Molecular basis of ETV6-mediated predisposition to childhood acute lymphoblastic leukemia.

There is growing evidence supporting an inherited basis for susceptibility to acute lymphoblastic leukemia (ALL) in children. In particular, we and others reported recurrent germline ETV6 variants linked to ALL risk, which collectively represent a novel leukemia predisposition syndrome. To understand the influence of ETV6 variation on ALL pathogenesis, we comprehensively characterized a cohort of 32 childhood leukemia cases arising from this rare syndrome. Of 34 nonsynonymous germline ETV6 variants in ALL, we identified 22 variants with impaired transcription repressor activity, loss of DNA binding, and altered nuclear localization. Missense variants retained dimerization with wild-type ETV6 with potentially dominant-negative effects. Whole-transcriptome and whole-genome sequencing of this cohort of leukemia cases revealed a profound influence of germline ETV6 variants on leukemia transcriptional landscape, with distinct ALL subsets invoking unique patterns of somatic cooperating mutations. 70% of ALL cases with damaging germline ETV6 variants exhibited hyperdiploid karyotype with characteristic recurrent mutations in NRAS, KRAS, and PTPN11. In contrast, the remaining 30% cases had a diploid leukemia genome and an exceedingly high frequency of somatic copy-number loss of PAX5 and ETV6, with a gene expression pattern that strikingly mirrored that of ALL with somatic ETV6-RUNX1 fusion. Two ETV6 germline variants gave rise to both acute myeloid leukemia and ALL, with lineage-specific genetic lesions in the leukemia genomes. ETV6 variants compromise its tumor suppressor activity in vitro with specific molecular targets identified by assay for transposase-accessible chromatin sequencing profiling. ETV6-mediated ALL predisposition exemplifies the intricate interactions between inherited and acquired genomic variations in leukemia pathogenesis.

Massively parallel variant characterization identifies NUDT15 alleles associated with thiopurine toxicity.

As a prototype of genomics-guided precision medicine, individualized thiopurine dosing based on pharmacogenetics is a highly effective way to mitigate hematopoietic toxicity of this class of drugs. Recently, NUDT15 deficiency was identified as a genetic cause of thiopurine toxicity, and NUDT15-informed preemptive dose reduction was quickly adopted in clinical settings. To exhaustively identify pharmacogenetic variants in this gene, we developed massively parallel NUDT15 function assays to determine the variants' effect on protein abundance and thiopurine cytotoxicity. Of the 3,097 possible missense variants, we characterized the abundance of 2,922 variants and found 54 hotspot residues at which variants resulted in complete loss of protein stability. Analyzing 2,935 variants in the thiopurine cytotoxicity-based assay, we identified 17 additional residues where variants altered NUDT15 activity without affecting protein stability. We identified structural elements key to NUDT15 stability and/or catalytical activity with single amino acid resolution. Functional effects for NUDT15 variants accurately predicted toxicity risk alleles in patients treated with thiopurines with far superior sensitivity and specificity compared to bioinformatic prediction algorithms. In conclusion, our massively parallel variant function assays identified 1,152 deleterious NUDT15 variants, providing a comprehensive reference of variant function and vastly improving the ability to implement pharmacogenetics-guided thiopurine treatment individualization.

Long-read HiFi sequencing of NUDT15: Phased full-gene haplotyping and pharmacogenomic allele discovery.

To determine the phase of NUDT15 sequence variants for more comprehensive star (*) allele diplotyping, we developed a novel long-read single-molecule real-time HiFi amplicon sequencing method. A 10.5 kb NUDT15 amplicon assay was validated using reference material positive controls and additional samples for specimen type and blinded accuracy assessment. Triplicate NUDT15 HiFi sequencing of two reference material samples had nonreference genotype concordances of >99.9%, indicating that the assay is robust. Notably, short-read genome sequencing of a subset of samples was unable to determine the phase of star (*) allele-defining NUDT15 variants, resulting in ambiguous diplotype results. In contrast, long-read HiFi sequencing phased all variants across the NUDT15 amplicons, including a *2/*9 diplotype that previously was characterized as *1/*2 in the 1000 Genomes Project v3 data set. Assay throughput was also tested using 8.5 kb amplicons from 100 Ashkenazi Jewish individuals, which identified a novel NUDT15 *1 suballele (c.-121G>A) and a rare likely deleterious coding variant (p.Pro129Arg). Both novel alleles were Sanger confirmed and assigned as *1.007 and *20, respectively, by the PharmVar Consortium. Taken together, NUDT15 HiFi amplicon sequencing is an innovative method for phased full-gene characterization and novel allele discovery, which could improve NUDT15 pharmacogenomic testing and subsequent phenotype prediction.

Adalimumab Immunogenicity Is Negatively Correlated with Anti-Hinge Antibody Levels in Patients with Rheumatoid Arthritis.

Patients with rheumatoid arthritis (RA) are frequently treated with anti-tumor necrosis factor-α immunoglobulin therapy but develop neutralizing antibodies against these drugs, necessitating therapeutic monitoring of drug concentrations and anti-drug antibodies. Patients with RA have multiple factors related to their autoimmune disposition that interfere with conventionally used methods to detect anti-drug antibodies. Currently deployed analytical methods have significant limitations that hinder clinical interpretation and/or routine use, and no method can detect immunogenicity and drug levels simultaneously to provide clinically meaningful recommendations. Given these limitations, the objective of this study was to identify sources of and associations with assay interference in patients with RA. We designed a modular immunogenicity and drug concentration detection technology to identify the factors that interfere with the detection of adalimumab and anti-adalimumab antibodies in a cohort of 206 patients with RA. Patients were included from the University of Pittsburgh Rheumatoid Arthritis Comparative Effectiveness Research registry. In this cohort, we analyzed clinical and plasma factors associated with anti-adalimumab and anti-hinge antibodies. A novel flow cytometry-based assay was developed and validated that simultaneously measures adalimumab and anti-adalimumab antibody concentrations, overcoming many of the interference factors that are limitations of conventional assays, including anti-fragment crystallizable (Fc) and anti-hinge antibodies. C-reactive protein (P = 0.035), Disease Activity Score-28 (DAS28) score (P = 0.002), and disease activity category (P = 0.009) were significantly associated with anti-adalimumab antibodies but not with anti-hinge antibodies (P > 0.05). Anti-hinge antibodies were inversely associated with drug-neutralizing antibodies (P = 0.002). In patients with RA, anti-hinge antibodies may have a potential protective effect against the development of anti-adalimumab antibodies. SIGNIFICANCE STATEMENT: Using a novel cytometric assay that simultaneously measures drug and anti-drug antibodies, we overcame many interferences that hinder the clinical interpretation of adalimumab immunogenicity testing. Our investigation in patients with RA demonstrated that immunogenicity impaired the pharmacological action of adalimumab via analysis of RA disease severity markers. We also demonstrate that patients with anti-hinge antibodies had lower anti-adalimumab antibody levels and decreased drug neutralization. Our results suggest that anti-hinge antibodies can predict adalimumab immunogenicity before the start of therapy.

Asparaginase-specific basophil recognition and activation predict Asparaginase hypersensitivity in mice.

Background: Asparaginase (ASNase) is a crucial part of acute leukemia treatment, but immune responses to the agent can reduce its effectiveness and increase the risk of relapse. Currently, no reliable and validated biomarker predicts ASNase-induced hypersensitivity reactions during therapy. We aimed to identify predictive biomarkers and determine immune cells responsible for anaphylaxis using a murine model of ASNase hypersensitivity. Methods: Our preclinical study uses a murine model to investigate predictive biomarkers of ASNase anaphylaxis, including anti-ASNase antibody responses, immune complex (IC) levels, ASNase-specific binding to leukocytes or basophils, and basophil activation. Results: Our results indicate that mice immunized to ASNase exhibited dynamic IgM, IgG, and IgE antibody responses. The severity of ASNase-induced anaphylaxis was found to be correlated with levels of IgG and IgE, but not IgM. Basophils from immunized mice were able to recognize and activate in response to ASNase ex vivo, and the extent of recognition and activation also correlated with the severity of anaphylaxis observed. Using a multivariable model that included all biomarkers significantly associated with anaphylaxis, independent predictors of ASNase-induced hypersensitivity reactions were found to be ASNase IC levels and ASNase-specific binding to leukocytes or basophils. Consistent with our multivariable analysis, we found that basophil depletion significantly protected mice from ASNase-induced hypersensitivity reactions, supporting that basophils are essential and can be used as a predictive marker of ASNase-induced anaphylaxis. Conclusions: Our study demonstrates the need for using tools that can detect both IC- and IgE-mediated hypersensitivity reactions to mitigate the risk of ASNase-induced hypersensitivity reactions during treatment.

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.

Rituximab administration in pediatric patients with newly diagnosed acute lymphoblastic leukemia.

Polyethylene glycol (PEG)-asparaginase (pegaspargase) is a key agent in chemotherapy for acute lymphoblastic leukemia (ALL), but recipients frequently experience allergic reactions. We hypothesized that by decreasing antibody-producing CD20-positive B cells, rituximab may reduce these reactions. Children and adolescents (aged 1-18 years) with newly diagnosed B-ALL treated on the St. Jude Total XVII study were randomized to induction therapy with or without rituximab on day 3 (cohort 1) or on days 6 and 24 (cohort 2). Patient clinical demographics, CD20 expression, minimal residual disease (MRD), rituximab reactions, pegaspargase allergy, anti-pegaspargase antibodies, and pancreatitis were evaluated. Thirty-five patients received rituximab and 37 did not. Among the 35 recipients, 16 (45.7%) experienced a grade 2 or higher reaction to rituximab. There were no differences between recipients and non-recipients in the incidence of pegaspargase reactions (P > 0.999), anti-pegaspargase antibodies (P = 0.327), or pancreatitis (P = 0.480). CD20 expression on day 8 was significantly lower in rituximab recipients (P < 0.001), but there were no differences in MRD levels on day 8, 15, or at the end of induction. Rituximab administration during induction in pediatric patients with B-ALL was associated with a high incidence of infusion reactions with no significant decrease in pegaspargase allergies, anti-pegaspargase antibodies, or MRD.

Clinical Pharmacogenetics Implementation Consortium Guideline for the Use of Aminoglycosides Based on MT-RNR1 Genotype.

Aminoglycosides are widely used antibiotics with notable side effects, such as nephrotoxicity, vestibulotoxicity, and sensorineural hearing loss (cochleotoxicity). MT-RNR1 is a gene that encodes the 12s rRNA subunit and is the mitochondrial homologue of the prokaryotic 16s rRNA. Some MT-RNR1 variants (i.e., m.1095T>C; m.1494C>T; m.1555A>G) more closely resemble the bacterial 16s rRNA subunit and result in increased risk of aminoglycoside-induced hearing loss. Use of aminoglycosides should be avoided in individuals with an MT-RNR1 variant associated with an increased risk of aminoglycoside-induced hearing loss unless the high risk of permanent hearing loss is outweighed by the severity of infection and safe or effective alternative therapies are not available. We summarize evidence from the literature supporting this association and provide therapeutic recommendations for the use of aminoglycosides based on MT-RNR1 genotype (updates at https://cpicpgx.org/guidelines/ and www.pharmgkb.org).

Association of Inherited Genetic Factors With Drug-Induced Hepatic Damage Among Children With Acute Lymphoblastic Leukemia.

Importance: Acute lymphoblastic leukemia (ALL) is the most common childhood cancer. Hepatotoxic effects, including hyperbilirubinemia and elevated alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, are common during all phases of therapy and are linked to several chemotherapeutic agents, including asparaginase, mercaptopurine, and methotrexate. Objective: To determine which genetic variants were associated with hyperbilirubinemia and elevated ALT and AST levels in children, adolescents, and young adults treated for ALL. Design, Setting, and Participants: This retrospective analysis of a multiethnic genome-wide association study was conducted between January 1, 2019, and April 15, 2022, including patients treated as part of Children's Oncology Group (COG) trials with centers in the United States, Canada, and Australia, which accrued data from December 29, 2003, to January 21, 2011 (AALL0232), and from January 22, 2007, to July 24, 2014 (AALL0434). Germline genotypes were interrogated using genome-wide arrays and imputed using a National Institutes of Health TOPMed Imputation server. Mixed-effects logistic regressions were used to account for multiple episodes for an individual patient. Genotype × treatment phase interaction was tested to uncover phase-specific genetic risk factors. Exposures: Total duration of multiagent protocol chemotherapy ranging from 2.5 to 3.5 years. Main Outcomes and Measures: The primary outcomes were National Cancer Institute Common Terminology Criteria for Adverse Events (version 4) hyperbilirubinemia of grade 3 or higher and elevated liver ALT and AST levels. Results: A total of 3557 participants were included in the analysis (2179 [61.3%] male; median age, 11.1 [range, 1-30] years). Among 576 known variants associated with these liver function test results in the general population, UGT1A1 variant rs887829 and PNPLA3 variant rs738409 were associated with increased risk of hyperbilirubinemia (odds ratio [OR], 2.18 [95% CI, 1.89-2.53]; P = 6.7 × 10-27) and ALT and AST levels (OR, 1.27 [95% CI, 1.15-1.40]; P = 3.7 × 10-7), respectively, during treatment for ALL. Corresponding polygenic risk scores were associated with hepatotoxic effects across all therapy phases and were largely driven by UGT1A1 and PNPLA3 variants. Genome-wide association analysis revealed an age-specific variant near the CPT1A gene that was only associated with elevated ALT and AST levels among patients younger than 10 years (OR, 1.28 [95% CI, 1.18-1.39]; P = 8.7 × 10-10). Conclusions and Relevance: These results suggest a strong genetic basis for interpatient variability in hyperbilirubinemia and aminotransferase level elevations during leukemia chemotherapy.

Pharmacogenomics for Drug Dosing in Children: Current Use, Knowledge, and Gaps.

Pharmacogenomics research ranges from the discovery of genetic factors to explain interpatient variability in drug exposure and response to clinical implementation of this knowledge to improve pharmacotherapy. Medications with actionable pharmacogenomic associations are frequently used in children, and therefore pharmacogenomics-guided precision medicine is readily applicable to the pediatric population. Although heritable genetics are considered immutable, the impact of genetic variation in pharmacogenes is modified by other factors such as age-dependent changes in gene expression. Early evidence has emerged indicating that the interaction between ontogeny and pharmacogenomics determines whether or how genetics-based dosing algorithms should be adjusted in children versus adults. However, there is still a paucity of data describing pharmacogenomic associations in patient populations across the life span. Future research is much needed to evaluate the impact of pharmacogenomics on drug dosing specific to the pediatric population, along with consideration of other developmental and physiological factors uniquely related to drug disposition in this population.

Amino Acid Metabolic Vulnerabilities in Acute and Chronic Myeloid Leukemias.

Amino acid (AA) metabolism plays an important role in many cellular processes including energy production, immune function, and purine and pyrimidine synthesis. Cancer cells therefore require increased AA uptake and undergo metabolic reprogramming to satisfy the energy demand associated with their rapid proliferation. Like many other cancers, myeloid leukemias are vulnerable to specific therapeutic strategies targeting metabolic dependencies. Herein, our review provides a comprehensive overview and TCGA data analysis of biosynthetic enzymes required for non-essential AA synthesis and their dysregulation in myeloid leukemias. Furthermore, we discuss the role of the general control nonderepressible 2 (GCN2) and-mammalian target of rapamycin (mTOR) pathways of AA sensing on metabolic vulnerability and drug resistance.

Mechanistic studies of PEG-asparaginase-induced liver injury and hepatic steatosis in mice.

PEGylated-l-asparaginase (PEG-ASNase) is a chemotherapeutic agent used to treat pediatric acute lymphoblastic leukemia (ALL). Its use is avoided in adults due to its high risk of liver injury including hepatic steatosis, with obesity and older age considered risk factors of the injury. Our study aims to elucidate the mechanism of PEG-ASNase-induced liver injury. Mice received 1500 U/kg of PEG-ASNase and were sacrificed 1, 3, 5, and 7 days after drug administration. Liver triglycerides were quantified, and plasma bilirubin, ALT, AST, and non-esterified fatty acids (NEFA) were measured. The mRNA and protein levels of genes involved in hepatic fatty acid synthesis, ß-oxidation, very low-density lipoprotein (VLDL) secretion, and white adipose tissue (WAT) lipolysis were determined. Mice developed hepatic steatosis after PEG-ASNase, which associated with increases in bilirubin, ALT, and AST. The hepatic genes Ppara, Lcad/Mcad, Hadhb, Apob100, and Mttp were upregulated, and Srebp-1c and Fas were downregulated after PEG-ASNase. Increased plasma NEFA, WAT loss, and adipose tissue lipolysis were also observed after PEG-ASNase. Furthermore, we found that PEG-ASNase-induced liver injury was exacerbated in obese and aged mice, consistent with clinical studies of ASNase-induced liver injury. Our data suggest that PEG-ASNase-induced liver injury is due to drug-induced lipolysis and lipid redistribution to the liver.

Medication Use as a Contributor to Fluid Overload in the PICU: A Prospective Observational Study.

In this prospective observational study, we explored the association of daily fluid intake from medication use with fluid overload in 75 children beginning 24 hours after intubation. The mean percent daily fluid intake from medications was 29% in the overall cohort. Excess intake and inadequate output contributed significantly to fluid overload. In the 28 patients who became ≥10% fluid overloaded, the mean percent daily fluid intake from medications was 34%, but just 23% in the patients who did not. Awareness of volume contribution and maximized concentration of parenteral medications when able may lessen the burden of fluid overload.

Glibenclamide Produces Region-Dependent Effects on Cerebral Edema in a Combined Injury Model of Traumatic Brain Injury and Hemorrhagic Shock in Mice.

Cerebral edema is critical to morbidity/mortality in traumatic brain injury (TBI) and is worsened by hypotension. Glibenclamide may reduce cerebral edema by inhibiting sulfonylurea receptor-1 (Sur1); its effect on diffuse cerebral edema exacerbated by hypotension/resuscitation is unknown. We aimed to determine if glibenclamide improves pericontusional and/or diffuse edema in controlled cortical impact (CCI) (5m/sec, 1 mm depth) plus hemorrhagic shock (HS) (35 min), and compare its effects in CCI alone. C57BL/6 mice were divided into five groups (n = 10/group): naïve, CCI+vehicle, CCI+glibenclamide, CCI+HS+vehicle, and CCI+HS+glibenclamide. Intravenous glibenclamide (10 min post-injury) was followed by a subcutaneous infusion for 24 h. Brain edema in injured and contralateral hemispheres was subsequently quantified (wet-dry weight). This protocol brain water (BW) = 80.4% vehicle vs. 78.3% naïve, p < 0.01) but was not reduced by glibenclamide (I%BW = 80.4%). Ipsilateral edema also developed in CCI alone (I%BW = 80.2% vehicle vs. 78.3% naïve, p < 0.01); again unaffected by glibenclamide (I%BW = 80.5%). Contralateral (C) %BW in CCI+HS was increased in vehicle (78.6%) versus naive (78.3%, p = 0.02) but unchanged in CCI (78.3%). At 24 h, glibenclamide treatment in CCI+HS eliminated contralateral cerebral edema (C%BW = 78.3%) with no difference versus naïve. By 72 h, contralateral cerebral edema had resolved (C%BW = 78.5 ± 0.09% vehicle vs. 78.3 ± 0.05% naïve). Glibenclamide decreased 24 h contralateral cerebral edema in CCI+HS. This beneficial effect merits additional exploration in the important setting of TBI with polytrauma, shock, and resuscitation. Contralateral edema did not develop in CCI alone. Surprisingly, 24 h of glibenclamide treatment failed to decrease ipsilateral edema in either model. Interspecies dosing differences versus prior studies may play an important role in these findings. Mechanisms underlying brain edema may differ regionally, with pericontusional/osmolar swelling refractory to glibenclamide but diffuse edema (via Sur1) from combined injury and/or resuscitation responsive to this therapy. TBI phenotype may mandate precision medicine approaches to treat brain edema.

NUDT15 polymorphisms alter thiopurine metabolism and hematopoietic toxicity.

Widely used as anticancer and immunosuppressive agents, thiopurines have narrow therapeutic indices owing to frequent toxicities, partly explained by TPMT genetic polymorphisms. Recent studies identified germline NUDT15 variation as another critical determinant of thiopurine intolerance, but the underlying molecular mechanisms and the clinical implications of this pharmacogenetic association remain unknown. In 270 children enrolled in clinical trials for acute lymphoblastic leukemia in Guatemala, Singapore and Japan, we identified four NUDT15 coding variants (p.Arg139Cys, p.Arg139His, p.Val18Ile and p.Val18_Val19insGlyVal) that resulted in 74.4-100% loss of nucleotide diphosphatase activity. Loss-of-function NUDT15 diplotypes were consistently associated with thiopurine intolerance across the three cohorts (P = 0.021, 2.1 × 10(-5) and 0.0054, respectively; meta-analysis P = 4.45 × 10(-8), allelic effect size = -11.5). Mechanistically, NUDT15 inactivated thiopurine metabolites and decreased thiopurine cytotoxicity in vitro, and patients with defective NUDT15 alleles showed excessive levels of thiopurine active metabolites and toxicity. Taken together, these results indicate that a comprehensive pharmacogenetic model integrating NUDT15 variants may inform personalized thiopurine therapy.