Abstract
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.
Highlights
Nucleophosmin mutations (NPM1m) occur in about onethird 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]
Among the 337 samples of AML diagnosis, the high-resolution melting (HRM) screening revealed the presence of NPM1 mutation in 88 of them (26.1%)
To confirm the absence of mutations and make sure that the new assay does not give false negative results, we investigated by direct sequencing 20 cases that were considered as NPM1 mutations (NPM1m) negative with the HRM analysis
Summary
Nucleophosmin mutations (NPM1m) occur in about onethird 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]. Several protocols and methods have been developed for the detection of NPM1m including DNA sequencing of different mutation-specific RT-PCR assays [10,11,12,13], denaturing high-performance liquid chromatography [14], capillary electrophoresis [15], locked nucleic acid-mediated polymerase chain reaction clamping [16], and high-resolution melting analysis [17]. These methods possess a high specificity to assess NPM1 mutational status at diagnosis, they always require direct sequencing for NPM1m characterization, a more expensive and time-consuming method. We investigated a new strategy where (i) mutational status, (ii) distinction between NPM1 mutation types, and (iii) quantitative value of the identified mutation at diagnosis would be rapidly obtained
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