Acute Lymphoblastic Leukemia Therapy Research Articles

Pilot study shows suppression of mineralocorticoid precursors under high-dose glucocorticoid therapy in pediatric acute lymphoblastic leukemia.

Glucocorticoids represent a key element in the treatment of pediatric acute lymphoblastic leukemia (ALL) and lead to adrenal suppression. We aimed to assess the differential response profile of adrenal steroids in children with ALL during BFM (Berlin-Frankfurt-Münster) induction treatment. Therefore, we performed liquid chromatography tandem-mass spectrometry (LC-MS/MS)-based steroid profiling of up to seven consecutive leftover morning serum samples derived from 11 patients (pts) with ALL before (day 0) and during induction therapy at days 1-5, 6-12, 13-26, 27-29, 30-35 and 36-40. 17-hydroxyprogesterone (17OHP), 11-deoxycortisol (11S), cortisol, 11-deoxycorticosterone (DOC), corticosterone and aldosterone were determined in parallel. Subsequently, steroid concentrations were normalized by multiples of median (MOM) to adequately consider pediatric age- and sex-specific reference ranges. MOM-cortisol and its precursors MOM-11S and MOM-17OHP were significantly suppressed by glucocorticoid treatment until day 29 (P < 8.06 × 10-10, P < 5.102 × 10-5, P < 0.0076, respectively). Cortisol recovered in one of four pts at days 27-29 and in two of five pts at days 36-40. Among the mineralocorticoids, corticosterone was significantly suppressed (P < 3.115 × 10-6). Aldosterone and DOC showed no significant changes when comparing day 0 to the treatment time points. However, two ALL patients with ICU treatment due to the sepsis showed significantly lower MOM-DOC (P = 0.006436) during that time and almost always the lowest aldosterone compared to all other time points. Suppression of mineralocorticoid precursors under high-dose glucocorticoid therapy suggests a functional cross talk of central glucocorticoid regulation and adrenal mineralocorticoid synthesis. Our data should stimulate prospective investigation to assess potential clinical relevance.

Gaps in Parental Understanding of Sleep Disturbances During Maintenance Therapy for Pediatric Acute Lymphoblastic Leukemia.

Background: Acute lymphoblastic leukemia (ALL) is the most common cancer in childhood, with survival rates approaching 90%. Sleep disturbance is common among ALL patients, often developing during the initial stages of chemotherapy treatment. While there have been significant efforts to understand and intervene in this issue during survivorship, there is far less research on children who are actively receiving treatment. In the current study, we sought to better understand the parent's experience in the sleep domain during maintenance therapy, including their perceptions of how their child's medical team had managed sleep disturbances, and recommendations for how to improve sleep management. Method: Fifteen parents of pediatric ALL patients (aged 4-12 years) completed semistructured interviews. Interview content was analyzed using a multistage thematic analysis. Results: Parents consistently expressed feeling unprepared to manage the sleep disruptions that arose during treatment, often reporting that they did not recall being told this would be a side effect. They were enthusiastic about learning how to improve their child's sleep, though they did not want pharmacotherapeutic interventions or additional medical/psychosocial appointments to address this. Conclusion: Despite consistent provider communication on sleep, parents report limited knowledge of the issue. This provides an obvious intervention target to improve treatment-related sleep disturbances. Clear messaging may help direct parents' attention and expectations regarding their child's treatment and potential for disturbed sleep, possibly in the form of a behavioral intervention that empowers parents with information about how to support their child's sleep health while they are undergoing treatment for ALL.

Analysis of non-alcoholic fatty liver disease during induction therapy for B-cell precursor acute lymphoblastic leukemia in children and adolescents

A prospective evaluation of non-alcoholic fatty liver disease (NAFLD) during induction therapy for acute lymphoblastic leukemia (ALL) has not been performed. Herein, we prospectively investigated the frequency, risk factors, and outcomes of NAFLD during induction therapy in children and adolescents with B-cell precursor ALL (BCP-ALL). This study enrolled 74 newly diagnosed BCP-ALL cases aged 1 year and older who were admitted to our department between January 2011 and December 2020. Median age was 6.6 years (1.3-17.5 years). Plain computed tomography (CT) of the upper abdomen was performed before induction therapy, and on days 15 and 29 after initiation of induction therapy. Patients with a liver/spleen CT ratio <0.9 were defined as having NAFLD. The frequency of NAFLD was 73%. Patients with NAFLD had a higher rate of hypertriglyceridemia. There was no significant difference in 5-year overall survival and event-free survival (EFS) between patients with and without NAFLD. However, after restricting the target age to 10 years and older, 5-year EFS was significantly higher in patients with NAFLD than in those without (88.5 vs. 42.9%, respectively, P=0.037). Similarly, 5-year cumulative incidence of relapse (CIR) was significantly lower in patients with NAFLD than in those without it (5-year CIR, 6.3 vs. 57.1%, respectively, P=0.013). Patients with NAFLD exhibit better outcomes including 5-year EFS and CIR. Further studies are necessary.

Current clinical outcome of CD19/CD22 dual-targeting CAR T-cell therapy in refractory or relapsed B-cell acute lymphoblastic leukemia

To enhance clinical outcomes for hematologic malignancies, dual-targeting CAR T-cell therapies were devised. This study seeks to generalize the safety and efficacy of CD19/CD22 dual-targeting CAR T-cell therapy for the treatment of refractory or relapsed B-cell acute lymphoblastic leukemia in published clinical studies to cast lights on current advances and future challenges of this therapeutic. Manual searches were performed on PubMed and Science Direct to identify relevant articles for inclusion in the report. Forest plots were utilized to display data from individual studies as well as pooled estimates derived based on random effect model for complete remission rate, relapse rate, overall survival rate, and incidences of adverse events, and all data were presented along with their respective 95% confidence intervals. As per result, the pooled estimates for complete remission rate (CR), CR with negative minimal residual disease, relapse rate, overall survival rate (OS), incidences of cytokine release syndrome (CRS), severe CRS, neurotoxicity (NT) and severe NT are as follows: 89% (95% CI: 83.4%, 93.1%), 86.5% (95% CI: 83.4%, 93.1%), 43.3% (95% CI: 26.4%, 60.3%), at most 64.1% (95% CI: 50.7%, 75.3%) at 1 year, 80.4% (95% CI: 66.4%, 91.1%), 10.7% (95% CI: 0%, 30.3%), 9% (95% CI: 2.9%, 15%), 6.5% (95% CI: 0%, 25.5%), suggesting that CD19/CD22 dual-targeting CAR T-cell therapy has a great potential for treating patients with refractory or relapsed B-cell acute lymphoblastic leukemia.

Resistance mechanism to Notch inhibition and combination therapy in human T-cell acute lymphoblastic leukemia

AbstractGain-of-function mutations in NOTCH1 are among the most frequent genetic alterations in T-cell acute lymphoblastic leukemia (T-ALL), highlighting the Notch signaling pathway as a promising therapeutic target for personalized medicine. Yet, a major limitation for long-term success of targeted therapy is relapse due to tumor heterogeneity or acquired resistance. Thus, we performed a genome-wide CRISPR-Cas9 screen to identify prospective resistance mechanisms to pharmacological NOTCH inhibitors and novel targeted combination therapies to efficiently combat T-ALL. Mutational loss of phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1) causes resistance to Notch inhibition. PIK3R1 deficiency leads to increased PI3K/AKT signaling, which regulates cell cycle and the spliceosome machinery, both at the transcriptional and posttranslational level. Moreover, several therapeutic combinations have been identified, in which simultaneous targeting of the cyclin-dependent kinases 4 and 6 (CDK4/6) and NOTCH proved to be the most efficacious in T-ALL xenotransplantation models.

Therapy of childhood acute lymphoblastic leukemia in resource-poor geospaces.

The therapy of children with acute lymphoblastic leukemia (ALL) in limited resource geospaces is challenging and must balance safety, efficacy, availability, and affordability. We modified the control arm of the St. Jude Total XI protocol for outpatient delivery including once-weekly daunorubicin and vincristine in initial therapy, postponing intrathecal chemotherapy until day 22, prophylactic oral antibiotics/antimycotics, use of generic drugs, and no central nervous system (CNS) radiation. Data were interrogated from 104 consecutive children ≤12 years (median, 6 years [interquartile range (IQR), 3, 9 years]. All therapies were given in an outpatient setting in 72 children. Median follow-up is 56 months (IQR 20, 126 months). A total of 88 children achieved a hematological complete remission. Median event-free survival (EFS) is 87 months [95% confidence interval (CI), 39, 60], 7.6 years in low-risk children (3.4, 8 years) whereas 2.5 years (1, 10 years) in high-risk children. The 5-year cumulative incidence of relapse (CIR) is 28% (18, 35%), 26% (14, 37%) in low-risk children and 35% (14, 52%) in high-risk children. Median survival for all subjects is not reached but must exceed 5 years. A total of 36 children relapsed at a median of 12 months (5, 23 months). Outcomes were comparable to those reported in the control arm of the Total Therapy XI study, but inferior to current treatment protocols in high-income countries. The average cost of the first 2 years of therapy was $28,500 USD compared with an average cost of approximately $150,000 USD in the US, an 80% saving. In conclusion, using an outpatient-based modification of the St. Jude Total XI protocol, we obtained good results with relatively few hospitalizations or adverse events and at a substantial saving. This model can be applied in other resource-poor geospaces.

Evaluation of FTO polymorphism in 6-mercaptopurine related intolerance in children with acute lymphoblastic leukemia.

Thiopurine drugs like 6-Mercaptopurine (6MP) are the cornerstone of maintenance therapy in acute lymphoblastic leukemia (ALL). A recently described variant in alpha-ketoglutarate dependent dioxygenase (FTO) gene has been reported to play an important role in thiopurine induced myelosuppression. In this study, we genotyped a coding variant (p.Ala134Thr, rs79206939) and an intronic variant (rs16952570) of FTO in 174 Indian children (age ≤ 12years) with ALL on maintenance phase of chemotherapy and examined correlation with the risk of thiopurine induced myelosuppression and hepatic toxicity. The prevalence of FTO-rs16952570 polymorphism was 18.4% (32/174) with 142 (82%) cases having TT genotype, 26 (15%) cases with TC genotype and 6 (3.4%) cases having CC genotype. FTO-rs79206939 was absent and non-polymorphic in our study group. The mean dose of 6-MP during 36weeks of maintenance of TT, TC and CC carriers of FTO-rs16952570 was 53.7, 53.6 and 54.1mg/m2/day. Number of patients tolerating starting dose of 60mg/m2/day was significantly higher in CC (50%) than TT/TC (14%) genotype carrying cases (p = 0.014). However, no statistical significance was observed for total leukocyte count (TLC), absolute neutrophil count (ANC) as well as for platelets counts in patients harboring FTO-rs16952570 TT/TC/CC genotype at 4, 8, 12, 24 and 36weeks after start of thiopurine therapy. Further, no significant correlation was noted between number of weeks of chemotherapy interruptions or episodes of febrile neutropenia and no evidence of hepatotoxicity was found with the genotype studied. Polymorphism in FTO-rs16952570 did not show any correlation with thiopurine related toxicity in ALL patients.

Differentiated, immunobased approach of anaplastic large cell lymphoma treatment in children and adolescents: literature review and protocol ALCL-NII DOG-2003 results

Background. Anaplastic large cell lymphoma (ALCL) is a rare variant of highly aggressive lymphomas in children and adolescents. ALCL is characterized by a heterogeneous clinical manifestation with nodal and extranodal lesions, various morphological variants, variable expression of T-cell markers and cytogenetic aberrations involving the ALK gene. Due to the rarity of this disease (up to 10–15 % of all non-Hodgkin’s lymphomas in children), there are no uniform standards for ALCL therapy. Block treatment schemes similar to those applied in the treatment of B-cell non-Hodgkin’s lymphomas are used; less often there can be used protocols similar to the therapy of acute lymphoblastic leukemia. Taking into account the unfavorable effect on the prognosis of T-cell markers expression in ALCL, we developed protocol ALCL-NII DOG-2003, which considered the immunological features of the tumor.Aim. To assess the effectiveness of differentiated immunobased ALCL therapy by domestic protocol ALCL-NII DOG-2003.Materials and methods. From 01.03.1997 to 01.10.2022, 60 patients with primary diagnosed ALCL were included in the study. The diagnosis was based on histological, immunohistochemical and cytogenetic criteria of WHO classification of hematopoietic and lymphoid tissues tumors. According to treatment program, patients were subdivided in 2 groups: patients treated by NHL-BFM 95 protocol (n = 21) and by ALCL-NII DOG-2003 (n = 39). The male:female ratio was 1,3:1 in group of patients with NHL-BFM 95 protocol and 1,4:1 – in ALCL-NII DOG 2003. The effectiveness assessment was based on overall, event-free and relapse-free survival with SPSS 21.0 computed program. The differences between groups were significant with p ≤0.05.Results. The use of risk-adopted, immuno-oriented protocol made it possible to obtain a 10‑year EFS in 97.4 ± 2.6 %, whereas results of the standard NHL-BFM 95 protocol are 63.9 ± 10 % (p = 0.001).Conclusion. The use of a differentiated approach to ALCL treatment, considering not only the stage and prognostic risk group, but also the immunological features of the tumor, is more effective, and allows achieving significantly higher survival rates, than standard program (NHL-BFM 95).

The role of minimal residual disease in therapy of pediatric acute lymphoblastic leukemia: a prospective cohort study

Background. During last 50 years there was a significant progress in understanding the nature of pediatric acute lymphoblastic leukemia (ALL). There were developed effective chemoradiotherapy regimens, new methods of diagnosis and emerged evaluation of treatment results. Determination of minimal residual disease (MRD) has become the most important factor in the patients stratification for risk-adapted treatment in the ALL IC-BFM 2009.&#x0D; Aim. To evaluate the survival rates of children with ALL according to the ALL IC-BFM 2009 protocol.&#x0D; Materials and methods. There were 136 people in the study of evaluating the effectiveness of therapy according to the ALL IC-BFM 2009 protocol: 69 boys and 67 girls (ratio 1.03:1). The median age is 4 years and 10 months. The observation period is from 26.01.2010 to 06.11.2022.&#x0D; Results. High survival rates of children with newly diagnosed ALL are achieved: overall survival (OS) is 91.2%, event-free (EFS) 82.4%, relapse-free (RFS) 88.6%. The best results are among patients of the standard risk group: OS, EFS and RFS reach 96.6%. In the intermediate group OS 96.2%, EFS 84.8% and RFS 88.3%. Worse results of survival are in the high risk group: OS 76.9%, EFS 65.4% and RFS 80.7%. Outcome analysis depending on the linear reveals a statistically insignificant difference in survival rates (for B-ALL OS 92.4%, EFS 83.1% and RFS 89.5%, for T-ALL OS 83.3%, EFS 77.8% and RFS 83.3%). It determines the tendency of improving the prognosis of pediatric T-ALL by optimizing the stratification of patients based on the indicators of MRD and the best direction of protocol.&#x0D; Conclusion. Results of survival rates of patients with ALL confirm high effectiveness of treatment according to the ALL IC-BFM 2009 protocol. MRD level on day 15 makes it possible to stratify patients and choose the optimal risk-adapted therapy.

Difference in Efficacy and Safety of Anti-CD19 Chimeric Antigen Receptor T-Cell Therapy Containing 4-1BB and CD28 Co-Stimulatory Domains for B-Cell Acute Lymphoblastic Leukemia.

This study quantified the differences in the efficacy and safety of different stimulation domains of anti-CD19 chimeric antigen receptor (CAR) T therapy for B-cell acute lymphoblastic leukemia (B-ALL). Clinical trials related to anti-CD19 CAR T-cell therapy for B-ALL were searched in public databases from database inception to 13 November 2021. The differences in overall survival (OS) and progression-free survival (PFS) of B-ALL patients treated with anti-CAR T-cell therapy containing 4-1BB and CD28 co-stimulatory domains were compared by establishing a parametric survival function. The overall remission rate (ORR), the proportion of people with minimal residual disease (MRD)-negative complete remission (CR), the incidence of cytokine release syndrome (CRS), and the neurotoxicity across different co-stimulatory domains was assessed using a random-effects model. The correlation between the ORR, MRD-negative CR, PFS, and OS was tested. The results showed that the median OS of anti-CAR T-cell treatment containing 4-1BB and CD28 co-stimulatory domains was 15.0 months (95% CI: 11.0-20.0) and 8.5 months (95% CI: 5.0-14.0), and the median PFS was 7.0 months (95% CI: 4.0-11.5) and 3.0 months (95% CI: 1.5-7.0), respectively. Anti-CD19 CAR T-cells in the 4-1BB co-stimulatory domain showed superior benefits in patients who achieved ORR. The incidence of neurotoxicity was significantly higher in the CD28 co-stimulatory domain of anti-CD19 CAR T-cells than in the 4-1BB co-stimulatory domain. In addition, the ORR and MRD-negative CR were strongly correlated with OS and PFS, and PFS and OS were strongly correlated. The 4-1BB co-stimulatory domain suggested a better benefit-risk ratio than the CD28 co-stimulatory domain in B-ALL.

DNA polymerase gamma variants and hepatotoxicity during maintenance therapy of childhood acute lymphoblastic leukemia: is there a causal relationship?

Hepatotoxicity is a frequent complication during maintenance therapy of acute lymphoblastic leukemia (ALL) with 6-mercaptopurine and methotrexate. Elevated levels of methylated 6-mercaptopurine metabolites (MeMP) are associated with hepatotoxicity. However, not all mechanisms are known that lead to liver failure in patients with ALL. Variants in the POLG gene, which encodes the catalytic subunit of mitochondrial DNA polymerase gamma (POLG1), have been related to drug-induced hepatotoxicity, for example, by sodium valproate. The association of common POLG variants with hepatotoxicity during maintenance therapy was studied in 34 patients with childhood ALL. Of the screened POLG variants, four different variants were detected in 12 patients. One patient developed severe hepatotoxicity without elevated MeMP levels and harbored a heterozygous POLG p.G517V variant, which was not found in the other patients.

Emerging molecular subtypes and therapies in acute lymphoblastic leukemia.

Tremendous strides have been made in the molecular and cytogenetic classification of acute lymphoblastic leukemia based on gene expression profiling data, leading to an expansion of entities in the recent International Consensus Classification (ICC) of myeloid neoplasms and acute leukemias and 2022 WHO Classification of Tumours: Haematolymphoid Tumors, 5th edition. This increased diagnostic and therapeutic complexity can be overwhelming, and this review compares nomenclature differences between the ICC and WHO 5th edition publications, compiles key features of each entity, and provides a diagnostic algorithmic approach. In covering B-lymphoblastic leukemia (B-ALL), we divided the entities into established (those present in the revised 4th edition WHO) and novel (those added to either the ICC or WHO 5th edition) groups. The established B-ALL entities include B-ALL with BCR::ABL1 fusion, BCR::ABL1-like features, KMT2A rearrangement, ETV6::RUNX1 rearrangement, high hyperdiploidy, hypodiploidy (focusing on near haploid and low hypodiploid), IGH::IL3 rearrangement, TCF3::PBX1 rearrangement, and iAMP21. The novel B-ALL entities include B-ALL with MYC rearrangement; DUX4 rearrangement; MEF2D rearrangement; ZNF384 or ZNF362 rearrangement, NUTM1 rearrangement; HLF rearrangement; UBTF::ATXN7L3/PAN3,CDX2; mutated IKZF1 N159Y; mutated PAX5 P80R; ETV6::RUNX1-like features; PAX5 alteration; mutated ZEB2 (p.H1038R)/IGH::CEBPE; ZNF384 rearranged-like; KMT2A-rearranged-like; and CRLF2 rearrangement (non-Ph-like). Classification of T-ALL is complex with some variability in how the subtypes are defined in recent literature. It was classified as early T-precursor lymphoblastic leukemia/lymphoma and T-ALL, NOS in the WHO revised 4th edition and WHO 5th edition. The ICC added an entity into early T-cell precursor ALL, BCL11B-activated, and also added provisional entities subclassified based on transcription factor families that are aberrantly activated.

Role of Cytogenetics and FISH in Laboratory Workup of B Cell Precursor Acute Lymphoblastic Leukemia

AbstractModern therapeutic protocols in acute leukemias risk stratify disease based on genetic characterization of the neoplastic cells and their response to treatment. Genetic characterization is routinely performed by cytogenetic testing of leukemic cells and is a standard component of modern risk-adapted therapy in acute lymphoblastic leukemia (ALL). High-throughput technologies like RNA sequencing have identified multiple novel subtypes in recent years. The cytogenetic strategy using GTG and fluorescent in-situ hybridization (FISH) has to be adapted to identify not only the primary principal chromosomal abnormalities but also the novel subtypes. In the review, we describe a systematic comprehensive cytogenetic strategy that integrates information from immunophenotyping, flow-based DNA ploidy, and karyotyping complemented by targeted FISH studies to identify more than 70% of genetic abnormalities described in B cell precursor ALL. The simplified strategy includes a four-probe FISH and flow ploidy strategy, ± karyotyping that identifies high risk (KMT2A, BCR::ABL1, hypodiploidy, iAMP21) and standard risk (ETV6::RUNX1 and high hyperdiploid) cytogenetic groups. The extended FISH panel includes probes targeting MEF2D, ZNF384, and CRLF2 rearrangements that are used intuitively on integrating the immunophenotyping features that characterize these entities. The strategy also includes a systematic approach to identify masked hypodiploidy integrating targeted FISH analysis directed toward identifying monosomies of chromosomes 7, 15, and 17 and flow cytometry-based DNA ploidy analysis. The recently described PH-like ALL is characterized by ABL class fusions and rearrangements of CRLF2 and JAK2 genes. FISH analysis using break-apart probes can be used to identify these aberrations. The cytogenetic approach also includes FISH analysis to identify intragenic and whole gene deletions of the IKZF1 genes that identify a subset of patients associated with high risk of treatment failure.

New Onset of Tuberculosis Complicating FDG PET/CT Evaluation in Patient With Recent Chimeric Antigen Receptor T-Cell Therapy.

A 16-year-old adolescent girl with CD19 chimeric antigen receptor (CAR) T-cell therapy for acute lymphoblastic leukemia experienced new onset of the fever. 18F-FDG PET/CT studies acquired at 1 and 2 months, respectively, after CAR-T, showed foci of abnormal activity in the mediastinal lymph nodes not seen on the study before therapy. However, these foci of abnormal activity were later proven due to newly developed tuberculosis after CAR T-cell therapy.

Practice of L-Asparaginase Usage: A Survey among Healthcare Providers Treating Children with Cancer in India

Introduction L-asparaginase is an essential chemotherapeutic agent in the therapy of acute lymphoblastic leukemia (ALL), which has led to improvement in survival. In low- and middle-income countries like India, the outcomes in ALL are inferior compared with the published literature, one of the causes of which is believed to be due to the inferior quality of bioequivalent asparaginase. Objective The following survey attempts to understand the practice of using this agent among oncologists treating children with cancer in our country. Methods The researchers designed a structured online questionnaire comprising 25 aspects of L-asparaginase usage in the study. The questionnaire was directed to the healthcare providers involved in treating children with cancer in India. Results Of the total 80 responses recorded, 51 (64%) respondents had more than 5 years of experience in pediatric oncology and were treating at least 5 to 10 newly diagnosed ALL patients per month. Forty-one (51%) respondents utilized native asparaginase, and 21 (26.3%) oncologists used PEGylated-asparaginase exclusively. The most common route of administration was the intramuscular route (66.3%). Seventy percent of respondents utilized native form at a dose of 10,000 IU/m2 and 20% at 6,000 IU/m2. The amounts used for PEGylated L-asparaginase were 1,000,IU/m2, 2,500,IU/m2, and variable doses in 48, 40, and 10% of responses, respectively. Though serum asparaginase assay (SAA) was not measured routinely in most of the centers, 39 (48.8%) healthcare providers perceived performing SAA helps to make the clinical decision. Conclusion This survey shows a wide variation in L-asparaginase usage among healthcare providers caring for children with cancer in our country. As L-asparaginase is the pivotal component of ALL therapy, uniformity in its usage and dosing with the possibility of monitoring SAA due to the quality of bioequivalent may be one of the critical steps toward improving outcomes in ALL in our country.

Abstract 909: Single-cell multi-omics reveals type-2 function and metabolic fitness in maintaining CAR T cell longevity associated with 8-year leukemia remission

Abstract Despite high response rate in chimeric antigen receptor (CAR) T therapy for acute lymphocytic leukemia (ALL), approximately half of the patients relapse within the first year, representing an urgent question to address in the next stage of cellular immunotherapy. To interrogate the molecular determinants of ultra-long CAR T persistence, we sequenced one million pre-infusion CAR T cells from 82 pediatric ALL patients and 6 healthy donors and obtained high quality multi-omics data from ~700k single cells at the basal level or upon antigen specific stimulation. We identified that elevated type-2 functionality in both CD4+ and CD8+ subsets was associated with five patients maintaining a median B-cell aplasia duration of 8.4 years (BCA-L group), which was independently validated using both intracellular flow cytometry and multiplexed secretomic assay. Further network analysis revealed several pathways promoting the activation of the Th2 master regulator GATA3 in BCA-L cells. Through ligand-receptor interaction analysis, type-2 cytokines were found to regulate a specific cluster of Tim-3+ terminal effector cells showing overactivation of cytotoxicity, impaired immune function, elevated exhaustion signature and diminished proliferative potentiality, and CAR T cells from BCA-L patients showed reduced dysfunctional hallmarks. In addition, activated BCA-L cells maintained a significantly higher level of oxidative phosphorylation metabolism that is likely associated with their observed central memory phenotype, whereas CAR T cells from patients who developed primary resistance or relapse exhibited imbalanced functional and metabolic programs. Specifically, CAR T products from initial non-responders showed hyperactive glycolysis metabolism, and the CD19-negative relapsed patients (RL- group) had significantly attenuated gene module expression of a broad spectrum of metabolic categories, including major energy-producing metabolisms. By direct comparison with BCA-L cells, our analysis identified a strikingly impaired mTOR signaling in RL- CAR T cells, along with reduced proliferation activities and a systematic deficiency of pathways orchestrating immune response. Although the mechanism is still unclear, our hypothesis is that the impaired mTOR signaling could only endow blunted functional and metabolic response in RL- cells, thereby conferring more opportunities to leukemic cells to escape the attack. In summary, our large-scale single-cell multi-omics dataset provides valuable insights for dissecting mechanistic basis of CAR T cell longevity in mediating 8-year leukemia remission, highlighting several therapeutic strategies to edit specific CAR T functionality to ameliorate clinical outcome, particularly boosting the type-2 immunity to mitigate dysfunction thereby extending persistence. Citation Format: Zhiliang Bai, J. Joseph Melenhorst, Stephan A. Grupp, Rong Fan. Single-cell multi-omics reveals type-2 function and metabolic fitness in maintaining CAR T cell longevity associated with 8-year leukemia remission [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 909.

Thiopurine Methyltransferase Intermediate Metabolizer Status and Thiopurine-Associated Toxicity During Maintenance Therapy in Childhood Acute Lymphoblastic Leukemia.

Mercaptopurine is a cornerstone of maintenance chemotherapy in childhood acute lymphoblastic leukemia (ALL). Its cytotoxic effects are mediated by 6-thioguanine nucleotides (TGNs) incorporation into lymphocyte DNA. Thiopurine methyltransferase (TPMT) inactivates mercaptopurine, and deficiency resulting from genetic variants increases TGN exposure and hematopoietic toxicity. Although mercaptopurine-dose reduction reduces toxicity risk without compromising relapse rate in patients with TPMT deficiency, dosing recommendations for those with moderately reduced activity (intermediate metabolizer (IM)) are less clear and their clinical impacts have yet to be established. This cohort study assessed the effect of TPMT IM status on mercaptopurine-associated toxicity and TGN blood exposure in pediatric patients with ALL initiated on standard dose mercaptopurine. Of 88 patients studied (mean age 4.8 years), 10 (11.4%) were TPMT IM, and all had undergone ≥ 3 cycles of maintenance therapy (80% completed). A larger proportion of TPMT IM than normal metabolizers (NM) had febrile neutropenia (FN) during the first two cycles of maintenance, reaching significance in the second cycle (57% vs. 15%, respectively; odds ratio=7.33, P < 0.05). Compared to NM, FN events occurred more frequently and with prolonged duration in IM in cycles 1 and 2 (adjusted P < 0.05). IM had a 2.46-fold increased hazard ratio for FN, and about twofold higher TGN level than NM (P < 0.05). Myelotoxicity was more common in IM than NM (86% vs. 42%, respectively) during cycle 2 (odds ratio=8.2, P < 0.05). TPMT IM initiated at a standard mercaptopurine dose are at greater risk for FN during early cycles of maintenance therapy, thus our findings support genotype-guided dose adjustment to reduce toxicity.

The influence of NUDT15 variants on 6-mercaptopurine-induced neutropenia in Vietnamese pediatric acute lymphoblastic leukemia.

6-Mercaptopurine (6-MP) serves as the backbone of maintenance therapy in acute lymphoblastic leukemia. The nucleoside diphosphate-linked moiety X-type motif 15 genes (NUDT15) affects the metabolism of 6-MP and thiopurine-related neutropenia in the Asian population. This study reports the influence of these variants on 6MP-induced neutropenia in children with acute lymphoblastic leukemia (ALL). A total of 102 children were enrolled in this retrospective cohort study. NUDT15 variants on exon 1 and exon 3 were identified by Sanger sequencing. We divided the intermediate metabolizer group and the normal metabolizer group base on NUDT15 diplotypes. During the first 3months of maintenance treatment, medical reports measured treatment-related toxicity (neutropenia) and 6-MP dose decreases. NUDT15 genotyping showed two categories of mutations: wild type (75.5%) and heterozygous variant (24.5%). Neutropenia during the early phase of maintenance therapy in the intermediate metabolizer group (68%) was significantly higher than the normal metabolizer group (18.2%) with 10-fold greater odds. Especially, the c.415C>T heterozygous variant was extremely associated with neutropenia compared with the C>C genotype (odds ratio [OR]: 12; 95% confidence interval [CI]: 3.5-41.7). The tolerated doses of 6-MP after the first 3months of maintenance therapy related to the intermediate metabolizer group and the normal metabolizer group were 48.7 and 64.3mg/m2/day, respectively (p<0.001). One-fourth of individuals had NUDT15 variations. All NUDT15 heterozygous mutations cause neutropenia and need 6-MP dose optimization. Given the frequency of NUDT15 mutations in Vietnamese children and their connection with early neutropenia, testing is indicated.

Artemis inhibition as a therapeutic strategy for acute lymphoblastic leukemia.

As effective therapies for relapse and refractory B-cell acute lymphoblastic leukemia (B-ALL) remain problematic, novel therapeutic strategies are needed. Artemis is a key endonuclease in V(D)J recombination and nonhomologous end joining (NHEJ) of DNA double-strand break (DSB) repair. Inhibition of Artemis would cause chromosome breaks during maturation of RAG-expressing T- and B-cells. Though this would block generation of new B- and T-cells temporarily, it could be oncologically beneficial for reducing the proliferation of B-ALL and T-ALL cells by causing chromosome breaks in these RAG-expressing tumor cells. Currently, pharmacological inhibition is not available for Artemis. According to gene expression analyses from 207 children with high-risk pre-B acute lymphoblastic leukemias high Artemis expression is correlated with poor outcome. Therefore, we evaluated four compounds (827171, 827032, 826941, and 825226), previously generated from a large Artemis targeted drug screen. A biochemical assay using a purified Artemis:DNA-PKcs complex shows that the Artemis inhibitors 827171, 827032, 826941, 825226 have nanomolar IC50 values for Artemis inhibition. We compared these 4 compounds to a DNA-PK inhibitor (AZD7648) in three patient-derived B-ALL cell lines (LAX56, BLQ5 and LAX7R) and in two mature B-cell lines (3301015 and 5680001) as controls. We found that pharmacological Artemis inhibition substantially decreases proliferation of B-ALL cell lines while normal mature B-cell lines are not markedly affected. Inhibition of DNA-PKcs (which regulates Artemis) using the DNA-PK inhibitor AZD7648 had minor effects on these same primary patient-derived ALL lines, indicating that inhibition of V(D)J hairpin opening requires direct inhibition of Artemis, rather than indirect suppression of the kinase that regulates Artemis. Our data provides a basis for further evaluation of pharmacological Artemis inhibition of proliferation of B- and T-ALL.

Isolation and Characterization of L-Asparaginase Producing Endophytic Fungi from Medicinal Plants of Rutaceae Family

Endophytic fungi synthesize a diverse set of enzymes and biomolecules with wide array of bioactivities. Thirty-five distinct fungal endophytes have been isolated from medicinal plants of Rutaceae and screened for their ability to produce various industrially important enzymes. Primary enzyme screening revealed that 65% of isolates produced L-Asparaginase, 62% of isolates produced cellulase, 60% of isolates produced amylase, 48% of isolates produced both lipase and pectinase, 45% of isolates produced laccase and 42% of isolates produced protease. Among the different enzymes screened, L-Asparaginase producing isolates were dominant and subjected to quantification using the nesslerization method. The endophytic isolates LCJ315, LCJ324, LCJ326 and LCJ335 were capable of producing high L-Asparaginase activity and their enzyme activity ranged between 7.58 U/ml to 8.84 U/ml. The four isolates were further subjected to molecular identification using 18S rRNA sequencing. The results were then subjected to BLAST analysis. The phylogenetic tree was created by MEGA software. The isolated endophytic fungal strains were identified as LCJ315 (Aspergillus sp.), LCJ326 (Colletotrichum sp.), LCJ324 and LCJ335 (Fusarium sp.). The fungal source of L-Asparaginase is preferred over other microbial sources of L-Asparaginase due to reduced side effects. Given the fact that L-Asparaginase is a crucial anticancer enzyme that is used as the initial therapy for acute lymphoblastic leukemia. Hence, the present study reveals that these endophytic fungal strains isolated from Rutaceae members can be used as an alternate source for L-Asparaginase production.