ELIANA and ENSIGN were phase 2, single-arm, multicenter trials of tisagenlecleucel in children and young adults with relapsed or refractory B-ALL. The trials had similar designs, eligibility criteria, and dosing, permitting pooling of data for characterization of tisagenlecleucel safety.

Eligibility criteria included age ≥3 years at screening and ≤21 years at initial diagnosis, relapsed/refractory B-ALL (second or greater bone marrow relapse, or any bone marrow relapse after allogeneic hematopoietic stem cell transplant (HSCT) and ≥6 months from HSCT at time of infusion, or refractory disease defined as not achieving CR after two cycles of standard chemotherapy after initial diagnosis or one cycle of standard chemotherapy after relapse), and ≥5% lymphoblasts in bone marrow at screening. Patients with active central nervous system (CNS) leukemia involvement defined as CNS-3 were excluded from ELIANA. In ENSIGN, patients with active CNS-3 (except for CNS parenchymal/ocular disease, cranial nerve involvement, or significant leptomeningeal disease) were eligible if reduced to CNS-1/2 and there was documented evidence of disease stabilization for ≥3 months preinfusion.

The study designs comprised screening; pretreatment (leukapheresis, tisagenlecleucel product preparation, bridging chemotherapy per physician discretion, and protocol-defined lymphodepleting chemotherapy); tisagenlecleucel infusion; and primary, secondary, and survival follow-up.

Leukapheresis material was collected from patients, cryopreserved within 24 hours, and shipped to the central manufacturing facilities. Tisagenlecleucel was manufactured ex vivo using autologous T cells (described previously).26 27 On final product release testing, cryopreserved tisagenlecleucel cells were shipped to clinical sites for infusion. In both studies, the protocol-specified dose range was 0.2×10⁶‒5.0×10⁶ CAR-positive viable T cells/kg for patients with ≤50 kg body weight, and 0.1×10⁸‒2.5×10⁸ CAR-positive viable T cells for patients >50 kg.4 28 Multidisciplinary teams at clinical trial sites completed extensive training and education on leukapheresis, tisagenlecleucel-specific product infusion, patient logistics, and AE management.

The safety population included all patients enrolled in ELIANA or ENSIGN who received tisagenlecleucel infusion. AEs that started or worsened after informed consent were recorded and assessed throughout the study until initiation of new therapy. After new therapy was initiated, AE collection was focused on the monitoring of delayed AEs per health authority guidance for AEs/serious AEs (ie, severe AEs potentially related to tisagenlecleucel, new incidence or exacerbation of pre-existing neurological disorders, rheumatologic or other autoimmune disorders, etc). Safety assessment and collection was the same for both trials and is continuing for up to 15 years postinfusion.

AEs were assessed per the Common Terminology Criteria for Adverse Events, V.4.03,29 except for CRS (graded according to the University of Pennsylvania grading scale),21 22 and graft-versus-host disease (GVHD; graded using protocol-defined criteria).30 31 Serious AEs were defined per protocol (online supplemental file). Certain AEs (CRS, febrile neutropenia, prolonged cytopenias based on hematologic laboratory parameters, infections, non-infectious neurologic events, and tumor lysis syndrome) occurring ≤8 weeks postinfusion were considered to be of interest based on experience from ongoing tisagenlecleucel clinical studies.23 25 CRS, neurologic events, and neurologic episodes were defined per protocol (online supplemental file), and CRS was managed according to a protocol-specified algorithm (online supplemental table 1). Neurologic events were managed by the treating physician; the studies did not include a recommendation for corticosteroids. Prolonged cytopenias are defined as grade 3/4 cytopenias that are not resolved to grade ≤2 by day 28 postinfusion. Cytopenias occurring before tisagenlecleucel infusion were considered to be persistent if all reported laboratory results prior to infusion indicated grade 3/4 severity, with the last planned assessment occurring prior to the administration of lymphodepleting chemotherapy.

Duration of events was analyzed using the Kaplan-Meier method with unresolved events censored using the data cut-off date, death date, or discontinuation date, whichever came first. Nominal p values were calculated using χ² tests for post hoc subgroup analyses, without adjusting for multiplicity. Time to resolution of grade 3/4 cytopenias to grade ≤2 were analyzed via the Kaplan-Meier method with unresolved events censored at last assessment, and 95% CIs were calculated using the log–log transformation within PROC LIFETEST (SAS V.9.3). Time from onset of remission to B cell recovery (≥1% CD19-positive cells in viable white cell count or ≥3% among lymphocytes in peripheral blood) was analyzed by the Kaplan-Meier method with continuing B cell aplasia censored at last assessment.

Patient disposition and dosing in ELIANA (data cut-off: April 13, 2018) and ENSIGN (data cut-off: October 6, 2017) are described in table 1; demographics and baseline characteristics are shown in table 2. This analysis included 137 patients who received tisagenlecleucel infusion at a median of 43 days after enrollment and with a median follow-up of 24 months (table 1).

Most patients (77%) experienced grade 3/4 AEs suspected to be related to tisagenlecleucel. The most common grade 3/4 tisagenlecleucel-related AEs were CRS (42%) and febrile neutropenia (28%). A complete listing of common (>20%) AEs suspected to be related to tisagenlecleucel is provided in online supplemental table 2. Certain AEs, such as grade 4 CRS, grade 3/4 neurologic events, and grade 3/4 infections, are of specific interest in this patient population (table 3). These complications were common in the first 8 weeks from infusion: grade 4 CRS developed in 22% of patients, grade 3/4 neurologic events occurred in 10% of patients, and grade 3/4 infections occurred in 19% of patients. There was no convincing evidence that the likelihood of these events depended on patient age, sex, or if the patient had previously undergone allogeneic HSCT (table 4). There were three patients who died due to AEs that were possibly related to tisagenlecleucel (cerebral hemorrhage in the setting of coagulopathy on day 15, systemic candidiasis associated with prolonged pancytopenia on day 62, and human herpesvirus-6 (HHV-6) encephalitis associated with a history of prolonged neutropenia and lymphopenia on day 53). An additional 35 (26%) patients died from leukemia progression and 5 (4%) patients died after treatment with additional anticancer therapy (table 1).

CRS occurred in 108 (79%) patients (table 3), with all but three (3%) of cases occurring within 14 days of infusion. The median time to CRS onset was 3 (range: 1–22) days and to resolution was 8 (range: 1–36) days. The median time to resolution of grade 3/4 CRS was 10 (range: 4–36) days (table 5). CRS recurred in four patients 2–5 days after the resolution of the first episode and at the same grade (grades 2–3) as the first episodes. CRS was managed using a protocol-specified algorithm (online supplemental table 1). Anti-cytokine therapy, including tocilizumab, was administered in 44 (41%) patients. Of the 43 patients who received tocilizumab, 42 (98%) had grade 3–4 CRS and 1 patient had grade 2 CRS. In the patients who received tocilizumab, the median onset of CRS was 2 (range: 1–22) days, median time to administration of tocilizumab from onset of CRS was 5 (range: 1–19) days, and the median duration of CRS was 11 (range: 5–33) days. There were no deaths attributed to CRS.

Grade 4 CRS occurred in 27% (25/94) of patients who had ≥50% bone marrow blasts at the time of enrollment and in 12% (5/43) of patients with <50% blasts (p=0.081), but these results need to be interpreted cautiously as tumor burden at the time of tisagenlecleucel infusion was not assessed and patients received bridging treatment between enrollment and infusion. Hypotension that required intervention developed in 56% of patients with CRS and high dose vasopressors were used in 31%. Fifty per cent of patients received oxygen supplementation and 17% were intubated. Hypofibrinogenemia was seen in 8% (9/108) of patients with CRS; hypofibrinogenemia occurred more frequently and was most severe in patients with grade 4 CRS (online supplemental table 3).

Within 8 weeks after infusion, 50 patients (36%) experienced 55 neurologic episodes, defined as overlapping or successive neurologic events, at a median of 8 (range: 2‒53) days from infusion. Common neurologic toxicities during this period are listed in table 3. The median time to resolution was 6 days for episodes of any grade and 10.5 days for grade 3/4 episodes. Most (91% (50/55)) of these episodes occurred in patients who also developed CRS but not necessarily at the same time. Three patients developed neurologic symptoms up to 2 days prior to CRS, 35 patients developed neurologic symptoms during CRS (median time to onset, 7 (range: 2‒29) days postinfusion), and 9 patients developed neurologic symptoms after resolution of CRS. The post-CRS symptoms developed at a median of 5 (range: 1‒39) days after CRS resolution. The time of onset of CRS and neurologic events in patients who experienced both events are shown in figure 1.

Neurologic events occurring ≤8 weeks postinfusion correlated with CRS severity (p<0.001; online supplemental figure 1 and table 4). These neurologic events occurred in 57% (13/23) of patients with a history of preinfusion neurologic events (eg, central nervous system hemorrhage, cerebrovascular accident, and non-infectious encephalopathy/delirium in medical history) versus 32% (37/114) of patients without such disease history (p=0.051). However, neither a prior diagnosis of CNS leukemia (yes: 37% vs no: 37%) nor prior history of CNS radiotherapy (yes: 31% vs no: 42%) correlated with frequency of neurologic events (although it should be noted that patients with CNS-3 leukemia were excluded). Neurologic events occurring >8 weeks postinfusion were rare (occurring in 4.3% of 116 patients >8 weeks to 1 year postinfusion and in 1.7% of 59 patients >1 year postinfusion). Neurologic events were managed with supportive care and anticonvulsants (online supplemental table 5).

All patients experienced cytopenias postinfusion. Median time to resolution of grade 3/4 cytopenia 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 (figure 2). Prolonged thrombocytopenia was more common in patients with thrombocytopenia before infusion compared with those who had normal platelet counts at the time of infusion (11/14 (79%) vs 47/123 (38%); p=0.009). Prolonged neutropenia was also more common in patients who were neutropenic prior to infusion compared with patients who were not (24/31 (77%) vs 57/106 (54%); p=0.03).

B cell aplasia was seen in all patients with CR/CR with incomplete hematologic recovery (CRi) and was managed with immunoglobulin replacement therapy per institutional guidelines. An estimated 66% of evaluable patients in ongoing response at both 12 and 24 months continued to have B cell aplasia (online supplemental figure 2). Since immunoglobulin replacement was often initiated prior to observation of hypogammaglobulinemia in patients with B cell aplasia, the rate of hypogammaglobulinemia (57/237 patients (42%)) is lower than that of B cell aplasia.

Infections developed in 49/137 (36%) patients ≤4 weeks postinfusion (grade 3/4, 14%) and in 18/126 (14%) patients 4‒8 weeks postinfusion (grade 3/4, 8%; table 3). Twenty-eight of 81 (35%) patients with prolonged grade 3/4 neutropenia developed grade 3/4 infections after day 28. Three patients in ELIANA had fatal infections (systemic candidiasis on day 62 in the setting of prolonged grade 3/4 pancytopenia, HHV-6 encephalitis on day 53 in the setting of prolonged grade 3/4 neutropenia and lymphopenia, and lower respiratory tract infection on day 506 following treatment with additional anticancer therapy). Among those with ≥1-year follow-up, 7/59 patients (12%) reported grade 3/4 infections ≥1 year postinfusion in the absence of leukemia relapse, all of which resolved. These include respiratory infections (upper respiratory tract infection, respiratory syncytial viral bronchiolitis, and pneumonia), otitis media, bacterial meningitis (pneumococcal), herpes zoster, sepsis, and Campylobacter and Clostridium difficile infection. All patients were receiving immunoglobulin. The case of bacterial meningitis occurred in a patient on regular immunoglobulin replacement and was caused by a pneumococcal serotype not covered by the 23 polyvalent vaccine.

Cardiac toxicities primarily developed during the first 8 weeks of the study (43/137, 31%; grade 3/4, 7%) and were rare thereafter (7/116 (6%) from 8 weeks to 1 year postinfusion; none thereafter). The most common cardiac toxicity was tachycardia (26%; online supplemental table 6); cardiac events of interest included arrhythmia, cardiac dysfunction, valvular dysfunction, and pericardial effusion. Importantly, cardiac events were transient and resolved in all survivors with the exception of one patient who had ongoing left ventricular dysfunction at the time of last contact.

Acute kidney injury developed in 32/137 (23%) patients (grade 3/4, 14%) and was largely transient. Twelve patients required dialysis during CRS (table 5). Reversible grade 3/4 liver function abnormalities including increased levels of aspartate aminotransferase (16%; all had CRS), alanine aminotransferase (14%; all had CRS), and total bilirubin (9%; 11/12 had CRS) were reported.

Among the 74 patients with prior HSCT, GVHD was uncommon following tisagenlecleucel infusion. Two cases (3%) were reported, including a case of grade 1 skin GVHD on day 26, and a case of grade 2 gastrointestinal GVHD on day 73, both of which resolved.

No replication-competent lentivirus was detected in any patient postinfusion. Secondary malignancies were reported in three patients, one with an underlying TP53 germline mutation who later developed glioblastoma multiforme and two with myelodysplastic syndrome (one of which was transient and completely resolved within 3.5 months). No cases of secondary malignancy related to insertional mutagenesis were observed.