Optimization of Analytical Performances and Routine Laboratory Implementation of NPM1 Mutation Detection by Digital...

Quantification and monitoring of NPM1 mutations represent a key tool in the management of acute myeloid leukemia (AML), offering strong prognostic value and serving as a marker of minimal residual disease (MRD). Digital PCR (dPCR) provides absolute quantification with high sensitivity and specificity, offering advantages over conventional qPCR-based methods, particularly for rare or atypical NPM1 mutations.

We adapted and evaluated a digital PCR strategy including both monoplex and multiplex assays targeting the most common NPM1 mutations (types A, B, D) as well as rare variants. A touch-down PCR protocol was optimized to harmonize the dPCR protocol and enhance specificity. Assay performance was evaluated in terms of limit of detection (LoD), limit of quantification (LoQ), linearity, and specificity, and compared to quantitative PCR (qPCR).

The multiplex dPCR assay allowed detection of all clinically relevant NPM1 mutations with an LoD of 0.01%, while monoplex type A, B and D assays reached 0.001% for type A, B and D. The method showed strong linearity (R ² > 0.99 for all targets) and high concordance with qPCR (R² = 0.9967). In patient samples, dPCR demonstrated improved sensitivity over qPCR, detecting low-level NPM1 mutations in samples previously negative by qPCR, and enabled early relapse detection.

The approach enabled early relapse prediction in selected patients and improved molecular follow-up, even for rare NPM1 mutations not covered by standard qPCR panels. It simplifies laboratory routine and improves MRD assessment, according to current ELN guidelines and the growing need for personalized molecular monitoring in AML.