New Quantitative Method to Identify NPM1 Mutations in Acute Myeloid Leukaemia

Somatic mutations in the NPM1 gene, which encodes for nucleophosmin, have been reported to be the most frequent genetic abnormalities found in acute myeloid leukaemia (AML). Their identification and quantification remain crucial for the patients’ residual disease monitoring. We investigated a new method that could represent a novel reliable alternative to sequencing for its identification. This method was based on high-resolution melting analysis in order to detect mutated patients and on an allele-specific oligonucleotide real-time quantitative polymerase chain reaction (ASO-RQ-PCR) for the identification and quantification of the transcripts carrying NPM1 mutations (NPM1m). Few patients carrying known NPM1m enabled us to set up a table with the different primers’ ΔCT values, identifying a profile for each mutation type. We then analysed a series of 337 AML patients' samples for NPM1 mutational status characterization and confirmed the ASO-RQ-PCR results by direct sequencing. We identified some mutations in 86 samples, and the results were fully correlated in 100% of the 36 sequenced samples. We also detected other rare NPM1m in two samples, that we confirmed by direct sequencing. This highly specific method provides a novel quick, useful, and costless tool, easy to use in routine practice. 1. Introduction Nucleophosmin mutations (NPM1m) occur in about one-third of acute myeloid leukaemias (AMLs) [1], and the current classification of myeloid neoplasms defined a recent entity of NPM1-mutated AML with distinct biological, clinical, and prognostic features [2]. Moreover, the detection and quantification of NPM1m represents a major specific marker for the molecular monitoring of minimal residual disease (MRD) in AML, since it appears as an early initiating event in leukaemogenesis [3, 4]. The expression of this marker is very stable during disease evolution, and the detection of increasing NPM1m expression levels seems strongly predictive for impending haematological relapse [5, 6]. Finally, patients’ stratification in international clinical protocols and the development of new targeted therapies rely on the NPM1 status in AML [7]. Thus, the identification of NPM1m is of critical importance for the AML patients’ admission process. Most of the NPM1m identified to date, as the type A mutation (75–80% of cases), are exon 12 frameshift mutations [1, 5, 8] leading to an aberrant accumulation of the protein in the cytoplasm [9]. Several protocols and methods have been developed for the detection of NPM1m including DNA sequencing of different mutation-specific