INTRODUCTION:Treatment for pediatric acute lymphoblastic leukemia includes asparaginase as an integral component of therapy. Hypersensitivity reactions or silent inactivation to Escherichia coli (E. coli) formulations may necessitate switching to Erwinia-based asparaginase (asparaginase Erwinia chrysanthemi [E-ASP] or recombinant asparaginase Erwinia chrysanthemi [R-ASP]). However, pharmacokinetic data for R-ASP in pediatrics remain limited, and recommended dosing schedules present logistical challenges. We investigated if adherence to approved dosing regimens for R-ASP is necessary to achieve optimal pharmacokinetic exposure in pediatric patients with leukemia or lymphoma. METHODS:We performed a single institution, retrospective review of pharmacokinetic data in patients who received R-ASP between October 1, 2021 and September 30, 2024. A one-compartment pharmacokinetic model with first-order absorption and elimination was used to evaluate asparaginase pharmacokinetic activity. The pharmacokinetic model was used to determine if the labeled dosing window for R-ASP is required to achieve effective asparagine depletion. RESULTS:Twenty-six patients with 115 measurements of serum asparaginase activity (SAA) were included in the analysis. Based on model simulations from our pediatric patient data, maintaining SAA ≥ 0.1 IU/mL in 90% of patients throughout the dosing interval with R-ASP 25/25/50 mg/m2 administered intramuscularly on Monday, Wednesday, and Friday requires precise timing. Specifically, the Friday 50 mg/m2 dose must be administered within 58 h of the Wednesday morning dose to sustain therapeutic activity. Additionally, only 62.5% of patients maintained SAA ≥ 0.1 IU/mL at 72 h after the last dose with R-ASP 25 mg/m2 administered intramuscularly on a Monday, Wednesday, and Friday dosing schedule. CONCLUSION:Overall, our findings support the importance of adhering closely to the United States Food and Drug Administration approved dosing schedule for R-ASP to ensure adequate asparaginase activity in pediatric patients with leukemia or lymphoma.

BACKGROUND:The prognosis for paediatric patients with relapsed or refractory acute myeloid leukaemia remains poor. Although encouraging, published paediatric data on venetoclax combined with intensive chemotherapy are scarce. We aimed to refine efficacy and toxicity estimates from the previously published dose-escalation phase of this study. METHODS:This multicentre, phase 1 study included two phases, an initial dose-escalation phase to identify the primary endpoint of recommended phase 2 dose (RP2D; previously reported), followed by an expansion cohort phase, the results of which are reported here. Paediatric patients (aged 2-24 years) with relapsed or refractory acute myeloid leukaemia from three US research hospitals were included. Patients had at least 5% blasts in the bone marrow by morphology or at least 1% blasts by flow cytometry. Treatment varied across cohorts. Patients received oral venetoclax 360 mg/m2 (maximum 600 mg) daily for 27 days after a 50% dose reduction on day 1 and intravenous cytarabine 1000 mg/m2 per dose every 12 h for eight doses on days 8-11 (cohort A), with intravenous idarubicin (12 mg/m2 on day 8; cohort B) and dexrazoxane or intravenous azacitidine (75 mg/m2 on days 1-7; cohort C). Here, we report results for all patients treated at the RP2D, including patients treated in the expansion cohort phase (cohort A and B) and patients in cohort C added by protocol amendment (Oct 13, 2020). This final report describes the key secondary endpoint of the rates of complete response with or without haematological recovery at the RP2D. Analyses were on the intention-to-treat population. This trial was registered with ClinicalTrials.gov (NCT03194932) and is completed. FINDINGS:From July 1, 2017, to July 22, 2022, 61 patients were enrolled and 44 patients were subsequently included and treated at the RP2D (21 [48%] female, 23 [52%] male; 22 [50%] in cohort A, 15 [34%] in cohort B, and seven [16%] in cohort C). The median follow-up was 5·3 years (IQR 4·2-6·3). After 1 cycle, 25 (57% [95% CI 41-72]) patients had a complete response with or without haematological recovery; 19 were negative for measurable residual disease. Common grade 3 or 4 adverse events included febrile neutropenia (23 [52%]), gastrointestinal disorders (14[32%]]) including colitis (four [9%]), and infections (11 [25%]). There were two grade 5 adverse events due to sepsis and multiorgan failure. INTERPRETATION:Venetoclax with high-dose cytarabine is active with acceptable safety in paediatric patients with relapsed or refractory AML. These findings support ongoing research of venetoclax with high-dose cytarabine in this population, including the randomised paediatric phase 3 trial (NCT05183035). FUNDING:US National Institutes of Health (NIH), American Lebanese Syrian Associated Charities (ALSAC), AbbVie, Gateway for Cancer Research.

Despite excellent survival and elimination of cranial radiation, patients treated for acute lymphoblastic leukemia (ALL) in childhood remain at increased risk for chronic conditions, including peripheral neuropathy and sarcopenia. This study aimed to evaluate the association between peripheral neuropathy and sarcopenia in survivors of childhood ALL without prior cranial radiation exposure. Additionally, we explore the effects of neuropathy and sarcopenia on physical function and exercise behavior. We included survivors of childhood ALL diagnosed between 1962 and 2012, aged ≥18 years without a history of cranial radiation from the St. Jude Lifetime Cohort Study (SJLIFE). Peripheral neuropathy was assessed using the Modified Total Neuropathy Score (mTNS). Sarcopenia was defined by low muscle mass (dual X-ray absorptiometry) and muscle weakness, with muscle strength assessed using hand grip and quadriceps strength tests. Physical function was evaluated with the Timed-Up-and-Go test and a 50-foot walk test. Physical activity was self-reported via the NHANES Physical Activity Questionnaire. Statistical analyses, including modified Poisson regression, were performed to examine associations. Among 537 survivors (median age: 28 years, range 18–52), 31.7

PURPOSE:BH3 mimetics targeting antiapoptotic BCL2 family proteins are promising therapeutics for T-cell acute lymphoblastic leukemia (T-ALL). However, their activity across genomic subtypes of this cancer and interactions with other antileukemic agents remain incompletely defined. EXPERIMENTAL DESIGN:We evaluated the ex vivo sensitivity of BCL2/BCL-XL dual, BCL2-, BCL-XL-, and MCL1-selective inhibitors across 58 xenografts derived from T-ALL, representing diverse molecular subtypes. The BCL2/BCL-XL dual inhibitor AZD4320 was further assessed in combination with selected antileukemic agents. Drug responses were quantified by dose-dependent induction of apoptosis and integrated with genomic and functional analyses. RESULTS:AZD4320 demonstrated subtype-specific cytotoxicity, with increased sensitivity in early T-cell precursor (ETP)-like T-ALL and resistance in TAL1 αβ-like T-ALL. Gene network analysis revealed subtype-dependent activation of distinct BCL2 family proteins, with AZD4320 response associated with BCL2 and MCL1 activity. Drug-drug interaction analysis using the Multi-dimensional Synergy of Combinations algorithm showed that AZD4320 synergized by potency-rather than maximal efficacy-with asparaginase and dasatinib, particularly broad interaction with asparaginase across subtypes. In vivo, AZD4320-asparaginase combination therapy conferred a survival benefit. Mechanistically, asparaginase-induced asparagine depletion promoted mitochondrial dysfunction, potentiating AZD4320-mediated cytotoxicity. CONCLUSIONS:These findings highlight the genomic context in shaping BH3 mimetic responses and point to the rational combination of this class of drugs with antileukemic agents such as asparaginase.

Ixazomib (MLN 9708) is an oral proteasome inhibitor, preclinically more potent than bortezomib, that is currently US Food and Drug Administration-approved for the treatment of multiple myeloma. We conducted a phase 1/2 study to estimate the maximum tolerated dose, recommended phase 2 dose (RP2D), and early efficacy of ixazomib when combined with chemotherapy in pediatric patients with relapsed/refractory (R/R) acute lymphoblastic leukemia (ALL) and lymphoblastic lymphoma (LL). Patients aged ≤21 years with R/R ALL/LL (including Down syndrome) were eligible. Ixazomib was combined with up to 3 different 28-day blocks of well-established, relapsed ALL chemotherapy. Ixazomib was tested at 2 dose levels (DL; DL1: 1.6 mg/m2 per dose; DL2: 2 mg/m2 per dose) using a 3+3 design. Dose-limiting toxicities (DLTs) during block 1 were used to make DL escalation decisions. Twenty-four patients enrolled, all with ALL (10 in phase 1, 14 in phase 2). The most common categories of grade ≥3 attributable adverse events were gastrointestinal disorders (n = 12) and febrile neutropenia (n = 9). Two patients experienced a DLT (both treated at DL2 in phase 2), however, DL2 was determined to be the RP2D. The complete response rate for evaluable patients was 67%; 64% (9/14) of whom were also flow minimal residual disease negative. The half-life was consistent across DLs and was comparable with that previously reported in adults. Oral capsule and liquid formulations were determined to be palatable. Ixazomib can be combined with chemotherapy with an acceptable safety profile and an encouraging early efficacy signal in pediatric R/R ALL. This trial was registered at www.clinicaltrials.gov as NCT03817320.

The 21st Ponte di Legno Working Group meeting convened in Orlando, USA, on December 4-5, 2025, bringing together leading childhood acute lymphoblastic leukemia (ALL) investigators from major global consortia. In response to the transformative advances in childhood ALL treatment, particularly the integration of immunotherapy into frontline therapy, the group revisited and updated its mission statement. The revised mission emphasizes collaborative studies on rare leukemia subsets, harmonized toxicity reporting, particularly for immunotherapy-related toxicities, and unrestricted worldwide collaboration. In addition, sharing data and strategies for integrating novel agents will be integral to optimizing future trial design. Key scientific topics included rare genetic subgroups, T-cell ALL genomics, treatment-related toxicity benchmarking, and central nervous system (CNS) disease management challenges. A major focus was immunotherapy integration into frontline therapy, particularly blinatumomab as an emerging standard of care and inotuzumab ozogamicin as an investigational agent, and their potential to enable chemotherapy de-escalation. Additional discussions addressed immunotherapy-specific toxicities. The integration of immunotherapy into frontline ALL therapy represents a paradigm shift with potential to improve outcomes while reducing treatment burden. However, careful attention to CNS disease control, emerging toxicities, and preservation of the remarkable achievements in childhood ALL therapy remains essential as the field advances.

Background Pharmacotyping, the ex vivo measurement of tumor cell responses to drugs, is particularly important for cancers lacking actionable genomic markers. However, current pharmacotyping methods are not clinically feasible due to prolonged drug incubations (days to weeks), extensive manual handling, and analytical limitations, including overlooking single-cell characteristics. Addressing these hurdles is critical for pediatric T cell acute lymphoblastic leukemia (T-ALL), an aggressive cancer with limited therapeutic options. Methods We developed μPharma, a pharmacotyping platform that predicts single-cell drug sensitivity without direct drug exposure by quantifying pretreatment biomarkers associated with therapeutic response. μPharma integrates an automated digital microfluidic immunofluorescence assay, optimized for suspension cells, with machine learning models trained on comprehensive single-cell features. We validated μPharma using T-ALL cell lines and patient-derived xenografts, predicting sensitivity to dasatinib and venetoclax by quantifying their target proteins, LCK and BCL2, respectively, including protein expression, phosphorylation status, spatial distribution, and cellular morphology. Findings We confirmed that phospho-LCK is predictive of dasatinib sensitivity, consistent with prior studies, and identified phospho-BCL2 as a previously unreported biomarker for venetoclax sensitivity. Integrating multiple biomarkers into machine learning models significantly enhanced predictive accuracy compared to single-marker analyses. Key informative features included spatial protein distribution and integrated protein-morphology metrics. Additionally, single-cell analysis revealed distinct cell subpopulations, suggesting intratumor heterogeneity in drug responses. Conclusions μPharma provides rapid (4-h assay), accurate, and automated prediction of drug sensitivity at single-cell resolution using minimal clinical samples, potentially enabling same-day precision oncology decision-making. Funding This work was supported by institutional start-up funds from the University of Utah, including internal supplements provided through the Immunology, Inflammation & Infectious Disease (3i) Initiative and the Diabetes & Metabolism Research Center (DMRC).

Hypersensitivity reaction rates of up to 27% have been reported for calaspargase pegol (CAL-PEG). We report desensitizing 9 patients across 11 doses of CAL-PEG. Our results demonstrate that 36.4% of the desensitized CAL-PEG doses were successful.

ABSTRACT:Children with ETV6::RUNX1 or high-hyperdiploid B-cell acute lymphoblastic leukemia (B-ALL) have favorable outcomes. The St. Jude (SJ) classification considers these patients low risk, regardless of their National Cancer Institute (NCI) risk classification, except when there is slow minimal residual disease (MRD) response or central nervous system/testicular involvement. We analyzed outcomes in children (aged 1-18.99 years) with these genotypes in the SJ Total XV/XVI studies (2000-2017). Patients with ETV6::RUNX1 (n = 222) or high-hyperdiploid (n = 296) B-ALL had 5-year event-free survival (EFS) of 97.7% ± 1.1% and 94.7% ± 1.4%, respectively. For ETV6::RUNX1, EFS was comparable between NCI standard-risk and high-risk patients and between SJ low-risk and standard-risk patients. Of the 40 NCI high-risk patients, 37 who received SJ low-risk therapy had excellent EFS (97.3% ± 2.8%). For high-hyperdiploid B-ALL, NCI high-risk patients had worse EFS than standard-risk patients (87.6% ± 4.5% vs 96.4% ± 1.3%; P = .016). EFS was similar for NCI standard-risk and high-risk patients classified as SJ low risk (96.0% ± 1.5% and 96.9% ± 3.2%; P = .719). However, EFS was worse for NCI high-risk patients than for NCI standard-risk patients receiving SJ standard/high-risk therapy (77.4% ± 8.2% vs 98.0% ± 2.2%; P = .004). NCI high-risk patients with ETV6::RUNX1 or high-hyperdiploid B-ALL who received SJ low-risk therapy had lower incidences of thrombosis (P = .013) and pancreatitis (P = .011) than those who received SJ standard/high-risk therapy. MRD-directed therapy yielded excellent outcomes, except for NCI high-risk high-hyperdiploid B-ALL patients with slow MRD response, who require new treatment approaches. Among NCI high-risk patients, 93% with ETV6::RUNX1 and 54% with high-hyperdiploid B-ALL experienced excellent outcomes with a low-intensity regimen. These trials were registered at www.clinicaltrials.gov as #NCT00137111 and #NCT00549848.

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer. Thiopurines such as 6-mercaptopurine (6MP) are essential in ALL maintenance therapy. However, dose-limiting toxicities can significantly disrupt treatment. While genetic variants in TPMT and NUDT15 are known to affect thiopurine response, many patients with normal function genotypes in these genes still experience adverse effects, suggesting that additional genes might be involved. We analyzed 663 pediatric ALL patients enrolled in the AALL03N1 trial to identify novel genetic determinants of 6MP sensitivity, focusing on individuals with normal function TPMT and NUDT15 genotypes. A transcriptome-wide association study (TWAS) was conducted to focus on expression quantitative trait loci (eQTLs). Findings were validated in two independent cohorts: St. Jude Total Therapy XV ( n  = 390) and XVI ( n  = 552). TWAS identified 31 genes associated with 6MP dose intensity ( q -value < 0.90). Of these, the imputed GNAQ expression was positively correlated with 6MP dose intensity and passed multiple testing thresholds in the validation cohorts. The rs60561071 variant, the eQTL in the GNAQ TWAS model, was associated with reduced gene expression and lower 6MP dose intensity. This study identifies GNAQ as a novel gene associated with thiopurine tolerance in ALL patients lacking known risk alleles in TPMT and NUDT15 . Moreover, this research highlighted the innovative use of TWAS, providing deeper insights into the molecular mechanisms that explain drug response variability.

2528 Background: By forming an immunological synapse between T cells and tumor antigen, bispecific T cell engagers (BiTEs) like blinatumomab have shown great promise in treating B-cell acute lymphoblastic leukemia (B-ALL). However, many relapsed and refractory (R/R) patients fail to achieve long-term survival, with 40% not surviving past 24 months. Prolonged T cell activation with blinatumomab therapy may lead to changes in differentiation that leave the T cell population unable to elicit a sustained anti-tumor response. A deeper understanding of the dynamics of the T cell compartment in R/R B-ALL patients will lead to improved treatment strategies and optimized patient selection for blinatumomab therapy. Methods: To characterize T cell persistence and response in this context, we assessed memory and exhaustion phenotypes in blinatumomab-treated T cells isolated from 10 R/R pediatric B-ALL patients treated with blinatumomab. CD8+ T cells were isolated from peripheral blood and bone marrow samples and analyzed for memory and exhaustion phenotypes via flow cytometry. Absolute lymphocyte counts were measured and linked to the sample flow cytometry data to assess expansion and contraction of T cell memory subsets throughout the course of therapy. Whole genome enzymatic methyl sequencing was performed on post-treatment PD-1 High and PD-1 Low CD8 T cells to determine the multipotency of the patient T cell compartment after blinatumomab treatment. Results: After 7 days of continued blinatumomab infusion, patient T cells demonstrated a significant expansion of terminally differentiated and effector memory T cells. Notably, we observed that non-responders had a high tumor burden at the start of the therapy and possessed a large population of naïve CD8 T cells that failed to expand. These CD8 T cells exhibited a significant increase in expression of TIM-3 and PD-1 compared to responders after the 7-day infusion. Methylation analysis of post-treatment CD8 T cells showed decreased methylation of exhaustion regulators IKZF1 and CD300a in non-responders compared to the responders. Additionally, in vitro treatment of T cells with blinatumomab induced T-cell exhaustion in a target-dependent manner. Conclusions: Blinatumomab therapy in pediatric B-ALL patients induced variable epigenetic and phenotypic changes to the T cell compartment indicative of exhaustion, corresponding to differences in T cell expansion and persistence between patients. Our study is the first to link epigenetic changes in exhaustion regulators with response variability in blinatumomab-treated patients. Furthermore, our findings highlight a potential role of baseline T cell composition and tumor burden in determining therapeutic outcomes. These insights provide a novel framework for improving patient stratification and treatment strategies to mitigate T cell exhaustion in blinatumomab therapy.

Relapse remains the leading cause of mortality in pediatric acute myeloid leukemia (AML), yet the genetic changes contributing to relapse remain incompletely defined. To address this gap, we performed whole-genome sequencing and targeted-capture sequencing on 39 diagnosis-relapse and 2 relapse-relapse pairs of pediatric AML. Mutational burden increased at relapse, largely reflecting spontaneous mutagenesis, whereas therapy-related signatures were rarely observed and only occasionally associated with pathogenic mutations. Although recurrently enriched mutations at relapse included those in FLT3, WT1, and TP53, relapse-fated subclones were frequently marked only by non-pathogenic or non-coding variants. Longitudinal deep sequencing in eight patients showed rapid depletion of major clones after induction therapy, whereas subclones often displayed variable chemosensitivity. Relapse-specific mutations emerged only late or remained undetectable during remission, suggesting that clonal selection of pre-existing clones is the predominant mechanism of relapse. Transcriptome analysis of paired RNA sequencing data revealed no differentially expressed genes, but gene set enrichment analysis and CIBERSORT deconvolution in each pair uncovered heterogeneous trajectories to relapse. Although relapse is often attributed to the emergence of stem-like phenotypes, our data demonstrate that transcriptional evolution is more diverse: some cases acquired stem-like features, whereas others showed partial differentiation, which was confirmed by re-analysis of a public single cell RNA sequence dataset. These changes were largely constrained by baseline differentiation states at diagnosis. Together, our data indicate that pediatric AML relapse arises through selection of pre-existing clones with diverse trajectories, underscoring the need to target both stem-like and differentiated populations to achieve durable cures.

Asparaginase is a critical component of modern therapy for acute lymphoblastic leukemia (ALL). Its use has improved cure rates for both children and adults with this disease. However, asparaginase therapy can be complicated by significant toxicities, including acute pancreatitis (AP). Such pancreatitis can be severe and result in life-threatening or fatal complications. Here, we describe five cases of severe asparaginase-induced pancreatitis in children receiving therapy for ALL. Despite maximal supportive care, these patients experienced severe symptoms and were unable to tolerate enteral nutrition (EN) initially after presentation. This case series highlights clinical features associated with intolerance to EN in this population. Providers should be mindful of these features when considering EN trials in patients with severe asparaginase-associated AP. Some patients may require total parenteral nutrition to adequately support nutrition and recovery in severe asparaginase-associated AP.

Background: Despite success in treating childhood acute lymphoblastic leukemia and lymphoma (ALL), modern multiagent chemotherapy regimens containing high-dose corticosteroids can result in osteonecrosis afflicting survivors, frequently involving the knee. Thus, we describe the usage of fresh osteoarticular allograft (FOAG) to treat steroid-induced osteonecrosis of the femoral condyle in pediatric cancer survivors. We assessed the efficacy of FOAGs to heal necrotic bone, provide long-term pain relief, while retrospectively reviewing quality of life and functional status. Methods: We recorded patient demographics, pain, and functional scores preoperatively, postoperatively, and at the most recent follow-up, additional surgical procedures, and radiographic outcomes at each timepoint. Anatomic locations and sizes of the grafts were noted using operative reports and intraoperative photographs. Function and pain scores were collected by direct patient communication or through survey, with data scored using the KOOS (Knee Injury and Osteoarthritis Outcome Score), PROMIS (Patient-Reported Outcomes Measurement Information System), and CTCAE (Common Terminology Criteria for Adverse Events) scoring tools. Results: Eighteen patients, (12 females) over a period of 8 years, underwent the procedure. One patient was excluded due to death before 12 months follow-up. The average age at diagnosis of femoral condyle osteonecrosis was 14.5 years. The average age at the time of surgery was 18.5 years (range, 13 to 25 y). All patients had at least 16 months follow-up (average 60 mo; range 16 to 99 mo). Grafts ranged in size from 18 to 27.5 mm. All patients reported a return to normal function of the knees with KOOS scores approaching normal, PROMIS scores averaging 46, and CTCAE outcomes improving at an average of 34 months post-surgery. Two patients experienced a partial graft failure, which was repaired with new allografts. Conclusions: Although historical reviews indicated unsatisfactory results, our experience highlights the successful usage of FOAG for the management of patients treated for childhood leukemia who develop steroid-induced osteonecrosis of the femoral condyle. This procedure proved effective in managing pain and improving function and quality of life, with good bone ingrowth despite large necrotic lesions. Levels of Evidence: Therapeutic Level IV.