Introduction Mature B-cell lymphoma (MBL) accounts for approximately 60% of all non-Hodgkin lymphoma (NHL) in children and adolescents (Cairo et al, Blood, 2007). MBL in children and adolescents is comprised of approximately 75-80% Burkitt lymphoma (BL), 15-20% diffuse large B cell lymphoma (DLBCL) and 1-2% primary mediastinal B-cell lymphoma (PMBL). However, despite the differences in histology they are treated uniformly in children and adolescents (Cairo, BJH, 2016). The use of short and intense multiagent chemotherapy alone and in combination with rituximab (R) results in ≥90% event-free survival (EFS) in children and adolescents with BL and DLBCL (Cairo et al, JCO, 2012; Goldman/Cairo Leukemia, 2013; BJH, 2014). However, similar chemotherapy approaches in adolescents and young adults with PMBL have only resulted in a 60-70% EFS (Gerrard/Cairo et al, Blood, 2013). Furthermore, patients with induction failure, recurrent or refractory MBL have a dismal prognosis with ≤30% overall survival (OS) with current re-induction platforms and autologous stem cell transplantation (Cairo et al, BJH, 2018). Newer therapeutic strategies are urgently needed for recurrent/refractory patients with MBL, especially those with the BL subtype and newly diagnosed and refractory patients with PMBL (Cairo, BJH, 2019). We previously demonstrated that Obinutuzumab (O), a new type II anti-CD20 monoclonal antibody, is an active agent against both BL and PMBL (Awasthi/Cairo et al, BJH, 2015). Additionally, we have previously demonstrated high expression of CD79b in children and adolescents with BL and PMBL (Miles/Cairo et al, BJH 2007). Polatuzumab vedotin (PV), an anti-CD79b antibody-drug conjugate, recently approved by the FDA in adults with recurrent/refractory DLBCL has been demonstrated to be an active agent alone and in combination with chemotherapy in all molecular forms of adult DLBCL (Pfeifer et al, Leukemia, 2015; Corrinna et al, Lancet Oncology, 2015; Morschauser et al, Lancet Onc, 2019; Tilly et al, Lancet Onc, 2019). However, the efficacy of PV alone or in combination with R or O against other subtypes of B-NHL, including BL and PMBL, is relatively unknown. Objective To determine the in-vitro cytotoxicity and changes in in-vivo survival in a BL NSG xenograft model of PV alone or in combination with O vs R against R-sensitive and R-resistant BL and PMBL. Methods We compared the cytotoxicity of PV±R (10 ug/ml) vs PV±O (10 ug/ml) against R-sensitive BL (Raji) and R-resistant BL (Raji 4RH) and PBML (Karpas-1106p) alone or with ex-vivo expanded NK cells (Chu/Cairo et al, Can Imm Res, 2015) (10:1 E:T) by the DELFIA cytotoxicity assay. PV and O were generously supplied by Genentech and Roche, respectively. Furthermore, NSG mice (NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ) were xenografted with luciferase positive BL (Raji, Raji 4RH) and PMBL cells (Karpas-1106p) and treated with PV (5 mg/kg) + O (20 mg/kg) vs PV + R (20 mg/kg) vs PV vs IgG isotype control vs PV + O + NK vs PV + R + NK vs PV + NK vs O + NK vs R + NK vs PBS (without tumor) and tumor burden was compared between treatment groups using the IVIS bioluminescence system and probability of OS by KM method as we have previously described (Chu/Cairo et al, Can Imm Res, 2015). Results PV + O + NK induced significantly more in-vitro cytotoxicity against BL Raji compared to PV + R + NK (p<0.007) vs PV, R or O + NK (p<0.0001), BL (Raji 4RH [R-resistant BL]) PV + O vs PV + R (p<0.004) and O, R or PV (p<0.002) and against PMBL (Karpas-1106p) PV + O + NK vs PV + R + NK (p<0.006) vs O, R, or PV + NK (p<0.001) (Fig. 1A, 1B, 1C, respectively). Most importantly, PV + O + NK vs PV + R + NK vs PV + NK + IgG isotype control significantly enhanced probability of survival in BL (Raji) and BL (Raji 4RH) NSG xenografts (p<0.03 and (p<0.05), (95.5 vs 50.4 days) (185 vs 47 days), respectively (Fig. 2A and 2B, respectively). Conclusions PV alone and in combination with O significantly induces cytotoxicity against BL, R-resistant BL and PMBL. Most importantly, PV + O + NK cells result in a significantly improved OS in BL and R-resistant BL NSG xenografts. These preclinical results suggest that PV should be considered for investigation in patients with both recurrent/refractory BL and PMBL and in patients with newly diagnosed disease with either high risk features or those with poor responses to induction therapy. Authors AA, MB & AA are all considered co-primary first authors Disclosures Klein: Roche: Employment, Equity Ownership, Patents & Royalties. Cairo:Jazz Pharmaceuticals: Other: Advisory Board, Research Funding, Speakers Bureau; Osuka: Research Funding; Miltenyi: Other: MTA.

Primary mediastinal large B-cell lymphoma (PMBL), a distinct mature B-cell lymphoma, expresses CD20 and has recently been successfully treated with the combination of a type I anti-CD20 monoclonal antibody, rituximab, with multiple combination chemotherapy regimens. Obinutuzumab is a glycoengineered type II anti-CD20 monoclonal antibody (mAb), recognizing a unique CD20 extracellular membrane epitope with enhanced antibody dependent cellular cytotoxicity (ADCC) vs rituximab. We hypothesize that obinutuzumab vs rituximab will significantly enhance in-vitro and in-vivo cytotoxicity against PMBL. PMBL cells were treated with equal dose of obinutuzumab and rituximab for 24 hours (1-100 μg/ml). ADCC were performed with ex-vivo expanded natural killer cells at 10:1 E: T ratio. Mice were xenografted with intravenous injections of luciferase expressing Karpas1106P cells and treated every 7 days for 8 weeks. Tumor burden was monitored by IVIS spectrum system. Compared with rituximab, obinutuzumab significantly inhibited PMBL cell proliferation (p = 0.01), promoted apoptosis (p = 0.05) and enhanced ADCC (p = 0.0002) against PMBL. Similarly, in PMBL xenografted NOD scid gamma mice, obinutuzumab significantly enhanced survival than rituximab when treated with equal doses (p = 0.05). Taken together our results suggest that obinutuzumab significantly enhanced natural killer cytotoxicity, reduced PMBL proliferation and prolonged the overall survival in humanized PMBL xenografted NOD scid gamma mice.

Background: Childhood, Adolescent and Young Adult (CAYA) B-NHL represents the third most common malignancies in children under the age of 15yrs (Hochberg/Cairo et al, BJH 2009; Miles/Cairo, BJH. 2012).The prognosis of mature-B-NHL (which include BL/PMBL) has significantly improved over the last 40 years through the use of short and intense multi-agent chemo-immunotherapy, however; a subset of patients with relapsed/refractory disease has chemoimmunotherapy resistant disease and a dismal prognosis (≤ 20% 5 yr. EFS, Cairo et al.Blood. 2007; Cairo et al. JCO.2012, Goldman/Cairo et al. Leukemia, 2013, Gerrard/Cairo et al. Blood. 2013). It is therefore critical to investigate and develop targeted translational strategies in BL/PMBL to reduce acute morbidities, decrease late effects, and provide new options for those with recurrent disease. Blinatumomab, targeting CD19, has demonstrated encouraging clinical activity against pre-B-ALL (Topp et al, Leukemia, 2018) and highly expressed in BL/PMBL. Objectives: To determine in-vitro activity of blinatumomab against rituximab sensitive/resistant BL and PMBL cell lines. Methods: BL; Raji, Raji-4RH, and PMBL: Karpas1106p/MedB-1 were cultured in RPMI with 10 or 20% FBS. Tumor cells were incubated with/without blinatumomab (generously supplied by Amgen) for 4 hr. with T-cells. CD3+ isolated and expanded T-cells were used for cytotoxicity assay. Cytotoxicity was determined by DELFIA®cytotoxicity assay at 10:1 E: T ratio and cytokines secretion was measured by multiples ELISA kit. CD3+/CD107a+, granzyme b and perforin T-cell expression level were measured by flow-cytometry. Results: Blinatumomab+T-cells compared to T-cells only elicited a significant increased cytotoxicity against, Raji 58.18±7.6% vs 27.3.81±11.2% (p=0.007), Raji-4RH 67.4±8.0% vs 27.6±2.5%, (p=0.004), Karpas1106p, 75.7±3.06 % vs. 17.86± 4.82% (p=0.003) and MedB1, 65.17±13.3% vs18.1±2.03%, respectively (p=0.05). Blinatumomab treated T-cells also increased IFN-γ secretion compared to IL2-T cells, Raji 1 (p=0.002) and Raji-4RH (p=0.02) respectively. Furthermore, CD107a, granzyme b and perforin expression were significantly enhanced with blinatumomab treated/activated T-cells against; Raji (p=0.03, p=0.003 & p=0.009), Raji-4RH (p=0.03, p=0.02 & p=0.03) and Karpas1106p (p=0.006, p=0.03 & p=0.02) respectively. Conclusion: Blinatumomab significantly enhances T-mediated in-vitro cytotoxicity and cytokine secretion against BL/PMBL. Further, blinatumomab treated and activated T-cells significantly enhanced CD107a, granzyme b and perforin expression. These preliminary studies demonstrate that BiTE (CD3/CD19) would be a novel agent to investigate as immunotherapy therapy in patients with relapse refectory BL/PMBL. Citation Format: Aradhana Awasthi Tiwari, Dina Edani, Janet Ayello, Mitchell S. Cairo. Blinatumomab enhanced anti-tumor activity against rituximab sensitive and resistant Burkitt Lymphoma (BL) and Primary Mediastinal B-cell Lymphoma (PMBL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2381.

Background: Natural killer (NK) cell allo-reactivity in haploidentical allogeneic hematopoietic cell transplantation (HCT) is associated with lower relapse without greater graft-versus-host disease (GVHD). Dimorphisms of the MHC class I chain-related gene A (MICA) alleles can influence NK cell triggering and T cell modulation. Studies evaluating donorrecipient MICA incompatibilities on outcomes after matched unrelated donor HCT show conflicting results, but its significance is unknown in haploidentical HCT. We hypothesized donor-recipient MICA incompatibilities may influence outcomes in haploidentical HCT. Methods: We conducted a single center, retrospective analysis of all haploidentical HCT for hematologic malignancies in which MICA data was available. Outcomes were compared between those with and without donor-recipient MICA matches with Cox analysis or Fine and Gray regression. A secondary analysis examined dimorphisms at the MICA-129 position, which has been categorized as weak (valine/valine: V/V), mixed (methionine/valine: V/M), or strong (methionine/methionine: M/M) receptor binding affinity. Results: From 2014 -2016, 38 patients underwent a haploidentical HCT, with 14 in the MICA matched cohort and 24 in the MICA mismatched cohort with no significant differences in baseline characteristics. Diagnoses included acute myeloid (N = 17) and lymphoblastic leukemia (N = 7), myelodysplastic syndromes (N = 4), and others (N = 10). Median age at HCT was 51 years (range, 7-72), with 79% Caucasian. Myeloablative HCT was performed in 53% of patients and 55% had a bone marrow graft source. Most patients received tacrolimus and mycophenolate with post-transplant cyclophosphamide as GVHD prophylaxis. There were no differences in HLA disparities between the two groups (P = .64). There was a trend towards greater acute GVHD in the MICA mismatched cohort (HR 3.06, P = .07) ( Figure 1). The MICA mismatched group also had a trend towards a lower risk of relapse mortality (HR .28, P = .06) ( Figure 2). There was no significant difference in relapse-free survival (HR .62, P = .32), nonrelapse mortality (HR 1.52, P = .63), or overall survival (HR .56, P = .24). The most common cause of death was relapse. In secondary analysis examining the MICA-129 genotype, patients with a V/V donor had higher risk of chronic GVHD (P = .006), though sample size was limited to 4 patients. No other outcome differences were seen relative to MICA-129 genotype. Conclusion: We conclude that donor-recipient MICA incompatibility may be associated with a higher incidence of acute GVHD after haploidentical HCT consistent with previous

Background: Mature B-NHL, including Burkitt lymphoma (BL) and primary mediastinal large B cell lymphoma (PMBL) express CD20+/CD79b+ and have an excellent prognosis, however, subset of patients relapse secondary to chemoimmunotherapy resistant disease and have a dismal prognosis (≤ 20% 5 yr. EFS, Cairo et al. Blood. 2007; Gerrard/Cairo et al., Blood, 2013, Goldman/Cairo et al. Leukemia, 2013). PV has been demonstrated to possess significant preclinical activity against indolent CD79b+NHL (Polson et. al.Can. Res.2009). We previously observed that obinutuzumab (Anti-CD20 mAb) significantly enhanced cell death and increased overall survival against BL (Awasthi/Cairo et al., BJH 2015) in xenografted NSG mice. However, additive/synergistic effects of PV with obinutuzumab against mature PMBL/BL are unknown. Objective: To determine the efficacy of the PV or obinutuzumab/RTX alone or in combination against PMBL and rituximab (RTX) sensitive/resistant BL cell lines. Methods: Raji4RH (provided by M. Barth, MD, Roswell Park Cancer Institute) and Raji/ Karpas1106P (ATCC, USA) were cultured in RPMI. Tumor cells were incubated with PV, and/or anti-CD79b, MMAE (generously supplied by Genentech Inc.) with obinutuzumab /rituximab (100ug/ml) for 4 hr with NK cells at 10:1 E: T ratio and cytotoxicity was determined by DELFIA cytotoxicity assay. Six to 8 week old female NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ), were divided into 5 groups: PBS, isotype control, PV, antiCD79B mAb and MMAE (5mg/kg). Mice were xenografted with intravenous injections of Luc+ BL and PMBL cells and tumor burden was monitored by IVIS spectrum system. Results: OS of mice receiving PV alone was significantly increased compared to antiCD79b or isotype control in Raji (35.5 vs.17 vs.19.5 days, p=0.0001, 0.0003), Raji4RH (50 vs.18 vs.18.5 days, p=0.0001, 0.0001) and Karpas1106P (150 vs 89 vs 64 days, p=0.03, 0.003), respectively. Obinutuzumab+NK, rituximab+NK compared to PV+NK cells significantly enhanced cell lysis in Raji, 65.9±2.4% vs. 38.9±5.4% vs. 44.24±8.1% (p=0.001 & p=0.001), Raji4RH, 52.8±9.4% vs. 16.04±7.2% vs.47.0±8.2% (p=0.03 & p=NS) and Karpas1106P, 66.10±5.3% vs.48.2±3.9% vs. 61.6±10.06% (p=0.004 & NS), respectively. PV+ obinutuzumab+NK, significantly improved cytotoxicity compared to PV+ rituximab+NK in Raji, 93.6±6.1% vs 79.9±5.3% (p=0.007), Raji4RH, 78.07±2.05% vs 63.5±0.16% (p=0.004) and Karpas1106P, 88.3±6.3 %vs.73.03±3.03% (p=0.003), respectively. Conclusion: Our preliminary data indicates that PV significantly increased survival in BL and PMBL NSG xenografts compared to anti-CD79b Ab alone. Furthermore, PV in combination with obinutuzumab significantly enhances cytotoxicity in BL and PMBL compared to obinutuzumab or PV alone. Citation Format: Aradhana Awasthi Tiwari, Dina Edani, Christeen Azmy, Janet Ayello, Christian Klein, Mitchell S. Cairo. Enhanced in vitro/in vivo cytotoxicity against Burkitt lymphoma/primary mediastinal large B cell lymphoma by polatuzumab vedotin (hu- anti-CD79b-vc-MMAE, PV) alone or in combination with obinutuzumab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1788.