Multiplex Droplet Digital PCR Covers 10 NPM1 Mutations for AML Measurable Residual Disease
A novel multiplex ddPCR assay using one generic probe and 10 mutation-specific primers was validated for detecting NPM1 mutations in acute myeloid leukemia, achieving a limit of detection of 0.01% VAF on genomic DNA. The study found that variant allele frequency better reflects tumor burden than mRNA expression, particularly in patients with co-occurring FLT3-ITD mutations. This flexible, expandable assay addresses the clinical gap of monitoring rare NPM1 variants beyond the three most common types.
The original study
Validation of a Multiplex mRNA- and gDNA-Based Droplet Digital PCR Assay in Acute Myeloid Leukemia Patients with an NPM1 Mutation.
- Authors
- Wierenga ATJ, Hesp LB, Simpelaar A, Morsink LM, Woolthuis CM, Schuringa JJ, et al.
- Journal
- Clinical chemistry
- Type
- Journal Article, Validation Study
- PMID
- 41206522
Original abstract
BACKGROUND: NPM1 is a disease-defining gene in the diagnosis of acute myeloid leukemia (AML) and is important for measurable residual disease (MRD) assessment. Over 50 different NPM1 mutations have been described, but only the 3 most common are routinely monitored during follow-up. METHODS: We developed a multiplex droplet digital polymerase chain reaction (PCR) assay for measurement of both variant allele frequencies (VAF) and mRNA transcripts of 10 different NPM1 mutations, using one generic probe, one generic NPM1 reverse primer, and 10 mutation-specific NPM1 forward primers. ABL1 expression and AP3B1 VAF were used as references. The performance of the assay was tested in diagnosis and follow-up samples from patients with an NPM1-mutated AML. RESULTS: Our assay shows negligible false-positive signals and high assay precision, leading to low limits of detection of at least 0.01%. The assay can easily be expanded to cover more NPM1 mutations by adding extra mutation-specific forward primers to the primer mix. Overall, a good correlation between mutant NPM1 expression and VAF was found. However, we also observed discrepant variable ABL1 expression levels, especially in AML patients with fms-related receptor tyrosine kinase 3-internal tandem duplications co-mutations. CONCLUSION: We developed a robust and extremely flexible mRNA- and gDNA-based multiplex droplet digital PCR NPM1 assay. Because the AML tumor load is better reflected by mutant NPM1 VAF than expression level, we recommend using the gDNA-based mutant NPM1 MRD assay with a VAF detection limit of 0.01%. For MRD signals below 0.01%, our more sensitive mRNA-based method can be used, although further research has to prove its clinical impact.