PURPOSE:The Children's Oncology Group (COG) protocol AALL0434 evaluated the safety and efficacy of multi-agent chemotherapy with Capizzi-based methotrexate/pegaspargase (C-MTX) in patients with newly diagnosed pediatric T-cell lymphoblastic lymphoma (T-LL) and gained preliminary data using nelarabine in high-risk patients. PATIENTS AND METHODS:The trial enrolled 299 patients, age 1-31 years. High-risk (HR) patients had ≥ 1% minimal detectable disease (MDD) in the bone marrow at diagnosis or received prior steroid treatment. Induction failure was defined as failure to achieve a partial response (PR) by the end of the 4-week induction. All patients received the augmented Berlin-Frankfurt-Muenster (ABFM) C-MTX regimen. HR patients were randomly assigned to receive or not receive 6 5-day courses of nelarabine incorporated into ABFM. Patients with induction failure were nonrandomly assigned to ABFM C-MTX plus nelarabine. No patients received prophylactic cranial radiation; however, patients with CNS3 disease (CSF WBC ≥ 5/μL with blasts or cranial nerve palsies, brain/eye involvement, or hypothalamic syndrome) were ineligible. RESULTS:At end-induction, 98.8% of evaluable participants had at least a PR. The 4-year event-free survival (EFS) and overall survival (OS) were 84.7% ± 2.3% and 89.0% ± 2.0%. The 4-year disease-free survival (DFS) from end-induction was 85.9% ± 2.6%. There was no difference in DFS observed between the HR and standard-risk groups (P = .29) or by treatment regimen (P = .55). Disease stage, tumor response, and MDD at diagnosis did not demonstrate thresholds that resulted in differences in EFS. Nelarabine did not show an advantage for HR patients. CNS relapse occurred in only 4 patients. CONCLUSION:COG AALL0434 produced excellent outcomes in one of the largest trials ever conducted for patients with newly diagnosed T-LL. The COG ABFM regimen with C-MTX provided excellent EFS and OS without cranial radiation.

The influence of genetic ancestry on genomics in T-cell acute lymphoblastic leukemia (T-ALL) has not been fully explored. We examined the impact of genetic ancestry on multiomic alterations, survival outcomes, and risk stratification. Among 1,309 children and young adults with T-ALL treated on the Children's Oncology Group trial AALL0434, the prognostic value of five commonly altered T-ALL genes varied by ancestry-including NOTCH1, which was associated with superior overall survival for patients of European ancestry but was nonprognostic among patients of African ancestry. Integrating genetic ancestry with published T-ALL risk classifiers, we identified that an X01 penalized Cox regression classifier stratified patients regardless of ancestry, whereas a European multigene classifier misclassified patients of certain ancestries. Overall, 80% of patients harbored a genomic alteration in at least one gene with differential prognostic impact in an ancestry-specific manner. These data demonstrate the importance of incorporating genetic ancestry into genomic risk classification. SIGNIFICANCE:There is a lack of studies examining the prognostic significance of genomic features by genetic ancestry in T-ALL, especially in non-European ancestral groups. In this study, we demonstrate how the prognostic value of individual alterations differs by genetic ancestry, warranting future studies to identify germline alleles affecting these associations. See related commentary by de Smith, p. xxx.

Abstract Asparaginase-associated toxicities (AAT) often compromise therapy for acute lymphoblastic leukemia (ALL), affecting relapse risk and survival. There are conflicting data on the contributions of older age, obesity by body mass index (BMI), and/or large body surface area (BSA) to AAT. We examined the association of these risk factors with AAT and the impact of AAT on disease response. Induction data were examined from 4925 patients aged between 1 and 30 years enrolled in the Children’s Oncology Group ALL trials AALL0232 and AALL0434, which included a single dose of pegaspargase (2500 IU/m2) without a maximum dose. The associations of age, BMI, and BSA with hyperbilirubinemia, elevated alanine aminotransferase (ALT), thrombosis, and pancreatitis were evaluated. The impact of AAT on minimal residual disease (MRD) positivity (≥0.01%) at the end of induction (EOI) was assessed. Increased risk of developing at least 1 AAT was observed in patients aged ≥10 years (P = .002) and in those with obesity and high BSA (P< .0001) but not with high BSA alone. Risks for hyperbilirubinemia, ALT elevations, and thrombosis were all increased in patients with obesity and high BSA (odds ratio [OR], 3.5; 95% confidence interval [CI], 2.2-5.7; OR, 3.3; 95% CI, 1.7-6.6; and OR, 3.1; 95% CI, 1.5-6.5, respectively). AAT were not associated with EOI MRD positivity. To our knowledge, we report the largest data set of AAT in children, adolescents, and young adults. Preventive strategies are indicated for older patients and for those with obesity and high BSA, but not with high BSA alone. These trials were registered at www.clinicaltrials.gov as NCT00075725 and NCT00408005.

Although B-cell acute lymphoblastic leukemia (B-ALL) is highly responsive to antigen-directed immunotherapies, treatment resistance remains a major barrier to achieving durable responses in patients. We recently developed a novel VpreB1 (CD179a)-directed antibody-drug conjugate with calicheamicin (VpreB1-ADC) that exploits the restricted expression of VpreB1 within the surrogate light chain in early B cells, including B-ALL. In the present work, we investigated mechanisms of resistance to the VpreB1-ADC. Mechanisms of resistance were evaluated using a TCF3::HLF B-ALL model, assessing target engagement parameters including VpreB1 surface expression and antibody internalization. The role of the multidrug resistance transporter ABCB1 (P-glycoprotein) was evaluated via pharmacologic inhibition, using tariquidar and zosuquidar, and enforced overexpression across multiple B-ALL cell lines. Sensitivity to alternative non-ABCB1 substrate payloads exatecan and PNU-159682 was also assessed. Resistant TCF3::HLF cells retained VpreB1 expression and efficient antibody internalization. Instead, resistance was driven by elevated ABCB1 expression and activity. ABCB1 inhibition with tariquidar or zosuquidar restored VpreB1-ADC sensitivity. Conversely, enforced ABCB1 overexpression conferred ADC resistance, which was reversed by ABCB1 inhibition. Cells with high ABCB1 activity remained fully sensitive to alternative payloads, including exatecan and PNU-159682, which are not ABCB1 substrates. ABCB1-mediated drug efflux drives intrinsic resistance to calicheamicin-conjugated ADCs in B-ALL. Combining ADCs with ABCB1 inhibitors or selecting payloads non-susceptible to ABCB1 efflux offer viable strategies to overcome resistance and optimize future ADC therapies.

Frequency of Genetic Alterations of Genes in the NOTCH Pathway by Ancestry.

ABSTRACT:We used single-cell genomics to characterize a patient with T-cell acute lymphoblastic leukemia treated in the Children's Oncology Group AALL0434 trial with poor clinical outcome despite favorable genomic features, identifying a STAT1-mediated interferon-related transcriptional signature and inflammatory microenvironment associated with sensitivity to small-molecule JAK inhibition.

T-cell Acute Lymphoblastic Leukemia Genomic Subtype Description.

T-cell Acute Lymphoblastic Leukemia Pathway Alterations by Categorical Genetic Ancestry

Association of Genetic Ancestry (25% interval) as Continuous Variables with Survival Outcomes.

Supplementary Figure S1. T-ALL subtype classifying driver genes among individuals of European, Admixed American, and African ancestry. Supplementary Figure S2. T-ALL subtype classifying driver genes among patients with Early T-cell Precursor (ETP)-like subtype among individuals of European, Admixed American and African ancestry. Supplementary Figure S3. Distribution of 15 genomically defined T-ALL subtypes described by Pölönen and colleagues (16) among individuals of African and Non-African ancestry. Supplementary Figure S4. 5-year event free survival for NOTCH1 and FBXW7 by ancestral group. Supplementary Figure S5. Prevalence of (A) coding versus non-coding alterations and (B) types of pathogenic variants in NOTCH1 among individuals of African versus non-African ancestry. Supplementary Figure S6. Event free survival among all patients, patients of European, Admixed American, and African ancestry by NOTCH1 alterations (no mutation, favorable, unfavorable). Supplementary Figure S7. NOTCH1 coding mutation types (frameshift, missense, nonsense) on a lollipop plot in patients of European, African, and Admixed American Ancestry. Supplementary Figure S8. 5-year event free survival for genes with at least 5% frequency for each ancestral group (CDKN2A, PHF6, PTEN, DNM2). Supplementary Figure S9. 5-year event free survival for genes with at least 5% frequency for each ancestral group (LEF1, MYB, MYC, 6q loss). Supplementary Figure S10. 5-year event free survival for genes with at least 5% frequency for each ancestral group (WT1, USP7, IL7R, N/KRAS). Supplementary Figure S11. Distribution of genetic ancestral composition of individuals by categorically defined genetic ancestry.

Infiltration of T cell acute lymphoblastic leukemia (T-ALL) into the meninges worsens prognosis, underscoring the need to understand mechanisms driving meningeal involvement. Here, we show that T-ALL cells expressing CXCR3 exploit normal T cell function to infiltrate the inflamed meninges. CXCR3 deletion hampered disease progression and extramedullary dissemination by reducing leukemic cell proliferation and migration. Conversely, forced expression of CXCR3 facilitated T-ALL trafficking to the meninges. We identified the ubiquitin-specific protease 7 as a key regulator of CXCR3 protein stability in T-ALL. Furthermore, we discovered elevated levels of CXCL10, a CXCR3 ligand, in the cerebrospinal fluid from patients with T-ALL and leukemia-bearing mice. Our studies demonstrate that meningeal stromal cells, specifically pericytes and fibroblasts, induce CXCL10 expression in response to leukemia and that loss of CXCL10 attenuated T-ALL influx into the meninges. Moreover, we report that leukemia-derived proinflammatory cytokines, TNF-α, IL-27, and IFN-γ, induced CXCL10 in the meningeal stroma. Pharmacological inhibition or deletion of CXCR3 or CXCL10 reduced T-ALL cell migration and adhesion to meningeal stromal cells. Finally, we reveal that CXCR3 and CXCL10 upregulated VLA-4/VCAM-1 signaling, promoting cell-cell adhesion and thus T-ALL retention in the meninges. Our findings highlight the pivotal role of CXCR3-CXCL10 signaling in T-ALL progression and meningeal colonization.

Association of NOTCH pathway alteration with Survival Outcomes stratified by categorical genetic ancestry.

Association of Genetic Ancestries (25% Interval) as Continuous Variables with Biologic Subtype (TAL1 DP-like as reference subtype) Using Multinomial Regression Model.

Association of Genetic Ancestries (25% Interval) as Continuous Variables with Pathway Alteration using Logistic Regression Models for Each Pathway.

Types of Reported Events by Genetic Ancestry.

T-cell Acute Lymphoblastic Leukemia subtype by Categorical Genetic Ancestry

B-lineage acute lymphoblastic leukemia (B-ALL) therapy is being transformed by therapies targeting antigens such as CD19, CD20, and CD22 on the surface of B-ALL cells. Moreover, having therapies targeting these different B-ALL antigens has helped address challenges associated with both intra- and inter-patient variability in targeted antigen expression levels and antigen loss as mechanisms of therapy resistances. To further expand the range of targetable antigens in B-ALL therapy, we developed a novel antibody-drug conjugate (ADC) that targets the VpreB1 (CD179a) component of the surrogate light chain. VpreB1 is expressed across most B-ALL molecular subtypes but otherwise has expression limited to precursor B cells, but not mature B cells. Our VpreB1 antibody demonstrated high affinity for its target protein and when conjugated to the toxin calicheamicin (VpreB1-ADC) exhibited significant in vitro toxicity against B-ALL cells harboring a range of genomic alterations. In vivo, the VpreB1-ADC was well tolerated in mice, with modest weight loss and decreased white blood cell counts. When tested against a B-ALL cell line and multiple B-ALL patient-derived xenograft models, the VpreB1-ADC significantly reduced leukemia burden, prolonged survival, and cured a subset of mice. These promising results support further investigation of the VpreB1 component of the surrogate light chain as a therapeutic target, including the VpreB1-ADC in preclinical and clinical trials, with the goal of expanding the arsenal of immunoconjugates available for the treatment of B-ALL.

Refractoriness to initial chemotherapy and relapse after remission are the main obstacles to curing T cell acute lymphoblastic leukemia (T-ALL). While tumor heterogeneity has been implicated in treatment failure, the cellular and genetic factors contributing to resistance and relapse remain unknown. Here we linked tumor subpopulations with clinical outcome, created an atlas of healthy pediatric hematopoiesis and applied single-cell multiomic analysis to a diverse cohort of 40 T-ALL cases. We identified a bone marrow progenitor (BMP)-like leukemia subpopulation associated with treatment failure and poor overall survival. The single-cell-derived molecular signature of BMP-like blasts predicted poor outcome across multiple subtypes of T-ALL and revealed that NOTCH1 mutations additively drive T-ALL blasts away from the BMP-like state. Through in silico and in vitro drug screenings, we identified a therapeutic vulnerability of BMP-like blasts to apoptosis-inducing agents including venetoclax. Collectively, our study establishes multiomic signatures for rapid risk stratification and targeted treatment of high-risk T-ALL. Tan and colleagues conducted a single-cell multiomic analysis of T cell acute lymphoblastic leukemia and identified a treatment-resistant subpopulation of bone marrow progenitor-like blasts associated with poor outcomes.

T-lineage acute lymphoblastic leukaemia (T-ALL) is a high-risk tumour1 that has eluded comprehensive genomic characterization, which is partly due to the high frequency of noncoding genomic alterations that result in oncogene deregulation2,3. Here we report an integrated analysis of genome and transcriptome sequencing of tumour and remission samples from more than 1,300 uniformly treated children with T-ALL, coupled with epigenomic and single-cell analyses of malignant and normal T cell precursors. This approach identified 15 subtypes with distinct genomic drivers, gene expression patterns, developmental states and outcomes. Analyses of chromatin topology revealed multiple mechanisms of enhancer deregulation that involve enhancers and genes in a subtype-specific manner, thereby demonstrating widespread involvement of the noncoding genome. We show that the immunophenotypically described, high-risk entity of early T cell precursor ALL is superseded by a broader category of 'early T cell precursor-like' leukaemia. This category has a variable immunophenotype and diverse genomic alterations of a core set of genes that encode regulators of hematopoietic stem cell development. Using multivariable outcome models, we show that genetic subtypes, driver and concomitant genetic alterations independently predict treatment failure and survival. These findings provide a roadmap for the classification, risk stratification and mechanistic understanding of this disease. Comprehensive genomic and transcriptomics analyses of more than 1,300 cases of childhood T-lineage acute lymphoblastic leukaemia identify 15 distinct subtypes that are associated with specific outcomes.