Development of an Amplicon-Based Next-Generation Sequencing Assay for Pan-type Detection of BCR::ABL1 Kinase Domain Mutations

BCR::ABL1 kinase domain (KD) mutations confer tyrosine kinase inhibitor (TKI) resistance in Philadelphia chromosome-positive (Ph+) leukemia, but conventional Sanger sequencing (SS) has limited sensitivity and most next-generation sequencing (NGS) assays cover only a single transcript type. We developed an amplicon-based NGS assay designed for pan-type amplification of major, minor, and micro BCR::ABL1 fusion transcript types, together with the entire KD, in a single multiplex long-range PCR. In 115 residual samples from 88 patients with Ph+ leukemia, transcript typing by NGS was completely concordant with reverse-transcription PCR for major, minor, and micro BCR::ABL1 transcripts, including a rare c3a2 variant. NGS detected low-level mutations missed by SS and raised the mutation-positivity rate from 19.7% (14/71) to 38.0% (27/71); newly detected mutations had a mean variant allele frequency (VAF) of 9.5% (range, 1.0-17.4). Compound and polyclonal mutations were resolved in four patients, and KD mutations were detected even at the BCR::ABL1 transcript levels around 0.1% International Scale or lower. The estimated limit of detection was approximately 1.0% VAF for both p.T315I and p.Y253H under the tested conditions, with no false positives at the limit of blank. This single-workflow assay is readily applicable to routine clinical testing of Ph+ leukemia.