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Clinical Laboratory Validation and Implementation of Quantitative, Real-Time PCR-based Detection of NPM1 Type A Mutation by Joelle Racchumi, Wayne Tam, Michael J. Kluk

Background: NPM1 mutations have prognostic significance in acute myeloid leukemia (AML) and monitoring mutant NPM1 levels during and after therapy has been described to predict relapse and survival. Despite the published significance of this molecular biomarker, routine monitoring for mutant NPM1 levels has not been widely adopted in academic clinical laboratories. Therefore, our objective was to validate a quantitative, reverse transcription-PCR assay for the detection of NPM1 Type A mutant transcripts for use in the clinical laboratory.
Methods: A quantitative, real-time, reverse-transcription PCR-based method for the detection of NPM1 Type A mutant transcripts was validated for use in routine clinical practice. Results from this assay were compared to results from orthogonal methods, including next generation sequencing and digital droplet PCR.
Results: This real-time, reverse-transcription PCR-based method is sensitive (limit of detection: 0.0150% NCN and reproducible (≤ 0.5 log10-fold variation). We summarize the rigorous validation results and share observations that will help other clinical laboratories that may wish to implement this testing. We show the superior sensitivity of this assay compared to other assays (e.g., 45 gene Myeloid Next Generation Sequencing panel) and present a representative case which highlights the assay’s utility in the pathologic assessment of cases with borderline morphologic or flow cytometric findings.
Conclusions: As molecular testing for residual disease in AML continues to expand, this sensitive and reproducible method will be an appropriate testing option for the detection of NPM1 Type A mutant transcripts in clinical practice.

DOI: 10.7754/Clin.Lab.2020.200104