NT5C2 mutations in relapsed pediatric BCP-ALL. (A) Schematic representation of the NT5C2 protein/gene. Mutations identified by Sanger sequencing and/or targeted next generation in relapses of 455 patients with BCP-ALL are described at the protein level. Multiple circles in the same amino acid position account for multiple patients with the same variant. NT5C2 exons are indicated by alternating light and dark gray boxes. Variant frequency refers to variant allele frequency for NGS-detected mutations and allelic peak ratio for Sanger-detected mutations. (B) Performance of the ASQ-PCR assays developed for sensitive detection of NT5C2 mutations p.R39Q and p.R367Q. The graphs represent exemplary amplification plots of standard dilution series of NT5C2 mutation-positive patient samples (blue) as well as of a mutation-negative DNA control (red). The distance between the 1E-03 dilution and the mutation-negative DNA control is larger than 3 C_T values for both assays as required by the guidelines for minimal residual disease detection in leukemia.³¹ (C) Range of NT5C2-mutant clone frequencies determined by ASQ-PCR for all p.R39Q and p.R367Q mutations. For this graphic, NT5C2-mutant clone frequencies were categorized into different quantitative levels in log₁₀ steps. The bar graphs represent the percentage of subclonal NT5C2 p.R39Q and p.R367Q mutations with a given quantitative level. The absolute number of mutations is displayed within the bar graphs. (D) Classification of relapsed patients with NT5C2 mutations after compilation of sequencing and ASQ-PCR data into 2 main groups: patients with clonal NT5C2 mutation (dark-red) and patients with subclonal NT5C2 mutation(s) only (light red). Approximately one-third of relapses harbored >1 NT5C2 mutation.