Abstract: Clinical trials frequently include multiple end points that mature at different times. The initial report, typically based on the primary end point, may be published when key planned co-primary or secondary analyses are not yet available. Clinical Trial Updates provide an opportunity to disseminate additional results from studies, published in JCO or elsewhere, for which the primary end point has already been reported. In the primary analysis of the global phase II ELIANA trial (ClinicalTrials.gov identifier: NCT02435849 ), tisagenlecleucel provided an overall remission rate of 81% in pediatric and young adult patients with relapsed or refractory B-cell acute lymphoblastic leukemia (R/R B-ALL), with 59% of responders remaining relapse-free at 12 months. Here, we report an update on efficacy, safety, and patient-reported quality of life in 79 pediatric and young adult patients with R/R B-ALL following a median follow-up of 38.8 months. The overall remission rate was 82%. The median event-free survival was 24 months, and the median overall survival was not reached. Event-free survival was 44% (95% CI, 31 to 57) and overall survival was 63% (95% CI, 51 to 73) at 3 years overall (most events occur within the first 2 years). The estimated 3-year relapse-free survival with and without censoring for subsequent therapy was 52% (95% CI, 37 to 66) and 48% (95% CI, 34 to 60), respectively. No new or unexpected long-term adverse events were reported. Grade 3/4 adverse events were reported in 29% of patients 〉 1 year after infusion; grade 3/4 infection rate did not increase 〉 1 year after infusion. Patients reported improvements in quality of life up to 36 months after infusion. These findings demonstrate favorable long-term safety and suggest tisagenlecleucel as a curative treatment option for heavily pretreated pediatric and young adult patients with R/R B-ALL.

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: Tisagenlecleucel, an anti-CD19 chimeric antigen receptor T cell therapy, has demonstrated efficacy in children and young adults with relapsed/refractory B cell acute lymphoblastic leukemia (B-ALL) in two multicenter phase 2 trials (ClinicalTrials.gov, NCT02435849 (ELIANA) and NCT02228096 (ENSIGN)), leading to commercialization of tisagenlecleucel for the treatment of patients up to age 25 years with B-ALL that is refractory or in second or greater relapse. Methods A pooled analysis of 137 patients from these trials (ELIANA: n=79; ENSIGN: n=58) was performed to provide a comprehensive safety profile for tisagenlecleucel. Results Grade 3/4 tisagenlecleucel-related adverse events (AEs) were reported in 77% of patients. Specific AEs of interest that occurred ≤8 weeks postinfusion included cytokine-release syndrome (CRS; 79% (grade 4: 22%)), infections (42%; grade 3/4: 19%), prolonged (not resolved by day 28) cytopenias (40%; grade 3/4: 34%), neurologic events (36%; grade 3: 10%; no grade 4 events), and tumor lysis syndrome (4%; all grade 3). Treatment for CRS included tocilizumab (40%) and corticosteroids (23%). The frequency of neurologic events increased with CRS severity (p 〈 0.001). Median time to resolution of grade 3/4 cytopenias to grade ≤2 was 2.0 (95% CI 1.87 to 2.23) months for neutropenia, 2.4 (95% CI 1.97 to 3.68) months for lymphopenia, 2.0 (95% CI 1.87 to 2.27) months for leukopenia, 1.9 (95% CI 1.74 to 2.10) months for thrombocytopenia, and 1.0 (95% CI 0.95 to 1.87) month for anemia. All patients who achieved complete remission (CR)/CR with incomplete hematologic recovery experienced B cell aplasia; however, as nearly all responders also received immunoglobulin replacement, few grade 3/4 infections occurred 〉 1 year postinfusion. Conclusions This pooled analysis provides a detailed safety profile for tisagenlecleucel during the course of clinical trials, and AE management guidance, with a longer follow-up duration compared with previous reports.

Abstract: Chimeric antigen receptor T-cell therapy (CAR-T) targeting CD19 has been associated with remarkable responses in paediatric patients and adolescents and young adults (AYA) with relapsed/refractory (R/R) B-cell precursor acute lymphoblastic leukaemia (BCP-ALL). Tisagenlecleucel, the first approved CD19 CAR-T, has become a viable treatment option for paediatric patients and AYAs with BCP-ALL relapsing repeatedly or after haematopoietic stem cell transplantation (HSCT). Based on the chimeric antigen receptor molecular design and the presence of a 4-1BB costimulatory domain, tisagenlecleucel can persist for a long time and thereby provide sustained leukaemia control. “Real-world” experience with tisagenlecleucel confirms the safety and efficacy profile observed in the pivotal registration trial. Recent guidelines for the recognition, management and prevention of the two most common adverse events related to CAR-T — cytokine release syndrome and immune-cell–associated neurotoxicity syndrome — have helped to further decrease treatment toxicity. Consequently, the questions of how and for whom CD19 CAR-T could substitute HSCT in BCP-ALL are inevitable. Currently, 40–50% of R/R BCP-ALL patients relapse post CD19 CAR-T with either CD19 − or CD19 + disease, and consolidative HSCT has been proposed to avoid disease recurrence. Contrarily, CD19 CAR-T is currently being investigated in the upfront treatment of high-risk BCP-ALL with an aim to avoid allogeneic HSCT and associated treatment-related morbidity, mortality and late effects. To improve survival and decrease long-term side effects in children with BCP-ALL, it is important to define parameters predicting the success or failure of CAR-T, allowing the careful selection of candidates in need of HSCT consolidation. In this review, we describe the current clinical evidence on CAR-T in BCP-ALL and discuss factors associated with response to or failure of this therapy: product specifications, patient- and disease-related factors and the impact of additional therapies given before (e.g., blinatumomab and inotuzumab ozogamicin) or after infusion (e.g., CAR-T re-infusion and/or checkpoint inhibition). We discuss where to position CAR-T in the treatment of BCP-ALL and present considerations for the design of supportive trials for the different phases of disease. Finally, we elaborate on clinical settings in which CAR-T might indeed replace HSCT.

Abstract: We assessed minimal residual disease (MRD) detection and B-cell aplasia after tisagenlecleucel therapy for acute lymphoblastic leukemia (ALL) to define biomarkers predictive of relapse (N = 143). Next-generation sequencing (NGS) MRD detection & gt;0 in bone marrow (BM) was highly associated with relapse. B-cell recovery [signifying loss of functional chimeric antigen receptor (CAR) T cells] within the first year of treatment was associated with a hazard ratio (HR) for relapse of 4.5 [95% confidence interval (CI), 2.03–9.97; P & lt; 0.001]. Multivariate analysis at day 28 showed independent associations of BMNGS-MRD & gt;0 (HR = 4.87; 95% CI, 2.18–10.8; P & lt; 0.001) and B-cell recovery (HR = 3.33; 95% CI, 1.44–7.69; P = 0.005) with relapse. By 3 months, the BMNGS-MRD HR increased to 12 (95% CI, 2.87–50; P & lt; 0.001), whereas B-cell recovery was not independently predictive (HR = 1.27; 95% CI, 0.33–4.79; P = 0.7). Relapses occurring with persistence of B-cell aplasia were largely CD19− (23/25: 88%). Detectable BMNGS-MRD reliably predicts risk with sufficient time to consider approaches to relapse prevention such as hematopoietic cell transplantation (HCT) or second CAR-T cell infusion. Significance: Detectable disease by BMNGS-MRD with or without B-cell aplasia is highly predictive of relapse after tisagenlecleucel therapy for ALL. Clonotypic rearrangements used to follow NGS-MRD did not change after loss of CD19 or lineage switch. High-risk patients identified by these biomarkers may benefit from HCT or investigational cell therapies. See related commentary by Ghorashian and Bartram, p. 2. This article is highlighted in the In This Issue feature, p. 1

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: Cytokine release syndrome (CRS) is a systemic inflammatory response associated with chimeric antigen receptor T-cell (CAR-T) therapies. In severe cases, CRS can be associated with coagulopathy and hypofibrinogenemia. We present our global multicenter experience with CRS-associated coagulopathy after tisagenlecleucel therapy in 137 patients with relapsed or refractory B-cell acute lymphoblastic leukemia from the ELIANA and ENSIGN trials. These trials included clinical guidelines for fibrinogen replacement during CRS-associated coagulopathy. Hypofibrinogenemia requiring replacement was observed only in patients with severe CRS. A higher percentage of patients who required replacement were & lt;10 years old, compared with those who did not require replacement. Twenty-three patients received replacement for hypofibrinogenemia ( & lt;1.5 g/L); 9 of them developed marked hypofibrinogenemia ( & lt;1 g/L). Very low fibrinogen levels ( & lt;1 g/L) were documented in patients before maximal CRS (n = 1), during maximal CRS (n = 7), and at CRS improvement (n = 1). Although hypofibrinogenemia was the most clinically significant coagulopathy, some patients also developed prolonged prothrombin time and activated partial thromboplastin time and increased international normalized ratio, further increasing the risk of bleeding. Hypofibrinogenemia was effectively managed using fibrinogen concentrate or cryoprecipitate replacement; severe (grade 4) bleeding events were rare (n = 2). CRS-associated coagulopathy with hypofibrinogenemia is manageable according to empiric guidelines of fibrinogen replacement for CAR-T trials. Fibrinogen concentrate should be used when cryoprecipitate is not reliably available. Monitoring fibrinogen levels in patients with moderate or severe CRS is essential for avoiding potentially fatal bleeding events. These trials were registered at www.clinicaltrials.gov as #NCT02435849 and #NCT02228096.