Detection of the nucleophosmin gene mutations in acute myelogenous leukemia through RT-PCR and polyacrylamide gel electrophoresis

Objectives: Mutations in the C‐terminal region of the nucleophosmin (NPM1) gene occur in approximately 60% of acute myeloid leukemia (AML) cases with normal karyotype and represent the most common genetic lesion presently known in this disease. Because of their frequency and favorable impact on prognostic outcome, screening for this aberration is currently recommended in routine diagnostic characterization of AML. Several techniques enabling to detect NPM1 mutation have been reported, but all require sophisticated equipment, which represent an obstacle particularly in countries with limited resources. Methods: We designed an RT‐PCR strategy to amplify NPM1 exon 12 followed by electrophoresis and fragment visualization on polyacrylamide gels to discriminate a 4–5 bp size difference resulting from mutations in this gene. A hemi‐nested method was designed to increase sensitivity for the study of minimal residual disease (MRD). Results: The assay enabled specific detection of NPM1 mutations in 12/36 patients. A 10^?2 sensitivity level was obtained using one amplification round, while the hemi‐nested PCR approach yielded a 10^?5 sensitivity level, therefore proving useful to assess MRD in patients carrying the mutation. The results were independently validated in 24 AML cases by sequencing analysis. Conclusions: This simple and low‐cost assay may integrate diagnostic work‐up of AML and could be used for assessment of response to therapy in patients with NPM1 mutations.