Quadriparesis and paraparesis following chimeric antigen receptor T-cell (CART) therapy in children and adolescents.

Immune effector cell-associated neurotoxicity syndrome (ICANS) is a common but potentially severe adverse event associated with chimeric antigen receptor T-cell (CART) therapy characterized by the development of acute neurologic symptoms following CART infusion. ICANS encompasses a wide clinical spectrum typified by mild to severe encephalopathy, seizures and/or cerebral edema. As more patients have been treated with CART new ICANS phenomenology has emerged. We present the clinical course of five children who developed acute onset of quadriparesis or paraparesis associated with abnormal brain and/or spine neuroimaging after infusion of CD19 or CD22-directed CART, adverse events not previously reported in children. Orthogonal data from autopsy studies, cerebrospinal fluid (CSF) flow cytometry and CSF proteomics/cytokine profiling demonstrated chronic white matter destruction, but a notable lack of inflammatory pathologic changes and cell populations. Instead, children with quadriparesis or paraparesis post-CART therapy had lower levels of pro-inflammatory cytokines such as interferon gamma (IFN), CCL17, CCL23, and CXCL10 than those who did not develop quadriparesis or paraparesis. Taken together, these findings imply a non-inflammatory source of this newly described ICANS phenomenon in children. The pathophysiology of some neurologic symptoms following CART may therefore have a more complex etiology than exclusive T-cell activation and excessive cytokine production.

How I use risk factors for success or failure of CD19 CAR T cells to guide management of children and AYA with B-cell ALL.

By overcoming chemotherapeutic resistance, chimeric antigen receptor (CAR) T cells facilitate deep, complete remissions and offer the potential for long-term cure in a substantial fraction of patients with chemotherapy refractory disease. However, that success is tempered with 10% to 30% of patients not achieving remission and over half of patients treated eventually experiencing relapse. With over a decade of experience using CAR T cells in children, adolescents, and young adults (AYA) to treat relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL) and 5 years since the first US Food and Drug Administration approval, data defining the nuances of patient-specific risk factors are emerging. With the commercial availability of 2 unique CD19 CAR T-cell constructs for B-ALL, in this article, we review the current literature, outline our approach to patients, and discuss how individual factors inform strategies to optimize outcomes in children and AYA receiving CD19 CAR T cells. We include data from both prospective and recent large retrospective studies that offer insight into understanding when the risks of CAR T-cell therapy failure are high and offer perspectives suggesting when consolidative hematopoietic cell transplantation or experimental CAR T-cell and/or alternative immunotherapy should be considered. We also propose areas where prospective trials addressing the optimal use of CAR T-cell therapy are needed.

Impact of poverty and neighborhood opportunity on outcomes for children treated with CD19-directed CAR T-cell therapy.

Children living in poverty experience excessive relapse and death from newly diagnosed acute lymphoblastic leukemia (ALL). The influence of household poverty and neighborhood social determinants on outcomes from chimeric antigen receptor (CAR) T-cell therapy for relapsed/refractory (r/r) leukemia is poorly described. We identified patients with r/r CD19+ ALL/lymphoblastic lymphoma treated on CD19-directed CAR T-cell clinical trials or with commercial tisagenlecleucel from 2012 to 2020. Socioeconomic status (SES) was proxied at the household level, with poverty exposure defined as Medicaid-only insurance. Low-neighborhood opportunity was defined by the Childhood Opportunity Index. Among 206 patients aged 1 to 29, 35.9% were exposed to household poverty, and 24.9% had low-neighborhood opportunity. Patients unexposed to household poverty or low-opportunity neighborhoods were more likely to receive CAR T-cell therapy with a high disease burden (>25%), a disease characteristic associated with inferior outcomes, as compared with less advantaged patients (38% vs 30%; 37% vs 26%). Complete remission (CR) rate was 93%, with no significant differences by household poverty (P = .334) or neighborhood opportunity (P = .504). In multivariate analysis, patients from low-opportunity neighborhoods experienced an increased hazard of relapse as compared with others (P = .006; adjusted hazard ratio [HR], 2.3; 95% confidence interval [CI], 1.3-4.1). There was no difference in hazard of death (P = .545; adjusted HR, 1.2; 95% CI, 0.6-2.4). Among children who successfully receive CAR T-cell therapy, CR and overall survival are equitable regardless of proxied SES and neighborhood opportunity. Children from more advantaged households and neighborhoods receive CAR T-cell therapy with a higher disease burden. Investigation of multicenter outcomes and access disparities outside of clinical trial settings is warranted.

Absolute lymphocyte count recovery following initial acute myelogenous leukemia therapy: Implications for adoptive cell therapy.

BACKGROUND: An adequate absolute lymphocyte count (ALC) is an essential first step in autologous chimeric antigen receptor (CAR) T-cell manufacturing. For patients with acute myelogenous leukemia (AML), the intensity of chemotherapy received may affect adequate ALC recovery required for CAR T-cell production. We sought to analyze ALC following each course of upfront therapy as one metric for CAR T-cell manufacturing feasibility in children and young adults with AML. PROCEDURE: ALC data were collected from an observational study of patients with newly diagnosed AML between the ages of 1 month and 21 years who received treatment between the years of 2006 and 2018 at one of three hospitals in the Leukemia Electronic Abstraction of Records Network (LEARN) consortium. RESULTS: Among 193 patients with sufficient ALC data for analysis, the median ALC following induction 1 was 1715 cells/µl (interquartile range: 1166-2388), with successive decreases in ALC with each subsequent course. Similarly, the proportion of patients achieving an ALC >400 cells/µl decreased following each course, ranging from 98.4% (190/193) after course 1 to 66.7% (22/33) for patients who received a fifth course of therapy. CONCLUSIONS: There is a successive decline of ALC recovery with subsequent courses of chemotherapy. Despite this decline, ALC values are likely sufficient to consider apheresis prior to the initiation of each course of upfront therapy for the majority of newly diagnosed pediatric AML patients, thereby providing a window of opportunity for T-cell collection for those patients identified at high risk of relapse or with refractory disease.

Presentation acuity, induction mortality, and resource utilization in infants with acute leukemia.

BACKGROUND: Treatment of infants with acute leukemia remains challenging, especially for acute lymphocytic leukemia (ALL). Infants have shown markedly higher rates of induction mortality compared with noninfants. There are limited data on presentation acuity and supportive care utilization in this age group. METHODS: In retrospective analyses of patients treated for new onset ALL or acute myeloid leukemia (AML) at pediatric hospitals contributing to the Pediatric Health Information System, we compared presentation acuity, induction mortality, and resource utilization in infants relative to noninfants less than 10 years at diagnosis. RESULTS: Analyses included 10 359 children with ALL (405 infants, 9954 noninfants) and 871 AML (189 infants, 682 noninfants). Infants were more likely to present with multisystem organ failure compared to noninfants for both ALL (12% and 1%, PR = 10.8, 95% CI: 7.4, 15.7) and AML (6% vs. 3%; PR = 2.0, 95% CI: 1.0, 3.7). Infants with ALL had higher induction mortality compared to noninfants, even after accounting for differences in anthracycline exposure and presentation acuity (2.7% vs. 0.5%, HR = 2.1, 95% CI: 1.0, 4.8). Conversely, infants and noninfants with AML had similar rates of induction mortality (3.2% vs. 2.1%, HR = 1.2, 95% CI: 0.3, 3.9), which were comparable to rates among infants with ALL. Infants with ALL and AML had greater requirements for blood products, diuretics, supplemental oxygen, and ventilation during induction relative to noninfants. CONCLUSIONS: Infants with leukemia present with higher acuity compared with noninfants. Induction mortality and supportive care requirements for infants with ALL were similar to all children with AML, and significantly higher than those for noninfants with ALL.

Long-term outcomes among 2-year survivors of autologous hematopoietic cell transplantation for Hodgkin and diffuse large b-cell lymphoma.

BACKGROUND: Autologous hematopoietic cell transplantation (auto-HCT) is a standard therapy for relapsed classic Hodgkin lymphoma (cHL) and diffuse large B-cell lymphoma (DLBCL); however, long-term outcomes are not well described. METHODS: This study analyzed survival, nonrelapse mortality, late effects, and subsequent malignant neoplasms (SMNs) in 1617 patients who survived progression-free for ≥2 years after auto-HCT for cHL or DLBCL between 1990 and 2008. The median age at auto-HCT was 40 years; the median follow-up was 10.6 years. RESULTS: The 5-year overall survival rate was 90% (95% confidence interval [CI], 87%-92%) for patients with cHL and 89% (95% CI, 87%-91%) for patients with DLBCL. The risk of late mortality in comparison with the general population was 9.6-fold higher for patients with cHL (standardized mortality ratio [SMR], 9.6) and 3.4-fold higher for patients with DLBCL (SMR, 3.4). Relapse accounted for 44% of late deaths. At least 1 late effect was reported for 9% of the patients. A total of 105 SMNs were confirmed: 44 in the cHL group and 61 in the DLBCL group. According to a multivariate analysis, older age, male sex, a Karnofsky score < 90, total body irradiation (TBI) exposure, and a higher number of lines of chemotherapy before auto-HCT were risk factors for overall mortality in cHL. Risk factors in DLBCL were older age and TBI exposure. A subanalysis of 798 adolescent and young adult patients mirrored the outcomes of the overall study population. CONCLUSIONS: Despite generally favorable outcomes, 2-year survivors of auto-HCT for cHL or DLBCL have an excess late-mortality risk in comparison with the general population and experience an assortment of late complications. Cancer 2018;124:816-25. © 2017 American Cancer Society.

Prospective, longitudinal assessment of quality of life in children from diagnosis to 3 months off treatment for standard risk acute lymphoblastic leukemia: Results of Children's Oncology Group study AALL0331.

Standard risk acute lymphoblastic leukemia (SR-ALL) has high cure rates, but requires 2-3 years of therapy. We aimed to (i) prospectively evaluate health-related quality of life (HRQOL) during and after SR-ALL therapy, and (ii) identify associated predictors. Parents of 160 SR-ALL patients enrolled on Children's Oncology Group (COG) therapeutic trial AALL0331 at 31 sites completed the Pediatric Quality of Life Inventory (PedsQL) 4.0 Generic Core Scales (physical, emotional and social functioning) and Family Assessment Device-General Functioning (FAD-GF) at 1, 6 and 12 months after diagnosis, and 3 months post-therapy. Mean PedsQL scores in physical, emotional and social functioning were impaired 1 month after diagnosis but steadily improved. Three months post-therapy, impaired physical and social functioning was observed in 27.8 and 25.8% of patients, respectively. In repeated-measures analysis, problematic family functioning predicted emotional (OR = 1.85, 95% CI 1.03-3.34) and social (OR = 1.99, 95% CI 1.21-3.27) impairment. Larger household size was associated with social impairment (OR = 1.21, 95% CI 1.02-1.45). Adverse neurological event(s) during therapy predicted post-therapy physical (OR = 5.17, 95% CI 1.61-16.63) and social (OR = 8.17, 95% CI 1.19-56.16) impairment. HRQOL 1 month after diagnosis was not predictive of HRQOL 3 months after therapy completion. In conclusion, children with SR-ALL experience considerable impairment in HRQOL at the end of induction, but rapidly improve. However, many still experience physical and social impairment 3 months post-therapy, suggesting a role for continued family and physical functioning support. Longer follow-up is needed to determine if post-therapy deficits change over time.

A prospective study of anxiety, depression, and behavioral changes in the first year after a diagnosis of childhood acute lymphoblastic leukemia: a report from the Children's Oncology Group.

BACKGROUND: The authors prospectively assessed anxiety, depression, and behavior in children with standard-risk acute lymphoblastic leukemia (SR-ALL) during the first year of therapy and identified associated risk factors. METHODS: A cohort study was performed of 159 children (aged 2 years-9.99 years) with SR-ALL who were enrolled on Children's Oncology Group protocol AALL0331 at 31 sites. Parents completed the Behavior Assessment System for Children, the General Functioning Scale of the Family Assessment Device, and the Coping Health Inventory for Parents at approximately 1, 6, and 12 months after diagnosis. RESULTS: Overall, mean scores for anxiety, depression, aggression, and hyperactivity were similar to population norms. However, more children scored in the at-risk/clinical range for depression than the expected 15% at 1 month (21.7%; P= .022), 6 months (28.6%; P< .001), and 12 months (21.1%; P= .032). For anxiety, more children scored in the at-risk/clinical range at 1 month (25.2% vs 15%; P= .001), but then reverted to expected levels. On adjusted analysis, unhealthy family functioning was found to be predictive of anxiety (odds ratio [OR], 2.24; P= .033) and depression (OR, 2.40; P= .008). Hispanic ethnicity was associated with anxiety (OR, 3.35; P= .009). Worse physical functioning (P= .049), unmarried parents (P= .017), and less reliance on social support (P= .004) were found to be associated with depression. Emotional distress at 1 month predicted anxiety (OR, 7.11; P= .002) and depression (OR, 3.31; P= .023) at 12 months. CONCLUSIONS: Anxiety is a significant problem in a subpopulation of patients with SR-ALL immediately after diagnosis, whereas depression remains a significant problem for at least 1 year. Children of Hispanic ethnicity or those with unhealthy family functioning may be particularly vulnerable. These data suggest that clinicians should screen for anxiety and depression throughout the first year of therapy.

Household income and health-related quality of life in children receiving treatment for acute myeloid leukemia: Potential impact of selection bias in health equity research.

OBJECTIVE: Examine the influence of household income on health-related quality of life (HRQOL) among children with newly diagnosed acute myeloid leukemia (AML). DESIGN: Secondary analysis of data prospectively collected from pediatric patients receiving treatment for AML at 14 hospitals across the United States. EXPOSURE: Household income was self-reported on a demographic survey. The examined mediators included the acuity of presentation and treatment toxicity. OUTCOME: Caregiver proxy reported assessment of patient HRQOL from the Peds QL 4.0 survey. RESULT: Children with AML (n = 131) and caregivers were prospectively enrolled to complete PedsQL assessments. HRQOL scores were better for patients in the lowest versus highest income category (mean ± SD: 76.0 ± 14 household income <$25,000 vs. 59.9 ± 17 income ≥$75,000; adjusted mean difference: 11.2, 95% CI: 2.2-20.2). Seven percent of enrolled patients presented with high acuity (ICU-level care in the first 72 h), and 16% had high toxicity (any ICU-level care); there were no identifiable differences by income, refuting mediating roles in the association between income and HRQOL. Enrolled patients were less likely to be Black/African American (9.9% vs. 22.2%), more likely to be privately insured (50.4% vs. 40.7%), and more likely to have been treated on a clinical trial (26.7% vs. 18.5%) compared to eligible unenrolled patients not enrolled. Evaluations of potential selection bias on the association between income and HRQOL suggested differences in HRQOL may be smaller than observed or even in the opposing direction. CONCLUSIONS: While primary analyses suggested lower household income was associated with superior HRQOL, differential participation may have biased these results. Future studies should partner with patients/families to identify strategies for equitable participation in clinical research.

Reinfusion of CD19 CAR T cells for relapse prevention and treatment in children with acute lymphoblastic leukemia.

ABSTRACT: Relapse after CD19-directed chimeric antigen receptor (CAR)-modified T cells remains a substantial challenge. Short CAR T-cell persistence contributes to relapse risk, necessitating novel approaches to prolong durability. CAR T-cell reinfusion (CARTr) represents a potential strategy to reduce the risk of or treat relapsed disease after initial CAR T-cell infusion (CARTi). We conducted a retrospective review of reinfusion of murine (CTL019) or humanized (huCART19) anti-CD19/4-1BB CAR T cells across 3 clinical trials or commercial tisagenlecleucel for relapse prevention (peripheral B-cell recovery [BCR] or marrow hematogones ≤6 months after CARTi), minimal residual disease (MRD) or relapse, or nonresponse to CARTi. The primary endpoint was complete response (CR) at day 28 after CARTr, defined as complete remission with B-cell aplasia. Of 262 primary treatments, 81 were followed by ≥1 reinfusion (investigational CTL019, n = 44; huCART19, n = 26; tisagenlecleucel, n = 11), representing 79 patients. Of 63 reinfusions for relapse prevention, 52% achieved CR (BCR, 15/40 [38%]; hematogones, 18/23 [78%]). Lymphodepletion was associated with response to CARTr for BCR (odds ratio [OR], 33.57; P = .015) but not hematogones (OR, 0.30; P = .291). The cumulative incidence of relapse was 29% at 24 months for CR vs 61% for nonresponse to CARTr (P = .259). For MRD/relapse, CR rate to CARTr was 50% (5/10), but 0/8 for nonresponse to CARTi. Toxicity was generally mild, with the only grade ≥3 cytokine release syndrome (n = 6) or neurotoxicity (n = 1) observed in MRD/relapse treatment. Reinfusion of CTL019/tisagenlecleucel or huCART19 is safe, may reduce relapse risk in a subset of patients, and can reinduce remission in CD19+ relapse.

INSPIRED Symposium Part 1: Clinical Variables Associated with Improved Outcomes for Children and Young Adults treated with Chimeric Antigen Receptor T cells for B cell Acute Lymphoblastic Leukemia.

Chimeric antigen receptor (CAR) T cell therapy (CAR-T) targeting the CD19 antigen on B cell acute lymphoblastic leukemia (B-ALL) has transitioned from a highly investigational therapy with limited access to a commercial therapy with established toxicities, response and survival rates, and access in numerous countries. With more than a decade of clinical study and 5 years of commercial access, data showing associations with success and failure have emerged. To address functional limitations of CAR-T and overcome constrained sample sizes when studying single-trial or single-center data, collaborative groups, including the Pediatric Real World CAR Consortium, the CAR-Multicenter Analysis, the Center for International Blood and Marrow Transplant Research, and the International BFM Study Group, among others, have been retrospectively interrogating the amassed clinical experience. The high patient numbers and varied clinical experiences compiled by these groups have defined clinical variables impacting CAR-T outcomes. Here we review published CAR-T trials and consortium/collaborative outcomes to establish variables associated with optimal response to CAR-T in children and young adults with B-ALL. We focus on findings with clinical relevance that have emerged, including data implicating pretreatment disease burden, presence of extramedullary disease, nonresponse to prior CD19 antigen targeting (blinatumomab therapy), CAR T cell dose, and fludarabine pharmacokinetics as factors impacting post-CAR-T survival. Additionally, we address the role of collaborative efforts going forward in guiding clinical practice evolution and further optimizing post-CAR-T outcomes.

Preinfusion factors impacting relapse immunophenotype following CD19 CAR T cells.

Relapse following chimeric antigen receptor (CAR) T-cell therapy directed against CD19 for relapsed/refractory B-acute lymphoblastic leukemia (r/r B-ALL) remains a significant challenge. Three main patterns of relapse predominate: CD19 positive (CD19pos) relapse, CD19 negative (CD19neg) relapse, and lineage switch (LS). Development and validation of risk factors that predict relapse phenotype could help define potential pre- or post-CAR T-cell infusion interventions aimed at decreasing relapse. Our group sought to extensively characterize preinfusion risk factors associated with the development of each relapse pattern via a multicenter, retrospective review of children and young adults with r/r B-ALL treated with a murine-based CD19-CAR construct. Of 420 patients treated with CAR, 166 (39.5%) relapsed, including 83 (50%) CD19pos, 68 (41%) CD19neg, and 12 (7.2%) LS relapses. A greater cumulative number of prior complete remissions was associated with CD19pos relapses, whereas high preinfusion disease burden, prior blinatumomab nonresponse, older age, and 4-1BB CAR construct were associated with CD19neg relapses. The presence of a KMT2A rearrangement was the only preinfusion risk factor associated with LS. The median overall survival following a post-CAR relapse was 11.9 months (95% CI, 9-17) and was particularly dismal in patients experiencing an LS, with no long-term survivors following this pattern of relapse. Given the poor outcomes for those with post-CAR relapse, study of relapse prevention strategies, such as consolidative hematopoietic stem cell transplantation, is critical and warrants further investigation on prospective clinical trials.

Rachitic changes, demineralization, and fracture risk in healthy infants and toddlers with vitamin D deficiency.

PURPOSE: To examine radiographic findings in children with vitamin D deficiency in comparison with biochemical marker levels and prevalence of fractures. MATERIALS AND METHODS: The parents or guardians of all participants provided written informed consent at the time of enrollment. The institutional review board approved the protocol, and HIPAA guidelines were followed. From a prospective sample of children seen for routine clinical care, 40 children with vitamin D deficiency (25-hydroxyvitamin D [25-OHD] level, ≤ 20 ng/mL) were identified, and high-detail computed radiographs of the wrists and knees were obtained. The children ranged in age from 8 to 24 months. Radiographs were scored by three readers with use of the 10-point Thacher score for rachitic changes and a five-point scale for demineralization. Serum calcium, phosphorus, alkaline phosphatase, and parathyroid hormone levels were determined. Fracture history was obtained for 35 of the 40 patients (88%). RESULTS: All readers identified rachitic changes at both readings in two patients (5%) and demineralization in two patients (5%). Interrater agreement was 65% for rachitic changes (κ = 0.33) and 70% for demineralization (κ = 0.37). When the majority of the raters determined that rachitic changes were absent at both readings, alkaline phosphatase levels were lower than those with other assessments (median, 267 vs 515 U/L [4.4589 vs 8.6005 µkat/L]; P = .01). When most raters determined that demineralization was present at both readings, serum 25-OHD levels were lower than those at other assessments (median, 9.0 vs 17.5 ng/mL [22.464 vs 43.68 nmol/L]; P = .02). No fractures were reported or identified radiographically. CONCLUSION: In infants and toddlers with vitamin D deficiency, rachitic changes and definite demineralization are uncommon and fracture risk is low.

Statistical Considerations for Analyses of Time-To-Event Endpoints in Oncology Clinical Trials: Illustrations with CAR-T Immunotherapy Studies.

Chimeric antigen receptor T-cell (CAR-T) therapy is an exciting development in the field of cancer immunology and has received a lot of interest in recent years. Many time-to-event (TTE) endpoints related to relapse, disease progression, and remission are analyzed in CAR-T studies to assess treatment efficacy. Definitions of these TTE endpoints are not always consistent, even for the same outcomes (e.g., progression-free survival), which often stems from analysis choices regarding which events to consider as part of the composite endpoint, censoring or competing risk in the analysis. Subsequent therapies such as hematopoietic stem cell transplantation are common but are not treated the same in different studies. Standard survival analysis methods are commonly applied to TTE analyses but often without full consideration of the assumptions inherent in the chosen analysis. We highlight two important issues of TTE analysis that arise in CAR-T studies, as well as in other settings in oncology: the handling of competing risks and assessing the association between a time-varying (post-infusion) exposure and the TTE outcome. We review existing analytical methods, including the cumulative incidence function and regression models for analysis of competing risks, and landmark and time-varying covariate analysis for analysis of post-infusion exposures. We clarify the scientific questions that the different analytical approaches address and illustrate how the application of an inappropriate method could lead to different results using data from multiple published CAR-T studies. Codes for implementing these methods in standard statistical software are provided.

Single-cell antigen-specific landscape of CAR T infusion product identifies determinants of CD19-positive relapse in patients with ALL.

A notable number of acute lymphoblastic leukemia (ALL) patients develop CD19-positive relapse within 1 year after receiving chimeric antigen receptor (CAR) T cell therapy. It remains unclear if the long-term response is associated with the characteristics of CAR T cells in infusion products, hindering the identification of biomarkers to predict therapeutic outcomes. Here, we present 101,326 single-cell transcriptomes and surface protein landscape from the infusion products of 12 ALL patients. We observed substantial heterogeneity in the antigen-specific activation states, among which a deficiency of T helper 2 function was associated with CD19-positive relapse compared with durable responders (remission, >54 months). Proteomic data revealed that the frequency of early memory T cells, rather than activation or coinhibitory signatures, could distinguish the relapse. These findings were corroborated by independent functional profiling of 49 patients, and an integrative model was developed to predict the response. Our data unveil the molecular mechanisms that may inform strategies to boost specific T cell function to maintain long-term remission.

Blinatumomab Nonresponse and High-Disease Burden Are Associated With Inferior Outcomes After CD19-CAR for B-ALL.

PURPOSE: CD19-targeted chimeric antigen receptor T cells (CD19-CAR) and blinatumomab effectively induce remission in relapsed or refractory B-cell acute lymphoblastic leukemia (ALL) but are also associated with CD19 antigen modulation. There are limited data regarding the impact of prior blinatumomab exposure on subsequent CD19-CAR outcomes. PATIENTS AND METHODS: We conducted a multicenter, retrospective review of children and young adults with relapsed or refractory ALL who received CD19-CAR between 2012 and 2019. Primary objectives addressed 6-month relapse-free survival (RFS) and event-free survival (EFS), stratified by blinatumomab use. Secondary objectives included comparison of longer-term survival outcomes, complete remission rates, CD19 modulation, and identification of factors associated with EFS. RESULTS: Of 420 patients (median age, 12.7 years; interquartile range, 7.1-17.5) treated with commercial tisagenlecleucel or one of three investigational CD19-CAR constructs, 77 (18.3%) received prior blinatumomab. Blinatumomab-exposed patients more frequently harbored KMT2A rearrangements and underwent a prior stem-cell transplant than blinatumomab-naïve patients. Among patients evaluable for CD19-CAR response (n = 412), blinatumomab nonresponders had lower complete remission rates to CD19-CAR (20 of 31, 64.5%) than blinatumomab responders (39 of 42, 92.9%) or blinatumomab-naive patients (317 of 339, 93.5%), P < .0001. Following CD19-CAR, blinatumomab nonresponders had worse 6-month EFS (27.3%; 95% CI, 13.6 to 43.0) compared with blinatumomab responders (66.9%; 95% CI, 50.6 to 78.9; P < .0001) or blinatumomab-naïve patients (72.6%; 95% CI, 67.5 to 77; P < .0001) and worse RFS. High-disease burden independently associated with inferior EFS. CD19-dim or partial expression (preinfusion) was more frequently seen in blinatumomab-exposed patients (13.3% v 6.5%; P = .06) and associated with lower EFS and RFS. CONCLUSION: With the largest series to date in pediatric CD19-CAR, and, to our knowledge, the first to study the impact of sequential CD19 targeting, we demonstrate that blinatumomab nonresponse and high-disease burden were independently associated with worse RFS and EFS, identifying important indicators of long-term outcomes following CD19-CAR.

Single-cell multiomics dissection of basal and antigen-specific activation states of CD19-targeted CAR T cells.

BACKGROUND: Autologous T cells engineered to express a chimeric antigen receptor (CAR) specific for CD19 molecule have transformed the therapeutic landscape in patients with highly refractory leukemia and lymphoma, and the use of donor-generated allogeneic CAR T is paving the way for further breakthroughs in the treatment of cancer. However, it remains unknown how the intrinsic heterogeneities of these engineered cells mediate therapeutic efficacy and whether allogeneic products match the effectiveness of autologous therapies. METHODS: Using single-cell mRNA sequencing in conjunction with CITE-seq, we performed multiomics characterization of CAR T cells generated from healthy donor and patients with acute lymphoblastic leukemia. CAR T cells used in this study were manufactured at the University of Pennsylvania through lentiviral transduction with a CD19-4-1BB-CD3ζ construct. Besides the baseline condition, we engineered NIH-3T3 cells with human CD19 or mesothelin expression to conduct ex vivo antigen-specific or non-antigen stimulation of CAR T cells through 6-hour coculture at a 1:1 ratio. RESULTS: We delineated the global cellular and molecular CAR T landscape and identified that transcriptional CAR tonic signaling was regulated by a mixture of early activation, exhaustion signatures, and cytotoxic activities. On CD19 stimulation, we illuminated the disparities of CAR T cells derived from different origins and found that donor CAR T had more pronounced activation level in correlation with the upregulation of major histocompatibility complex class II genes compared with patient CAR T cells. This finding was independently validated in additional datasets from literature. Furthermore, GM-CSF(CSF2) expression was found to be associated with functional gene productions, but it induced little impact on the CAR T activation. CONCLUSIONS: Through integrated multiomics profiling and unbiased canonical pathway analyses, our results unveil heterogeneities in the transcriptional, phenotypic, functional, and metabolic profiles of donor and patient CAR T cells, providing mechanistic basis for ameliorating clinical outcomes and developing next-generation 'off- the-shelf' allogeneic products.

Integrative Bulk and Single-Cell Profiling of Premanufacture T-cell Populations Reveals Factors Mediating Long-Term Persistence of CAR T-cell Therapy.

The adoptive transfer of chimeric antigen receptor (CAR) T cells represents a breakthrough in clinical oncology, yet both between- and within-patient differences in autologously derived T cells are a major contributor to therapy failure. To interrogate the molecular determinants of clinical CAR T-cell persistence, we extensively characterized the premanufacture T cells of 71 patients with B-cell malignancies on trial to receive anti-CD19 CAR T-cell therapy. We performed RNA-sequencing analysis on sorted T-cell subsets from all 71 patients, followed by paired Cellular Indexing of Transcriptomes and Epitopes (CITE) sequencing and single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) on T cells from six of these patients. We found that chronic IFN signaling regulated by IRF7 was associated with poor CAR T-cell persistence across T-cell subsets, and that the TCF7 regulon not only associates with the favorable naïve T-cell state, but is maintained in effector T cells among patients with long-term CAR T-cell persistence. These findings provide key insights into the underlying molecular determinants of clinical CAR T-cell function. SIGNIFICANCE: To improve clinical outcomes for CAR T-cell therapy, there is a need to understand the molecular determinants of CAR T-cell persistence. These data represent the largest clinically annotated molecular atlas in CAR T-cell therapy to date, and significantly advance our understanding of the mechanisms underlying therapeutic efficacy.This article is highlighted in the In This Issue feature, p. 2113.

Risk-Adapted Preemptive Tocilizumab to Prevent Severe Cytokine Release Syndrome After CTL019 for Pediatric B-Cell Acute Lymphoblastic Leukemia: A Prospective Clinical Trial.

PURPOSE: To prospectively evaluate the effectiveness of risk-adapted preemptive tocilizumab (PT) administration in preventing severe cytokine release syndrome (CRS) after CTL019, a CD19 chimeric antigen receptor T-cell therapy. METHODS: Children and young adults with CD19-positive relapsed or refractory B-cell acute lymphoblastic leukemia were assigned to high- (≥ 40%) or low- (< 40%) tumor burden cohorts (HTBC or LTBC) based on a bone marrow aspirate or biopsy before infusion. HTBC patients received a single dose of tocilizumab (8-12 mg/kg) after development of high, persistent fevers. LTBC patients received standard CRS management. The primary end point was the frequency of grade 4 CRS (Penn scale), with an observed rate of ≤ 5 of 15 patients in the HTBC pre-defined as clinically meaningful. In post hoc analyses, the HTBC was compared with a historical cohort of high-tumor burden patients from the initial phase I CTL019 trial. RESULTS: The primary end point was met. Seventy patients were infused with CTL019, 15 in the HTBC and 55 in the LTBC. All HTBC patients received the PT intervention. The incidence of grade 4 CRS was 27% (95% CI, 8 to 55) in the HTBC and 3.6% (95% CI, 0.4 to 13) in the LTBC. The best overall response rate was 87% in the HTBC and 100% in the LTBC. Initial CTL019 expansion was greater in the HTBC than the LTBC (P < .001), but persistence was not different (P = .73). Event-free and overall survival were worse in the HTBC (P = .004, P < .001, respectively). In the post hoc analysis, grade 4 CRS was observed in 27% versus 50% of patients in the PT and prior phase I cohorts, respectively (P = .18). CONCLUSION: Risk-adapted PT administration resulted in a decrease in the expected incidence of grade 4 CRS, meeting the study end point, without adversely impacting the antitumor efficacy or safety of CTL019.