Evaluation of Methotrexate Pharmacogenomic Variants to Predict Acute Neurotoxicity in Children with Acute Lymphoblastic Leukemia

Background: Methotrexate is an important component of curative therapy in childhood acute lymphoblastic leukemia (ALL), but exposure to intrathecal (IT) and intravenous (IV) methotrexate results in acute neurotoxicity in approximately 10% of patients. While methotrexate-related neurotoxicity is a significant source of morbidity in children with ALL and potentially compromises treatment efficacy, little is known about factors impacting susceptibility. While numerous pharmacogenomics variants have been implicated in methotrexate efficacy, toxicity, and pharmacokinetics, the role of these variants in neurotoxicity susceptibility in children with ALL is not well-established. Therefore, we evaluated the association between suspected methotrexate pharmacogenomic variants and methotrexate-related neurotoxicity in a multi-institutional cohort of children treated for ALL.

Methods: This study included children (age <20 years) diagnosed with ALL (2005-2019) at six treatment centers in the southwest US. Clinical information was abstracted from medical records. Suspected neurotoxic events occurring within 21 days of IT and/or IV methotrexate delivered between the end of induction and start of maintenance therapy were independently reviewed by at least two pediatric oncologists. Germline DNA was extracted and genotyped using the Illumina Infinium Global Screening Array. A total of 97 annotated methotrexate pharmacogenomic variants with at least grade 3 evidence were identified using the Pharmacogenomics Knowledge Base (PharmGKB). Associations between variants and neurotoxicity were assessed by logistic regression to estimate odds ratios (ORs) and corresponding 95% confidence intervals (CI). The cohort was randomly split (80/20) and random forest was constructed with downsampling and 5-repeat, 10-fold cross validation to estimate the ability of the variants to correctly classify neurotoxicity compared to a clinical model considering sex, race, ethnicity, and age alone.

Results: A total of 763 eligible patients were evaluated. The population was 46% female, 57.5% Latino, and diagnosed at a mean age of 7 years. Overall, 8.2% (n=63) developed methotrexate-associated neurotoxicity. Most cases presented with stroke-like symptoms (n=41), followed by seizures (n=12), tremors/numbness (n=4), altered mental status (n=4), syncopal episodes (n=1), or aphasia (n=1). Older age at diagnosis (OR=1.21 per year, 95% CI: 1.15-1.28) and Latino ethnicity (OR=2.11, 95% CI: 1.73-2.07) were independently associated with an increased likelihood of neurotoxicity. In logistic models, none of the 97 available pharmacogenomic variants reached statistical significance after correcting for multiple comparisons, but two variants, rs17222723 in ABCC2 (OR=2.83, 95% CI: 1.20-6.15) and rs1045642 in ABCB1(OR=0.66, 95% CI: 0.44-0.98), were nominally associated (p-value<0.05) with neurotoxicity susceptibility. A random forest model that incorporated pharmacogenomic variants in addition to clinical factors (AUC=0.73, sensitivity=0.69, specificity=0.65) did not perform significantly better than a model that considered clinical factors alone (AUC=0.74, sensitivity=0.70, specificity=0.65).

Conclusion: This study is among the largest to evaluate pharmacogenomic predictors of methotrexate-related neurotoxicity among children with ALL. Overall, our study confirmed Latino ethnicity and older age as risk factors for neurotoxicity, and demonstrated that, while some annotated methotrexate pharmacogenomic variants may be modestly associated with neurotoxicity susceptibility, these variants contributed relatively little to overall neurotoxicity risk among children with ALL. These findings suggest that additional factors beyond currently identified pharmacogenomic variants may impact methotrexate-related neurotoxicity among children diagnosed with ALL. Specifically, these results underscore the need to identify novel genetic and non-genetic factors associated with neurotoxicity susceptibility following methotrexate chemotherapy, particularly among highly vulnerable populations such as Latino children with ALL.