Abstract: Despite numerous advances in the identification of risk factors for the development of severe coronavirus disease 2019 (COVID-19), factors that promote recovery from COVID-19 remain unknown. Natural killer (NK) cells provide innate immune defense against viral infections and are known to be activated during moderate and severe COVID-19. Killer immunoglobulin-like receptors (KIR) mediate NK cell cytotoxicity through recognition of an altered MHC-I expression on infected target cells. However, the influence of KIR genotype on outcome of patients with COVID-19 has not been investigated so far. We retrospectively analyzed the outcome associations of NK cell count and KIR genotype of patients with COVID-19 related severe ARDS treated on our tertiary intensive care unit (ICU) between February and June 2020 and validated our findings in an independent validation cohort of patients with moderate COVID-19 admitted to our tertiary medical center. Results Median age of all patients in the discovery cohort ( n  = 16) was 61 years (range 50–71 years). All patients received invasive mechanical ventilation; 11 patients (68%) required extracorporeal membrane oxygenation (ECMO). Patients who recovered from COVID-19 had significantly higher median NK cell counts during the whole observational period compared to patients who died (121 cells/µL, range 16–602 cells/µL vs 81 cells/µL, range 6–227 cells/µL, p -value = 0.01). KIR2DS5 positivity was significantly associated with shorter time to recovery (21.6 ± 2.8 days vs. 44.6 ± 2.2 days, p -value = 0.01). KIR2DS5 positivity was significantly associated with freedom from transfer to ICU (0% vs 9%, p -value = 0.04) in the validation cohort which consisted of 65 patients with moderate COVID-19. Conclusion NK cells and KIR genotype might have an impact on recovery from COVID-19.

Abstract: Seroprevalence studies of SARS‐CoV‐2 have shown that there is a high number of undiagnosed missing cases. Seroprevalence of SARS‐CoV‐2 in people living with HIV (PLWH) is lacking. Therefore, we conducted a prospective cross‐sectional study to estimate the seroprevalence of SARS‐CoV‐2 among PLWH without known diagnosis of COVID‐19 in the south‐west of Germany. Methods Serological testing for SARS‐CoV‐2 immunoglobulin G (IgG) antibodies based on two assays was performed in PLWH who visited the outpatient HIV centre of two hospitals from April to June 2020. Additionally, patients had to answer questionnaires about possible COVID‐19‐related symptoms and predefined risk factors. Moreover, we tested 50 non‐HIV‐infected patients receiving post‐ or pre‐exposure (PEP/PrEP) HIV prophylaxis. Results In all, 594 (488 male, 106 female) PLWH (median age 51 years) and 50 PEP/PrEP‐users were included in the study. The estimated seroprevalence of the PLWH cohort was 1.85% (11/594), with 11 positive tested cases in the cohort. Among all patients, only five had COVID‐19‐related symptoms. One PCR‐positive patient did not show any antibody response in repeatedly carried out tests. None of the patients was hospitalized due to COVID‐19. Three PrEP users were tested positive. Three patients had been previously diagnosed with SARS‐COV‐2 infection before inclusion. The used questionnaire did not help to detect SARS‐CoV‐2 positive patients. Conclusions Despite the limitation of being only a snapshot in time because of the ongoing pandemic, to our knowledge this is the largest study so far on seroprevalence of SARS‐CoV‐2 in PLWH in Germany. Our study suggests that the seroprevalence of SARS‐CoV‐2 in PLWH is comparable to those previously reported for parts of the general German population and that the questionnaire used here might not be the best tool to predict COVID‐19 diagnosis.

Abstract: Background Chronic antigenic stimulation of the B-cell receptor (BCR) seems to play a critical role in the pathogenesis of B-cell lymphomas. We recently identified ARS2 and LRPAP1 as the autoantigenic targets of the B-cell receptors of approximately 25% of diffuse large B cell lymphomas (DLBCLs) of the ABC type and 45% of mantle cell lymphomas (MCLs), respectively. These BCR antigens can be used to target lymphoma cells in an approach we designated as BAR (B-cell receptor antigens for reverse targeting). The optimal therapeutic format BARs can be integrated in has yet to be found. Since the most established approach to deliver therapeutic payloads to specific targets are antibodies which have well-defined pharmacokinetics, we constructed and tested an antibody like construct (BAR-body) incorporating the DLBCL-BAR ARS2 in substitution for the variable domains of the heavy and light chains. Material and methods To create the ARS2 BAR-body, we exchanged the heavy and light chain variable region sequences of an IgG1 antibody with a sequence of similar length (approximately 120 amino acids) of the ARS2 protein (aa 343 - 466) containing the DLBCL reactive epitope (aa 343 - 375). The construct was assembled in a pCR2.1 vector, then transferred to a pSfi FLAG Tag vector for fusion with the FLAG tag and transfected into HEK293 cells for production. Purification of the BAR-body was performed via anti-FLAG antibody affinity chromatography. The BAR-body was detected by western blot analysis and binding capacity to the ARS2-reactive lymphoma cell lines U2932 and OCI-Ly3 and the not ARS2-reactive control DLBCL cell line TMD8 was assessed by flow cytometry. ARS2 BAR-body induced cytotoxicity of lymphoma cells with an ARS2 reactive BCR was measured by LDH release assays with human PBMCs as effector cells at an E:T ratio of 10:1. Results We cloned, expressed and characterized an ARS2 containing BAR-body incorporating 4 molecules of the lymphoma-reactive epitope of ARS2 resulting in an antibody like construct using a BAR (ARS2) as binding moiety instead of normal variable regions. The ARS2 BAR-body could successfully be cloned and expressed as confirmed by western blot analysis, which showed the construct at approximately 150 kD as was to be expected. The BAR-body bound specifically to the ARS2-reactive lymphoma cell lines U2932 and OCI-Ly3 and did not bind to the DLBCL cell line TMD8, which has a B-cell receptor of different specificity or to lymphoma cell lines of different entities. In LDH release assays with 5 x 104 PBMCs and 5 x 103 lymphoma cells (E:T ratio of 10:1) the ARS2 BAR-body induced PBMC mediated specific lysis of the ARS2 reactive lymphoma cell lines U2932 and OCI-Ly3 but not the control DLBCL cell line TMD8 starting at a concentration of 0,1µg/ml. Cytotoxic effects were dose dependent, reached a maximum of 50% specific lysis at a concentration of 1µg/ml and did not increase at concentrations of 10µg/ml. Conclusion Here, we show that BARs can substitute for the variable domains as binding moiety in antibody like constructs to target the BCR of B-cell lymphomas. Because approaches using their specific cognate antigen for targeting the malignant B cells have an exclusive specificity for the BCR of the malignant clone, they can be expected to be less toxic than the currently available antibody derived therapies targeting B-cells, because they leave normal B-lymphocytes unaffected. By incorporating BARs into the well-known format of an antibody we hope to capitalize on years of experience with this therapeutic format from conducting and interpreting in vivo experiments to the translation of the BAR approach into the clinic. Disclosures Stilgenbauer: Genentech: 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; Hoffmann La-Roche: 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; Janssen: 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; GSK: 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; Celgene: 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; Boehringer-Ingelheim: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: 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; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Abstract: Background: Refractory or early relapsed ( 〈 1 year) diffuse large B-cell lymphoma has a dismal prognosis especially for those not responding to salvage chemotherapy (Van den Neste; Bone Marrow Transplant. 2016 Jan;51(1):51-7). Allogeneic stem cell transplantation is potentially curative (Glass et al; Lancet Oncology 2014 Jun;15(7):757-66). Even though this is less likely in those not responding or having frank progression pre-transplantation. Methods: At our institution we identified all patients with aggressive B-cell lymphoma (diffuse-large B-cell lymphoma and blastoid mantle cell lymphoma) who were refractory or progressive to salvage chemotherapy with R-DHAP and who had peripheral blood stem cells ( 〉 2x10^6 CD34+/kg body weight) collected after the 1st or 2nd cycle. These patients were eligible and planned to receive salvage high-dose melphalane and autologous stem cell transplantation as remission induction before allogeneic stem cell transplantation. Allogeneic transplantation was performed from sibling donors or matched unrelated or mismatched unrelated donors: The conditioning regimen with fludarabine, busulfane, cyclophosphamide and anti-thymocyte globuline as published (Glass et al; Lancet Oncology 2014 Jun;15(7):757-66) was used in all but 2 patients. Statistical analysis was performed with Prism©; GraphPad Software; (LaJolla CA) Results: From2006 to 2015 we identified 28 patients (21 male , 7 female, age 18-67 years, median age 47 years), 25 had diffuse large B-cell lymphoma and 3 had blastoid mantle cell lymphoma. 22 had relapsed within 1 year after primary diagnosis and 6 within a median of 25.3 months. After high-dose melphalane and autologous stem cell transplantation 13 patients had a partial and 6 a complete remission. 1 patient died due to neutropenic infection, 2 patients died due to progressive disease leading to a transplant related mortality of 3.5 %. Median progression-free survival after autologous transplantation was 4.6 months. 24 proceeded to allogeneic stem cell transplantation. 8 patients had a matched related sibling, 9 had a matched unrelated donor and 7 had a mismatched unrelated donor. Transplant related mortality was 42 % in this heavily pretreated population. 2-year overall survival of all patients intended for treatment is 21 %. One of these patients with relapsed mediastinal lymphoma after allogeneic transplantation was cured by salvage radiotherapy and is in long-term remission ( 〉 2 years). Conclusions: Salvage high-dose melphalane and autologous peripheral blood stem cell transplantation for diffuse large B-cell lymphoma as a bridge to allogeneic transplantation is potentially curative for a minor fraction of these patients. However, the remission rate of 79 % (46% PR, 21% CR) and progression-free survival of 4.6 months after high-dose melphalane and autologous stem cell transplantation provides a window of opportunity to use new drugs and cellular therapies in these poor prognosis patients. Disclosures No relevant conflicts of interest to declare.

Abstract: BACKGROUND: According to the Lugano classification (Cheson et al., JCO 2014, 32: 3059-3067) fludeoxyglucose positron emission tomography combined with computed tomography (FDG PET/CT) is the standard for evaluation and staging of aggressive non-Hodgkin Lymphoma (NHL). It is a matter of debate whether FDG PET/CT is sufficient to determine bone marrow (BM) involvement. We performed a pooled analysis of data from the PETAL (EudraCT 2006-001641-33) and OPTIMAL 〉 60 trials (EudraCT 2010-019587-36) as prospective, open-label, randomized, multicenter Phase-III trials to determine whether initial staging with FDG PET/CT would allow non-invasive diagnosis of BM involvement and thus could avoid established but potentially painful and unpleasant BM biopsy (BMB). PATIENTS AND METHODS: Patients treated within the trials were included if both FDG PET/CT and BMB were performed during initial staging. Only patients with a biopsy-proven, centrally confirmed diagnosis of diffuse large B-cell lymphoma, primary mediastinal B-cell lymphoma or follicular lymphoma grade 3b were included. FDG PET/CT images and BM findings were blinded for central review by board certified PET/CT readers. Discordant findings were documented and resolved after unblinding by interdisciplinary discussion using findings of complementary imaging and/or subsequent PET examinations. Based on literature (Berthet et al., J Nucl Med 2013), a newly defined gold standard was used to confirm BM involvement. It includes a positive BMB or a positive FDG PET confirmed by targeted biopsy, complementary CT imaging or targeted MRI, or concurrent disappearance of focal FDG-avid BM lesions with other lymphoma manifestations during immunochemotherapy. RESULTS: Out of 1,976 patients a total of 930 patients met the inclusion criteria. BMB confirmed BM involvement in 85 of 930 patients (9%). According to FDG PET/CT, BM was involved in 185 out of 930 patients (20%) with 36 discordances (4%) between negative initial FDG PET/CT and positive BMB ("false negatives" with respect to previous reference standard, PRS). Discordances between positive initial FDG PET/CT and negative BMB ("false positives" by PRS) occurred in 136 patients (15%). If only BMB is used as in the PRS, the negative predictive value (NPV) of FDG PET/CT was 709/745=95% (95% confidence interval [95%CI]: 93%-97%) with a sensitivity (Sens) of 58% (95%CI: 46%-68%) and a specificity (Spec) of 84% (95%CI: 81%-86%). After discussion of these cases, 185 out of 221 cases with BM involvement were identified as true positive, resulting in a Sens of 84% for FDG PET/CT (95%CI: 78%-88%). By means of FDG PET/CT 745 cases were negative of which 709 were confirmed as true negatives, resulting in an NPV of 95% (95%CI: 93%-97%). All 185 cases with positive FDG PET/CT were evaluated as true positive for BM involvement in the unblinded synopsis of all findings, resulting in a positive predictive value (PPV) of 100% (95%CI: 98%-100%). All 709 negative FDG PET/CT findings were finally confirmed as such, so Spec was 100% (95%CI: 99%-100%). Thus, the prevalence in our total cohort analyzed was 24%, since 221 out of 930 cases had confirmed BM involvement. Diagnostic performance parameters for BMB as compared to the newly defined gold standard were Sens 85/221=38% (95%CI: 32%-45%), Spec 709/709=100% (95%CI: 99%-100%), PPV 85/85=100% (95%CI: 96%-100%) and NPV 709/845=84% (95%CI: 81%-86%), respectively. Differences between the PRS in the diagnosis of BM involvement by only BMB and the newly defined gold standard result mainly from false negative BMB due to sampling errors, which are detected by FDG PET/CT. DISCUSSION: In the majority of patients with aggressive B-cell lymphoma, routine BMB does not give any additional relevant diagnostic or prognostic information over FDG PET/CT alone and could therefore be omitted. BMB should only be performed if results would have direct therapeutic impact e.g. in patients with limited stage disease and lack of further risk factors according to the international prognostic index (IPI). Disclosures Held: MSD: Consultancy; Bristol-Myers Squibb: Consultancy, Other: Travel support, Research Funding; Roche: Consultancy, Other: Travel support, Research Funding; Amgen: Research Funding; Acrotech: Research Funding. Stilgenbauer:Gilead: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; AbbVie: Consultancy, Honoraria, Research Funding, Speakers Bureau; GSK: Consultancy, Honoraria, Research Funding, Speakers Bureau; Hoffmann La-Roche: Consultancy, Honoraria, Research Funding, Speakers Bureau; AstraZeneca: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau. Duehrsen:Amgen: Consultancy; University Hospital Essen, Germany: Employment; Takeda: Consultancy; Novartis: Consultancy; Gilead: Honoraria; Janssen: Honoraria; Gilead: Consultancy; AbbVie: Consultancy; Celgene: Research Funding; CPT: Consultancy; Takeda: Honoraria; Novartis: Honoraria; Amgen: Research Funding; Roche: Research Funding; AbbVie: Honoraria; Amgen: Honoraria; CPT: Honoraria. Hüttmann:University Hospital Essen: Employment; Takeda: Honoraria; Gilead: Honoraria. Poeschel:Amgen: Other: Travel, accommodations, expenses; Abbvie: Other: Travel, accomodations, expenses; Roche: Other: Travel, accomodations, expenses; Hexal: Speakers Bureau.