Background and Aims

Carboxypeptidase g2 (CPDG2 or glucarpidase) is a rescue drug for patients who develop delayed clearance or kidney injury from high-dose methotrexate (MTX). As there are no strategies for predicting patients who will require this additional treatment, we evaluated the role of demographic, clinical, and genetic factors for CPDG2 use.

Methods

Cases who received CPDG2 and controls were identified in a chart review of ALL patients who received MTX doses between 1000-5000mg/m2 at Texas Children’s Cancer Center. Multivariable Bayesian logistic regression was performed to evaluate the probability of CPDG2 use considering the following factors: self-reported race/ethnicity, MTX dose, body mass index (BMI), ALL subtype, and age at diagnosis. Additionally, the effects of 49 genetic variants associated with MTX metabolism were also evaluated in a subset of patients who underwent genotyping.

Results

A total of 423 patients were identified who received 1592 doses of MTX. Of the 18 patients who received CPDG2, 17 (94%) were Hispanic. No patients who received 1000 or 2000mg/m2 of MTX received CPDG2. Hispanic ethnicity demonstrated an odds ratio (OR) of 3.46 (95% compatibility interval [CI]:1.27–8.04) and older age an OR of 1.80 per 1 SD increase in age (95%CI:1.11-2.79). Of the 177 patients in the genomic cohort, 11 received CPDG2. Each additional allele G of rs7317112 in ABCC4, which codes for an MTX efflux transporter in renal cells, demonstrated an OR of 1.77 (95%CI:1-3.13). Five other loci (GATA3/rs3824662, NTRK1/rs10908, ARID5B/rs10821936, GATA3/rs3824662, and TSG1/rs9345389) also demonstrated probabilities of association between 82-92%.

Conclusions

Overall, we demonstrated that certain demographic characteristics, including Hispanic ethnicity and age, are strongly associated with CPDG2 use. Additionally, we provide evidence that inherited genetic variation could be used to predict those at risk of requiring CPDG2. If validated in independent populations, this information could be leveraged to improve risk-stratification strategies for children with ALL.

Background and Aims

Nucleoside diphosphate–linked moiety X-type motif (NUDT15) an enzyme involved in the metabolism of 6-MP, the cornerstone of maintenance therapy in acute lymphoblastic leukemia (ALL), is associated with myelosuppression. Inadequate information exists on the correlation of these variants with clinical observations. This study evaluates the frequency of NUDT15 415 CT (R139C) allele & its association with clinical myelotoxicity.

Methods

One hundred forty seven children with ALL (5.19±2.9 years) were evaluated for NUDT15 415 CT (R139C) allele prior to maintenance therapy (MT) (Jan 2016 to Oct 2019), including 100 cases in which complete NUDT15 gene sequencing was performed. A combination of RFLP-PCR and Sanger sequencing was used. Data pertaining to 6-MP dosing was noted from case record files.

Results

Eighteen patients (18/147) had 415CT SNP (12.2%). 6-MP dose in SNP positive (mut+) group was 49±8.75mg/m2/day vs. 53.25±6.63 mg/m2/day in SNP negative (mut-) group (p=0.016). No difference was observed with methotrexate dose in the 2 groups. One hundred twenty nine (129) patients were evaluable for a minimum of 6 months of MT. (Excluded: death: 6; relapse: 1; 10 not completed 6 months MT & one with CMV infection); the mean dose in the mut+ & mut- groups being 49±8.6 mg/m2/day and 53.25 ±6.5 mg/m2/day (p=0.048). Chemotherapy interruptions were higher in the mut+ (4.64 ± 2.97 weeks) vs the mut- group(2.91 ± 2.29 weeks) (p=0.044) as were episodes of febrile neutropenia : 1.55± 2.67 in mut+ & 0.81±1.02 in the mut- group (p=0.044).

Conclusions

Polymorphisms in TPMT & ITPA198 CA are linked to myelotoxicity. This study provides conclusive evidence of the relationship of the dose of 6MP with myelotoxicity in individuals with the NUDT415CT variant allele with the prevalence of the polymorphism being 12% in our population. Large geographic area based pharmacogenetic studies exploring the pharmacokinetics of 6MP across various ethnic groups are required for precision therapy.

Background and Aims

We recently found that pediatric acute lymphoblastic leukemia (ALL) relapses have increased mutational loads, sometimes resulting in a hypermutation phenomenon, which appear to occur across childhood ALL subtypes, particularly in second relapses, and can be caused by different mutational mechanisms, including abberant AID/APOBEC activity and mismatch repair deficiency¹.

Mutations arise in single cells which subsequently grow out into (sub)clones and become detectable. Since ALL is a multiclonal disease, different mechanisms can be active simultenously or subsequently, which arise endogenously or be induced by therapy.

Methods

We performed whole genome sequencing of 33 samples (diagnosis, relapses and complete remissions) from six leukemia patients and clustered the mutations that followed the same dynamics over time. These mutational clusters were used to identify endogenous and/or external mutational mechanisms in different subclones within each patient.

Results

We defined up to six clusters per patient, with an average cluster size of 1,067 mutations [63-5.767] . We idenified at least five mutational mechanisms, including abberant activity of AID/APOBEC deaminases, and an incompletely understood mutational pattern associated with UV exposure previously also reported by others in ALL². In four patients we observed mutational mechanisms only becoming active in rising clones at first or second relapse. In one Down-syndrome patient who developed two relapses, we identified at least two different mutational mechanisms that were active at different stages of disease. One of these mechanisms (AID/APOBEC) was active in a falling clone at diagnosis and in rising subclones at first and second relapse, suggesting that this aberrant AID/APOBEC activity gave rise to new clones during the course of disease.

Conclusions

We have shown dynamic clonal evolutionary trajectories driven by multiple mutational mechanisms in children with ALL and mutliple relapses. Understanding of the causes and consequences of the mutational mechanisms driving relapse in ALL may provide opportunities for treatment.

Background and Aims

The utility of miRNA as a prognostic biomarker for childhood ALL is a subject of on-going research. We report next-generation sequencing-based miRNA expression profile in ALL relapse.

Methods

The cross-sectional study included children (≤12-years) with B-lineage ALL. Cases included patients with relapsed disease. Survivors in remission for >4-years formed the control group. Total RNA was extracted from peripheral blood samples. miRNA library preparation (TruSeq small RNA kit) and sequencing (MiSeq platform, Illumina) were performed. The differentially expressed miRNAs were identified using DeSeq2 software (Cut-off criteria: fold change ±2, p-adj <0.05). Targets for differentially expressed miRNAs were retrieved by TargetScan/Diana Tools.

Results

Six cases and an equal number of controls were enrolled. RT-PCR was negative for t(1;19), t(4;11), t(9;22) and t(12;21) in all. Sequencing was performed in 4 cases and 5 controls (mean age: 4.25±4.9 years) in whom the extracted RNA fulfilled the recommended criteria (RNA integrity number ≥8, RNA concentration ≥200 ng/μl). A total of 1224 miRNAs were identified, among which the expression of 25 miRNAs varied significantly between cases and controls. Twelve miRNAs (miR-34a,miR-10b,miR-96,miR-502-3p,miR-221*,miR-99b,miR-100,miR-1271,miR-183,miR-182,miR-500a*,miR-484) were up-regulated and 13 (miR-381,miR-127-3p,miR-29b,miR-342-5p,miR-409-3p,miR-889,miR-136*,miR-410,miR-485-3p,miR-376c,miR-487b,miR-370,miR-433) down-regulated in cases as compared to controls.

In silico analysis revealed the up-regulated miRNAs in cases had predicted mRNA targets responsible for limiting the neoplastic transformation. The targets included tumor suppressors (e.g.-RB1, TP53, PTEN), cell-cycle regulatory genes (cyclin E/B, MDM2), apoptotic genes (CASP3, CASP9) and genes involved in metastasis inhibition (SERPINE, THBS1). Conversely, the down-regulated miRNAs had predicted targets responsible for promoting neoplasm, including proliferation/survival genes (e.g.-AKT2, CTNNB1, NOTCH2), genes involved in angiogenesis (HIF1A) and fatty-acid biosynthesis (FASN, ACSL3).

Conclusions

A distinct profile of miRNAs associated with relapse in pediatric B-lineage ALL is reported. The differentially expressed miRNAs can be exploited as novel prognostic markers. Further, modulating the expression of signature miRNAs by antimiR/mimic based therapies could be explored in patients with relapse.