Ultra-Sensitive DNA-Based NPM1 Mutation Detection Using superRCA on Flow Cytometry

Background: NPM1 mutations are established biomarkers for measurable residual disease (MRD) monitoring in acute myeloid leukemia (AML). RT-qPCR is widely used for NPM1 MRD assessment, but sensitive DNA-based methods offer advantages in sample stability and workflow. superRCA is a novel mutation detection technology that combines genotyping with rolling circle amplification to convert target molecules into fluorescent particles that can be enumerated by flow cytometry. Here, we evaluated the performance of superRCA for detection of the most common NPM1 variants, MutA, MutB, and MutD. Methods: Analytical performance was assessed by determining limit of blank (LoB), limit of detection (LoD), limit of quantification (LoQ), precision, linearity, and cross-reactivity. Method comparison was performed using 83 biobanked clinical samples from patients with NPM1 MutA-positive AML previously analyzed by RT-qPCR, qPCR, ddPCR, and deep sequencing. Correlation, agreement, and positive and negative percent agreement (PPA/NPA) versus RT-qPCR were evaluated. Results: The superRCA assay demonstrated LoD below 0.005% variant allele frequency (VAF) for all three NPM1 variants. Linearity was tested and demonstrated over 0.01–10.24% VAF. superRCA showed strong correlation with the other methods. Using LoD as the positivity threshold, PPA and NPA versus RT-qPCR detectable signal were 64% and 100%, respectively; using LoB, PPA was 89% and NPA 92%. Several superRCA-positive samples were negative by lower-input DNA methods. Conclusions: superRCA introduces ultra-sensitive molecular testing by combining sequence-specific DNA genotyping and flow cytometric enumeration. The NPM1 superRCA assay demonstrated low background, high sensitivity, precision, and broad linearity, with strong correlation with established molecular methods. These findings support further evaluation of superRCA for NPM1 MRD monitoring in AML.