Additive effects of TPMT and NUDT15 on thiopurine toxicity in children with acute lymphoblastic leukemia across multiethnic populations.

BACKGROUND: Thiopurines such as mercaptopurine (MP) are widely used to treat acute lymphoblastic leukemia (ALL). Thiopurine-S-methyltransferase (TPMT) and Nudix hydrolase 15 (NUDT15) inactivate thiopurines, and no-function variants are associated with drug-induced myelosuppression. Dose adjustment of MP is strongly recommended in patients with intermediate or complete loss of activity of TPMT and NUDT15. However, the extent of dosage reduction recommended for patients with intermediate activity in both enzymes is currently not clear. METHODS: MP dosages during maintenance were collected from 1768 patients with ALL in Singapore, Guatemala, India, and North America. Patients were genotyped for TPMT and NUDT15, and actionable variants defined by the Clinical Pharmacogenetics Implementation Consortium were used to classify patients as TPMT and NUDT15 normal metabolizers (TPMT/NUDT15 NM), TPMT or NUDT15 intermediate metabolizers (TPMT IM or NUDT15 IM), or TPMT and NUDT15 compound intermediate metabolizers (TPMT/NUDT15 IM/IM). In parallel, we evaluated MP toxicity, metabolism, and dose adjustment using a Tpmt/Nudt15 combined heterozygous mouse model (Tpmt+/-/Nudt15+/-). RESULTS: Twenty-two patients (1.2%) were TPMT/NUDT15 IM/IM in the cohort, with the majority self-reported as Hispanics (68.2%, 15/22). TPMT/NUDT15 IM/IM patients tolerated a median daily MP dose of 25.7 mg/m2 (interquartile range = 19.0-31.1 mg/m2), significantly lower than TPMT IM and NUDT15 IM dosage (P < .001). Similarly, Tpmt+/-/Nudt15+/- mice displayed excessive hematopoietic toxicity and accumulated more metabolite (DNA-TG) than wild-type or single heterozygous mice, which was effectively mitigated by a genotype-guided dose titration of MP. CONCLUSION: We recommend more substantial dose reductions to individualize MP therapy and mitigate toxicity in TPMT/NUDT15 IM/IM patients.

Prognostic and Pharmacotypic Heterogeneity of Hyperdiploidy in Childhood ALL.

PURPOSE: High hyperdiploidy, the largest and favorable subtype of childhood ALL, exhibits significant biological and prognostic heterogeneity. However, factors contributing to the varied treatment response and the optimal definition of hyperdiploidy remain uncertain. METHODS: We analyzed outcomes of patients treated on two consecutive frontline ALL protocols, using six different definitions of hyperdiploidy: chromosome number 51-67 (Chr51-67); DNA index (DI; DI1.16-1.6); United Kingdom ALL study group low-risk hyperdiploid, either trisomy of chromosomes 17 and 18 or +17 or +18 in the absence of +5 and +20; single trisomy of chromosome 18; double trisomy of chromosomes 4 and 10; and triple trisomy (TT) of chromosomes 4, 10, and 17. Additionally, we characterized ALL ex vivo pharmacotypes across eight main cytotoxic drugs. RESULTS: Among 1,096 patients analyzed, 915 had B-ALL and 634 had pharmacotyping performed. In univariate analysis, TT emerged as the most favorable criterion for event-free survival (EFS; 10-year EFS, 97.3% v 86.8%; P = .0003) and cumulative incidence of relapse (CIR; 10-year CIR, 1.4% v 8.8%; P = .002) compared with the remaining B-ALL. In multivariable analysis, accounting for patient numbers using the akaike information criterion (AIC), DI1.16-1.6 was the most favorable criterion, exhibiting the best AIC for both EFS (hazard ratio [HR], 0.45; 95% CI, 0.23 to 0.88) and CIR (HR, 0.45; 95% CI, 0.21 to 0.99). Hyperdiploidy and subgroups with favorable prognoses exhibited notable sensitivities to asparaginase and mercaptopurine. Specifically, asparaginase sensitivity was associated with trisomy of chromosomes 16 and 17, whereas mercaptopurine sensitivity was linked to gains of chromosomes 14 and 17. CONCLUSION: Among different definitions of hyperdiploid ALL, DI is optimal based on independent prognostic impact and also the large proportion of low-risk patients identified. Hyperdiploid ALL exhibited particular sensitivities to asparaginase and mercaptopurine, with chromosome-specific associations.

Associations of T-Cell Receptor Repertoire Diversity with L-Asparaginase Allergy in Childhood Acute Lymphoblastic Leukemia.

Asparaginase is a critical component of therapy for childhood acute lymphoblastic leukemia (ALL), but it is commonly associated with allergy, which results in morbidity and poorer outcomes. The underlying basis of this allergy is undoubtedly immune-mediated, but the exact components of T-cell immunity have yet to be characterized. We performed longitudinal TCR sequencing of 180 bone marrow samples from 67 children with B-ALL treated as part of the Ma-Spore-ALL-2010 trial, and we evaluated the associations of TCR profile with asparaginase hypersensitivity, with functional validation of asparaginase activity in a separate cohort of 113 children. We found that a more diverse and dynamically changing TCR repertoire was associated with increased risk of clinical hypersensitivity and decreased L-asp activity. Allergic patients had a higher proportion of infrequent clonotypes, as well as a significantly lower degree of shared clonotypes amongst the cohort. Allergic patients also had significantly higher longitudinal variability of clonotypes across timepoints, where a higher dissimilarity between diagnosis and week 5 represented an 8.1-fold increased risk of an allergic event. After an allergy had occurred, there was shaping and convergence of the TCR repertoire towards a common antigen. Understanding the immunological basis of T-cell responses in allergy lays the groundwork for developing predictive biomarkers or strategies to mediate this common toxicity in childhood ALL.

Pharmacotypes across the genomic landscape of pediatric acute lymphoblastic leukemia and impact on treatment response.

Contemporary chemotherapy for childhood acute lymphoblastic leukemia (ALL) is risk-adapted based on clinical features, leukemia genomics and minimal residual disease (MRD); however, the pharmacological basis of these prognostic variables remains unclear. Analyzing samples from 805 children with newly diagnosed ALL from three consecutive clinical trials, we determined the ex vivo sensitivity of primary leukemia cells to 18 therapeutic agents across 23 molecular subtypes defined by leukemia genomics. There was wide variability in drug response, with favorable ALL subtypes exhibiting the greatest sensitivity to L-asparaginase and glucocorticoids. Leukemia sensitivity to these two agents was highly associated with MRD although with distinct patterns and only in B cell ALL. We identified six patient clusters based on ALL pharmacotypes, which were associated with event-free survival, even after adjusting for MRD. Pharmacotyping identified a T cell ALL subset with a poor prognosis that was sensitive to targeted agents, pointing to alternative therapeutic strategies. Our study comprehensively described the pharmacological heterogeneity of ALL, highlighting opportunities for further individualizing therapy for this most common childhood cancer.

Maintenance therapy for acute lymphoblastic leukemia: basic science and clinical translations.

Maintenance therapy (MT) with oral methotrexate (MTX) and 6-mercaptopurine (6-MP) is essential for the cure of acute lymphoblastic leukemia (ALL). MTX and 6-MP interfere with nucleotide synthesis and salvage pathways. The primary cytotoxic mechanism involves the incorporation of thioguanine nucleotides (TGNs) into DNA (as DNA-TG), which may be enhanced by the inhibition of de novo purine synthesis by other MTX/6-MP metabolites. Co-medication during MT is common. Although Pneumocystis jirovecii prophylaxis appears safe, the benefit of glucocorticosteroid/vincristine pulses in improving survival and of allopurinol to moderate 6-MP pharmacokinetics remains uncertain. Numerous genetic polymorphisms influence the pharmacology, efficacy, and toxicity (mainly myelosuppression and hepatotoxicity) of MTX and thiopurines. Thiopurine S-methyltransferase (encoded by TPMT) decreases TGNs but increases methylated 6-MP metabolites (MeMPs); similarly, nudix hydrolase 15 (encoded by NUDT15) also decreases TGNs available for DNA incorporation. Loss-of-function variants in both genes are currently used to guide MT, but do not fully explain the inter-patient variability in thiopurine toxicity. Because of the large inter-individual variations in MTX/6-MP bioavailability and metabolism, dose adjustments are traditionally guided by the degree of myelosuppression, but this does not accurately reflect treatment intensity. DNA-TG is a common downstream metabolite of MTX/6-MP combination chemotherapy, and a higher level of DNA-TG has been associated with a lower relapse hazard, leading to the development of the Thiopurine Enhanced ALL Maintenance (TEAM) strategy-the addition of low-dose (2.5-12.5 mg/m2/day) 6-thioguanine to the 6-MP/MTX backbone-that is currently being tested in a randomized ALLTogether1 trial (EudraCT: 2018-001795-38). Mutations in the thiopurine and MTX metabolism pathways, and in the mismatch repair genes have been identified in early ALL relapses, providing valuable insights to assist the development of strategies to detect imminent relapse, to facilitate relapse salvage therapy, and even to bring about changes in frontline ALL therapy to mitigate this relapse risk.

Life-threatening infections during treatment for acute lymphoblastic leukemia on the Malaysia-Singapore 2003 and 2010 clinical trials: A risk prediction model.

AIM: Life-threatening infections significantly impact the care of children undergoing therapy for acute lymphoblastic leukemia (ALL) who are at risk of severe sepsis due to both host and treatment factors. Our aim was to develop a life-threatening infection risk prediction model that would allow remote rapid triage of patients to reduce time to first dose of antibiotics and sepsis-related mortality. METHODS: A retrospective analysis of 2068 fever episodes during ALL therapy was used for model building and subsequent internal validation. RESULTS: Three hundred and seventy-seven patients were treated for ALL in two institutions with comparable critical and supportive care resources. A total of 55 patients accounted for 71 admissions to the critical care unit for sepsis that led to eight septic deaths during a 16-year study period. A retrospective analysis of risk factors for sepsis enabled us to build a model focused on 13 variables that discriminated admissions requiring critical care well: area under the receiver operating characteristic curve of .82; 95% CI .76-.87, p<.001, and Brier score of .033. Significant univariate predictors included neutropenia, presence of symptoms of abdominal pain, diarrhea, fever during induction or steroid-based phases, and the lack of any localizing source of infection at time of presentation. CONCLUSION: We have developed a risk prediction model that can reliably identify ALL patients undergoing treatment who are at a higher risk of life-threatening sepsis. Clinical applicability can potentially be extended to low-middle income settings, and its utility should be further studied in real-world settings.

Association of Genetic Ancestry With the Molecular Subtypes and Prognosis of Childhood Acute Lymphoblastic Leukemia.

IMPORTANCE: Racial and ethnic disparities persist in the incidence and treatment outcomes of childhood acute lymphoblastic leukemia (ALL). However, there is a paucity of data describing the genetic basis of these disparities, especially in association with modern ALL molecular taxonomy and in the context of contemporary treatment regimens. OBJECTIVE: To evaluate the association of genetic ancestry with childhood ALL molecular subtypes and outcomes of modern ALL therapy. DESIGN, SETTING, AND PARTICIPANTS: This multinational, multicenter genetic association study was conducted from March 1, 2000, to November 20, 2020, among 2428 children and adolescents with ALL enrolled in frontline trials from the United States, South East Asia (Singapore and Malaysia), and Latin America (Guatemala), representing diverse populations of European, African, Native American, East Asian, and South Asian descent. Statistical analysis was conducted from February 3, 2020, to April 19, 2021. MAIN OUTCOMES AND MEASURES: Molecular subtypes of ALL and genetic ancestry were comprehensively characterized by performing RNA sequencing. Associations of genetic ancestries with ALL molecular subtypes and treatment outcomes were then evaluated. RESULTS: Among the participants in the study, 1340 of 2318 (57.8%) were male, and the mean (SD) age was 7.8 (5.3) years. Of 21 ALL subtypes identified, 8 were associated with ancestry. East Asian ancestry was positively associated with the frequency of somatic DUX4 (odds ratio [OR], 1.30 [95% CI, 1.16-1.45]; P < .001) and ZNF384 (OR, 1.40 [95% CI, 1.18-1.66]; P < .001) gene rearrangements and negatively associated with BCR-ABL1-like ALL (OR, 0.79 [95% CI, 0.66-0.92]; P = .002) and T-cell ALL (OR, 0.80 [95% CI, 0.71-0.90]; P < .001). By contrast, occurrence of CRLF2 rearrangements was associated with Native American ancestry (OR, 1.48 [95% CI, 1.29-1.69]; P < .001). When the percentage of Native American ancestry increased, ETV6-RUNX1 fusion became less frequent (OR, 0.80 [95% CI, 0.70-0.91]; P < .001), with the opposite trend observed for ETV6-RUNX1-like ALL. There was a marked preponderance of T-cell ALL in children of African descent compared with those with a high percentage of Native American ancestry (African: OR, 1.22 [95% CI, 1.07-1.37]; P = .003; Native American: OR, 0.53 [95% CI, 0.40-0.67]; P < .001). African ancestry was also positively associated with the prevalence of TCF3-PBX1 (OR, 1.49 [95% CI, 1.25-1.76]; P < .001) and negatively associated with DUX4 rearrangements (OR, 0.70 [95% CI, 0.48-0.93]; P = .01) and hyperdiploidy (OR, 0.77 [95% CI, 0.68-0.86]; P < .001). African and Native American ancestries as continuous variables were both associated with poorer event-free survival (for every 25% increase in ancestry: hazard ratio [HR], 1.2; 95% CI, 1.1-1.4; P = .001 for African ancestry; HR, 1.3; 95% CI, 1.0-1.6; P = .04 for Native American ancestry) and overall survival (for every 25% increase in ancestry: HR, 1.2; 95% CI, 1.1-1.5; P = .01 for African ancestry; HR, 1.4; 95% CI, 1.0-1.8; P = .03 for Native American ancestry). Even after adjusting for biological subtypes and clinical features, Native American and African ancestries remained associated with poor prognosis. CONCLUSIONS AND RELEVANCE: This study suggests that ALL molecular subtypes and prognosis are associated with genetic ancestry, potentially pointing to a genetic basis for some of the racial and ethnic disparities in ALL. Therefore, molecular subtype-driven treatment individualization is needed to help address racial and ethnic gaps in outcomes.

Curing the Curable: Managing Low-Risk Acute Lymphoblastic Leukemia in Resource Limited Countries.

Although childhood acute lymphoblastic leukemia (ALL) is curable, global disparities in treatment outcomes remain. To reduce these global disparities in low-middle income countries (LMIC), a paradigm shift is needed: start with curing low-risk ALL. Low-risk ALL, which accounts for >50% of patients, can be cured with low-toxicity therapies already defined by collaborative studies. We reviewed the components of these low-toxicity regimens in recent clinical trials for low-risk ALL and suggest how they can be adopted in LMIC. In treating childhood ALL, the key is risk stratification, which can be resource stratified. NCI standard-risk criteria (age 1-10 years, WBC < 50,000/uL) is simple yet highly effective. Other favorable features such as ETV6-RUNX1, hyperdiploidy, early peripheral blood and bone marrow responses, and simplified flow MRD at the end of induction can be added depending on resources. With limited supportive care in LMIC, more critical than relapse is treatment-related morbidity and mortality. Less intensive induction allows early marrow recovery, reducing the need for intensive supportive care. Other key elements in low-toxicity protocol designs include: induction steroid type; high-dose versus low-dose escalating methotrexate; judicious use of anthracyclines; and steroid pulses during maintenance. In summary, the first effective step in curing ALL in LMIC is to focus on curing low-risk ALL with less intensive therapy and less toxicity.

Genetic Alterations in Childhood Acute Lymphoblastic Leukemia: Interactions with Clinical Features and Treatment Response.

Acute lymphoblastic leukemia (ALL) is the most common cancer among children. This aggressive cancer comprises multiple molecular subtypes, each harboring a distinct constellation of somatic, and to a lesser extent, inherited genetic alterations. With recent advances in genomic analyses such as next-generation sequencing techniques, we can now clearly identify >20 different genetic subtypes in ALL. Clinically, identifying these genetic subtypes will better refine risk stratification and determine the optimal intensity of therapy for each patient. Underpinning each genetic subtype are unique clinical and therapeutic characteristics, such as age and presenting white blood cell (WBC) count. More importantly, within each genetic subtype, there is much less variability in treatment response and survival outcomes compared with current risk factors such as National Cancer Institute (NCI) criteria. We review how this new taxonomy of genetic subtypes in childhood ALL interacts with clinical risk factors used widely, i.e., age, presenting WBC, IKZF1del, treatment response, and outcomes.

Genome-Wide Association Study of Susceptibility Loci for TCF3-PBX1 Acute Lymphoblastic Leukemia in Children.

Acute lymphoblastic leukemia (ALL) is the most common cancer in children. TCF3-PBX1 fusion defines a common molecular subtype of ALL with unique clinical features, but the molecular basis of its inherited susceptibility is unknown. In a genome-wide association study of 1494 ALL cases and 2057 non-ALL controls, we identified a germline risk locus located in an intergenic region between BCL11A and PAPOLG: rs2665658, P = 1.88 × 10-8 for TCF3-PBX1 ALL vs non-ALL, and P = 1.70 × 10-8 for TCF3-PBX1 ALL vs other-ALL. The lead variant was validated in a replication cohort, and conditional analyses pointed to a single causal variant with subtype-specific effect. The risk variant is located in a regulatory DNA element uniquely activated in ALL cells with the TCF3-PBX1 fusion and may distally modulate the transcription of the adjacent gene REL. Our results expand the understanding of subtype-specific ALL susceptibility and highlight plausible interplay between germline variants and somatic genomic abnormalities in ALL pathogenesis.

Successful toxicity reduction during delayed intensification in the non-high-risk arm of Malaysia-Singapore Acute Lymphoblastic Leukaemia 2010 study.

In non-high-risk (non-HR) patients, the Malaysia-Singapore Acute Lymphoblastic Leukaemia 2003 (MS2003) study achieved good outcomes. However, its delayed-intensification (DI) phase, comprising repeated blocks of protocol III (2003-PIII), was toxic and caused significant treatment delays. The successor MS2010 study attempted to lower DI toxicity by replacing myelosuppressive drugs (doxorubicin, cytarabine) with vincristine and asparaginase. PATIENTS AND METHODS: We analysed 1748 admissions for fever in 315 Singapore children with non-HR acute lymphoblastic leukaemia (ALL) (MS2003, n = 183; MS2010, n = 132), comprising 76% of the total cohort (n = 413), to study the impact of these changes. RESULTS: The new 2010-PVa which has no doxorubicin, was associated with significantly fewer hospitalisations due to fever (0.08 versus 0.30 admissions per block [A/blk], p < 0.001), as than 2003-PIIIa. Similarly in 2010-PIIIb and PVb, where one block of cytarabine was replaced by two doses of vincristine, admissions for fever were also fewer (0.47 versus 0.74 A/blk, p < 0.001) than in 2003-PIIIb. However, the addition of single doses of vincristine and asparaginase in 2010-PIIIa, even with a mandatory seven-day rest, led to more hospitalisations (0.45 A/blk, p < 0.001), increased risk of bacteraemia (relative-risk (RR) = 7.66, p = 0.005) and critical-care admissions (RR = 4.31, p = 0.13). Despite this, overall treatment-related mortality decreased from 2.7% to 0.8%. Taken together, the reduced phase delays allowed earlier completion of the intensive phase of treatment (standard risk: 38.1 versus 49.4 weeks, p < 0.001; intermediate risk: 50.9 versus 58.8 weeks, p < 0.001), while maintaining excellent 10-year event-free survival of 95.4% and overall survival of 96.2%. CONCLUSIONS: In non-HR ALL, replacing doxorubicin/cytarabine with vincristine/asparaginase during some DI blocks is effective in reducing toxicity without compromising outcomes. CLINICAL TRIAL INFORMATION: NCT0289464.

Pharmacogenomics in acute lymphoblastic leukemia.

Pharmacogenomics is a fast-growing field of personalized medicine using a patient's genomic profile to determine drug disposition or response to drug therapy, in order to develop safer and more effective pharmacotherapy. Childhood acute lymphoblastic leukemia (ALL), being the most common malignancy in childhood, which is treated with uniform and standardized clinical trials, is remarkably poised for pharmacogenomic studies. In the last decade, unbiased genome-wide association studies have identified multiple germline risk factors that strongly modify host response to drug therapy. Some of these genomic associations (e.g. TPMT, NUDT15 and mercaptopurine dosing) have accumulated a significant level of evidence on their clinical utility such that they are warranted as routine clinical tests to guide modification of treatment. Most of these germline associations however, have not yet reached such actionability. Insights have also been gathered on germline factors that affect host susceptibility to adverse effects of antileukemic agents (eg, vincristine, asparaginase, methotrexate). Further large-scale studies are required, along with the assimilation of both germline and somatic variants, to precisely predict host drug response and drug toxicities, with the eventual aim of executing genomic-based precision-pharmacotherapy in the treatment of ALL.