as determined by dose limiting toxicities (DLTs). Methods This was a 6+3 trial design with two predefined dose levels (DL1 1x106 and DL2 2.5x106 CAR T-cells/kg body weight, respectively). Secondary endpoints included adverse events (AEs) and best overall response rate (ORR). Pharmacodynamic assessments included maximum concentration (Cmax) of CAR T-cells, time to peak expansion (tmax), AUC (d0 to d28), and persistence. MB-CART2019.1 was produced by lentiviral transduction of autologous fresh leukapheresis in the closed automated CliniMACS Prodigy® System (Miltenyi Biotec, Bergisch Gladbach, Germany). Re-infusion (Day 0) of fresh MB-CART2019.1 was scheduled 14 days after leukapheresis. Fludarabine/cyclophosphamide lymphodepleting chemotherapy was administered from day -5 to -3. Results A total of 12 patients, 6 per dose level have been enrolled and treated between February and December 2019, 5 female and 7 male patients. Median age was 72 y (range 20, 78 y), with 10 patients & gt;65 y and 8 & gt;70 y. Histologies included aggressive B-NHL (11) and mantle cell lymphoma (1). Five (5) patients had refractory disease at study entry and IPI was ≥3 in 7 patients. Median time from leukapheresis to re-infusion was 14 d (range 13, 14 d). No DLT and no cytokine release syndrome (CRS) or neurotoxicity grade ≥3 were observed. One patient in dose level 1 experienced a grade 5 AE, which was due to disease progression. CRS grade 1 occurred in 3/6 patients on DL1 and DL2 each, and CRS grade 2 in 2 patients on DL2. Tocilizumab was given in 1 patient. Neurotoxicity grade 1 occurred in 1 patient on DL2. The above described CRS and neurotoxicity resolved completely. Mean Cmax of MB-CART2019.1 was 348.3 cells/µl (range 3.9, 830.4 cells/µl) on DL1 and 692 cells/µl (range 5.3, 3147.8 cells/µl) on DL2. Mean tmax was 15.8 d (range 9, 21 d) on DL1 and 11.5 d (range 9, 14 d) on DL2. Mean AUC was 3155 d*cells/µl (DL1) and 4339 d*cells/µl (DL2). Persistence of MB-CART2019.1 was observed in 12/12 patients until data cut-off. Altogether 9/12 patients (ORR 75%) responded to MB-CART2019.1 with 5/12 CRs. In DL1 3/6 patients responded (ORR 50%) and in DL2 6/6 patients (ORR 100%). The 3 patients without response to MB-CART2019.1 had a mean AUC0-28 of 870 d*cells/µl, whereas mean AUC0-28 in 9 responders was 4843 d*cells/µl reflecting the correlation between the pharmacodynamic parameters and the clinical response. Responses are ongoing in 5/9 patients, with a maximum duration of response of 330 days at data cut-off. Summary/Conclusions In this first-in-human dose finding study of MB-CART2019.1 no DLT and no severe (grade ≥3) CRS or neurotoxicity were observed. Feasibility and safety were very good in this cohort of elderly r/r B-NHL patients. The sustained expansion of tandem CAR T-cells was accompanied by efficacy: all patients (6/6) treated on DL2 responded and all 5 patients with CR (5/5) are in ongoing remission by the time of this report. Based on the promising risk-to-benefit ratio observed in our study, evaluation of MB-CART2019.1 at a dose of 2.5x106/kg body weight in clinical phase II and phase III trials for patients with relapsed aggressive B-NHL is underway. Disclosures Borchmann: Miltenyi Biotec B.V. & Co. KG: Honoraria. Balke-Want:Miltenyi Biotec B.V. & Co. KG: Honoraria. Ayuk:Celgene: Consultancy, Honoraria; Kite/Gilead: Honoraria; Therakos/Mallinckrodt: Honoraria, Research Funding; Neovii: Research Funding; Novartis: Honoraria. Holtkamp:Miltenyi Biomedicine GmbH: Current Employment. Preussner:Miltenyi Biomedicine GmbH: Ended employment in the past 24 months. Zadoyan:Miltenyi Biomedicine GmbH: Current Employment. Hanssens:Miltenyi Biomedicine GmbH: Current Employment. Kaiser:Miltenyi Biotec B.V. & Co. KG: Current Employment. Jurk:Miltenyi Biotec B.V. & Co. KG: Current Employment. Bürger:Miltenyi Biotec B.V. & Co. KG: Current Employment. Schneider:Lentigen Technology Inc., A Miltenyi Company: Current Employment, Patents & Royalties. Dropulic:Lentigen Technology Inc., A Miltenyi Company: Current Employment. Overstijns:Miltenyi Biomedicine GmbH: Current Employment, Membership on an entity's Board of Directors or advisory committees; Miltenyi Biotec B.V. & Co. KG: Current Employment, Membership on an entity's Board of Directors or advisory committees. Scheid:Novartis: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; BMS: Honoraria; Amgen: Honoraria; Takeda: Honoraria, Research Funding. Holtick:Miltenyi Biotec B.V. & Co. KG: Honoraria. Miltenyi:Miltenyi Biomedicine GmbH: Current Employment, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Lentigen Technology Inc., A Miltenyi Company: Current Employment, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Miltenyi Biotec B.V. & Co. KG: Current Employment, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Abstract: Acquired immunoglobulin G (IgG)‐mediated thrombotic thrombocytopenic purpura ( TTP ) has not yet been described in non‐twin siblings. We report two cases of acquired TTP in Caucasian sisters with inactive ADAMTS 13 metalloprotease due to ADAMTS 13 autoantibodies suggesting a role of genetic determinants in this life‐threatening disease. However, human leucocyte antigen ( HLA ) class II types presumably associated with acquired TTP were not identified in the patients, indicating that HLA class II typing may not be useful in acquired TTP risk assessment of family members.