Sensitive NPM1 mutation quantitation in acute myeloid leukemia using ultradeep next-generation sequencing in the diagnostic laboratory

Abstract

Context.—Detection of measurable residual disease after therapy is an important predictor of outcome in acute myeloid leukemia. Objective.—To investigate the feasibility of using next-generation sequencing (NGS) in the diagnostic laboratory to perform quantitative NPM1 mutation assessment using ultradeep (approximately 300 0003–500 0003) sequencing (NGS-qNPM1) as a method of assessing residual disease burden in patients with acute myeloid leukemia. Design.—A flexible NGS-based assay for the detection and quantitation of NPM1 mutations was developed by polymerase chain reaction amplification of target DNA sequences, sequencing on an Illumina (San Diego, California) MiSeq, and analyzing data with an in-house–designed bioinformatic pipeline. NGS-qNPM1 was compared with current NPM1 quantitation methods (real-time quantitative-polymerase chain reaction and multiparame-ter flow cytometry). Results.—The NGS-qNPM1 assay had a sensitivity of between 104 and 105 and showed high concordance and correlation with reference methodologies. Moreover, the NGS-qNPM1 assay was able to be integrated into the laboratory’s existing, targeted amplicon-based sequencing workflow. Conclusions.—An NGS-based, quantitative NPM1-mu-tation assessment can be used to monitor patients with acute myeloid leukemia, and it has some practical advantages over existing modalities.