Technological Advances: CEBPA and FLT3 Internal Tandem Duplication Mutations Can be Reliably Detected by Next Generation Sequencing

Background: The detection of CEBPA and FLT3 mutations by next generation sequencing (NGS) is challenging due to high GC content and Internal Tandem Duplications (ITDs). Recent advances have been made to surmount these challenges. In this study, we compare three commercial kits and evaluate the performance of these more advanced hybrid-capture and AMP-chemistry based methods. Methods: Amplicon-based TSM 54-Gene Panel (Illumina) was evaluated against hybridization-capture SOPHiA Genetics MSP, OGT SureSeq, and AMP chemistry-based VariantPlex (Archer) for wet-lab workflow and data-analysis pipelines. Standard kit directions and commercial analysis pipelines were followed. Seven CEBPA and 10 FLT3-positive cases were identified that previously were missed on an amplicon NGS assay. The average reads, coverage uniformity, and the detection of CEBPA or FLT3 mutations were compared. Results: All three panels detected all 10 CEBPA mutations and all 10 FLT3 ITDs with 100% sensitivity. In addition, there was high concordance (100%) between all three panels detecting 47/47 confirmed variants in a set of core myeloid genes. Conclusions: The results show that the NGS assays are now able to reliably detect CEBPA mutations and FLT3 ITDs. These assays may allow foregoing additional orthogonal testing for CEBPA and FLT3.

Automated summary

This study compares the performance of three commercial kits in detecting CEBPA and FLT3 mutations. The results show that all three kits have 100% sensitivity in detecting CEBPA and FLT3 mutations, and there is high concordance in detecting confirmed variants in core myeloid genes. These findings indicate that NGS assays can now reliably detect CEBPA and FLT3 mutations.