Pediatric B-cell Acute Lymphoblastic Leukemia Patients Research Articles

The safety and efficacy of blinatumomab in the treatment of B-cell acute lymphoblastic leukemia in children

To explore the safety and efficacy of blinatumomab in the treatment of CD19 positive (CD19+) B-cell acute lymphoblastic leukemia (B-ALL) in children. A retrospective analysis was conducted on the clinical data of pediatric B-ALL patients who received blinatumomab treatment from Hematology & Blood Diseases Hospital of Chinese Academy of Medical Sciences from August 2021 to October 2023. Based on their disease status, the patients were divided into refractory/relapsed(RR) group, minimal residual disease clearance (MC) group, and chemotherapy intolerance (IC) group. Clinical data of the children were collected to evaluate the adverse drug reactions, therapeutic efficacy and survival of the children. In total, 35 patients were included, with 20 males and 15 females, aged from 0.6 to 16.4 (9.9±4.2) years old. There were 10 cases in the RR group, 20 cases in the MC group and 5 cases in the IC group. A total of 56 cycles of infusion were completed, with one cycle in 24 cases, two cycles in 5 cases, three cycles in 2 cases and four cycles in 4 cases. The median infusion time [M (Q1, Q3)] from the first to the fourth cycle was 14 (14, 28) days, 28 (28, 28) days, 28 (28, 28) days and 28 (26, 28) days, respectively. In terms of adverse reactions, the incidence of grade 1-2 cytokine release syndrome(CRS) was 57.1% (32/56), with grade 1 CRS accounting for 84.4% (27/32). The incidence rate of immune effector cell-associated neurotoxicity syndrome(ICANS) (grade 4) was 1.8% (1/56). In the RR group, 6 cases were treated effectively, and minimal residual disease(MRD) turned negative, before treatment, MRD levels were all less than 20%. Among them, 3 cases had MRD turning positive again 14 to 42 days after discontinuation of Belintoumab. Four cases were treated ineffectively, with MRD >20% before treatment. All MRD positive cases in MC group turned negative and all MRD negative cases in the IC group remained negative after treatment. The median follow-up time of RR group was 5.7 (3.8, 9.4) months, and 1 year median survival rate and event-free survival rate were 40.0%±21.9% and 33.3%±19.2%, respectively. The median follow-up time for MC and IC group patients was 6.7 (5.2, 12.5) months and 7.1 (5.1, 7.6) months, respectively, with an event free survival rate of 100%. The safety and efficacy of using belintoumab in partial RR, MRD clearance, and chemotherapy intolerance are good.

Identification of a circular RNA isoform of WASHC2A as a prognostic factor for high-risk paediatric B-ALL patients

Pediatric B-cell acute lymphoblastic leukemia (B-ALL) is a serious disease for which a better understanding of prognostic factors and new therapeutic targets is needed. Circular RNAs (circRNAs) are promising markers due to their stability and differential expression patterns in various diseases. However, their role in pediatric B-ALL patients, particularly in risk stratification and relapse prediction, remains poorly understood. In this study, we comprehensively examined the circRNA landscape in pediatric B-ALL patients, focusing on both high-risk and standard-risk patients. Using advanced sequencing techniques and sophisticated bioinformatics tools, we identified thousands of circRNAs, including a novel circRNA derived from the WASHC2A gene, termed circWASHC2A. CircWASHC2A showed differential expression between high-risk and standard-risk patients and exhibited potential for predicting relapse in high-risk patients. Functional experiments highlighted a role for circWASHC2A in regulating cell cycle progression and mitochondrial respiratory activity in leukaemic cells. Transcriptomic analysis further supported these findings, suggesting the involvement of circWASHC2A in signalling pathways relevant to leukaemia pathogenesis. This study provides in-depth insights into the circRNA landscape of pediatric B-ALL patients and identifies circWASHC2A as a potential biomarker for risk stratification and relapse prediction, with significant implications for tailoring diagnostic and therapeutic strategies in this patient population.

Molecular characterization and biomarker identification in pediatric B-cell acute lymphoblastic leukemia.

6524 Background: B-cell acute lymphoblastic leukemia (B-ALL) is the most prevalent hematologic malignancy in children and a leading cause of mortality. Despite high cure rates, the management of B-ALL remains challenging due to its high heterogeneity and propensity for relapse. This study aimed to delineate molecular features of pediatric B-ALL and explore the clinical utility of circulating tumor DNA (ctDNA) as a biomarker in B-ALL. Methods: The study analyzed 146 primary pediatric B-ALL patients who were diagnosed from August 2020 to April 2023 and received systemic chemotherapy in Wuhan Children's Hospital. Baseline bone marrow (BM) and plasma samples were collected for next-generation sequencing (NGS) to profile the mutational landscape. BM samples collected on day 19 of the treatment were utilized for minimal residual disease (MRD) testing to assess treatment efficacy. Transcriptomic profiling of baseline BM samples was performed via bulk RNA sequencing. Fisher's exact test and the Wilcoxon rank sum test were utilized to compare categorical and continuous variables between groups, respectively. A two‐tailed P-value<0.05 was considered statistically significant unless indicated otherwise. Results: Transcriptomic analysis revealed that 86.3% of patients (126 out of 146) could be categorized into 13 distinct molecular subtypes, with hyperdiploidy emerging as the predominant subtype. Baseline BM NGS identified gene fusions in 61% of patients, including 37 novel fusions previously unreported in B-ALL. Specifically, the KMT2A-TRIM29 novel fusion was detected and validated in a male child diagnosed with the KMT2A-rearranged subtype. Despite initially responding well to therapy, the patient experienced disease progression after a year, indicating a poor prognosis. We also found that elevated mutant counts and maximum-variant-allele-frequency (maxVAF) in baseline BM were associated with significantly poorer response to chemotherapy ( P=0.0012 and 0.028, respectively). Additionally, MRD-negative patients exhibited upregulated expression of immune-related pathways ( P<0.01) and increased CD8+ T cell infiltration ( P=0.047), indicating a more active immune microenvironment in better responding tumors. Baseline plasma ctDNA, which exhibited high mutational concordance with paired baseline BM samples, also demonstrated significant associations with chemotherapy efficacy, highlighting its potential as a non-invasive tool for disease monitoring and treatment outcome prediction. Conclusions: This study elucidated novel gene fusions and potential biomarkers for treatment response in pediatric B-ALL. Importantly, both baseline plasma ctDNA and BM samples offer promising prognostic insights into chemotherapy responses, paving the way for non-invasive monitoring strategies in the management of pediatric B-ALL.

Abstract 3952: Multiomic single-cell tumor evolution models of minimal residual disease in pediatric B-cell acute lymphoblastic leukemia

Abstract Detection of minimal residual disease (MRD) after initial diagnosis (IDX) and therapy is a strong prognostic marker for relapse in pediatric B-cell acute lymphoblastic leukemia (B-ALL). Previous studies have explored clonal genetic and gene expression changes between initial diagnosis and relapse. However, studying tumor cells present at MRD is challenging due to their low abundance. Examining the genomic profiles of leukemia cells that resist induction chemotherapy and understanding their evolutionary relationship to cells present at diagnosis may lead to a better understanding of the cells that eventually seed relapse. We analyzed somatic point mutations, indels, and copy number variants from 41 pediatric B-ALL patients with high-risk B-ALL. We specifically focused on patients from the following four common high-risk subtypes: KMT2A-rearranged, Ph+, Ph-like, and iAMP21, who had detectable disease at the end of induction. Leukemic burden at end-induction (day 29) ranged from 0.03% to 66.00%. Data were generated in partnership with the National Cancer Institute’s Human Tumor Atlas Network and the Children’s Oncology Group. Our data set of 41 patients includes 39 with paired initial diagnostic and end-induction samples that we used to create tumor evolution models based on single cells across disease stages. We produced single-cell multiome gene expression (scRNA) and chromatin accessibility (scATAC) profiles (10x Genomics) with a median of 7428 leukemic blast cells at IDX (range: 2490-14219) and 1907 at MRD (range: 240-10167) after filtering. We called somatic mutations from scRNA and scATAC using SComatic and Monopogen, and we detected copy number changes using Numbat and EpiAneufinder. We also designed a targeted DNA sequencing panel including 335 variants based on B-ALL literature and database searches to produce scDNA (Mission Bio) for 24 patients (16 IDX-MRD pairs) with a median of 2346 cells profiled at IDX (range: 340-8849) and 1828 at MRD (range: 596-5133). We built tumor phylogenies based on somatic SNVs and CNVs with COMPASS. Using multiple tools and complementary single-cell omics data types to detect somatic changes improved our confidence in the events used for tumor evolution tree construction. Our approach allowed us to define recurrent genomic events and patterns across pediatric B-ALL subtypes and to describe the clonal evolution of cells measured at initial diagnostic and end-induction disease stages. We were also able to link cell-specific mutation events to their transcriptional and epigenomic signatures, including mapping mutation events along the B-cell development trajectory and within differentially expressed gene pathways. Some cases showed dramatic shifts in subclonal structure between IDX and MRD that may be related to therapy-induced clonal selection, such as a loss of NRAS hotspot mutation. Citation Format: Steven M. Foltz, Avi Loren, Changya Chen, Rushabh Mehta, Elizabeth Li, Jason Xu, Fatemeh Alikarami, Kathrin M. Bernt, Kai Tan. Multiomic single-cell tumor evolution models of minimal residual disease in pediatric B-cell acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3952.

Short-Term Efficacy and Safety of Blinatumomab in Early Postinduction Intensification Therapy for Pediatric B-Cell Acute Lymphoblastic Leukemia

Background: Multidrug chemotherapy is the main treatment for children with B-cell acute lymphoblastic leukemia (B-ALL), which has been associated with multiple long-term complications and high rates of treatment non-completion due to toxicities. Recently, blinatumomab has proven to be effective and well-tolerated in the treatment of relapsed or refractory pediatric B-ALL. However, there is still a lack of reports about the real-world application of blinatumomab in pediatric B-ALL who achieved first complete remission (CR). O bjectives: To assess the short-term efficacy and safety of blinatumomab in early postinduction intensification therapy for children with B-ALL who had achieved first complete remission in a real-world setting. M ethods: A single-center retrospective analysis was performed to collect data on pediatric B-ALL patients (pts) who received blinatumomab monotherapy during early postinduction intensification therapy. All pts had undergone China Children's Leukemia Group (CCLG)-2018 induction chemotherapy until achieving CR. Blinatumomab treatment was initiated after obtaining consent from patients and caregivers, following detailed counseling about the potential risks and benefits. Efficacy was assessed based on minimal residual disease (MRD) negativity (<0.01%) using multiparameter flow cytometry (MFC). Adverse events (AEs) were assessed by Common Terminology Criteria for Adverse Events (CTCAE) version 5.0. R esult: From September 2022 to July 2023, A total of 11 pts were included in this study due to chemotherapy intolerance: median age was 3 years (range, 1 to 11 years) with 54.5% (n=6) being male. Before blinatumomab therapy, MRD was detected in four pts with values of 0.024%, 0.037%, 10%, and 0.09%, respectively.The remaining pts tested negative for MRD. After one cycle of blinatumomab therapy, all MRDpos pts showed a decline in MRD, and 75% achieved undetectable MRD. The median follow-up time was 3 months. A total of 28 AEs were reported, with 75% classified as grade 1-2 and the remaining 25% as grade 3-4. The most frequently reported AEs were grade 1-2 fever (90.9%), observed within 7 days of treatment. Grade 3-4 hematological AEs included decreased white blood cell count and decreased neutrophil count. Additionally, four cases of infection, two cases of transient rash and one case of grade 2 diarrhea were observed. No pts experienced immune effector cell-associated neurotoxicity syndrome (ICANS), grade 3-4 cytokine release syndrome (CRS) or unexpected serious adverse events (SAEs). Furthermore, none of the AEs resulted in treatment delay, dose reduction or discontinuation. All pts experiencing treatment-associated toxicities recovered and proceeded to consolidation therapy. C onclusion: The use of blinatumomab in early intensification demonstrated promising short-term efficacy and a manageable safety profile in pediatric B-ALL pts. However, further confirmation of these results requires larger, randomized clinical trials.

Oncogenic RAS Signaling Promotes an Immunosuppressive Niche in Acute Lymphoblastic Leukemia

Emerging evidence suggests that genomic aberrations in cancer cells may impact the composition of the tumor microenvironment leading to a tumor-specific supporting niche. Previous genomic profiling studies have identified somatic alterations in RAS pathway genes that drive treatment resistance in B-cell acute lymphoblastic leukemia (B-ALL): yet it is unknown whether such genetic events may specifically hijack the leukemic niche. Here, we analyzed bulk RNA-sequencing data of bone marrow mononuclear cells from 185 pediatric B-ALL patients entering our center and performed mutation calling of RAS pathway genes, namely KRAS, NRAS, BRAF and PTPN11. Transcriptomic analysis revealed attenuated immune responses and decreased T cell activation in samples harboring RAS mutations (n= 62) compared with wild type cases (n= 123). To further explore this finding at single-cell resolution, we sorted bone marrow mononuclear cells from 8 primary B-ALL pediatric patient samples (4 RAS mutant and 4 wild type), enriching on the hematopoietic and non-hematopoietic compartments, followed by single-cell RNA-sequencing using the 10x Genomics platform. Twelve cell populations were identified by the expression of bona fide markers, mapping different immune and stromal cells in the ALL landscape. Specifically, the RAS-mutant microenvironment was defined by dysfunctional CD8+ T-cells, characterized by high expression of TIM-3, TIGIT and LAG-3, as well as regulatory T cells, displaying expression of CTLA-4, ICOS and FOXP3, suggesting a RAS-mediated immunosuppressive mechanism. In light of these changes, we examined flow cytometry data of bone marrow mononuclear cells collected at diagnosis from our cohort of 185 patient samples. Whereas the overall proportion of lymphoid subsets (T, B and NK cells) was not significantly changed among RAS mutant and wild type groups, we detected a decrease in myeloid subsets (neutrophils, eosinophils, and monocytes) in RAS mutant ALL, which was more prominent in monocytes. Cross comparison of the transcriptional state of monocytes in our single-cell data unravel that monocytes in the RAS mutant niche displayed a non-classical phenotype, characterized with high expression of the immune checkpoint TIM-3, which is a major driver of T cell exhaustion. Taken together, the bone marrow microenvironment of ALL patients harbouring oncogenic RAS lymphoblasts is defined by dysfunctional immune cell populations. Further studies on the cellular dynamics in the niche will provide the basis for the development of therapeutic opportunities to target the immune micro-environment in RAS mutant ALL.

Probiotic-Derived Heptelidic Acid Demonstrates Therapeutic Efficacy Against Pediatric B-Cell Acute Lymphoblastic Leukemia

Introduction Pediatric B-cell acute lymphoblastic leukemia (B-ALL) is known to be sensitive to chemotherapy. However, approximately 15-20% of pediatric B-ALL patients experience relapse, and survival rates after relapse can drop to 5-10%. Therefore, new drugs need to be developed. Probiotics are live microorganisms with potential health benefits. Heptelidic acid (HA) from the probiotic Aspergillus oryzae has been shown to exert antitumor effects against pancreatic cancer, lung cancer, thyroid cancer, breast cancer and melanoma in vivo as well as in vitro. In this study, we assessed the antitumor effects of HA in B-ALL, which is the most common type of pediatric cancer. Methods Two B-ALL cell lines (JM1 and Reh), four CD34 + hematopoietic stem cells (HSCs) and three high-risk patient-derived B-ALL cells (s90: age < 1 year old, s96: age > 10 years old, s98: relapse) collected from the spleen or bone marrow of a patient-derived xenograft (PDX) model were used in this study. Cytotoxicity of HA was assessed by MTS assay. GAPDH activity and intracellular ATP contents were measured, as HA binds to cysteine 152 of GAPDH, a crucial region for its enzymatic activity and ATP production in glycolysis. The synergistic effects of HA with therapeutics, vincristine (VCR), doxorubicin (DXR) and L-asparaginase (L-Asp), were evaluated. GAPDH is involved not only in glucose metabolism but also in amino acid and fatty acids for energy production. Therefore, we analyzed whether HA exerted cytotoxicity under glucose, amino acid or fatty acid-deprived conditions in Reh cells. To analyze the underlying mechanism for HA's cytotoxicity against B-ALL, cell cycle analysis by flowcytometry, western blotting of cleaved PARP, caspase assays, and inhibitory assays using specific inhibitors for PCD mechanisms, such as RIPK-1 inhibitor, MLKL inhibitor, and autophagy inhibitor, were conducted. In vivo studies were done using a B-ALL PDX model. Oral administration of 0.5mg/kg HA was carried out for 21 days, while intraperitoneal injection of 0.15mg/kg VCR was performed once per week for 3 cycles in the HA-VCR combinational therapeutic efficacy study. Results HA exhibited significant cytotoxicity in JM1 (IC50: 169 ng/ml), Reh (IC50: 126.5 ng/ml), and three samples obtained from PDX mice (s90 IC50: 66.6 ng/ml, s96 IC50: 89.9 ng/ml, and s98 IC50: 275.6 ng/ml), while sparing CD34 + HSCs. GAPDH activity of CD34+ HSCs was significantly lower than both B-ALL cell lines and patient-derived cells. The cytotoxic effect of HA was associated with the reduction of GAPDH activity and ATP contents, suggesting glycolysis inhibition. Glucose deprivation induced significant reduction of cell viability, and HA showed the cytotoxicity in amino acid and fatty acid deprived media in Reh cells. Oral daily administration of HA prolonged the survival of the B-ALL PDX mice (34.5 days, n=8) compared to control mice (28 days, n=7) without causing body weight loss (p<0.05) (Figure 1A). The effects of over 0.5mg/kg of HA was confirmed by the reduction in GAPDH activity observed in leukemia cells isolated from the bone marrow. HA demonstrated synergistic effects when combined with chemotherapeutic agents, especially VCR. The combination therapy of HA and VCR resulted in improved survival outcomes in the B-ALL PDX model (49 days, n=5) compared to single-agent treatments (HA: 28 days, n=5, VCR: 40 days, n=5) and control groups (26 days, n=5) (p<0.05) (Figure 1B). Mechanistic studies revealed that HA induced PCD in B-ALL cells. HA treatment caused G2/M arrest (Control: 18.8%, HA: 27.6% in JM1; Control: 13.3%, HA: 18.3% in Reh), and this was accompanied by increased cleavage of PARP, a hallmark of PCD, in both cell lines. Moreover, the cytotoxicity of HA was attenuated by RIPK1 inhibition in JM1 and Reh. Conclusion We have demonstrated that B-ALL cells utilize glucose for their survival and HA is cytotoxic in B-ALL cells by disrupting glycolysis through the inhibition of GAPDH. The therapeutic efficacy of HA is further enhanced when combined with chemotherapeutic agents. The underlying mechanisms of HA involve the induction of RIPK-1 mediated PCD. This is the first study to demonstrate HA as a novel therapeutic agent for B-ALL.

Poor outcome of pediatric B-cell acute lymphoblastic leukemia associated with high level of CRLF2 gene expression in distinct molecular subtypes.

Overexpression of the cytokine receptor-like factor 2 (CRLF2) gene is the most common feature in the Philadelphia chromosome (Ph)-like subtype of B-cell acute lymphoblastic leukemia (B-ALL). However, the predictive value of CRLF2 overexpression for the prognosis of pediatric B-ALL patients remain controversial. The molecular mechanisms that upregulate CRLF2 expression level in patients has not been fully elucidated. In this study, the prognostic impact of CRLF2 expression level on molecular types of B-ALL in pediatric patients from Zhujiang Hospital (n = 111) was retrospectively analyzed. Youden index analysis was used to categorize CRLF2 expression into 3 groups, and these categories more precisely described the differences in the prognosis of patients with varying expression levels of CRLF2 in both the Zhujiang Hospital cohort and the TARGET cohort. We used the Zhujiang Hospital cohort as a discovery cohort to determine the cutoff value of CRLF2 expression. CRLF2-high patients accounted for approximately 6%. In addition, the percentage of bone marrow blast cells and initial white blood cell count in CRLF2-high patients were higher than those in CRLF2-low patients, and MRD turned negative slower. The results were validated in the TARGET cohort and indicated that CRLF2 overexpression could be subdivided by CRLF2 expression levels into 2 categories: CRLF2-high with a poor survival and CRLF2-medium with a good OS and EFS. Such heterogeneity was attributed to the different molecular mechanisms leading to CLRF2 upregulation, where the CRLF2 overexpression level was high in Ph-like B-ALL and medium in high hyperdiploid B-ALL. This study highlights the importance of the molecular mechanisms of the upregulation of CRLF2 expression in predicting the prognosis of pediatric B-ALL patients.

Defining the Basal and Immunomodulatory Mediator-Induced Phosphoprotein Signature in Pediatric B Cell Acute Lymphoblastic Leukemia (B-ALL) Diagnostic Samples.

It is theorized that dysregulated immune responses to infectious insults contribute to the development of pediatric B-ALL. In this context, our understanding of the immunomodulatory-mediator-induced signaling responses of leukemic blasts in pediatric B-ALL diagnostic samples is rather limited. Hence, in this study, we defined the signaling landscape of leukemic blasts, as well as normal mature B cells and T cells residing in diagnostic samples from 63 pediatric B-ALL patients. These samples were interrogated with a range of immunomodulatory-mediators within 24 h of collection, and phosflow analyses of downstream proximal signaling nodes were performed. Our data reveal evidence of basal hyperphosphorylation across a broad swath of these signaling nodes in leukemic blasts in contrast to normal mature B cells and T cells in the same sample. We also detected similarities in the phosphoprotein signature between blasts and mature B cells in response to IFNγ and IL-2 treatment, but significant divergence in the phosphoprotein signature was observed between blasts and mature B cells in response to IL-4, IL-7, IL-10, IL-21 and CD40 ligand treatment. Our results demonstrate the existence of both symmetry and asymmetry in the phosphoprotein signature between leukemic and non-leukemic cells in pediatric B-ALL diagnostic samples.

The prognostic significance of hematogones in childhood B-cell acute lymphoblastic leukemia.

Recent studies have demonstrated hematogones (HGs) expansion to be associated with favorable outcomes in hematological diseases, especially in patients with acute myeloid leukemia and patients undergoing hematopoietic stem cell transplantation. Acute lymphoblastic leukemia (ALL) is the most common form of cancer in children. As of now, minimal residual disease (MRD) remains the most compelling independent prognostic factor in childhood ALL. There is need for more prognostic tools for evaluating relapse risk. The goal of this study was to assess the prognostic value of HGs on relapse-free survival (RFS) and overall survival (OS) in childhood ALL. In this prospective cohort study, a total of 122 subjects with definitive diagnosis of precursor B lymphoblastic leukemia were evaluated. Flow cytometric HG detection was performed in bone marrow aspirates after induction and consolidation therapy. The median follow-up period of patients was 35.5±9.4 (SD) months. Patients who had at least 1.0% HGs had a significantly better RFS (p=.023). Moreover, univariate and multivariate analyses confirmed that positive HGs were independently associated with longer RFS (unadjusted model: hazard ratio=0.33, 95% CI=0.12-0.91, p=.031; adjusted model: hazard ratio=0.30, 95% CI=0.11-0.82, p=.020). Along with the role of MRD, our study shows the significance of HGs as an independent prognostic factor. The results indicate the independent prognostic value of HGs on RFS after adjustment for other prognostic factors, and can be beneficial for risk stratification and treatment modifications amongst pediatric B-cell ALL patients.

Blinatumomab for treating pediatric B-lineage acute lymphoblastic leukemia: A retrospective real-world study.

Blinatumomab was shown to be safe and effective for consolidation therapy in B-cell acute lymphoblastic leukemia (B-ALL). This study aimed to investigate the effectiveness and safety of blinatumomab in pediatric B-ALL patients in a real-world setting. This was a retrospective, observational study that included patients who initiated blinatumomab treatment between October 1, 2020 and June 20, 2022. Patients with B-ALL diagnosis, age below 18 years, and at least one blinatumomab treatment cycle were included. Treatment-related toxicities were assessed. Totally 23 pediatric patients were included in this study, with a median age of 6 years (range, 2 to 11 years). Blinatumomab therapy was applied for MRD-positive (disease ≥0.01%, n = 3) or chemotherapy-ineligible (n = 20) B-ALL cases. The median follow-up time was 9 months, and all evaluable patients achieved complete molecular remission with undetectable MRD. Four relapsed B-ALL cases proceeded to hematopoietic stem cell transplantation (HSCT) without further bridging therapy, while the others underwent maintenance chemotherapy after blinatumomab treatment. Grade ≥3 febrile neutropenia, white blood cell decrease and seizure were observed in 57%, 48% and 4.3% of patients, respectively. One case discontinued therapy due to neurologic toxicities. Elevated cytokine levels were observed in 4 patients. In all 23 patients, increased T-cell and low B-cell counts (<10/μl) were detected during blinatumomab therapy. These encouraging results suggest blinatumomab in pediatric B-ALL patients with MRD+ or chemotherapy-related toxicities is effective and safe in the short run, although long-term follow-up is still needed.

Abstract 3910: Cellular profiling of leukapheresis and infusion products of patients enrolled in CD19 CAR T cell therapy identifies biomarkers associated with clinical outcomes

Abstract CD19 CAR T cell therapy has emerged as a promising therapy in the treatment of B cell malignancies, such as B cell acute lymphoblastic leukemia (B-ALL). However, questions remain regarding the heterogeneity of the CAR T cell infusion products that lead to differential clinical outcomes and side effects, including cytokine release syndrome and neurotoxicity. The purpose of this study is to identify biomarkers and T cell subpopulations within 1) leukapheresis samples of patients receiving CD19 CAR T cell therapy and 2) pre-infusion manufactured CAR T cell products to identify correlates with clinical outcomes. We hypothesize that different manufacturing practices used to generate CAR T cells from the patient’s leukapheresis T cells drive heterogeneity within the infusion products that drive differential patient outcomes, and that cell intrinsic properties within the leukapheresis T cells drive phenotypic dispositions observed in the infusion products. Leukapheresis samples and pre-infusion CAR T cells were obtained from pediatric and adult B-ALL patients enrolled in CD19 CAR T cell clinical trials. Briefly, leukapheresis T cells from pediatric patients were CD4/8 sorted and then stimulated with CD3/CD28 beads followed by CAR transduction. Pediatric CD4 CAR T cells were cultured with IL-7 + IL-15 while CD8 CAR T cells were cultured with IL-2 + IL-15. CAR T cells from adults were stimulated with LCLs expressing CD19. To identify T cell subpopulations and biomarkers leukapheresis T cells and CAR T cells from each patient were stained with two mass cytometry panels: 1) a phenotype panel of antibodies to characterize surface markers, transcription factors and chemokine receptors and 2) a functional panel of antibodies specific for cytokines to broadly characterize the secretion profile of leukapheresis and CAR T cells upon stimulation with PMA and Ionomycin. Additional multi-omic profiling was performed with CITE-seq and 10X Chromium on leukapheresis samples and CAR T cells of select adult patients to provide in depth analysis on transcriptomic drivers of phenotypes observed within CAR T cell infusion products. We hierarchically clustered leukapheresis T cells and CAR T cells and correlated the abundance of T cell subclusters within each patient to their neurotoxicity grade (0-5). We determined that CD4 CAR T cells that secrete elevated levels of IL-3 upon stimulation correlate with an increased risk of severe neurotoxicity (designated as grades 3-5) within our pediatric cohort. Identification of IL-3 as a driver of neurotoxicity within the CD4 infusion products may inform strategies to improve manufacturing practices used to generate safer CAR T cell products. Citation Format: Tony Chour, Alexandre Hirayama, Ye Zheng, Alyssa Sheih, Summer Zhuang, Ashley Wilson, Vicky Wu, Raphael Gottardo, Cameron Turtle, Rebecca Gardner, Evan Newell. Cellular profiling of leukapheresis and infusion products of patients enrolled in CD19 CAR T cell therapy identifies biomarkers associated with clinical outcomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3910.

Elevated blood MxA protein levels in children with newly diagnosed B-ALL: A prospective case-control study

Background/Aim: Although leukemia is thought to be triggered or initiated by viral infections, it is not clear which viruses are the causative agents for which stage of the disease. Previous studies have shown that the MxA protein is expressed from blood mononuclear cells in reply to inducement of type I interferons in viral infections. Viral infections may trigger childhood B-cell acute lymphoblastic leukemia (B-ALL), and the hypothesis of this study was the detection of the presence of viral infection by measuring MxA expression in blood mononuclear cells of recently diagnosed pediatric B-ALL patients as a surrogate viral marker. Methods: This study consisted two groups; the study group consisted of 30 newly diagnosed B-ALL and the control group consisted of 29 healthy asymptomatic children of similar age. Proven bacterial infection and COVID-19 PCR positivity were exclusion criteria. Bacterial culture of peripheral blood, complete blood count, plasma CRP levels and whole blood MxA levels detected by ELISA (Enzyme-Linked ImmunoSorbent Assay) method were taken. Results: The patients’ mean age was 7.42 years in the leukemia group (previously mentioned as study group) and 7.25 years in the control group. Routine serologic studies for newly diagnosed leukemia patients (CMV, EBV VCA and Hepatitis B IgM, anti-HCV and anti-HIV) were negative in all patients without any bacterial infection detected. The MxA levels were found significantly higher in children with B-ALL than in control group (5.84 (2.18-199.38) and 2.45 (1.17-88.65) ngr/ml, respectively, with P

Clinical impacts of copy number variations in B-cell differentiation and cell cycle control genes in pediatric B-cell acute lymphoblastic leukemia: a single centre experience

BackgroundIKZF1 gene deletions have been identified as a poor prognostic factor in pediatric B-cell acute lymphoblastic leukemia (B-ALL), especially in the presence of co-occurring deletions (IKZF1plus profile). This study aimed to determine the frequency of IKZF1 deletions and deletions in other B-cell differentiation and cell cycle control genes, and their prognostic impact in Slovenian pediatric B-ALL patients.Patients and methodsWe studied a cohort of 99 patients diagnosed with B-ALL from January 2012 to December 2020 and treated according to the ALL IC-BFM 2009 protocol. Eighty-eight bone marrow or peripheral blood samples were analysed for copy number variations (CNVs) using the SALSA MLPA P335 ALL-IKZF1 probemix.ResultsAt least one CNV was detected in more than 65% of analysed samples. The most frequently altered genes were PAX5 and CDKN2A/B (30.7%, 26.1%, and 25.0%, respectively). Deletions in IKZF1 were present in 18.2% of analysed samples and were associated with an inferior 5-year event-free survival (EFS; 54.8% vs. 85.9%, p = 0.016). The IKZF1plus profile was identified in 12.5% of the analysed samples, and these patients had an inferior 5-year EFS than those with deletions in IKZF1 only and those without deletions (50.8% vs. 75.0% vs. 85.9%, respectively, p = 0.049). Overall survival (OS) was also worse in patients with the IKZF1plus profile than those with deletions in IKZF1 only and those without deletions (5-year OS 76.2% vs. 100% vs. 93.0%, respectively). However, the difference between the groups was not statistically significant.ConclusionsOur results are in concordance with the results obtained in larger cooperative clinical trials. Copy number variations analysis using the SALSA MLPA kit is a reliable tool for initial diagnostic approach in children with B-ALL, even in smaller institutions in low- and middle-income countries.

A Novel Anti-CD19 Chimeric Antigen Receptor T Cell Product Targeting a Membrane-Proximal Domain of CD19

Introduction: CD19-directed chimeric antigen receptor T (CART19) cell therapies have shown impressive clinical outcomes in CD19+ B-cell malignancies. All the FDA-approved CART19, including tisagenlecleucel, axicabtagene ciloleucel, brexucabtagene autoleucel, and lisocabtagene maraucel use an anti-CD19 single-chain variable fragments (scFv) derived from the FMC63 antibody that binds to a CD19 epitope that is located in the membrane-distal portion of CD19. While this CART19 products are very effective in the clinic, the majority of patients still fails treatment or eventually relapses due to several mechanisms of resistance, including CAR T cell dysfunction in the immunosuppressive microenvironment. Novel strategies to enhance the activity of CART cells are critically needed. We and others recently demonstrated that modifications of the binding region of the CAR (scFv) (Singh N., Nat Med, 2021) can drastically change the interaction between the CAR T cell and the cancer cells, potentially improving the anti-tumor effect. In this study, we aimed to develop a novel anti-CD19 CAR that binds to a membrane-proximal domain of CD19 with the goal of improving CART functions. Moreover, we hypothesized that such a CART product would be active against B-cell acute lymphoblastic leukemia (B-ALL) blasts that present aberrant expression of the CAR19(FMC63) on the surface as we described in two pediatric B-ALL patients relapsed after CART19 (CTL019) at our Institution (Ruella M., Nat Med, 2018).Methods and Results: To these goals, we screened a chicken immune scFv library against the CD19 extracellular domain. We selected an scFv clone (1218) that was not competing with FMC63 for CD19 binding in competition enzyme immunoassays and real-time interaction analysis. The chicken 1218 scFv was humanized by CDR-grafting to human germline genes and backmutations (h1218 scFv) and was confirmed to be specific for CD19 in cell microarray assays and flow cytometry analyses. In order to define the epitope that was recognized by the 1218 scFv, we performed mutagenesis assays replacing several residues of human CD19 with monkey CD19 residues showed that, as compared to FMC63, the epitope of the h1218 scFv is localized in a membrane-proximal location shown by three-dimensional modeling (Teplyakov A., Proteins, 2018) (Fig. 1A). Key residues needed for the binding of the h1218 scFv were identified as T51, S53, E55, K59 and K63, while the membrane-distal amino acid H218 is recognized as a key residue for FMC63 binding.Therefore, we designed a novel CAR construct using the h1218 scFv, a 4-1BB costimulatory and CD3z signaling domains, using a lentiviral backbone. This CAR(h1218) construct was very active in vitro in human T cells demonstrating cytotoxicity, cytokine release, and proliferation upon encounter with CD19+ leukemic and lymphoma cells (NALM6, RAJI, and OCI-Ly18) (data not shown). Moreover, CART19(h1218) showed enhanced long-term (12 days) tumor control as compared to CART19(FMC63) against wild type NALM6 (data not shown). In vivo, CART19(h1218) cells (1.5x10 6 CAR+ cells/mouse) demonstrated tumor clearance against both Raji (CD19+ B-cell lymphoma) and Nalm6 (CD19+ B-cell leukemia) xenografts (Fig. 1B). Remarkably, in a more challenging stress- test model with lower CART doses (0.75x10 6 CAR+ cells/mouse) CART19(h1218) demonstrated stronger tumor control against Nalm6 cells as compared to CART19(FMC63) (Fig. 1C). Furthermore, our group recently described the occurrence of a B-ALL relapse characterized by the expression of the CAR19 itself on the cell surface due to accidental transduction of leukemic cells during manufacturing. The CART19(h1218) product but not CART19(FMC63) was able to recognize and kill CAR19(FMC63)-expressing leukemic cells in vitro at varying E:T ratios. Of note, in a long-term (12 days) in vitro killing assay using a low, challenging E:T ratio of 0.125:1 we observed potent and prolonged tumor control (Fig. 1D).Conclusions: These results indicate that the development of a CAR binder able to recognize an alternative and membrane-proximal epitope of CD19 might lead to improved anti-leukemia and lymphoma activity and can recognize CAR19(FMC63)+ blasts that are not recognized by CART19(FMC63). This novel CART19(h1218) product will be tested in phase I-II clinical trials for patients with B-cell malignancies. [Display omitted] DisclosuresPatel: AbClon Inc.: Research Funding. Kim: Abclon Inc.: Current Employment. Lee: AbClon Inc.: Research Funding. Guruprasad: AbClon Inc.: Research Funding. Kim: AbClon Inc.: Current Employment. Choi: AbClon Inc.: Current Employment. Kim: AbClon Inc.: Current Employment. Kim: AbClon Inc.: Current Employment. Lee: AbClon Inc.: Current Employment. Lee: AbClon Inc.: Current Employment. Park: AbClon Inc.: Current Employment. Lee: AbClon Inc.: Current Employment. Cui: AbClon Inc.: Current Employment. Yoon: AbClon Inc.: Current Employment. Kim: AbClon Inc.: Research Funding. Kim: AbClon Inc.: Research Funding. Hwang: AbClon Inc.: Current Employment. Lee: AbClon Inc.: Current Employment. Lee: AbClon Inc.: Current Employment. Chung: AbClon Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding. Ruella: Novartis: Patents & Royalties; BMS, BAYER, GSK: Consultancy; AbClon: Consultancy, Research Funding; Tmunity: Patents & Royalties; viTToria biotherapeutics: Research Funding.

Siglec-15 Is a Novel Immunomodulatory Protein and Therapeutic Target in Acute Lymphoblastic Leukemia

Immunotherapies have recently shown efficacy in treatment of aggressive, refractory pediatric B cell acute lymphoblastic leukemia (B-ALL), which remains one of the leading causes of cancer-related death in children. The immune evasion mechanisms of B-ALL are still being explored to discover new therapeutic targets and improve patient outcomes.Recent reports have implicated a role for the molecule Siglec-15 (Sig15) in regulating immune response in solid tumor-infiltrating macrophages. Our lab has found higher expression of SIGLEC15 at the RNA level in primary pediatric B-ALL as compared to healthy donor controls, as well as at the RNA and protein levels across a panel of B-ALL, T cell acute lymphoblastic leukemia (T-ALL), and diffuse large B cell lymphoma (DLBCL) cell lines compared to healthy donor PBMCs. Higher expression of SIGLEC15 in pediatric B-ALL samples from the TARGET database correlates with markers of PKC and NFκB activation known to drive B-ALL leukemogenesis, which we have demonstrated to regulate Sig15 RNA and protein expression in vitro.Knockout of Siglec15 expression in a BCR-ABL1 + murine model of B-ALL engrafted in immunocompetent and Rag1 -/- immunodeficient recipients resulted in leukemia clearance in immunocompetent, but not immunodeficient, recipients and 100% survival (Figure A, p=0.01 Sig15 KO into WT vs. Rag1 -/-). Further study indicates that Siglec15 expression on these leukemia cells suppresses T cell effector and memory population expansion at 7 days post-engraftment (Figure B) and correlates with higher levels of IL-10 and lower levels of CCL17 present in the bone marrow, representing a more immunosuppressive bone marrow milieu. These data suggest a prominent role for Sig15 in the suppression of adaptive immune response to B-ALL as well as other hematological malignancies.We have also reported for the first time the release of a soluble form of Sig15 (sSig15), which we have demonstrated to circulate at higher levels in the plasma of pediatric B-ALL patients compared to healthy donors (Figure C, ****P≤0.0001). Detection of this sSig15 negatively correlated with circulating levels of IL-12 and IL-1α/β (Figure D, depicting correlations of cytokines using Pearson's r), suggesting sSig15 levels correspond to a systemically immunosuppressive phenotype. Flow cytometry of fresh pediatric B ALL cells demonstrates expression of surface Sig15 in a subset of cases. Thus, Sig15 has the capacity to promote immunosuppressive effects at both marrow-localized and systemic levels.Together, these results suggest Siglec-15 is a novel, potent immunosuppressive molecule active in leukemia progression that may be targeted therapeutically to activate T lymphocytes against leukemia cells. [Display omitted] DisclosuresAbukharma: NextCure Inc.: Current Employment. Liu: NextCure: Current Employment, Current holder of stock options in a privately-held company.

Single-Cell Decoding of Minimal Residual Disease in Pediatric B Cell Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer, of which B cell ALL (B-ALL) accounts for up to 85% of all cases. Although risk-stratified chemotherapy significantly improves the clinical outcome of affected pediatric patients with B-ALL, relapse still occurs in approximately 10% of children, representing the main cause of pediatric cancer deaths. Minimal residual disease (MRD) that persists after chemotherapy was the most valuable prognostic marker for hematological malignancies and solid cancers. Unfortunately, our understanding of the resistance mechanisms elicited in MRD is limited due to the rarity and heterogeneity of these residual cells. In our study, we employed paired scRNA-seq and single-cell BCR sequencing (scBCR-seq) to study the distinct features of leukemic cells from longitudinal samples obtained at the diagnosis, residual and relapsed stages at the single-cell level.By performing unsupervised clustering of the scRNA-seq data from 16,543 bone marrow CD19 -CD34 +cells and 20,392 CD19 +cells of healthy donors, we successfully defined the cell clusters of different B cell development stages sequentially, from hematopoietic stem cell / lymphoid-primed multipotential progenitors (HSC/LMPP) to common lymphoid progenitor (CLP), proB, preBI, preBII, immature/mature B cells and finally, activated B cells.By referring to the landmarks of normal cells, we then employed scRNA-seq and scBCR-seq to further dissect the phenotypic complexities within and across 4 pediatric B-ALL diagnostic-relapsed pairs at the single cell level. Our study found that BCR states can be used to distinguish leukemic cells and normal cells. From the four relapsed pediatric B-ALL patients, we obtained a total of 104,055 CD19 +cells with paired scRNA-seq and scBCR-seq data encompassing the three stages of B cell differentiation that passed the quality control. By examining the scBCR-seq data at the diagnosis stage to distinguish leukemic and normal (or non-leukemic) B cells based on genome-wide expression patterns rather than a few marker genes, we employed a machine learning approach. To train the classifier, CD19 +cells with non-clonal BCRs in D19 samples were selected as “non-leukemic”, and CD19 +cells with clonal BCRs (B265) and without detected BCRs (B590, B069 and B887) in D19 samples were selected as “leukemic”.Using this strategy, we compared the gene expression profiles of the leukemic cells at the diagnosis and relapse for each patient, with the aim to identify specific features of relapse stage leukemia cells at the single-cell level. We found that the cell composition profile tended to shift to early differentiation stages in the relapsed samples in all four patients. By analyzing the differentiation stage, cell cycle and gene expression characteristics of relapsed cell samples at the single-cell level, we obtained some unique findings, such as significantly increased expression of CDKN1A in relapsed cells.To understand the basis of such specific differentiation stage and cell cycle transition during chemotherapy, we performed differential expression analysis to identify genes specifically altered at D19 compared to diagnosis and relapse. Then, pathway enrichment analysis applied to the differentially expressed genes revealed that the hypoxia pathway was one of the top hits, being significantly upregulated during intensified chemotherapy. We obtained experimental support by validating the efficacy of the HIF-1a inhibitor PX478 combined with chemotherapy drugs in two B-ALL cell lines and two primary B-ALL cells.In summary, our study leveraged single-cell transcriptomic analysis with paired BCR repertoire profiling to decode the molecular aberrations across the phenotypically heterogeneous disease, B-ALL. We also applied a powerful B cell development classifier and an innovative machine learning model for B-ALL cell identification at single-cell resolution to determine the clonal signatures, and to track residual cell evolution in a longitudinal manner from diagnosis, to post-treatment, and finally relapse. We propose that hypoxia signaling pathway activation might serve as valuable MRD therapeutic target. While this study provided great insights into the transcriptomic characteristics of MRD cells, practically, our analytical approach could readily be applied to other hematological malignancies and solid cancers. DisclosuresNo relevant conflicts of interest to declare.

Increase in polymorphonuclear myeloid-derived suppressor cells and regulatory T-cells in children with B-cell acute lymphoblastic leukemia

Our study aimed to evaluate the levels of MDSCs and Tregs in pediatric B-cell acute lymphoblastic leukemia (B-ALL), their relation to patients’ clinical and laboratory features, and the impact of these cells on the induction response. This study included 31 pediatric B-ALL patients and 27 healthy controls. All patients were treated according to the protocols of the modified St. Jude Children’s Research Hospital total therapy study XIIIB for ALL. Levels of MDSCs and Tregs were analyzed using flow cytometry. We observed a reduction in the levels of CD4 + T-cells and an increase in both the polymorphonuclear MDSCs (PMN-MDSCs) and Tregs. The frequencies of PMN-MDSCs and Tregs were directly related to the levels of peripheral and bone marrow blast cells and CD34 + cells. Complete postinduction remission was associated with reduced percentages of PMN-MDSCs and Tregs, with the level of PMN-MDCs in this subpopulation approaching that of healthy controls. PMN-MDSCs and Tregs jointly play a critical role in maintaining an immune-suppressive state suitable for B-ALL tumor progression. Thereby, they could be independent predictors of B-ALL progress, and finely targeting both PMN-MDSCs and Tregs may be a promising approach for the treatment of B-ALL.

Cross-Sectional Physician Survey on the Use of Minimal Residual Disease (MRD) Testing in the Management of Pediatric and Adult Patients with Acute Lymphoblastic Leukemia (ALL)

Introduction:Acute lymphoblastic leukemia (ALL) is a rare disease that progresses quickly and may be fatal within a few months if untreated. Although the majority of adults and pediatric patients with ALL achieve complete remission (CR) with current chemotherapy regimens, a significant proportion will ultimately have a relapse, particularly in the adult population. Minimal residual disease (MRD) has been demonstrated as a prognostic factor; higher levels of MRD indicate a higher likelihood of relapse. New techniques have been developed to quantitatively detect the presence of MRD in patients who achieve CR. The objective of this study was to explore use of MRD testing in the adult B-cell ALL patient population compared to its use in pediatric B-cell ALL patients, as well as to compare its use in academic versus community settings.Methods:This cross-sectional, non-interventional study used a quantitative survey to explore use of MRD testing among US physicians who treat ALL. Prior to implementation, 2 rounds of qualitative interviews were conducted to develop the content of the quantitative survey (4 in-person interviews and 2 online/phone interviews). Following the qualitative phase, a 20-minute quantitative online survey was conducted with US-based academic hematologists who actively monitor or treat ≥5 B-cell ALL patients and use MRD testing in practice. Physicians self-reported their primary hospital affiliation as being academic- or community-based and were classified as adult or pediatric treaters based on their reported proportion of adult versus pediatric B-cell ALL patients. Descriptive analyses (frequencies and percentages) of survey responses were conducted and Pearson's chi square test was used to assess differences by physician practice setting (academic versus community-based) and by primary patient population (adult versus pediatric).Results:One hundred fifty adult treaters (100 community adult treaters and 50 academic adult treaters) and 30 pediatric treaters (10 community pediatric treaters and 20 academic pediatric treaters) participated in the survey, for a total of 180 physician participants. Physicians used a variety of MRD tests, with flow cytometry being the most frequent choice among both adult and pediatric treaters, followed by polymerase chain reaction and next generation sequencing testing.The use of MRD testing was found to be higher among physicians who primarily treat pediatric patients (93%) than those who treat adult patients (73%) (p<0.05). Among the physicians who primarily treat adults (n=150), the number of patients receiving MRD testing was higher among those receiving treatment from an academic center compared to those from a community setting (84% and 67%, respectively; p<0.01). Academic adult treaters were also more likely to give patients ≥5 tests through the course of disease compared to community adult treaters (16% and 5%, respectively; p<0.05) (Table 1).Pediatric patients commonly received an MRD test during/after induction or upon relapse, while there was no consensus on the timing of MRD tests for adult patients. MRD testing was most common following maintenance treatment phase rather than during treatment (Figure 1). Collectively, physicians often conduct MRD testing after induction or consolidation of treatment.When asked to identify barriers that affected their decisions to conduct MRD tests, a greater proportion of academic adult treaters (32%) reported no barriers to conducting MRD testing compared to community adult treaters (13%) (p<0.01). Community adult treaters were more likely to indicate cost (50%) and lack of insurance coverage (42%) as barriers to MRD testing compared to academic adult treaters (24% each) (p<0.05) (Table 2; Figure 2).Conclusion:In this survey of physicians who treat patients with B-cell ALL and have used any MRD tests, physicians who primarily treat pediatric patients were more likely to conduct MRD testing than physicians who primarily treat adults. Physicians who treat patients with ALL in academic settings were also more likely to perform MRD testing compared with physicians who treat patients in community settings; some of the reasons for differences in MRD testing rates were cost and insurance barriers. Resolving these issues would be critical to improving detection of a molecular relapse in patients with ALL, most importantly among adults and in community settings. [Display omitted] DisclosuresChia:Amgen, Inc.: Employment, Equity Ownership. Delaney:Amgen, Inc.: Consultancy. McNamara:Amgen, Inc.: Consultancy. Kim:Amgen, Inc.: Employment, Equity Ownership. Romanov:Amgen, Inc.: Employment, Equity Ownership.

Pharmacokinetics and Pharmacodynamics of Tocilizumab for the Management of Cytokine Release Syndrome (CRS) in Pediatric and Young Adult Patients with Relapsed/Refractory (R/R) B-Cell Acute Lymphoblastic Leukemia (B-ALL) Treated with CAR T-Cell Therapy, CTL019

Background: ELIANA (NCT02435849) is a pivotal multicenter study to test the efficacy of CTL019 (tisagenlecleucel; anti-CD19 CAR-T) in children and young adult patients (pts) with relapsed/refractory B-ALL. Tocilizumab (toci) has been used for the management of moderate/severe (grade 3/4) CRS in ≈ 38% of pts treated with CTL019 (Buechner J et al, EHA 2017) at the same doses used in approved non-oncologic pediatric indications (<30 kg received 12 mg/kg and ≥30 kg received 8 mg/kg [800 mg max dose]). CRS onset generally occurred at a median of 3 days (range 1-22 days) after CTL019 infusion, requiring administration of 1-3 doses of toci in some patients per the Penn CRS treatment algorithm. Toci is a humanized mAb that binds to and blocks IL-6 receptor (IL-6R) signaling. The pharmacokinetics (PK) and pharmacodynamics (PD) of toci in pediatric B-ALL patients with CAR-associated CRS has not previously been described.Objectives: To characterize the PK/PD of toci for the management of CRS following CTL019 infusion in B-ALL in the ELIANA study. To describe the impact of toci on CTL019 cellular kinetics and expansion.Methods: Pharmacokinetics of toci and pharmacodynamics of soluble IL-6R (sIL-6R) concentrations (conc) were determined from serum and quantified using validated assays to provide mechanistic support for the use of toci in the management of CRS in B-ALL. Maximum toci conc (Cmax) was derived using non-compartmental methods and compared with levels in approved indications. sIL-6R, proinflammatory cytokines and time to CRS resolution were characterized to describe the pharmacodynamic effects of toci. Summary statistics and graphical analyses of CTL019 exposure by number of toci doses were performed for patients that responded to CTL019 infusion to describe the impact of toci on CTL019 expansion and persistence.Results: A total of 28 out of 58 pts with CRS received their first dose of toci at a median of 5 days (range 1-18) after CRS onset. 17 pts received 1 IV dose of toci; 8 pts 2 doses; 3 pts 3 doses. First dose of toci ranged from 6.9 to 12 mg/kg; second dose of toci ranged from 8 to 12 mg/kg. Following the first and second dose of toci, the mean Cmax (SD) were ≈ 111 (30.6) µg/mL and 265 (376) µg/mL, respectively (Table 1). Individual patient PK/PD concentration-time profiles in B-ALL, show increased sIL-6R after the first toci dose that remained elevated following the second dose (Figure 2). The median time to CRS resolution (including fever resolution) was 5 days (range, 2-29 days) after toci administration. CRS onset coincided with CTL019 expansion, and was followed by a peak in serum cytokines, including IL-6. Maximal expansion of the CTL019 transgene (determined by qPCR) was 159% higher in patients treated with toci (n=14) compared with patients that did not receive toci (n=28) demonstrating tocilizumab does not negatively impact expansion (Figure 3).Conclusions: Toci administration resulted in resolution of CRS symptoms within a median of 5 days after administration. Notably, levels of toci achieved in B-ALL were similar to levels published in pediatric non-oncologic indications [tocilizumab label], resulting in concentration- and time-dependent increases in sIL-6R. CTL019 transgene expanded and persisted following tocilizumab administration. Together these data support the use of toci for the management of CRS, showing the compound to be 1) pharmacologically active, 2) able to achieve levels consistent with those achieved in other disorders in spite of CRS and 3) able to be given without impairing CTL019 expansion. [Display omitted] DisclosuresLee:Novartis Pharmaceuticals Corporation: Other: Post-Doctoral Fellow; The State University of New Jersey: Other: Post doctoral fellow. Bittencourt:Novartis Pharmaceuticals Corporation: Consultancy; Amgen Inc.: Consultancy; Jazz Pharmaceuticals: Consultancy, Honoraria, Other: Travel Grant. Rives:Novartis Pharmaceuticals Corporation: Consultancy; Jazz Pharmaceutical: Other: Travel expenses; Shire: Consultancy. Boyer:Novartis Pharmaceuticals Corporation: Honoraria. Pulsipher:Adaptive: Other: Advisory board 6/17; CSL Behring: Other: advisory board Feb 2017; Jazz Pharmaceuticals: Other: advisory board, education; Chimerix: Other: Advisory board Dec 2015; Novartis Pharmaceuticals Corporation: Consultancy, Other: Phase II steering committee. Jaitner:Novartis Pharma AG: Employment. Yi:Novartis Pharmaceuticals Corporation: Employment. Lund:Novartis Pharmaceuticals Corporation: Employment. Leung:Novartis Pharmaceuticals Corporation: Employment. Awasthi:Novartis Pharmaceuticals Corporation: Employment. Wood:Novartis Pharmaceuticals Corporation: Employment, Equity Ownership. Maude:Novartis Pharmaceuticals: Consultancy, Other: Medical Advisory Boards. Grupp:Novartis Pharmaceuticals Corporation: Consultancy, Other: grant; Adaptimmune: Consultancy; Jazz Pharmaceuticals: Consultancy; University of Pennsylvania: Patents & Royalties. Mueller:Novartis Pharmaceuticals Corporation: Employment.