NPM1 Mutation Detection in Acute Myeloid Leukemia: A Method Comparison Study

Mutations in the nucleophosmin (NPM1) gene have been used as molecular biomarkers for prognostication of patients with adult acute myeloid leukemia (AML).

We designed a rapid and sensitive method using the allele-specific-refractory mutation system-polymerase chain reaction (ARMS-PCR) to detect the most common mutations of NPM1 gene, which are mostly four base pair insertions and compared its efficacy with direct sequencing and capillary electrophoresis which served as the gold standards.

The incidence of mutation was 22% (33% of patients with normal karyotypes had mutation compared with 16% of patients with abnormal karyotypes) based on the results obtained with capillary electrophoresis analysis and direct sequencing. All of the specimens determined to be mutation-positive by the gold standard tests were also positive by the ARMS-PCR method. Significantly, the ARMS-PCR test also helped determine the mutation status of an extra set of patients who had low call rates on capillary electrophoresis and appeared normal on direct sequencing.

The low mutation rate in some patients hindered its detection in the gold standard assays because of the interference of the mutation signal by high background noise. The low sensitivity of the gold standard assays for detecting low copy number mutations rates thus increase their risk of producing false negative results that adversely affects prognostication and therapy. Our results suggest that the mutation detection rate of the ARMS-PCR assay is better than existing tests. This is most probably because of the fact that in an ARMS-PCR-based method, the mutated variant is specifically amplified, based on a mutation-specific primer.

We conclude that the high sensitivity of the ARMS-based method together with its rapidity and low expense should make it a suitable choice for clinical laboratories.