Evaluation of methods for detection of NPM1 gene mutations in acute myeloid leukemia

The purpose of this study was to establish real-time based methods for detection of NPM1 gene mutation in acute myeloid leukemia(AML).Primers/probes were designed according to the clustered region of NPM1 mutations on exon 12.Two real-time PCR assays,including high resolution melting curve(HRM) and allele-specific PCR(AS-PCR),were developed and clinically evaluated with 89 AML samples,which were parallelly detected by capillary electrophoresis(CE) and sequencing.The results showed that a total of 17 mutation-positive samples were detected,including type A(15 cases),type B(1 case) and type Nm(1 case).HRM assay could detect all mutant types,and the analytical sensitivity was around 5%.In contrast,AS-PCR assay detected only 95% mutant types,but its sensitivity was as high as 0.01%.It is concluded that considering the characteristics of each method as well as the clinical evaluation results,HRM may be used for screening of NPM1 mutations at diagnosis,while the AS-PCR can be used for the MRD quantification during follow-up.