Abstract: Introduction Detection of minimal residual disease (MRD) is an important predictor of patient outcome following treatment of B-cell acute lymphoblastic leukemia (B-ALL). We assessed concordance between two MRD assays, with different assay sensitivities, to determine which MRD detection method could support early relapse detection. Immunoglobulin next generation sequencing (Ig NGS) and flow cytometry (FC) were tested in samples from two clinical trials ELIANA (NCT02435849) and ENSIGN (NCT02228096) for pediatric relapsed and refractory B-ALL patients treated with tisagenlecleucel. We also assessed whether using blood with Ig NGS would be comparable to BM testing with FC. Finally we analyzed whether clonal evolution, as detected by Ig NGS, occurred during of the course of therapy for both CD19+ and CD19- relapse patients. Methods In this analysis, bone marrow and peripheral blood specimens at screening (pre-tisagenlecleucel infusion), post-infusion and relapse were tested. Ig NGS was performed in 300 samples from 88 patients. 237 samples from 83 patients also had FC MRD results available. MRD was measured on fresh blood and bone marrow using a 3-tube FC assay (CD10, CD19, CD13, CD20, CD22, CD33, CD34, CD38, CD45, CD58, CD123). The FC MRD assay has a lower limit of sensitivity of 0.01% of white blood cells. Ig NGS detection of MRD was performed using the Adaptive Biotechnology's NGS MRD assay. MRD quantitative values, along with the qualitative MRD calls at each assay sensitivity level (10-4, 10-5 and 10-6) were reported. At baseline, 85 out of 88 samples had informative clones. Results and Conclusions To examine the comparability of flow cytometry and Ig NGS methods in assessing MRD, baseline and post-treatment samples were tested. Baseline samples, which had a high disease burden, showed 100% MRD concordance between both assays. However, post-treatment, where the leukemic burden was dramatically reduced, Ig NGS detected a greater number of MRD positive samples compared to FC, at each sensitivity level tested (10-4, 10-5 and 10-6). At the highest sensitivity level of 10-6, Ig NGS was able to detect 18% more MRD positive post-treatment samples. Importantly, Ig NGS was able to detect MRD positivity 1-4 months ahead of clinical relapse in a small number of relapsed patients, whether relapse was CD19+ or CD19-. This may provide an important window of opportunity for pre-emptive treatment while a patients' tumor burden is still low. In B-ALL, it has previously been described that MRD levels can be one to three logs lower in blood compared to bone marrow (VanDongen JJ et al. Blood 2015). Our results support these findings whereby MRD burden in bone marrow was higher than in blood using both FC and Ig NGS. We next set out to determine if the increased sensitivity afforded by the Ig NGS assay could provide a level of MRD detection in the blood comparable to FC in the bone marrow. In patients with matching data available, Ig NGS was able to detect more MRD positive blood samples than FC MRD positive bone marrow samples. This suggests that monitoring of MRD using Ig NGS in the blood holds the potential to be used as a surrogate for FC MRD in bone marrow. The relationship between MRD and prognosis was examined. Patients who were MRD negative by both Ig NGS and FC at the end of first month post-infusion had better progression-free survival and overall survival compared to those with detectable MRD. Tumor clonality will be further analyzed to understand sub-clone composition at baseline and clonal evolution following tisagenlecleucel treatment. Taken together, these results highlight the importance of using a highly sensitive assay, such as Ig NGS, when monitoring for MRD. MRD detection by Ig NGS holds the potential to identify early response/relapse in patients, which could provide a window of opportunity for additional intervention before morphological relapse. Ongoing studies with larger patient groups will provide further information on the applicability of Ig NGS MRD detection and its association with long-term outcome in tisagenlecleucel-treated pediatric r/r B-ALL patients. Disclosures Pulsipher: Novartis: Consultancy, Honoraria, Speakers Bureau; CSL Behring: Consultancy; Amgen: Honoraria; Adaptive Biotech: Consultancy, Research Funding. Han:Novartis Pharmaceuticals Corporation: Employment, Equity Ownership. Quigley:Novartis Pharmaceuticals Corporation: Employment. Kari:Adaptimmune LLC: Other: previous employment within 2 years; Novartis Pharmaceuticals Corporation: Employment. Rives:Shire: Consultancy, Other: Symposia, advisory boards ; Amgen: Consultancy, Other: advisory board ; Novartis Pharmaceuticals Corporation: Consultancy, Other: Symposia, advisory boards ; Jazz Pharma: Consultancy, Other: Symposia, advisory boards . Laetsch:Bayer: Consultancy; Eli Lilly: Consultancy; Pfizer: Equity Ownership; Novartis Pharmaceuticals Corporation: Consultancy; Loxo Oncology: Consultancy. Myers:Novartis Pharmaceuticals Corporation: Consultancy, Honoraria, Research Funding, Speakers Bureau. Qayed:Novartis: Consultancy. Stefanski:Novartis Pharmaceuticals Corporation: Consultancy, Honoraria, Speakers Bureau. Baruchel:Shire: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy; Servier: Consultancy; Roche: Consultancy; Jazz Pharmaceuticals: Consultancy, Honoraria, Other: Travel, accommodations or expenses; Celgene: Consultancy. Bader:Cellgene: Consultancy; Riemser: Research Funding; Medac: Patents & Royalties, Research Funding; Neovii: Research Funding; Novartis: Consultancy, Speakers Bureau. Yi:Novartis Pharmaceuticals Corporation: Employment. Kalfoglou:Novartis Pharmaceuticals Corporation: Employment. Robins:Adaptive Biotechnologies: Consultancy, Employment, Equity Ownership, Patents & Royalties. Yusko:Adaptive Biotechnologies: Employment, Equity Ownership. Görgün:Novartis Pharmaceuticals Corporation: Employment. Bleickardt:Novartis Pharmaceuticals Corporation: Employment. Wong:Novartis Pharmaceuticals Corporation: Employment, Equity Ownership. Grupp:Novartis Pharmaceuticals Corporation: Consultancy, Research Funding; Jazz Pharmaceuticals: Consultancy; Adaptimmune: Consultancy; University of Pennsylvania: Patents & Royalties.

Abstract: A single-center trial of CD19 directed, lentiviral transduced chimeric antigen receptor (CAR) T cells (CTL019) for relapsed and refractory (r/r) B-ALL pediatric patients showed rates of CR 〉 90% with prolonged CAR T cell persistence/CR without further therapy in the majority of patients infused (Maude NEJM 2014). We report here the feasibility, safety and efficacy of the first multicenter global pivotal registration CAR T cell trial. Features of this trial include: i) the first trial in which industry-manufactured cells were provided to all patients; ii) enrollment across 25 centers in the US, EU, Canada, Australia, and Japan; iii) successful transfer and manufacturing of cells in a global supply chain; and iv) successful implementation of cytokine release syndrome (CRS) management across a global trial. All patients had CD19 positive B-ALL with morphologic marrow tumor involvement at registration ( 〉 5% blasts), and were either primary refractory; chemo-refractory after first relapse, relapsed after second line therapy; or ineligible for allogeneic SCT. CTL019 was manufactured from patient PBMC under GMP conditions in the US, at a centralized "sponsor-owned" manufacturing facility, and supplied to all sites. The primary endpoint of overall remission rate (CR+CRi) within 3 months and secondary endpoints (EFS, DOR, OS and safety) were assessed by an independent review committee. Based on preliminary data as of March 2016, 57 patients were enrolled. There were 3 manufacturing failures (5%), 5 patients were not infused due to death or adverse events (9%), and 15 patients were pending infusion at the data cut off. Following fludarabine/cyclophosphamide lymphodepleting chemotherapy in the majority of the patients, 34 patients (median age 11 [3-23], 50% with prior HSCT) were infused with a single dose of CTL019 at a median dose of 2.9 x106 transduced CTL019 cells/kg (0.2 to 4). Among 29 patients reaching D28 prior to the data cutoff, 83% (24/29) achieved CR or CRi by local investigator assessment, all of which were MRD-negative. Two early deaths occurred prior to initial disease assessment, one due to disease progression and one due to intracranial hemorrhage. Two patients did not respond. One patient was in CR by BM at D28, but CSF was not assessed, therefore this patient was classified as "incomplete" assessment. Safety was managed by a protocol-specified CRS algorithm with no cases of refractory CRS. Using the Penn CRS grading scale, 82% of patients experienced CRS, with 7 grade 3 (21%) and 8 grade 4 (24%) events. 44% patients with CRS required anti-cytokine therapy; all received tocilizumab with or without other anti-cytokine therapy, with complete resolution of CRS. Besides CRS, the most common grade 3 and 4 non-hematologic AEs were febrile neutropenia (29%), increased bilirubin (21%), increased AST (21%), and hypotension (21%). 21% of patients experienced grade 3 or 4 neuropsychiatric events including confusion, delirium, encephalopathy, agitation and seizure; no cerebral edema was reported. CTL019 in vivo cellular kinetics by qPCR demonstrated transgene persistence in blood in responding patients at and beyond 6 months. Overall exposure (AUC 0-28d) and maximal expansion (Cmax) of CTL019 DNA measured by qPCR was higher in responding compared with non-responding patients. In summary, this pivotal global study in pediatric and young adult patients with r/r B-ALL receiving CTL019, confirms a high level of efficacy and a similar safety profile to that shown in the prior single center experience. Safety was effectively and reproducibly managed by appropriately trained investigators. The study has completed accrual. At the meeting, updated data from a planned formal interim analysis including safety, efficacy (primary and selected secondary endpoints), cellular kinetics, and impact of anti-cytokine therapy will be presented for more than 50 patients infused at 25 global sites. Disclosures Grupp: Jazz Pharmaceuticals: Consultancy; Novartis: Consultancy, Research Funding; Pfizer: Consultancy. Laetsch:Novartis: Consultancy; Loxo Oncology: Consultancy. Bittencourt:Seattle Genetics: Consultancy; Jazz Pharmaceuticals: Consultancy, Other: Educational Grant. Maude:Novartis: Consultancy. Myers:Novartis Pharmaceuticals: Consultancy. Rives:Novartis: Consultancy; Jazz Pharma: Consultancy. Nemecek:Medac, GmbH: Research Funding; Novartis: Consultancy; National Marrow Donor Program: Membership on an entity's Board of Directors or advisory committees. Schlis:Novartis: Honoraria. Martin:Jazz Pharmaceuticals: Other: One time discussion panel; Novartis: Other: Support of clinical trials. Bader:Medac: Consultancy, Research Funding; Riemser: Research Funding; Neovii Biotech: Research Funding; Servier: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Peters:Novartis: Consultancy; Jazz: Speakers Bureau; Amgen: Consultancy; Pfizer: Consultancy; Medac: Consultancy. Biondi:Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Advisory Board; Cellgene: Other: Advisory Board; BMS: Membership on an entity's Board of Directors or advisory committees. Baruchel:Servier: Consultancy; Novartis: Consultancy; Celgene: Consultancy; Jazz: Consultancy; Baxalta: Research Funding. June:University of Pennsylvania: Patents & Royalties; Johnson & Johnson: Research Funding; Celldex: Consultancy, Equity Ownership; Pfizer: Honoraria; Immune Design: Consultancy, Equity Ownership; Novartis: Honoraria, Patents & Royalties: Immunology, Research Funding; Tmunity: Equity Ownership, Other: Founder, stockholder . Sen:Novartis: Employment. Zhang:Novartis: Employment. Thudium:Novartis: Employment. Wood:Novartis Pharmaceuticals: Employment, Other: Stock. Taran:Novartis: Employment. Pulsipher:Chimerix: Consultancy; Jazz Pharmaceutical: Consultancy; Novartis: Consultancy, Other: Study Steering Committee; Medac: Other: Housing support for conference.

Abstract: BACKGROUND Tisagenlecleucel is an FDA approved chimeric antigen receptor (CAR)-T cell therapy that reprograms T cells to eliminate CD19+ B cells. ELIANA (NCT02435849) is a phase 2 pivotal study of tisagenlecleucel in pediatric/young adult patients (pts) with CD19+ r/r B-cell acute lymphoblastic leukemia (ALL), the first global trial of a CAR-T cell therapy. The primary objective was met, with an overall remission rate (ORR) of 81% (complete remission [CR] + CR with incomplete blood count recovery [CRi] ). Here we present an update of ELIANA, with additional pts and additional 11 mo follow-up from the previous report (Maude et al. N Engl J Med 2018). METHODS Eligible pts were aged ≥3 y at screening and ≤21 y at diagnosis and had ≥5% leukemic blasts in bone marrow. Tisagenlecleucel was centrally manufactured at 2 sites (Morris Plains, NJ, USA and Leipzig, Germany) by lentiviral transduction of autologous T cells with a vector encoding for a second generation 4-1BB anti-CD19 CAR and expanded ex vivo. Tisagenlecleucel was provided to pts at 25 study centers in 11 countries on 4 continents using cryopreserved apheresed mononuclear cells, central production facilities, and a global supply chain. The primary endpoint, ORR within 3 mo and maintained for ≥28 d among infused pts, was assessed by an independent review committee. Secondary endpoints included duration of remission (DOR), overall survival (OS), safety, and cellular kinetics. RESULTS As of April 13, 2018, 113 pts were screened and 97 enrolled. There were 8 manufacturing failures (8%) and 10 pts (10%) were not infused due to death or adverse events (AEs). Following lymphodepleting chemotherapy in most pts (76/79; fludarabine/cyclophosphamide [n=75]), 79 pts were infused with a single dose of tisagenlecleucel (median dose, 3.0×106 [range, 0.2-5.4×106] CAR-positive viable T cells/kg), and all had ≥3 mo of follow-up or discontinued earlier (median time from infusion to data cutoff, 24 mo [range, 4.5-35 mo]). Median age was 11 y (range, 3-24 y); 61% of pts had prior hematopoietic stem cell transplant (SCT). Among the 65 pts with CR/CRi, 64 (98%) were MRD- within 3 mo. Median DOR by K-M analysis was not reached (Figure): responses were ongoing in 29 pts (max DOR, 29 mo and ongoing); 19 pts relapsed before receiving additional anticancer therapy (13 died subsequently); 8 pts underwent SCT while in remission, 8 received additional anticancer therapy (non-SCT) and 1 discontinued while in remission. The probability of relapse-free survival at 18 mo was 66% (95% CI, 52%-77%). Median OS was not reached; OS probability at 18 mo was 70% (95% CI, 58%-79%). Cytokine release syndrome (CRS) occurred in 77% of pts (grade [G] 3/4; 48%; graded using the Penn scale); 39% of pts received tocilizumab for treatment of CRS with or without other anti-cytokine therapies; 48% of pts required ICU-level care for CRS, with a median ICU stay of 7 d. All cases of CRS were reversible. Most common G 3/4 nonhematologic AEs ( 〉 15%) other than CRS were neutropenia with a body temperature 〉 38.3°C (62% within 8 wk of infusion), hypoxia (20%), and hypotension (20%). 13% of pts experienced G 3 neurological events, with no G 4 events or cerebral edema. Based on laboratory results, 43% and 54% of pts had G 3/4 thrombocytopenia and neutropenia not resolved by d 28; the majority of events resolved to G ≤2 by 3 mo. 25 post-infusion deaths were reported: 2 within 30 d (1 disease progression, 1 cerebral hemorrhage); 23 after 30 d of infusion (range, 53-859 d; 18 disease progression, 1 each due to encephalitis, systemic mycosis, VOD [hepatobiliary disorders related to allogeneic-SCT], bacterial lung infection, and an unknown reason after study withdrawal). Tisagenlecleucel expansion in vivo correlated with CRS severity, and persistence of tisagenlecleucel along with B-cell aplasia in peripheral blood was observed for ≥2.5 y in some responding pts. Analysis of B-cell recovery and correlation with relapse will be presented. CONCLUSIONS With longer follow-up, the ELIANA study continues to confirm the efficacy of a single infusion of tisagenlecleucel in pediatric and young adults with ALL without additional therapy. AEs were effectively and reproducibly managed globally by appropriately trained personnel at study sites. The achievement of high overall response rates and deep remissions, in combination with the median duration of response and overall survival not being reached, further corroborate previously reported results. Figure. Figure. Disclosures Grupp: Novartis Pharmaceuticals Corporation: Consultancy, Research Funding; Jazz Pharmaceuticals: Consultancy; Adaptimmune: Consultancy; University of Pennsylvania: Patents & Royalties. Maude:Novartis Pharmaceuticals Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees. Rives:Shire: Consultancy, Other: Symposia, advisory boards ; Jazz Pharma: Consultancy, Other: Symposia, advisory boards ; Novartis Pharmaceuticals Corporation: Consultancy, Other: Symposia, advisory boards ; Amgen: Consultancy, Other: advisory board . Baruchel:Celgene: Consultancy; Amgen: Consultancy; Roche: Consultancy; Jazz Pharmaceuticals: Consultancy, Honoraria, Other: Travel, accommodations or expenses; Novartis: Membership on an entity's Board of Directors or advisory committees; Shire: Research Funding; Servier: Consultancy. Bittencourt:Novartis Pharmaceuticals Corporation: Consultancy; Jazz Pharmaceuticals: Consultancy, Honoraria. Bader:Riemser: Research Funding; Cellgene: Consultancy; Medac: Patents & Royalties, Research Funding; Neovii: Research Funding; Novartis: Consultancy, Speakers Bureau. Laetsch:Bayer: Consultancy; Pfizer: Equity Ownership; Eli Lilly: Consultancy; Novartis Pharmaceuticals Corporation: Consultancy; Loxo Oncology: Consultancy. Stefanski:Novartis Pharmaceuticals Corporation: Consultancy, Honoraria, Speakers Bureau. Myers:Novartis Pharmaceuticals Corporation: Consultancy, Honoraria, Research Funding, Speakers Bureau. Qayed:Novartis: Consultancy. Pulsipher:CSL Behring: Consultancy; Amgen: Honoraria; Adaptive Biotech: Consultancy, Research Funding; Novartis: Consultancy, Honoraria, Speakers Bureau. Martin:Novartis Pharmaceuticals Corporation: Research Funding; Jazz Pharmaceuticals: Research Funding. Nemecek:Novartis Pharmaceuticals Corporation: Other: advisory boards. Boissel:Servier: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceuticals Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees. Leung:Novartis Pharmaceuticals Corporation: Employment. Eldjerou:Novartis Pharmaceuticals Corporation: Employment. Yi:Novartis Pharmaceuticals Corporation: Employment. Mueller:Novartis Institutes for Biomedical Research: Employment; Novartis Pharmaceuticals Corporation: Equity Ownership, Other: Patent pending. Bleickardt:Novartis Pharmaceuticals Corporation: Employment.