Analytical Validation of an Annotation Tool for WGS-Based Pharmacogenomics: Preparing for Clinical Implementation in Pediatric Oncology

Abstract

Whole genome sequencing (WGS) is increasingly accessible in clinical practice, enabling pharmacogenomics (PGx) integration, including in pediatric oncology. However, the lack of validated software to accurately annotate clinically actionable PGx variants from WGS limits routine implementation. We therefore aimed to identify and validate a PGx annotation tool suitable for clinical use in pediatric oncology. We evaluated several tools for technical performance and clinical integration. The Pharmacogenomics Clinical Annotation Tool (PharmCAT) was selected for its alignment with expert-reviewed PGx guidelines, inclusion of relevant gene-drug pairs and prescribing recommendations. PharmCAT was validated using an in silico dataset by introducing known diplotypes into the Genome in a Bottle (GIAB) reference sample, complemented by four clinically confirmed diplotypes from three patients. Diplotype and phenotype outputs from WGS were compared against the GIAB and patient reference data. We tested 71 diplotypes across seven genes (TPMT, NUDT15, CYP3A5, CYP2C9, CYP2C19, DPYD, UGT1A1), using ≥ 95% sensitivity and specificity as validation criteria. CYP2D6 was excluded from this validation due to genotyping limitations from the input data used by PharmCAT. The tool was integrated into our WGS analysis pipeline using containerization for consistent, reproducible execution. Diplotype and phenotype results from PharmCAT fully matched the in silico GIAB set and patient samples, achieving 100% sensitivity and specificity. These findings confirm PharmCAT as a reliable tool for preemptive PGx annotation, supporting implementation in pediatric oncology. Its clinical integration supports individualized dosing, reducing adverse effects and improving efficacy. Further validation of additional gene-drug pairs will broaden its clinical utility.