Clinical Validation and Implementation of a Measurable Residual Disease Assay for NPM1 in Acute Myeloid Leukemia by Error-Corrected Next-Generation Sequencing

Mol Diagn Ther. 2019 Dec;23(6):791-802. doi: 10.1007/s40291-019-00436-8.


Background: Nucleophosmin 1 (NPM1) is one of the most commonly mutated genes in acute myeloid leukemia, with mutations observed in approximately 30% of all adult cases. The persistence of NPM1 mutations following chemotherapy is associated with a greater risk of relapse as well as a lower rate of survival, making NPM1 measurable residual disease (MRD) an informative clinical target.

Methods: Herein, we have developed a straightforward unique molecular identifier (UMI)-based amplicon next-generation sequencing method for the detection of NPM1-mutated MRD that addresses some of the limitations present in other assays.

Results: The NPM1 assay allowed for accurate counting of individual mutant and wild-type molecules down to 0.01% variant allelic frequency. In silico contamination experiments highlighted the ability of this UMI methodology to maximize specificity through dramatic reductions in sequencing/demultiplexing bleed-through error.

Conclusion: Performance and clinical utility of the NPM1 MRD assay are established via both validation experiments and analyses of live performance over 1.5 years of routine clinical service.

MeSH terms

  • Biomarkers, Tumor / blood
  • Biomarkers, Tumor / genetics
  • High-Throughput Nucleotide Sequencing / methods*
  • Humans
  • Leukemia, Myeloid, Acute / diagnosis
  • Leukemia, Myeloid, Acute / genetics*
  • Limit of Detection
  • Mutation
  • Neoplasm, Residual / diagnosis*
  • Neoplasm, Residual / genetics
  • Nuclear Proteins / blood
  • Nuclear Proteins / genetics*
  • Nucleophosmin
  • Recurrence
  • Sensitivity and Specificity
  • Sequence Analysis, DNA


  • Biomarkers, Tumor
  • NPM1 protein, human
  • Nuclear Proteins
  • Nucleophosmin